Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02728605 2011-01-18
MEASUREMENT OF ANTI-BETA AMYLOID ANTIBODIES IN HUMAN
BLOOD
BACKGROUND OF THE INVENTION
Introduction
[00011 Misfolding and aggregation of the amyloid beta peptide (A[3) is central
to the
pathogenesis of Alzheimer's disease (AD). The human immunoglobulin G (IgG)
repertoire
contains endogenous antibodies against the A[3 peptide that arise in the
absence of vaccination or
passive immunization. Endogenous anti-An antibody activity has been detected
in the blood of
normal adults of various ages and patients with AD (Weksler et al., Exp
Gerontol., 37:943-948
(2002); Hyman et al., Ann Neurol., 49:808-810.5 (2001); Mruthinti et at.,
Neurobiol Aging.,
25:1023-1032 (2004); Nath et al., Neuromolecular Med., 3:29-39 (2003); Sohn et
al., Frontiers
in Bioscience., 14:3879-3883 (2009)). The human plasma-derived antibody
preparation known
as intravenous immunoglobulin (IVIG) has been reported to contain elevated
levels of
endogenous anti-amyloid antibodies and is under study as a potential treatment
for AD (Dodel et
al., JNeurol Neurosurg Psychiatry., 75:1472-1474 (2004); Hyman et al., Ann
Neurol., 49:808-
810.5 (2001); Mruthinti et al., Neurobiol Aging., 25:1023-1032 (2004)).
[00021 Published studies provide widely disparate estimates of the relative
titers of anti-Ali
antibodies in AD patients versus aged normal controls. Initial studies of
intact plasma specimens
by standard ELISA methods ascribed lower titers of endogenous antibodies
against A[3
monomers to AD patients than aged-matched non-demented controls (Weksler et
al., Exp
Gerontol., 37:943-948 (2002)). Subsequent studies reported equal or increased
titers of
circulating anti-A(3 monomers antibodies in AD patients (Mruthinti et al.,
Neurobiol Aging,
25:1023-1032 (2004)) based on ELISAs performed on plasma immunoglobulin eluted
from
Protein G columns at low pH (Akerstrom et al., JBiol Chem., 261:10240-10247
(1986)). The
higher titers obtained by this method were hypothesized to reflect the
presence of a pool of
bound anti-amyloid antibodies that were undetected in assays of whole plasma
and measurable
when freed from antigen by acidification in the course of protein G
purification. However in
murine models, exposure of polyclonal IgG to low pH results in partial
denaturation of
antibodies and generates large artifactual increases in apparent anti-A(3
antibody titers (Li et al.,
BMC Neurosci., 8:22 (2007)). Assays that employ IgG isolated from human plasma
by protein
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CA 02728605 2011-01-18
G chromatography and acid elution may therefore result in artifactually
elevated measures of
anti-amyloid activity.
100031 The measurement of endogenous anti-A(3 antibody activity is further
complicated by
the existence of multiple classes of human antibodies that recognize linear as
well as
conformational neo-epitopes on aggregated forms of A. Reports to date have
identified
endogenous human antibodies against A[3 fibrils (O'Nuallain et al., Jlmmunol.,
176:7071-7078
(2006)), A[3 oligomers (Moir et al., JBiol Chem., 280:17458-17463 (2005);
Relkin et al.,
Alzheimer's and Dementia, 3:5196, X (2008); O'Nuallain et al., Biochemistry,
47:12254-12256
(2008)) and conformational epitopes on A0 monomers (Baumketner A et al., Prot
Sci., 15:420-
428 (2006)). Other types of amyloid binding antibodies and even catalytic
antibodies against A[3
have been reported (Taguchi H et al., JBiol Chem., 284:4714-4722 (2008)).
Conventional
ELISAs may under- or over-estimate total anti-amyloid activity if they fail to
take into account
the diversity of antibody types present as well as such phenomena as cross-
reactivity and
polyvalency.
[00041 Polyvalent antibodies to both foreign and self-antigens are part of the
natural antibody
repertoire of humans (Djoumerska et al., Scand J of Immunol., 61: 357-363
(2005)). Most
polyvalent antibodies have germ line hypervariable regions, display lower
affinity for their
antigens compared to monovalent affinity-maturated antibodies and have more
flexible antigen-
binding sites. They are thought to serve as a defense mechanism against
pathogens. An
interesting feature of some polyvalent antibodies is that they can be
generated from monovalent
antibodies by mild disruptive treatments, e.g., exposure to pH below about 4
or disruptive agents
(Bouvet et al., JAutoimmun., 16:163-172 (2001); Dimitrov et al., Molec
Immunol, 44:1854-1863
(2007)).
[00051 Methods to increase sensitivity and signal to noise of anti-An antibody
assays have
been described, including a radio-immunoprecipitation assay (Brettschneider et
al., Biol
Psychiatry., 57:813-816 (2005)) and a novel peptide microarray method that was
recently used to
characterize endogenous human anti-A(3 antibodies in plasma as a function of
age and AD
(Britshgi et al., Proc. Natl. Acad. Sci. USA., 106:12145-12150 (2009)).
Although the latter
approach yielded improved signal to noise ratios compared to ELISA, its use of
whole plasma
may be problematic owing to the effects of other plasma proteins and
macromolecules on anti-
A[3 antibody measurements.
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CA 02728605 2011-01-18
[00061 These and other factors that may confound measurements of endogenous
anti-A(3
antibody levels have important implications for studies of treatment of AD by
IVIG (intravenous
immunoglobulin preparation). IVIG was first reported to contain anti-A(3
monomer antibodies
(Dodel et al., JNeurol Neurosurg Psychiatry., 75:1472-1474 (2004)) at
relatively low levels.
The reported paucity of anti-An antibodies brought into question the potential
for IVIG to exert
anti-amyloid effects in AD patients. Nevertheless, human clinical trials of
IVIG in AD patients
resulted in positive clinical outcomes as well as altered levels of A(3 in
plasma and cerebrospinal
fluid (Dodel et al., JNeurol Neurosurg Psychiatry., 75:1472-1474 (2004);
Relkin et al.,
Neurobiol. ofAging, 30:1728-1736 (2009)). Although endogenous anti-amyloid
antibody
activity provided the original rationale for testing IVIG in AD, it is not
known with certainty
whether this is the major or exclusive mechanism of action of IVIG in this
disease. The question
of whether true endogenous anti-amyloid antibodies are present in human plasma
at levels
sufficient to exert biologically meaningful effects has yet to be answered.
100071 As such, improved methods of assaying anti-amyloid activity in human
plasma are
needed to better understand the possible role of endogenous antibodies in the
pathogenesis and
treatment of AD and other amyloid-associated disorders. The present invention
provides
improved detection methods that address problems associated with the detection
and quantitation
of amyloid-specific antibodies, and consequently, with the detection of
amyloid. The invention
thus provides for more accurate diagnosis of diseases associated with amyloid
proteins, patient
selection for amyloid-related treatment, and other amyloid related
applications.
BRIEF SUMMARY OF THE INVENTION
[00081 The invention provides improved methods for preparing and assaying
endogenous anti-
amyloid antibodies in human plasma and intravenous immunoglobulin
preparations. These
novel methods show that human plasma contains polyvalent antibodies that bind
to A(3 with
lower avidity, as well as endogenous anti-amyloid antibodies with moderate to
high avidity for
A[3 and its aggregates.
[00091 In one aspect, the invention provides a method for detecting high
avidity anti-amyloid
antibodies present in a sample. In some embodiments, the method comprises the
steps of (a)
contacting the biological sample with a plurality of amyloid antigens under
suitable conditions to
form: (i) a complex comprising an amyloid antigen and a low-affinity or non-
specific antibody;
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CA 02728605 2011-01-18
and (ii) a complex comprising an amyloid antigen and a high avidity anti-
amyloid antibody; (b)
washing the amyloid antigen complexes formed in step (a) with a solution
comprising a
chaotropic agent to dissociate at least one complex comprising an amyloid
antigen and a low-
affinity or non-specific antibody; and (c) detecting the presence or level of
the remaining
complexes.
[00101 In some embodiments, the biological sample is blood or a fraction
thereof (e.g., plasma
or serum). In some embodiments, the biological sample is separated using
thiophilic
chromatography prior to step (a). In some embodiments, the biological sample
is separated using
thiophilic chromatography after step (b). In some embodiments, the thiophilic
chromatography
is used to remove non-immunoglobulin substances from the biological sample, to
yield an
enriched immunoglobulin preparation.
[00111 In a second aspect, the invention provides a method detecting a high
avidity anti-
amyloid antibody present in a biological sample, the method comprising the
steps of (a)
contacting the biological sample with a thiophilic resin to bind the anti-
amyloid antibody; (b)
eluting the antibody from the thiophilic resin at a non-denaturing pH to form
an eluate; (c)
contacting the eluate with an amyloid antigen to form a complex comprising the
amyloid antigen
and the anti-amyloid antibody; and (d) detecting the presence or level of the
complex.
[00121 In some embodiments, the biological sample is blood or a fraction
thereof (e.g., plasma
or serum). In some embodiments, the complex formed between the amyloid antigen
and the
anti-amyloid antibody is washed with a solution containing a chaotropic salt
prior to detecting
the presence or level of the complex.
[00131 In a third aspect, the invention provides a method for identifying a
subject who is a
candidate for treatment of an amyloid-related disease or condition. In some
embodiments, the
method comprises the steps of (a) contacting a biological sample from the
subject with a
plurality of amyloid antigens under suitable conditions to form: (i) a complex
comprising an
amyloid antigen and a low-affinity or non-specific antibody; and (ii) a
complex comprising an
amyloid antigen and a high avidity anti-amyloid antibody; (b) washing the
amyloid antigen
complexes formed in step (a) with a solution containing a chaotropic agent to
dissociate at least
one complex comprising an amyloid antigen and a low-affinity or non-specific
anti-amyloid
antibody; (c) detecting the level of the remaining complexes; and (d)
determining whether the
subject would benefit from a treatment comprising administration of an
immunoglobulin
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CA 02728605 2011-01-18
preparation by comparing the level of the anti-amyloid antibody to a control
level. In one
embodiment, the treatment comprises the administration of an immunoglobulin
preparation.
[00141 In some embodiments of the aspects described herein, the control is
from an individual
or group of individuals that do not have an amyloid-related disease, such that
an increased level
of the remaining complex relative to control is indicative of a candidate for
treatment. In some
embodiments, the control is from an individual or group of individuals that do
have an amyloid-
related disease, such that a similar level of the remaining complex relative
to control is indicative
of a candidate for treatment. One of skill will understand how to select at
least one appropriate
control and interpret the results accordingly.
[00151 In a fourth aspect, the invention provides a method for identifying a
subject who is a
candidate for treatment of an amyloid-related disease or condition. In some
embodiments, the
method comprises the steps of (a) contacting the biological sample with a
thiophilic resin to bind
an anti-amyloid antibody present in the sample; (b) eluting the antibody from
the thiophilic resin
at a non-denaturing pH to form an eluate; (c) contacting the eluate with an
amyloid antigen to
form a complex comprising the amyloid antigen and the anti-amyloid antibody;
(d) detecting the
presence or level of the complex; and (e) determining whether the subject
would benefit from a
treatment comprising administration of an immunoglobulin preparation by
comparing the level
of the anti-amyloid antibody to a control level. In one embodiment, the
treatment comprises the
administration of an immunoglobulin preparation. As explained above, one of
skill will
understand how to select at least one appropriate control for diagnosing an
amyloid-associated
disorder and interpret the results accordingly.
[00161 In a fifth aspect, the invention provides a method for diagnosing a
disease associated
with an amyloid protein in a subject. In some embodiments, the method
comprises the steps of
(a) contacting a biological sample from the subject with a plurality of
amyloid antigens under
suitable conditions to form: (i) a complex comprising an amyloid antigen and a
low-affinity or
non-specific antibody; and (ii) a complex comprising an amyloid antigen and a
high avidity anti-
amyloid antibody; (b) washing the amyloid antigen complexes formed in step (a)
with a solution
containing a chaotropic agent to dissociate at least one complex comprising an
amyloid antigen
and a low-affinity or non-specific antibody; (c) detecting the level of the
remaining complexes;
and (d) diagnosing the subject by comparing the level of the anti-amyloid
antibody to a control
level. In some embodiments, the invention provides methods for diagnosing
Alzheimer's
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disease, or a likelihood of developing the disease, in a subject. As explained
above, one of skill
will understand how to select at least one appropriate control for diagnosing
an amyloid-
associated disorder and interpret the results accordingly.
[0017] In a sixth aspect, the invention provides a method for diagnosing a
disease associated
with an amyloid protein in a subject. In some embodiments, the method
comprises the steps of
(a) contacting the biological sample with a thiophilic resin to bind the anti-
amyloid antibody; (b)
eluting the antibody from the thiophilic resin at a non-denaturing pH to form
an eluate; (c)
contacting the eluate with an amyloid antigen to form a complex comprising the
amyloid antigen
and the anti-amyloid antibody; (d) detecting the presence or level of the
complex; and (e)
diagnosing the subject by comparing the level of the anti-amyloid antibody to
a control level. As
explained above, one of skill will understand how to select at least one
appropriate control for
diagnosing an amyloid-associated disorder and interpret the results
accordingly.
[0018] In a seventh aspect, the invention provides a method for providing a
prognosis for the
progression of a disease associated with an amyloid protein in a subject. In
some embodiments,
the method comprises the steps of (a) contacting a biological sample from the
subject with a
plurality of amyloid antigens under suitable conditions to form: (i) a complex
comprising an
amyloid antigen and a low-affinity or non-specific antibody; and (ii) a
complex comprising an
amyloid antigen and a high avidity anti-amyloid antibody; (b) washing the
amyloid antigen
complexes formed in step (a) with a solution containing a chaotropic agent to
dissociate at least
one complex comprising an amyloid antigen and a low-affinity or non-specific
antibody; (c)
detecting the level of the remaining complexes; and (d) providing a prognosis
for the progression
of the disease by comparing the level of the anti-amyloid antibody to a
control level. In some
embodiments, the invention provides methods for providing a prognosis for the
progression of
Alzheimer's disease in a subject. As explained above, one of skill will
understand how to select
at least one appropriate control for providing a prognosis for an amyloid-
associated disorder and
interpret the results accordingly.
[0019] In an eighth aspect, the invention provides a method for providing a
prognosis for the
progression of a disease associated with an amyloid protein in a subject. In
some embodiments,
the method comprises the steps of (a) contacting the biological sample with a
thiophilic resin to
bind the anti-amyloid antibody; (b) eluting the antibody from the thiophilic
resin at a non-
denaturing pH to form an eluate; (c) contacting the eluate with an amyloid
antigen to form a
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complex comprising the amyloid antigen and the anti-amyloid antibody; (d)
detecting the
presence or level of the complex; and (e) providing a prognosis for the
progression of the disease
by comparing the level of the anti-amyloid antibody to a control level. In
some embodiments,
the invention provides methods for providing a prognosis for the progression
of Alzheimer's
disease in a subject. As explained above, one of skill will understand how to
select at least one
appropriate control for providing a prognosis for an amyloid-associated
disorder and interpret the
results accordingly.
[00201 In a ninth aspect, the invention provides a method for providing a
prognosis for
treatment of a disease associated with an amyloid protein, for example a known
therapy or
preventative strategy for AD. In some embodiments, the treatment can comprise
administration
of an immunoglobulin preparation such as IVIG in a subject. In some
embodiments, the method
comprises the steps of (a) contacting a biological sample from the subject
with a plurality of
amyloid antigens under suitable conditions to form: (i) a complex comprising
an amyloid antigen
and a low-affinity or non-specific antibody; and (ii) a complex comprising an
amyloid antigen
and a high avidity anti-amyloid antibody; (b) washing the amyloid antigen
complexes formed in
step (a) with a solution containing a chaotropic agent to dissociate at least
one complex
comprising an amyloid antigen and a low-affinity or non-specific antibody; (c)
detecting the
level of the remaining complexes; and (d) providing a prognosis for treatment
of the disease by
comparing the level of the anti-amyloid antibody to a control level. In some
embodiments, the
invention provides methods for providing a prognosis for the treatment of
Alzheimer's disease in
a subject. As explained above, one of skill will understand how to select at
least one appropriate
control for diagnosing an amyloid-associated disorder and interpret the
results accordingly.
[00211 In a tenth aspect, the invention provides a method for providing a
prognosis for
treatment of a disease associated with an amyloid protein, for example a known
therapy or
preventative strategy for AD. In some embodiments, the treatment can comprise
administration
of an immunoglobulin preparation such as IVIG in a subject. In some
embodiments, the method
comprises the steps of (a) contacting the biological sample with a thiophilic
resin to bind the
anti-amyloid antibody; (b) eluting the antibody from the thiophilic resin at a
non-denaturing pH
to form an eluate; (c) contacting the eluate with an amyloid antigen to form a
complex
comprising the amyloid antigen and the anti-amyloid antibody; (d) detecting
the presence or
level of the complex; and (e) providing a prognosis for treatment of the
disease by comparing the
level of the anti-amyloid antibody to a control level. In some embodiments,
the invention
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CA 02728605 2011-01-18
provides methods for providing a prognosis for the treatment of Alzheimer's
disease in a subject.
As explained above, one of skill will understand how to select at least one
appropriate control for
diagnosing an amyloid-associated disorder and interpret the results
accordingly.
BRIEF DESCRIPTION OF THE DRAWINGS
[00221 Figure 1. IgG in human plasma binds significantly to empty ELISA wells
as well as to
A(3. The relative binding to A[3 and blank plates is variable across
individuals. Human plasma
samples (n=23) were assayed by ELISA using plates with blank well and plates
bearing A[3
monomer peptide. The resultant absorbance values (A450õ,,,) are plotted as
histograms (blank
wells, black; A[3 wells, grey) for each individual.
[00231 Figure 2. Binding to empty wells occurs through the antigen binding
region (Fab) of
the IgG molecule. Commercially available Fab, Fab2' and F, fragments, isolated
from pooled IgG
of five individuals, were bound to blank and amyloid-bearing plates by
standard ELISA
methodology. Bound Fab and Fab2' fragments were detected using an anti-human
kappa
secondary antibody; F, fragments using an anti-human IgG secondary antibody
specific for the
F, portion of the molecule. Binding to blank plates is clearly mediated
through the Fab and Fab2'
regions of the IgG molecule rather than the F, region.
[00241 Figure 3. Cross-reactivity of anti-An antibodies in IVIG is exemplified
by
thyroglobulin's effects on the binding properties of anti-An antibodies (a =
A[3 plate, b = A[3
plate with thyroglobulin competition, c = thyroglobulin plate, d =
thyroglobulin plate with
thyroglobulin competition). ELISA measurements were made on using 3-fold
serial dilutions of
IVIG, starting at I mg/ml with and without the addition of 40 g/ml
thyroglobulin on plates
bearing either thyroglobulin (triangles) or A(3 monomer peptide (circles).
This level of
thyroglobulin completely eliminates the binding of anti-thyroglobulin
antibodies in IVIG to
thyroglobulin plates and reduces the binding to A(3 plates by approximately
50%.
[00251 Figure 4. The avidity of IVIG antibodies bound to ELISA plates bearing
A[342
oligomers is greater than those bound to empty ELISA wells. To measure
avidity, IVIG was
bound to ELISA plates bearing A042 oligomers or blank plates bearing no
antigen.
Quadruplicate wells were then incubated with increasing concentrations of the
chaotropic salt
ammonium thiocyanate, as indicated, and the amount of bound human antibody
determined by
standard ELISA methods. The results shown are the means and standard
deviations of two
8
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independent determinations. Antibodies binding to A0 oligomers have a greater
avidity for their
ligand than those that bind to empty wells.
[00261 Figure 5. 4M thiocyanate treatment improves the ratio of binding to
wells containing
A042 oligomers vs binding to empty wells. Shown is a standard ELISA experiment
in which the
solid triangles indicate the binding to A042 oligomer plates and the solid
circles, the binding to
blank plates. Open symbols indicate the same binding experiment followed by
incubation with
4M thiocyanate. The ratio of AR42 oligomer to blank plate binding using
purified human IgG
(I mg/ml) from control subjects is substantially increased from approximately
3, without 4M
thiocyanate treatment, to between 7 and 10 following this treatment. Thus,
treatment with the
chaotropic agent permits the measurement of the higher avidity anti-A(3
oligomer antibodies in
mixtures of purified human IgG, independent of low avidity antibodies that
bind to empty
ELISA wells.
[00271 Figure 6. Coomassie-stained 10% SDS:NuPAGE gel of protein samples
reduced with
R mercaptoethanol and loaded at equal mass shows IgG purified by
chromatography on
Thiophilic Adsorbant is nearly completely free of contaminating plasma
proteins. The lanes are
(a) IVIG, (b) IgG purified by chromatography on protein G, (c) IgG purified by
chromatography
on Thiophilic Adsorbant and (d) the original plasma sample. Also indicated are
the molecular
weight markers (M) and their sizes in kD.
100281 Figure 7. Binding of whole plasma and antibodies isolated from plasma
by Thiophilic
or Protein G chromatography to an A(3 oligomer SPR sensor. SPR sensogram shows
arbitrary
response units (y-axis) as a function of time using an A042 oligomer sensor.
Sample injection
was begun at 0 and ended at 125 seconds; SPR buffer flow was continued until
250 seconds
before stripping and reconstitution of the sensor. The red line showing
binding response of
plasma is near zero as a result of interference from other plasma proteins and
macromolecules.
The bottom (pink) line shows the response to IgG isolated using protein G and
acid elution. The
result appears negative because of induced polyvalency and the resulting
increased binding to the
control sensor. The top (blue) line is the response of IgG prepared using
Thiophilic Adsorbant.
The initial 125 seconds reflect total anti-oligomer antibody binding activity
(high and low
affinity) whereas the portion of the sensogram after buffer wash (after 125
seconds) is principally
attributable to antibodies with high affinity binding activity.
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DETAILED DESCRIPTION OF THE INVENTION
Introduction
[00291 The diagnosis of Alzheimer's disease is difficult owing to the fact
that the disease,
especially in the early stages, presents with symptoms common to various other
psychological
conditions. Definitive diagnosis may be achieved by the visualization of AR
tangles and plaques
formed in the brain. Likewise, other amyloid-related diseases and conditions
are commonly
diagnosed with the help of tissue biopsies, on which amyloid deposits can be
stained using the
Congo red procedure (Linke, R.P. (2006) "Protein misfolding, aggregation and
conformational
diseases" (V.N. Uversky and A.L. Fink, eds.), Protein Reviews, Volume 4, (M.Z.
Atassi, ed.);
Chapter 11.1, pp. 239-276; Springer). However, due to the risks and
complications involved
with performing biopsies of organs such as the brain and heart, this is not a
practical solution for
the diagnosis of various amyloid-related diseases, including, Alzheimer's
disease, Creutzfeldt-
Jakob disease, and cardiac amyloidosis.
[00301 Advantageously, the present invention provides non-invasive methods for
the
diagnosis, or for aiding in the diagnosis, of amyloid-related diseases,
through the specific
detection and/or quantification of amyloid proteins in blood and plasma
samples. In certain
embodiments, the methods provided herein are useful for aiding in the
diagnosis of amyloid-
related disorders, wherein the method further comprises using a second
diagnostic method. For
example, the methods provided herein may be used in combination with other
diagnostic tools to
provide a diagnosis or prognosis for an amyloid protein related disorder. In
one embodiment, the
methods provided herein can be used in combination with tests and evaluations
used to diagnose
Alzheimer's disease, which include without limitation, cognitive tests,
psychosocial histories,
mental state examinations, neuropsychological testing, familial history, CT
scans,
electroencephalography (EEG), Positron Emission Tomography (PET) scans, Single
Photon
Emission Computed Tomography (SPECT) Scans, Magnetic Resonance Spectroscopy
Imaging
(MRSI), tests that rule out other disorders (e.g., physical exam, chest X-ray,
magnetic resonance
imaging, electrocardiogram (ECG)), and the like. These methods overcome
significant hurdles
associated with the detection of these proteins, such as the non-specific
binding of low avidity
and/or polyvalent antibodies to amyloid proteins.
100311 Similarly, the present invention also provides methods for aiding in
treatment decisions.
The non-invasive methods for detecting high avidity anti-amyloid antibodies
provided herein
allow for the assignment and/or administration of a therapy for an amyloid-
related disorder. For
CA 02728605 2011-01-18
example, the present invention provides methods for identifying a patient
likely to benefit from a
treatment for an amyloid-related disorder, diagnosing an amyloid-related
disorder, providing a
prognosis for an amyloid-related disorder. Accordingly, in certain
embodiments, the methods
further comprise a step of assigning and/or administrating a treatment for an
amyloid-related
disorder (e.g., IVIG administration or other known therapy) to the subject.
[00321 The human immunoglobulin G (IgG) repertoire contains endogenous
antibodies against
beta amyloid (A[3) that arise in the absence of immunization. These antibodies
can be used for
diagnosis and treatment of amyloid-related diseases, such as Alzheimer's
disease (AD). The
present invention addresses problems that complicate the measurement of these
antibodies in
plasma and pool immunoglobulin preparations such as IVIG (immunoglobulin
preparation from
pooled plasma). These problems include relatively high background binding to
empty ELISA
wells, assay interference attributable to other plasma proteins, and the
confounding effects of
polyvalency. Addressing the confounding issues that call antibody specificity
into question also
improves the reliability of amyloid detection using amyloid-specific
antibodies.
100331 The present invention provides, among other aspects, methods that
improve the
measurement of anti-A(3 antibodies in plasma. One factor that can confound
accurate of anti-All
antibodies measurements is the presence of polyvalent IgG in human plasma that
binds to blank
ELISA plates (as well as unfilled areas on A[3 plates). Blank plate binding is
variable across
individuals and not effectively eliminated by standard ELISA blocking agents
or background
subtraction.
[00341 Quantification of anti-A[3 antibodies in human plasma or serum has been
carried out in
most instances on ELISA plates bearing either monomeric or aggregated
(oligomeric or fibrillar)
All peptide. Plasma samples from normal human donors assayed by this method
bind
significantly to blank ELISA wells (wells not bearing any added antigen).
Extensive, non-
specific blank plate binding occurs despite the use of traditional blocking
agents such as skim
milk, fetal calf serum, albumin and commercial blocking preparations (Klaver
et al., J. Neurosci.
Meth. 187, 263-269).
[00351 The presence of antibodies that have the capacity to bind to blank
ELISA wells
complicates measurement of anti-Al oligomer antibodies in individuals since
the observed
absorbance values obtained for empty wells can be comparable or even greater
than those
obtained for amyloid-bearing plates (Figure 1).
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CA 02728605 2011-01-18
[00361 It is demonstrated herein that blank plate binding can be significantly
reduced by
exposure of the antigen-antibody complex to a chaotropic salt solution of
suitable molarity to
dislodge weakly bound polyvalent antibodies without significantly reducing the
binding of
specific anti-A(3 antibodies.
[00371 Another potential source of inaccuracy in anti-A(3 antibody
measurements is binding
interference by other proteins and macromolecules present in whole plasma.
Isolation of IgG
from plasma can eliminate this interference but exposure to low pH during
isolation by Protein G
chromatography generates polyvalency that artifactually inflates measured
levels of anti-A[3
antibodies.
[00381 Using the improved methods provided herein, surface plasmon resonance
(SPR)
measurements confirm that human blood (and IVIG) contains polyvalent
antibodies with
relatively low avidity for A[3, as well as antibodies with moderate to high
avidity for
conformational epitopes on A[3 monomers and aggregates (e.g., oligomers,
filaments).
[00391 It is demonstrated herein that more accurate binding measurements can
be obtained by
avoiding exposure of IgG to denaturing conditions during isolation. Assays of
IgG subjected to
thiophilic chromatography yield titers of anti-amyloid antibodies that are
essentially unchanged
from the starting material, indicating that no artifactual polyvalency has
been created. In
contrast, IgG subjected to acid-elution from a Protein G column artifactually
gains as much as an
order of magnitude or more of anti-amyloid activity. IgG produced by passage
over Thiophilic
absorbant columns can include antibodies bound to antigen. Thus, the measured
titer is that of
free antibody. Thiophilic chromatography removes a majority of the plasma
proteins, some of
which interfere with measurements of anti-amyloid antibodies in whole plasma.
Measurements
such as the microarray method using whole plasma may be confounded the
presence of other
proteins that can vary across individuals of different ages and disease states
(Britshgi M et al.,
Proc. Natl. Acad. Sci. USA., 106:12145-12150 (2009)).
[00401 As explained above, the complexities involved in measuring human anti-
amyloid
antibodies have created uncertainty as to whether endogenous amyloid-specific
IgG actually
exists or if such antibodies represent naturally-occurring or artifactually-
induced polyvalent
antibodies. The results presented herein show that endogenous human anti-
amyloid antibodies
can indeed be isolated from IVIG by affinity chromatography. These antibodies
show specificity
for either linear or conformational epitopes of beta amyloid with binding
constants in the
12
CA 02728605 2011-01-18
hundreds of namomolar range. This is within an order of magnitude of the Kd
measured for
murine monoclonal anti-amyloid antibodies using the same SPR sensors, and
represents
considerably higher binding avidity than would be expected were this activity
to have arisen
from a non-specific, polyvalent antibody-antigen interaction.
[0041] Moreover, anti-fibril antibodies isolated by sequential passage over
beta amyloid and
IAPP fibril affinity columns show less cross-reactivity with monomeric and
oligomeric amyloid
antigens, as well as blank plates. These results suggest that conformational
neo-epitopes that
differ across stages of amyloid assembly are recognized by the human IgG
repertoire.
[0042] It is shown herein that high avidity endogenous anti-amyloid antibodies
represent less
than 0.1 % of the total mass of plasma-derived human IgG. The greater amyloid
binding capacity
of IVIG relative to individual plasma samples could reflect the clonal
diversity of IVIG inherent
in its derivation from thousands of plasma donors. Another source of increased
anti-A[3 activity
could be less specific binding that arises as a consequence of the chemical
and physical
processes involved in IVIG production.
[0043] Naturally-occurring auto-antibodies can regulate the uptake of self
antigens and
promote specific CD4+ T cell responses (Nielsen, Eur Jlmmunol., 3 1:2660-2668
(2001)).
Polyvalent, anti-A(3 auto-antibodies and/or those generated by exposure to
mildly denaturing
conditions may have effects on the levels of A[3 peptide in circulation or the
central nervous
system. Thus, endogenous anti-An antibodies may be responsible for the
therapeutic benefits of
I V IG in AD patients.
[0044] Advantageously, the methods provided herein allow for the specific
detection and
quantitation of high avidity anti-amyloid antibodies. Specifically, the
methods provided herein
allow for the detection and quantification of a heterogenous population of
antibodies that bind an
amyloid protein of interest with high affinity, e.g., with a Kd in the range
of a monoclonal
antibody that was specifically raised against the amyloid protein of interest.
Definitions
[0045] As used herein, a "chaotropic agent" refers to a chemical that
destabilizes the three-
dimensional structure of proteins. In certain embodiments, a chaotropic agent
can refer to an
ionic chaotrope (e.g., a chaotropic ion or chaotropic salt) or alternatively
to a non-ionic
chaotrope. Non-limiting examples of chaotropic salts include: guanidinium
salts, e.g.,
13
CA 02728605 2011-01-18
guanidinium chloride, guanidinium nitrate, guanidinium thiocyanate;
thiocyanate salts, e.g.,
ammonium thiocyanate, potassium thiocyanate, sodium thiocyanate, lithium
thiocyanate,
calcium thiocyanate, guanidinium thiocyanate; perchlorate salts, e.g.,
ammonium perchlorate,
sodium perchlorate, lithium perchlorate, magnesium perchlorate, calcium
perchlorate; iodide
salts, e.g., ammonium iodide, potassium iodide, sodium iodide, lithium iodide,
magnesium
iodide, calcium iodide; chlorate salts, e.g., sodium chlorate, lithium
chlorate, magnesium
chlorate, calcium chlorate; and the like. Non-ionic chaotropes include,
without limitation, urea
and thiourea.
[0046] "Thiophilic," in reference to chromatography and similar affinity-based
methods, refers
to affinity selection based on a sulfur-containing ligand for a protein, in
particular, for
immunoglobulins, in high concentrations of certain salts. The protein can be
eluted by removal
of the salts. Thiophilic affinity chromatography (TAC) has a broad specificity
for
immunoglobulins of different classes and species, and offers advantages over
Protein A or G
purification because of the relatively mild conditions, reduced cost of
materials, and broad range
of specificity. Exemplary salts for use with TAC include ammonium, sodium, and
potassium
sulfate, though others are commonly used. TAC reagents and gels are
commercially available,
e.g., from Millipore . For a review, see Matejtschuk (2004) Methods in Mol.
Biol. 244:195-204.
[0047] The terms "antibody," "immunoglobulin," and like terms refer to a
polypeptide
substantially encoded by an immunoglobulin gene or immunoglobulin genes, or
fragments
thereof, which specifically bind and recognize an analyte (antigen). The
recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon
and mu constant
region genes, as well as the myriad immunoglobulin variable region genes.
Light chains are
classified as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or
epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD,
and IgE,
respectively.
100481 An exemplary immunoglobulin (antibody) structural unit is composed of
two pairs of
polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy"
chain (about 50-
70 kD). The N-terminus of each chain defines a variable region of about 100 to
110 or more
amino acids primarily responsible for antigen recognition. The terms variable
light chain (VL)
and variable heavy chain (VH) refer to these light and heavy chains
respectively. The C terminal
14
CA 02728605 2011-01-18
ends of each heavy chain are disulfide bonded together, and form the constant
region of the
antibody.
[0049] The term "specifically bind" refers to a molecule (e.g., targeting
agent, antibody) that
binds to a target (antigen, epitope, etc.) with at least 2-fold greater
affinity than non-target
compounds, e.g., at least 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-
fold, 20-fold, 25-fold,
50-fold, or 100-fold greater affinity.
[0050] The terms "antigen," "analyte," "antibody target," etc. are used
interchangeably herein.
In some cases, the site of interaction of an antibody with its target is
defined, e.g., the targeted
site, binding interaction site, or "epitope." In some cases, the epitope is a
three-dimensional
moiety. Thus, for example, where the target is a protein, the epitope can be
comprised of
consecutive amino acids, or amino acids from different parts of the protein
that are brought into
proximity by protein folding (e.g., a discontinuous epitope). The same is true
for other types of
target molecules that form three-dimensional structures.
[00511 Thus, the term "amyloid antigen" can refer to an amyloid monomer,
oligomer, or fibril,
or can instead refer to an amyloid fragment or single epitope. One of skill
will appreciate that a
given amyloid protein or aggregate will typically comprise a plurality of
amyloid antigens or
epitopes.
[0052] Antibody affinity is the strength of the reaction between a single
antigenic determinant
and a single combining site on the antibody. It is the sum of the attractive
and repulsive forces
operating between the antigenic determinant and the combining site of the
antibody. Affinity is
typically expressed in terms of a dissociation constant (KD, Kd, etc.). The
higher the affinity of
an antibody for an antigen, the lower the Kd.
[0053] As used herein, the term "low affinity" and "non-specific" are relative
terms. The
cutoffs between high, medium, low, and no affinity can be set by the user
depending on the
desired stringency, and the range of affinity measurements obtained in a
particular individual.
Typically, a high affinity antibody will have a Kd less than about 10 nM. In
certain
embodiments, a high affinity antibody will have a Kd less than about 10 nM or
less than about 9
nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, I nM, 900 pM, 800 pM, 700 pM,
600 pM,
500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5 pM, 1 pM, or
lower.
Typically, a low affinity antibody will have a Kd greater than about 100 nM.
In certain
CA 02728605 2011-01-18
embodiments, a low affinity antibody will have a Kd greater than about 100 nM
or greater than
about 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, I PM, 2
PM, 3
M, 4 M, 5 M, 6 pM, 7 M, 8 M, 9 M, 10 M, or greater.
[00541 Avidity is a measure of the overall strength of binding of an antigen
with many
antigenic determinants and multivalent antibodies. Avidity is influenced by
both the valence of
the antibody and the valence of the antigen. Avidity, however, is more than
the sum of the
individual affinities. That is, affinity refers to the strength of binding
between a single antigenic
determinant and an individual antibody combining site whereas avidity refers
to the overall
strength of binding between multivalent antigens and antibodies. In
embodiments of the
methods provided herein, an anti-amyloid antibody being detected has high
avidity for the target
amyloid protein. In certain embodiments, an anti-amyloid antibody being
detected has both high
avidity and high affinity for a target amyloid epitope. In other embodiments,
an anti-amyloid
antibody being detected has high avidity for a target amyloid protein yet low
affinity for an
individual amyloid epitope.
[00551 As used herein, an "amyloid protein" refers to a polypeptide capable of
polymerizing to
form a cross-beta structure in vivo or in vitro. These proteins are abnormally
deposited in organs
and/or tissues of individuals suffering from conditions characterized by
amyloidosis (e.g.,
Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, etc.).
Accordingly, an anti-
amyloid antibody is an antibody that specifically recognizes an amyloid
protein. Anti-amyloid
antibodies specifically recognize one or more oligomeric state of a particular
amyloid protein, for
example, an monomeric, dimeric, or oligomeric amyloid protein. Non-limiting
examples of
amyloid proteins include Beta amyloid (A[3; Abeta), Islet amyloid polypeptide
(IAPP; Amylin),
Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin (HD), Calcitonin
(CCP), Atrial
natriuretic factor (ANF), Apolipoprotein Al (Apo-A 1), Serum amyloid A protein
(SAA), Medin
amyloid (fragment of Milk fat globule-EGF factor 8 protein; MFG-E8), Prolactin
(PRL),
Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase C), Beta 2
microglobulin
([32M), Gelsolin (AGEL), Transforming growth factor-beta-induced protein ig-h3
(Beta ig-h3;
Keratoepithelin), Cystatin C (CST3), and Immunoglobulin light chain (AL). Any
of these or
other amyloid proteins can be detected by using the methods provided herein.
100561 As used herein, a "biological sample" refers to a cell or population of
cells or a quantity
of tissue or fluid from a subject. Most often, the sample has been removed
from an animal, e.g.,
16
CA 02728605 2011-01-18
a human. "Biological sample" includes blood and blood fractions (e.g., plasma,
serum, etc.),
other biological fluids (e.g., urine, saliva, lymph fluids, Cerebrospinal
fluid (CSF), etc.), sections
of tissues such as biopsy and autopsy samples, frozen sections taken for
histologic purposes,
lymph tissue, cultured cells, and the like.
[0057] As used herein, the term "about" denotes an approximate range of plus
or minus 10%
from a specified value. For instance, the language "about 20%" encompasses a
range of 18-22%.
As used herein, about also includes the exact amount. Hence "about 20%" means
"about 20%"
and also "20%." As used herein, "about" refers to a range of at or about the
specified value.
100581 The terms "dose" and "dosage" are used interchangeably herein. A dose
refers to the
amount of active ingredient given to an individual at each administration. The
dose will vary
depending on a number of factors, including frequency of administration; size
and tolerance of
the individual; severity of the condition; risk of side effects; and the route
of administration. One
of skill in the art will recognize that the dose can be modified depending on
the above factors or
based on therapeutic progress. The term "dosage form" refers to the particular
format of the
pharmaceutical, and depends on the route of administration. For example, a
dosage form can be
in a liquid, e.g., a saline solution for injection.
[0059] A "therapeutically effective" amount or dose or "sufficient/effective"
amount or dose,
is a dose that produces effects for which it is administered. The exact dose
will depend on the
purpose of the treatment, and will be ascertainable by one skilled in the art
using known
techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3,
1992); Lloyd, The
Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar,
Dosage
Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th
Edition, 2003,
Gennaro, Ed., Lippincott, Williams & Wilkins).
[0060] As used herein, the term "prevent" refers to a decreased likelihood or
reduced
frequency of cognitive symptoms or amyloid plaques in a patient. The
prevention may be
complete or partial, such that fewer symptoms are observed in a patient than
would have
occurred without the present invention.
[00611 The terms "therapy," "treatment," and "amelioration" refer to any
reduction in the
severity of symptoms or amount of amyloid aggregation, or improvement in
cognitive function.
As used herein, the terms "treat" and "prevent" are not intended to be
absolute terms. Treatment
17
CA 02728605 2011-01-18
can refer to any delay in onset, amelioration of symptoms, improvement in
patient survival,
increase in survival time or rate, etc. The effect of treatment can be
compared to an individual or
pool of individuals not receiving the treatment.
[0062] A "control" sample or value refers to a sample that serves as a
reference, usually a
known reference, for comparison to a test sample. For example, a test sample
can be taken from
a patient of unknown amyloid status and compared to control samples from a
patient diagnosed
with amyloidosis (e.g., Alzheimer's disease, Parkinson's disease, Creutzfeldt-
Jakob disease, etc.)
and/or an individual not suffering from amyloidosis or known to have low anti-
amyloid antibody
titre. A control can also represent an average value gathered from a
population of similar
individuals, e.g., Alzheimer's disease patients, Parkinson's disease patients,
Creutzfeldt-Jakob
disease patients, etc. with a similar medical background, or of the same age,
weight, etc. A
control value can also be obtained from the same individual, e.g., from an
earlier-obtained
sample, prior to symptoms, or before or at different therapeutic time points.
In some cases,
controls can include comparisons within an individual or between individuals,
e.g., comparison
of anti-amyloid antibody titres with the titre of one or more known
antibodies.
[0063] One of skill in the art will understand which controls are valuable in
a given situation
and be able to analyze data based on comparisons to control values. Controls
are also valuable
for determining the significance of data. For example, if values for a given
parameter are widely
variant in controls, variation in test samples will not be considered as
significant.
Alzheimer's Disease and Other Amyloid-Related Disorders
[0064] Amyloid proteins and abnormal protein aggregates play a role in a
number of different
diseases and conditions. The invention provides methods for detecting specific
high-avidity
auto-antibodies against various forms of amyloid proteins or certain protein
aggregates which
can be useful for diagnosis and determining a course of therapy.
[0065] Amyloid related disorders include not only Alzheimer's Disease (AD),
but also
Parkinson's Disease, Familial Amyloid Polyneuropathy (FAP), various forms of
amyloidosis
(e.g., senile systemic, Finnish type, Icelandic type, leptomeningeal, familial
visceral, cutaneous,
dialysis-related, etc.), and aoric medial amyloid. Additional disorders
related to abnormal
protein aggregation include Type II diabetes mellitus, transmissible
spongiform encephalopathy,
Creutzfeldt-Jakob disease, Huntington's Disease, medullary thyroid carcinoma,
atherosclerosis,
18
CA 02728605 2011-01-18
rheumatoid arthritis, prolactinomas, Frontotemporal Dementias, Down's
syndrome. Lewy body
disease, and similar diseases
[00661 Accordingly, the invention can be used to detect and/or isolate auto-
antibodies against
specific forms of amyloid and amyloid aggregates found in amyloid-related
disorders. In
addition, the methods disclosed herein can be applied to detection of
antibodies against abnormal
protein aggregates formed from Beta amyloid (A[3; Abeta), Islet amyloid
polypeptide (IAPP;
Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin (HD),
Calcitonin
(CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-A1), Serum
amyloid A protein
(SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8 protein; MFG-
E8), Prolactin
(PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase C), Beta
2
microglobulin (p2M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), proteins
having polyQ repeats, Tau protein (Tau), and other amyloid proteins.
[00671 Detection of specific auto-antibodies, e.g., for A[3 fibrils in the
case of AD and
Parkinson's, can be used in diagnosis or risk factor assessment. The
diagnostic methods of the
invention can be applied to individuals considered at risk for developing an
amyloid-related
disorder, e.g., based on age, family history, cognitive symptoms, etc., as can
be determined by
one of skill in medicine.
[00681 Risk factors and symptoms of amyloid-related disorders will be best
determined by a
skilled medical practitioner. Risk of developing these disorders increases
with age, and correlate
with family history. In many cases, an absolutely definitive diagnosis is
considered unfeasible.
Observable symptoms of AD include disruptive memory loss, differences in
ability to solve
problems, confusion as to time or place, trouble completing routine tasks,
social withdrawal, and
mood changes. These correlate with physical manifestations of the disease such
as increased
amyloid plaque formation and overall decrease in brain volume.
Detection and Isolation of Anti-Amyloid Immunoglobulins
[0069] Low levels of auto-antibodies can be found in the blood of individuals
that have not
been inoculated with a given antigen. For example, plasma preparations pooled
from a number
of individuals include a small fraction of IgG antibodies that bind to amyloid
proteins such as
All As explained herein, inaccurate methods determining antibody specificity
and harsh
19
CA 02728605 2011-01-18
methods of antibody isolation have caused uncertainty as to whether the
amyloid-specific
antibodies actually exist, or instead represent artifacts or non-specific
interactions.
[00701 The present invention provides methods of detecting and/or isolating
antibodies that are
specific for various forms of amyloid, including monomeric, oligomeric, and
fibrillar amyloid
proteins (i.e., A(3). For example, the small population of A(3-specific IgGs
can be isolated from a
biological sample by removing low-affinity and non-specific antibodies using a
mild chaotropic
wash. In some embodiments, the methods include first separating IgG from other
proteins in the
sample using thiophilic affinity-based methods, and removing low-affinity and
non-specific
antibodies using a mild chaotropic wash. These steps can be performed in any
order. Typically,
the biological sample is blood plasma or serum.
100711 One of skill in the art will appreciate that the chaotropic agent
should be present in an
amount sufficient to disrupt the weak binding interactions of low affinity or
non-specific
antibodies without significantly disrupting the binding specificity of high-
affinity antibodies.
For example, while urea is a chaotropic agent, a high concentration of urea
can generate
polyvalency. This is not fatal to the detection methods of the invention, as
any amyloid-specific
affinity generated by the chaotropic agent will typically be low.
[00721 The antibodies can be detected using standard methods, e.g., using a
detectably labeled
antibody binding agent such as an anti-human secondary antibody, antigen
(e.g., A(3), Protein A,
Protein G, etc. Such methods include ELISAs, Western blots,
immunofluorescence, and other
protein detection methods known in the art (see, e.g., U.S. Patents 4,366,241;
4,376,110;
4,517,288; and 4,837,168). For a review of the general immunoassays, see also
METHODS IN
CELL BIOLOGY, VOL. 37, Asai, ed. Academic Press, Inc. New York (1993); BASIC
AND CLINICAL
IMMUNOLOGY 7TH EDITION, Stites & Terr, eds. (1991). Immunological binding
assays (or
immunoassays) typically utilize a ligand (e.g., amyloid, A(3, or a particular
form of A(3) to
specifically bind to and often immobilize an antibody.
[00731 Immunoassays also often utilize a labeling agent to specifically bind
to and label the
binding complex formed by the ligand and the antibody. The labeling agent can
itself be one of
the moieties comprising the antibody/analyte complex, e.g., detectably labeled
analyte.
Alternatively, the labeling agent may be a third moiety, such as another
antibody, that
specifically binds to the antibody/analyte complex.
CA 02728605 2011-01-18
[00741 A competitive assay can be used, wherein the labeling agent is a second
anti-amyloid
antibody bearing a label. The two antibodies then compete for binding to the
immobilized
amyloid antigen. Alternatively, in a non-competitive format, the anti-amyloid
antibody lacks a
label, but a second antibody specific to antibodies of the species from which
the anti-amyloid
antibody is derived, e.g., human, and which binds the anti-amyloid antibody,
is labeled.
100751 Other proteins capable of specifically binding immunoglobulin constant
regions, such
as Protein A or Protein G can also be used as the label agent (see, generally
Kronval, et al., J.
Immunol. 111:1401-1406 (1973); and Akerstrom, et al., J. Immunol. 135:2589-
2542 (1985)).
[00761 Throughout the assays, incubation and/or washing steps may be required
after each
combination of reagents. Incubation steps can vary from about 5 seconds to
several hours, e.g.,
from about 5 minutes to about 24 hours. However, the incubation time will
depend upon the
assay format, antibody, volume of solution, concentrations, and the like.
Usually, the assays will
be carried out at ambient temperature, although they can be conducted over a
range of
temperatures, such as 10 C to 40 C.
[00771 If desired, the user can set a cutoff range for high, medium, and low
affinity/avidity,
and detect antibodies that fall within these ranges. Affinity assays can be
used to determine the
dissociation constant (Kd) of the antibody. The phrase "dissociation constant"
refers to the
affinity of an antibody for an antigen. Specificity of binding between an
antibody and an antigen
exists if the dissociation constant (KD = 1 /K, where K is the affinity
constant) of the antibody is
< 1 M, with increasing specificity and affinity antibodies often having a KD
< 100 nM or < 10
nM. KD = [Ab-Ag]/[Ab][Ag] where [Ab] is the concentration at equilibrium of
the antibody,
[Ag] is the concentration at equilibrium of the antigen and [Ab-Ag] is the
concentration at
equilibrium of the antibody-antigen complex. Typically, the binding
interactions between
antigen and antibody include reversible non-covalent associations such as
electrostatic attraction,
Van der Waals forces and hydrogen bonds. This method of defining binding
specificity applies
to single heavy and/or light chains, CDRs, fusion proteins or fragments of
heavy and/or light
chains.
[00781 The resulting population of A[3-specific antibodies is heterogenous,
but binds to A[3
with high affinity, e.g., with a Kd in the range of a monoclonal antibody that
was specifically
raised against All
21
CA 02728605 2011-01-18
Therapies for Amyloid Related Disorders and Alzheimer's Disease
[00791 Treatments for amyloid-related diseases and conditions, such as AD, are
typically
focused on cognitive and mood symptoms of the disease. Early treatment and
prevention
regimes include physical and social activity, memory games, and puzzle and
problem solving.
Pharmaceutical therapies for symptomatic individuals include cholinesterase
inhibitors (to
address reduced acetylcholine), partial glutamate antagonists (e.g.,
Memantine), and psychiatric
drugs (e.g., antipsychotics, sleep aides, anxiolytics, and beta-blockers).
Cholinesterase inhibitors
include Aricep t (donepezil hydrochloride), Exelon (rivastigmine), Razadyne
(galantamine),
and Cognex (tacrine).
100801 It has also been observed that pooled immunoglobulin preparations can
be effective for
improving AD symptoms. Such preparations, called IVIG (intravenous
immunoglobulin), are
prepared as described, e.g., in USSN 12/789,345 (filed May 27, 2010) and USSN
12/789,365
(filed May 27, 2010). Methods of treating AD, Parkinson's, and other protein
aggregation
disorders using IVIG are disclosed in US Pub. Nos. 2009/0148463 and
2009/0221017.
[00811 Briefly, IVIG is formed by pooling intravenous blood from more than one
individual,
separating the plasma fraction, and enriching for IgG immunoglobulin using a
combination of
chromatography, ultrafiltration, and diafiltration. IVIG is typically
administered by intravenous
infusion.
Methods for the Detection of Anti-Amyloid Antibodies
[00821 In one aspect, the present invention provides improved methods for
detecting anti-
amyloid antibodies in a biological sample. For example, methods are provided
herein for the
measurement of high avidity anti-amyloid antibodies present in the blood of a
mammal. In one
embodiment, the present invention provides methods for the accurate
quantification of antibodies
against aggregated or non-aggregated amyloid proteins present in biological
fluids. These
antibodies are emerging as significant tools for prediction, diagnosis and
treatment of human
amyloidosis diseases including Alzheimer's disease (AD) and other
neurodegenerative disorders.
Antibodies may also develop in other mammalian species particularly those used
as models of
these diseases. As such, the present invention improves the quantification and
characterization
of antibodies against aggregated amyloid proteins as well as unaggregated
amyloid monomers
when the level of monomers is low in the fluid being studied.
22
CA 02728605 2011-01-18
[0083] Thus, the present invention provides methods of detecting anti-amyloid
antibodies, e.g.,
from a biological sample from an individual. Typically, the anti-amyloid
antibody will be
soluble in the biological sample, e.g., serum, plasma, cerebrospinal fluid,
lymph, urine, or other
biological fluid or fraction. However, anti-amyloid antibodies can be detected
in tissue biopsies
as well.
[0084] In preferred methods, a biological sample (e.g., blood, serum, or
plasma sample) is
collected or a biopsy is performed and the collected biological sample is
tested in vitro. In
certain embodiments, immunoglobulins present in the sample are enriched prior
to detection. In
other embodiments, immunoglobulins are not further enriched prior to
detection. The tissue or
fluid is then contacted with an amyloid antigen and any immune complexes that
result indicate
the presence of an anti-amyloid antibody in the sample. To facilitate such
detection, the amyloid
antigen can be radiolabeled or coupled to an effector molecule which is a
detectable label, such
as a fluorescent label.
[0085] Anti-amyloid antibodies and amyloid proteins and/or aggregates can be
detected using
standard immunoassay methods. Standard methods include, for example,
radioimmunoassay,
sandwich immunoassays (including ELISA), immunofluorescence assays, Western
blot, affinity
chromatography (affinity ligand bound to a solid phase), and in situ detection
with labeled
antibodies. Detectable labels suitable for such use include any composition
detectable by
spectroscopic, photochemical, biochemical, immunochemical, electrical, optical
or chemical
means. Useful labels in the present invention include magnetic beads (e.g.,
DYNABEADS),
fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine,
green fluorescent
protein, and the like), radiolabels (e.g., 3H 1251, 35S, 14C, or 32P), enzymes
(e.g., horse radish
peroxidase, alkaline phosphatase and others commonly used in an ELISA), and
colorimetric
labels such as colloidal gold or colored glass or plastic (e.g. polystyrene,
polypropylene, latex)
beads. In some embodiments, a secondary detection agent is employed to detect
anti-amyloid
antibody binding (e.g., goat anti-human FITC). A general overview of the
applicable technology
can be found in Harlow & Lane, Antibodies: A Laboratory Manual (1988).
[0086] Methods currently employed for measuring anti-amyloid antibodies
extracted from
biological specimens suffer from artifactual gains or losses of anti-amyloid
activity as a result of
exposure to denaturing conditions. For example, some methods employ
acidification either
during enrichment of antibodies (Ig) using Protein A or G affinity
chromatography or to liberate
23
CA 02728605 2011-01-18
antibodies from bound antigen. Exposure to low pH causes certain mammalian
immunoglobulins to wholly or partially denature. This leads either to loss of
function or
increased polyvalency, both of which phenomena have deleterious effects on
measuring levels of
specific anti-amyloid antibodies. Advantageously, methods provided herein
separate Ig from
plasma and dissociate weakly bound antigen molecules from the anti-amyloid
antibodies without
exposure to acids or other denaturing conditions.
[00871 In one embodiment, the present invention provides methods for
quantifying levels of
antibodies against amyloid-forming proteins in mammalian plasma and other
biological fluids.
Unlike existing methods, the present invention circumvents confounding effects
of non-specific
antibody binding, interference from plasma macromolecules and artifactual
generation of
binding activity by denaturation of immunoglobulin during purification.
100881 For instance, previous assays that employed whole plasma to measure
endogenous anti-
amyloid activity are artifactually altered by interference from other, as yet
unidentified plasma
macromolecules. Advantageously, the methods provided herein eliminates the
interfering
activity of these unidentified plasma macromolecules in a manner that
preserves the anti-amyloid
antibodies of interest.
[00891 Measurements made on conventional glass, metal, or plastic substrates
are affected by
non-specific binding of antibodies to the amyloid protein and to the assay
plates. Human plasma
contains relatively large amounts of polyvalent antibodies that have low
avidity for amyloid
aggregates. The presence of such antibodies as well as antibodies hat bind non-
specifically to
assay substrates artifactually alters the measured anti-amyloid activity in
various assay methods.
Advantageously, the methods provided herein are able to distinguish specific
from non-specific
antibodies on the basis of differences in avidity of binding.
Methods Employing a Chaotropic Wash Step
100901 In one embodiment, the present invention provides methods for
quantifying levels of
antibodies against amyloid-forming proteins in mammalian plasma and other
biological fluids.
Unlike existing methods, the present invention circumvents confounding effects
of non-specific
antibody binding, interference from plasma macromolecules and artifactual
generation of
binding activity by denaturation of immunoglobulin during purification.
24
CA 02728605 2011-01-18
[00911 For instance, measurements made on conventional glass, metal, or
plastic substrates are
affected by non-specific binding of antibodies to the amyloid protein and to
the assay plates.
Human plasma contains relatively large amounts of polyvalent antibodies that
have low avidity
for amyloid aggregates. The presence of such antibodies as well as antibodies
hat bind non-
specifically to assay substrates artifactually alters the measured anti-
amyloid activity in various
assay methods. Advantageously, the methods provided herein are able to
distinguish specific
from non-specific antibodies on the basis of differences in avidity of
binding.
100921 Accordingly, in one aspect, the invention provides a method for
detecting high avidity
anti-amyloid antibodies present in a sample. In some embodiments, the method
comprises the
steps of (a) contacting the biological sample with a plurality of amyloid
antigens under suitable
conditions to form: (i) a complex comprising an amyloid antigen and a low-
affinity or non-
specific antibody; and (ii) a complex comprising an amyloid antigen and a high
avidity anti-
amyloid antibody; (b) washing the amyloid antigen complexes formed in step (a)
with a solution
comprising a chaotropic agent to dissociate at least one complex comprising an
amyloid antigen
and a low-affinity or non-specific antibody; and (c) detecting the presence or
level of the
remaining complexes.
100931 In one embodiment, the biological sample is a biological fluid, for
example, blood,
plasma, urine, lymph, synovial fluid, etc. In other embodiments, the
biological sample may be a
tissue sample or biopsy. In a preferred embodiment, the biological sample is a
blood sample or
fraction thereof (e.g., plasma or plasma fraction). In another preferred
embodiment, the
biological sample is an immunoglobulin preparation or enrichment. In a
specific embodiment,
the immunoglobulin enrichment is performed by thiophilic chromatography. In
another specific
embodiment, the immunoglobulin enrichment is performed by mixed mode ligand
chromatography.
[00941 In one embodiment, the anti-amyloid antibody being detected is an
antibody specific
for an amyloid protein selected from Beta amyloid (A[3; Abeta), Islet amyloid
polypeptide
(IAPP; Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin
(HD),
Calcitonin (CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-A
1), Serum amyloid
A protein (SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8
protein; MFG-E8),
Prolactin (PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase
C), Beta 2
microglobulin ((32M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
CA 02728605 2011-01-18
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), and an
amyloid protein having a polyQ repeat. In one embodiment, the anti-amyloid
antibody is
specific for amyloid protein monomers. In another embodiment, the anti-amyloid
antibody is
specific for amyloid protein oligomers (i.e., dimers, trimers, etc). In yet
another embodiment,
the anti-amyloid antibody is specific for amyloid protein fibrils.
[0095] In a preferred embodiment, the anti-amyloid antibody being detected is
an antibody
specific for Beta amyloid (Al); Abeta). In one embodiment, the anti-AO
antibody is specific for
Al) monomers. In another embodiment, the anti-Ali antibody is specific for Al)
oligomers (i.e.,
dimers, trimers, etc). In yet another embodiment, the anti-A(3 antibody is
specific for Al) fibrils.
[0096] In one embodiment of the methods provided herein, the low-affinity or
non-specific
antibody is a low avidity anti-amyloid antibody. In a specific embodiment, the
low avidity anti-
amyloid antibody is a low avidity anti-AR antibody.
[0097] The chaotropic wash step employed in the methods provided herein may be
performed
with any known chaotropic agent. When a chaotropic salt is employed, the salt
will generally
comprise either an anion having a chaotropic effect equal or greater than a
chlorate ion (Cl03-) or
a cation having a chaotropic effect greater than a calcium ion.
[0098] In one embodiment, the chaotropic salt is a guanidinium salt. Non-
limiting examples of
guanidinium salts that may be used in conjunction with the methods provided
herein include
guanidinium chloride, guanidinium nitrate, and guanidinium thiocyanate.
[0099] In another embodiment, the chaotropic salt is a thiocyanate salt. Non-
limiting
examples of thiocyanate salts that may be used in conjunction with the methods
provided herein
include ammonium thiocyanate, potassium thiocyanate, sodium thiocyanate,
lithium thiocyanate,
calcium thiocyanate, and guanidinium thiocyanate. In a specific embodiment,
the chaotropic salt
is ammonium thiocyanate. In one embodiment, ammonium thiocyanate is used at a
wash
concentration between about 0.5 M and about 4.0 M. In another embodiment,
ammonium
thiocyanate is used at a wash concentration between about 1.0 M and about 3.0
M. In yet
another embodiment, ammonium thiocyanate is used at a wash concentration
between about 1.5
M and about 2.5 M.
[0100] In yet another embodiment, the chaotropic salt is a perchlorate salt.
Non-limiting
examples of perchlorate salts that may be used in conjunction with the methods
provided herein
26
CA 02728605 2011-01-18
include ammonium perchlorate, sodium perchlorate, lithium perchlorate,
magnesium perchlorate,
and calcium perchlorate.
[0101] In yet another embodiment, the chaotropic salt is an iodide salt. Non-
limiting examples
of iodide salts that may be used in conjunction with the methods provided
herein include
ammonium iodide, potassium iodide, sodium iodide, lithium iodide, magnesium
iodide, and
calcium iodide.
[0102] In yet another embodiment, the chaotropic salt is a chlorate salt. Non-
limiting
examples of chlorate salts that may be used in conjunction with the methods
provided herein
include sodium chlorate, lithium chlorate, magnesium chlorate, and calcium
chlorate.
[0103] The concentration of the chaotropic agent used in the wash step of
methods provided
herein should be selected such that it is sufficient to disrupt interactions
between non-specific
(i.e., low avidity) anti-amyloid antibodies and amyloid antigens, yet does not
disrupt interactions
between specific (i.e. high avidity) anti-amyloid antibodies and amyloid
antigens. The absolute
concentration of chaotropic agent employed will depend upon a number of
factors, including, the
strength of the particular chaotropic agent and the strength of the antibody-
amyloid antigen
interactions formed in the particular system being employed (e.g., anti-An
antibody ELISA
assay). The skilled artisan will know how to readily determine the optimal
concentration of
chaotropic agent to use for their particular system. In one embodiment, the
chaotropic agent is
used at a wash concentration between about 0.5 M and about 4.0 M. In another
embodiment,
chaotropic agent is used at a wash concentration between about 1.0 M and about
3.0 M. In yet
another embodiment, chaotropic agent is used at a wash concentration between
about 1.5 M and
about 2.5 M.
[0104] In certain embodiments of the methods provided herein, immunoglobulins
found in the
biological sample (i.e., blood, plasma, urine, lymph, etc.) are enriched prior
to the step of
contacting the biological sample with one or more amyloid antigens.
Preferably, the
immunoglobulins are enriched by a method that does not partially or completely
denature the
antibodies. In one embodiment, the methods provided herein for detecting high
avidity anti-
amyloid antibodies comprise a step of enriching immunoglobulins by a
chromatographic method
that does not result in denaturation of the antibodies. In one embodiment, the
chromatographic
method is performed with a thiophilic resin. In another embodiment, the
chromatographic
method is performed with mixed mode ligand chemistry (Upfront Chromatography
A/S). In
27
CA 02728605 2011-01-18
other embodiments, the methods provided herein for detecting high avidity anti-
amyloid
antibodies comprise a step of enriching immunoglobulins by a non-
chromatographic method that
does not result in denaturation of the antibodies.
Methods Employing a Thiophilic Enrichment Step
[01051 In one embodiment, the present invention provides methods for
quantifying levels of
antibodies against amyloid-forming proteins in mammalian plasma and other
biological fluids.
Unlike existing methods, the present invention circumvents confounding effects
of non-specific
antibody binding, interference from plasma macromolecules and artifactual
generation of
binding activity by denaturation of immunoglobulin during purification.
[01061 For instance, previous assays that employed whole plasma to measure
endogenous anti-
amyloid activity are artifactually altered by interference from other, as yet
unidentified plasma
macromolecules. Advantageously, the methods provided herein eliminates the
interfering
activity of these unidentified plasma macromolecules in a manner that
preserves the anti-amyloid
antibodies of interest.
[01071 Methods currently employed for measuring anti-amyloid antibodies
extracted from
biological specimens suffer from artifactual gains or losses of anti-amyloid
activity as a result of
exposure to denaturing conditions. For example, some methods employ
acidification either
during enrichment of antibodies (Ig) using Protein A or G affinity
chromatography or to liberate
antibodies from bound antigen. Exposure to low pH causes certain mammalian
immunoglobulins to wholly or partially denature. This leads either to loss of
function or
increased polyvalency, both of which phenomena have deleterious effects on
measuring levels of
specific anti-amyloid antibodies. Advantageously, methods provided herein
separate Ig from
plasma and dissociate weakly bound antigen molecules from the anti-amyloid
antibodies without
exposure to acids or other denaturing conditions.
[01081 Accordingly, in one aspect, the invention provides a method detecting a
high avidity
anti-amyloid antibody present in a biological sample, the method comprising
the steps of (a)
contacting the biological sample with a thiophilic resin to bind the anti-
amyloid antibody; (b)
eluting the antibody from the thiophilic resin at a non-denaturing pH to form
an eluate; (c)
contacting the eluate with an amyloid antigen to form a complex comprising the
amyloid antigen
and the anti-amyloid antibody; and (d) detecting the presence or level of the
complex.
28
CA 02728605 2011-01-18
[0109] In one embodiment, the anti-amyloid antibody being detected is an
antibody specific
for an amyloid protein selected from Beta amyloid (A[3; Abeta), Islet amyloid
polypeptide
(IAPP; Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin
(HD),
Calcitonin (CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-A1),
Serum amyloid
A protein (SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8
protein; MFG-E8),
Prolactin (PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase
C), Beta 2
microglobulin (02M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), and an
amyloid protein having a polyQ repeat. In one embodiment, the anti-amyloid
antibody is
specific for amyloid protein monomers. In another embodiment, the anti-amyloid
antibody is
specific for amyloid protein oligomers (i.e., dimers, trimers, etc). In yet
another embodiment,
the anti-amyloid antibody is specific for amyloid protein fibrils.
[0110] In a preferred embodiment, the anti-amyloid antibody being detected is
an antibody
specific for Beta amyloid (A[3; Abeta). In one embodiment, the anti-AR
antibody is specific for
A[3 monomers. In another embodiment, the anti-A(3 antibody is specific for A[3
oligomers (i.e.,
dimers, trimers, etc). In yet another embodiment, the anti-A(3 antibody is
specific for A[3 fibrils.
[0111] In one embodiment of the methods provided herein, the low-affinity or
non-specific
antibody is a low avidity anti-amyloid antibody. In a specific embodiment, the
low avidity anti-
amyloid antibody is a low avidity anti-AR antibody.
[0112] In certain embodiments of the methods provided herein, the complex
comprising the
amyloid antigen and the anti-amyloid antibody is washed with a solution
containing a chaotropic
salt prior to detecting the presence or level of said complex in order to
dissociate interactions
between non-specific antibodies (i.e., low avidity antibodies) and the amyloid
antigen. The
chaotropic wash step employed in the methods provided herein may be performed
with any
known chaotropic agent. When a chaotropic salt is employed, the salt will
generally comprise
either an anion having a chaotropic effect equal or greater than a chlorate
ion (C1O3-) or a cation
having a chaotropic effect greater than a calcium ion. In one embodiment, the
chaotropic agent
is selected from urea, thiourea, a guanidinium salt, a thiocyanate salt, a
perchlorate salt, an iodide
salt, and a chlorate salt. In a specific embodiment, the chaotropic salt is
ammonium thiocyanate.
In one embodiment, ammonium thiocyanate is used at a wash concentration
between about 0.5
M and about 4.0 M. In another embodiment, ammonium thiocyanate is used at a
wash
29
CA 02728605 2011-01-18
concentration between about 1.0 M and about 3.0 M. In yet another embodiment,
ammonium
thiocyanate is used at a wash concentration between about 1.5 M and about 2.5
M.
101131 The concentration of the chaotropic agent used in the wash step of
methods provided
herein should be selected such that it is sufficient to disrupt interactions
between non-specific
(i.e., low avidity) anti-amyloid antibodies and amyloid antigens, yet does not
disrupt interactions
between specific (i.e. high avidity) anti-amyloid antibodies and amyloid
antigens. The absolute
concentration of chaotropic agent employed will depend upon a number of
factors, including, the
strength of the particular chaotropic agent and the strength of the antibody-
amyloid antigen
interactions formed in the particular system being employed (e.g., anti-A(3
antibody ELISA
assay). The skilled artisan will know how to readily determine the optimal
concentration of
chaotropic agent to use for their particular system. In one embodiment, the
chaotropic agent is
used at a wash concentration between about 0.5 M and about 4.0 M. In another
embodiment,
chaotropic agent is used at a wash concentration between about 1.0 M and about
3.0 M. In yet
another embodiment, chaotropic agent is used at a wash concentration between
about 1.5 M and
about 2.5 M.
Diagnostic and Prognostic Methods
[01141 In certain aspects the methods provided herein for detecting anti-
amyloid antibodies
can be used to for diagnosis and prognosis of individuals at risk of, or
suffering from, amyloid
related diseases as described above. For example, high levels of amyloid, in
particular, A(3, in
serum and certain tissues can indicate an increased likelihood of developing
the amyloid plaques
associated with Alzheimer's Disease. High levels of amyloid-specific auto-
antibodies also
indicate that an individual is at risk for AD, or a worsening of AD-related
symptoms.
Methods for Diagnosing and/or Selecting a Suitable Treatment
[01151 Thus, in some embodiments, the invention provides methods for diagnosis
and/or
selecting a course of treatment for a subject having or suspected of having an
amyloid-related
disorder. In some embodiments, the methods comprise detection and/or
measurement of auto-
antibodies specific for an amyloid protein. Methods for detecting endogenous
antibodies having
high avidity for amyloid proteins are outlined herein.
[01161 In one embodiment, the methods for detecting anti-amyloid antibodies
can comprise
obtaining an antibody-containing sample from a subject, conveniently a blood
or plasma sample.
CA 02728605 2011-01-18
The sample can be further separated, e.g., into plasma and cellular matter, to
isolate the antibody-
containing fraction, although this step is not necessary. The sample can also
be exposed to size
filtration and/or chromatography methods. In some embodiments, the sample is
exposed to
thiophilic chromatography to remove non-immunoglobulin proteins from the
sample. The
sample, or eluent, can then be contacted with an amyloid antigen in a system
designed to detect
an interaction between the antibody and the antigen, for example an antigen
conjugated to a
convenient matrix, e.g., an ELISA plate or chromatography matrix. The amyloid
antigen can be
in any form, either heterogenous amyloid, or largely homogenous amyloid
monomer, oligomer,
or fibril. After sufficient incubation to allow formation of amyloid-antibody
complexes, the
amyloid-antibody complexes are exposed to a wash comprising a chaotropic
agent, to remove
low-affinity and non-specific antibodies. After the chaotropic wash, the
presence or level of
antibody complexed with amyloid antigen is detected. The antibody can be
eluted from the
antigen prior to detection if desired, or competed away with a labeled amyloid
antigen for ease of
detection. The antibody can be detected as described above.
101171 In some embodiments, at least one control is run alongside the sample,
and compared
for amount of antibody and/or level of binding. In other embodiments, the
result of the assay
may be compared to a control level previously established for the system of
interest. For
example, a positive control can include the same biological sample obtained
from an individual
or group of individuals that is known to have an amyloid-related disease.
Another example of a
suitable positive control is a known anti-amyloid monoclonal antibody, e.g.,
as a high affinity
and/or avidity comparison. An exemplary negative control can include the same
biological
sample obtained from an individual or group of individuals that have low risk
of developing the
amyloid related disease. Another example of a suitable negative control is a
known antibody
specific for a non-amyloid antigen or having low avidity for the amyloid
antigen.
[01181 Using such methods, and correlating a relatively high level of anti-
amyloid antibodies
with an increased likelihood that the subject has or is at high risk of
developing an amyloid-
related disorder, one of skill can diagnose an amyloid-related disease in the
subject.
101191 The above methods can be applied to selecting a patient group for an
amyloid-related
disease therapy. For example, the level of anti-amyloid antibodies can be
determined in a
plurality of individuals. As explained above, those individuals determined to
have relatively
high levels of anti-amyloid antibodies can be selected for treatment. In some
embodiment, the
31
CA 02728605 2011-01-18
level of anti-amyloid antibodies is detected periodically over a course of
treatment. In some
embodiments, the treatment comprises administration of IVIG.
Methods Employing a Chaotropic Wash Step
[0120] In one aspect, the present invention provides methods for identifying a
subject who is a
candidate for treatment of an amyloid-related disease or condition, comprising
quantifying levels
of antibodies against amyloid-forming proteins in mammalian plasma and other
biological fluids,
wherein a chaotropic wash step is employed to reduce the signal associated
with non-specific and
low-avidity anti-amyloid antigen binding. In a preferred embodiment, the
treatment comprises
administration of an immunoglobulin preparation. In a specific embodiment, the
amyloid-related
disease is Alzheimer's disease.
[0121] In a related aspect, the present invention provides methods for
diagnosing a disease
associated with an amyloid protein, comprising quantifying levels of
antibodies against amyloid-
forming proteins in mammalian plasma and other biological fluids, wherein a
chaotropic wash
step is employed to reduce the signal associated with non-specific and low-
avidity anti-amyloid
antigen binding. In a preferred embodiment, the method is for diagnosing
Alzheimer's disease.
[0122] Unlike existing methods, the present invention circumvents confounding
effects of non-
specific antibody binding, interference from plasma macromolecules and
artifactual generation
of binding activity by denaturation of immunoglobulin during purification.
[01231 For instance, measurements made on conventional glass, metal, or
plastic substrates are
affected by non-specific binding of antibodies to the amyloid protein and to
the assay plates.
Human plasma contains relatively large amounts of polyvalent antibodies that
have low avidity
for amyloid aggregates. The presence of such antibodies as well as antibodies
hat bind non-
specifically to assay substrates artifactually alters the measured anti-
amyloid activity in various
assay methods. Advantageously, the methods provided herein are able to
distinguish specific
from non-specific antibodies on the basis of differences in avidity of
binding.
[0124] Accordingly, in a specific embodiment, the invention provides a method
for identifying
a subject who is a candidate for treatment of an amyloid-related disease or
condition. In some
embodiments, the method comprises the steps of (a) contacting a biological
sample from the
subject with a plurality of amyloid antigens under suitable conditions to
form: (i) a complex
comprising an amyloid antigen and a low-affinity or non-specific antibody; and
(ii) a complex
32
CA 02728605 2011-01-18
comprising an amyloid antigen and a high avidity anti-amyloid antibody; (b)
washing the
amyloid antigen complexes formed in step (a) with a solution containing a
chaotropic agent to
dissociate at least one complex comprising an amyloid antigen and a low-
affinity or non-specific
anti-amyloid antibody; (c) detecting the level of the remaining complexes; and
(d) determining
whether the subject would benefit from a treatment for an amyloid-related
disease or condition
by comparing the level of the anti-amyloid antibody to a control level. In a
preferred
embodiment, the treatment comprises the administration of an immunoglobulin
preparation.
[0125] In some embodiments of the methods for identifying a subject who is a
candidate for
treatment, the control is from an individual or group of individuals that do
not have an amyloid-
related disease, such that an increased level of the remaining complex
relative to control is
indicative of a candidate for treatment. In some embodiments, the control is
from an individual
or group of individuals that do have an amyloid-related disease, such that a
similar level of the
remaining complex relative to control is indicative of a candidate for
treatment. In yet another
embodiment, the control is from an individual or group of individuals that
have been diagnosed
with an amyloid-related disease and who have responded favorably to a
particular course of
treatment. In another embodiment, the control is from an individual or group
of individuals that
have been diagnosed with an amyloid-related disease and who have not responded
favorably to a
particular course of treatment. One of skill will understand how to select at
least one appropriate
control and interpret the results accordingly.
[0126] In certain embodiments, the methods for identifying a subject who is a
candidate for
treatment further comprise a step of assigning a course of treatment or
administering a treatment
to the subject. Generally, this course of treatment will be assigned when the
level of anti-
amyloid antibodies in the biological sample is above a control threshold
(e.g., a threshold
indicating that the subject likely has an amyloid-related condition or is
likely to respond to a
particular treatment), or more closely resembles a positive control (e.g., a
control level from a
group or individual having an amyloid-related condition or a control level
from a group or
individual who has responded favorably to a particular treatment) than to a
negative control (e.g.,
a control level from a group or individual not having an amyloid-related
condition or a control
level from a group or individual who has not responded favorably to a
particular treatment). In a
preferred embodiment, the treatment comprises administration of an
immunoglobulin
preparation.
33
CA 02728605 2011-01-18
[0127] In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises identifying a subject who is a candidate for
treatment of
Alzheimer's disease.
[01281 In a related embodiment, the present invention provides a method for
diagnosing a
disease associated with an amyloid protein in a subject. In some embodiments,
the method
comprises the steps of (a) contacting a biological sample from the subject
with a plurality of
amyloid antigens under suitable conditions to form: (i) a complex comprising
an amyloid antigen
and a low-affinity or non-specific antibody; and (ii) a complex comprising an
amyloid antigen
and a high avidity anti-amyloid antibody; (b) washing the amyloid antigen
complexes formed in
step (a) with a solution containing a chaotropic agent to dissociate at least
one complex
comprising an amyloid antigen and a low-affinity or non-specific antibody; (c)
detecting the
level of the remaining complexes; and (d) diagnosing the subject by comparing
the level of the
anti-amyloid antibody to a control level.
[01291 In some embodiments of the methods for diagnosing a disease associated
with an
amyloid protein in a subject, the control is from an individual or group of
individuals that do not
have an amyloid-related disease, such that an increased level of the remaining
complex relative
to control is indicative of a high likelihood that the subject has an amyloid-
related condition. In
some embodiments, the control is from an individual or group of individuals
that do have an
amyloid-related disease, such that a similar level of the remaining complex
relative to control is
indicative of a high likelihood that the subject has an amyloid-related
condition. One of skill
will understand how to select at least one appropriate control and interpret
the results
accordingly.
[01301 In certain embodiments, the methods for diagnosing a disease associated
with an
amyloid protein in a subject further comprise a step of assigning a course of
treatment or
34
CA 02728605 2011-01-18
administering a treatment to the subject. Generally, this course of treatment
will be assigned
when the level of anti-amyloid antibodies in the biological sample is above a
control threshold
(e.g., a threshold indicating that the subject likely has an amyloid-related
condition or is likely to
respond to a particular treatment), or more closely resembles a positive
control (e.g., a control
level from a group or individual having an amyloid-related condition or a
control level from a
group or individual who has responded favorably to a particular treatment)
than to a negative
control (e.g., a control level from a group or individual not having an
amyloid-related condition
or a control level from a group or individual who has not responded favorably
to a particular
treatment). In a preferred embodiment, the treatment comprises administration
of an
immunoglobulin preparation.
[01311 In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises diagnosing Alzheimer's disease.
[01321 In one embodiment of the methods for identifying a subject who is a
candidate for
treatment or method for diagnosing a disease associated with an amyloid
protein in a subject, the
biological sample is a biological fluid, for example, blood, plasma, urine,
lymph, synovial fluid,
etc. In other embodiments, the biological sample may be a tissue sample or
biopsy. In a
preferred embodiment, the biological sample is a blood sample or fraction
thereof (e.g., plasma
or plasma fraction). In another preferred embodiment, the biological sample is
an
immunoglobulin preparation or enrichment. In a specific embodiment, the
immunoglobulin
enrichment is performed by thiophilic chromatography. In another specific
embodiment, the
immunoglobulin enrichment is performed by mixed mode ligand chromatography.
[01331 In one embodiment, the anti-amyloid antibody being detected is an
antibody specific
for an amyloid protein selected from Beta amyloid (A[3; Abeta), Islet amyloid
polypeptide
(IAPP; Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin
(HD),
CA 02728605 2011-01-18
Calcitonin (CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-Al),
Serum amyloid
A protein (SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8
protein; MFG-E8),
Prolactin (PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase
C), Beta 2
microglobulin ((32M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), and an
amyloid protein having a polyQ repeat. In one embodiment, the anti-amyloid
antibody is
specific for amyloid protein monomers. In another embodiment, the anti-amyloid
antibody is
specific for amyloid protein oligomers (i.e., dimers, trimers, etc). In yet
another embodiment,
the anti-amyloid antibody is specific for amyloid protein fibrils.
[01341 In one embodiment, the disease or condition associated with an amyloid
protein is
selected from the group consisting of Alzheimer's disease, Type 2 diabetes
mellitus, Parkinson's
disease, Transmissible spongiform encephalopathy, Huntington's Disease,
Medullary carcinoma
of the thyroid, Cardiac arrhythmias, Atherosclerosis, Rheumatoid arthritis,
Aortic medial
amyloid, Prolactinomas, Familial amyloid polyneuropathy, Hereditary non-
neuropathic systemic
amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice
corneal dystrophy,
Cerebral amyloid angiopathy, Cerebral amyloid angiopathy (Icelandic type), and
systemic AL
amyloidosis. In a preferred embodiment, the disease or condition is
Alzheimer's disease. In a
particularly preferred embodiment, the disease or condition is Alzheimer's
disease and the anti-
amyloid antibodies being detected are anti-Beta amyloid (A(3; Abeta)
antibodies.
[01351 In certain embodiments of the methods provided herein, detection of the
presence or
level of a particular amyloid protein is useful to diagnose a particular
disease or condition, or to
select a candidate for the treatment of a particular disease or condition. Non-
limiting examples
of amyloid-amyloid disease combinations that are well suited for use in the
methods provided
herein can be found in Table 1. In certain embodiments, the detection of the
presence or level of
a high-avidity antibody specific for the amyloid protein listed in Table I
will be diagnostic of the
corresponding disease listed.
Table 1. Non-limiting examples of amyloid proteins associated with specific
diseases.
Disease Amyloid Protein GenBank UniProt
Accession Accession
Alzheimer's disease Beta amyloid (A13; Abeta) NP 000475 P05067
Type 2 diabetes mellitus Islet amyloid polypeptide NP 000406 P10997
(IAPP; Amylin) -
Parkinson's disease Alpha-synuclein (SNCA) NP 000336 P37840
Transmissible spongiform encephalopathy (e.g., Major Prion Protein (PrP) NP
000302 P04156
Creutzfeldt-Jakob disease) -
36
CA 02728605 2011-01-18
Huntington's Disease Huntingtin (HD) NP 002102 P42858
Medullary carcinoma of the thyroid Calcitonin (CCP) NP_001029124 P01258
Cardiac arrhythmias, Isolated atrial amyloidosis Atrial natriuretic factor
(ANF) NP 006163 P01160
Atherosclerosis Apolipoprotein Al (Apo-A1) NP_000030 P02647 Serum Rheumatoid
arthritis Serum amyloid A protein NP_954630 P02735
Medin amyloid (fragment of
Aortic medial amyloid Milk fat globule-EGF factor 8 NP_005919 Q08431
protein; MFG-E8)
Prolactinomas Prolactin (PRL) NP000939 P01236
Familial amyloid polyneuropathy Transthyretin (ATTR) NP 000362 P02766
Hereditary non-neuropathic systemic amyloidosis Lysozyme C (1,4-beta-N- NP
000230 P61626
acetylmuramidase C)
Dialysis related amyloidosis Beta 2 microglobulin (132M) NP_004039 P61769
Finnish amyloidosis Gelsolin (AGEL) NP 000168 P06396
Transforming growth factor-
Lattice corneal dystrophy beta-induced protein ig-h3 NP_000349 Q15582
(Beta ig-h3; Keratoepithelin)
Cerebral amyloid angiopathy Beta amyloid (AP; Abeta) NP_000475 P05067
Cerebral amyloid angiopathy (Icelandic type) Cystatin C (CST3) NP 000090
P01034
systemic AL amyloidosis Immunoglobulin light chain
(AL)
[0136] In a preferred embodiment, the anti-amyloid antibody being detected is
an antibody
specific for Beta amyloid (A(3; Abeta). In one embodiment, the anti-A[3
antibody is specific for
AP monomers. In another embodiment, the anti-A[3 antibody is specific for A(3
oligomers (i.e.,
dimers, trimers, etc). In yet another embodiment, the anti-A(3 antibody is
specific for AR fibrils.
[0137] In one embodiment of the methods provided herein, the low-affinity or
non-specific
antibody is a low avidity anti-amyloid antibody. In a specific embodiment, the
low avidity anti-
amyloid antibody is a low avidity anti-A(3 antibody.
[0138] The chaotropic wash step employed in the methods provided herein may be
performed
with any known chaotropic agent. When a chaotropic salt is employed, the salt
will generally
comprise either an anion having a chaotropic effect equal or greater than a
chlorate ion (CI03-) or
a cation having a chaotropic effect greater than a calcium ion.
[0139] In one embodiment, the chaotropic salt is a guanidinium salt. Non-
limiting examples of
guanidinium salts that may be used in conjunction with the methods provided
herein include
guanidinium chloride, guanidinium nitrate, and guanidinium thiocyanate.
[0140] In another embodiment, the chaotropic salt is a thiocyanate salt. Non-
limiting
examples of thiocyanate salts that may be used in conjunction with the methods
provided herein
include ammonium thiocyanate, potassium thiocyanate, sodium thiocyanate,
lithium thiocyanate,
calcium thiocyanate, and guanidinium thiocyanate. In a specific embodiment,
the chaotropic salt
37
CA 02728605 2011-01-18
is ammonium thiocyanate. In one embodiment, ammonium thiocyanate is used at a
wash
concentration between about 0.5 M and about 4.0 M. In another embodiment,
ammonium
thiocyanate is used at a wash concentration between about 1.0 M and about 3.0
M. In yet
another embodiment, ammonium thiocyanate is used at a wash concentration
between about 1.5
M and about 2.5 M.
[01411 In yet another embodiment, the chaotropic salt is a perchlorate salt.
Non-limiting
examples of perchlorate salts that may be used in conjunction with the methods
provided herein
include ammonium perchlorate, sodium perchlorate, lithium perchlorate,
magnesium perchlorate,
and calcium perchlorate.
101421 In yet another embodiment, the chaotropic salt is an iodide salt. Non-
limiting examples
of iodide salts that may be used in conjunction with the methods provided
herein include
ammonium iodide, potassium iodide, sodium iodide, lithium iodide, magnesium
iodide, and
calcium iodide.
[01431 In yet another embodiment, the chaotropic salt is a chlorate salt. Non-
limiting
examples of chlorate salts that may be used in conjunction with the methods
provided herein
include sodium chlorate, lithium chlorate, magnesium chlorate, and calcium
chlorate.
[01441 The concentration of the chaotropic agent used in the wash step of
methods provided
herein should be selected such that it is sufficient to disrupt interactions
between non-specific
(i.e., low avidity) anti-amyloid antibodies and amyloid antigens, yet does not
disrupt interactions
between specific (i.e. high avidity) anti-amyloid antibodies and amyloid
antigens. The absolute
concentration of chaotropic agent employed will depend upon a number of
factors, including, the
strength of the particular chaotropic agent and the strength of the antibody-
amyloid antigen
interactions formed in the particular system being employed (e.g., anti-A[3
antibody ELISA
assay). The skilled artisan will know how to readily determine the optimal
concentration of
chaotropic agent to use for their particular system. In one embodiment, the
chaotropic agent is
used at a wash concentration between about 0.5 M and about 4.0 M. In another
embodiment,
chaotropic agent is used at a wash concentration between about 1.0 M and about
3.0 M. In yet
another embodiment, chaotropic agent is used at a wash concentration between
about 1.5 M and
about 2.5 M.
38
CA 02728605 2011-01-18
[0145] In certain embodiments of the methods provided herein, immunoglobulins
found in the
biological sample (i.e., blood, plasma, urine, lymph, etc.) are enriched prior
to the step of
contacting the biological sample with one or more amyloid antigens.
Preferably, the
immunoglobulins are enriched by a method that does not partially or completely
denature the
antibodies. In one embodiment, the methods provided herein for detecting high
avidity anti-
amyloid antibodies comprise a step of enriching immunoglobulins by a
chromatographic method
that does not result in denaturation of the antibodies. In one embodiment, the
chromatographic
method is performed with a thiophilic resin. In another embodiment, the
chromatographic
method is performed with mixed mode ligand chemistry (Upfront Chromatography
A/S). In
other embodiments, the methods provided herein for detecting high avidity anti-
amyloid
antibodies comprise a step of enriching immunoglobulins by a non-
chromatographic method that
does not result in denaturation of the antibodies.
Methods Employing a Thiophilic Enrichment Step
[0146] In one aspect, the present invention provides methods for identifying a
subject who is a
candidate for treatment, comprising administration 'of an immunoglobulin
preparation
comprising quantifying levels of antibodies against amyloid-forming proteins
in mammalian
plasma and other biological fluids, wherein a thiophilic enrichment step is
employed to reduce
the signal associated with non-specific antibody binding, interference from
plasma
macromolecules and artifactual generation of binding activity by denaturation
of
immunoglobulin during purification.
[0147] In a related aspect, the present invention provides methods for
diagnosing a disease
associated with an amyloid protein, comprising administration of an
immunoglobulin preparation
comprising quantifying levels of antibodies against amyloid-forming proteins
in mammalian
plasma and other biological fluids, wherein a thiophilic enrichment step is
employed to reduce
the signal associated with non-specific antibody binding, interference from
plasma
macromolecules and artifactual generation of binding activity by denaturation
of
immunoglobulin during purification.
[0148] Unlike existing methods, the present invention circumvents confounding
effects of non-
specific antibody binding, interference from plasma macromolecules and
artifactual generation
of binding activity by denaturation of immunoglobulin during purification.
39
CA 02728605 2011-01-18
[01491 For instance, previous assays that employed whole plasma to measure
endogenous anti-
amyloid activity are artifactually altered by interference from other, as yet
unidentified plasma
macromolecules. Advantageously, the methods provided herein eliminates the
interfering
activity of these unidentified plasma macromolecules in a manner that
preserves the anti-amyloid
antibodies of interest.
[01501 Methods currently employed for measuring anti-amyloid antibodies
extracted from
biological specimens suffer from artifactual gains or losses of anti-amyloid
activity as a result of
exposure to denaturing conditions. For example, some methods employ
acidification either
during enrichment of antibodies (Ig) using Protein A or G affinity
chromatography or to liberate
antibodies from bound antigen. Exposure to low pH causes certain mammalian
immunoglobulins to wholly or partially denature. This leads either to loss of
function or
increased polyvalency, both of which phenomena have deleterious effects on
measuring levels of
specific anti-amyloid antibodies. Advantageously, methods provided herein
separate Ig from
plasma and dissociate weakly bound antigen molecules from the anti-amyloid
antibodies without
exposure to acids or other denaturing conditions.
[01511 Accordingly, in a specific embodiment, the invention provides a method
for identifying
a subject who is a candidate for treatment of an amyloid-related disease or
condition. In some
embodiments, the method comprises the steps of (a) contacting the biological
sample with a
thiophilic resin to bind an anti-amyloid antibody present in the sample; (b)
eluting the antibody
from the thiophilic resin at a non-denaturing pH to form an eluate; (c)
contacting the eluate with
an amyloid antigen to form a complex comprising the amyloid antigen and the
anti-amyloid
antibody; (d) detecting the presence or level of the complex; and (e)
determining whether the
subject would benefit from a treatment comprising administration of an
immunoglobulin
preparation by comparing the level of the anti-amyloid antibody to a control
level. In a preferred
embodiment, the treatment comprises the administration of an immunoglobulin
preparation.
[01521 In some embodiments of the methods for identifying a subject who is a
candidate for
treatment, the control is from an individual or group of individuals that do
not have an amyloid-
related disease, such that an increased level of the remaining complex
relative to control is
indicative of a candidate for treatment. In some embodiments, the control is
from an individual
or group of individuals that do have an amyloid-related disease, such that a
similar level of the
remaining complex relative to control is indicative of a candidate for
treatment. In yet another
CA 02728605 2011-01-18
embodiment, the control is from an individual or group of individuals that
have been diagnosed
with an amyloid-related disease and who have responded favorably to a
particular course of
treatment. In another embodiment, the control is from an individual or group
of individuals that
have been diagnosed with an amyloid-related disease and who have not responded
favorably to a
particular course of treatment. One of skill will understand how to select at
least one appropriate
control and interpret the results accordingly.
[0153] In certain embodiments, the methods for identifying a subject who is a
candidate for
treatment further comprise a step of assigning a course of treatment or
administering a treatment
to the subject comprising administration of an immunoglobulin preparation.
Generally, this
course of treatment will be assigned when the level of anti-amyloid antibodies
in the biological
sample is above a control threshold (e.g., a threshold indicating that the
subject likely has an
amyloid-related condition or is likely to respond to a particular treatment),
or more closely
resembles a positive control (e.g., a control level from a group or individual
having an amyloid-
related condition or a control level from a group or individual who has
responded favorably to a
particular treatment) than to a negative control (e.g., a control level from a
group or individual
not having an amyloid-related condition or a control level from a group or
individual who has
not responded favorably to a particular treatment). In a preferred embodiment,
the treatment
comprises administration of an immunoglobulin preparation.
[0154] In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises identifying a subject who is a candidate for
treatment of
Alzheimer's disease.
[0155] In a related embodiment, the present invention provides a method for
diagnosing a
disease associated with an amyloid protein in a subject. In some embodiments,
the method
comprises the steps of (a) contacting the biological sample with a thiophilic
resin to bind the
41
CA 02728605 2011-01-18
anti-amyloid antibody; (b) eluting the antibody from the thiophilic resin at a
non-denaturing pH
to form an eluate; (c) contacting the eluate with an amyloid antigen to form a
complex
comprising the amyloid antigen and the anti-amyloid antibody; (d) detecting
the presence or
level of the complex; and (e) diagnosing the subject by comparing the level of
the anti-amyloid
antibody to a control level.
[0156] In some embodiments of the methods for diagnosing a disease associated
with an
amyloid protein in a subject, the control is from an individual or group of
individuals that do not
have an amyloid-related disease, such that an increased level of the remaining
complex relative
to control is indicative of a high likelihood that the subject has an amyloid-
related condition. In
some embodiments, the control is from an individual or group of individuals
that do have an
amyloid-related disease, such that a similar level of the remaining complex
relative to control is
indicative of a high likelihood that the subject has an amyloid-related
condition. One of skill
will understand how to select at least one appropriate control and interpret
the results
accordingly.
[0157] In certain embodiments, the methods for diagnosing a disease associated
with an
amyloid protein in a subject further comprise a step of assigning a course of
treatment or
administering a treatment to the subject. Generally, this course of treatment
will be assigned
when the level of anti-amyloid antibodies in the biological sample is above a
control threshold
(e.g., a threshold indicating that the subject likely has an amyloid-related
condition or is likely to
respond to a particular treatment), or more closely resembles a positive
control (e.g., a control
level from a group or individual having an amyloid-related condition or a
control level from a
group or individual who has responded favorably to a particular treatment)
than to a negative
control (e.g., a control level from a group or individual not having an
amyloid-related condition
or a control level from a group or individual who has not responded favorably
to a particular
treatment). In a preferred embodiment, the treatment comprises administration
of an
immunoglobulin preparation.
[0158] In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
42
CA 02728605 2011-01-18
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises diagnosing Alzheimer's disease.
[01591 In one embodiment of the methods for identifying a subject who is a
candidate for
treatment or method for diagnosing a disease associated with an amyloid
protein in a subject, the
biological sample is a biological fluid, for example, blood, plasma, urine,
lymph, synovial fluid,
etc. In other embodiments, the biological sample may be a tissue sample or
biopsy. In a
preferred embodiment, the biological sample is a blood sample or fraction
thereof (e.g., plasma
or plasma fraction). In another preferred embodiment, the biological sample is
an
immunoglobulin preparation or enrichment. In a specific embodiment, the
immunoglobulin
enrichment is performed by thiophilic chromatography. In another specific
embodiment, the
immunoglobulin enrichment is performed by mixed mode ligand chromatography.
[01601 In one embodiment, the anti-amyloid antibody being detected is an
antibody specific
for an amyloid protein selected from Beta amyloid (A[3; Abeta), Islet amyloid
polypeptide
(IAPP; Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin
(HD),
Calcitonin (CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-A1),
Serum amyloid
A protein (SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8
protein; MFG-E8),
Prolactin (PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase
C), Beta 2
microglobulin ((32M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), and an
amyloid protein having a polyQ repeat. In one embodiment, the anti-amyloid
antibody is
specific for amyloid protein monomers. In another embodiment, the anti-amyloid
antibody is
specific for amyloid protein oligomers (i.e., dimers, trimers, etc). In yet
another embodiment,
the anti-amyloid antibody is specific for amyloid protein fibrils.
[01611 In one embodiment, the disease or condition associated with an amyloid
protein is
selected from the group consisting of Alzheimer's disease, Type 2 diabetes
mellitus, Parkinson's
disease, Transmissible spongiform encephalopathy, Huntington's Disease,
Medullary carcinoma
of the thyroid, Cardiac arrhythmias, Atherosclerosis, Rheumatoid arthritis,
Aortic medial
amyloid, Prolactinomas, Familial amyloid polyneuropathy, Hereditary non-
neuropathic systemic
amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice
corneal dystrophy,
43
CA 02728605 2011-01-18
Cerebral amyloid angiopathy, Cerebral amyloid angiopathy (Icelandic type), and
systemic AL
amyloidosis. In a preferred embodiment, the disease or condition is
Alzheimer's disease. In a
particularly preferred embodiment, the disease or condition is Alzheimer's
disease and the anti-
amyloid antibodies being detected are anti-Beta amyloid (A[3; Abeta)
antibodies.
[01621 In certain embodiments of the methods provided herein, detection of the
presence or
level of a particular amyloid protein is useful to diagnose a particular
disease or condition, or to
select a candidate for the treatment of a particular disease or condition. Non-
limiting examples
of amyloid-amyloid disease combinations that are well suited for use in the
methods provided
herein can be found in Table 1. In certain embodiments, the detection of the
presence or level of
a high-avidity antibody specific for the amyloid protein listed in Table I
will be diagnostic of the
corresponding disease listed.
[01631 In a preferred embodiment, the anti-amyloid antibody being detected is
an antibody
specific for Beta amyloid (A(3; Abeta). In one embodiment, the anti-A[3
antibody is specific for
A[3 monomers. In another embodiment, the anti-A(3 antibody is specific for A[3
oligomers (i.e.,
dimers, trimers, etc). In yet another embodiment, the anti-A(3 antibody is
specific for A(3 fibrils.
[01641 In one embodiment of the methods provided herein, the low-affinity or
non-specific
antibody is a low avidity anti-amyloid antibody. In a specific embodiment, the
low avidity anti-
amyloid antibody is a low avidity anti-A(3 antibody.
[01651 In certain embodiments of the methods provided herein, the complex
comprising the
amyloid antigen and the anti-amyloid antibody is washed with a solution
containing a chaotropic
salt prior to detecting the presence or level of said complex in order to
dissociate interactions
between non-specific antibodies (i.e., low avidity antibodies) and the amyloid
antigen. The
chaotropic wash step employed in the methods provided herein may be performed
with any
known chaotropic agent. When a chaotropic salt is employed, the salt will
generally comprise
either an anion having a chaotropic effect equal or greater than a chlorate
ion (Cl03-) or a cation
having a chaotropic effect greater than a calcium ion. In one embodiment, the
chaotropic agent
is selected from urea, thiourea, a guanidinium salt, a thiocyanate salt, a
perchlorate salt, an iodide
salt, and a chlorate salt. In a specific embodiment, the chaotropic salt is
ammonium thiocyanate.
In one embodiment, ammonium thiocyanate is used at a wash concentration
between about 0.5
M and about 4.0 M. In another embodiment, ammonium thiocyanate is used at a
wash
44
CA 02728605 2011-01-18
concentration between about 1.0 M and about 3.0 M. In yet another embodiment,
ammonium
thiocyanate is used at a wash concentration between about 1.5 M and about 2.5
M.
[0166] The concentration of the chaotropic agent used in the wash step of
methods provided
herein should be selected such that it is sufficient to disrupt interactions
between non-specific
(i.e., low avidity) anti-amyloid antibodies and amyloid antigens, yet does not
disrupt interactions
between specific (i.e. high avidity) anti-amyloid antibodies and amyloid
antigens. The absolute
concentration of chaotropic agent employed will depend upon a number of
factors, including, the
strength of the particular chaotropic agent and the strength of the antibody-
amyloid antigen
interactions formed in the particular system being employed (e.g., anti-Ap
antibody ELISA
assay). The skilled artisan will know how to readily determine the optimal
concentration of
chaotropic agent to use for their particular system. In one embodiment, the
chaotropic agent is
used at a wash concentration between about 0.5 M and about 4.0 M. In another
embodiment,
chaotropic agent is used at a wash concentration between about 1.0 M and about
3.0 M. In yet
another embodiment, chaotropic agent is used at a wash concentration between
about 1.5 M and
about 2.5 M.
Methods for Providing a Prognosis
[0167] In some embodiments, the detection of anti-amyloid antibody levels can
provide a
prognosis for a subject. The subject can be untreated or undiagnosed, or
currently undergoing
treatment or taking preventative measures against an amyloid-related disease.
Thus, the level of
anti-amyloid antibodies can be periodically detected to monitor the subject
over time. In a
specific embodiment, the detection of high levels of anti-amyloid antibodies
can provide a
prognosis for the subject.
[0168] In some embodiments, the methods of detecting anti-amyloid antibodies
can be used to
decide on a therapeutic regime. In cases where there appears to be relatively
low risk of
developing an amyloid-related disease, the subject can be advised to take
preventative measures.
In some cases, however, where the risk of developing an amyloid related
disease is determined to
be higher, a treatment can be prescribed. The treatment can comprise
administration of an
immunoglobulin preparation, e.g., a pooled immunoglobulin preparation such as
IVIG or other
forms of immunotherapy.
CA 02728605 2011-01-18
[0169] Methods to detect amyloid are outlined above. For example, the method
can include
obtaining from a subject a biological sample, contacting the sample with an
amyloid protein or
antigen thereof, detecting the presence or absence of anti-amyloid antibody
binding, and
diagnosing an increased likelihood of developing an amyloid-related disorder
based on levels of
high avidity anti-amyloid antibodies in the sample as compared to a control.
In some
embodiments, the method further comprises selecting a course of treatment
appropriate to the
subject. In some embodiments, the treatment comprises administering IVIG as
described herein
to the subject.
Methods Employing a Chaotropic Wash Step
[0170] In one aspect, the present invention provides methods for providing a
prognosis for the
progression of a disease or condition associated with an amyloid protein,
comprising quantifying
levels of antibodies against amyloid-forming proteins in mammalian plasma and
other biological
fluids, wherein a chaotropic wash step is employed to reduce the signal
associated with non-
specific and low-avidity anti-amyloid antigen binding.
[0171] In a related aspect, the present invention provides methods for
providing a prognosis
for treatment of a disease or condition associated with an amyloid protein,
comprising
quantifying levels of antibodies against amyloid-forming proteins in mammalian
plasma and
other biological fluids, wherein a chaotropic wash step is employed to reduce
the signal
associated with non-specific and low-avidity anti-amyloid antigen binding.
[0172] Unlike existing methods, the present invention circumvents confounding
effects of non-
specific antibody binding, interference from plasma macromolecules and
artifactual generation
of binding activity by denaturation of immunoglobulin during purification.
[0173] For instance, Measurements made on conventional glass, metal, or
plastic substrates are
affected by non-specific binding of antibodies to the amyloid protein and to
the assay plates.
Human plasma contains relatively large amounts of polyvalent antibodies that
have low avidity
for amyloid aggregates. The presence of such antibodies as well as antibodies
hat bind non-
specifically to assay substrates artifactually alters the measured anti-
amyloid activity in various
assay methods. Advantageously, the methods provided herein are able to
distinguish specific
from non-specific antibodies on the basis of differences in avidity of
binding.
46
CA 02728605 2011-01-18
[01741 Accordingly, in a specific embodiment, the invention provides a method
for providing a
prognosis for the progression of a disease associated with an amyloid protein
in a subject. In
some embodiments, the method comprises the steps of (a) contacting a
biological sample from
the subject with a plurality of amyloid antigens under suitable conditions to
form: (i) a complex
comprising an amyloid antigen and a low-affinity or non-specific antibody; and
(ii) a complex
comprising an amyloid antigen and a high avidity anti-amyloid antibody; (b)
washing the
amyloid antigen complexes formed in step (a) with a solution containing a
chaotropic agent to
dissociate at least one complex comprising an amyloid antigen and a low-
affinity or non-specific
antibody; (c) detecting the level of the remaining complexes; and (d)
providing a prognosis for
the progression of the disease by comparing the level of the anti-amyloid
antibody to a control
level.
[01751 In some embodiments of the methods for providing a prognosis for the
progression of a
disease associated with an amyloid protein, the control is from an individual
or group of
individuals that experienced progression of the disease, such that a similar
or increased level of
the remaining complex relative to control is indicative of a high likelihood
or progression of the
disease. In some embodiments, the control is from an individual or group of
individuals that did
not experience progression of the disease, such that a similar level of the
remaining complex
relative to control is indicative of a low likelihood of progression of the
disease. In yet another
embodiment, the control is from the same patient taken at an earlier time,
such that an increased
level of the remaining complex relative to control is indicative of a high
likelihood or
progression of the disease. In certain embodiments, the previous sample may
have been taken
about 1 month prior, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16,
1 7, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 month prior, or 4, 5, 6, 7, 8,
9, 10 or more years
prior. One of skill will understand how to select at least one appropriate
control and interpret the
results accordingly.
[01761 In certain embodiments, the methods for providing a prognosis for the
progression of a
disease associated with an amyloid protein further comprise a step of
assigning a course of
treatment or administering a treatment to the subject Generally, this course
of treatment will be
assigned when the level of anti-amyloid antibodies in the biological sample is
above a control
threshold (e.g., a threshold indicating a high likelihood or progression of
the disease), or more
closely resembles a positive control (e.g., a control level from a group or
individual having
experienced progression of the disease) than to a negative control (e.g., a
control level from a
47
CA 02728605 2011-01-18
group or individual not having experienced progression of the disease). In a
preferred
embodiment, the treatment comprises administration of an immunoglobulin
preparation.
[0177] In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises diagnosing Alzheimer's disease.
[0178] In a related embodiment, the present invention provides a method for
providing a
prognosis for treatment of a disease associated with an amyloid protein, for
example a known
therapy or preventative strategy for AD. In some embodiments, the treatment
can comprise
administration of an immunoglobulin preparation such as IVIG in a subject. In
some
embodiments, the method comprises the steps of (a) contacting a biological
sample from the
subject with a plurality of amyloid antigens under suitable conditions to
form: (i) a complex
comprising an amyloid antigen and a low-affinity or non-specific antibody; and
(ii) a complex
comprising an amyloid antigen and a high avidity anti-amyloid antibody; (b)
washing the
amyloid antigen complexes formed in step (a) with a solution containing a
chaotropic agent to
dissociate at least one complex comprising an amyloid antigen and a low-
affinity or non-specific
antibody; (c) detecting the level of the remaining complexes; and (d)
providing a prognosis for
treatment of the disease by comparing the level of the anti-amyloid antibody
to a control level.
[0179] In some embodiments of the methods for providing a prognosis for
treatment of a
disease associated with an amyloid protein, the control is from an individual
or group of
individuals that responded favorably to a particular treatment, such that a
similar level of the
remaining complex relative to control is indicative of a good prognosis for
the treatment of the
disease. In some embodiments, the control is from an individual or group of
individuals that did
not responded favorably to a particular treatment, such that a similar level
of the remaining
complex relative to control is indicative of a poor prognosis for the
treatment of the disease. One
48
CA 02728605 2011-01-18
of skill will understand how to select at least one appropriate control and
interpret the results
accordingly.
[01801 In certain embodiments, the methods for providing a prognosis for
treatment of a
disease associated with an amyloid protein further comprise a step of
assigning a course of
treatment or administering a treatment to the subject. Generally, this course
of treatment will be
assigned when the level of anti-amyloid antibodies in the biological sample
more closely
resembles a positive control (e.g., a control level from a group or individual
that responded
favorably to a particular treatment) than to a negative control (e.g., a
control level from a group
or individual that did not responded favorably to a particular treatment). In
a preferred
embodiment, the treatment comprises administration of an immunoglobulin
preparation.
[01811 In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises diagnosing Alzheimer's disease.
[01821 In one embodiment of the methods for providing a prognosis for the
progression of a
disease associated with an amyloid protein or for providing a prognosis for
treatment of a disease
associated with an amyloid protein, the biological sample is a biological
fluid, for example,
blood, plasma, urine, lymph, synovial fluid, etc. In other embodiments, the
biological sample
may be a tissue sample or biopsy. In a preferred embodiment, the biological
sample is a blood
sample or fraction thereof (e.g., plasma or plasma fraction). In another
preferred embodiment,
the biological sample is an immunoglobulin preparation or enrichment. In a
specific
embodiment, the immunoglobulin enrichment is performed by thiophilic
chromatography. In
another specific embodiment, the immunoglobulin enrichment is performed by
mixed mode
ligand chromatography.
[01831 In one embodiment, the anti-amyloid antibody being detected is an
antibody specific
for an amyloid protein selected from Beta amyloid (A(3; Abeta), Islet amyloid
polypeptide
49
CA 02728605 2011-01-18
(IAPP; Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin
(HD),
Calcitonin (CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-A1),
Serum amyloid
A protein (SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8
protein; MFG-E8),
Prolactin (PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase
C), Beta 2
microglobulin (132M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), and an
amyloid protein having a polyQ repeat. In one embodiment, the anti-amyloid
antibody is
specific for amyloid protein monomers. In another embodiment, the anti-amyloid
antibody is
specific for amyloid protein oligomers (i.e., dimers, trimers, etc). In yet
another embodiment,
the anti-amyloid antibody is specific for amyloid protein fibrils.
[01841 In one embodiment, the disease or condition associated with an amyloid
protein is
selected from the group consisting of Alzheimer's disease, Type 2 diabetes
mellitus, Parkinson's
disease, Transmissible spongiform encephalopathy, Huntington's Disease,
Medullary carcinoma
of the thyroid, Cardiac arrhythmias, Atherosclerosis, Rheumatoid arthritis,
Aortic medial
amyloid, Prolactinomas, Familial amyloid polyneuropathy, Hereditary non-
neuropathic systemic
amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice
corneal dystrophy,
Cerebral amyloid angiopathy, Cerebral amyloid angiopathy (Icelandic type), and
systemic AL
amyloidosis. In a preferred embodiment, the disease or condition is
Alzheimer's disease. In a
particularly preferred embodiment, the disease or condition is Alzheimer's
disease and the anti-
amyloid antibodies being detected are anti-Beta amyloid (A[3; Abeta)
antibodies.
[01851 In certain embodiments of the methods provided herein, detection of the
presence or
level of a particular amyloid protein is useful to provide a prognosis for the
progression of a
disease or provide a prognosis for the treatment of a disease associated with
an amyloid protein.
Non-limiting examples of amyloid-amyloid disease combinations that are well
suited for use in
the methods provided herein can be found in Table 1. In certain embodiments,
the detection of
the presence or level of a high-avidity antibody specific for the amyloid
protein listed in Table 1
will be diagnostic of the corresponding disease listed.
[01861 In a preferred embodiment, the anti-amyloid antibody being detected is
an antibody
specific for Beta amyloid (A[3; Abeta). In one embodiment, the anti-A(3
antibody is specific for
A(3 monomers. In another embodiment, the anti-A(3 antibody is specific for A(3
oligomers (i.e.,
dimers, trimers, etc). In yet another embodiment, the anti-A(3 antibody is
specific for A[3 fibrils.
CA 02728605 2011-01-18
[01871 In one embodiment of the methods provided herein, the low-affinity or
non-specific
antibody is a low avidity anti-amyloid antibody. In a specific embodiment, the
low avidity anti-
amyloid antibody is a low avidity anti-AP antibody.
[01881 The chaotropic wash step employed in the methods provided herein may be
performed
with any known chaotropic agent. When a chaotropic salt is employed, the salt
will generally
comprise either an anion having a chaotropic effect equal or greater than a
chlorate ion (C1O3") or
a cation having a chaotropic effect greater than a calcium ion.
101891 In one embodiment, the chaotropic salt is a guanidinium salt. Non-
limiting examples of
guanidinium salts that may be used in conjunction with the methods provided
herein include
guanidinium chloride, guanidinium nitrate, and guanidinium thiocyanate.
[01901 In another embodiment, the chaotropic salt is a thiocyanate salt. Non-
limiting
examples of thiocyanate salts that may be used in conjunction with the methods
provided herein
include ammonium thiocyanate, potassium thiocyanate, sodium thiocyanate,
lithium thiocyanate,
calcium thiocyanate, and guanidinium thiocyanate. In a specific embodiment,
the chaotropic salt
is ammonium thiocyanate. In one embodiment, ammonium thiocyanate is used at a
wash
concentration between about 0.5 M and about 4.0 M. In another embodiment,
ammonium
thiocyanate is used at a wash concentration between about 1.0 M and about 3.0
M. In yet
another embodiment, ammonium thiocyanate is used at a wash concentration
between about 1.5
M and about 2.5 M.
[01911 In yet another embodiment, the chaotropic salt is a perchlorate salt.
Non-limiting
examples of perchlorate salts that may be used in conjunction with the methods
provided herein
include ammonium perchlorate, sodium perchlorate, lithium perchlorate,
magnesium perchlorate,
and calcium perchlorate.
[01921 In yet another embodiment, the chaotropic salt is an iodide salt. Non-
limiting examples
of iodide salts that may be used in conjunction with the methods provided
herein include
ammonium iodide, potassium iodide, sodium iodide, lithium iodide, magnesium
iodide, and
calcium iodide.
101931 In yet another embodiment, the chaotropic salt is a chlorate salt. Non-
limiting
examples of chlorate salts that may be used in conjunction with the methods
provided herein
include sodium chlorate, lithium chlorate, magnesium chlorate, and calcium
chlorate.
51
CA 02728605 2011-01-18
[0194] The concentration of the chaotropic agent used in the wash step of
methods provided
herein should be selected such that it is sufficient to disrupt interactions
between non-specific
(i.e., low avidity) anti-amyloid antibodies and amyloid antigens, yet does not
disrupt interactions
between specific (i.e. high avidity) anti-amyloid antibodies and amyloid
antigens. The absolute
concentration of chaotropic agent employed will depend upon a number of
factors, including, the
strength of the particular chaotropic agent and the strength of the antibody-
amyloid antigen
interactions formed in the particular system being employed (e.g., anti-Ap
antibody ELISA
assay). The skilled artisan will know how to readily determine the optimal
concentration of
chaotropic agent to use for their particular system. In one embodiment, the
chaotropic agent is
used at a wash concentration between about 0.5 M and about 4.0 M. In another
embodiment,
chaotropic agent is used at a wash concentration between about 1.0 M and about
3.0 M. In yet
another embodiment, chaotropic agent is used at a wash concentration between
about 1.5 M and
about 2.5 M.
[0195] In certain embodiments of the methods provided herein, immunoglobulins
found in the
biological sample (i.e., blood, plasma, urine, lymph, etc.) are enriched prior
to the step of
contacting the biological sample with one or more amyloid antigens.
Preferably, the
immunoglobulins are enriched by a method that does not partially or completely
denature the
antibodies. In one embodiment, the methods provided herein for detecting high
avidity anti-
amyloid antibodies comprise a step of enriching immunoglobulins by a
chromatographic method
that does not result in denaturation of the antibodies. In one embodiment, the
chromatographic
method is performed with a thiophilic resin. In another embodiment, the
chromatographic
method is performed with mixed mode ligand chemistry (Upfront Chromatography
A/S). In
other embodiments, the methods provided herein for detecting high avidity anti-
amyloid
antibodies comprise a step of enriching immunoglobulins by a non-
chromatographic method that
does not result in denaturation of the antibodies.
Methods Employing a Thiophilic Enrichment Step
[0196] In one aspect, the present invention provides methods for providing a
prognosis for the
progression of a disease or condition associated with an amyloid protein,
comprising quantifying
levels of antibodies against amyloid-forming proteins in mammalian plasma and
other biological
fluids, wherein a thiophilic enrichment step is employed to reduce the signal
associated with
52
CA 02728605 2011-01-18
non-specific antibody binding, interference from plasma macromolecules and
artifactual
generation of binding activity by denaturation of immunoglobulin during
purification.
[01971 In a related aspect, the present invention provides methods for
providing a prognosis
for treatment of a disease or condition associated with an amyloid protein,
comprising
quantifying levels of antibodies against amyloid-forming proteins in mammalian
plasma and
other biological fluids, wherein a thiophilic enrichment step is employed to
reduce the signal
associated with non-specific antibody binding, interference from plasma
macromolecules and
artifactual generation of binding activity by denaturation of immunoglobulin
during purification.
[01981 Unlike existing methods, the present invention circumvents confounding
effects of non-
specific antibody binding, interference from plasma macromolecules and
artifactual generation
of binding activity by denaturation of immunoglobulin during purification.
[01991 For instance, previous assays that employed whole plasma to measure
endogenous anti-
amyloid activity are artifactually altered by interference from other, as yet
unidentified plasma
macromolecules. Advantageously, the methods provided herein eliminates the
interfering
activity of these unidentified plasma macromolecules in a manner that
preserves the anti-amyloid
antibodies of interest.
102001 Methods currently employed for measuring anti-amyloid antibodies
extracted from
biological specimens suffer from artifactual gains or losses of anti-amyloid
activity as a result of
exposure to denaturing conditions. For example, some methods employ
acidification either
during enrichment of antibodies (Ig) using Protein A or G affinity
chromatography or to liberate
antibodies from bound antigen. Exposure to low pH causes certain mammalian
immunoglobulins to wholly or partially denature. This leads either to loss of
function or
increased polyvalency, both of which phenomena have deleterious effects on
measuring levels of
specific anti-amyloid antibodies. Advantageously, methods provided herein
separate Ig from
plasma and dissociate weakly bound antigen molecules from the anti-amyloid
antibodies without
exposure to acids or other denaturing conditions.
[02011 Accordingly, in a specific embodiment, the invention provides a method
for providing a
prognosis for the progression of a disease associated with an amyloid protein
in a subject. In
some embodiments, the method comprises the steps of (a) contacting the
biological sample with
a thiophilic resin to bind the anti-amyloid antibody; (b) eluting the antibody
from the thiophilic
53
CA 02728605 2011-01-18
resin at a non-denaturing pH to form an eluate; (c) contacting the eluate with
an amyloid antigen
to form a complex comprising the amyloid antigen and the anti-amyloid
antibody; (d) detecting
the presence or level of the complex; and (e) providing a prognosis for the
progression of the
disease by comparing the level of the anti-amyloid antibody to a control
level.
[0202] In some embodiments of the methods for providing a prognosis for the
progression of a
disease associated with an amyloid protein, the control is from an individual
or group of
individuals that experienced progression of the disease, such that a similar
or increased level of
the remaining complex relative to control is indicative of a high likelihood
or progression of the
disease. In some embodiments, the control is from an individual or group of
individuals that did
not experience progression of the disease, such that a similar level of the
remaining complex
relative to control is indicative of a low likelihood of progression of the
disease. In yet another
embodiment, the control is from the same patient taken at an earlier time,
such that an increased
level of the remaining complex relative to control is indicative of a high
likelihood or
progression of the disease. In certain embodiments, the previous sample may
have been taken
about I month prior, or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 month prior, or 4, 5, 6, 7, 8,
9, 10 or more years
prior. One of skill will understand how to select at least one appropriate
control and interpret the
results accordingly.
[0203] In certain embodiments, the methods for providing a prognosis for the
progression of a
disease associated with an amyloid protein further comprise a step of
assigning a course of
treatment or administering a treatment to the subject. Generally, this course
of treatment will be
assigned when the level of anti-amyloid antibodies in the biological sample is
above a control
threshold (e.g., a threshold indicating a high likelihood or progression of
the disease), or more
closely resembles a positive control (e.g., a control level from a group or
individual having
experienced progression of the disease) than to a negative control (e.g., a
control level from a
group or individual not having experienced progression of the disease). In a
preferred
embodiment, the treatment comprises administration of an immunoglobulin
preparation.
[0204] In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
54
CA 02728605 2011-01-18
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises diagnosing Alzheimer's disease.
[02051 In a related embodiment, the present invention provides a method for
providing a
prognosis for treatment of a disease associated with an amyloid protein, for
example a known
therapy or preventative strategy for AD. In some embodiments, the treatment
can comprise
administration of an immunoglobulin preparation such as IVIG in a subject. In
some
embodiments, the method comprises the steps of (a) contacting the biological
sample with a
thiophilic resin to bind the anti-amyloid antibody; (b) eluting the antibody
from the thiophilic
resin at a non-denaturing pH to form an eluate; (c) contacting the eluate with
an amyloid antigen
to form a complex comprising the amyloid antigen and the anti-amyloid
antibody; (d) detecting
the presence or level of the complex; and (e) providing a prognosis for
treatment of the disease
by comparing the level of the anti-amyloid antibody to a control level.
102061 In some embodiments of the methods for providing a prognosis for
treatment of a
disease associated with an amyloid protein, the control is from an individual
or group of
individuals that responded favorably to a particular treatment, such that a
similar level of the
remaining complex relative to control is indicative of a good prognosis for
the treatment of the
disease. In some embodiments, the control is from an individual or group of
individuals that did
not responded favorably to a particular treatment, such that a similar level
of the remaining
complex relative to control is indicative of a poor prognosis for the
treatment of the disease. One
of skill will understand how to select at least one appropriate control and
interpret the results
accordingly.
102071 In certain embodiments, the methods for providing a prognosis for
treatment of a
disease associated with an amyloid protein further comprise a step of
assigning a course of
treatment or administering a treatment to the subject. Generally, this course
of treatment will be
assigned when the level of anti-amyloid antibodies in the biological sample
more closely
resembles a positive control (e.g., a control level from a group or individual
that responded
favorably to a particular treatment) than to a negative control (e.g., a
control level from a group
CA 02728605 2011-01-18
or individual that did not responded favorably to a particular treatment). In
a preferred
embodiment, the treatment comprises administration of an immunoglobulin
preparation.
[0208] In certain embodiments, the treatment is selected from a cognitive
treatment, such as
physical and/or social activity, memory games, and puzzle and/or problem
solving; a
pharmaceutical therapy, such as a cholinesterase inhibitor (to address reduced
acetylcholine), a
partial glutamate antagonists (e.g., Memantine), and a psychiatric drugs
(e.g., antipsychotics,
sleep aides, anxiolytics, and beta-blockers). Cholinesterase inhibitors
include, without
limitation, Aricept (donepezil hydrochloride), Exelon (rivastigmine),
Razadyne
(galantamine), and Cognex (tacrine), and combination thereof. In a preferred
embodiment, the
treatment comprises the administration of an immunoglobulin preparation. In a
specific
embodiment, the method comprises diagnosing Alzheimer's disease.
[0209] In one embodiment of the methods for providing a prognosis for the
progression of a
disease associated with an amyloid protein or for providing a prognosis for
treatment of a disease
associated with an amyloid protein, the biological sample is a biological
fluid, for example,
blood, plasma, urine, lymph, synovial fluid, etc. In other embodiments, the
biological sample
may be a tissue sample or biopsy. In a preferred embodiment, the biological
sample is a blood
sample or fraction thereof (e.g., plasma or plasma fraction). In another
preferred embodiment,
the biological sample is an immunoglobulin preparation or enrichment. In a
specific
embodiment, the immunoglobulin enrichment is performed by thiophilic
chromatography. In
another specific embodiment, the immunoglobulin enrichment is performed by
mixed mode
ligand chromatography.
[0210] In one embodiment, the anti-amyloid antibody being detected is an
antibody specific
for an amyloid protein selected from Beta amyloid (A[3; Abeta), Islet amyloid
polypeptide
(IAPP; Amylin), Alpha-synuclein (SNCA), Major Prion Protein (PrP), Huntingtin
(HD),
Calcitonin (CCP), Atrial natriuretic factor (ANF), Apolipoprotein Al (Apo-A1),
Serum amyloid
A protein (SAA), Medin amyloid (fragment of Milk fat globule-EGF factor 8
protein; MFG-E8),
Prolactin (PRL), Transthyretin (ATTR), Lysozyme C (1,4-beta-N-acetylmuramidase
C), Beta 2
microglobulin ((32M), Gelsolin (AGEL), Transforming growth factor-beta-induced
protein ig-h3
(Beta ig-h3; Keratoepithelin), Cystatin C (CST3), Immunoglobulin light chain
(AL), and an
amyloid protein having a polyQ repeat. In one embodiment, the anti-amyloid
antibody is
specific for amyloid protein monomers. In another embodiment, the anti-amyloid
antibody is
56
CA 02728605 2011-01-18
specific for amyloid protein oligomers (i.e., dimers, trimers, etc). In yet
another embodiment,
the anti-amyloid antibody is specific for amyloid protein fibrils.
[02111 In one embodiment, the disease or condition associated with an amyloid
protein is
selected from the group consisting of Alzheimer's disease, Type 2 diabetes
mellitus, Parkinson's
disease, Transmissible spongiform encephalopathy, Huntington's Disease,
Medullary carcinoma
of the thyroid, Cardiac arrhythmias, Atherosclerosis, Rheumatoid arthritis,
Aortic medial
amyloid, Prolactinomas, Familial amyloid polyneuropathy, Hereditary non-
neuropathic systemic
amyloidosis, Dialysis related amyloidosis, Finnish amyloidosis, Lattice
corneal dystrophy,
Cerebral amyloid angiopathy, Cerebral amyloid angiopathy (Icelandic type), and
systemic AL
amyloidosis. In a preferred embodiment, the disease or condition is
Alzheimer's disease. In a
particularly preferred embodiment, the disease or condition is Alzheimer's
disease and the anti-
amyloid antibodies being detected are anti-Beta amyloid (A[3; Abeta)
antibodies.
[02121 In certain embodiments of the methods provided herein, detection of the
presence or
level of a particular amyloid protein is useful to provide a prognosis for the
progression of a
disease or provide a prognosis for the treatment of a disease associated with
an amyloid protein.
Non-limiting examples of amyloid-amyloid disease combinations that are well
suited for use in
the methods provided herein can be found in Table 1. In certain embodiments,
the detection of
the presence or level of a high-avidity antibody specific for the amyloid
protein listed in Table I
will be diagnostic of the corresponding disease listed.
[0213] In a preferred embodiment, the anti-amyloid antibody being detected is
an antibody
specific for Beta amyloid (A[3; Abeta). In one embodiment, the anti-A(3
antibody is specific for
A(3 monomers. In another embodiment, the anti-A[3 antibody is specific for A[3
oligomers (i.e.,
dimers, trimers, etc). In yet another embodiment, the anti-A(3 antibody is
specific for A[3 fibrils.
[02141 In one embodiment of the methods provided herein, the low-affinity or
non-specific
antibody is a low avidity anti-amyloid antibody. In a specific embodiment, the
low avidity anti-
amyloid antibody is a low avidity anti-A(3 antibody.
[02151 In certain embodiments of the methods provided herein, the complex
comprising the
amyloid antigen and the anti-amyloid antibody is washed with a solution
containing a chaotropic
salt prior to detecting the presence or level of said complex in order to
dissociate interactions
between non-specific antibodies (i.e., low avidity antibodies) and the amyloid
antigen. The
57
CA 02728605 2011-01-18
chaotropic wash step employed in the methods provided herein may be performed
with any
known chaotropic agent. When a chaotropic salt is employed, the salt will
generally comprise
either an anion having a chaotropic effect equal or greater than a chlorate
ion (C1O3-) or a cation
having a chaotropic effect greater than a calcium ion. In one embodiment, the
chaotropic agent
is selected from urea, thiourea, a guanidinium salt, a thiocyanate salt, a
perchlorate salt, an iodide
salt, and a chlorate salt. In a specific embodiment, the chaotropic salt is
ammonium thiocyanate.
In one embodiment, ammonium thiocyanate is used at a wash concentration
between about 0.5
M and about 4.0 M. In another embodiment, ammonium thiocyanate is used at a
wash
concentration between about 1.0 M and about 3.0 M. In yet another embodiment,
ammonium
thiocyanate is used at a wash concentration between about 1.5 M and about 2.5
M.
102161 The concentration of the chaotropic agent used in the wash step of
methods provided
herein should be selected such that it is sufficient to disrupt interactions
between non-specific
(i.e., low avidity) anti-amyloid antibodies and amyloid antigens, yet does not
disrupt interactions
between specific (i.e. high avidity) anti-amyloid antibodies and amyloid
antigens. The absolute
concentration of chaotropic agent employed will depend upon a number of
factors, including, the
strength of the particular chaotropic agent and the strength of the antibody-
amyloid antigen
interactions formed in the particular system being employed (e.g., anti-A(3
antibody ELISA
assay). The skilled artisan will know how to readily determine the optimal
concentration of
chaotropic agent to use for their particular system. In one embodiment, the
chaotropic agent is
used at a wash concentration between about 0.5 M and about 4.0 M. In another
embodiment,
chaotropic agent is used at a wash concentration between about 1.0 M and about
3.0 M. In yet
another embodiment, chaotropic agent is used at a wash concentration between
about 1.5 M and
about 2.5 M.
Pharmaceutical Compositions and Dosages
102171 A pharmaceutical composition comprising immunoglobulin, e.g. an
enriched
immunoglobulin preparation comprising heterogenous human antibodies can be
administered by
a variety of methods known in the art. The route and/or mode of administration
vary depending
upon the desired results, but will typically be intravenous, intramuscular,
intraperitoneal, or
subcutaneous. The pharmaceutical composition can include an acceptable carrier
suitable for
intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal
administration (e.g., by
injection or infusion).
58
CA 02728605 2011-01-18
[0218] Proper fluidity of the composition can be maintained, for example, by
use of coating
such as lecithin, by maintenance of required particle size in the case of
dispersion and by use of
surfactants. In some cases, it is preferable to include isotonic agents, for
example, sugars,
polyalcohols such as mannitol or sorbitol, and sodium chloride in the
composition. Long-term
absorption of the injectable compositions can be brought about by including in
the composition
an agent which delays absorption, for example, aluminum monostearate or
gelatin.
[0219] Pharmaceutical compositions of the invention can be prepared in
accordance with
methods well known and routinely practiced in the art. See, e.g., Remington:
The Science and
Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and
Controlled
Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New
York, 1978.
Pharmaceutical compositions are preferably manufactured under GMP conditions.
Typically, a
therapeutically effective dose or efficacious dose of the immunoglobulin
preparation is employed
in the pharmaceutical compositions of the invention. The pharmaceutical
composition can be
formulated into dosage forms by conventional methods known to those of skill
in the art.
Dosage regimens are adjusted to provide the optimum desired response (e.g., a
therapeutic
response). For example, a single bolus may be administered, several divided
doses may be
administered over time or the dose may be proportionally reduced or increased
as indicated by
the exigencies of the therapeutic situation. It can be advantageous to
formulate parenteral
compositions in dosage unit form for ease of administration and uniformity of
dosage. Dosage
unit form as used herein refers to physically discrete units suited as unitary
dosages for the
subjects to be treated; each unit contains a predetermined quantity of active
compound calculated
to produce the desired therapeutic effect in association with the required
pharmaceutical carrier.
102201 Actual dosage levels can be varied so as to obtain an amount of the
active ingredient
which is effective to achieve the desired therapeutic response for a
particular patient without
being toxic to the patient. A physician can start doses of the pharmaceutical
composition at
levels lower than that required to achieve the desired therapeutic effect and
gradually increase
the dosage until the desired effect is achieved. In general, effective doses
vary depending upon
many different factors, including the specific disease or condition to be
treated, its severity,
physiological state of the patient, other medications administered, and
whether treatment is
prophylactic or therapeutic. An exemplary treatment regime entails
administration once per
every two weeks or once a month or once every 3 to 6 months.
59
CA 02728605 2011-01-18
[0221] The composition can be administered on multiple occasions. Intervals
between single
dosages can be weekly, monthly or yearly. Intervals can also be irregular as
indicated by
measuring therapeutic progress in the patient. Dosage and frequency can vary
depending on the
half-life of the antibodies in the patient.
[0222] In the case of an immunoglobulin preparation, intravenous
immunoglobulin (IVIG) is
commonly used. The IVIG formulation is designed for administration by
injection. Because the
IgG preparation of this invention has achieved an exceptionally high
immunoglobulin
concentration (10% w/v or higher), which significantly reduces the volume for
a therapeutically
effective dose, the composition of the present invention are particularly
advantageous for
subcutaneous and/or intramuscular administration to a patient, as well as more
commonly used
intravenous administration.
[0223] The term "effective amount" refers to an amount of an immunoglobulin,
particularly
IgG, preparation that results in an improvement or remediation of a medical
condition being
treated in the subject (e.g., Alzheimer's disease, Parkinson's disease, etc.).
An effective amount
to be administered to the subject can be determined by a physician with
consideration of
individual differences in age, weight, disease severity, route of
administration (e.g., intravenous
v. subcutaneous) and response to the therapy. In certain embodiments, an
immunoglobulin
preparation of this invention can be administered to a subject at about 5
mg/kilogram to about
2000 mg/kilogram each day. In additional embodiments, the immunoglobulin
preparation can be
administered in amounts of at least about 10 mg/kilogram, at last 15
mg/kilogram, at least 20
mg/kilogram, at least 25 mg/kilogram, at least 30 mg/kilogram, or at least 50
mg/kilogram. In
additional embodiments, the immunoglobulin preparation can be administered to
a subject at
doses up to about 100 mg/kilogram, to about 150 mg/kilogram, to about 200
mg/kilogram, to
about 250 mg/kilogram, to about 300 mg/kilogram, to about 400 mg/kilogram each
day. In other
embodiments, the doses of the immunoglobulin preparation can be greater or
less. Further, the
immunoglobulin preparations can be administered in one or more doses per day.
Clinicians
familiar with the diseases treated by IgG preparations can determine the
appropriate dose for a
patient according to criteria known in the art.
[0224] In certain embodiments, a concentrated IgG preparation can be
administered to a
subject at dose of about 5 mg/kilogram to about 2000 mg/kilogram per
administration. In certain
embodiments, the dose may be at least about 5 mg/kg, or at least about 10
mg/kg, or at least
CA 02728605 2011-01-18
about 20 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90
mg/kg, 100
mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 350
mg/kg, 400
mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650 mg/kg, 700 mg/kg, 750
mg/kg, 800
mg/kg, 850 mg/kg, 900 mg/kg, 950 mg/kg, 1000 mg/kg, 1100 mg/kg, 1200 mg/kg,
1300 mg/kg,
1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, or at
least about
2000 mg/kg.
[0225] In accordance with the present invention, the time needed to complete a
course of the
treatment can be determined by a physician and may range from as short as one
day to more than
a month. In certain embodiments, a course of treatment can be from I to 6
months.
Kits
102261 The invention further provides kits for the detection and/or isolation
of high affinity
anti-amyloid antibodies. The kits can be used for diagnosis of amyloid-related
disorders, and
selection of an individual for appropriate therapy, such as with IVIG.
[0227] Kits will typically include instructions for use in written or
electronic format, and
standard reagents, solutions and buffers for the desired assay. The kit can
optionally include
standard controls or consumable labware, such as ELISA plates, chromatography
tools,
containers, and reaction vessels. The kit can also include devices for
collection of a biological
sample, e.g., syringes and blood fractionation devices.
[0228] The kits of the invention can include materials for the detection of
endogenous, high-
avidity antibodies that are specific for amyloid. The kit can include
thiophilic chromatography
reagents and appropriate wash and elution buffers to separate IgG from other
proteins in the
sample.
[0229] The kit can also include materials to separate high avidity amyloid-
specific antibodies
from low avidity, or non-specific antibodies. Such materials can include a
solid support
conjugated to the desired form of amyloid, e.g., monomeric, oligomeric
(globular), or fibrillar
amyloid (e.g., A[3). The solid support can be a bead, a chromatography
stationary phase (e.g.,
agarose, silica, etc.), an ELISA plate, etc. The materials can also include at
least one chaotropic
wash buffer, to separate and remove low affinity antibodies from the amyloid-
conjugated
support. The kit can advantageously include a known high avidity (positive)
and low avidity
(negative) controls, for comparison to the test sample.
61
CA 02728605 2011-01-18
[02301 The kits of the invention can further comprise reagents for detection
of high avidity
anti-amyloid antibodies, as described herein.
[02311 In one embodiment, the present invention provides a method for
preparing a biological
sample for detecting a high avidity anti-amyloid antibody, the method
comprising the steps of-
(a) contacting a biological sample from the subject with a plurality of
amyloid antigens under
suitable conditions to form: (i) a complex comprising an amyloid antigen and a
low-affinity or
non-specific antibody; and (ii) a complex comprising an amyloid antigen and a
high avidity anti-
amyloid antibody; and (b) washing the amyloid antigen complexes formed in step
(a) with a
solution containing a chaotropic agent to dissociate at least one complex
comprising an amyloid
antigen and a low-affinity or non-specific antibody. In a specific embodiment,
the biological
sample is an immunoglobulin composition prepared by thiophilic chromatography.
In another
specific embodiment, the non-specific antibody is a low avidity anti-amyloid
antibody.
102321 In one embodiment, the present invention provides a method for
preparing a biological
sample for detecting a high avidity anti-amyloid antibody, the method
comprising the steps of:
(a) contacting the biological sample with a thiophilic resin to bind the anti-
amyloid antibody; (b)
eluting the antibody from the thiophilic resin at a non-denaturing pH to form
an eluate; and (c)
contacting the eluate with an amyloid antigen to form a complex comprising the
amyloid antigen
and the anti-amyloid antibody. In a specific embodiment, the method further
comprises a step of
washing the complex formed in step (c) with a solution containing a chaotropic
agent.
[02331 In one embodiment, the present invention provides a kit for detecting a
high avidity
anti-amyloid antibody present in a biological sample, which comprises at least
one selected from
the group consisting of a chaotropic agent and a thiophilic chromatographic
adsorbant. In one
embodiment, the kit comprises both a chaotropic agent and a thiophilic
chromatographic
adsorbant.
[02341 In another embodiment, the present invention provides the use of a
chaotropic agent
and/or a thiophilic chromatographic adsorbant for the manufacture of a kit for
detecting a high
avidity anti-amyloid antibody present in a biological sample.
[02351 In another embodiment, the present invention provides the use of a
chaotropic agent
and a thiophilic chromatographic adsorbant in combination for the manufacture
of a kit for
detecting a high avidity anti-amyloid antibody present in a biological sample.
62
CA 02728605 2011-01-18
Examples
Example 1
[02361 Collection of human plasma and IgG preparation. Blood samples from
healthy adults
and patients with AD were obtained under protocols approved by the Weill
Medical College
Institutional Review Board and collected in EDTA tubes for the production of
plasma by
standard methods. Experiments using whole plasma were carried out on specimens
stored at -
80 C and thawed once by exposure to room temperature for 30 minutes. IgG was
purified from
plasma by two methods: (1) standard chromatography on Protein G Sepharose
(Akerstrom et al.,
JBiol Chem., 261:10240-10247 (1986) and (2) preparative chromatography on
Thiophilic
Absorbant, as specified by the manufacturer (Pierce, Rockford, IL). For
Protein G
chromatography, IgG was eluted with 0.1 M glycine-HC1, pH 2.8, and collected
in tubes
containing 2M Tris base to reduce time of exposure to low pH. Eluted IgG from
either column
was dialyzed against PBS prior to use.
Example 2
[02371 Preparation of various oligomeric species of AR. A[3 monomer (Biosource
International, Camarillo, CA) was dissolved in 50% acetonitrile, incubated for
30 minutes at
37 C and lyophilized. The resulting pellet was dissolved in 1,1,1,3,3,3-
hexafluoro-2-propanol,
HFIP, incubated at 37 C for 20-30 minutes and then dried under argon.
[0238] A(3 oligomers were prepared as previously described (Barghorn et al.,
JNeurochem.,
95:834-847 (2005)). The dried A(3 peptide film was then dissolved at 5 mM in
dry DMSO and
sonicated in a bath sonicator for 30 minutes to ensure complete disruption of
A[3 structures. The
A[3 was adjusted to 400 gM with PBS, l/10`h volume of 2% SDS was added and the
mixture was
incubated at 37 C for 6 hours. The mixture was then diluted with 3 volumes of
water and
incubated for 18 hours at 4 C. The resultant globular oligomers were
characterized by
SDS:PAGE and SEC and stored at 4 C for up to four weeks.
[02391 A[3 fibrils were prepared by a modification of the method of O'Nuallain
(ONuallain et
al., Jlmmunol., 176:7071-7078 (2006)), HFIP-treated A(3 monomer was dissolved
in freshly
made 2 mM NaOH. After gentle agitation, the solution was centrifuged at 10,000
x g for 60 min
to remove large clumps or fibrils. The supernatant was then adjusted to I x
PBS containing
63
CA 02728605 2011-01-18
0.05% sodium azide and incubated with agitation at 37 C for 10 to 14 days.
Fibril structure was
confirmed by transmission electron microscopy of negatively stained grids.
Example 3
102401 ELISA of human anti-AD antibodies. Maxisorp ELISA plate (Nunc,
Roskilde,
Denmark) were coated with 0.1 ml of 0.1 M, pH 9.6, NaHCO3 buffer containing
0.1 .tg total AP,
either monomer (I mg/ml in formic acid) or premade A(3 globulomer for 1 hour
at 37 C. A13
fibril plates were made as previously described (O'Nuallain et al.,
,Ilmmunol., 176:7071-7078
(2006)). The plates were used immediately or stored at 4 C. After removing AP
coating buffer,
the plates were washed 3 times with 0.2 ml of PBS (137 mM NaCl, 2.7 mM KCI, 10
mM
Na2HPO4, pH 7.4) containing 0.05% Tween 20 (PBST). After the last wash, the
plates were
blocked with 0.2 ml/well of I% BSA in PBST for 1 hour at 37 C. The blocking
solution was
removed and the plates were washed 3 times with PBST as described above.
102411 Each ELISA plate was used to assay 10 plasma samples, diluted 1:10 with
PBST,
respectively, as well as a negative PBST control and a positive control of
IVIG at 10 mg/ml
(Gammagard Liquid, Baxter). When complete titers were done, each sample was
analyzed as
serial three fold dilutions in PBST. The plates were incubated for 1 hr at 37
C, washed 3 times
with 0.2 ml PBST, and incubated with 0.1 ml of a 1:10,000 dilution of
horseradish peroxidase-
conjugated goat, affinity-purified anti-human IgG antibody (Biosource
International, Camarillo,
CA) in PBST for 30 minutes at 37 C. The plates were washed 4 times with PBST
and developed
by adding 0.1 ml/well TMB (Invitrogen, Carlsbad, CA) for 15-30 minutes at room
temperature.
At the first indication that the negative control wells were changing color,
0.1 ml of I M HC1 was
added to each well to terminate the reaction. The OD at 405 nm was measured
using a Synergy
HT ELISA reader (Bio-Tek, Winooski, VT).
102421 The anti-A(3 antibody titers were calculated using the KC4 Signature
software to fit a
four parameter sigmoid curve to the ODs obtained from the serial dilutions of
plasma or IVIG.
Prior to curve fitting, the average OD of the secondary antibody-only controls
was subtracted
from the OD of wells with plasma, or IVIG. If necessary, the dilution of the
sample was reduced
so that the maximum OD of the least dilute sample was less than the half-max
of the IVIG
standard. The titer of the anti-A(3 antibody in a sample was taken as the
reciprocal of the dilution
64
CA 02728605 2011-01-18
at which the OD was equal to one-half the maximum OD (usually 2.0-3.0) of the
IVIG standard
diluted to 10 mg/ml.
Example 4
[0243] SPR measurements. A carboxylated sensor was loaded into a SensiQ SPR
apparatus
ICX Nomadics, Oklahoma City, OK), rinsed with a 1 minute injection of 0.1M
phosphoric acid
to remove surface contaminants and activated by injection of 2 mM EDC, 0.5 mM
NHS into
both control and experimental channels. Neutravidin (25 .ig/ml) in 10 mM
acetate buffer pH 4.7
was injected into the experimental channel followed by a 50 g/mL solution of
biotin-tagged All
monomer, oligomer or fibril in 10 mM HEPES buffer (pH 7.4), 150 mM NaCl, 1.34
mM EDTA,
and I% Tween 20. Coupling was terminated by the injection of I M ethanolamine
(pH 8) into
both channels. SPR antibody binding assays were done using serial two-fold
dilutions of plasma
or purified IgG preparations. Analysis of binding was done using Qdat software
(ICX
Nomadics, Oklahoma City, OK).
Example 5
[0244] Selective antigen-antibody dissociation by chaotrophic salt. Conditions
for selective
dissociation were determined by first measuring the avidity of antibodies
binding to blank plates
and Ap-coated plates. We then determined the molarity of the chaotropic salt
(ammonium
thiocyanate) necessary to provide the optimum dissociation between binding to
blank or amyloid
plates (Pullen et al., Jlmmunol Methods., 86:83-87 (1986)). Briefly, an All-
coated ELISA plate
was incubated with 1:10 dilution of human plasma or murine monoclonal anti-All
antibodies (1
ug/ml) diluted in PBST as described above. The wells were emptied and 0.2 ml
of 50 mM Na
phosphate buffer, pH 6.0, or this buffer containing increasing concentrations
of ammonium
thiocyanate was added in quadruplicate to each set of wells. The plates were
incubated with for
minutes at 25 C, washed three times with PBST and processed as described above
for
25 standard ELISA.
Example 6
[0245] Isolation of Anti-amyloid antibodies by Affinity Purification. Resins
for affinity
purification of anti-AR monomer antibodies that couple AR peptide to the resin
at the amino or
carboxyl terminus were obtained commercially (Alpha Diagnostic Intl., San
Antonio, TX).
30 Resins for anti-An oligomer and anti-An fibril antibodies were prepared by
coupling AR
oligomers and fibrils, made as described above, to N-hydroxysuccinimide-
activated Sepharose 4
CA 02728605 2011-01-18
Fast Flow as recommended by the manufacturer (GE Healthcare, Uppsala, Sweden).
The
columns were equilibrated in PBS and 5 g/ml IVIG diluted to 10 mg/ml in PBS
was passed over
the columns at 0.5 ml/min for a total of at least 5 complete passes of 500 ml
before washing with
PBS and standard elution with 0.1 M glycine, pH 2.5. The eluted IgG was
immediately
neutralized, concentrated by centrifugation on ultrafiltration spin columns
and dialyzed against
PBS.
Example 7
[02461 Blank plate binding during anti-AD nti-AD antibody measurements.
Quantification of anti-An
antibodies in human plasma or serum has been carried out in most instances on
ELISA plates
bearing either monomeric or aggregated (oligomeric or fibrillar) A(3 peptide.
Plasma samples
from normal human donors assayed by this method bind significantly to blank
ELISA wells
(wells not bearing any added antigen). Extensive, non-specific blank plate
binding occurs
despite the use of traditional blocking agents such as skim milk, fetal calf
serum, albumin and
commercial blocking preparations (Klaver et al., J. Neurosci. Meth. 187, 263-
269). The extent
of binding to blank ELISA plates is variable across plasma donors (Figure 1).
102471 Binding to blank ELISA plates also occurs with purified IgG isolated
from plasma
samples and pooled immunoglobulin preparations such as IVIG. IVIG binds in a
saturable,
concentration-dependant fashion to blank, AP monomer, A(3 oligomer and A(3
fibril plates. The
maximum binding values in A450 nm units was 4.54 (blank), 2.7 (monomer), 2.88
(oligomer), and
2.82 (fibril), respectively. Based on the best fit of the ELISA curves, the
maximum binding for
IVIG in absorbance units is approximately 1.6-fold greater on blank plates
than on any of the
amyloid bearing plates. Similar results were obtained for IgG isolated from
plasma of normal
individuals or AD patients. The absolute values of the maximum binding
differed depending on
the individual. The substantial binding of IVIG to the blank plate indicates a
greater number of
potential antibody binding sites on the blank plates relative to the amyloid-
bearing plates.
[02481 Despite the difference in maximum binding amplitude, the apparent
titers of antibodies
binding to blank ELISA wells were generally lower than those binding to wells
containing A[3
peptides or its assemblies. Blank plate half maximum binding titers for IVIG
were 17.6-, 3.9-
and 6.5-fold lower than that for the anti-An monomer, A(3 oligomer and A(3
fibril antibodies,
respectively. Typical half maximum titer values were 2.44 (blank plate), 42.84
(monomer), 9.42
(oligomer), and 15.85 (fibril). Similar results were obtained with IgG
isolated from the plasma
66
CA 02728605 2011-01-18
of most individual donors. The extent of blank plate binding varied across
individuals but the
maximal binding values derived from fitting the ELISA curves were always
higher than for
blank plates than the anti-amyloid antibodies. This suggests that measurements
of anti-amyloid
antibody levels based on a single dilution rather than a titer curve are
likely to be over-
estimations because of the added effects of blank plate binding.
[0249] The presence of antibodies that have the capacity to bind to blank
ELISA wells
complicates measurement of anti-A(3 oligomer antibodies in individuals since
the observed
absorbance values obtained for empty wells can be comparable or even greater
than those
obtained for amyloid-bearing plates (Figure 1). This does not mean that the
plate-binding
antibodies are identical. In fact, the absorbance obtained with empty wells at
the concentrations
used were greater than that for wells containing amyloid in six of the twenty-
two individuals
shown in Figure 1 suggesting that the antibodies do not all have the same
specificities. Blank
plate binding may thus reflect the presence of polyvalent antibodies that in
some cases are
capable of binding to both blank plates and amyloid-bearing plates.
Example 8
[0250] Blank plate binding is Fab mediated. To determine the nature of the
plate binding
interaction, we examined binding of a commercially available pool of IgG's
from 5 individuals,
as well as Fab, Fab2', and Fc fragments isolated from this same pool. We found
that binding of
antibodies to blank ELISA wells was the result of binding to the antigen-
binding region (Fab) of
the IgG molecule, as opposed to non-specific interactions involving other
domains. Figure 2
shows the antigen-binding region (Fab and Fab2' fragments) of IgG is
responsible for the binding
to the empty wells rather than a non-specific interaction through other
domains of the
immunoglobulin molecule.
Example 9
[0251] Depletion of blank plate binding antibodies. Isolation of IgG from
plasma samples
does not eliminate the binding of antibodies to blank ELISA plates. It was
tested whether
passage of IVIG over columns of polystyrene and/or agarose would deplete IgG
molecules that
readily bound to these substrates and possibly reduce the binding of IVIG to
blank ELISA wells.
The depletion was not specific for blank plate binding antibodies. The
absolute ratio of the
absorbance obtained with blank plates and A[3-bearing plates remained
relatively constant
regardless of column chromatography. A further drawback to this approach was
that the total
67
CA 02728605 2011-01-18
amount of anti-A(3-binding antibody was substantially reduced by all of the
depletion procedures,
suggesting that some of the antibodies that bind to AP can also adhere to
agarose or polystyrene.
Such binding is most likely to be attributable to low affinity, polyvalent
antibodies that bind to
multiple, unrelated epitopes, including A[3 and polystyrene.
[0252] More direct evidence for polyvalency of certain anti-An antibodies
comes from a
competition study using the plasma protein, thyroglobulin (Figure 3). The
binding of IVIG to
A[3 peptide-bearing ELISA wells was reduced when thyroglobulin was used as the
competitor,
confirming that a large pool of anti-A(3 antibodies in IVIG are polyvalent and
can bind to both of
these proteins. As seen in Figure 3, a concentration of thyroglobulin which
completely
eliminated binding of IVIG to thyroglobulin-bearing ELISA plates reduces the
binding of IVIG
to A[3 plates to approximately 50% of control values.
Example 10
[0253] Reduction of blank plate binding by chaotropic salt. The potential to
separate
antibodies that bound to blank wells from those bound to A[3 based on
differences in avidity was
examined. Avidity measurements were performed using increasing concentrations
of the
chaotropic salt ammonium thiocyanate to dissociate antibody/antigen complexes
formed on
ELISA plates. The binding of IVIG to ELISA plates whose wells were either
blank or were
coated with globular A[3 oligomer were compared. Figure 4 shows a typical
experiment
indicating that the avidity of anti-globular A(3 oligomer antibodies in IVIG
is clearly greater than
that seen for the binding of IVIG to blank plates. On the blank plates, 50% of
the binding seen
for phosphate buffer control conditions occurs when plates are incubated in
approximately 2.5M
ammonium thiocyanate. For the A(3 oligomer-bearing plates, reduction to 50%
binding is not
achieved even at 4M ammonium thiocyanate, the highest concentration used in
this study. A¾
oligomers prepared by several independent methods, including cross-linked and
non cross-linked
methods (Barghorn et al., JNeurochem., 95:834-847 (2005); Kayed et al.,
Molecular
Neurodegeneration, 2:18-29 (2007)), gave essentially identical results.
[0254] These results suggest that at least some anti-A(3 oligomer antibodies
can be
differentiated from antibodies that bind to blank plates on the basis of
binding avidity. To
further test this hypothesis, we depleted IVIG of high affinity anti-A(3
oligomer antibodies by
passage over an affinity column bearing A[3 globulomers. For the anti-A(3
oligomer-depleted
IVIG, the concentration of chaotropic salt required to achieve 50% maximal
binding of globular
68
CA 02728605 2011-01-18
A(3 oligomer was reduced from >4M to 1.3M ammonium thiocyanate. Similarly,
there was a
reduction from 2.5M to 0.9M ammonium thiocyanate for empty wells. These
results suggest that
some antibodies in the human repertoire interact with both A(3 oligomers and
empty wells.
However there are also antibodies that have a high avidity for A(3 oligomers
specifically.
Example 11
102551 To confirm that incubation in ammonium thiocyanate following antibody
binding
would allow discrimination between high affinity anti-A(3 oligomer antibodies
and those that
bind more promiscuously to empty ELISA wells, the avidity of IgG in plasma and
purified IgG
from four individuals, two young and two elderly controls was determined
(Figure 5). Purified
IgG, at I mg/ml, from these individuals exhibited consistent binding to empty
wells. The
observed absorbance for blank plate binding is reduced by -80% following 4M
thiocyanate
treatment; similar to the result obtained with IVIG in Example 10. In
contrast, the relative
binding to wells bearing A042 oligomers was reduced by only -20%. As with A(3
oligomer,
both anti-A(3 monomer and anti-An fibril antibodies are more resistant to
ammonium thiocyanate
dissociation than antibodies binding to blank plates. Thus, it appears
possible to define a set of
conditions that permits the measurement of anti-An antibodies in mixtures of
purified human
antibodies with minimal interference from antibodies that have the capacity to
bind to empty
ELISA wells.
Example 12
[02561 Generation of polyvalent antibodies by acids and destabilizing agents.
Exposure of
serum to even mild acidic conditions has been shown to increase the titers of
polyvalent
antibodies, many of which target auto-antigens (Bouvet et al., J Autoimmun.,
16:163-172 (2001);
Djoumerska et at., Scand J of Immunol., 61: 357-363 (2005)). The generation of
polyvalency by
mild acidification has also been reported for a specific monoclonal antibody
in which the pH
change resulted in increased polyvalent binding through a partial denaturation
of antigen binding
site (Dimitrov et al., Molec Immunol, 44:1854-1863 (2007)). We tested several
lots of IVIG
(Gammaguard, Baxter) and found that acidification at pH 3.0 led to an increase
in A[3 monomer
titers, ranging from 3.7- to 9.8-fold. This increase in the titer was reversed
by a 24 hour
incubation period at neutral pH and room temperature. Generation of
polyvalency by incubation
at pH 2.0 was not fully reversible. This suggests that incubation at very low
pH irreversibly
alters the structural integrity of antibodies, which nevertheless retain the
capacity to bind non-
specifically to a variety of antigens.
69
CA 02728605 2011-01-18
[02571 Exposure to 6 M urea, a more stringent denaturation treatment, also
increases the
apparent titer of a number of auto-antibodies present in IVIG (Bouvet et al.,
JAutoimmun.,
16:163-172 (2001)). IVIG at 10 mg/ml in PBS was dialyzed against PBS alone or
with 6M urea.
The increase in antibody titer was calculated by dividing the antibody titer
after urea treatment
by the antibody titer of the PBS control (Wardemann et al., Science, 301:1374-
1377 (2003). The
results shown in Table 1 are an average of a minimum of two experiments for
each autoantigen.
Table I shows that 6M urea treatment increased binding of antibodies in IVIG
to a set of auto-
antigens including A(3. The increases ranged from 4- to 23-fold after urea
treatment compared to
control IVIG preparations, with the greatest effect seen for A[3 peptide.
Therefore, partial
denaturation of IgG particularly increases the titer of serum anti-A(3
antibodies, presumably by
altering the antigen binding site to allow more promiscuous binding.
Table 1. Increase in Autoantibody Titer in IVIG Following Dialysis against 6M
Urea*
Auto-antigen Fold Increase in Titer
A042 Peptide Monomer 23
Thyroglobulin 9
LPS+ 4
ssDNA+ 6
dsDNA+ 5
Insulin+ 5
102581 The artifactual generation of A[3 binding activity by exposure to acids
or other
denaturing conditions has important implications for the measurement of anti-
An antibody titers
in purified human IgG preparations. For example, in normal controls the half-
max titer for blank
plate binding by IgG purified by the Protein G method, averaged > than 100-
fold higher than that
for the original plasma. In these same individuals, a 50-fold increase was
seen after acidification
for binding to oligomer plates. Purification of human IgG by methods that can
at least partially
denature IgG, e.g., acid elution from Protein G resins, results in
artificially high titers due to
acid-induced polyvalency.
CA 02728605 2011-01-18
Example 13
[0259] Effects of non-denaturing ITgG purification. We then investigated
whether isolation of
lgG by thiophilic absorbant chromatography would avoid the creation of
polyvalency associated
with acid elution. Thiophilic absorbent chromatographic exploits the binding
of IgG to a moiety
in which a sulphone group is in close proximity to a thioether group. As shown
Figure 6, IgG
prepared by Thiophilic chromatography removes a majority of non-immunoglobulin
proteins
present in plasma. While Thiophilic Absorbant is somewhat less effective in
removing other
plasma constituents than Protein G chromatography, it does not subject the
immunoglobulin to
denaturing conditions that can foster generation of polyvalency. Consistent
with this assertion,
repurification of IgG from IVIG using Thiophilic Absorbant does not alter the
titer of antibodies
binding to either AR or blank plates.
[0260] To compare the binding of anti-A13 antibodies in whole plasma with
those purified by
various methods, surface plasmon resonance (SPR) was employed as described
above. We
compared the binding of whole plasma from a normal control to IgG purified
from the same
plasma by either Thiophilic Absorbant or Protein G chromatography.
Measurements were
carried out on a AP oligomer sensor. The A[3 oligomer sensor comprised a
biotinylated A(3
oligomer bound to Neutravidin, which was coupled covalently to PEG chains on
the sensor.
[0261] SPR response curves for comparable concentrations of the three
preparations are shown
in Figure 7. IgG purified by thiophilic reagent yields a more positive
deflection than whole
plasma. This difference likely reflects decreased interference by plasma
proteins with A[3
binding. When IgG was isolated by Thiophilic chromatography, the increase in
the half-max
titer for blank plate binding was approximately 3-fold greater than plasma and
that for the
binding to oligomer plates almost 5-fold greater. Thus, the specific binding
to A(3 oligomer
plates is improved with the Thiophilic isolation method, most likely
reflecting the elimination of
interfering plasma proteins.
[0262] The IgG purified by Protein G chromatography showed a strikingly
different, negative
response that indicates greater binding to the control sensor than the antigen-
bearing plate. The
control sensor has a coating of PEG with alanine at the terminus, and the
negative response after
Protein G chromatography most likely results from the creation of new
polyvalent specificities in
the IgG after acid exposure, at least some of which recognize the PEG coating
on the control
sensor.
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CA 02728605 2011-01-18
Example 14
[0263] Measurement of plasma anti-amyloid activity. To determine whether anti-
amyloid
activity measured in human plasma was exclusively the result of natural or
induced polyvalency,
affinity chromatography was used to separate human anti-amyloid antibodies of
various
specificities from IVIG, including anti-A(3 monomer, A[3 oligomer and AR
fibril antibodies. In
the case of anti-A(3 monomer antibodies, two types of antibodies were
isolated. The first set of
antibodies bound to A[3 monomer coupled to the affinity resin at the amino
terminus (anti-APN)
and the second bound to A[3 monomer coupled at the carboxyl terminus (anti-
A(3C).
[0264] We determined the dissociation constants for the two types of anti-A(3
monomer and the
anti-oligomer antibodies by SPR analysis. Table 2 shows the Kd measurements
for the anti-
amyloid antibodies we isolated, and for 6E10 a monoclonal anti-Ali antibody,
which binds to an
A(3 epitope located near the amino terminus. The Kd values indicate that the
affinity purified
antibodies have different specificities, as the binding affinities were higher
to sensors containing
the same form of amyloid as was used for the affinity purification. There may,
however, be
some overlapping specificity. All affinity purified antibody preparations
showed binding to
wells of blank ELISA plates. The percent of the OD seen on the various amyloid
plates that was
obtained for blank wells binding varied with the different affinity-purified
antibody preparations.
For instance, in the case of anti-A(3N antibody, binding to blank wells was
equal to
approximately 28% of the binding to monomer wells, 15% of the binding to
fibril wells and
essentially all of the binding to the oligomer wells.
Table 2. Kd's of affinity-purified anti-amyloid antibodies from IVIG
Sensor Antibody Kd (nM)
C-terminal monomer 650
C-terminal monomer N-terminal monomer 24,000
Oligomer 20,000
6E10 47
C-terminal monomer 1,900
N-terminal monomer N-terminal monomer 450
Oligomer 4,000
6E10 158
C-terminal monomer 4,400
Oligomer N-terminal monomer 2,300
Oligomer 590
6E10 69
72
CA 02728605 2011-01-18
[02651 Table 3 summarizes the blank plate binding properties of the affinity
purified anti-A(3
antibodies isolated from IVIG. In the case of anti-fibril antibodies, two
different preparations
were assayed. The first antibody preparation was affinity purified on an A[342
fibril column, and
includes antibodies to A[3 peptide and structural neo-epitopes associated with
fibrillization. The
second preparation of anti-A(3 fibril antibody was affinity purified a second
time on an IAPP
(islet amyloid polypeptide) fibril column, and presumably contains only those
antibodies to the
neo-epitopes associated with fibrils (ONuallain et al., .Ilmmunol., 176:7071-
7078 (2006)). The
latter fraction showed decreased blank plate binding.
Table 3. Blank plate binding of affinity purified anti-A(3 antibodies. Ratio
of blank plate
binding to amyloid plate binding.
Affinity-purified antibodies ELISA Plates
Monomer Oligomer Fibril
anti-A N monomer 0.28 1.00 0.15
anti-A[3 C monomer 0.56 0.36 0.49
anti-A oligomer 0.58 0.73 0.62
anti-A(3 fibril # 1 * 0.72 1.00 0.62
anti-A fibril #2** 0.27 0.63 0.13
* Purified by passage over an fibrillar A(i42 affinity resin
** Purified over an fibrillar A042 affinity resin followed by repurification
on an fibrilar LAPP resin
[02661 It is understood that the examples and embodiments described herein are
for illustrative
purposes only and that various modifications or changes in light thereof will
be suggested to
persons skilled in the art and are to be included within the spirit and
purview of this application
and scope of the appended claims. All publications, patents, and patent
applications cited herein
are hereby incorporated by reference in their entirety for all purposes.
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