Note: Descriptions are shown in the official language in which they were submitted.
CA 02203142 1997-04-18
WO 96/12544 PCT/L1S94/11895
A Diagnostic Assay for Alzheimer's Disease: Assessment of
A~8 Abnormalities
y Background of the Invention
Statement as to Rights to Inventions Made Under
Federally-Sponsored Research and Development
Part of the work performed during the development of this invention
utilized U.S. Government Funds under Grants Nos. ROl NS3048-03 and RO1
AG11899-O1 from The National Institutes of Health (NIH). The government
may have certain rights in this invention.
Field of the Invention
The purpose of this invention is to assay the quantity and quality of A(3
peptide in Alzheimer's disease (AD) and A~i amyloidotic disorders related to
Alzheimer's disease. Specifically, the invention proposes to achieve this end
by enriching the peptide by capturing it from biological fluids such as
plasma,
serum, cerebrospinal fluid or urine with a zinc- or copper-chelated microwell
plate, and then measuring the amounts of captured A(3 with specific anti-A,~
antibodies in an ELISA.
Related Art
Alzheimer's disease is characterized pathologically by the accumulation
in the brain of A(3 protein. The A~3 proteina is a small peptide that is also
found cerebrospinal fluid and plasma. Much evidence implicates the
accumulation of A/3 in the pathogenesis of the disease, either as the
neurotoxic
agent itself or as a hallmark which accompanies neurotoxicity in the disorder.
A/3 accumulates as a highly insoluble deposit within neuronal tissues. It is
desirable to discover a treatment which would reverse the deposition and
relieve or arrest clinical deterioration.
CA 02203142 1997-04-18
WO 96112544 PCTIUS94/11895
-2-
Aggregation of A~i in the brain is believed to contribute to the
progressive dementia, characteristic of Alzheimer's disease (AD) and to the
premature AD observed among Down's syndrome patients. A~i, a 4.3-kDa
peptide, is the principal constituent of the cerebral amyloid deposits, a
~5 pathological hallmark of Alzheimer's disease (AD) (Masters et al. , Proc.
Natl.
Acad. Sci. USA 82:4245-4249 (1985); Glenner & Wong, Biochem. Biophys.
Rev. Commun. 120:885-890 (1984)). A~3 is derived from the much larger
amyloid protein precursor (APP) (Kung et al., Nature 325:733-736 (1987);
Tanzi et al. , Science 235: 880-884 (1987); Robakis et al. , Proc. Natl. Acad.
Sci. USA 84:4190-4194 (1987); Goldgaber et al., Science 235:877-880
(1987)), whose physiological function remains unclear. The cause of
Alzheimer's disease remains elusive; however, the discovery of mutations of
APP close to or within the A/3 domain (Goate et al. , Nature 349:704-706
(1991); Levy et al., Science 248:1124-1126 (1990); Murrell et al., Science
254:97-99 (1991); Hendricks et al., Nature Genet. 1:218-221 (1992), linked
to familial AD (FAD) (E. Levy et al. , Science 248:1124 (1990); A~3 Goate et -
al. , Nature 349:704 (1991); M. Chartier-Harlin et al. , Nature 353:844
(1991);
J. Murrell, M. Farlow, B. Ghetti, M.D. Benson, Science 254:97 (1991); L.
Hendricks et al. , Nature Genet. 1:218 (1992); M. Mullan et a1. , Nature
Genet. 1:345 (1992)), indicates that the metabolism of A(3 and APP is likely
to be intimately involved with the pathophysiology of this disorder.
Alzheimer's disease affects 10 % of individuals over the age of 60,
however, the existence of A(3 deposits in 40 % of the brains of normal
individuals in their forties suggests an even larger subclinical prevalence.
Hence, the disease process is likely to be very common, with individual
thresholds of neuronal and functional reserve being responsible for the
varying
onset of clinical symptoms. The disease is debilitating, chronic, incurable
and
very expensive to treat and an effective prevention or therapy would have an
enormous commercial market. However, there are no reliable biochemical
markers for AD.
FAD patients with the "Swedish" APP mutation overproduce the
soluble, secreted form of A~3 and suffer from early onset ( < 60 years) AD
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-3-
(Citron et al., Nature 360:672-674 (1992)). A potential neuropathogenic
mechanism has been reported by Younkin and colleagues (Society for
Neuroscience, Mol. Genet. Med. 3:95-137 (1993)) for the APP 717 mutations
which account for 90% of the APP mutations causing FAD. These mutations
apparently lead to an increase in the ratio of "long" A(3 (1-42) to A(3 (1-
40).
A(3 (1-40) is the predominant species in the cerebrospinal fluid (CSF), and is
a relatively soluble peptide. A(3 (1-42) is significantly more amyloidogenic,
and its overproduction relative to the 1-40 species appears to lead to early-
onset AD in these patients. Therefore, levels of A/3 (1-40) and A(3 (1-42) in
the cerebrospinal fluid (CSF), plasma, serum or urine may be expected to
correlate with cerebral pathology in sporadic AD cases, the predominant
clinical form of the disorder.
Two protocols currently exist for the estimation of A(3 levels in
biological fluids. The first involves the immunoprecipitation of A(3 with
specific anti-A~3 antibodies (e.g. Haass et al., Nature 359:322-325 (1992);
Citron et al. , Nature 360:672-674 (1992)), a technique which is, at best,
semiquantitative. This technique was used in combination with western
blotting to measure A/3 levels in CSF (Shoji et al. , Science 258:126-129
(1992)) but found no gross differences between AD and control specimens.
A double antibody capture ELISA using monoclonal antibodies raised against
A~3 appears to give specific Aa quantification with a sensitivity Iimit at
about
0.6 nM (Seubert et al. , Nature 359:325-327 (1992)).
The double antibody ELISA is more widely used and is the only
described means of accurately quantifying A(3. It has two important
limitations. It requires an abundance of expensive antibody to coat the wells
of microwell plates in order to capture the A~3 from the biological fluid. A
second anti-A~3 antibody, at a higher dilution, is used to detect captured
A/3.
The second limitation of the double-antibody capture ELISA technique for A(3
assay is that it requires a fluorescence-generating enzyme-conjugated
detection
antibody and a fluorescence microwell plate reader for the final step of the
assay.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-4-
Fluorescence plate readers are highly specialized and expensive (about
$30,000, Millipore Cytofluor), which limits the accessibility of the
technique.
Fluorescence has been preferred over more versatile, and cheaper,
chromogenic assays (e.g., horse radish peroxidase-conjugated detection
antibody acting on a chromogenic substrate), because it lowers the limit of
sensitivity allowing the measurement of A~3 at the levels found in biological
fluids. No A~3 assay has been described where the development of a
chromogenic substrate was measured by a visible-light microwell plate reader,
a far less expensive instrument (e.g., $8,000).
The A(3 species assay of the present invention will provide a rational
basis to monitor response to putative treatments for AD, as well as providing
early diagnostic information if clinical outcome studies validate the
correlation
of the A~3 levels in the blood or CSF with disease progression.
Summary of the Invention
It has now been found that A(3 strongly and specifically binds zinc and
copper in a pH dependent manner. These binding properties of A(3 have been
exploited in this invention to create a novel means of capturing A(3 from
biological fluids with a zinc- or copper-treated microwell plate, as well as a
novel means for the bulk chromatographic purification of A~3 from biological
fluids.
An advantage of this new ELISA technique over the previously
described double antibody capture ELISA is that it obviates the need for a
capture antibody (saving reagents and expense) and, because zinc- and copper-
mediated capture appears to be more efficient than immobilized antibody
capture, it is over an order of magnitude more sensitive than the reported
sensitivity of double antibody capture ELISA. Hence, the assay results with
biological fluids can be achieved using cheaper chromogenic substrates, in
conjunction with a visible-light microwell plate reader.
In the present invention, an assay is designed to quantify the amount
of A/3 peptide present in a solution such as a biological fluid. To do so, a
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-S-
solid substrate is used to which a zinc (II) and/or copper (II) complex is
immobilized. Preferably, the metal is complexed with immobilized
nitriloacetic acid. The substrate is then contacted with the biological fluid.
The free coordination sites on the zinc or copper atom act as a capture trap
for A~3 peptide which can then be detected and quantified in a number of
different ways.
The levels of A(3 are believed to correlate with the cerebral pathology
of AD. However, the more highly amyloidogenic 1-42 species of A~i may be
more important in AD pathology than other species such as the 1-40 form.
The present invention allows levels of the different species of A(3 to be
measured by use of antibodies that are specific to 1-4.2 species and do not
recognize the 1-40 form. Such antibodies are produced preferrably from mice
(although they may be produced from Guinea pigs, rabbits, rats, goats, sheep,
horses,et cetera.) by injection with peptides containing the unique 40-4.2
region of the 1-42 species (peptides comprising the A(3 sequence from residue
42 to any residue less than 38 are suitable immunogens). A monoclonal
antibody that is specific to the 1-42 species of A/3 may be selected by
testing
for immunoreactivity to different A~3 species that have been immobilized on
a zinc or copper treated microwell plate.
Therefore, the first aspect of the invention relates to a diagnostic assay
for detecting and/or quantifying A/3 peptide which may be present in a
candidate solution, comprising:
(a) contacting the candidate solution with a solid support
with a heavy metal cation immobilized thereon to capture A(3 peptide on the
surface of the solid support, thereby forming a first complex which comprises
solid supportlheavy metal cation/A(3 peptide;
(b) blocking all exposed metal binding sites remaining after
A~i capture with a blocker;
(c) contacting the first complex, which has been passed
through step (b), with an antibody specific for A~i peptide to form a second
complex which comprises solid support/heavy metal cation/A~3 peptide/
antibody specific for A~3 peptide;
CA 02203142 1997-04-18
WO 96/12544 PCT/US94111895
-6-
(d) labelling the second complex to form a detectable third
complex which comprises solid support/heavy metal cation/A/3 peptide/
antibody specific for A(3 peptide/label; and
(e) detecting the third complex, and quantifying A~3 peptide
which may be present in the candidate solution.
A second aspect of the invention relates to a diagnostic assay
for detecting and/or quantifying A/3 peptide which may be present in a
candidate solution, comprising:
(a) contacting the candidate solution with a solid support
with a heavy metal cation immobilized thereon to capture A(3 peptide on the
surface of the solid support, thereby forming a first complex which comprises
solid support/heavy metal cation/A(3 peptide;
(b) blocking all exposed metal binding sites remaining after
A~3 capture with a blocker;
(c) contacting the first complex, which has been passed
through step (b), with an antibody specific for A(3 peptide, called A(3
antibody, to form a second complex which comprises solid support/heavy
metal cadon/A(3 peptide/A(3 antibody; ,
(d) contacting said second complex with one or more anti-
antibodies specific to the A(3 antibody to form a third complex which
comprises solid support/heavy metal cation/A/3 peptide/A(3 antibody/one or
more anti-antibodies;
(e) labelling said third complex to form a detectable fourth
complex which comprises solid support/heavy metal cation/A(3 peptide/A~3
antibody/one or more anti-antibodies/label; and
(f) detecting the fourth complex, thereby quantifying A(3
peptide which may be present in the candidate solution.
The preferred heavy metal cations used in the practice of the present
invention are zinc (II) or copper (II) complexed to nitriloacetic acid. Other
organic ligands which may be used to complex the heavy metal, e.g.
copper and zinc, are, but not limited to, iminodiacetic acid, tris(carboxy-
methyl)ethylenediamine,N, N, N, N, N-carboxy(methyl)tetraethylenepentamine,
CA 02203142 1997-04-18
WO 96/12544 PCT/LTS94/11895
and methionine-polyethyleneglycol (for other such compounds, see F.H.
Arnold, Biotechnology 9:151-156 (1991), e. g., at page 154).
The preferred antibodies used in the practice of the invention are those
that are either specific to A(31.~2 which do not cross react with A(3,~ or
specific to A~31~ which do not cross react with A~31~2.
In the preferred embodiments of the invention, the antibodies specific
to A(3 protein are labelled with a radioisotope (radioactive isotope), which
can
then be determined by such means as the use of a gamma counter or a
scintillation counter. Isotopes which are particularly useful for the purpose
of the present invention are: 3H, '~I, 13~I, 3zP, 3sS~ IaC~ s~Cr9 36C1~ s~Co,
sgCo,
s9Fe and'sSe. In other prefered embodiments of the invention, the antibodies
specific to A(3 protein are labelled by conjugating them to enzymes which can
be detected when conjugated to said antibody, such as, but not limited to,
fluorescence-generating enzymes, as well as chromogenic enzymes like
alkaline phosphatase, urease, and horseradish peroxidase.
The body fluids that are assayed by the diagnostic assays of the present
invention, are preferably pretreated as described in the Examples.
Another aspect of the invention relates to kits for carrying out the
aforementioned assays which comprise a carrier means, compartmentalized in
close confinement therein to receive one or more container means, which
comprises a first container means containing a solid support having a heavy
metal cation immobilized thereon and a second container means containing an
antibody specific for A(3 peptide.
A further aspect of the invention relates to kits for carrying out the
above-mentioned assays, which comprise a carrier means, compartmentalized
in close confinement therein to receive one or more container means, which
comprises a first container means containing a solid support having a heavy
metal cation immobilized thereon, a second container means containing an
' antibody specific for A(3 protein, and a third container means containing an
anti-antibody which is specific for the antibody in the second container
means.
Preferably, the anti-antibody is detectably labeled.
CA 02203142 1997-04-18
WO 96/12544 PCTIUS94/11895
_g_
A further aspect of the invention relates to kits preferrably used for
carrying out the above-mentioned assays with biological fluids, which
comprise a carrier means, compartmentalized in close confinement therein to
receive one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing an antibody specific for A(3 protein, a
third container means containing an anti-antibody which is specific for the
antibody in the second container means, and a fourth container means
containing a methylating compound.
Another aspect of the invention relates to kits preferrably used for
carrying out the above-mentioned assays with biological fluids, which
comprise a carrier means, compartmentalized in close confinement therein to
receive one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing an antibody specific for A(3 protein, a
third container means containing an anti-antibody which is specific for the
antibody in the second container means, a fourth container means containing
a methylating compound, and a fifth container means containing magnisium
chloride.
Another aspect of the invention relates to kits preferrably used for
carrying out the above-mentioned assays with biological fluids, which
comprise a carrier means, compartmentalized in close confinement therein to
receive one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing an antibody specific for A(3 protein, a
third container means containing an anti-antibody which is specific for the
antibody in the second container means, a fourth container means containing
a methylating compound, a fifth container means containing magnisium
chloride, and a sixth container means containing a blocker.
Yet, another aspect of the invention relates to kits for carrying out the
assays of the present invention which comprises a carrier means,
compartmentalized in close confinement therein to receive , one or more
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
_g_
container means, which comprises a first container means containing a solid
support having a heavy metal cation immobilized thereon and a second
container means containing a labelled antibody specific for A(3 protein.
Another aspect of the invention relates to kits preferrably used for
carrying out the assays of the present invention with biological fluids which
comprises a carrier means, compartmentalized in close confinement therein to
receive one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing a labelled antibody specific for A~3
protein, and a third container means containing a methylating compound.
A further aspect of the invention relates to kits preferrably used for
carrying out the assays of the present invention with biological fluids which
comprises a carrier means, compartmentalized in close confinement therein to
receive one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing a labelled antibody specific for A~i
protein, a third container means containing a methylating compound, and a
fourth container means containing magnesium chloride.
Another aspect of the invention relates to kits preferrably used for
carrying out the assays of the present invention with biological fluids which
comprises a carrier means, compartmentalized in close confinement therein to
receive one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing a labelled antibody specific for A~3
protein, a third container means containing a methylating compound, a fourth
container means containing magnesium chloride, and a fifth container means
containing a blocker.
The next aspect of the invention relates to kits for carrying out the
aforementioned assays which comprise a carrier means, compartmentalized in
close confinement therein to receive one or more container means, which
comprises a first container means containing a solid support having a heavy
CA 02203142 1997-04-18
WO 96/12544 PCT/LTS94l11895
-10-
metal cation immobilized thereon and a second container means containing an
antibody specific for A(3 protein bound to a labelled anti-antibody.
Another aspect of the invention relates to kits preferrably used for
carrying out the aforementioned assays with biological fluids which comprise
a carrier means, compartmentalized in close confinement therein to receive
one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing an antibody specific for A~3 protein
bound to a labelled anti-antibody, and a third container means containing a
methylating compound.
A further aspect of the invention relates to kits preferrably used for
carrying out the aforementioned assays with biological fluids which comprise
a carrier means, compartmentalized in close confinement therein to receive
one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing an antibody specific for A~i protein
bound to a labelled anti-antibody, a third container means containing a
methylating compound, and a fourth container means containing magnesium
chloride.
A further aspect of the invention relates to kits preferrably used for
carrying out the aforementioned assays with biological fluids which comprise
a carrier means, compartmentalized in close confinement therein to receive
one or more container means, which comprises a first container means
containing a solid support having a heavy metal cation immobilized thereon,
a second container means containing an antibody specific for A~i protein
bound to a labelled anti-antibody, a third container means containing a
methylating compound, a fourth container means containing magnesium
chloride and a fifth container means containing a blocker.
Another aspect of the invention relates to a method for purification of
A(3 peptide from biological fluids containing one or more proteins which
comprises:
, , CA 02203142 2004-06-14
-Il-
(a) methylating cysteine groups of the proteins in the
biological fluid;
(b) acidifying the biological fluid obtained from step (a);
(c) applying the biological fluid obtained from step (b) to
a copper-charged chelating-Sepharose column;
(d) washing the column with equilibration buffer to obtain
an eluate solution; and
(e) collecting the eluate solution, thereby obtaining purified
Aa peptide.
Another aspect of the invention relates to a method for purification of
AiB peptide from biological fluids containing one or more proteins which
comprises:
(a) methylating cysteine groups of the proteins in the
biological fluid;
IS (b) acidifying the biological fluid obtained from step (a);
(c) adding to the biological fluid obtained from step (b), a
free copper-charged chelating slurry to form a mixture;
(d) centrifuging the mixture obtained from step (c) to obtain
a pellet;
(e) washing the pellet obtained from step (d) with
equilibration buffer, thereby obtaining purified p,~ peptide.
A further aspect of the invention relates to a kit for carrying out the
method for bulk purification of A,B peptide in biological fluids which
comprises a carrier means compartmentalized in close confinement therein to
receive one or more container means which comprises a first container means
containing a copper charged chelating-Sepharose column and a second
container means containing an antibody speck for A(3 peptide which may be
used to confirm presence of purified A(3 peptide.
Finally, another aspect of the invention relates to a kit for carrying out
the method of purifying A/3 peptide from biological fluids which comprises a
carrier means compartmentalized in close confinement therein to receive one
or more container means which comprises a first container means containing
*Trade-mark
CA 02203142 1997-04-18
WO 96!12544 PCT/US94l11895
-12-
free copper-charged chelating-Sepharose and a second container means
containing an antibody specific for A(3 peptide which may be used to confirm
presence of purified A~i peptide.
Brief Description of the Figures
FIGS. la, 1b, lc, 1d and 1e depict graphs showing analyses of
ssZn2+ binding to A~. Values shown are means t S.D., n > 3. (la)
Scatchard plot. Aliquots of A(3 were incubated (60 min) with 65Znz+ in the
presence of varying concentrations of unlabeled Znz+ (0.01-50 yvt total). The
proportion of 65Znz+ binding to immobilized peptide (1.0 nmol) described two
binding curves as shown. The high-affinity binding curve has been corrected
by subtracting the low-affinity component, and the low-affinity curve has had
the high-affinity component subtracted. (1b) Bar graph showing the
specificity of the Znz+ binding site for metals. A(3 was incubated (60 min)
with ~Znz+ (157 nM, 138,000 cpm) and competing unlabeled metal ions (50 -
~,M total). (lc) Bar graph showing ~Znz+ (74 nNt, 104,000 cpm) binding to
negative (aprotinin, insulin a-chain, reverse peptide 40-1) and positive
(bovine
serum albumin (BSA)) control proteins and A~3 fragments (identified by their
residue numbers within the Aa sequence, glnll refers to A~3~-zs where residue
11 is glutamine). Percent binding of total counts 65Znz+/min added is
corrected for the amounts (in nanomoles) of peptides adhering to the
membrane. (1d) Scatchard plot. As for (la), with Aril-zs peptide substituting
for A(31.~. 157 nM ~Zn (I38,000 cpm) is used in this experiment to probe
immobilized peptide (1.6 nmol). (1e) Graph showing the pH dependence of
6sZnz+ binding to A(3m.
FIGs. 2a, 2b and 2c depict graphs showing effect of Znz+ and other
metals on A~ polymerization using G50 gel filtration chromatography.
Results shown are indicative of n > 3 experiments where SS ~,g of A(3 is
applied to the column and eluted in 15 ml, monitored by 254 nm absorbance.
(2a) A graph showing the chromatogram of A(3 in the presence of EDTA, 50
~cM, Znz+, 0.4 ~.tvt; Znz+, 25 ~clvt; and Cuz+, 25 ~,M. The elution points of
CA 02203142 1997-04-18
WO 96/12544 PCT/I1S94/11895
-13-
molecular mass standards and relative assignments of A(3 peak elutions are
indicated. Mass standards were blue dextran (2 x 106 daltons, Vo = void
volume), BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c (12.4
kDa), and aprotinin (6.5 kDa). The mass of A~3 is 4.3 kDa. (2b) Bar graph
showing the relative amounts (estimated from areas under the curve) of
soluble A(3 eluted as monomer, dimer, or polymer in the presence of various
metal ions (25 ~,tvt), varying concentrations of Zn2+ or Cu2+ (the likelihood
of
Tris chelation is indicated by upper limit estimates), and EDTA. Data for
experiments performed in the presence of copper were taken from 214 nm
readings and corrected for comparison. (2c) Bar graph showing the effects
of pre-blocking the chromatography column with BSA upon the recovery of
A(3 species in the presence of zinc (25 ~,M), copper (25 ~.tvt), or chelator.
FIGs. 3a and 3b depict bar graphs showing A~i binding to kaolin
(aluminum silicate): effects of zinc (25 ptv~), copper (25 ~.tvt), and EDTA
(50
~,ivt). (3a) Bar graph showing the concentration (by 214 nm absorbance) of
A~i remaining in supernatant after incubation with 10 mg of G50 Sephadex.
(3b) Bar graph showing the concentration (by 214 nm absorbance) of A(3
remaining in supernatant after incubation with 10 mg of kaolin, expressed as
percent of the starting absorbance.
FIGs. 4a and 4b depict a blot and a bar graph showing the effect of
Zn2+ upon A(3 resistance to tryptic digestion. (4a) A blot of tryptic digests
of A~3 (13.9 ~,g) after incubation with increasing concentrations of zinc
(lane
labels, in micromolar), stained by Coomassie Blue. Digestion products of 3.6
kDa (A(3~), and 2.1 kDa (AJ31~.~), as well as undigested A~31~ (4.3 kDa),
are indicated on the left. The migration of the low molecular size markers
(STD) are indicated (in kilodaltons) on the right. (4b) A bar graph showing
6sZna+ binding to A/3 tryptic digestion products. The blot was incubated with
6sZn2+, the visible bands excised, and the bound counts for each band
- determined. These data are typical of n = 3 replicated experiments.
FIG. 5 depicts a graph showing a scatchard analysis of 65Zn
binding to rat A~1~. Dissolved peptides (1.2 nMol) were dot-blotted onto
0.20 ~, PVDF membrane (Pierce) and competition analysis performed as
~ 02203142 2004-06-14
-14-
described in Example 1 (FIG. 1). Rat A(31.~ and human AQI,~ were
synthesized by solid-phase Fmoc chemistry. Purification by reverse-phase
HPLC and amino acid sequencing confirmed the synthesis. The regression
line indicates a K" of 3.8 pM. Stoichiometry of binding is 1:1. Although the
data points for the Scatchard curve are slightly suggestive of a biphasic
curve,
a biphasic iteration yields association constants of 2 a~ 9 p,M, which does
not
justify an interpretation of physiologically separate binding sites.
FIGS. 6a, 6b, 6c and 6d depict graphs showing the effect of zinc
upon human, 'uI-human and rat A.~,.,o aggregation into > 0.2 a particles.
Stock human and rat A~B,.,o peptide solutions (16 ~cM) in water were pre
filtered (Spin X * Costar, 0.2 ~c cellulose acetate, 700g), brought to 100 mM
NaCI, 20 mM Tris-HCI, pH 7.4 (buffer 1) t EDTA (50 uM) or metal
chloride salts, incubated (30 minutes, 37°C) and then filtered again
(700g, 4
minutes). The fraction of the Aster in the filtrate was calculated by the
ratio
of the filtrate ODZ" (the response of the OD2,4, titrated against human and
rat
A(31~ concentrations (up to 20 ~cM in the buffers used in these experiments),
was determined to be linear) relative to the ODzl4 of the unfiltered sample.
All data points are in triplicate, unless indicated. (6a) A graph showing the
proportions of A~i,.~, incubated t Zn2+ (25 ~uM) or EDTA {50 ~cM) and then
filtered through 0.2 ~c, titrated against peptide concentration. (6b) A graph
showing the proportion of A~1~ (1.6 ~cM) filtered through 0.2 ~., titrated
against Zn2+ concentration. 1'~I-human Aim (luI-human A~i,.~ was Prepared
according to the method in Mantyh et al., J. Neurochem 61:1171 (1993)
(15,000 CPM, the kind gift of Dr. John Maggio, Harvard Medical School)
was added to unlabeled Aal~ (1.6 ~,M) as a tracer, incubated and filtered as
described above. The CPM in the filtrate and retained on the excised filter
were measured by a ~y-counter. (6c) A bar graph showing the proportion of
A~(31.~ {1.6 ~,M) filtered through 0.2 ~c following incubation with various
metal
ions (3 ~cM). The atomic number of the metal species is indicated. (6d) A
graph showing the effects of Zn2+ (25 ~cM) or EDTA (50 ~.M) upon kinetics
of human A/31~ aggregation measured by 0.2 ~ filtration. Data points are in
duplicate.
*Trade-mark
' ' CA 02203142 2004-06-14
-15-
FIGS. 7a, 7b, 7c and 7d depict bar graphs showing the size
_ estimation of zinc-induced AFB aggregates. (7a and 7b) Bar graphs showing
the proportion of A~B,~ (1.6 ~cM in 100 mM NaCI, 20 mM Tris-HCI, pH 7.4
(buffer 1), incubated ~ Zn2+ (25 ~lVn or EDTA (50 N,M) and then filtered
through filters of indicated pore sizes (Durapore* filters (Ultrafree-MC
Millipore) were used for this study, hence there is a slight discrepancy
between the values obtained with the 0.22 p, filters in this study compared to
values obtained in FIG. 6 using 0.2 p. Costar filters). (7c) A bar graph
' showing ~ZnCl2 (130,000 CPM, 74 nM) used as a tracer of the assembly of
the zinc-induced aggregates of human A~1,~ produced in FIGs. 7a and 7b.
By determining the amounts of Aa,~ and ~Zn in the filtrate, the quantities
retarded by the filters could be determined, and the stoichiometry of the
zinc:
A~ assemblies estimated. (7d) Bar graph. Following this procedure, the
filters, retaining Zn: A~ assemblies, were washed with buffer 1 (100 mM
1S NaCI, 20 mM Tris-HCI, pH 7.4) + EDTA (50 ~cM x 300 p,1, 700g, 4
minutes). The amounts of zinc-precipitated A/3,.,a resolubilized in the
filtrate
fraction were determined by OD2,4, and expressed as a percentage of the
amount originally retained by the respective filters. ~Zn released into the
filtrate was measured by y-counting.
y'IGs. 8a and 8b are photographs showing zinc-induced tinctorial
amyloid formation. (8a) Zinc-induced human A~,~ precipitate stained with
Congo Red. The particle diameter is 40 ~c. A(i,~ (200 ~cl x 25 ~M in buffer
1 (i00 mM NaCI, 20 mM Tris-HCI, pH 7.4)) was incubated (30 minutes,
37°C) in the presence of 25 ~cM Zn2+. The mixture was then centrifuged
2S (16,000g x 15 minutes), the pellet washed in buffer 1 (100 mM NaCI, 20 mM
Tris-HCI, pH 7.4) + EDTA (50 p.M), pelleted again and resuspended in
Congo Red (1 % in 50% ethanol, 5 minutes). Unbound dye was removed, the
pellet washed with buffer 1 (100 mM NaCI, 20 mM Tris-HCI, pH 7.4) and
' mounted for microscopy. (8b) The same aggregate visualized under
polarized light, manifesting green birefringence. The experiment was repeated
with EDTA (50 ~,M) substituted for Znz+ and yielded no visible material.
*Trade-mark .
CA 02203142 1997-04-18
WO 96/12544 PCT/ITS94/11895
=16-
FIG. 9 depicts a graph showing the effect of zinc and copper upon
human,125I-human and rat A~1.,~ aggregation into > 0.2 w particles. Stock
human and rat A(31.4o peptide solutions (16 ~cM) in water were pre-filtered
(Spin-X, Costar, 0.2 ~c cellulose acetate, 700g), brought to 100 mM NaCI, 20
mM Tris-HCI, pH 7.4 (buffer 1) t EDTA (50 ~cM) or metal chloride salts,
incubated (30 minutes, 37°C) and then filtered again (700g, 4 minutes).
The
fraction of the A(3,.4o in the filtrate was calculated by the ratio of the
filtrate
OD214 (the response of the OD214, titrated against human and rat A(31.,,~
concentrations (up to 20 ~,M in the buffers used in these experiments), was
determined to be linear) relative to the OD2la of the unfiltered sample. All
data points are in triplicate, unless indicated. (FIG. 9) The graph shows the
proportions of A/3,.~0, incubated ~ Zn2+ (25 ~cM) or Cu2+ or EDTA (50 p,M)
and then filtered through 0.2 ~,, titrated against peptide concentration.
FIG. 10 depicts the amino acid sequence of human A~ peptide.
The amino acid sequence of human A(3 peptide is depicted and amino acid
positions are numbered.
FIG. 11 depicts a standard curve graph for increasing A~
concentration on an ELISA using a copper coated 96-well plate for solid
phase capture. Values are shown t S.D. n = 3. Increasing concentrations
of A~3 were prepared in coating buffer (Tris 20 mM, pH 7.4 and NaCI
150 mM). Aliquots (200 ~d) were transferred to the wells of a copper-treated
96-well plate ((copper (II) was immobilized on the well surface with
nitriloacetic acid) and incubated for 2 h at 37°C. The solution in the
wells
was removed and replaced with 300 ~,1 per well of blocking buffer (2 % gelatin
in Tris 20 mM, pH 8 and NaCI 150 mM) and the plate incubated at 37°C
for
a further 2 h. The wells were washed two times with 300 ~,1 aliquot's of
washing buffer (Tris 20 mM, pH 8 and NaCI 150 mM) before being incubated
(2 h at 37°C) with a primary antibody (200 ~cl per well of antibody
diluted
1/1000 with blocking buffer containing a reduced gelatin concentration
(0.2 % )) directed at the N-terminus of Aa (a now commercially available
mouse monoclonal antibody supplied by Dr. S.K. Kim of the NY State
Institute for Basic Reseaich in Developmental Disabilities). The wells were
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-17-
washed three times with washing buffer before the addition of anti-mouse-
antibody-HRPO conjugate (200 ~,1 per well of a 1/1000 dilution in 0.2%
gelatin blocking buffer) and a final incubation at 37°C for 2 h. The
wells
were washed three times with washing buffer and one final rinse in water
before the addition of 200 ~,1 per well of HRPO substrate solution (Pierce,
34024). After a 30 minute incubation at room temperature (18-22°C) a 25
~,1
aliquot of 2 M HZS04 was added to each well and absorbance of the plate
measured at 450 nm. The average background absorbance (wells containing
no A~3) was subtracted from the absorbance of A/3 standards and the resulting
values plotted against peptide concentration.
Other features and advantages of the invention will be apparent from
the following detailed description, and from the claims.
Detailed Description of the Preferred Embodiments
A~3,.~, the major component of Alzheimer's disease cerebral amyloid
is relatively soluble at high concentrations ( <_ 3 .7 mM) and has been
detected
in CSF and blood. Physiological factors which decrease solubility and induce
A/3 amyloid formation may be important in the pathogenesis of the disease.
It has been discovered that human A(3 specifically and saturably binds zinc,
and that concentrations of this metal ion above 300 nM rapidly destabilize
human A(31,.~ solutions, inducing tinctorial amyloid formation. High-affinity
binding (KA = 107 nM) compatible with normal CSF zinc levels, and low-
affinity binding (K" = 5.2 ~,M) have now been shown. In contrast, rat A~31~
binds zinc less avidly and does not aggregate in its presence, suggesting a
possible explanation for lack of cerebral Aa amyloid in these animals.
Collectively, these data suggest a potentially critical role for cerebral
,zinc
metabolism in the neuropathogenesis of Alzheimer's disease.
Further, it has been observed that abnormalities of zinc homeostasis
occur in AD and DS patients. Cerebral zinc homeostasis, which has been
reported to be abnormal in AD (D. Wenstrup, W.D. Ehmann, W.R.
Markesbery, Brain Res. 533:125 (1990); J. Constantinidis, Encephale 16:231
CA 02203142 2004-06-14
18
(1990); F.M. Corrigan, G.P. Reynolds, N.I. Ward, Biometals 6:149 (1993);
C.O. Hershey et al., Neurology 33:1350 (1983)) may be important for the
metabolic fate of AFB since increased concentrations of zinc promote the
peptide's adhesiveness and resistance to proteolytic digestion. Moreover, oral
zinc supplementation has recently been shown to have an acutely adverse
effect on cognition in AD subjects, but not age-matched controls indicated
that
environmental or nutritional zinc exposure may be a contributing factor to AD
pathophysiology.
The present findings have indicated that Aa strongly and specifically
binds zinc in a pH dependent manor. In the brain milieu, these metal ions
are present in sufficient concentration to exert these effects on binding and
solubility. A decrease in A~3 solubility occurs in the presence of
concentrations of zinc as low as 0.3 tcM. Occupation of the zinc binding site
on A/3 increases the resistance of the peptide to tryptic digestion at the a
secretase site, a-Secretase is an, as yet, unident~ed protease which has been
observed to cleave the precursor molecule of A~, the Amyloid Protein
Precursor (APP) within the Aa domain, rendering A/3 incapable of
accumulating. Hence, occupation of the zinc binding site on A(3 will increase
the biological half life of the peptide and so increase its availability for
deposition.
The diagnostic assays of the present invention are carried out as
exemplified in Example 14, below. In general, a commonly used protocol for
an ELISA is followed. The AQ peptide acts as the antigen of a conventional
direct ELISA. The plates used are coated with zinc (II) or copper (II), hence,
enabling the relatively stable binding of the A/3 peptide to the surface of
the
plate. Preferably, the zinc (I1] or copper (II) is complexed to a ligand which
is immobilized on the plate. Examples of such ligands include nitriloacetic
acid and iminodiacetic acid. The complexes are prepared by disolving the
ligand in an organic solvent such as ether, depositing the solution on a solid
support, letting the solvent evaporate, and then adding an equeous solution of
a zinc (B) or copper (I>) salt (such as the chloride). The solid support may
then be washed with additional solution to remove unreacted ligaud and metal
salt. Preferred solid supports include but are not limited to nitrocellulose,
*Trade-mark
CA 02203142 1997-04-18
WO 96/12544 PCTlUS94111895
-19-
diazocellulose, microtiter plates, glass, plastic, polystyrene, polyvinyl,
polyvinylchloride, polypropylene, polyethylene, dextran, affinity support gels
' such as Sepharose or agar, starch, and nylon. Those skilled in the art will
note that many other suitable carriers for binding the ligand exist, or will
be
able to ascertain the same by use of routine experimentation.
In a different embodiment of the diagnostic assays, the A~3 peptide in
a solution can be detected by using solid support particles. The particles,
beads or pieces of a solid support, are coated with zinc (II) or copper (II)
form of nitriloacetic acid, thus, enabling the relatively stable binding of
the
A(3 peptide to the surface of the particle. The candidate solution is added to
the particles and incubated as before-described to allow binding of the A(3
peptides to the surfaces) of the particles. Labelled antibodies, particularly
radiolabelled ones, are used to bind to the A(3 peptides. Alternatively,
antibodies specific for A(3 are added to bind to the A~3 peptides, and then
labelled anti-antibodies, particularly radiolabelled ones, which are specific
for
the A~3 antibodies, are added to bind to the A(3 antibodies, thereby allowing
detection and quantification of the A(3 peptides. The A(3 peptides are
detected
and/or quantified with the appropriate means, e.g., scintillation counter.
The bulk purification of A(3 from biological fluids is best achieved with
copper charged chelating-Sepharose (Pharmacia, catalog no. 17-0575-O1).
The cysteine groups in the sample proteins are first methylated with N-methyl
maleimide, about 1-20 mM, preferrably, about 10-20 mM, and most
preferrably, about 10 mM for about 1-2 hours, preferrably about 1 hour,
(other appropriate compounds, such as, iminodiacetic acid, may be used
instead of maleimide in simillar concentrations and for simillar periods of
time), then acidified by titrating pH to about 4.9-5.0, preferrably to about
5.0,
using about 1-2 M, preferrably about 1M, sodium acetate, pH about 3-4,
preferrably about 3.5, and the total NaCI concentration increased by about
~ 450-550 mM, preferrably by about 500 mM, with about 4-5 M, preferrably
about SM NaCI. The sample is then applied to a copper-charged chelating
Sepharose column (e.g., 250 ~,l bed volume for about 15 ml of CSF) or free
copper-charged chelating-Sepharose slurry (about 50-60 ~.1, preferrably about
CA 02203142 1997-04-18
WO 96/12544 PCT/LTS94/11895
-20-
50 ~,1 of about 50 % v/v) is added to the sample if the volume is less than
about 4 ml. Equilibration buffer is about 450-550 mM, preferrably about 500
mM NaCI about 25-100 mM, preferrably about 50 mM MES, pH about 4.0-
5.1, preferrably about 5.0 and is used to wash the column or the Sepharose
pellet following centrifugation (preferably, low speed centrifugation (about
1,200-1,800 g, preferrably about 1,500 g, for about 2-4. minutes, preferrably
about 3 minutes)). It should be noted that as the speed of centrifugation
increases, the centrifugation time decreases. The Sepharose pellet is
developed with SDS sample buffer containing 50 mM EDTA if the sample is
to be applied in entirety to western blot analysis. Alternatively, the
Sepharose
can be developed with about 450-550 mM, preferrably about 500 mM NaCI,
50 mM EDTA, pH about 7.0-9.0, preferrably about 8.0, alone and the eluate
sampled for western blot analysis. The treatment of 15 ml of CSF by this
method enriched both soluble APP as well as 4.3 and 3.6 kDa species of A(3
(identified by an antibody that identifies an epitope in the first 16 residues
of
A(3; commercially available). In order to bind copper or zinc, the peptide
requires an intact domain from residues 6-28. 4G8 only recognized the two
A/3 species and not APP, confirming that the APP captured by the Sepharose
was post-secretase cleaved soluble APP. The use of specific anti-Aa
antibodies as described above on western blot analysis of these products can
confirm the specificity of the ELISA immunoreactivity.
DEFINITIONS
A(3 peptide is also known in the art as A~3, (3 protein, ~3-A4 and A4.
Amyloid as is commonly known in the art, and as is intended in the
present specification, is a form of aggregated protein.
Similarly, A~i Amyloid is an aggregated A(3 peptide. It is found in the
brains of patients afflicted with AD and DS and may accumulate following
head injuries and in Guamanian amyotrophic lateral sclerosis/Parkinson's
dementia (GALS/PDC).
CA 02203142 2004-06-14
-21-
Tinctorial amyloid is referred to amyloid that in addition to being
insoluble in aqueous buffer can be stained with Congo Red, and has positive
birefringence in polarized light.
Anti-amyloidotic agent refers to a compound that inhibits formation of
amyloid.
Zinc-induced A/3 aggregates are, like tinctorial amyloid, insoluble in
aqueous buffer and stain with Congo Red.
A~ amyloidosis, as is commonly known in the art and intended in the
present specification, refers to the pathogenic condition in humans and other
animals which is characterized by formation of AS amyloid in neural tissue
such as brain.
Pre-filtering and pre-filtered as used in the present specification means
passing a solution, e.g. AS peptide in aqueous solution, through a pomus
membrane by any method, e.g. centrifugation, drip-through by gravitational
force, or by application of any form of pressure, such as gaseous pressure.
Physiological solution as used in the present specification means' a
saline solution which comprises compounds at physiological pH, about 7.4,
which closely represents a bodily or biological fluid, such as CSF, blood,
plasma, et cetera.
Heavy metal chealating agent refers to any agent, e.g., compound or
molecule, which chelates heavy metals, i.e., binds the heavy metal very
tightly and can inhibit or stop interaction with other agents. Examples of
such
heavy metal chealating agents are EDTA or Desferrioxamine.
In the present invention, the heavy metal salts are of auy heavy metal
or any transition metal, in any foam, soluble or insoluble, e.g. the chloride,
bromide, or iodide salts.
A blocker of heavy metal rations as used in the present invention refers
to any compound that binds to all exposed metal binding sites remaining on
the heavy metal rations, which are conjugated to the solid support, after A(3
capture. Examples of such blockers are, but are not limited to, gelatin
(Biorad, catalog no. 170-6537) and S~perBlock (Pierce, catalog no. 375-35).
*Trade-mark
CA 02203142 1997-04-18
WO 96/12544 PCT/LTS94/11895
-22-
In the present specification, unless otherwise indicated, zinc means
salts of zinc, i.e., Zn2+ in any form, soluble or insoluble.
Biological fluid means fluid obtained from a person or animal which
is produced by said person or animal. Examples of biological fluids include
but are not limited to cerebrospinal fluid (CSF), blood, serum, urine, and
plasma. In the present invention, biological fluid includes whole or any
fraction of such fluids derived by purification by any means, e. g. , by
ultrafiltration or chromatography.
Neat sample of a biological fluid means that the biological fluid has not
been altered by, for example, dilution.
Control human subject refers to a healthy person who is not afflicted
with amyloidosis.
Synthetic peptide standard in the present invention means an assembly
of amino acids linked by peptide bonds that is synthesized in a laboratory.
Methods for making synthetic peptides include, although are not restricted to,
such procedures as solid phase P~ chemistry.
A candidate solution in the present invention means a solution which
is suspected of containing A(3 peptide.
An anti-antibody in the present invention means an antibody that binds
specifically to another antibody. Generally such antibodies are obtained by
immunizing an animal with the antibody from another animal. Thus, one can
obtain goat anti-IgG polyclonal antibodies in this way.
A solid support in the present invention means any solid material to
which the heavy metal cations can be complexed, and which can be used to
make and use the invention. Examples of such solid support are, but not
limited to, microtitre plates, petri dishes, bottles, slides, and other such
containers made of plastic, glass, polyvinyl, polystyrene, and other solid
materials which do not interfere with the formation of complexes and allow
detection of labelled antibodies.
More specifically, solid support particles which may be used in the
present invention are irregular shaped solid supports such as beads,
particles,
CA 02203142 1997-04-18
WO 96/12544 PCT/ITS94/11895
- 23 -
and pieces of the aforementioned solid materials which may be used for the
practice of the present invention.
Persons skilled in the art are able to screen for and determine the
usefulness of a solid support material by parallel testing and comparison
between the material in question and a known solid support material such as
polyvinyl or polystyrene,.
In the present invention, the A~3 peptide may be comprised of any
sequence of the A(3 peptide as long as it contains at least the amino acids
corresponding to positions 6 through 28 of A(3 peptide which comprise the
binding site for zinc, the most preferred heavy metal cation capable of
binding
to a polypeptide comprising at least amino acids 6 to 28 of A(3. The preferred
embodiments of the invention make use of peptides A~31_39. Ay-ao. Aa~-an
A/31~z, and A(31.~3. The most preferred embodiment of the invention makes
use of A~31.,~. However, any of the A(3 peptides which comprises at least
amino acids 6 to 28 of AJ3 may be employed according to the present
invention. The sequence of A~3 peptide, including amino acids 6 to 28, is
found in Fig. 10 (C. Hilbich et al., J. Mol. Biol. 228:460-473 (1992)).
In the present method, the A(3 peptide is directly detected by using
optical spectrophotometry. This is possible because a direct correlation
exists
between concentration of the peptide and OD214 measurements. Although the
preferred wave length for the OD measurements is about 214 nm, the
measurements may be carried out for the purpose of the present invention at
wave lengths from about 190 to about 500 nm. Preferred wave lengths are,
however, from about.208 to about 280 nm.
Further, the A(3 peptide may be detected by radiolabelling the peptide
and measuring the compounds per minute (CPM) of the filtrates andlor the
pellets. A preferred radiolabelled A/3 peptide in the present invention is 3H
A(3. Other radiolabels which can be used in the present invention are 14C and
' 35S .
. The labelled antibodies and anti-antibodies are detected by using
visible-light microwell plate reader (for chromogenic enzymes), fluorescence
microwell plate reader (for fluorescence-generating enzymes), and
scintillation
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-24-
counter (for radioisotopes). The types of labels and the appropriate means for
detectioin of the labels, however, are not limited to those specifically
mentioned herein.
Other heavy metal canons capable of binding to a polypeptide
comprising at least amino acids 6 to 28 of A(3 which may be used in the
practice of the invention include metallochloride salts, preferably of zinc,
copper, or mercury. The most preferred embodiment of the invention,
however, makes use of zinc chloride.
In the preferred embodiments of the invention, the antibodies specific
to A~3 protein are labelled with a radioisotope (radioactive isotope), which
can
then be determined by such means as the use of a gamma counter or a
scintillation counter. Isotopes which are particularly useful for the purpose
of the present invention are: 3H, i'~I, 13'I, 32p~ ssS~ ~aC~ s~Cr~ 36C1~ s7Co,
sBCo,
s9Fe and'sSe.
Another way in which the antibody of the present invention can be
detectably labeled is by linking or conjugating the same to an enzyme. This
enzyme, in turn, when later exposed to its substrate, will react with the
substrate in such a manner as to produce a chemical moiety which can be
detected as, for example, by spectrophotometric, fluorometric or visual
means. Examples of enzymes which can be used to detectably label the
antibody of the present invention include malate dehydrogenase,
staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol
dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate
isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-VI-
phosphate dehydrogenase, glucoamylase and acetylcholine esterase. Avidin-
biotin binding may be used to facilitate the enzyme labeling.
It is also possible to label the antibody with a fluorescent compound.
When the fluorescently labeled antibody is exposed to light of the proper wave
length, its presence can then be detected due to the fluorescence of the dye.
Among the most commonly used fluorescent labelling compounds are
CA 02203142 1997-04-18
WO 96/12544 PCTlL1S94/11895
- 25 -
fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin,
allophycocyanin, o-phthaldehyde and fluorescamine.
The antibody of the invention can also be detectably labeled using
fluorescent emitting metals such as lszEu, or others of the lanthanide series.
These metals can be attached to the antibody molecule using such metal
chelating groups as diethylenetriaminepentaacetic acid (DTPA) or
ethylenediaminetetraacetic acid (EDTA).
The antibody of the present invention also can be detectably labeled by
coupling it to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting the
presence of luminescence that arises during the course of a chemical reaction.
Examples of particularly useful chemiluminescent labeling compounds are
luminol, isoluminol, theromatic acridinium ester, imidazole, acridinium salt
and oxalate ester.
Likewise, a bioluminescent compound may be used to label the
antibody of the present invention. Bioluminescence is a type of
chemiluminescence found in biological systems in which a catalytic protein
increases the efficiency of the chemiluminescent reaction. The presence of a
bioluminescent antibody is determined by detecting the presence of
luminescence. Important bioluminescent compounds for purposes of labeling
are luciferin, luciferase and aequorin.
Another technique which may also result in greater sensitivity when
used in conjunction with the present invention consists of coupling the
antibody of the present invention to low molecular weight haptens. The
haptens can then be specifically detected by means of a second reaction. For
example, it is common to use such haptens as biotin (reacting with avidin) or
dinitrophenyl, pyridoxal and fluorescamine (reacting with specific anti-hapten
antibodies) in this manner.
In addition, the sensitivity of the assay may be increased by use of
amplification strategies including substrate cycling and enzyme channeling as
taught by Mosbach (Lindbladh et al. , Trends in Biochem. Sci. 18:279-283
{1993).
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-26-
The pH of the reaction mixtures for A(3 capture to immobilized metal
ions is, unless otherwise indicated, preferably close to neutral (about 7.4).
The pH, therefore, may range from about 6.8 to about 8.5, preferably from
about 7 to about 7.8, and most preferably about 7.4. The pH' of other
incubations, including antibody and anti-antibody incubations is between 7-9,
preferrably at 8.
Buffers which can be used in the methods of the present invention
include, but are not limited to, Tris-chloride and Tris-base, MOPS, HEPES,
bicarbonate, Krebs, and Tyrode's. The concentration of the buffers may be
between about 10 mM and about 500 mM. However, considering that these
buffers chelate zinc, the concentration of the buffers should be kept as low
as
possible without compromising the results.
The present invention permits use of very low concentrations of A/3
peptide, e.g. from about 0.1 nM to 3.7 mM (upper limit of solubility). A
preferred embodiment of the invention employs about 0.8 nM concentration
of A(3 peptide, the lowest detectable concentration of A(3 previously reported
for an ELISA type assay was 0.5 nM (Schubert et al. , Nature 359:325-327
(1992)).
The present invention may be practiced at temperatures ranging from
about 1 degree centigrade to about 99 degrees centigrade. The preferred
temperature range is from about 4 degrees centigrade to about 40 degrees
centigrade. The most preferred temperature for the practice of the present
invention is about 37 degrees centigrade. Therefore, an advantage of the
present invention is the greatest sensitivity over previous detection systems.
The A~3 peptide is trapped by the free coordination sites on the zinc or
copper atoms (binds to the zinc or copper atoms) at near-instantaneous rate.
However, defusion rates are a limiting factor in the absorption of the peptide
and antibodies to the solid phase. In a preferred embodiment of the invention,
the incubations are carried out for about 90-240, . preferably about 120,
minutes to maximize capture.
To determine whether A/3 binds zinc, a synthetic peptide representing
secreted A~31~ was incubated with 65Zn2+ . Rapid binding (60 % B",~ at
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-27-
1 min), which plateaued at 1 h, was observed. Scatchard analysis of 65Zn2+
binding describes two saturable binding curves, a high affinity curve (KQ
< 107 nNt), and a lower affinity curve (KQ < 5.2 ~,ivt) (Fig. la). The
affinity
constant estimates might be skewed by assuming that the Tris buffer does not
bind zinc. In fact, Tris-HCl binds zinc and copper with stability constants of
4.0 and 2.6, respectively (Dawson et al. , Data for Biochemical Research,
Oxford University Press (1986)). Incubating A/3 in the presence of higher
concentrations of Tris (150 and 500 mM) abolishes 65Zn2+ binding to A/3
( ~ 50 % and ~ 95 % , respectively), indicating that Tris-induced Zn2+
chelation
cannot be excluded. Our calculated affinity constants are therefore upper
limit
estimates.
6sZnz+ binding is very specific, with Zn2+ being the only unlabeled
metal ion tested that is capable of competing off the label (Fig. 1b). To
determine the specific region of A~3 involved in zinc binding and to validate
the dot-blot binding system, equivalent amounts of various peptides
representing fragments of A(3,_,~ and peptide controls were assayed for ~Zn2+
binding in this system (Figs. lc and 1d).
The reverse sequence (40-1) control peptide only binds 50% of B",a,
compared with A(31~ (Fig. lc), indicating that zinc binding is not merely a
consequence of the presence of favorable residues. A/31_28 bound 30% of
B",a,~,
indicating that the carboxyl terminus plays an important role in promoting
zinc
binding. Glutamine substitution for the glutamate at position 11 of A(31_Z8,
in
accordance with the Down's syndrome A/3 sequence reported by Glenner and
Wong, Biochem. Biophys. Res. Commun. 120:885-890 (1984), does not
interfere with 65Zn2+ binding. The Scatchard plot of 65Zn2+ binding to A~31_2s
reveals similar low-affinity (KQ < 15 yvt) and high-affinity (KQ < 334 nNt)
binding associations (Fig. lad to those of A~31~, but overall the A~i,_28
peptide
binds zinc less avidly. Although the A(31_2$ peptide clearly binds zinc,
peptides
' overlapping this region (1-17 and 12-28) do not individually bind zinc.
Additionally, a peptide covering a region of the carboxyl terminus (25-35)
also
is unable to bind zinc (Fig. lc).
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-28-
The calculated stoichiometry of high-affinity Znz+-binding to A(3,
derived from the x-intercepts on the Scatchard plots (Fig. l, a and ~, is
0.7:1
(A/31.~) and 1:4 (A~31_z8). For low-affinity binding, the Znz+:A(3 ratio is
2.5:1
(A/3i.,~) and 4:1 (A(31_zs)~
65Znz+ binding of sequenced tryptic digest products of A/3 ((Fig. 4b)
indicates that the 6-40 fragment binds zinc, but that the other visible digest
fragment 17-40 (Fig. 4b), equivalent to the post-secretase (Esch et al. ,
Science
248:1122-1124 (1990); Sisodia et al., Science 248:492-495 (1990)) carboxyl-
terminal product produced in vivo, does not bind zinc. The contribution of
histidines (residues 6, 13, and 14) to Znz+ binding is indicated by the
deterioration of binding with lower pH (30% of B",~ at pH 6.0, Fig. 1e).
Taken together, these data indicate that zinc coordination requires the
contiguous sequence between residues 6 and 28, a region containing all
3 histidine residues, and that optimal zinc binding also requires the presence
of the carboxyl-terminal domain.
Further experiment investigated whether zinc binding could affect A~3
conformation as assayed by migration on gel-filtration chromatography.
Major A~3 species believed to correspond to monomeric, dimeric, and
polymeric forms were observed (Fig. 2a). Total concentrations of Znz+ as
low as 0.4 ~,Ivt decrease recovery of A~3 compared with elution profiles
obtained in the presence of EDTA and other metals (Figs. 2a and 2b). At 25
~clvt total Znz+, < 20 % of the A(3 applied to tthe column is eluted. The
greatest loss occurred among high order polymer and dimeric species. The
relative amount of monomeric A(3 is less affected. A systematic assessment
of several metals indicates that the reduction of A~i recoverable by
chromatography is most sensitive to Znz+, with related transition metals Coz+
Niz+, and Fez+ (at 25 ~,M) displaying similar effects on chromatography to
those obtained with only 10 ~,M Znz+ (Fig. 2b). Other transition metals,
heavy metals, and Al3+ (25 ~,lvt) have partial effects on A/3 solubility
comparable with 3 ~,M total Znz+. Meanwhile, Baz+, Agz+, Mgz+, and Caz+
(25 ~,M) have the least effect on A~3 compared with the EDTA profile,
although a 60 % reduction in eluted peptide was observed in the presence of
CA 02203142 1997-04-18
WO 96/12544 PCT/US94l11895
-29-
these metal ions. Pb2+ (25 p,M) most strongly promotes the elution of the
monomeric peptide, abolishing high order polymers; overall recovery is
similar to that obtained with 0.4 ~.M total Zn2+. In making comparisons of the
effects of these metal ions, it is again important to consider the
differential
metal ion chelating effects of Tris previously mentioned.
A dramatic increase in A(3 dimerization is observed with Cu2+ (25 p,M
total). This metal also induces exaggerated A~3 absorbance (4-fold) at 254 nm
when compared with 214 nm absorbance and induces the monomeric species
to apparently fluoresce at 254 nm causing negative readings (Fig. 2a) which
are proportionally positive at 214 nm (Fig. 2b). A higher concentration of
Cuz+ (80 ~,wt total) promotes increased recovery of A(3, indicating that the
presence of Cu2+ at relatively low concentrations (less than 25 ~tvt) favors
solubility in this system.
The metal ions which most favored A~3 solubility (Mg2+, 25 pwt and
total Cu2+, 25 yvt) were tested for their ability to stabilize A~3 in a
soluble
state in the presence of 25 p.M total Zn2+. These combinations neither rescue
nor worsen Zn2+-induced loss of A~3 recovery (Fig. 2b). Overall, these data
suggest that Zn2+ reduces the recovery of A(3, whereas a chelating agent
attenuates this effect.
Chromatography of A(3 was performed under various conditions to
determine if zinc-induced loss of A~3 could be blocked. Pretreating the
column with 3 % BSA as an adsorption blocker significantly increased the
amounts of A(3 recovered from the column, and suggest that on untreated
apparatus the peptide precipitates onto a column component (Fig. 2c).
Blocking the column results in a 200 % increase in the recovery of A(3 in the
presence of Zn2+ (25 ~cNt total), a 75 % increase in recovery in the presence
of Cu2+ (25 ~,tvt total), but only a 10% increase in the presence of EDTA (50
This indicates that precipitation onto the column is most specifically
accelerated by zinc.
To determine the part of the column onto which A(3 was precipitating,
A(3 solutions were incubated with various column components and assayed by
UV absorption before and after incubation. Replicating the chromatography
' CA 02203142 2004-06-14
-30-
experimental conditions, AS (100 ~clvt in equilibration buffer) was incubated
for 1 h in plastic reaction vessels with or without the presence of Sephadex.
Loss to the plastic accounts for < 5 % of the observed precipitation, to
siliconized plastic < 1 % , and binding to Sephadex* < 1 % . Hence, AS
precipitates are unlikely to be adsorbing to the Sephadex or plastic support.
However, similar incubations in borosilicate glass test tubes result in 20%
adsorption, which increase to 35 % in the presence of zip (25 ~ct~t).
The glass in the Bio-Rad Econo Columns* is made of 7740 Pyrex
(Corning; Park Ridge, IL) and is composed of Si02, 80.6%; B203, 13.096;
NazO, 4.0%; and A1z03, 2.3%. Previous workers have found evidence
linking aluminosilicates with ~-amyloid deposition (Masters et al. , EMBO J.
4:2757-2763 (1985a); Candy et al., Lancet 1:354-357 (1986)). In light of
these reports, experiments were performed to further investigate the
phenomena which were observed for precipitation of A~ on 7740 Pyrex glass.
Rapid and extensive binding of AS to kaolin, an insoluble hydrated aluminum
silicate was observed. Moreover, incubation of A/3 (0.4 mg/ml) with
Sephadex (5 °~, v/v) in the presence of zinc, copper, or EDTA
causes only
small changes in solubility, some of which is probably due to binding of the
peptide to the plastic reaction vessels (Fig. 3a): Incubation of Aa (0.4
mg/ml)
with kaolin (5 % , vlv, 5 min, room temperature), causes precipitation of up
to 87 % of the peptide present. This precipitation is greatest in the presence
of zinc (25 ~cM) where the amount of Aa recovered from the zinc incubation
supernatant is nearly half of the amount recovered from the EDTA incubation
supernatant (Fig. 3b). The effect of copper (25 ~,lvt) upon kaolin-induced A(3
precipitation is similar to the effect of EDTA (Fig. 3b). The binding of A(3
to kaolin is not reversible to subsequent treatment with 10 mHt EDTA, but can
be eluted by 2 M NaOH.
To further test whether zinc induces irreversible precipitation of Aa in
the absence of kaolin, A~i incubated with ZnZ+ (200 uM, 1-24 h, 20°C)
was
subjected to SDS Tris/Tricine gel electrophoresis. The monomeric species
was the major band detected on Coomassie-stained gels and migrated
*Trade-mark
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-31-
identically to unincubated A~3, indicating that zinc does not induce covalent
or
SDS-resistant polymerization of A~3.
The APPa-secretase site at Lys-16 (Esch et al. , Science 248:1122-1124
(1990); Sisodia et al., Science 248:492-495 (1990)) in A~3 is within the
obligatory zinc binding region. The ability of Zn2+ to protect A~3 from
secretase-type cleavage was investigated using the serine-protease trypsin,
whose activity is unaffected by zinc. Amino-terminal sequence on A~i tryptic
digestion products transferred to polyvinylidene difluoride membrane
following SDS-polyacrylamide gel electrophoresis indicated two detectable
fragments corresponding to residues 6-40 and 17-40 (F~g. 4a). The predicted
tryptic cleavage product representing residues 29-40 did not appear on the
blot
and may not be retained by the polyvinylidene difluoride membrane during
transfer and treatment. Digestion is inhibited by the presence of increasing
concentrations of Zn2+. At 200 ~,M, Zn2+ causes complete inhibition of A(3
hydrolysis; however, at this zinc level, tryptic activity is also slightly
inhibited. Probing the blot with ~Zn2+ confirmed the zinc binding identity of
the peptide fragments and facilitated quantification of the hydrolysis of the
zinc binding site (Fig. 4b). The rate of digestion of A/31.~ and the A~3~o
fragment is inhibited by the presence of zinc, whereas the digestion of the
A~31.,~ fragment is not inhibited by increasing zinc concentrations. Hence,
only the peptides possessing the intact zinc binding domain of A~3 (residues 6-
28), and therefore capable of binding Zn2+ (Fig. 4b), have their rates of
digestion inhibited by zinc in this experiment. These data indicate that
secretase-type cleavage of A~i can be inhibited by Zn2+ binding to the A(3
substrate. The results of the preceding experiments can be summarized as
follows.
Firstly, the data indicates that soluble A~3,~ possesses high and low
affinity zinc binding sites. Secondly, the zinc binding site on A(3 maps to
residues 6-28, with possibly conformational- and histidine-dependent
properties. Thirdly, the affinity constants for zinc binding indicate that
both
binding associations are within physiological zinc concentrations. The binding
of zinc may inhibit the action of «-secretase type cleavage of the peptide.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-32-
Furthermore, occupancy of the low affinity binding site may be associated
with accelerated precipitation of A/3 by aluminum silicate (kaolin). Occupancy
of the high affinity site appears to have little effect on A(3 precipitation
and is
very highly specific, although the data cannot exclude the possibility of
specific binding sites for alternative metals elsewhere on A~i. Finally,
copper's strong conformational interaction (dimerization and fluorescence)
with A(3 indicates that it may also directly interact with the peptide and may
have a role in preventing A~3 precipitation onto aluminum silicate.
Extracellular zinc may modify the adhesiveness of APP to extracellular
matrix elements (Bush et al., J. Biol. Chem. 268:16109-16112 (1993)) and
thus be an important factor in the physiology of the protein. Although the
physiological function of APP remains unclear, the protein is thought to play
a role in cell adhesiveness (Shivers et al. , EMBO J. 7:1365-1370 (1988)) and
neurite outgrowth (Milward et al., Neuron 9:129-137 (1992)). Physiological
function of the A(3-zinc interaction is also unclear, however, increased
resistance of A(3 to proteolytic cleavage in the presence of zinc would
increase
the peptide's biological half life, and the resulting increase in adhesiveness
may also promote its binding to extracellular matrix elements. It has heen
reported recently that A~i also promotes neurite outgrowth by complexing with
laminin and fibronectin in the extracellular matrix (Koo et al. , Proc. Natl.
Acad. Sci. USA 90:4748-4752 (1993)). Hence, both APP and A(3 may interact
with the extracellular matrix to modulate cell adhesion. The possibility that
zinc is a local environmental cofactor modulating this interaction merits
further investigation.
APP is abundant in platelets and brain (Bush et al. , J. Biol. Chem.
265:15977-15983 (1990)) where zinc is also highly concentrated (Baker et al. ,
Thromb. Haemostasis 39:360-365 (1978); Frederickson, C.J., Int. Rev.
Neurobiol. 31:145-328 (1989)). Although APP is concentrated in vesicles in
both of these tissues (Bush et al., J. Biol. Chem. 265:15977-15983 (1990);
Schubert et al. , Brain Res. 563:184-194 (1991)), and zinc is actively taken
up
(Wolf et al., Neurosci. Lett. 51:277-280 (1984)) and stored in synaptic
vesicles in nerve terminals throughout the telencephalon (Perez-Clausell and
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-33-
Danscher, Brain Res. 3371:91-98 (1985), the colocalization of APP with zinc
in these vesicles has yet to be demonstrated. Vesicular zinc storage is
thought
to play a role in stabilizing functional molecules such as nerve growth factor
(NGF) and insulin as insoluble intravesicular precipitates (Frederickson et
al. ,
J. Histochem. Cytochem. 35:579-583 (1987)). Zinc may similarly play a role
in stabilizing APP and A(3.
The well characterized interaction between insulin and zinc has several
striking parallels to the interaction of A~3 and zinc. Like A~3, insulin
exhibits
histidine-dependent high-affinity (KQ = 5 tcM) and low-affinity (Ka = 140 yvt)
zinc binding with stoichiometries of 1:1 (insulin:zinc) and 1:2, respectively
(Goldman and Carpenter, Biochemistry 13:4566-4574 (1974)). Additionally,
metal-free insulin exhibits a pH-dependent polymerization pattern consisting
of monomer, dimer, tetramer, hexamer, and higher aggregation states, in
dynamic equilibrium. At neutral pH, zinc and other divalent metal ions shift
the equilibrium toward the higher aggregation states. At stoichiometric ratios
of Zn2+:insulin in excess of 0.33, the peptide precipitates (Fredericq, E.,
Arch. Biochem. Biophys. 65:218-228 (1956)), reminiscent of zinc's effects
upon A~3 observed in the current studies.
AJ3 chelates zinc with such high affinity that reports of its neurotoxic
effects in neuronal cultures (Yankner et al. , Science 250:279-282 (1990); Koh
et al., Brain Res. 533:315-320 (1990)) might be explained by a disturbance
of zinc homeostasis. A(3 accumulates most consistently in the hippocampus,
where extreme fluctuations of zinc concentrations occur (0.15-300 tcM)
(Frederickson, C.J., Int. Rev. Neurobiol. 31:145-328 (1989)), e.g., during
synaptic transmission (Assaf and Chung, Nature 308:734-736 (1984; Howell
et al., Nature 308:736-738 (1984); Xie and Smart, Nature 349:521-524
(1991)). Choi and co-workers (Weiss et al., Nature 338:212 (1989)) have
proposed that this trans-synaptic movement of zinc may have a normal
' signaling function and may be involved in long term potentiation. The
hippocampus is the region of the brain that both contains the highest zinc
concentrations (Frederickson et al., Brain Res. 273:335-339 (1983)) and is
most severely and consistently affected by the pathological lesions of
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-34-
Alzheimer's disease (Hyman et al. , Ann. Neurol. 20:472-481 (1986)). One
of the prominent neurochemical deficits in Alzheimer's disease is cholinergic
deafferentation of the hippocampus, which has been shown to raise the
concentration of zinc in this region (Stewart et al. , Brain Res. 290:43-51
(1984)).
The rapid zinc-accelerated precipitation of A/3 by aluminum silicate
(kaolin) is significant because of the candidacy of aluminum as a pathogenic
agent in AD (Pert and Brody, Science 208:297-299 (1980)). Recent reports
of Zn2+- and A13+-induced sedimentation of A(3 (Mantyh et al. , J. Neurochem.
61:1171-1174 (1993)), and the nucleation of A~3 precipitation by
aluminosilicate (Candy et al. , Biochem. Soc. Traps. 21:53S (Abstract) (1992))
also support these observations.
Evidence for altered zinc metabolism in AD includes decreased
temporal lobe zinc levels (Wenstrup et al., Brain Res. 533:125-131 (1990);
Constantinidis, Encephala 16:231-239 (1990); Corrigan et al. , Biometals
6:149-154 (1993)), elevated (80%) cerebrospinal fluid levels (Hershey etal.,
Neurology 33:1350-1353 (1983)), increased hepatic zinc with reduced zinc
bound to metallothionein (Lui et al. , J. Am. Geriatr. Soc. 38:633-639
(1990)),
a Zn2+-modulated abnormality of APP in AD plasma (Bush et al. , Ann.
Neurol. 32:57-65 (1992)), an increase in extracellular Zn2+-metalloproteinase
activities in AD hippocampus (Backstrom et al. , J. Neurochem. 58:983-992
(1992)), and decreased levels of astrocytic growth inhibitory factor, a
metallothionein-like protein which chelates zinc (Uchida et al. , Neuron 7:337-
347 (1991)). Collectively, these reports indicate that there may be an
abnormality in the uptake or distribution of zinc in the AD brain causing high
extracellular concentrations and low intracellular concentrations. Meanwhile,
environmentally induced elevations of brain concentrations of both zinc
(Duncan et al. , J. Neurosci. 12:1523-1537 (1992)) and aluminum (Garruto et
al. , Proc. Natl. Acad. Sci. USA 81:1875-1879 (1984); Perl et al. , Science
217:1053-1055 (1982)) have been implicated in the pathogenesis of
GALS/PDC complex, a disease also characterized by neurofibrillary tangles
(Guiroy et al., Proc. Natl. Acad. Sci. USA 84:2073-2077 (1987)).
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-35-
Interestingly, a pervasive abnormality of zinc metabolism manifested by
immunological and endocrine dysfunction has been described as a common
complication of Down's syndrome (Franceschi et al. , J. Ment. Defzc. Res.
32:169-181 (1988); Bjorksten et al., Acta. Pediatr. Scand. 69:183-187
(1980)), a condition characterized by the invariable onset of presenile A(3
deposition and Alzheimer's disease (Rumble et al. , N. Engl. J. Med.
320:1446-1452 (1989)).
These results indicate that abnormally high zinc concentrations increase
A(3 resistance to secretase-type cleavage and also accelerate A(3
precipitation
onto aluminosilicates. Zinc-induced accumulation of A(3 in the neuropil may,
in turn, invoke a glial inflammatory response, free radical attack, and
oxidative cross-linking to form an, ultimately, "mature" amyloid.
Collectively, these findings support the biochemical rationale for the
chelation
approach in the therapy of Alzheimer's disease (Crapper McLachlan et al. ,
Lancet 337:1304-1308 (1991)), since reduction of cerebral concentrations of
both aluminum and zinc could potentially decelerate the precipitation of
A(3. The assay of the present invention is ideally suited for the preparation
of a kit. Such a kit may comprise a carrier means being compartmentalized
to receive in close confinement therein one or more container means, such as
vials, tubes, and the like, each of said container means comprising one of the
separate elements of the assay to be used in the method. For example, there
may be provided a container means containing standard solutions of the A(3
peptide or lyophilized A(3 peptide and a container means containing a standard
solution or varying amounts of a heavy metal cation capable of binding to the
peptide comprising at least amino acids 6 to 28 of A~3 peptide, in any form,
i. e. , in solution or dried, soluble or insoluble, in addition to further
carrier
means containing varying amounts and/or concentrations of reagents used in
the present methods, e. g. , standard solutions or varying amounts of
chealators
of heavy metal cations in any form, in solution or dried. Standard solutions
of A(3 peptide preferably have concentrations above about 10 tcM, more
preferably from about 10 to about 25 tcM or if the peptide is provided in its
lyophilized form, it is provided in an amount which can be solubilized to said
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-36-
concentrations by adding an aqueous buffer or physiological solution.
Standard solutions of heavy metal cations preferably have concentrations
above 300 nM, more preferably about 25 p. M. The standard solutions of
analytes may be used to prepare control and test reaction mixtures for
comparison, according to the methods of the present invention for determining
whether a compound inhibits formation of A~i amyloid.
These studies show that A(3 binds zinc in a saturable and specific
manner. Moreover, they demonstrate that physiological concentrations of
Zn2+ increase the resistance of the peptide to proteolytic catabolism and
promote A~3 precipitation by aluminosilicate. Based on these findings, it is
possible that excessive zinc concentrations accelerate A~3 deposition in AD
and
related pathological conditions.
Further, the effects of physiological concentrations of zinc upon the
stability of synthetic human A~il~o in solution were studied, using the
rat/mouse species of the peptide ("rat A~3") for comparison. Soluble A(31.~ is
produced by rat neuronal tissue (C. Haass and D.J. Selkoe, personal
communication), however, A~i amyloid deposition is not a feature of aged rat
brains (D.W. Vaughan and A. Peters, J. Neuropathol. Exp. Neurol. 40:472
(1981)). (3-amyloidogenesis occurs in other aged mammals possessing the
human A/3 sequence, which is strongly conserved in all reported animal
species, except rat and mouse (E.M. Johnstone, M.O. Chaney, F.H. Norris,
R. Pascual, S.P. Little, Mol. Brain Res. 10:299 (1991)). The rat/mouse A~3
substitutions (Arg->Gly, Tyr-~Phe and His-~Arg at positions 5, 10 and 13,
respectively [B.D. Shivers et al., EMBO J. 7:1365 (1988)]) appear to cause
a specific change in the peptide's physicochemical properties sufficient to
confer upon the peptide its relative immunity to amyloid formation. Since
zinc binding to human Aa,~o is histidine-mediated, the altered zinc binding
properties of rat A/3 are entirely consistent with the proposed mechanism and
binding site of the human peptide.
The binding affinity of zinc to rat A~i,~o was studied in a 65Zn
competitive assay system as described in Example 1 (FIG. 1), to measure the
KA of zinc binding to human A(31~o. In contrast to human Aa,~,o, the
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-37-
Scatchard analysis of zinc binding to rat A(31~ reveals only one binding
association (KA = 3.8 p,M), with 1:1 stoichiometry (FIG. 5).
It was observed that the recovery of human A/3,~ in filtration
chromatography is dramatically reduced in the presence of zinc, due, in part,
to increased adhesiveness of A(3. To determine whether the aggregation of
human A~31.~ is also enhanced in the presence of zinc, the peptide was
incubated with various concentrations for 30 minutes with Zn2+ (25 ~,M) or
EDTA and then filtered the solutions through 0.2 ~, filters. Zinc caused up
to 80% of the available peptide to aggregate into > 0.2 ~c particles (FIG.
6A).
(Incubation of A(31.~ solutions in the filter devices, without actual
filtration,
indicated that there was no non-specific loss of peptide to the plastic or
membrane surfaces.) There appears to be a shallow negative log-linear
relationship between human A(3 peptide concentration and the proportion of
filterable peptide in 25 ~cM Zn2+, but even at the lowest concentration tested
(0. 8 ~cM), > 70 % of the human Aal~ solution aggregated. In contrast, the
effect of Zn2+ on rat A(3,,~ was unremarkable, with no aggregation of a
0.8 ~,M peptide solution detected under the same conditions, and only 25
aggregation of a 4 ~M solution. Meanwhile, in the presence of EDTA,
human and rat A/31~ solutions behaved indistinguishably, with no detectable
aggregation observed at 0. 8 ~M, and ~ 15 % aggregation at higher peptide
concentrations.
Next, the formation of > 0.2 ~ A~3 particles was titrated against
increasing zinc concentrations (FIG. 6B), and a shallow response curve for
human A,131~ (1.6 ~cM) was observed until the zinc concentration reached 300
nM, corresponding to the saturation of high-affinity binding. At zinc
concentrations above 300 nM, corresponding to low-affinity binding, human
A~ii-,~ dramatically aggregates. In contrast, rat A~31~ remains stable in the
presence of up to 10 ~,M zinc, and only at 25 ~.M zinc was aggregation
observed.
To determine the effects of zinc on A(31~ at physiological peptide
concentrations requires an assay more sensitive than spectroscopy. (Human
A(31~o at 0.8 ~cM in buffer 1 corresponds to 0.090 absorbance units at 214 nm.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-38-
Aggregation studies of peptides at lower starting concentrations would involve
readings at the limits of setlsitivity). Thus, the effects of zinc on 1'~I-
human
A(31~ used as a tracer in the presence of unlabeled peptide was characterized.
Unlike its unlabeled precursor, 1'~I-A/31.~ (at 1.6 ~,M total peptide)
remained
stable in the presence of increasing zinc concentrations, indicating that 1~I-
A(31~ is not a suitable tracer (FIG. 6B). The tracer is iodinated on the
tyrosine residue at position 10, which is a phenylalanine in the rat peptide.
Thus, the tyrosine residue may be critical to the stability of the human
peptide. These data may also explain why a recent report required relatively
high concentrations of Zn2+ (1 mM) to precipitate 1~I-human A~31~ in
centrifugation studies (P.W. Mantyh et al.,. J. Neurochem. 61:1171 (1993)).
Extrapolating the curve in FIG. 6A to 0.6 nM currently provides the best
estimate of the effect of zinc upon physiological A(3 concentrations (M. Shoji
et al. , Science 258:126 (1992); P. Seubert et al. , Nature 359:325 (1992)),
and
indicates that 25 % of the peptide would aggregate into > 0.2 p, particles
under
these conditions. The specific vulnerability of human A(31~ for Zn2+ is
indicated by the observation that Znz+ is the only one of several metal ions
tested on an equimolar basis, including Al3+, to induce significant
aggregation
of human A/31~ in this system (FIG. 6C).
Next, the kinetics of the assembly of zinc-induced human A~31~
aggregates (FIG. 6D) was investigated. (In order to achieve time point
measurements of less than 1 minute, the procedure was modified so that
samples were centrifuged at 2500g, allowing the sample volume to be
completely filtered in 40 seconds.) The data obtained indicate that following
the addition of stock A(31~ in water (15.9 ~M, pH 5.6) to Zn2+ (25 ~,M) in
saline buffer (pH 7.4) there is a near-instantaneous aggregation of the
peptide
(1.6 ~cM final concentration) into filterable particles with two phases
observed
over two hours. The initial phase is rapid, with a half maximal assembly rate
of ~ 0.4 ~cM/min. The steady state of the second phase is achieved within
about 2 minutes, whereupon particle assembly proceeds at a rate of 3.2
nM/min with no evidence of saturation within 2 hours. At this rate, the
available peptide is exhausted within five hours of initiation. Although the
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-39-
addition of EDTA buffer caused the near-instantaneous aggregation of 20
of the 1.6 ~,M A~3l~o solution into > 0.2 tc particles, no further particle
' assembly was observed over the time course of the experiment. In
comparison, human A~il~,o (20 tcM in PBS, pH 7.4) has been reported to be
stable for 10 days (J.T. Jarrett, E.P. Berger, P.T. Lansbury, Biochemistry
32:4693 (1993)), and seeding the solution with A~31~2 (2 tcM), the more
amyloidogenic A(3 species, induced aggregation of this solution which was
half maximal only after 4-5 days. Thus, the results presented here represent
a major advance among attempts to induce amyloid formation in vitro using
the wild-type form of the main species of secreted A(3 (A(3,~o).
To estimate the size of the A(3 aggregates formed in the presence of
zinc, A(31~o (1.6 ~,M) was incubated with Zn2+ (25 tcM) or EDTA and then
passed through filters with various pore sizes (FIGs. 7a and 7b). Following
incubation in EDTA, human A~il~o assembled into populations of
heterogeneous particle sizes, > 0.1/x,: 47 % , > 0.22.: 40 % , > 0.65: 32 % .
The comparable proportions of filtered rat A(31~,o particles were, > 0. ltc:
36 % ,
> 0.22~c: 27 % , > 0.65,: 25 % . Upon incubation with Zn2+ (25 ~cM), the
proportion of > 0.65 rat peptide particles increased only slightly, however
the proportion of > 0.65, human peptide particles dramatically increased,
recruiting 82 % of the available peptide. Interestingly, the proportions of
> 0.1~, and > 0.22, particles formed from the human A~il~,o also increased by
SO and 55 % , respectively, following incubation with Zn2+, however, the same
reaction induced only a 20 % and 30 % increase, respectively, in the amounts
of these particles assembled from rat peptide. Remarkably, only 4 % of the
human A(3,~,o incubated with Znz+ remained in solution following 0.1 ~,
filtration. Collectively, these data indicate that the human species of A~i,~o
differs from the rat species both in the extent and size of zinc-induced
particle
formation.
The stoichiometry of zinc:human A(3 in these aggregates is at least l: l
(FIG. 7c), but increases to 1.3:1 with the smaller (0.1 ~,) pore size filters.
Because the stoichiometries for high- and low- affinity Zn: Aa binding are
1:1 and .= 2:1 respectively, these data indicate that formation of > 0.65
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-40-
A(3 aggregates is mediated by high-affinity zinc interaction, whereas low-
affinity zinc interaction most likely contributes to the formation of smaller
( < 0.22.) aggregates. Interestingly, when the retained aggregates are washed
with EDTA, only 22 % of the peptide is recovered from > 0. 65 aggregates,
although the complexed zinc (using 65Zn as tracer) is completely recovered
(FIG. 7d). This indicates that zinc-induced A~i aggregation is largely
irreversible by chelation. The amount of < 0.221c peptide resolubilized by
EDTA treatment is 7 % greater, which may reflect the increased contribution
of low-affinity zinc binding to the smaller, chelation-reversible, A(3
particle
formation.
Sedimentation of zinc-induced A~3 particles by centrifugation resulted
in an abundant precipitate of human A j3l~o which stained with Congo Red
(FIG. 8a) and manifested green birefringence under polarized light (FIG. 8b),
meeting the criteria for tinctorial amyloid formation. However, following
incubation with Zn2+ under the same conditions, the rat peptide formed
significantly fewer and smaller particles, with minimal birefringence. No rat
Aj3 amyloid was induced by Zn2+ concentrations of less than 10 ~cM, whereas,
by tinctorial criteria, human A~3 axnyloid was induced by Zn2+ concentrations
as low as 3 ~M. In neither case was Congo Red-stained material detected
following incubation with EDTA-containing buffer.
Taken together, these data indicate that soluble human A(31~ has a
dramatically greater propensity than rat A(31~ to form amyloid in the presence
of physiological zinc concentrations. The tinctorial amyloid aggregates are
frequently as large as the amorphous amyloid plaque cores purified from AD
brain tissue (C.L. Masters et al., Proc. Natl. Acad. Sci. USA 82:4245
(1985)). Meanwhile, the small degree (10-20%) of >0.2 ~ A~3l~o particle
assembly observed following the incubation of A/31~o with EDTA probably
reflects the relatively slow aggregation which occurs in the presence of
neutral
pH (S. Tomski and R.M. Murphy, Arch. Biochem. Biophys. 294:630 (1992))
and NaCI (C. Hilbich, B. Kisters-Woike, J. Reed, C.L. Masters, K.
Beyreuther, J. Mol. Biol. 218:149 (1991)). Hence, the specific vulnerability
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-41 -
of human A(3 to zinc-induced amyloid formation is a promising explanation
for aspects of the pathology of AD and related pathological conditions.
The cerebral cortex, and especially the hippocampus, contains the
highest concentrations of zinc in the body (C.J. Frederickson, M.A. Klitenick,
W.I. Manton, J.B. Kirkpatrick, Brain Res. 273:335 (1983)), and is exposed
to extreme fluctuations of extracellular zinc levels (0.15 to 300 ~,M,
C.J. Frederickson, Int. Rev. Neurobiol. 31:145 (1989)), e.g. during synaptic
transmission (S.Y. Assaf and S.-H. Chung, Nature 308:734 (1984); G.A.
Howell, M.G. Welch, C.J. Frederickson, Nature 308:736 (1984)). The
cortical vasculature contains an intraluminal zinc concentration of 20 ~cM
(LJ.T. Davies, M. Musa, T.L. Dormandy, J. Clin. Pathol. 21:359 (1968)),
but the perivascular interstitial zinc concentration is 0.15 ~cM (C.J.
Frederickson, Int. Rev. Neurobiol. 31:145 (1989)). Both sites of high zinc
concentration gradients are severely and consistently affected by the
pathological lesions of AD (B. T. Hyman, G. W . Van Hoesen, L. J. Kroner,
A.R. Damasio, Ann. Neurol. 20:472 (1986); G.G. Glenner and C.W. Wong,
Biochem. Biophys. Res. Commun. 120:885 (1984)). Interestingly, a prominent
neurochemical deficit in AD is cholinergic deafferentation of the hippocampus,
which raises the concentration of zinc in this region (G.R. Stewart, C.J.
Frederickson, G.A. Howell, F.H. Gage, Brain Res. 290:43 (1984)).
Additional evidence for altered cerebral zinc metabolism in AD include
decreased temporal lobe zinc levels (D. Wenstrup, W.D. Ehmann, W.R.
Markesbery, Brain Res. 533:125 (1990); J. Constantinidis, Encephale 16:231
(1990); F.M. Corrigan, G.P. Reynolds, N.I. Ward, Biometals 6:149 (1993)),
elevated (80% ) CSF levels (CØ Hershey et al. , Neurology 33:1350 (1983)),
an increase in extracellular Zn2+-metalloproteinase activities in AD
hippocampus (J.R. Backstrom, C.A. Miller, Z.A. Tokes, J. Neurochem.
58:983 (1992)), and decreased levels of astrocytic growth-inhibitory factor,
a metallothionein-like protein which chelates zinc (Y. Uchida, K. Takio, K.
Titani, Y. Ihara, M. Tomonaga, Neuron 7:337 (1991)). Recently, a clinical
study assayed the effects of oral zinc supplementation (6.7-fold the
recommended daily allowance, a dose commonly found in nutritional
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-42-
supplements) upon cognition and plasma APP levels in AD subjects and age-
matched controls. Five sequentially-studied AD subjects each experienced an
acute decline in cognition within forty-eight hours of ingesting the zinc
dose.
Under the same conditions, age-matched control subjects remained unaffected
by the dose. Among the abnormal changes of neuropsychological
measurements taken of the AD group was a 31 % drop in Mini-Mental State
Examination (M.F. Folstein, S.E. Folstein, P.R. McHugh, J. Psychiatr. Res.
12:189 (1975)) scores, after four days of zinc supplementation. This
represented a deterioration which, in the ordinary course of the disease,
would
only be expected after two to four years (Galasko et al. , JAGS 39:932
(1991)). Plasma APP levels also rose significantly in response to zinc in both
the AD and the control groups. All changes were rapidly reversible following
cessation of the four day supplementation. Collectively, these reports
indicate
that there may be an abnormality in the uptake or distribution of zinc in the
AD brain. Pervasive abnormalities of zinc metabolism, and premature AD
pathology, are also common clinical complications of Down's syndrome (C.
Franceschi et al. , J. Ment. Defzc. Res. 32:169 (1988); B. Rumble et al. , N.
Engl. J. Med. 320:1446 (1989)).
The data presented here indicate that stability in the presence of
physiological concentrations of zinc clearly differentiates the propensity of
human and rat A/31.~o peptide species to form amyloid. The rapid induction
of tinctorial human A(3 amyloid, under physiologically relevant conditions, at
peptide concentrations more than an order of magnitude lower than the lowest
levels achieved previously for A/3,.~o aggregation, and within two minutes of
incubation, establishes a novel assay system for the study of A/3 amyloidosis.
More importantly, these findings can have profound implications for the
potential role of zinc in Alzheimer-associated neuropathogenesis.
The following examples are provided by way of illustration to further
describe certain preferred embodiments of the invention, and are not intended
to be limiting of the present invention, unless specified.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
- 43 -
Examples
t Experimental Procedures
Unless otherwise indicated, the following experimental procedures
materials, and reagents were used in the present invention:
. Reagents--Precautions taken to avoid zinc contamination included using
analytical-grade reagents, electrophoresis-grade Tris-HCl (Bio-Rad), and
highly deionized water. A(3,_" was synthesized by the Biopolymers
Laboratory, MIT. A~i~l (reverse peptide) was purchased from Bachem
(Torrance, CA). Other reagents were from Sigma. A/31~o and A~31_2g results
were replicated with peptides from Bachem and Sigma. A(3,~o results were
also replicable with peptide synthesized by W. M. Keck Foundation
Biotechnology Resource Laboratory, Yale University. 65Zn was purchased
from Amersham Corp.
6sZna+ Binding Studies--Dissolved peptides (1.2 nMol, unless other-
wise stated) were dot-blotted onto polyvinylidene difluoride membrane (0.2-
tcm pore size; Pierce Chemical Co.), washed twice with chelating buffer (200
td x 100 mM NaCI, 20 mnt Tris-HCI, 1 mnt EDTA, pH 7.4), then five times
with blocking buffer (200 ~,1 x 100 mNt NaCI, 201nM Tris-HCI, 1 mNt MnCl2,
pH 7.4), and then incubated (60 min, 20°C) with 65Zn (unless otherwise
stated
130,000 cpm, 74 mlvt 65ZnC12 in 200 ~cl of blocking buffer ~ competing metal
ion chloride). The dot-blot was then washed with blocking buffer (5 x 200
td), the dot excised, placed in a test tube, and assayed by ~y-counting (11
efficiency). The equilibration volume for stoichiometry estimates was
regarded as 6 x 200 ~cl. The 214 nm UV absorbance of the unbound flow-
through was assayed to determine the total amount of peptide remaining bound
onto the membrane. Peptide stock concentrations were confirmed by amino
acid analysis. To alter the pH, the 65Zn incubation was carried out in the
presence of 100 mNt buffer: MOPS (pH 6.5-7.0), MES (pH 5.0-6.0), acetate
CA 02203142 2004-06-14
- L
(pH 3.5-4.5). The dot-blot apparatus was washed with detergent and EDTA
(50 mM) then rinsed and siliconized between use.
A~ Chromatography--AS (55 ~cg) was incubated with metal salt
solution or EDTA in siliconized 1.5-ml plastic reaction vessels in 100 mM
NaCI, 20 mM Tris-HCI, pH 7.4 ("TBS," I00 ~d, 1 h, 37°C). AFB was
stored
in aliquots of 0.52 mg/mI in water at -20°C, then kept at 4°C
when thawed.
Reagents were mixed without vortex mixing. The incubated A~8 was directly
applied to a G50 SF (Pharmacia, Uppsala, Sweden) column (Bio-Rad Econo-
Column, 30 x 0.7 cm) pre-equilibrated with metal salt solution or EDTA (50
p,M) in TBS at 20°C and eluted at 8 ml/h (Wiz peristaltic pump, Isco,
Lincoln, NE). Absorbance was measured at 254 and 214 nm (Type 6 optical
unit, Isco). The amount of A~ eluting at various peaks was estimated from
the area under the curve. This was possible because the relationship of UV
absorbance was determined to be linear over the range of AS diiutions used
in these studies, indicating that absorbance is proportional to the amount of
peptide present despite polymerization state (see below). The maximum
recovery of Aa occurs in the presence of EDTA. Because the sample eluted
in a volume of approximately 15 ml, the average concentration of the peptide
on the column was 0.8 ~cM. ,
To study the effects of protein blocking upon adsorption of A/3 to the
chromatography column, a Sephadex G50 SF column which had been
characterized previously for A/3 behavior was eluted with 3 % bovine serum
albumin (BSA) in TBS (50 ml) and equilibrated with non-BSA-containing
buffer, subsequent to repeating the A~g experiments.
Spectroscopic Assay--Measurements were performed on a Hewlett-
Packard 8452A diode array spectrophotometer using a 1-cm path length quartz
cuvette. Concentration versus absorbance curves were performed at 214 nm,
254 nm, 280 nm, and full spectrum. 214 nm readings were SO-fold more
sensitive in detecting the peptide than 254 nm readings, whereas the 280 nm
readings of low micromotar A~3 solutions were below sensitivity limits and
*Trade-mark
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
- 45 -
hence could not be used in these studies. The standard curves generated were
linear at concentrations below 0.1 mg/ml. In addition, the effects of Cu2+,
Zn2+, EDTA, and TBS upon absorbance were examined. At concentrations
below 0.1 mg/ml, adjusting the peptide in water to TBS caused = 15
quenching. Cu2+-, Zn2+_, and EDTA-containing A(3 solutions were studied
for artifactual absorbance over the linear range of the 214 nm absorbance
curve. 1 mHt EDTA caused 60% quenching, hence 50 ~tvt EDTA was
employed, contributing a similar degree of quenching to that observed with
Cu2+ and Zn2+.
A~ Binding to Kaolin (Aluminum Silicate)--Kaolin suspension was
prepared in high performance liquid chromatography water (Fisher), defined,
and adjusted to 50% (v/v). A(3 (40 tcg) was incubated in siliconized reaction
vessels with either kaolin or Sephadex G50 SF (10 tcl x 50% (v/v)) in Cu2+,
Zn2+, or EDTA (100 td in TBS, 5 min, room temperature). The suspension
was then pelleted (1500 x g, 3 min) and the supernatant removed and diluted
20-fold with water to bring the UV absorbance readings into the linear range.
Samples were assayed at 214 nm bcfore and after incubation with kaolin or
Sephadex.
T~yptic Digestion of A,~ A~l",~ (13.9 tcg) was incubated with Znz+
(12 tcl in blocking buffer, 1 h, 37°C) and then digested with trypsin
(12 ng,
3 h, 37°C). The reaction was stopped by adding SDS sample buffer
containing phenylinethylsulfonyl fluoride (1 mnt), boiling the samples (5
min),
and applying the samples to Tris/Tricine gel electrophoresis and transfer. The
blot was washed with EDTA, Coomassie-stained, incubated with 65Zn2+,
individual bands were excised, assayed for 65Zn2+ binding, and N-terminal
sequenced to confirm the identity of the digestion products. The effects of
Zn2+ (up to 100 yvt in TBS) on the activity of trypsin, itself, were assayed
by
assay of Z-Arg-amido-4-methylcoumarin (Sigma) fluorescent cleavage product
and determined to be negligible. It was found that 200 ~,wt Zn2+, however,
inhibited tryptic activity by 12 % .
CA 02203142 1997-04-18
WO 96/12544 PCTIUS94/11895
-46=
Zinc- or Copper-treated microwell plate
Any standard microtitre plate, for example a Costar catalog no. 9017,
can be used for making the heavy metal cation substrate which can trap A(3
protein. The plate is coated with a solvated nitrilotriacetic acid. Next, the
divalent metal ion of choice, for example, zinc or copper metal cation, is
added, which complexes to the surface of the plate. A preferred metal cation
microtiter plate is available from Xenopore, Saddle Brook, New Jersey
(catalog number for zinc plates: ZCP00100, catalog number for copper plates:
CCP00100). It is preferred that like the zinc and copper plates made by
Xenopore, there be at least two free coordination sites available for binding
to A/3 protein. In this way, the A(3 protein can competitively attach to and
stay bound to the substrate via the heavy metal cation.
Chelating-Sepharose Chromatography
The chelating-Sepharose resin (250 td) was poured into a disposable
polystyrene column (Pierce, 29920) and packed between two porous
polyethylene discs. In the following steps solutions were allowed to drain
through the gel bed by gravity. The gel was first pre-equilibrated with 5 ml
of equilibration buffer (MES 50 mM, pH 5.0 and NaCI 500 mM). The
sample (10-15 ml) was then loaded on to the column. The gel was washed
with 5 ml of equilibration buffer before bound protein was recovered by
applying 1 ml of elution buffer to the gel (EDTA 50 mM, MES 50 mM, pH
5.0 and NaCI 500 mM) and collecting the eluate.
Example 1: Analyses of 6fZn2+ binding to A~
Aliquots of A~i were incubated (60 min) with 65Zn2+ in the presence of
varying concentrations of unlabeled Zn2+ (0.01-50 ~M total). The proportion
of 65Zn2+ binding to immobilized peptide (1.0 nmol) described two binding
curves as shown in Figure la (Scatchard plot). Values shown are means +
S.D., n > 3. The high-affinity binding curve has been corrected by
subtracting the low-affinity component, and the low-affinity curve has had the
high-affinity component subtracted. (FIG. 1b) depicts specificity of the Zn2+
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
- 47 -
binding site for various metals. A(3 was incubated (60 min) with 65Zn2+ (157
nNt, 138,000 cpm) and competing unlabeled metal ions (50 yvt total).
(FIG. lc) depicts 65Zn2+ (74 nM, 104,000 cpm) binding to negative (aprotinin,
insulin a-chain, reverse peptide 40-1) and positive (bovine serum albumin
A
(BSA)) control proteins and A/3 fragments (identified by their residue numbers
within the A(3 sequence, glnll refers to A(31_28 where residue 11 is
glutamine).
Percent binding of total counts 65Zn2+/min added is corrected for the amounts
(in nanomoles) of peptides adhering to the membrane. (FIG. 1d) depicts as
for la, except with A~31_Zg peptide substituting for A~31~. 157 nM 65Zn
(138,000 cpm) is used in this experiment to probe immobilized peptide (1.6
nmol). (FIG. 1e) depicts pH dependence of 65Zn2+ binding to A(3i,~.
Example 2: Effect of Zn2+ and other metals on A~ polymerization using
G50 gel filtration chromatography
Results shown are indicative of n > 3 experiments where 55 ~g of A(3
is applied to the column and eluted in 15 ml, monitored by 254 nm
absorbance. (FIG. 2a) depicts chromatogram of A(3 in the presence of
EDTA, 50 tctvt, Zn2+, 0.4 yvt; Zn2+, 25 tcwt; and Cu2+, 25 ~,M. The elution
points of molecular mass standards and relative assignments of A~3 peak
elutions are indicated. Mass standards were blue dextran (2 x 106 kDa, vv =
void volume), BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c
(12.4 kDa), and aprotinin (6.5 kDa). The mass of A~3 is 4.3 kDa. (FIG. 2b)
depicts relative amounts (estimated from areas under the curve) of soluble A(3
eluted as monomer, dimer, or polymer in the presence of various metal ions
(25 ~,M), varying concentrations of Zn2+ or Cu2+ (the likelihood of Tris
chelation is indicated by upper limit estimates), and EDTA. Data for
experiments performed in the presence of copper were taken from 214 nm
readings and corrected for comparison. (FIG. 2c) depicts effects of pre-
blocking the chromatography column with BSA upon the recovery of A~3
species in the presence of zinc (25 ~,M), copper (25 ,uM), or chelator.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-48-
Example 3: A~ binding to kaolin (aluminum silicate): effects of zinc
(25 tcNr), copper (25 tcM), and EDTA (50 wM)
(FIG. 3a) depicts concentration (by 214 nm absorbance) of A/3
remaining in supernatant after incubation with 10 mg of G50 Sephadex.
(FIG. 3b) depicts concentration (by 214 nm absorbance) of A(3 remaining in
supernatant after incubation with 10 mg of kaolin, expressed as percent of the
starting absorbance.
Example 4: Effect of Zn2+ upon A~ resistance to tryptic digestion
(FIG. 4a) depicts a blot of tryptic digests of A(3 (13.9 tcg) after
incubation with increasing concentrations of zinc (lane labels, in
micromolar),
stained by Coomassie Blue. Digestion products of 3.6 kDa (A(3~), and 2.1
kDa (A(31~"~), as well as undigested A(31~ (4.3 kDa), are indicated on the
left.
The migration of the low molecular size markers (STD) are indicated (in
kilodaltons) on the right. (FIG. 4b) depicts 65Zn2+ binding to A~3 tryptic
digestion products. The blot in 4a was incubated with 65Znz+, the visible
bands excised, and the bound counts for each band determined. These data
are typical of n = 3 replicated experiments.
Example 5: Scatchard analysis of 65Zn binding to rat A~1~
Dissolved peptides (1.2 nmol) were dot-blotted onto 0.20 ~. PVDF
membrane (Pierce) and competition analysis performed as described in
Example 1 to measure the KA of zinc binding to human A~31,~ (Figure 1).
In the present invention, rat A~31,~ and human A/31~o were synthesized
by solid-phase Fmoc chemistry. Purification by reverse-phase HPLC and
amino acid sequencing confirmed the synthesis. The tabulated results are
presented in Figure 5. The regression Iine indicates a K" of 3.8 tcM.
Stoichiometry of binding is 1:1. Although the data points for the Scatchard
curve are slightly suggestive of a biphasic curve, a biphasic iteration yields
association constants of 2 and 9 ~cM, which does not justify an interpretation
of physiologically separate binding sites.
CA 02203142 1997-04-18
WO 96/12544 PCT/LTS94/11895
-49-
Example 6: Effect of zinc upon human, Izsl human and rat A~l~o
aggregation into > 0.2 tc panicles
- Stock human and rat A(31~o peptide solutions (16 tcM) in water were
prefiltered (Spin-X, Costar, 0.2 ~. cellulose acetate, 700g), brought to 100
mM
NaCI, 20 mM Tris-HCI, pH 7.4 (buffer 1) ~ EDTA (50 ~cM) or metal
chloride salts, incubated (30 minutes, 37°C) and then filtered again
(700g, 4
minutes). The fraction of the A(31~o in the filtrate was calculated by the
ratio
of the filtrate OD2la (the response of the OD214, titrated against human and
rat
A(31.~"o concentrations (up to 20 ~,M in the buffers used in these
experiments),
was determined to be linear relative to the OD2la of the unfiltered sample.
The results are tabulated in Figure 6. All data points are in triplicate,
unless
indicated. (FIG. 6a) Proportions of A/31,~0, incubated t Zn2+ (25 tcM) or
EDTA (50 tcM) and then filtered through 0.2 tc, titrated against peptide
concentration. (FIG. 6b) Proportion of A,~l~,o (1.6 ~.M) filtered through 0.2
tc, titrated against Zn2+ concentration. 1uI-human A~3l~o (1'~I-human A~3,~o
was prepared according to the method in J.E. Maggio, PNAS USA 89:5462-
5466 (1992) (15,000 CPM, the kind gift of Dr. John Maggio, Harvard
Medical School) was added to unlabeled A~3l~o (I.6 tcM) as a tracer, incubated
and filtered as described above. The CPM in the filtrate and retained on the
excised filter were measured by a ~y-counter. (FIG. 6c) Proportion of A/31~o
(1.6 tcM) filtered through 0.2 tc following incubation with various metal ions
(3 ~,M). The atomic number of the metal species is indicated. (FIG.
6d) Effects of Zn2+ (25 ~M) or EDTA (50 tcM) upon kinetics of human A~il~o
aggregation measured by 0.2 ~, filtration. Data points are in duplicate.
Example 7: Size estimation of zinc-induced A,8 aggregates
(FIGs. 7a and 7b) Proportion of A~3l~o (1.6 ~,M in buffer 1 (100 mM
NaCI, 20 mM Tris-HCI, pH 7.4)), was incubated ~ Zn2+ (25 ~,M) or EDTA
(50 tcM) and was then filtered through filters of indicated pore sizes
(Durapore
filters (Ultrafree-MC, Millipore) were used for this study, hence there is a
slight discrepancy between the values obtained with the 0.22 tc filters in
this
study compared to values obtained in FIG. 2 using 0.2 ~. Costar filters).
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/1189~
-50-
(FIG. 7c) 65ZnC1z (130,000 CPM, 74 nM) was used as a tracer of the
assembly of the zinc-induced aggregates of human A(31,~o produced in FIG.
3A. By determining the amounts of A(31~o and 65Zn in the filtrate, the
quantities retarded by the filters could be determined, and the stoichiometry
of the zinc: A(3 assemblies estimated. (FIG. 7d) Following this procedure,
the filters, retaining Zn: A~i assemblies, were washed with buffer 1 (100 mM
NaCI, 20 mM Tris-HCI, pH 7.4) + EDTA (50 ~uM x 300 td, 700g, 4
minutes). The amounts of zinc-precipitated A~il~o resolubilized in the
filtrate
fraction were determined by OD214, and expressed as a percentage of the
amount originally retained by the respective filters. 65Zn released into the
filtrate was measured by ~y-counting.
Example 8: Zinc-induced tinctorial amyloid formation
(FIG. 8a) depicts Zinc-induced human A~il,~o precipitate stained with
Congo Red. The particle diameter is 40 tc. A(3,.40 (200 td x 25 ~,M in buffer
1 (100 mM NaCI, 20 mM Tris-HCI, pH 7.4)) was incubated (30 minutes,
37°C) in the presence of 25 ~,M Zn2+. The mixture was then centrifuged
(16,000g x 15 minutes), the pellet washed in buffer 1 (100 mM NaCI, 20 mM
Tris-HCI, pH 7.4) + EDTA (50 tcM), pelleted again and resuspended in
Congo Red (1 % in 50% ethanol, 5 minutes). Unbound dye was removed, the
pellet washed with buffer 1 (100 mM NaCI, 20 mM Tris-HCI, pH 7.4) and
mounted for microscopy. (FIG. 8b) The same aggregate visualized under
polarized light, manifesting green birefringence. The experiment was repeated
with EDTA (50 ~M) substituted for Zn2+ and yielded no visible material.
Example 9: Effect of zinc and copper upon human, IlSI human and rat
A~r-oo aggregation into > 0.2 tc particles
Stock human and rat A(3~~o Peptide solutions (16 tcM) in water were
pre-filtered (Spin-X, Costar, 0.2 ~. cellulose acetate, 700g), brought to 100
mM NaCI, 20 mM Tris-HCI, pH 7.4 (buffer 1) ~ EDTA (50 ~cM) or metal
chloride salts, incubated (30 minutes, 37°C) and then filtered again
(700g, 4
minutes). The fraction of the A~ii~o in the filtrate was calculated by the
ratio
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
- S1 -
of the filtrate ODZIa (the response of the OD214, titrated against human and
rat
A~il~o concentrations (up to 20 ~,M in the buffers used in these experiments),
was determined to be linear) relative to the ODZi4 of the unfiltered sample.
All data points are in triplicate, unless indicated. (FIG. 9) A graph showing
the proportions of A(3,~, incubated t Zn2+ (25 lcM) or Cu2+ or EDTA (50
tcM) and then filtered through 0.2 ~., titrated against peptide concentration.
Example 10: Effect of zinc upon A~ produced in cell culture
A cell culture, preferably mammalian cell culture, expressing,
preferably overexpressing, human APP is established according to well-known
methods in the art, e.g. N. Suzuki et al., Science 264:1336-1340 (1994); X-D
Cai et al., Science 259:514-516 (1993); F.S. Esch et al., Science 248:1122-
1124 (I990). Next, zinc is added to the culture medium to final concentration
from about 200 nM to about 5 tcM. Then the cell cultures, containing zinc,
are incubated from about 15 minutes to as long as they can survive in the
culture. Preferably, the cells are incubated for 3 to 4 days. While fresh
media may be added to the cultures, no spent medium should be taken out
since it contains amyloid or zinc-induced A(3 aggregates.
The media which can be used are isotonic or physiological media, at
physiological pH (about 7.4). Preferably Tyrode's buffer is used with
calcium, magnesium, and potassium, as well as glucose. Any medium used
must be devoid of cysteine, glutamate, aspartate, and histidine since these
amino acids chelate zinc. Basically, any isotonic buffer or physiological
medium which minimizes constituents which chelate zinc may be used. For
example, Krebs Mammalian Ringer Solutions, in Data for Biochemical
Research, 3d Edition by Dawson et al. , Oxford Science Publications, pp.446
(N.Y. 1986), and page 447 for Balanced Salt Solutions, provide recipes for
making various useful media. The constituents that should be left out are
serum and the four amino acids mentioned above.
The cell culture should be incubated at about 37 degrees centigrade
with air or OZ/COZ (the maximum concentration of COZ is 5 % ).
CA 02203142 2004-06-14
-52-
Next, the cells and the medium are harvested together. A detergent
such as Triton (at concentrations of about 1-2~b v:v) is added and the mixture
is incubated for about 3 minutes to overnight. Preferably, however, it is
incubated for about 1 to 2 hours.
After incubation, the cell debris as well as amyloid and zinc-induced
Aa aggregates are pelleted by centrifugation. The pellet is suspended in
pepsin (about 2~) or in any other peptidase, and it is incubated from about
1 hour to overnight to allow digestion of the cell debris.
Again, it is pelleted, washed with PBS or any other appropriate salt
solution, stained with Congo Red, washed again, pelleted to remove any
unbound Congo Red, and resuspended in aqueous solution. At this point, a
sample can be visually inspected under a microscope. Further, it can be
quantitated using a grid.
Example 11: Assay for predicting the effectiveness of candidate reagents in
cell culture
The assay is set up in duplicate as described in Example 10. However,
a candidate reagent is added to one of the two cell cultures and EDTA is
added to the other cell culture. After the final step in Example 10, the
amount of amyloid and zinc-induced ASS aggregates are compared under the
microscope. The probability and level of effectiveness of the candidate
reagent is assessed based on the degree decrease in formation of amyloid and
zinc-induced A~3 aggregates in the cell culture.
Example 12: Rapid assay for detection of AJ3 amyloid forn~n in
biological fluid
Cerebrospinal fluid (CSF) is obtained from a healthy human subject
(control) and. a human patient suspected of amyloidosis. Both samples of CSF
are titrated by serial dilutions, e. g. , neat, 1:2, 1:4, 1:6, . . . ;
dilutions may be
made up to 1:10,000.
*Trade-mark
CA 02203142 1997-04-18
WO 96112544 PCT/LTS94/11895
-53-
To each of the samples, an equal amount of A13 peptide in water is
added to the final concentration of above about 10 tcM, preferably about 10
to about 25 tcM.
Next, a solution which contains a heavy metal cation capable of
binding to a peptide comprising at least amino acids 6 to 28 of A13,
preferably
Zn2+, plus NaCI and a buffer, e. g. , Tris at pH 7.4, is added to the final
heavy
metal cation, e. g. , Zn2+, to a final concentration of about above 300 nM,
preferably 25 tcM.
Then, the samples are centrifuged to form pellets. Pellets are stained
with an amyloid-staining dye, e. g. , Congo Red, and observed under a
microscope, thereby comparing levels of A13 amyloid in the control versus the
sample from the patient with amyloidosis. If quantification of amyloid is
desired, a grid can be used.
Example 13: Rapid assay for detection of AJ3 amyloid formation in
biological fluid using 3H AJ3
The assay is set up as explained in Example 12, except that the A13
peptide added is labelled beforehand by tritium. Moreover, after
centrifugation, the pellets are counted in a scintillation counter.
The preferred method of detecting the amyloid, however, is by using
filtration techniques as described above instead of centrifugation. After the
samples are passed through a filter, the filters are added to scintillation
fluid
and the counts are determined
Comparing the CPM from control samples with samples of the
suspected amyloidosis patient, it can be determined whether the patient is in
fact afflicted with amyloidosis. That is, an elevated CPM count in the patient
. samples compared to the control samples is indicative of amyloidosis.
Example 14: ELISA for detection and/or quanh; fication of A~ peptides
A(3-specific antibody of the enzyme-antibody conjugate binds to A(3
peptide bound to the heavy metal canon which is bound to the microtiter well
surface. The conjugated enzyme cleaves a substrate to generate a colored
CA 02203142 1997-04-18
WO 96/12544 PCT/L1S94/11895
-54-
reaction product that can be detected spectrophotometrically. The absorbance
of the colored solution in individual microtiter wells is proportional to the
amount of A/3 peptide.
This assay is optimized for detection and quantitation of A(3 peptide in
neat body fluids or in a partially purified or purified A~i peptide
preparation.
Pretreatment of Samples Before ELISA
The body fluid or sample of partially purified A~3 may be treated prior
to transfer to the 96-well plate to increase the efficiency of A~3 absorption
to
the solid-phase support. Treatments can include, but are not restricted to:
pre-incubation with methylating agents such as N-methyl malemide (1-10 mM
for 1-2 hr) that disrupt protein metal binding sites involving a cysteine
residue
(the A~3 peptide does not contain a cysteine residue); the addition of soluble
metal salts such as soluble MgCl2 (0.5-5 mM) that block non-specific metal
binding sites on proteins; the addition of compounds such as CuCl2
(0.2-2 mM) which can change the polymerization state of A(3; and the addition
of buffers to acidify the solution.
Materials
A/3 peptide (purified or partially purified) or neat body fluid
and controls (synthetic peptide standard)
Coating buffer (Tris 20 mM, pH 7.4, 150 mM NaCI)
Diluting buffer (Tris 20 mM, pH 7.4, 150 mM NaCI)
Blocking buffer (2 % gelatine, Tris 20 mM, pH 8.0, 150 mM NaCI)
Wash buffer (Tris 20 mM, pH 8.0, 150 mM NaCI)
Normal saline (150 mM NaCI)
10 mM diethanolamine, pH 9.5, containing 0.5 mM MgCl2
Urease-, HRPO-, or alkaline phosphatase-A(3 peptide conjugate
(prepared as described in UNIT 11.l of Current Protocols in
Molecular Biology, Vol. 2, Ausubel et al. , editors, (Greene
Publishing Associates and Wiley-Interscience, publishers), New
York. -
CA 02203142 2004-06-14
- 55 ,.
Urease substrate solution {Allelix #1001 100), peroxidase substrate
solution (Kirkegaard and Perry #50-62-00), or alkaline
phosphatase substrate solution
Zinc (Xenopore ZCP 00100) or Copper (Xenopore CCP 00100)
96-well microtiter plates (or other heavy metal ration bound plates as
described in Materials and Methods)
Multichannel pipet
Adhesive covers or tape for covering microtiter plates
Microtiter plate spectrophotometer with 590-nm and/or 405-run filters
1. Dissolve purified or partially purified A(3 peptide and controls in
coating buffer at about 0.2-2.0 tcglml.
Depending on the a,~nity of the antibody fvr the A/3 peptide, it
may be necessary to increase or decrease the amount of AJ3
peptide or neat body, fluid in coating bu,,~er.
For specificity testing, include closely related corarol antigens
which the antibody should not recognize.
2. Fill columns 2 through 12 of a 96-well microtiter plate with 0.1 ml
coating buffer.
A 96-well plate is divided into 12 columns (labeled 1-12) and
8 rows (labeled A-H).
3. Starting in column 1 of a 96-well microtiter plate, serially dilute A/3
peptide in coating buffer. Place 0.2 ml of Ap peptide solution in each
well in column 1. Remove 0.1 mI from each well with a multichannel
pipet and transfer to each well in column 2, which contains 0.1 m1 of
coating buffer. Pipet material in column 2 five times up and down.
Remove 0.1 ml from each well in column 2 and transfer to column 3.
Repeat this procedure through column 11. Remove 0.1 ml from
column 11 and discard. Leave column 12 blank. Prepare two
identical plates for duplicate assays. For controls prepare plates as per
steps 2 and 3, using control material in place of A,f3 solution. An
example of a control Material is a coating buffer without Ap peptide.
* Trade-mark
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-56-
This will give a range of dilutions in each of 8 rows (A to H)
from 1:1 through l: l, 024 ji. e. , column number (dilution);
1 (1:1), 2 (1:2), 3 (1:4), 4 (1:8), 5 (1:16), 6 (1:32), 7 (1:64),
8 (1:128), 9 (1:256), 10 (1:512), and 11 (1:1, 024)J.
4. Cover the plates with adhesive covers or tape and incubate for 2 hours
at 37°C.
5. Remove A(3 peptide solution by shaking into a sink and fill all wells
with 0.3 ml blocking buffer. Incubate for 2 h at 37°C.
HRPO is inactivated by sodium azide. Do not use buffers
containing sodium azide with HRPO-antibody conjugates.
Filter sterilize buffers used routinely (i.e., diluting buffer) and
store at 4 °C.
6. Remove blocking buffer by shaking into a sink and add 0.3 ml of
washing buffer. Empty wells and refill with washing buffer. Repeat
one more time.
7. Remove washing buffer by shaking into a sink and then fill rows B to
H with 0.1 ml diluting buffer.
8. Add 0.2 ml of enzyme-antibody conjugate diluted in diluting buffer to
row A of each plate. Recommended starting dilution of conjugate is
1:100. Serially dilute conjugate from row A to row H by transferring
0.1 ml to the well in the next row, as described in step 3. Final
volume of conjugate in each well should be 0.1 ml.
This will give a range of dilution from 1:100 through 1:12, 800
jrow (dilution): A (1:100, B (1:200), C (1:400), D (1:800), E
(1:1, 600), F (l: 3, 200), G (1: 6, 400), and H (1:12, 800)J.
9. Cover the plates with adhesive covers or tape and incubate for a set
length of time at a controlled temperature.
Time and temperature of incubation are determined empirically.
Generally, 30 to 90 min at 37°C is sufficient. Longer times of
incubation may increase sensitivity, but nonspecific binding may
also increase. An example is the monoclonal mouse antibody
6E10. For example, the mouse mAb 6E10 has an optimal
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/1189~
-57-
incubation of 2 h at 37°C or overnight at room temperature
(18-20 °C).
10. Shake out the plates into a sink. Wash plates with wash buffer twice
(0.3 ml each time) for urease- or alkaline phosphatase-antibody
conjugates and four times for HRPO-antibody conjugates by filling
well and shaking out the wash buffer into a sink. If an urease-
antibody conjugate was used, rinse plates an additional three times
with normal saline. If an alkaline phosphatase-antibody conjugate was
used, rinse plates twice with 10 mM diethanolamine, pH 9.5,
containing 0.5 mM MgCl2. Pat plates dry by inverting on a paper
towel.
11. Add 0.2 ml of either urease, peroxidase, or alkaline phosphatase
substrate solution, depending on the enzyme-antibody conjugate used.
For example, a high sensitivity substrate for HRPO is a TMB solution
(Pierce catalog no. 34024) and that is measured at 450 nm.
Appropriate absorbances include 590 nm (urease), 405 nm (HRPO or
alkaline phosphatase), and 450 (HRPO when TMB is used as the
substrate) using a microtiter plate spectrophotometer.
For alkaline phosphatase-based assays, add 100 p,l of 0.1 M
EDTA to each well at the end of the incubation in order to stop
the reaction. For HPRO assays using TMB substrate solutions,
the reaction is terminated after incubation by the addition of 25
~,l of sulfuric acid (1-3 M).
12. Plot absorbance versus (A~i) antigen concentration [Ag) on semilog
paper for analysis of each dilution of enzyme-antibody conjugate. For
working dilution of conjugate, choose a concentration that provides
maximum sensitivity over a linear range of [Ag) and minimum binding
(below 0.05 absorbance units) to control antigens (synthetic peptide
standards) .
13. Serially dilute individual body fluid and controls or partially purified
or purified A(3 peptide preparations, as described in step 3. Use two
columns per sample.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-58-
14. Repeat steps 4-6.
15. Shake out diluting buffer into a sink and add 0.1 ml per well of
enzyme-antibody conjugate diluted in diluting buffer at the
concentration determined in step 12.
16. Cover the plates with adhesive covers and incubate under the same
conditions as used in step 9.
17. Repeat steps 10 and 11.
18. Compare the absorbance of the unknown to the standard curve for the
enzyme-antibody conjugate concentration that was plotted in step 12 in
order to determine the quantity of antigen expressed per volume of a
body fluid or a sample of partially purified A~3 peptide.
Example I5: A method for bulk purification of A~ peptide from biological
fluids
The bulk purification of A/3 from biological fluids is best achieved with
copper charged chelating-Sepharose (Pharmacia catalog no. 17-0575-O1). The
cysteine groups in the sample proteins are first methylated with a maleimide
compound (e.g., N-methyl maleimide (Sigma catalog no. 930-88-1), about
1-10 mM, about 1 hour; also see Yomomote and KeKine, Anal. Biochem.
90:300-308 (1978)).
The methylated sample is then acidified by titrating pH to about 5.0
using about 1M sodium acetate, pH about 3.5, and the total NaCI
concentration increased by about 500 mM with about SM NaCI. The pH of
the sample is monitored with a glass pH detector or pH indicator paper while
sodium acetate is added dropwise with gentle stirring until the required pH is
obtained.
The sample is then applied to a copper-charged chelating-Sepharose
column (e.g., about 250 ~cl bed volume for about 15 ml of CSF) as described
above, in the section entitled Experimental Procedures. Equilibration buffer
is about 500 mM NaCI about 50 mM MES pH about 5.0 and is used to wash
the column. The Sepharose can be developed with about 500 mM NaCI, 50
mM EDTA, pH 8.0 alone and the eluate sampled for western blot analysis.
CA 02203142 1997-04-18
WO 96/12544 PCT/US94/11895
-59-
The treatment of 15 ml of CSF by this method enriched both soluble APP as
well as 4.3 and 3.6 kD a species of A~3 (identified by an antibody that
identifies an epitope in the first 16 residues of A~i; commercially
available).
- In order to bind copper or zinc, the peptide requires an intact domain
from residues 6-28. 4G8 only recognized the two A~i species and not APP,
confirming that the APP captured by the Sepharose was post-secretase cleaved
soluble APP. The use of specific anti-A~3 antibodies as described above on
western blot analysis of these products can confirm the specificity of the
ELISA immunoreactivity.
Example 16: A method for purification of A~ peptide when the volume of
the biological material is less than about 4 ml
The cysteine groups in the sample proteins are first methylated with a
maleimide compound (e.g., N-methyl maleimide (Sigma catalog no. 930-88-1)
about 1-10 mM, about 1 hour; also see Yomomote and KeKine, Anal.
Biochem. 90:300-308 (1978)).
The methylated sample is then acidified by titrating pH to about 5.0
using about 1M sodium acetate, pH about 3.5, and the total NaCI
concentration increased by about 500 mM with about SM NaCI. The pH of
the sample is monitored with a glass pH detector or pH indicator paper while
sodium acetate is added dropwise with gentle stirnng until the required pH is
obtained.
Free copper-charged chelating-Sepharose slurry (about 60 ~cl of about
50% v/v) is added to the sample.
Following centrifugation (preferably, low speed centrifugation (about
1,500 g, for about 3 minutes, equilibration buffer is used to wash the
Sepharose pellet. Equilibration buffer is about 500 mM NaCI about 50 mM
MES, pH about 5Ø
The Sepharose can be developed (protein is eluted by the addition of
the eluting buffer) with about 500 mM NaCI, 50 mM EDTA, pH 8.0 alone
and the eluate sampled for western blot analysis.
CA 02203142 2004-06-14
Having now fully described this invention, it will be understood by
those of skill in the art that it can be performed within any wide range of
equivalent modes of operation as well as other parameters without affecting
the scope of the invention or any embodiment thereof.
