Note: Descriptions are shown in the official language in which they were submitted.
~3~)2~5~3
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DIAGNOSTIC FOR ALZHEIM~R'S DISEASE
This invention was made with support under
grants from the National Institute of Health. The
United States Government has certain rights in this
invention.
The present invention is directed to laboratory
tests indicative of ~lzheimer's Disease and more
particularly to assays for the levels of Alzheimer's
Disease-associated antigenic materials in cerebral
spinal fluid.
BACKGROUND OF THE INVENTION
A neurological degenerative disorder receiving
increasing public attention is Alzheimer's Disease (AD)
which is the cause of "senility" in a large number of
older persons. This disease is characterized by
alterations in the neurofibrils. In addition,
argentophilic deposits, known as senile plaques, are
observed.
Degeneration of the neurofibrils is evidenced
by neurofibrillary tanglesl which are made of paired
helical filaments, H. M. Wisniewski, et al. in: Marotta
CA, ed. Neurofilaments, Minneapolis: University of
Minnesota Press 1983: 196-221. The neurofibrillary
tangles are found in neuronal perikaryon and in
dystrophic neurites of the neuritic (senile) plaques in
the cerebrum of a person suffering from AD.
The biochemical origin and composition of
paired helical filaments have remained obscure.
A heterogeneous group of closely related
microtubule-associated proteins, called "tau," with
molecular weights on SDS-polyocrylamide gels mostly
between about 55,000 and 62,000 daltons, has long been
known from studies of various tissues, including
:
~302;;:5~
mammalian brain. See, e.g., Weingarten et al., Proc.
Nat'l. Acad. Sci. (U.S.A.) 72, 1858-1862 (1975);
Cleveland et al., J. Mol. Biol. 116, 227-247 (1977); and
Lindwall and Cole, J. Biol. Chem. 259, 12241-12245
(1984). In vitro, tau is known to promote microtubule
assembly and to interconnect actin filaments, and both
activities are known to be influenced by the degree of
phosphorylation of tau. Selden and Pollard, J. Biol.
Chem. 258, 7064-7071 (1983); and Lindwall and Cole/ J.
Biol. Chem. 259, 5301-5305 (1984). The role of tau in
vivo remains unclear.
The cross-reactivity of anti-paired helical
filament antibodies with normal brain microtubules
assembled in vitro has been reported.
Grundke Iqbal et al., Ann. Neurol. 6, 532-537 (1979);
and Grundke-Iqbal et al., Lancet I, 578-580 (1979~.
Antisera to brain microtubules are known to label
isolated PHF which are washed repeatedly with SDS and
neurofibrillary tangles and plaque neurites, but not
amyloid, in tissue sections of Alzheimer's brain
(i.e., brain from Alzheimer's Disease patients).
Wisniewski et alu, J. Neuropathol. Exp. Neurol. 43,
543-565 (1984). High molecular weight
microtubule-associated proteins and various
neurofilament peptides from normal brain are also known
to be immuno-cross-reactive with isolated paired helical
filament. Ishii et al., Acta. Neuropathol. 48, 105-112
(1979); Anderton et al., Nature 298, 84-86 (1982);
Dahl et al., J. Neurosci. (Baltimore) 2, 113-119 (1982);
Gambetti et al., J. Neuropathol. Exp. Neurol. 42, 69-79
(1983); Perry et al., Proc. Nat'l. Acadu Sci. (U.S.A.)
82, 3916-3920 (1985); and Kosik et al., Proc. Nat'l.
Acad. Sci. (U.S.A.) 81, 7941-7945 (1984).
A monoclonal antibody specifically reactive
with paired helical filaments has been reported by G. P.
Wang, et al., Acta Neuropathol 62: 268-75 (1984), and
the distribution of reactivity of the antibody in brain
~:311)22S~
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tissue from deceased Alzheimer's Disease patients has
confirmed the localized high concentrations of paired
helical filaments in neuronal perikaryon and dystrophic
neurites of neuritic plaques in the cerebrum.
Alzheimer's Disease in living persons is now
diagnosed primarily according to behavioral symptoms.
With present methods for diagnosing AD, it is
known that a large fraction of patients are misdiagnosed
as having the disease. There is clearly a need for
improvement in methods for diagnosing the disease.
There is no known laboratory test of a body
fluid or tissue which gives quantitative results that,
alone or in conjunction with results of other tests,
such as the tests based on behavioral symptoms now being
used, are indicative of AD in a living patient.
Although paired helical filaments are known to occur in
high concentration in certain locations in AD patients'
brains, levels of paired helical filaments in brain
tissue cannot be measured in living patients.
Thus, to improve the clinical usefulness and
reliability of AD diagnoses, it would be highly
desirable to have laboratory tests, to be conducted with
body fluids or tissue of the living, which give
quantitative results indicative of presence or absence
of AD.
SUMMARY OF THE INVENTION
It has now been discovered that cerebral spinal
fluid (CSF) contains trace levels of paired helical
filament antigens ~referred to herein as PHFA), which
are antigens that are cross-reactive with antibodies
known to be reactive with isolated paired helical
filaments (referred to herein as "PHF"), and that there
is an indicative correlation between Alzheimer's Disease
and high levels of PHFA in CSF. Thus, assays for P~IFA
in the CSF are provided, based upon binding of anti-PHF
antibody to PHFA in CSF specimens. The assays according
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22~;~
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to the invention are applicable to living patients and
provide objective, quantitative results that are
indicative of whether a patient suffers from AD. High
levels of PHFA in CSF have been found to be indicative
of, and can also be employed to confirm diagnoses of,
AD. A preferred assay is a two-step, competitive
enzyme-linked immunosorbent assay (ELISA), in which PHFA
in a spinal fluid specimen compete with surface-bound
PHF for PHF-reactive antibody.
Further, it has been discovered that a major
component of P~F in neurofibrillary tangles and of PHFA
in CSF of persons suffering from Alzheimer's Disease is
abnormally phosphorylated tau protein. Thus, the
present invention also provides assays, preferably
immunoassays, for the Alæheimer's Disease-associated
abnormally phosphorylated tau in CSF. The presence of
said abnormally phosphorylated protein in the CSF of a
person indicates that the person suffers from AD. A
finding of such presence, employing an assay of the
invention for abnormally phosphorylated tau protein, can
also be used to confirm a diagnosis of AD based on other
techniques.
DETAILED DESCRIPTION OF THE INVENTION
The invention is based on the discovery that
PHFA is found in the CSF of humans and that an elevated
level of PHFA in the CSF is indicative of Alzheimer's
Disease. Because CSF specimens can be relatively easily
obtained, the present invention provides methods for
indicating whether a living person suffers from AD by
assaying for PHFA in a specimen of CSF from the person.
Prior to the discovery which underlies the present
invention~ it was not known to be possible to test for
AD-indicative PHFA in living persons, because it had
only been established that elevated levels of PHF were
found in localized areas of brain tissue which could
only be examined after a patient had died.
o2~25~
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One class of AD-indicative assays in accordance
with the invention are immunoassays for PHFA in CSF.
In another aspect, it has been discovered that
a major constituent of PHF in brains of persons who have
died of Alzheimer's Disease and of the PHFA in the CSF
of persons suffering from the disease is abnormally
phosphorylated tau proteins. Thus, the present
invention also entails methods for indicating whether a
person is suffering from Alzheimer's Disease by assaying
a specimen of the person's CSF for these abnormally
phosphorylated tau proteins.
The immunoassays according to the invention
include solution-phase and solid-phase, and competitive
as well as non competitive. They include enzyme-linked
immunosorbent assays (ELISAIs) and radioimmunoassays
(RIA's). The immunoassays are carried out by methods
known in the immunoassay art for determining the
concentration of an antigen (e.g., PHFA or abnormally
phosphorylated tau protein) in a specimen using antibody
(polyclonal or monoclonal) known to be reactive with the
antigen.
One particular type of AD-indicative
immunoassay according to the invention is a two-step,
competitive solid-phase assay wherein a quantity of
prepared PHF and an unknown quantity of PHFA in a CSF
specimen compete for binding by a PHF-specific
antibody. Either the prepared PHF or the PHFA in the
CSF is bound to a solid support (e.g., the inside wall
of a microtiter plate well) while the other is held in
solution. In the assay, an excess of the anti-PHF
antibody is first incubated with and complexed with the
entire amount of either the support-bound antigen or the
antigen in solution and the antibody remaining unbound
is then incubated with the other of the antigens. The
complexing of antibody with either the PHF or the PHFA
in the first step results in "inhibiting" antibody
binding with the other of the antigens in the second
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step. The preferred of these competitive solid-phase
immunoassays are ELISA's. Thus, as understood in the
immunoassay art, the amount of antibody not complexed in
the first step, but complexed in the second, whether the
amount is support-bound or in solution, is measured
by 1) reacting it with an anti-antibody~ preferably a
polyclonal, subclass non-specific antibody, that reacts
with immunoglobulin of the same class (e.g., IgG) and
from the same species (e.g., mouse) as the anti-PHF
antibody and that has been previously linked to or
complexed with an enzyme, 2) separating the portion of
immunoglobulin-enzyme complex that binds to the
PHF-antibody or PHFA-antibody complex from that portion
which does not bind, and 3) determining the amount of
enzyme in either the bound or unbound portion by
exposing the same to a reagent or reagents which undergo
a chromogenic reaction catalyzed by the enzyme.
Heretofore, there have been no reports of the
presence of PHFA in the CSF, and there have been no
reports of substances specifically associated with
Alzheimer's Disease in the CSF. The discoveries of the
presence of PHFA in the spinal fluid and the correlation
of elevated spinal fluid PHFA levels with Alzheimer's
Disease are highly significant because CSF is accessible
to assay for diagnostic purposes with living patients.
Indeed, it may be expected that an assay for PHFA in
CSF, such as the competitive ELISA described herein or
other immunoassays, will eventually be performed on any
patient suspected of having or being in a high risk
category for Alzheimer's Disease.
More specifically, the immunoassay that we have
developed for PHFA-level measurement in CSF is a
two-step, competitive enzyme-linked immunosorbent assay
(ELISA). The assay is based upon a sandwich technique,
E. Engvall, et al., Method Enzymol., 70: 419-439
(1980), such as that which has been used previously to
determine titers and specificity of antisera as well as
' -` , -
2~1)
to measure antigens both qualitatively and
quantitatively. In the assay that we have developed,
PHFA in CSF specimens is measured quantitatively through
an antibody inhibition step.
Isolated (i.e., purified) paired helical
filaments are obtained according to the method of K.
Iqbal, et al., Acta Neuropathol, 62, 167-177 (1984).
Briefly, by a standard technique, microtiter
plate wells are coated with a fixed quantity of
sonicated PHF per well. As the inhibition step, in two
separate sets of vials, aliquots of a solution
containing PHF-reactive antibody (preferably monoclonal
antibody) are incubated with various known
concentrations of fragmented (sonicated) PHF (in the set
of vials for establishing a standard curve), as well as
with CSF specimens (for dilutions thereof) containing
unknown quantities of PHFA, for a period of -time that is
sufficient to permit substantially complete reaction of
the anti-PHF antibody with the available PHF or PHFA.
Thereafter, an equal volume of the incubation mixture
from each vial is added to the PHF-coated plate wells
and incubated for a period of time sufficient for
antibody, which was not prereacted (inhibited) with the
PHF (or PHFA) in the solution in the vial, to react with
the PHF that is coated on the plates. Then the plates
are washed thoroughly to remove all antibody, PHF, and
antibody-PHF or antibody-PHFA complex that is not bound
to the plate.
For assays, such as competitive assays, which
require that the PHF be dissolved in or stably suspended
in aqueous medium, it is necessary to fragment the PHF
so that it may be carried by the aqueous medium, intact
PHF being substantially insoluble in aqueous medium. In
accordance with an important aspect of the invention,
fragmented PHF is prepared for use in assays by
suspension and ultrasonication of the suspended,
purified P~F. Chemical fragmentation of PHF in aqueous
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medium is more cumbersome and much less desirable than
ultrasonic fragmentation because chemical fragmentation
requires use of protein denaturants, which must be
completely removed from the fragmented PHF before the
PHF is suitable for use in an assay but which is very
difficult to remove without causing reaggregation of the
PHF. PHF fragments in the presence of a denaturant will
not bind efficiently to microtiter plate wells or
anti-PHF antibody in an assay. Further, as denaturant
is separated from a solution of such PHF ~ragments, the
fragments tend to form large, insoluble aggregates that
are unsuitable for assay purposes.
The conditions of ultrasonication of PHF are
not considered particularly critical, as long as the
combination of intensity and exposure time are
sufficient to fragment substantially all of the
particles to a size where they are not visible under a
light microscope, e.g., so that the fragments have
greatest dimensions less than about 50 nm. E'ragments
produced by ultrasonication are known to be stable in
solution for at least about 24 hours, and it is expected
that they are stable for considerably longer periods at
temperatures approaching the freezing point; however,
PHF fragment solutions should not be frozen, as freezing
causes undesirable aggregation.
As a means of measuring the amount of antibody
that is bound to the plate, the plate is exposed to an
excess of a second antibody that reacts with the
anti-PHF antibody and which is appropriately labeled
with an enzyme. The second antibody is an
anti-immunoglobulin which is reactive with
immunoglobulins of the species from which the anti-PHF
~ antibody is derived and is usually non-subclass or
- non-class specific. For example, if the first antibody
is mouse anti-PHF monoclonal IgG antibody, the enzyme
label may be linked to anti-mouse IgG or anti-mouse
immunoglobulin that is not specific as to class. A
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preferred means of labeliny the second antibody is by
linkage to an enzyme, such as alkaline phosphatase,
which cataly~es the conversion of p-nitrophenyl
phosphate to p-nitrophenol, which reaction is
chromogenic, being observable by a rise in the optical
density of the solution at 405 nm. The rate of increase
in optical density of the solution per unit time is
related to the amount of enzyme linked to the second
antibody in the system, and measurement of this rate
(the increase in optical density after a speciied
period of time) is the basis for measuring the amount of
first antibody bound to PHF in the microtiter well.
Other enzymatic reporter systems, such as the peroxidase
reporter system, described in the Journal of Biological
Chemistry 257: 14173-1~180 (1982), may be substituted
for the alkaline phosphatase reporter system as a means
for visualizing PHF-bound antibodies.
In this particular assay system, the more PHFA
that is present in the CSF specimen, the less color
change that develops per unit time. Although the rate
of color development decreases as a function of PHFA
concentration in the CSF specimen, it is not a linear
relationship. Accordingly, it is necessary to prepare a
standard curve, using solutions of known PHF
concentrations, against which the color development from
a specimen with an unknown concentration of PHFA may be
compared. The PHF for the solutions used to establish
the standard curve can be Eragmented, purified PHE that
is used to coat microtiter plate wells as described
above. The standard curve for this assay will be
approximately linear over only a limited PHF
concentration range; and, in order that the level of PHF
in an unknown CSF specimen may be read with accuracy
from a standard curve, it is helpful in some cases,
particularly in the case of abnormally high PHFA levels,
to perform the assay on serial dilutions of a CSF
specimen to ensure that the concentrations of PHFA in
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one or more of the serial dilutions o~ the specimen
falls within the linear range. However, assay of a
single CSF concentration is generally adequate to
determine an abnormally high PHFA level; and, because a
very high PHFA concentration is indicative of AD,
precision of numerical values of highly elevated PHFA
levels will not be important in many cases.
A variation on the ELISA assay described above
is to bind anti-PHE` antibody, rather than PHF, to the
solid support. Also required in this case is
enzyme-labeled PHF (PHF that is linked either directly
or through a linking moiety, such as any bifunctional
linker known in the art to covalently join protein
molecules stably, to a suitable enzyme)~ A known
quantity of the enzyme-linked PHF is added to CSF
specimens of known volume, and aliquots of such mixtures
are placed in the antibody-coated wells. PHFA in the
CSF specimens compete for antibody binding sites with
the enzyme-labeled PHFo After an appropriate amount of
time for PHF enzyme-labeled and PHFA in CSF to bind to
the anti-PHF antibody, the wells are washed and
subsequently exposed to a reagent system that undergoes
a chromogenic reaction catalyzed by the enzyme linlced to
the PHF. In such an assay, greater rate of color
development indicates less PHFA from CSF in the reaction
mixture. This assay system has the advantage of being
somewhat simpler to perform, requiring only antibody
linked to a plate, an enzyme-linked PHF solution, and
substrate solution for enzymatic catalyzed reaction.
This assay system may be more suitable than the
two-step, competitive ELISA for providing in kit form to
a medical laboratory.
Radioimmunoassays represent another suitable
technique whereby PHFA in a CSF specimen may be
quantified. For example~ employing the well known
chloramine-T reaction with fragmented, purified PHF, the
labeling of PHF can be with a radioisotope, 125I,
rather than an enzyme, and the amount of labeled PHF
measured (via radioactive emission) as bound to the
microtiter plate wells will decrease as the amount of
PHFA in CSF being assayed increases. However,
enzyme-linked or other non-radioactive chromogenic assay
procedures are preferred to radioimmunoassay techniques
from the standpoint of safety considerations and reagent
stability.
It is noteworthy, however, that this invention
entails assay of PHFA in CSF by any available technique
in the art, immunoassay or other. With respect to
immunoassay techniques, the invention is not limited to
any particular form that an immunoassay may take.
The development of an assay sensitive to levels
of PHFA in CSF was made possible, in part, through the
development of monoclonal antibodies specifically
reactive with P~F. The first hybridoma cell line, which
produces PHF-specific monoclonal antibody, was developed
by~Wang, et al., supra. This hybridoma cell line is
deposited at the American Type Culture Collection,
Rockville, Maryland, U.S.A. (ATCC), where it is assigned
accession number HB 9039. The deposit with the ATCC has
been made under the terms of the Budapest Treaty on the
~eposit of Microorganism for the Purposes of Patent
Procedure and the Regulations promulgated thereunder and
samples of the cell line are and will be available from
the ATCC to industrial property offices and other
persons legally entitled to receive them in accordance
with said Treaty and Regulations and in accordance with
the patent laws and regulations of every country and
international organization in which an application
corresponding to this application is filed or a patent
based on any such application is granted.
Reference herein to the hybridoma line with
ATCC accession number HB9039 also encompasses
subcultures of that line, as well as lines of mutants of
cells from that line, or subcultures thereof, provided
~31[)Z2~
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that the subcultures and lines of such mutants produce
the monoclonal antibody reactive with PHF that is
provided by the line with ATCC accession number HB9039.
The invention encompasses hybridoma cell lines
which produce monoclonal antibodies which have specific
activity for PHF, and the anti-PHF monoclonal antibodies
produced by such cell lines. In particular this
invention encompasses the cell line HB 9039 and the
monoclonal antibody produced thereby. With the
monoclonal antibody produced by the HB 9039 cell line,
selection for hybridoma cell lines which produce
antibodies reactive with the same antigenic determinant
of PHF is greatly facilitated, and the invention
encompasses other hybridoma cell lines which produce
antibodies that react with the same antigenic
determinant on PHF as the antibody produced by hybridoma
line HB9039. ~inding of a monoclonal antibody to the
same antigenic determinant is established if prior
binding to PHF of the monoclonal antibody produced by
the HB 9039 cell line blocks subsequent binding of the
other monoclonal antibody.
As indicated above, it has been discovered that
a major component of PHF isolated from Alzheimer's
brains and and PHFA in CSF of persons suffering from
Alzheimer's Disease is abnormally phosphorylated tau
protein.
Consequently, the presence of the abnormally
phosphorylated tau protein in a specimen of CSE from a
person indicates that the person is afflicted with
Alzheimer's Disease. The presence of abnormally
phosphorylated tau in CSF can be determined by any of
the immunoassay techniques described above, in
connection with PHFA, using polyclonal or monoclonal
antibody reactive with the abnormally phosphorylated
tau. As positive control or as standard to establish
standard curves in the assays, and as competitor in
competitive assays, abnormally phosphorylated tau
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protein prepared by electrophoresis or by immunoaffinity
chromatography as described below, is employed~
Alternatively, employing polyclonal or
monoclonal antibody that differs in affinity for
abnormally phosphorylated and normal tau protein, the
concentration of tau proteins in a CSE specimen can be
measured by immunoassay both before and after subjecting
the specimen, or portions thereof, to treatment, such as
treatment with alkaline phosphatase, that
dephosphorylates any abnormally phosphorylated tau
protein. A difference in the amount of tau detected
before and after dephosphorylation indicates the
presence of abnormally phosphorylated tau in the
specimen.
Microtubule-associated proteins t known as tau
proteins, are known, as described above.
Also known and widely distributed among
practitioners in the art is a tau-specific monoclonal
antibody known as tau-l. Binder et al., J. Cell
Biol. 101, 1371-1378 ~1985); Grundke-Iqbal et al., J.
Biol. Chem. 261, 6084-6089 (1986); Wood et al.l Proc.
Nat'1. Acad. Sci. (U.S.A.) 83, 4040-4043 (1986). Tau-1
binds to tau proteins from calf, rat and normal human
brain and the epitope it recoynizes seems to be confined
virtually exclusively to tau proteins.
Abnormally phosphorylated tau protein was
identified as a component of PHF both by
immonocytochemical analysis of neuro~ibrillary tangles
in tissue section of Alzheimer's brains both before and
after dephosphorylation with alkaline phosphatase and by
immunoblots of polypeptides from isolated PHF, prepared
as described by Iqbal et al., Acta ~europathol. 62,
; 167-177 (1984), both before and after dephosphorylation
with alkaline phosphatase.
~ 35 Dephosphorylation of PHF (or PHFA in CSF) is
; accomplished with calf alkaline phosphatase at 40-50 ug/ml, pH 7.5-8Ø
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It was found that tau-l bound only weakly, if
at all, to the neurofibrillary tangles, and the isolated
PHF in immunoblots, without dephosphorylation, although
it does bind significantly with tau proteins from normal
brain without dephosphorylation. However, after
dephosphorylation, the tau-l bound to the PHF, in
tangles and in immunoblots, nearly as if the PHF were
composed of normal tau protein. (In the immunoblots,
the tau proteins from PHF migrate to essentially the
same positions as tau proteins isolated from normal
brain.)
The tau-l monoclonal antibody, or any
monoclonal antibody or polyclonal anti-serum which binds
to abnormally phosphorylated tau and normal tau with
different affinities, can be employed to assay CSF
specimens for abnormally phosphorylated tau,
characteristic of PHF and Alzheimer's disease, by simply
assaying one portion of the CSF specimen without
dephosphorylation and another portion with
dephosphorylation. If abnormally phosphorylated tau is
- present in the specimen, the two assays will yield
different concentrations for tau. Otherwise/ the assays
will yield the same concentration for tau~
Abnormally phosphorylated tau protein can be
isolated from normal tau protein in Alzheimer brain
preparations by standard electrophoretic techniques,
such as agarose gel electrophoresis, on the basis of the
additional negative charges, due to the additional
phosphate groups, on the abnormally phosphorylated
protein.
Alternatively, the abnormally phosphorylated
protein can be isolated by standard immunoaffinity
chromatography techniques from isolated PHF or from
preparations of Alzheimer brain tissue. Firs~, antibody
which recognizes tau, both phosphorylated and not, is
used to isolate tau proteins. Many such antibody
preparations are known and are available in the art.
:~3~D2~Si~
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See Grundke-Iqbal et al., J. Biol. Chem. 261, 6084-6089
(1986); Drubin et al., J. Cell Biol. 98, 1090-1097
(1984); and Kosik et al., Proc. Nat'l. Acad. Sci.
(U.S.A.) 83, 4044-4048 (1986). Then either an antibody
specific for normal tau protein, such as tau-l, or an
antibody specific for abnormally phosphorylated tau, is
employed to isolate the abnormally phosphorylated from
the normal tau.
With purified abnormally phosphorylated tau
available, polyclonal antibody preparations and
monoclonal antibodies specific for the abnormally
phosphorylated protein can be prepared by standard, well
known techniques. In the preparation of monoclonal
antibodies, hybridoma cultures are screened for antibody
that recognizes abnormally phosphorylated tau but does
not recognize normal tau (as derived, e.g., from brain
tissue of persons younger than about 50 years old who
have died but did not suffer from Alzheimer's Disease).
EXAMPLE I
CSF specimens were collected from nine patients
with Alzheimer's Disease as determined by the criteria
of J. Eisdorfer, et al. J. Fam. Pract. 11: 553-57
(1980), (mean age 64+10 SD years) and from nine patients
(57+19 SD years) with non-AD neurological conditions,
including stroke, seizures, multiple sclerosis, and
other neurological conditions. The specimens were coded
before analysis for PHF antigen.
Paired helical filaments were isolated at
necropsy from the cerebral corte~ from patients with
Alzheimer's Disease and senile dementia of the Alzheimer
type according to the method of Iqbal, et al., supra.
Isolated paired helical filaments were fragmented to
particles of sizes not visible by light microscopy by
suspension in 0.32 M sucrose followed by ultrasonication
of the suspension using a Branson sonicator at
10% pulse, 10 watts output and 4 second on-off cycle for
.
~3~2;~5~
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30 min. The protein concentration was determined by
amino acid analysis and adjusted to 75 ug/ml with
phosphate-buffered saline (PBS), pH 7.2.
A two-step compe~itive ELISA was done in
duplicate. Microtiter plate wells were coated with P~F
by incubation overnight at 4C in a humid chamber with
100 ul per well of a dilution of the sonicated P~F
suspension to 0.5 ug/ml PHF with 0.05 M Na
carbonate-bicarbonate buffer, pH 9.6. After the
incubation~ the wells were washed three times with PBS
* *
containing 0.05% "Tween-20" (PBS-Tween).
Mouse anti-PHF-specific monoclonal antibody,
Wang, et al., supra., from hybridoma line HB 9039, at an
initial concentration of 30 mg/ml protein, was diluted
1:20,000 with PBS-Tween. 100 ul aliquots of the diluted
antibody were mixed 1:1 with 1) PBS-Tween as a negative
control, 2) serial dilutions of sonicated PHF suspension
in PBS-Tween for establishing a standard curve and 3)
the CSF specimens. The mixtures were incubated for
one hour at 22C. The monoclonal antibody solutions
were then transferred to the wells of the microtiter
plate with bound PHF. The plate was incuba~ed at 22C
for three hours. After washing three times with
PBS-Tween, 100 ul of alkaline phosphatase conju~ated to
goat anti-mouse IgG (0.25 ug/ml) was added to each of
the wells and incubated at 22C for three hours.
After again washing three times with PBS-Tween,
100 ul of substrate (p-nitrophenyl phosphate, 1 ug/ml)
was added and reacted for 90 and 120 minute periods at
room temperature. Optical density was measured at both
times at 405 nm in a standard ELISA reader. A standard
curve was established, and PHFA levels in CSF specimens
were determined with reference to the standard curveO
An arbitrary unit of PHF antigen was
established as the inhibition of the rate of color
development in the ELISA (from the rate with PBS-Tween
negative control) obtained with 180 ng protein of PHF
* trade mark
13~2;~S~
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suspension (i.e., the inhibition with the PHF serial
dilution containing fragmented PHF at 1.8 ug/ml PHF
protein and treated as described above). The PHF
levels, in these arbitrary units, of the nine CSF
specimens obtained from patients with Alzheimer's
Disease were as follows: .01, .22, .23, .26, .34, .47,
.48, .55, and .64; the mean and standard deviation being
0.35 ~ 0.06. The PHF levels, in the arbitrary units, of
the nine specimens obtained from patients without
Alzheimer's Disease were as follows: .01, .08, .12~
.16, .18, .19, .28, .39, and .40, the mean and standard
deviation being 0.21 ~ .05.
It can be seen that the Alzheimer's Disease
patients, as a group, have substantially higher levels
of PHF antigen in their CSF than that of other
neurological patients (p less than 0.05, one-tailed t
test). It is also appreciated that there is
considerable overlap of the PHFA levels in the CSFs of
these two groups. Thus, while an elevated level of PHFA
in a CSF specimen is indicative oE Alzheimer's Disease,
it cannot be said to be conclusive. Nevertheless, the
indicative nature of this test, along with other
clinical symptoms, has important utility in establishing
whether or not a patient has Alzheimer's Disease. For
example, any PHFA level above about 0.4 unit in the CSF
of a patient is highly indicative of Alzheimer's Disease
in the patient. A level of below about 0.2 unit is
indicative of the patient's being healthy or having a
neurological disorder other than Alzheimer's Disease.
As noted above, the numerical unit by which the
two groups were compared is arbitrary and other units
may be used. For example, if an antigen from a
non-human animal is found to be cross-reactive with an
antibody that reacts with human PHF, whether or not the
antibody has similar degree of affinity for the antigens
from the different species, a PHF unit may be
established relative to the animal antigen; or
~3~
- 18 -
alternatively, tests using such a non-human antigen may
be correlated with tests using the less readily
available human PHF so that results may be expressed in
terms of human PHF activity.
Although the assay is preferably performed with
a PHF-specific monoclonal antibody, the test may be
performed using polyclonal antiserum generated against
PHF (Grundke-Iqbal, et al. Acta Neuropathol.,
62:259~267, (1984); Grundke-Iqbal, et alO, Acta
Neuropathol., 66:52-81 (1985). Monoclonal antibody has
the advantage of uniformity, allowing better
standardization of results~ a result that is highly
desirable when kits for the assay are to be prepared for
commercial distribution. It is inherently possible,
however, to practice the invention using PHF from a
variety of sources and with a variety of antibodies,
setting up standard curves and establishing normal and
abnormal values in each case.
The PHF levels in the above example are
determined for a given volume of CSF. It will be
appreciated that values may be otherwise calculated,
e.g., relative to a measured level of total protein per
volume in each CSF specimen.
It should be appreciated that the
non-Alzheimer's Disease controls in the above Example
were not normal, healthy persons, but that each of the
controls had a neurological disorder. The comparison in
the example was between Alzheimer's Disease and
non-Alzheimer's Disease patients suffering from various
neurological disorders because CSF specimens are seldom
taken from patients without compelling medical reason.
Therefore~ normal, CSF specimens from healthy adult
controls are not as readily available as are CSF
specimens from non-Alzheimer's Disease neurological
patients from which CSF is frequently obtained for a
variety of diagnostic purposes.
. .
~3~2:~0
-- 19 --
Because the non-Alzheimer's Disease controls
exhibit neurological de~eneration for other reasons, it
is expected that their PHFA levels may well prove to be
elevated relative to a normal, healthy population, and
that the difference in mean PHFA levels in Alzheimer's
Disease and normal, healthy adults may prove to be even
greater than the difference found in the Example.
Nevertheless, the comparison in the Example is
meaningful because it is expected that the test will
initially be used in helping to decide with what
neurological disorder a patient is afflicted rather than
to distinguish an Alzheimerls Disease patient from a
normal, healthy person.
While the invention has been described with
some specificity, modifications apparent to one with
ordinary skill in the art may be made without departing
from the spirit and scope of the invention.
Various features of the invention are set forth
in the following claims.
;
.
