U.S. patent application number 10/308150 was filed with the patent office on 2003-07-31 for monoclonal antibodies directed against the microtubule-associated protein tau, hybridomas secreting these antibodies, antigen recognition by these monoclonal antibodies and their applications.
This patent application is currently assigned to INNOGENETICS S.A.. Invention is credited to Mercken, Marc, Van De Voorde, Andre, Vandermeeren, Marc, Vanmechelen, Eugeen.
Application Number | 20030143760 10/308150 |
Document ID | / |
Family ID | 27442475 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030143760 |
Kind Code |
A1 |
Vandermeeren, Marc ; et
al. |
July 31, 2003 |
Monoclonal antibodies directed against the microtubule-associated
protein tau, hybridomas secreting these antibodies, antigen
recognition by these monoclonal antibodies and their
applications
Abstract
The invention relates to a monoclonal antibody which forms an
immunological complex with an epitope of an antigen belonging to
normal human tau protein as well as abnormally phosphorylated human
tau protein, with said tau protein being liable to be obtained from
a brain homogenate, itself isolated from human cerebral cortex. The
monoclonal antibodies of the invention can be used to detect tau
and abnormally phosphorylated tau in brain extracts and in
unconcentrated cerebrospinal fluid.
Inventors: |
Vandermeeren, Marc; (Geel,
BE) ; Vanmechelen, Eugeen; (Nazareth-Eke, BE)
; Mercken, Marc; (Turnhout, BE) ; Van De Voorde,
Andre; (Lokeren, BE) |
Correspondence
Address: |
Charles A. Muserlian
c/o Bierman, Muserlian and Lucas
600 Third Avenue
New York
NY
10016
US
|
Assignee: |
INNOGENETICS S.A.
|
Family ID: |
27442475 |
Appl. No.: |
10/308150 |
Filed: |
December 2, 2002 |
Related U.S. Patent Documents
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Application
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Filing Date |
Patent Number |
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10308150 |
Dec 2, 2002 |
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09790148 |
Feb 21, 2001 |
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09790148 |
Feb 21, 2001 |
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09348952 |
Jul 7, 1999 |
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6232437 |
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09348952 |
Jul 7, 1999 |
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08403917 |
Jan 19, 1995 |
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6010913 |
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08403917 |
Jan 19, 1995 |
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08403916 |
Jan 19, 1995 |
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08403916 |
Jan 19, 1995 |
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08244951 |
Jan 19, 1995 |
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5843779 |
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08244951 |
Jan 19, 1995 |
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PCT/EP93/03499 |
Dec 10, 1993 |
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Current U.S.
Class: |
436/543 ;
435/338; 435/70.21; 530/388.26 |
Current CPC
Class: |
C07K 16/18 20130101;
C07K 2317/34 20130101; Y10S 436/811 20130101; C07K 14/4711
20130101 |
Class at
Publication: |
436/543 ;
435/70.21; 435/338; 530/388.26 |
International
Class: |
G01N 033/531; C12P
021/04; C12N 005/06; C07K 016/40 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 14, 1992 |
EP |
92.403 403.6 |
Claims
1. Monoclonal antibody which forms an immunological complex with an
epitope of an antigen belonging to human normal as well as
abnormally phosphorylated tau protein, with said tau protein being
liable to be obtained from a brain homogenate, itself isolated from
the human cerebral cortex, characterized by the fact that: it does
not form an immunological complex with other phosphorylated
proteins such as MAP-1, MAP-2 and neurofilaments which share part
of their sequence with tau protein, as determined by means of an
ELISA, and it is liable to detect human normal as well as
abnormally phosphorylated tau protein in cerebrospinal fluid (CSF),
with said tau protein being at a concentration as low as 1.0 pg/ml.
and it is liable to detect said tau proteins with 100% recovery
upon the addition of a fixed amount of tau proteins in
tau-protein-negative CSF.
2. Monoclonal antibody according to claim 1, characterized by the
fact that it forms an immunological complex: either with an epitope
located within the following amino acid of human tau protein:
11 (SEQ ID NO 1) 155 160 165 NH.sub.2-Arg Gly Ala Ala Pro Pro Gly
Gln Lys Gly Gln 170 175 Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro
Pro 180 185 Ser Ser Glu Glu Pro Pro Lys Ser Gly Glu Pro Pro 190 195
200 Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly 205 210 Ser Pro
Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro 215 220 Ser Leu Pro Thr Pro
Pro Thr Arg
more specifically with an epitope located within the following
amino acid sequence:
12 (SEQ ID NO 2) 199 200 205 210 Ser Pro Gly Ser Pro Gly Thr Pro
Tyr Ser Arg Ser 215 220 Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg
Glu 225 230 231 Pro Lys Lys Val Ala Val Val Arg Thr
more specifically with an epitope located within the following
amino acid sequence:
13 (SEQ ID NO 3) 207 210 215 Gly Ser Arg Ser Arg Thr Pro Ser Leu
Pro Thr Pro Pro
most specifically with an epitope located within the following
amino acid sequence:
14 218 224 (SEQ ID NO 4) Pro Pro Thr Arg Glu Pro Lys
or with any other peptide capable of forming an immunological
complex with a monoclonal antibody, which is capable of forming a
complex with an epitope located in a tau protein regions as shown
in any of SEQ ID NO 1 to 4.
3. Monoclonal antibody according to claims 1 or 2 secreted by the
hybridoma deposited at ECACC on Oct. 8, 1992 under No.
92100853.
4. Hybridoma, which secretes a monoclonal antibody according to
anyone of claims 1 to 3, more particularly the hybridoma deposited
at ECACC on Oct. 8, 1992 under No. 92100853.
5. Peptides which can be obtained from a brain homogenate, itself
isolated from the human cerebral cortex or from cerebral cortex
obtained from a patient having Alzheimer's disease and which forms
an immunological complex with the monoclonal antibody according to
anyone of claims 1 to 3.
6. Peptides liable to form an immunological complex with any of the
monoclonal antibodies, according to anyone of claims 1 to 3, which
are contained in, or are constituted by parts of the sequence as
shown in SEQ ID NO 1 to 4, which contain or are constituted by the
sequence of the peptides liable to form an immunological complex
with a monoclonal antibody according to anyone of claims 1 to 3,
which itself is liable to form a complex with the epitope located
in the tau protein region as shown in SEQ ID NO 1 to 4.
7. Peptides of about 100 amino acids which contain the sequence as
shown in SEQ ID NO 1 to 4, which contain the sequence of the
peptides liable to form an immunological complex with a monoclonal
antibody according to anyone of claims 1 to 3, which itself is
liable to form a complex with a peptide within sequence as shown in
SEQ ID NO 1 to 4.
8. Peptides according to anyone of claims 5 to 7, which are liable
to generate a monoclonal antibody according to anyone of claims 1
to 3 upon immunization.
9. Peptides which are contained in the brain, in the cerebrospinal
fluid, or the serum of a patient having Alzheimer's disease or any
brain disease involving PHF or normal tau protein and which forms
an immunological complex with a monoclonal antibody according to
anyone of claims 1 to 3.
10. Process for obtaining and isolating a hybridoma according to
claim 4, secreting a monoclonal antibody according to anyone of
claims 1 to 3, characterized in that it involves: starting from
spleen cells of an animal, e.g. mouse or rat, previously immunized
in vivo, or from spleen cells of such cells previously immunized in
vitro with an antigen recognized by the monoclonal antibody
deposited at ECACC on Oct. 8, 1992 under No. 92100853, or with a
peptide containing or which are constituted by parts of any of the
sequences as represented in SEQ ID NO 1 to 4; fusing said immunized
cells with myeloma cells under hybridoma-forming conditions; and
selecting those of the hybridomas which secrete the monoclonal
antibodies which specifically recognize an epitope of the antigen
according to any of claims 5 to 8 and which form an immunological
complex with said epitope.
11. Process for producing monoclonal antibodies according to anyone
of claims 1 to 3 which involves: culturing the selected hybridomas
according to claim 4, in an appropriate medium culture; and
recovering the monoclonal antibodies excreted by said selected
hybridomas; or alternatively: implanting the selected hybridomas of
claim 4 into the peritoneum of a mouse and, when ascites has been
produced by the animal, recovering the monoclonal antibodies then
formed from said ascites.
12. Process for the detection or diagnosis in vitro of brain
disease involving either or both PHF-tau or normal tau protein,
e.g. Alzheimer's disease, which involves: contacting a monoclonal
antibody according to anyone of claims 1 to 3, with a preparation
of NFT or a detergent-extracted brain homogenate isolated from a
patient having had Alzheimer's disease or any other disease
involving tau protein or abnormally phosphorylated tau protein
under conditions suitable for producing an antigen-antibody
complex; and separating the antigen from said complex and
recovering the antigen sought in a purified form.
13. Process for the detection or diagnosis in vitro of brain
disease involving abnormally phosphorylated tau protein, e.g.
Alzheimer's disease, which includes: bringing a sample of brain
homogenate, or of cerebrospinal fluid, or of serum from a patient
suspected of suffering from a neurological disorder involving tau
protein or PHF, more particularly Alzheimer's disease, into contact
under in vitro conditions with a monoclonal antibody according to
anyone of claims 1 to 3, with said conditions being suitable for
producing an antigen-antibody complex; and detecting the
immunological binding of said antibody to said sample of brain
homogenate, or of cerebrospinal fluid, or of serum.
14. Process for the detection or diagnosis in vitro of brain
diseases involving PHF and/or normal tau protein, e.g. Alzheimer's
disease, comprising the steps of: bringing a sample of
unconcentrated cerebrospinal fluid sample isolated from a patient
suspected to suffer from a neurological disorder involving normal
or abnormally phosphorylated tau protein, more particulary
Alzheimer's disease, into contact under in vitro conditions with a
monoclonal antibody according to anyone of claim 1 to 3, under
conditions suitable for producing an antigen-antibody complex; and,
detecting the immunological binding of said antibody to said sample
of cerebrospinal fluid by means of a sandwich ELISA, preferably by
applying the catalysed reporter diagnosis enhancement (CARD)
procedure.
15. Kit for the diagnosis in vitro of one of the following
diseases: Alzheimer's disease, Down's syndrome, Pick's disease,
SSPE and other neurological disorders in which normal tau protein
or abnormally phosphorylated tau protein or paired helical
filaments are implicated, characterized in that the kit comprises:
at least a microplate for deposition thereon of any monoclonal
antibody according to anyone of claims 1 to 3; a preparation
containing the sample to be diagnosed in vitro, possibly together
with a labeled peptide containing the epitope of the invention and
preferably with a peptide lying with as shown in SEQ ID NO 1 to 4;
a second antibody which can be a monoclonal antibody recognizing an
epitope of normal tau, or of abnormally phosphorylated tau protein,
or of any peptide according to anyone of claims 5 to 9, with said
epitope being different from the one of the invention, or which can
be a polyclonal antibody recognising normal tau or abnormally
phosphorylated tau or a peptide according to anyone of claims 5 to
9, with said polyclonal antibody being liable to form an
immunological complex with epitopes which are all different from
the epitope of the invention, with said polyclonal antibody being
preferably purified by immunoaffinity chromatography using
immobilized tau protein, or a marker either for specific tagging or
coupling with said second antibody; appropriate buffer solutions
for carrying out the immunological reaction between the monoclonal
antibody of the invention and a test sample on the one hand, and
the bound second antibody and the marker on the other hand,
possibly a peptide according to anyone of claims 5 to 9 for
standard purposes, or for competition purposes with respect to the
antigen which is sought.
Description
[0001] The invention relates to new monoclonal antibodies directed
against the human microtubule-associated protein tau, to the
hybridomas secreting these monoclonal antibodies, and to the
antigen recognition by these monoclonal antibodies and their
applications. The invention also relates to a process for
diagnosing brain diseases involving the particular epitope (of the
tau protein) which is recognized by said monoclonal antibodies.
[0002] Alzheimer's disease (AD) is the most common form of
adult-onset dementia. At present, no biochemical test is available
for antemortem diagnosis of AD. The disease is therefore clinically
diagnosed primarily by exclusion of other forms of dementia. The
illness is characterized neuropathologically by the presence of
neuritic (senile) plaques and neurofibrillary tangles (NFT).
[0003] Neurofibrillary tangles consist of paired helical filaments
(PHF), of which the main protein component is a modified form of
the microtubule-associated protein tau (Brion et al., 1985;
Greenberg and Davies, 1990; Lee et al., 1991), which under normal
circumstances promotes microtubule assembly and stability
(Weingarten et al., 1975; Br and Karsenti, 1990), which is
synthesized in the neurons of several species, including humans
(Kosik et al., 1989) and which is abundantly present in the axonal
compartment of these neurons (Binder et al., 1985).
[0004] The protein exists as a family of different isoforms of
which 4 to 6 isoforms are found in normal adult brain but only 1
isoform is detected in fetal brain (Goedert et al., 1989). The
diversity of the isoforms is generated from a single gene by
alternative mRNA splicing (Himmler, 1989). The most striking
feature of tau protein, as predicted from molecular cloning, is a
stretch of 31 or 32 amino acids occurring in the carboxy-terminal
part of the molecule that is repeated 3 or 4 times. Additional
diversity is generated through 29 or 58 amino acid-long insertions
in the NH.sub.2-terminal part of the molecules (Goedert et al.,
1989).
[0005] Tau variants of 64 and 69 kDa, which are abnormally
phosphorylated, as revealed by the apparent increase in their
molecular mass observed after alkaline phosphatase treatment, have
been detected exclusively in brain areas showing neurofibrillary
tangles and senile plaques (Flament et al., 1989, 1990). The sites
of phosphorylation by 4 different kinases have been mapped in the
C-terminal microtubule-binding half of tau, and it could be shown
that the action of a calcium calmodulin-dependent kinase on
bacterially expressed tau resulted in the phosphorylation of
Ser(405) which induced a lower electrophoretical mobility (Steiner
et al., 1990). Tau present in paired helical filaments, called
PHT-tau is abnormally phosphorylated (Lee et al., 1991). This
abnormal phosphorylation causes a conformational change in tau,
resulting probably in self-association and the formation of PHFs.
PHF-tau in AD is phosphorylated at several sites, one of which is
the phosphoserine 199 and/or 202. This site is specifically
recognized by a mAb called AT8 (Biernat et al., 1992). Therefore,
AT8 is a discriminative marker for PHF-tau (Goedert et al.,
1992).
[0006] Several antibodies have been reported that show reactivity
to human tau either because they are directed to non-specific
phosphorylated epitopes present on neurofilament and subsequently
shown to cross-react with normal and abnormally phosphorylated tau
(Nukina et al., 1987; Ksiezak-Reding et al., 1987) or because they
recognized specific epitopes on normal and abnormally
phosphorylated tau (Kosik et al., 1988). In addition to the tau
antibodies directed towards non-specific epitopes, antibodies
directed specifically to phosphorylated tau epitopes have been
described (Mercken et al., 1992b).
[0007] Although overall tau mRNA levels are only slightly modulated
in Alzheimer-affected brain regions (Goedert et al., 1988; Barton
et al., 1990) it has been shown that total tau protein levels may
differ at least 6-fold (Khatoon et al., 1992). This has been
demonstrated by polyclonal antibodies against tau (Flament and
Delacourte, 1990) and by monoclonal antibodies directed to
well-defined epitopes. The Alz 50 monoclonal antibody recognizing a
phosphate-independent epitope in the N-terminus of the tau molecule
(Goedert et al., 1991) has been used in a sandwich immunoassay on
brain homogenates and it has been shown that tau levels are higher
in Alzheimer's patient brains (Ghanbari et al., 1990; patent
application EP 444 856).
[0008] An antibody named "423", raised against pronase-treated PHFs
and specifically reactive with a 9.5 kDa and a 12 kDa fragment was
also used to measure tau protein in Alzheimer's disease (patent
application WO 89/03993). Similarly, it was found that increased
mAb 423 immunoreactivity was observed in Alzheimer brain
homogenates as compared with control brain homogenates.
[0009] Mercken et al. (1992b) describe a range of monoclonal
antibodies which are either specific for a phosphatase-sensitive
epitope (AT8) or which react-with PHF-tau as well as with normal
tau (AT1, AT4, AT6, AT9, AT11, AT12 and AT14) in Western
blotting.
[0010] Moreover, the antibody tau 1 (Wischik et al. 1988;
Harrington et al., 1990) was also used to measure tau in brain
homogenates. In one case when tau levels were specifically measured
in Alzheimer-affected brain sections, tau levels were eight-fold
higher as compared with levels in normal brain homogenates (Khatoon
et al., 1992).
[0011] In a first attempt to diagnose Alzheimer disease in
cerebrospinal fluid, the PHF-tau-specific monoclonal antibody AT8
(Mercken et al., 1992b), was used. However, no PHF tau antigen
could be demonstrated.
[0012] Thus far, none of the monoclonals that have been described
have been successful in detecting tau in non-concentrated
cerebrospinal fluid (CSF), although the presence of tau was
observed in 100-fold concentrated CSF (Wolozin and Davies, 1987) or
in CSF samples using polyclonal antibodies (Delacourte and
Vermersch, 1991).
[0013] The aim of the present invention is therefore to provide
monoclonal antibodies which allow reliable and sensitive detection
of normal and abnormally phosphorylated tau present in brain
extracts and in unconcentrated cerebrospinal fluid. The invention
also aims at providing the hybridoma which secretes the above-said
monoclonal antibodies.
[0014] The invention furthermore aims at providing the epitope of
the tau protein present in brain homogenates or in body fluids such
as cerebrospinal fluid, which is recognized by said monoclonal
antibodies.
[0015] The invention aims at providing a process for the detection
or diagnosis in vitro of brain diseases involving tau protein.
[0016] The monoclonal antibodies of the invention are characterized
by the fact that they react with an epitope which is present in
both normal and abnormally phosphorylated human tau protein. The
monoclonal antibodies are furthermore characterized by the fact
that they form an immunological complex with an epitope or an
antigen belonging to normal and abnormally phosphorylated human tau
protein. The monoclonal antibodies of the invention are also
characterized by the fact that they do not form an immunological
complex with other phosphorylated proteins such as MAP-1, MAP-2 and
neurofilaments which share part of their sequence with tau protein
(Nukina et al., 1987; Lewis et al., 1988) as determined by means of
an ELISA. The monoclonal antibodies of the invention are also
characterized by the fact that they are liable to detect human tau
protein in CSF, with said tau protein being at a concentration as
low as 1.0 pg/ml and with said tau protein being detected at 100%
recovery upon the addition of a fixed amount of tau protein in tau
protein-negative CSF (100% spiking recovery).
[0017] The monoclonal antibodies of the invention also enable the
diagnosis of Alzheimer's disease (AD) on the basis of CSF, i.e., to
detect tau and modified forms of tau in CSF. The problem associated
herewith is that this antigen is present in a very low amount in
CSF, therefore the detection assay must be very sensitive. This
problem can be resolved by using the combination of the monoclonal
antibody of the invention together with the catalysed reporter
deposition amplification technique (CARD, Bobrow et al., 1989),
allowing a tau-specific CARD ELISA with a higher sensitivity.
Alternatively, a mixture or combinations of labeled monoclonal
antibodies, each recognizing epitopes different from AT120 epitope,
could be used as detector antibodies.
[0018] The results obtained with the monoclonal antibody secreted
by the hybridoma AT120 of the invention indicate that elevated tau
levels are not only found in AD, but also in other neurological
diseases where neuronal death or damage occurs.
[0019] The expression "form an immunologically complex with" means
that the monoclonal antibody of the invention binds to the
above-said antigen under one of the following conditions as
mentioned in the techniques below:
[0020] Light Immunomicroscopy
[0021] Brain tissue samples, obtained at surgery or autopsy, are
fixed by immersion in 4% formalin or Bouin's fixative and embedded
in paraffin for sectioning. The monoclonal antibodies of the
invention are applied in conjunction with a technique to visualize
the formed immune complexes such as the avidin-biotinylated
peroxidase complex technique (Hsu et al., 1981) using
3,3'-diaminobenzidine tetrahydrochloride for development of color.
Sections are counterstained with Harris haematoxylin stain.
[0022] Immunoelectron Microscopy in Tissue Sections
[0023] Brain tissue samples, obtained at surgery or autopsy are
fixed in either Bouin's fixative or 10% buffered formalin before
sectioning without embedding (Vibratome). The monoclonal antibody
of the invention is used for immunostaining by the indirect
immunogold method after which the sections are fixed, embedded and
sectioned for electron microscopy, all according to standard
protocols known to those skilled in the art (Brion et al.,
1985).
[0024] Immunoblotting Procedures
[0025] For immunoblotting, fractions enriched in tau are prepared
as described (Lindwall and Cole, 1984). Typically, 50 g of brain
tissue is cut into small pieces with scissors and homogenized 1:1
(wt/vol) in buffer A (20 mM 2-[N-morpholino]ethanesulfonic acid, 80
mM NaCl, 2 mM EDTA, 0.1 mM EGTA, 1 mM .beta.-mercaptoethanol, pH
6.75) with a Potter homogenizer equipped with a Teflon plunger. The
homogenate is centrifuged for 1 h at 150,000 g at 4.degree. C., and
the supernatant is heated for 5 min in boiling water and chilled
again for 10 min on ice. The slurry is centrifuged for 2 h at
150,000 g at 4.degree. C., and the supernatant is collected. The
heat-stable cytosolic extract is added to 2.5% perchloric acid and
centrifuged for 1 h at 150,000 g at 4.degree. C., after which tie
supernatant is neutralized with 3 M Tris. The supernatant is then
dialyzed in water and concentrated in a Centriprep concentrator
(Amicon, Lausanne, Switzerland).
[0026] SDS-polyacrylamide electrophoresis is performed under
reducing conditions on 12% gels (Laemmli, 1970). After
electrophoresis, the proteins are either fixed and stained with
Coomassie brilliant blue, or transferred (Towbin et al., 1979) to
nitrocellulose sheets (Hybond-C, Amersham) or Immobilon filters
(Millipore).
[0027] After transfer, the filters are presoaked in PBS containing
0.05% (v/v) Tween 20 (Tween-PBS) and then incubated for 1 h in
Tween-PBS containing 5% (w/v) skimmed dried milk and 10% (v/v)
newborn calf serum (blocking buffer). Next, the filters are treated
overnight at 4.degree. C. with a monoclonal antibody according to
the invention appropriately diluted in blocking buffer.
[0028] The filters are then washed three times in Tween-PBS and
treated for 1.5 h at room temperature with horseradish
peroxidase-labeled rabbit anti-mouse IgG (Dakopatts, Denmark)
diluted {fraction (1/3000)} in blocking buffer. After three washes
in Tween-PBS, streptavidine-biotinyla- ted horseradish peroxidase
complex (Amersham), diluted {fraction (1/250)} in blocking buffer,
is applied for 1.5 h at room temperature. Thereafter, the filters
are washed three times in Tween-PBS and once in PBS. The filters
are then incubated in PBS containing 0.05% (w/v) diaminobenzidine
and 0.03% (v/v) hydrogen peroxide until background staining
develops.
[0029] It should be clear that the formation of an immunological
complex between the monoclonal antibodies and the antigen is not
limited to the precise conditions described above, but that all
techniques that respect the immunochemical properties of the
antibody and antigen binding will produce similar formation of an
immunological complex.
[0030] Human normal tau is a class of at least six tau proteins
ranging in molecular weight from 58 to 64 kDa which are
specifically expressed in the somatodendritic domain of all neurons
(Papasozomenos and Binder, 1987). Moreover Alzheimer
(tangle)-specific tau forms have been described which occur in the
degenerating cortical neurons of Alzheimer's disease or Down's
Syndrome and of which the lower electrophoretic mobility can be
attributed to abnormal phosphorylation (Flament et al., 1989;
Delacourte et al., 1990).
[0031] According to an advantageous embodiment of the invention,
the monoclonal antibody forms an immunological complex with all
forms of tau described above, with said human tau protein being
liable to be obtained from a brain homogenate, itself isolated from
the cerebral cortex of a patient suffering from a neurological
disease.
[0032] A "brain homogenate" and tau protein can be obtained by the
man skilled in the art according to standard methods such as the
method of Lindwall and Cole (1984).
[0033] According to an advantageous embodiment, the monoclonal
antibodies of the invention form an immunological complex:
[0034] either with an epitope located within the following amino
acid sequence of human tau protein:
1 155 160 165 (SEQ ID NO 1) NH.sub.2- Arg Gly Ala Ala Pro Pro Gly
Gln Lys Gly Gln Ala Asn Ala 170 175 180 Thr Arg Ile Pro Ala Lys Thr
Pro Pro Ser Ser Glu Glu Pro Pro 185 190 195 Lys Ser Gly Glu Pro Pro
Lys Ser Gly Asp Arg Ser Gly Tyr Ser 200 205 210 Ser Pro Gly Ser Pro
Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro 215 220 Ser Leu Pro Thr Pro
Pro Thr Arg
[0035] more specifically with an epitope located within the
following amino acid sequence:
2 199 200 205 210 (SEQ ID NO 2) Ser Pro Gly Ser Pro Gly Thr Pro Gly
Ser Arg Ser Arg Thr Pro 215 220 225 Ser Leu Pro Thr Pro Pro Thr Arg
Glu Pro Lys Lys Val Ala Val 230 231 Val Arg Thr
[0036] more specifically with an epitope located within the
following amino acid sequence:
3 207 210 215 (SEQ ID NO 3) Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro
Thr Pro Pro
[0037] most specifically with an epitope located within the
following amino acid sequence:
4 218 224 (SEQ ID NO 4) Pro Pro Thr Arg Glu Pro Lys
[0038] or with any other peptide capable of forming an
immunological complex with a monoclonal antibody, which is capable
of forming a complex with an epitope located in a tau protein
regions as shown in any of SEQ ID NO 1 to 4.
[0039] The sequences as shown in SEQ ID NO 1 to 4 will be hereafter
designated as containing "the epitope" of the invention. Amino acid
sequence 1 spans the amino acids 155-221 of human tau using the
numbering of human tau 40, amino acid sequence 2 spans the amino
acids 199-231 of human tau, amino acid sequence 3 spans the amino
acids 207-219 of human tau, and amino acid sequence 4 spans the
amino acids 218-224 of human tau (Goedert et al., 1989).
[0040] The peptides capable of forming an immunological complex
with a monoclonal antibody, which itself is liable to form a
complex with the above-mentioned peptide will be defined as the
"variant peptides".
[0041] A preferred monoclonal antibody of the invention is secreted
by the hybridoma deposited at ECACC (European Collection of Animal
Cell Cultures, Vaccine Research and Production Laboratory, Public
Health and Laboratory Service (PHLS), Centre for Applied
Microbiology and Research, Proton Down, GB-Salisbury, Wiltshire SP4
OJG) on Oct. 8, 1992 under No. 92100853.
[0042] This hybridoma will be hereafter designated as "hybridoma
AT120" and the secreted monoclonal antibody will be designated as
"monoclonal antibody AT120".
[0043] The invention also relates to the hybridoma which secretes a
monoclonal antibody according to the invention, and particularly
the hybridoma filed at ECACC on Oct. 8, 1992 under No.
92100853.
[0044] The above-mentioned monoclonal antibodies are obtained by a
process involving obtention and isolation of hybridomas which
secrete these monoclonal antibodies.
[0045] A process for obtaining the hybridoma involves:
[0046] starting from spleen cells of an animal, e.g. mouse or rat,
previously immunized in vivo, or from spleen cells of such cells
previously immunized in vitro with an antigen recognized by the
monoclonal antibody deposited at ECACC on Oct. 8, 1992 under No.
92100853, or with a peptide containing or which are constituted by
parts of any of the sequences as represented in SEQ ID NO 1 to
4;
[0047] fusing said immunized cells with myeloma cells under
hybridoma-forming conditions; and
[0048] selecting those hybridomas which secrete the monoclonal
antibodies which specifically recognize an epitope of the
above-said antigen and which form an immunological complex with
normal tau or the abnormally phosphorylated form of tau protein or
with the peptide comprising the epitope of tau recognized by the
monoclonal antibody of the invention.
[0049] A process for producing the corresponding monoclonal
antibodies involves:
[0050] culturing the selected hybridoma as indicated above in an
appropriate culture medium; and
[0051] recovering the monoclonal antibodies secreted by said
selected hybridoma; or alternatively
[0052] implanting the selected hybridoma into the peritoneum of a
mouse and, when ascites has been produced in the animal;
[0053] recovering the monoclonal antibodies then formed from said
ascites.
[0054] The monoclonal antibodies of the invention can be prepared
by conventional in vitro techniques such as the culturing of
immobilized cells using e.g. hollow fibers or microcapsules or such
as the culturing of cells in homogeneous suspension using e.g.
airlift reactors or stirred bioreactors.
[0055] The invention also relates to a peptide (antigen), which can
be obtained from a human brain homogenate itself being isolated
from the human cerebral cortex obtained from a patient having
Alzheimer's disease, and which forms an immunological complex with
the monoclonal antibody of the invention.
[0056] The invention also relates to peptides (antigens) which are
liable to form an immunological complex with anyone of the
monoclonal antibodies of the invention and
[0057] which are contained in or are constituted by parts of the
sequence as shown in SEQ ID NO 1, 2, 3, or 4;
[0058] which contain or are constituted by the sequence of the
variant peptides defined above.
[0059] It is to be recalled that variant peptides are those
peptides able to form an immunological complex with a monoclonal
antibody, which itself is liable to form a complex with an epitope
located in the tau protein region as shown in SEQ ID NO 1 to 4.
[0060] The invention also relates to polypeptides (antigens) of
about 100 amino acids
[0061] which contain the sequence as shown in SEQ ID NO 1 to 4,
or
[0062] which contain the sequence of the variant peptides defined
above.
[0063] The invention also relates to the above- mentioned peptides
which are liable to generate monoclonal antibodies of the
invention.
[0064] The invention also relates to a peptide (antigen) which is
contained in the brain, in the cerebrospinal fluid, or in the serum
of a patient having Alzheimer's disease or any brain disease
involving normal human tau protein and which forms an immunological
complex with a monoclonal antibody of the invention.
[0065] The invention also relates to a peptide (antigen) which is
contained in the brain, in the cerebrospinal fluid, or in the serum
of a patient having Alzheimer's disease or any brain disease
involving PHF or abnormally phosphorylated human tau protein and
which forms an immunological complex with a monoclonal antibody of
the invention.
[0066] A method for preparing the peptides of the invention is
characterized in that, preferably starting from the C-terminal
amino acid, the successive aminoacyls in the requisite order, or
aminoacyls and fragments formed beforehand and already containing
several aminoacyl residues in the appropriate order, or
alternatively several fragments prepared in this manner beforehand,
are coupled successively in pairs, it being understood that care
will be taken to protect all the reactive groups carried by these
aminoacyls or fragments beforehand except for the amine groups of
one and carboxyl groups of the other, or vice versa, which must
normally participate in peptide bond formation, in particular after
activation of the carboxyl group, according to methods known in
peptide synthesis, and so on, proceeding stepwise up to the
N-terminal amino acid.
[0067] The antigen of the invention, which can be prepared by
methods known to those skilled in the art (Lindwall and Cole, 1984)
starting from the human cerebral cortex is characterized by its
ability to form an immunological complex with the monoclonal
antibody of the invention as defined above, advantageously with the
monoclonal antibody secreted by the hybridoma AT120 deposited at
the ECACC under No. 92100853 on Oct. 8, 1992.
[0068] The antigen of the invention is advantageously contained in
the brain, in the cerebrospinal fluid or the serum of a patient
having Alzheimer's disease, Down syndrome, Pick's disease, subacute
sclerosing panencephalitis (SSPE) or other neurological diseases in
which the normal tau or abnormally phosphorylated tau protein are
implicated; this antigen provokes an immunological reaction with
the monoclonal antibody of the invention.
[0069] The invention also relates to a process for the detection or
the diagnosis in vitro of brain disease involving tau protein, i.e.
Alzheimer's disease, which involves:
[0070] bringing the monoclonal antibody of the invention into
contact with a preparation of NFT containing tau protein or a
detergent-extracted brain homogenate containing tau protein
isolated from a patient having had Alzheimer's disease or any other
disease involving tau protein or abnormally phosphorylated tau
protein under conditions suitable for producing an antigen-antibody
complex; and,
[0071] separating the antigen from said complex and recovering the
antigen sought in a purified form.
[0072] The preparation of tau can be carried out according to
Lindwall and Cole (1984).
[0073] Advantageously, the monoclonal antibodies used are in an
immobilized state on a suitable support such as a resin. The
process for the detection of the antigen can then be carried out as
follows:
[0074] bringing the supernatant containing proteins and
polypeptides obtained as a result of an extraction procedure
starting from brain tissues or cerebrospinal fluid known to those
skilled in the art (Iqbal et al., 1984; Greenberg and Davies, 1990)
into contact with said monoclonal antibody, under such conditions
as to allow the formation of an immunological complex;
[0075] washing the immobilized antibody-antigen complex then
formed;
[0076] treating this complex with a solution (e.g. 3 M potassium
thiocyanate, 2.5 M magnesium chloride, 0.2 M citrate-citric acid,
pH 3.5 or 0.1 M acetic acid) capable of producing the dissociation
of the antigen- antibody complex; and;
[0077] recovering the antigen in a purified form.
[0078] The process of the invention for the detection or diagnosis
in vitro of brain disease involving tau protein and abnormally
phosphorylated tau protein, as e.g. in Alzheimer's disease,
includes:
[0079] bringing a sample of a brain homogenate, or of cerebrospinal
fluid, or of serum from a patient suspected of suffering from a
neurological disorder involving tau protein or PHF, more
particularly Alzheimer's disease, into contact under in vitro
conditions with the monoclonal antibody of the invention, with said
conditions being suitable for producing an antigen-antibody
complex; and
[0080] detecting the immunological binding of said antibody to said
sample of brain. homogenate, or of cerebrospinal fluid, or of
serum.
[0081] The detection of the immunologically bound monoclonal
antibody can be achieved by conventional technology.
Advantageously, the monoclonal antibody of the invention itself
carries a marker or a group for direct or indirect coupling with a
marker as exemplified hereafter. Also, a polyclonal antiserum can
be used which was raised by injecting the antigen of the invention
in an animal, preferably a rabbit, and recovering the antiserum by
immunoaffinity purification in which said polyclonal antibody is
passed over a column to which said antigen is bound and eluting
said polyclonal antibodies by conventional technology.
[0082] Detection can also be achieved by competition binding of the
antigen with a labeled peptide comprising the epitope of the
invention.
[0083] A particularly advantageous embodiment of the process of the
invention for the detection or diagnosis in vitro of brain diseases
involving PHF and/or normal tau protein, e.g. Alzheimer's disease,
comprises the steps of:
[0084] bringing a sample of unconcentrated cerebrospinal fluid
sample isolated from a patient suspected of suffering from a
neurological disorder involving normal or abnormally phosphorylated
tau protein, more particulary Alzheimer's disease, into contact
under in vitro conditions with a monoclonal antibody according to
the invention, under conditions suitable for producing an
antigen-antibody complex; and,
[0085] detecting the immunological binding of said antibody to said
sample of cerebrospinal fluid by means of a sandwich ELISA,
preferably by applying the catalysed reporter diagnosis enhancement
(CARD) procedure.
[0086] The invention also relates to a kit for the diagnosis in
vitro of one of the following diseases: Alzheimer's disease, Down's
syndrome, Pick's disease, subacute sclerosing panencephalitis
(SSPE) and other neurodegenerative disorders in which normal tau
protein or abnormally phosphorylated tau protein are implicated.
Such a kit would contain:
[0087] at least one microplate for deposition thereon of any
monoclonal antibody of the invention;
[0088] a preparation containing the sample to be diagnosed in
vitro, possibly together with a labeled peptide containing the
epitope of the invention and preferably, a peptide lying in the
peptide sequence as shown in SEQ ID NO 1 to 4,
[0089] a second antibody
[0090] which can be a monoclonal antibody recognizing another
epitope of normal or abnormally phosphorylated tau protein, or of
any peptide of the invention, with said epitope being different
from the one of the invention, or
[0091] which can be a polyclonal antibody directed against normal
or abnormally phosphorylated tau, or against a peptide of the
invention, with said polygonal antibody being liable to form an
immunological complex with epitopes which are all different from
the epitope of the invention, with said polyclonal antibody being
preferably purified by immunoaffinity chromatography using
immobilized tau protein, or
[0092] a marker either for specific tagging or coupling with said
second antibody;
[0093] appropriate buffer solutions for carrying out the
immunological reaction between the monoclonal antibody of the
invention and a test sample on the one hand, and the bound second
antibody and the marker on the other hand.
[0094] The labeled peptide mentioned above can be a peptide which
has been labeled by any means known for the man skilled in the art.
Moreover, the marker specific for tagging and coupling can be any
marker known to the man skilled in the art.
[0095] The invention also relates to a kit, as described above,
also containing the antigen of the invention, with said antigen of
the invention being either a standard (for quantitative
determination of the antigen which is sought) or a competitor, with
respect to the antigen which is sought, for the kit to be used in a
competition dosage process.
BRIEF DESCRIPTION OF THE FIGURES
[0096] FIG. 1
[0097] Western blotting detection of normal tau or PHF-tau using
the monoclonal antibodies Tau-1, (Binder et al., 1985) AT8 (Mercken
et al., 1992b) and AT120. Lanes 1, 3 and 5: PHF-tau isolated
according to Greenberg and Davies (1990). Lanes 2, 4 and 6: Normal
affinity purified human tau according to Mercken et al. (1992a).
Lane 7: Molecular weight markers.
[0098] Lanes 1 and 2 were developed using AT8, lanes 3 and 4, using
AT120 and lanes 5 and 6, using Tau-1 monoclonal antibody.
[0099] FIG. 2 and FIG. 2B
[0100] Detection of tau protein by immunochemistry
[0101] FIG. 2A: section from hippocampus of a patient with
Alzheimer's disease. Magnification 212.times..
[0102] FIG. 2B: section from another hippocampus of a patient with
Alzheimer's disease with abundant tangles. Magnification
212.times..
[0103] FIG. 3
[0104] Titration of normal (0) and PHF tau (.box-solid.), spiked in
a tau-negative CSF pool using the amplified (CARD) AT120 sandwich
ELISA. All dilutions were tested in duplicate and the data
presented as optical density (OD) units.
[0105] FIG. 4
[0106] Western blotting of several deletion mutants constructed as
indicated in example V, and stained with AT120 as indicated in
Example I. Mutants comprise the following amino acids (AA); Lane 1,
full length tau 34 (Goedert et al., 1989); Lane 2, amino terminus
of tau 34 up to AA.sub.154; Lane 3, from AA.sub.155 to the
carboxyterminus of tau 34; Lane 4; aminoterminus of tau 34 up to
AA.sub.242; Lane 5, aminoterminus tau 34 up to AA.sub.221; Lane 6,
from AA.sub.222 to the carboxyterminus of tau 34.
[0107] FIG. 5
[0108] Epitope recognition sites of the monoclonal antibodies HT7,
BT2, AT8 are depicted on the epitope of the invention (SEQ ID NO 1)
shown in the one letter amino acid code. Epitopes are boxed. The
star "*" designates the fact that the AT8 epitope recognition needs
phosphorylation of serine residue 202.
[0109] FIG. 6
[0110] Complete sequence of the mTHFMPH-tau1 fusion protein (SEQ ID
NO 23) with indication of relevant restriction sites.
EXAMPLES
Example 1
Preparation of the Monoclonal Antibody AT120 (IgG1, Subtype
Kappa)
[0111] 1. Preparation of the Antigen for Immunization
[0112] PHF-tau was partially purified by a modification of the
method of Greenberg and Davies (1990). Postmortem tissue,
consisting mostly of gray matter from the frontal and temporal
cortex, was obtained from histologically confirmed Alzheimer
patients. This Alzheimer gray matter brain sample (5-10 g) was
homogenized with 10 volumes of cold buffer H (10 mM Tris/1 mM
EGTA/0.8 M NaCl/10% sucrose, pH 7.4) in a Teflon/glass Potter S
(Braun, Germany) homogenizer. After centrifugation of the
homogenate in a 60 Ti MSE rotor at 27,000 .times. g for 20 min at
4.degree. C., the pellet was removed and the supernatant was
adjusted to 1% (wt/vol) N-laurosylsarcosine and 1% (vol/vol)
2-mercaptoethanol and incubated while rotating on a mixer (Swelab,
Sweden) for 2.5 hours at 37.degree. C. The supernatant mixture was
centrifuged at 108,000 .times. g for 35 min at 20.degree. C. The
PHF-tau containing pellet was gently washed with PBS and finally
suspended in 1 ml of the same buffer.
[0113] The antigen preparation was evaluated by a 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, followed by
Western blotting using immunoblotting with polyclonal rabbit
antihuman normal tau antiserum (Mercken et al., 1992b).
[0114] 2. Immunization Protocol and Fusion Procedure
[0115] Balb/c mice were primed subcutaneously with 100 .mu.g
partially purified PHF-tau in complete Freund's adjuvant and
boosted intraperitoneally 3 times thereafter at 3-week intervals
with 100 .mu.g of the same antigen in incomplete Freund's adjuvant.
On days 3 and 2 before the fusion, mice were boosted with 100 .mu.g
PHF-tau in saline.
[0116] Mouse spleen cells were fused with SP2/0 myeloma cells,
using a modified procedure of Kohler and Milstein (1975), with PEG
4000.
[0117] The cells of the fusion experiment were suspended at a
density of 4.5.times.10.sup.4 spleen cells/well on 96-well plates
preseeded with mouse peritoneal macrophage cells as a feeder layer.
These wells were screened after 12 days for anti-tau antibody
production in a sandwich ELISA either specific for normal tau or
for PHF-tau as discussed in section 3. below.
[0118] Hybridoma growth was in Dulbecco's modified Eagle's medium
(DMEM) supplemented with 20% fetal calf serum, sodium pyruvate (1
mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100
mg/ml), and nonessential amino acids. All products were purchased
from Gibco, (Paisley, U.K.). Cells were incubated in a humidified
CO.sub.2-air incubator.
[0119] 3. Sandwich ELISA for Antibody Screening
[0120] The screening ELISA used for the detection of anti-tau
monoclonal antibodies was a sandwich ELISA system with
affinity-purified polyclonal rabbit anti-human tau antibodies
(Mercken et al; 1992a) in the coating phase. To this end, purified
human normal tau, prepared as described in Mercken et al. (1992a)
was used for the preparation of an immuno-affinity column using
cyanogen bromide-activated Sepharose (Pharmacia, LKB Sweden). The
affinity-bound anti-tau fraction was eluted from this column with a
0.1 M citric acid buffered solution at pH 2.5. After
neutralization, the anti-tau-containing fractions were pooled and
coated overnight (1 .mu.g/ml) at 4.degree. C. on high-binding
microtiter plates (Nunc, Gibco, Paisley, UK) in coating buffer (10
mM Tris, 10 mM NaCl, 10 mM NaN.sub.3, pH 8.5). After overcoating
for 30 min with 125 .mu.l 10%-saturated casein in PBS to reduce
non-specific binding, the plates were incubated with 100 .mu.l of
an appropriately diluted PHF-tau preparation and incubated for 60
min at 37.degree. C. The plates were washed 3 times with PBS-0.05%
Tween 20 (v/v); 100 .mu.l hybridoma supernatant was added and
incubation was continued for 1 h at 37.degree. C. After washing,
the bound monoclonal antibodies were detected with
peroxidase-conjugated rabbit anti-mouse serum (Dakopatts, Glostrup,
Denmark). All reagents were diluted in PBS with 10% casein. After
final washing, 100 .mu.l 0.42 mM 3,5,3',5'-tetramethylbenzidine,
0.003% H.sub.2O.sub.2 v/v in 100 mM citric acid, 100 mM disodium
hydrogen phosphate, pH 4.3, was added as peroxidase substrate. The
reaction was stopped with 50 .mu.l of a 2 M H.sub.2SO.sub.4
solution. Absorbance was read in a Titertek Multiscan (Flow
Laboratories, Eflab, Oy, Finland) at 450 nm.
[0121] The cross-reactivity of the monoclonal antibodies with
normal tau in ELISA was tested in a sandwich ELISA identical to the
screening assay, except that affinity-purified normal tau was used
as the antigen instead of PHF-tau.
[0122] At the first selection of positive hybridoma cultures, most
positive cultures were initially composed of mixed clones as seen
by visual inspection of the wells (3-4 clones per well). These
positive cultures were arbitrarily designated AT1 to AT24 (some of
these hybridoma cultures, i.e., AT1 to AT14 were described by
Mercken et al., 1992b). After this initial screening round,
hybridoma cultures were subcloned by limiting dilution, a technique
well-know to those skilled in the art, finally resulting in pure
hybridoma clones secreting antibodies with a homogeneous idiotype.
These pure hybridoma clones were further tested with respect to
their reactivity pattern on normal and PHF-tau in ELISA, Western
blotting and immunohistochemistry, and to their capability to
diagnose neurological diseases by means of their affinity for tau
protein present in an undiluted sample of cerebrospinal fluid.
Based on these criteria, the monoclonal antibody AT120 was selected
and further characterized as shown in the following examples.
[0123] 4. Determination of the Antibody Class and Subclass
[0124] The antibody class and subclass was determined by Inno-LIA
(Innogenetics, Ghent, Belgium). The antibody of the invention,
AT120, appeared to be of the IgG1, kappa subtype.
Example II
Detection of Pathological Tau and Normal Tau in ELISA and by
Western Blotting
[0125] 1. Detection of Normal Tau in ELISA Using AT120
[0126] Protein G-purified monoclonal antibody AT120, obtained from
serum-free hybridoma AT120 conditioned medium, was coated on ELISA
plates and reacted with different dilutions of affinity-purified
human normal tau as described in Mercken et al. (1992a), prepared
in a solution of PBS and 10% casein.
[0127] The purity of normal tau was determined by SDS-PAGE. Tau
samples were analyzed on an 420 A/H amino acid analyzer (Applied
Biosystems B.V., Maarssen, The Netherlands) according to the
manufacturer's instructions and the protein showed the expected
amino acid composition. From the amino acid composition and by
comparison with a standard peptide the concentration of normal tau
was determined.
[0128] After incubation of the ELISA plates with different
concentrations of tau spiked in tau- and PHF-tau-negative CSF for 1
h at room temperature, the plates were washed and incubated with
0.2 .mu.g/ml biotynilated BT2 and HT7, each recognizing an epitope
different from the AT120 epitope and present on normal term. After
washing, complexed biotynilated antibodies were detected with
horseradish peroxidase conjugated streptavidine (Jackson) and color
development as specified in example I. The results are shown in
Table I and FIG. 3.
5TABLE I Detection of normal tau in ELISA ABSORBANCE (expressed as
CONCENTRATION milliabsorbance units) (pg/ml) PHF-tau normal tau 160
1682 1609 80 901 970 40 566 678 20 257 256 10 143 154 0 92 87
[0129] 2. Detection of Pathological Tau and of Normal Tau in
Western Blotting Using AT120
[0130] Purified normal human tau and PHF-tau were applied to 10%
SDS-polyacrylamide gels and run under denaturing conditions
according to Laemmli (1970). After SDS-PAGE, the transfer to
nitrocellulose (Hybond-C, Amersham, Brussels, Belgium) was carried
out in 10 mM NaHCO.sub.3, 3 mM Na.sub.2CO.sub.3, pH 9.9 for 120 min
at 55 V with cooling. After blotting, the nitrocellulose was
equilibrated to phosphate buffered saline (PBS), and protein
binding sites were blocked with blot buffer (PBS supplemented with
5% w/v skimmed dried milk and 10% v/v newborn calf serum). Blotted
proteins were incubated overnight at 4.degree. C. with AT120 as
primary antibody. After 3 washings with PBS-0.05% Tween 20(v/v),
horseradish peroxidase-labeled rabbit anti-mouse immunoglobulins
(Dakopatts, Glostrup, Denmark) were used at a dilution of {fraction
(1/3000)} and were incubated for 90 min at room temperature. All
antisera were diluted in blot buffer. The blots were then washed
three times in PBS/Tween and developed with substrate solution
(PBS, 0.05% w/v 3,3'-diaminobenzidine, 0.03% v/v H.sub.2O.sub.2)
after which the reaction was stopped in H.sub.2O. Results shown in
FIG. 1 indicate that the AT120 antibody recognizes all tau
isoforms. By contrast, the Tau-1 antibody (Binder et al., 1985)
reacts solely with normal tau, and the AT8 antibody (Mercken et
al., 1992b) only with PHF-tau.
[0131] AT120, AT8 and Tau-1 mAbs were tested for phosphatase
sensitivity of their epitopes in ELISA and in Western Blot on
PHF-tau antigen. The reactivity of the AT120 antibody with PHF tau
was not sensitive to phosphatase treatment either in ELISA (data
not shown) or on Western blots (data not shown). The reactivity of
AT8 was almost completely abolished after alkaline phosphatase
treatment of the PHF-tau antigen in ELISA. Dephosphorylation of
PHF-tau enhanced Tau-1 immunoreactivity, as described previously
(Binder et al., 1985).
Example III
Detection of Tau by Immunohistochemistry
[0132] Paraffin sections of formalin-fixed brain tissue from
neocortex, hippocampus, cerebellum, pons, and spinal cord of
several Alzheimer patients and age-matched controls were prepared,
as well as sections of peripheral nerve from one control
patient.
[0133] Cryostat sections from Alzheimer and age-matched control
brain were also prepared. Tissues were immunostained either with
the peroxidase-antiperoxidase (PAP) technique (Steinberger et al.,
1970) or with the avidin-biotin complex (ABC) technique (Hsu et
al., 1981) using Dakopatts (Denmark) and Amersham (UK) reagents,
respectively.
[0134] Briefly, after blocking non-specific interactions with
normal swine serum (Dakopatts X90 1) diluted 1:25 in Tris-buffered
saline (TBS) containing 1% bovine serum albumin (BSA), sections
were incubated overnight with the AT120 primary antibody
appropriately diluted in TBS/BSA. Secondary antibody and peroxidase
complex were then applied for 30 min each, with intermediate
rinsing in TBS. Color was developed with
3,3'-diaminobenzidinetetrahydrochloride (Sigma). Sections were
counterstained with Harris' hematoxylin, dehydrated, coverslipped,
and viewed under a light microscope.
[0135] FIGS. 2A and 2B clearly indicate that AT120 produces
abundant staining of NFT, dystrophic neurites in plaques, and
dispersed staining of neuropil (neuropil threads).
Example IV
Detection of Tau in Cerebrospinal Fluid Samples
[0136] Cerebrospinal Fluid Samples
[0137] CSF samples from patients were collected at the Department
of Neurology of the University Hospital of Antwerp. All samples
were obtained by lumbar puncture performed for routine diagnostic
purposes. CSF samples were frozen and kept at -75.degree. C. in
small aliquots until use.
[0138] The patients were divided into 3 different groups: 27
patients diagnosed with probable AD according to McKhann et al.
(1984), mentally healthy control patients, who underwent lumbar
puncture for radiculopathy and patients suffering from other
neurological diseases (OND). The OND group included inflammatory,
vascular, and other diseases, including patients with
neurodegenerative diseases such as adenoleukodystrophy, frontal
lobe degeneration, cerebellar atrophy, olivo-ponto-cerebellar
atrophy, and amyotrophic lateral scelerosis. The age, sex and
diagnosis were noted for each patient.
[0139] AT120 Assay
[0140] AT120 monoclonal antibodies purified from serum-free
conditioned medium by Protein G column chromatography were coated
overnight at 4.degree. C. on high-binding microtiter plates (Nunc,
Gibco, Paisley, UK) in coating buffer at 3 .mu.g/ml (10 mM Tris, 10
mM NaCl, 10 mM NaN.sub.3, pH 8.5). After overcoating for 30 min
with 150 .mu.l 10%-saturated casein in PBS to reduce non-specific
binding, the plates were incubated with 25 .mu.l CSF and 75 .mu.l
conjugate mixture containing 0.2 .mu.g/ml of biotynilated BT.sub.2
and an equal amount of HT7 in 5% Tween 20, 10% saturated casein in
PBS. The plates were left overnight at room temperature and after
washing peroxidase conjugated streptavidine (Jackson) ({fraction
(1/15000)}) was added for 30 minutes at room temperature.
[0141] Following an additional washing, 100 .mu.l 0.42 mM
3,5,3',5'-tetramethylbenzidine 0.003% H.sub.2O.sub.2 v/v in 100 mM
citric acid, 100 mM disodium hydrogen phosphate, pH 4.3, were added
as perocidase substrate. The reaction was stopped with 50 .mu.l of
a 2M H.sub.2SO.sub.4 solution. Absorbance was read in a Titertech
Multiscan (Flow laboratories, Eflab, Oy, Finland) at 450 nm.
[0142] Absorbance values obtained with AT120 from the CSF samples
were compared with standard curves generated from known quantities
of affinity purified normal human tau and this comparison allowed
the result to be expressed as pg tau/ml.
[0143] A summary of these results are compiled in Table II, where
patients ID, diagnosis, age and tau values expressed in pg/ml CSF
are listed. From these results, it is obvious that levels of
control patients are substantially lower (mean: 16.4 pg/ml) as
compared to the group of patients suffering from various
neurological diseases (OND; mean value: 26.4 pg/ml). For patients
with Alzheimer's disease the mean value is clearly elevated above
those of control and OND samples (mean Alzheimer patient: 50.8
pg/ml).
[0144] If a cut-off level of 27 pg/ml is adopted, 8% of the control
samples are positive, while for the OND group and the Alzheimer
group these values are 27% and 80% respectively.
6TABLE II Tau levels as determined with the AT120 ELISA assay,
grouped according to control patients, Alzheimer patients (AD) and
other neurological diseases (OND) and according to age cohort,
expressed in pg/ml. Mean and Std (= SD) is expressed as pg/ml per
age cohort. Number Diagnosis Age pg/ml mean Std 3 AD (early onset
AD) 35 *56.5 33.3 12.3 260 A.D. 41 *31.2 113 A.D. 44 *42.6 161 A.D.
(possibly Creutzfeld) 57 *33.8 81 A.D. 58 *14.5 126 A.D. 59 *24.8
421 A.D. 59 *29.8 338 A.D. 64 *51.2 61.68 35.4 254 A.D. 66 *80.2
209 A.D. 67 *74.4 383 Primary degenerative 67 *32.5 dementia 38
A.D. 67 *68.7 229 A.D. 73 *70.9 132 A.D. 76 *51.9 88 A.D. 76 *25.3
65 Dementia 77 *80.1 71 A.D. 78 *53.9 28 A.D. (early onset A.D.) 78
*48.7 11 A.D. 85 *14 39 A.D. 86 *150 386 control 5 *17.5 17.3 4.37
108 control 20 *14.3 402 control 26 *14 106 control 27 *14 424
control 28 *14 355 control 28 *20.1 399 control 29 *14 373 control
30 *17.4 381 control 31 *14 372 control 32 *19.2 379 control 32
*18.3 241 control 32 *14 415 control 32 *14 428 control 33 *16.6
118 control 34 *21.5 224 control 36 *15.7 24 control 37 *25.2 369
control 38 *23.9 425 control 39 *14 145 control 40 *31.9 61 control
40 *17.3 377 control 40 *14 366 control 41 *17.7 401 control 41
*20.1 400 control 42 *14 417 control 42 *14 354 control 43 *16.8 34
control 43 *25.8 217 control 43 *14 364 control 44 *14 134 control
45 *16.7 367 control 45 *14 427 control 45 *14 394 control 48 *14
361 control 48 *14 237 control 50 *17.2 396 control 52 *16.4 100
control 54 *23.9 411 control 54 *14 371 control 55 *18.4 423
control 56 *14.9 99 control 56 *27.9 192 control 60 *19.9 24.83
15.17 387 control 61 *14 389 control 65 *19.5 426 control 66 *15.4
141 control 67 *14 368 control 67 *15.4 60 control 77 *43.5 348
control 80 *56.9 419 Hydrocephalus 0 *150 29.35 26.78 413
Adenoleukodystrofy 13 *23.1 376 Paresthesia 14 *21.1 239 Epilepsy,
encephalitis 16 *29.8 7 SSPE 17 *48.1 139 Cerebellitis (Mycoplasma
p.) 18 *19.7 180 Herpes encephalitis 19 *14.1 206 GBS 20 *15.9 140
TC ? 21 *14.2 228 Ishernic cerebral infarct 22 *150 197 Kawasaki 24
*27.8 133 MS 24 *37.1 212 Alcohol PNP 25 *14 66 MS ? 25 *14 143
Myopathy 25 *18.3 82 MS 26 *20.2 258 Guillian-Barre 26 *14.4 105 MS
26 *18.4 169 Dementia ? 27 *37.5 351 Viral meningitis 28 *17.3 213
MS 28 *15.7 253 Encephalitis viral ? 28 *38.1 236 Migraine 29 *17.3
405 Guillian-Barre 29 *47.6 72 MS 29 *14 234 MS 29 *19.9 128 GBS 29
*14 138 PNP 32 *17.6 218 Empty sella 35 *14 135 MS 35 *16.3 346
Neuritis optico 35 *14 117 Trigeminus neurology 36 *14 350 MS 37
*14 384 Guillian-Barre 37 *14 123 CVA 38 *14 56 TIA (transient
ischemic 39 *14.9 attack) 172 CVA ? psych. 40 *21.7 189 Hemicranial
headache 41 *15.9 119 Amyloidosis 42 *15.3 69 Meningitis 42 *26.5
231 Contuno alcohol 42 *15.5 130 External oftalmoplegia 43 *53.6
357 CVA 44 *16.6 391 MS 44 *14 62 MS44 21.2 124 Syphilis 45 *20.4
89 PNP 45 *27.1 122 Cauda equina syndroom etiol. 46 *14 249 MS 47
*14 112 MS ? 48 *14.9 363 Encephalitis 48 *35.9 125 MS 48 *24.9 205
GBS 49 388 MS 50 *14 418 Tetanos 50 *14 207 GBS ? 50 *70.3 114
Cerebellar atrofy 51 *51.5 121 Syphilis 51 *28.1 35 MS + PNP
(diabetic) 51 *17.3 215 OLM 52 *14 101 Brain infarct 53 *52.8 255
PNP + (MS ?), diabetic 53 *18.4 173 Lyme disease 54 *17.5 360
(Borrelia) (MS-like) 54 *34.7 374 ALS 54 *65.3 179 Lyme disease 54
*16.4 50 Epilipsy-alcoholism 55 *21.5 184 Epilepsia 55 *16.7 210 MS
56 *16.2 58 CVA 57 *25.3 137 Pick ? 57 *77.4 131 Meningial bleeding
57 *66.7 398 Meningoencephalis 58 *117.8 349 Meningoencephalis 58
*35.6 244 Facialis parese 58 *18.5 219 Pseudobullair syndrome 58
*39.6 64 MS 58 *40.7 36 TIA 59 *14 240 Guillian-Barre 68? *14 25.06
15.69 70 N. ulnaris/Parkinson (?) 60 *23.8 166 tbc 60 *14 85
Aneurism 60 *18.3 204 Bipyramidal idiopathy 60 *16.8 167 Cerebellar
atrophy 61 *14 157 Bulbar paralysis 62 *17.4 30 Steele Richardson
62 *19.2 153 Lymfoma CSZ 62 *22.2 414 Subacute polyneuropathy 62
*14 37 OPCA 63 *26.7 109 Pick; ALS 63 *40.5 277 COLD 64 *14 195 E,
dialysis 64 *14 148 MS 65 *15.4 375 Polyneuropathy, Charot- 65
*15.5 Marie-Tooth 182 ALS 65 *14 230 Parkinson 66 *21.1 409
Multiple vascular (pons.) 66 170 Temp. E 66 *24.2 186 ALS 66 *21.2
59 Menigeal aneurysma 67 *28.4 233 Korsakow-like post trauma 67
*29.6 120 ALS 67 *25.7 550 PNP, CVA, diabetes 67 *64.2 110 Diabetes
67 *14 259 Polyvascular syndrome + 68 demyelinisation 248 (?) 68
*14 235 CVA 68 *77.9 362 Guillain-Barre 68 *14 208 PNP-zona 68
*28.8 115 Subacute combined 68 *14 degeneration 98 TIA 68 *34.9 193
GBS 68 *44.1 222 Guillain-Barre 69 *14.1 102 PNP 70 *14 242
Bipyramidal syndrome PNP 72 *20.8 251 Trauma cerebri commoti 77 *14
422 Infarct 78 *20.7 42 Multi-infarct dementia 78 *38.9 93
Diabetes, PNP, radiculopathy 85 *61.9 53 Mixed dementia, Parkinson
85 *59.3
[0145] Abbreviations
[0146] SSPE: Subacute sclerosing panencephalitis, GBS:
Guillain-Barr syndrome, TC: Meningeal tuberculosis, MS: Multiple
sclerosis, PNP: Polyneuropathy, CVA: cerebrovascular amyloidosis,
ALS: Amyotrophic lateral sclerosis, TIA: Transient ischemic attack,
OPCA: Olivo-ponto cerebellar atrophy, COLD: Chronic obstructive
lung disease.
Example V
Definition of the AT120 Epitope
[0147] Since AT120 reacts equally well with all isoforms of tau
(Goedert et al., 1989), the smallest recombinant tau form was used
for deletion mapping. To this, two sites were used for deletion
construction, the SacII site at position 155 of the human tau 34
sequence and the SmaI site at position 220 (Goedert et al., 1989).
A mTNF-fusion vector pmTNF(MPH) (Innogenetics, Ghent, Belgium), in
which the smallest tau open reading frame was fused to 25 acids of
mouse tumor necrosis factor, was cut with ApaI-SacII, blunted with
T.sub.4 DNA polymerase and ligated. After cutting the ligated
material with Sac II and Apa I to reduce non-mutant background, the
mixture was transformed into MC1061 pc 1587 (Casadaban & Cohen,
(1980)). Each selected clone was further characterized by
restriction digestion and by its reactivity with anti-tau
antibodies.
[0148] The same mouse TNF fusion tau vector was used to insert a
frame shift mutation in the Xmal restriction site. The vector was
cut with SmaI, blunted with T.sub.4 DNA polymerase and the ligated
mixture was retreated with SmaI before transformation in order to
reduce non-mutant background. The reactivity pattern of each of the
mutants was checked with AT120 monoclonal antibodies by means of
Western Blotting. This allowed to localize the epitope of AT120 in
a first approximation to the region of 65 amino acids spanning
amino acid 155-221 which is represented in SEQ ID NO 1 (FIG. 4).
However, since in this region also two other antibodies, BT2
(Mercken et al, 1992a) and HT7 (Mercken, Ph.d. Thesis) show
reactivity it was mandatory to prove that no competition binding
was observed between the latter monoclonal antibodies and
AT120.
[0149] Therefore, a competition ELISA with each of these antibodies
was performed. To this, affinity purified rabbit anti-human tau
polyclonal antibodies were coated overnight at 4.degree. C. in
coating buffer (10 mM Tris pH 8.6 , 10 mM NaCl, 10 mM NaN.sub.3),
and after blocking with 0.1% caseine in PBS, 100 .mu.l/well of pure
PHF-tau was added for 1 h at 37.degree. C. After washing, 50 .mu.l
of either AT8, BT2 or AT120 unlabeled monoclonal antibody was added
at a concentration of 10 .mu.g/ml and incubation was continued for
30 min at 37.degree. C. Next, 50 .mu.l of biotinylated monoclonal
antibody was added. Each of these biotinylated antibodies was used
in a preset concentration, which in a tau Sandwich ELISA (as
described in Example II) gave 50% of the maximal OD value. After a
subsequent incubation of 1 h at 37.degree. C., the plates were
further treated as described in Example II.
7TABLE III Competition ELISA using monoclonal antibodies AT8, AT120
and BT2. Unlabeled competitor Absorbance BIOTINYLATED AT8 AT120 BT2
AT8 0.001 0.372 N.D. AT120 0.476 0.001 N.D. BT2 N.D. 0.543
0.054
[0150] N.D. refers to not determined absorbances
[0151] The results, shown in Table III clearly indicate that the
epitope recognized by the monoclonal antibody AT120 is different
from the epitopes recognized by the monoclonal antibodies BT2 and
AT8. Consequently, solid phase immobilized synthetic nonapeptides,
the sequence of which was derived from the epitope of the invention
as depicted in SEQ ID NO 1, were incubated with each of these
monoclonal antibodies. The complexes were visualised as in Example
I, section 3. On the basis of their respective reactivity patterns
it was confirmed that the epitope recognized by AT120 is different
from that of each of the other monoclonal antibodies (Table IV and
FIG. 5)
8TABLE IV Reactivity of monoclonal antibodies BT2, HT7 and AT120
with solid phase bound nonapeptides. The nonapeptides are
designated in the one-letter amino acid code. Detecting Monoclonal
antibody Absorbance Nonapeptides HT7 BT2 AT120 GAAPPGQKG 3.00 0.168
0.089 (SEQ ID NO 5) GDRSGYSSP 0.493 3.00 0.553 (SEQ ID NO 6)
[0152] In addition to these experiments, the AT120 epitope was also
confirmed by peptide mapping. Hereto, a total of 20 .mu.g of
mTNF-MPHtau1 (see FIG. 6) was lyophilized and redissolved in 56
.mu.l of 100 mM Tris HCl containing 10% acetonitrile. To this was
added 14 .mu.l of endoprotease Asp N (0.04 .mu.g/ml) and the
mixture was incubated for 8 hours at 37.degree. C. Following
cleavage, the mixture was divided equally over 5 sample wells and
subjected to SDS-polyacrylamide gel electrophoresis. Following
electrophoresis, the peptides in the gel were blotted onto a PVDF
membrane. A strip of the PVDF membrane corresponding to one lane on
the gel was removed. Free binding sites on the membrane were
blocked by incubation with 0.5% casein in PBS, and the strip was
incubated with the monoclonal antibody AT120 to determine if
protein fragments were present which were capable of being
recognized. The presence of bound antibody was detected using an
alkaline phosphatase-labeled rabbit anti-mouse conjugate followed
by incubation with 5-bromo-4-chloro-3-indolyl phosphate and nitro
blue tetrazolium. The remainder of the PVDF membrane was stained
with Amido Black, which revealed the presence of 5 bands, all with
a molecular size smaller than that of the original protein. One of
these bands was visible on the portion of the blot which was
incubated with AT120. The band which was specifically recognized by
AT120 corresponded to the largest of the Amido Black-stained bands
with an apparent molecular size of approximately 6 kDa. This band
was excised from the Amido Black-stained blot and subjected to
amino terminal sequence analysis (Edman degradation). The analysis
revealed that the band recognized by AT120 began with Asp193 in the
amino acid sequence of tau, close to the C-terminus of the tau
sequence contained within the mTNF-MPHtau1 recombinant protein.
[0153] It thus appeared probable, that additional amino acid
sequences located downstream with respect to Arg221 might be
required for binding of the monoclonal antibody AT120. In order to
test this hypothesis in an attempt to further delineate the
boundaries of the epitope, peptide (tau5B) was synthesized:
9 tau5B 208 210 215 (SEQ ID NO 7) Bio-Gly Gly Ser Arg Ser Arg Thr
Pro Ser Leu Pro Thr 220 225 230 231 Pro Pro Thr Arg Glu Pro Lys Lys
Val Arg Val Val Arg Thr
[0154] Since it cannot be excluded that the second glycine residue
of the spacer sequence is functionally equivalent to Gly207, the
position of the epitope bound by AT120 must lie between Gly207 and
Thr231.
[0155] In order to localize the epitope within the sequence of
peptide tau5B even more precisely, overlapping 12-mers spanning the
tau5B sequence were synthesized in quadruplicate as defined spots
on Whatman 1 MM paper. The paper was first derivatized with two
residues of .beta.-alanine to provide the paper with a handle on
which peptide synthesis could take place. The first of these
.beta.-alanine residues was coupled as a symmetric anhydride in the
presence of 1 equivalent of dimethylaminopyridine. The second
.beta.-alanine residue was coupled following in situ activation
using 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetra- methyluronium
tetrafluoroborate in the presence of 1-hydroxybenzotriazole. The
coupling of all additional amino acids was performed using the same
chemistry (Knorr et al., 1989). The peptides synthesized and their
recognition by AT120 were as follows:
10 REACTION WITH PEPTIDE SEQUENCE AT120 GGSRSRTPSLPT (SEQ ID NO 8)
- GSRSRTPSLPTP (SEQ ID NO 9) - SRSRTPSLPTPP (SEQ ID NO 10) -
RSRTPSLPTPPT (SEQ ID NO 11) - SRTPSLPTPPTR (SEQ ID NO 12) -
RTPSLPTPPTRE (SEQ ID NO 13) - TPSLPTPPTREP (SEQ ID NO 14) -
PSLPTPPTREPK (SEQ ID NO 15) +++ SLPTPPTREPKK (SEQ ID NO 16) +++
LPTPPTREPKKV (SEQ ID NO 17) +++ PTPPTREPKKVA (SEQ ID NO 18) +++
TPPTREPKKVAV (SEQ ID NO 19) +++ PPTREPKKVAVV (SEQ ID NO 20) +++
PTREPKKVAVVR (SEQ ID NO 21) - TREPKKVAVVRT (SEQ ID NO 22) -
[0156] From this experiment, it is evident that the epitope
recognized by AT120 is a linear sequence comprised of amino acids
Pro218 to Lys224 as specified in SEQ ID NO 4.
References
[0157] Barton A, Harrison P, Najlerahim A, Heffernan J, McDonald B,
Robison J, Davies D, Harrison W, Mitra P, Hardy J, Pearson R,
(1990) Increased tau messenger RNA in Alzheimer's disease
hippocampus. Am J Pathol, 137:497-502.
[0158] Baudier J, Cole R (1987) Phosphorylation of Tau proteins to
a state like that in Alzheimer's brain is catalyzed by a
calcium/calmodulin-depen- dent kinase and modulated by
phospholipids. J Biol Chem 262:17577-17583.
[0159] Biernat J, Milandelkow M, Schoter C, Lichtenberg-Kraag B,
Steiner B, Berling B, Meyer H, Mercken M, Vandermeeren M, Mandelkow
E, The switch of tau protein to an Alzheimer-like state includes
the phosphorylation of two serine-proline motifs upstream of the
microtubule binding region. EMBO J, 1992, 11:1593-1597.
[0160] Binder L, Frankfurter A, Rebhun L (1985) The distrubution of
Tau in the mammalian central nervous system. J Cell Biol
101:1371-1378.
[0161] Bobrow M, Harris T, Shaughnessy K, Litt G (1989) Catalyzed
reporter deposition, a novel method of signal amplification.
Application to immunoassays. J Immunol Meth 125:279-285.
[0162] Br M, Karsenti E (1990) Effects of brain
microtubule-associated proteins on microtubule dynamics and the
nucleating activity of centrosomes. Cell Motil Cytoskeleton
15:88-98.
[0163] Brion J, Couck A, Passareiro E, Flament-Durand J (1985)
Neurofibrillary tangles of Alzheimer's disease: an
immunohistochemical study. J Submicrosc Cytol 17:89-96.
[0164] Casadaban M, Cohen S (1980), Analysis of gene control
signals by DNA fusion and cloning in Eschericia coli. J Mol Biol,
138:179-207.
[0165] De Bont H, Van Boom J, Liskamp R (1990) Automatic synthesis
of phosphopeptides by phosphorylation on the solid phase.
Tetrahedron Lett 31:2497-2500.
[0166] Delacourte A, Vermersch P (1992) Abnormally phosphorylated
tau proteins in Alzheimer's disease: a diagnostic test ?
Breakthroughs in Alzheimer's disease, 5-6 March, London, p.
1-4.
[0167] Delacourte A, Flament S, Dibe E, Hublau P, Sablonniere B,
Hemon B, Sherrer V, Defossez A (1990) Pathological proteins Tau64
and 69 are specifically expressed in the somatodendritic domain of
the degenerating cortical neurons during Alzheimer's disease. Acta
Neuropathol 80:111-117.
[0168] Flament S, Delacourte A, Hemon B, Defossez A (1989)
Characterization of two pathological Tau protein variants in
Alzheimer brain cortices. J Neurol Sci 92:133-141.
[0169] Flament S, Delacourte A (1990) Tau Marker? Nature
346:6279.
[0170] Flament S, Delacourte A, Mann D (1990) Phosphorylation of
tau proteins: a major event during the process of neurofibrillary
degeneration. A comparitive study between Alzheimer's disease and
Down's syndrome. Brain Res 516:15-19.
[0171] Ghanbari H, Kozuk T, Miller B, Riesing S (1990) A sandwich
enzyme immunoassay for detecting and measuring Alzheimer's
disease-associated proteins in human brain tissue. J Clin
Laboratory Anal 4:189-192.
[0172] Goedert M, Wishik C, Crowther R, Walker J, Klug A, Cloning
and sequencing of the cDNA encoding a core protein of the paired
helical filament of Alzheimer disease: identification as the
microtubuli-associated protein tau. Proc Natl Acad Sci USA, 1988,
85, 4051-4055
[0173] Goedert M, Spillantini M, Jakes R, Rutherford D, Crowther R
(1989) Multiple isoforms of human microtubule-associated protein
tau: sequences and localization in neurofibrillary tangles of
Alzheimer's disease. Neuron 3:519-526.
[0174] Goedert M, Spillantini M, Jakes R (1991) Localization of the
Alz-50 epitope in recombinant human microtubule-associated protein
tau. Neurosci Lett. 126:149-154.
[0175] Goedert M, Cohen E, Jakes R, Cohen P (1992) p42 Map kinase
phosphorylation sites in microtubule-associated protein tau one
dephosphorylated by protein phosphatase 2A1: implications for
Alzheimer's disease. FEBS Lett. 312:95-99.
[0176] Greenberg S, Davies P (1990) A preparation of Alzheimer
paired helical filaments that displays distinct tau proteins by
polyacrylamide gel electrophoresis. Proc Natl Acad Sci USA
87:5827-5831.
[0177] Harrington C, Edwards P, Wischik C (1990) Competitive ELISA
for the measurement of tau protein in Alzheimer's disease. J
Immunol Methods 134:261-271.
[0178] Himmler A (1989) Structure of the bovine Tau gene:
alternatively spliced transcripts generate a protein family. Mol
Cell Biol 9:1389-1396.
[0179] Hsu S, Raine L, Fanger H (1981) Use of
avidin-biotin-peroxidase complex (ABC) in immunoperoxidase
techniques: a comparison between ABC and unlabeled antibody (PAP)
procedures. J Histochem Cytochem 29:577-580.
[0180] Iqbal K, Zaidi T, Thompson C, Merz P, Wisniewski H (1984)
Alzheimer paired helical filaments: bulk isolation, solubility, and
protein composition. Acta Neuropathol 62:167-177.
[0181] Ishiguro K, Ihara Y, Uchida T, Imahori K (1988) A novel
tubulin-dependent protein kinase forming a paired helical filament
epitope on tau. J Biochem 104:319-321.
[0182] Khatoon S, Grundke-Iqbal I, Iqbal K (1992) Brain levels of
microtublule-associated protein tau are elevated in Alzheimer's
disease: a radioimmuno-slot-blot assay for nanograms of the
protein. J Neurochem 59:750-753.
[0183] Knorr R, Trzeciak A, Bannwarth W, Gillessen D (1989) New
coupling reagents in peptide chemistry. Tetrahedron Letters
30:1927-1930.
[0184] Kosik K, Orecchio L, Binder L, Trojanowski J, Lee V, Lee G
(1988) Epitopes that span the Tau molecule are shared with paired
helical filaments. Neuron 1:817-825.
[0185] Kosik K, Candall J, Mufson E, Neve R (1989) Tau in situ
hybridization in normal and Alzheimer brain: A predominant
localization in the neuronal somatodendritic compartment. Ann
Neurol 26:352-361.
[0186] Ksiezak-Reding H, Dickson D, Davies P, Yen S (1987)
Recognition of tau epitopes by anti-neurofilament antibodies that
bind to Alzheimer neurofibrillary tangles. Proc Natl Acad Sci USA
84:3410-3414.
[0187] Ksiezak-Reding H, Chien C, Lee V, Yen S (1990) Mapping of
the Alz50 epitope in microtubule-associated proteins tau. J
Neurosci Res 25:412-419.
[0188] Kohler G, Milstein C (1975) Continuous cultures of fused
cells secreting antibody of predefined specificity. Nature
256:495-497.
[0189] Laemmli U (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-685.
[0190] Lee V, Balin B, Otvos L, Trojanowski J (1991) A68: a major
subunit of paired helical filaments and derivatized forms of normal
tau. Science 251:675-678.
[0191] Lewis S, Wang D, Cowan N (1988) Microtubule-associated
protein MAP2 shares a microtubule binding motif with Tau protein.
Science 242:936-939.
[0192] Lindwall G, Cole R (1984) The purification of tau proteins
and the occurrence of two phosphorylation states of tau in brain. J
Biol Chem 259:12241-12245.
[0193] Mehta P, Thal L, Wisniewski H, Grundke-Iqbal I, Iqbal K
(1985) Paired helical filament antigen in CSF. The Lancet 2:35.
[0194] Mercken M, Vandermeeren M, Lubke U, Six J, Boons J,
Vanmechelen E, Van de Voorde A, Gheuens J (1992a) Affinity
purification of human tau proteins and the construction of a
sensitive sandwich enzyme-linked immunosorbent assay for human tau
detection. J Neurochem 58:548-553.
[0195] Mercken M, Vandermeeren M, Lubke U, Six J, Boons J, Van de
Voorde A, Martin J J, Gheuens J (1992b) Monoclonal antibodies with
selective specificity for Alzheimer Tau are directed against
phosphatase-sensitive epitopes. Acta Neuropathol 84:265-272.
[0196] Nukina N, Kosik K S, Selkoe D (1987) Recognition of
Alzheimer paired helical filaments by monoclonal neurofilament
antibodies is due to crossreaction with tau protein. Proc Natl Acad
Sci USA 84:3415-3419.
[0197] Nukina N, Kosik K, Selkoe D (1988) The monoclonal antibody,
Alz 50, recognizes tau proteins in Alzheimer's disease brain.
Neurosci Lett 87:240-246.
[0198] Papasozomenos S, Binder L (1987) Phosphorylation determines
two distinct species of tau in the central nervous system. Cell
Motility Cytoskeleton 8:210-226.
[0199] Sternberger L A, Hardy P H, Cuculis P H, Meyer H G (1970)
The labeled antibody enzyme method of immunohistochemistry:
preparation and properties of soluble antigen-antibody complex
(horseradish peroxidase-antihorseradish peroxidase) and its use in
identification of spirochetes. J Histochem Cytochem 18:315-333.
[0200] Steiner B, Mandelkow E, Biernat J, Gustke N, Meyer H,
Schmidt B, Mieskes G, Soling H, Drechsel D, Kirschner M, Goedert M,
Mandelkow E (1990) Phophorylation of microtubule-associated protein
tau: identification of the site for Ca.sup.2+-calmodulin dependent
kinase and relationship with tau phosphorylation in Alzheimer
tangles. The EMBO J 9:3539-3544.
[0201] Towbin H, Staehelin T, Gordon J (1979) Electrophoretic
transfer of proteins form polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc Natl Acad Sci USA
76:4350-4354.
[0202] Vallee R (1982) A taxol-dependent procedure for the
isolation of microtubules and microtubule-associated proteins
(MAPs). J Cell Biol 92:435-442.
[0203] Weingarten M, Lockwood A, Hwo S, Kirschner M (1975) A
protein factor essential for microtubule assembly. Proc Natl Acad
Sci USA 72: 1858-1862.
[0204] Wischik C, Novak M, Edwards P, Klug A, Tichelaar W, Crowther
R (1988) Structural characterization of the core of the paired
helical filament of Alzheimer disease. Proc Natl Acad Sci USA
85:4884-4888.
[0205] Wolozin B, Davies P (1987) Alzheimer-related neuronal
protein A68: specificity and distribution. Ann Neurol 22:521-526.
Sequence CWU 1
1
* * * * *