U.S. patent application number 10/641416 was filed with the patent office on 2004-02-26 for monoclonal antibodies specific for phf-tau, hybridomas secreting them, antigen recognition by these antibodies and their applications.
This patent application is currently assigned to Innogenetics N.V.. Invention is credited to Vandermeeren, Marc, Vanmechelen, Eugeen, Voorde, Andre Van De.
Application Number | 20040038430 10/641416 |
Document ID | / |
Family ID | 8214780 |
Filed Date | 2004-02-26 |
United States Patent
Application |
20040038430 |
Kind Code |
A1 |
Vandermeeren, Marc ; et
al. |
February 26, 2004 |
Monoclonal antibodies specific for PHF-TAU, hybridomas secreting
them, antigen recognition by these antibodies and their
applications
Abstract
The present invention relates more particularly to a monoclonal
antibody which forms an immunological complex with a phosphorylated
epitope of an antigen belonging to abnormally phosphorylated tau
(PHF-tau) residing in the region spanning positions (143-254), and
with said monoclonal antibody being characterized by the fact that
it is capable of specifically detecting abnormally phosphorylated
tau protein (PHF-tau) in cerebrospinal fluid (CSF).
Inventors: |
Vandermeeren, Marc; (Geel,
BE) ; Vanmechelen, Eugeen; (Nazareth, BE) ;
Voorde, Andre Van De; (Lokeren, BE) |
Correspondence
Address: |
Lawrence J. Crain
Greer, Burns & Crain, Ltd.
Suite 2500
300 South Wacker Drive
Chicago
IL
60606
US
|
Assignee: |
Innogenetics N.V.
|
Family ID: |
8214780 |
Appl. No.: |
10/641416 |
Filed: |
August 14, 2003 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10641416 |
Aug 14, 2003 |
|
|
|
10247853 |
Sep 20, 2002 |
|
|
|
10247853 |
Sep 20, 2002 |
|
|
|
09432285 |
Nov 2, 1999 |
|
|
|
6500674 |
|
|
|
|
09432285 |
Nov 2, 1999 |
|
|
|
08666360 |
Jun 20, 1996 |
|
|
|
6008024 |
|
|
|
|
Current U.S.
Class: |
436/518 ;
530/388.1 |
Current CPC
Class: |
C07K 14/4711 20130101;
C07K 16/18 20130101 |
Class at
Publication: |
436/518 ;
530/388.1 |
International
Class: |
G01N 033/543; C07K
016/18 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 1993 |
EP |
93 403 133.7 |
Claims
1. A monoclonal antibody which forms an immunological complex with
a phosphorylated epitope of an antigen belonging to abnormally
phosphorylated tau protein (phf-tau) residing in the region
spanning positions 143-254 with the following amino acid
sequence:
5 143 150 (SEQ ID NO 1) NH.sub.2- Lys Gly Ala Asp Gly Lys Thr Lys
Ile Ala Thr Pro Arg 160 170 Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln
Ala Asn Ala Thr Arg 180 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr
Pro Pro Ser Ser 190 200 Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly
Tyr Ser Ser Pro 210 Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr
Pro Ser Leu 220 230 Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala
Val Val Arg 240 Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu
Gln Thr 250 Ala Pro Val Pro Met Pro Asp Leu Lys COOH
with said monoclonal antibody being characterized by the the fact
that it is capable of specifically detecting abnormally
phosphorylated tau protein (PHF-tau) in cerebrospinal fluid
(CSF).
2. A monoclonal antibody according to claim 1, characterized by the
fact it forms an immunological complex: either with a
phosphorylated epitope located within the sequence defined in claim
1 (SEQ ID NO 1), or with any other phosphorylated peptide capable
of forming an immunological complex with a monoclonal antibody,
which itself is capable of forming a complex with a phosphorylated
epitope located within the tau protein region as defined in claim 1
(SEQ ID NO 1).
3. A monoclonal antibody AT180 according to any of claims 1 or 2
secreted by the hybridoma deposited at ECACC on Dec. 22, 1992 under
No. 92122204.
4. A monoclonal antibody AT270 according to any of claims 1 or 2
secreted by the hybridoma deposited at ECACC on Jul. 7, 1993 under
No. 93070774.
5. A hybridoma, which secretes a monoclonal antibody according to
anyone of claims 1 to 4, more particularly the hybridoma deposited
at ECACC on Dec. 22, 1992 under No. 92122204, or the monoclonal
antibody deposited at ECACC on Jul. 7, 1993 under No. 93070774.
6. A peptide containing from 6 to 100 amino acids, and with said
peptide specifically forming an immunological complex with any of
the monoclonal antibodies, according to anyone of claims 1 to 4,
with said peptide being in the phosphorylated form, and with the
sequence of said peptide comprising, or consisting of
phosphorylated parts of the sequence as shown in SEQ ID NO 1, or,
with the sequence of said peptide comprising or consisting of the
sequence of the peptides capable of forming an immunological
complex with a monoclonal antibody according to anyone of claims 1
to 4.
7. A peptide according to claim 6, characterized in that its amino
acid sequence comprises or consists of its amino acid sequence the
following sequence: Val-Arg-Thr-Pro-Pro (SEQ ID NO 2), with said
Thr being phosphorylated and with said peptide being characterized
in that it specifically forms an immunological complex with the
monoclonal antibody according to claim 3.
8. A peptide according to claim 6 consisting of or comprising in
its amino acid sequence the following sequence: Pro-Lys-Thr-Pro-Pro
(SEQ ID NO 3), with said Thr being phosphorylated and with said
peptide being characterized in that it specifically forms an
immunological complex with the monoclonal antibody according to
claim 4.
9. A phosphorylated peptide according to any of claims 6 to 8,
which is capable of generating a monoclonal antibody according to
anyone of claims 1 to 4 upon immunization.
10. A process for obtaining and isolating a hybridoma according to
claim 5, secreting a monoclonal antibody according to anyone of
claims 1 to 4, characterized in that it involves: starting from the
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, preferably abnormally phosphorylated tau
(PHF-tau), or a phosphorylated peptide according to claims 6 to 9,
recognized by a monoclonal antibody as defined in any of claims 1
to 4; fusing said immunized cells with myeloma cells under
hybridoma-forming conditions; and, selecting those hybridomas which
secrete monoclonal antibodies which specifically recognize a
phosphorylated epitope of abnormally phosphorylated tau (PHF-tau)
in cerebrospinal fluid (CSF).
11. Monoclonal antibodies according to any of claims 1 to 4 such as
obtained by a process according to claim 10, characterized in that
it involves: starting from the spleen cells of a mouse previously
immunized in vitro with abnormally phosphorylated tau (PHF-tau), or
a phosphorylated tau peptide according to any of claims 6 to 9,
recognized by a monoclonal antibody according to any of claims 1 to
4; fusing said immunized spleen cells with myeloma cells under
hybridoma-forming conditions, selecting those hybridomas which
secrete monoclonal antibodies which specifically recognize PHF-tau
and which are capable of specfically detecting PHF-tau in CSF.
12. A process for producing monoclonal antibodies according to
anyone of claims 1 to 4 which involves: culturing the selected
hybridomas according to claim 5 or the hybridomas such as obtained
through the process of claim 9, in an appropriate medium culture;
and recovering the monoclonal antibodies excreted by said selected
hybridomas; or alternatively: implanting the selected hybridomas of
claim 5, or the hybridomas obtained through the process of claim 9,
into the peritoneum of a mouse and, when ascites has been produced
by the animal, recovering the monoclonal antibodies then formed
from said ascites.
13. Process for the post-mortem detection or diagnosis of
brain/neurological disease involving PHF-tau protein, such as
Alzheimer's disease, which comprises at least the following steps:
contacting a monoclonal antibody according to anyone of claims 1 to
4, 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 abnormally phosphorylated tau
(PHF-tau) under conditions suitable for producing an
antigen-antibody complex; and detecting the immunological binding
of said antibody to said brain homogenate, and possibly separating
the antigen from said complex and recovering the antigen sought in
a purified form.
14. Process for the detection or diagnosis in vitro of brain
disease involving abnormally phosphorylated tau protein, such as
Alzheimer's disease, which includes: bringing a sample of CSF,
preferably unconcentrated CSF, or of serum from a patient suspected
of suffering from a neurological disorder involving PHF-tau, more
particularly Alzheimer's disease, or proteins or polypeptides
extracted therefrom, into contact under in vitro conditions with a
monoclonal antibody according to anyone of claims 1 to 4, with said
conditions suitable for producing an antigen-antibody complex; and,
detecting the immunological binding of said antibody to said sample
of cerebrospinal fluid, or of serum, or extract thereof.
15. Kit for the diagnosis in vitro of one of the following
diseases: Alzheimer's disease, Down's syndrome, Pick's disease and
other neurological disorders in which abnormally phosphorylated tau
protein or paired helical filaments are implicated, characterized
in that the kit comprises: at least one monoclonal antibody
according to anyone of claims 1 to 4, deposited on a microplate; a
preparation containing the sample (CSF, serum, or proteins
extracted therefrom) to be diagnosed in vitro, a second antibody
which can be a monoclonal antibody recognizing an epitope of
abnormally phosphorylated tau protein, or an epitope of any
phosphorylated tau peptide carrying an epitope of PHF-tau, with
said epitopes being different from the one of the invention, or
which can be a polyclonal antibody recognizing abnormally
phosphorylated tau or a polyclonal antibody recognizing a peptide
carrying an epitope of PHF-tau, with said polyclonal antibody being
capable of forming an immunological complex with epitopes which are
different from the epitope of the invention, with said polyclonal
antibody being preferably purified by immunoaffinity chromatography
using immobilized tau protein; 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 carrying an epitope of PHF-tau comprised in the
region as defined in claim 1 (SEQ ID NO 1) for standard purposes,
or for competition purposes with respect to the antigen which is
sought.
16. Method or kit according to any of claims 13 to 15, for the in
vitro detection or diagnosis of brain/neurological disease
involving abnormally phosphorylated tau protein, such as
Alzheimer's disease, comprising at least one of the following
combinations of monoclonal antibodies: a mixture comprising at
least two monoclonal antibodies according to any of claims 1 to 4.
a mixture comprising at least one monoclonal antibody according to
any of claims 1 to 4 as well as at least one other monoclonal
antibody recognizing tau or PHF-tau.
17. Method or kit according to claim 16, further characterized in
that it involves a sandwich detection format comprising coating and
detecting antibodies, with said coating antbodies consisting of at
least one antibody according to any one of claims 1 to 4, and with
said detecting antibodies consisting of at least one monoclonal
antibody capable of detecting both normal and/or abnormally
phosphorylated human tau of which the epitope is different from the
epitope of any of the monoclonal antibodies according to any of
claims 1 to 4.
Description
[0001] The invention relates to new monoclonal antibodies specific
for PHF-tau, to the hybridomas secreting these monoclonal
antibodies, and to the antigen recognition pattern of these
monoclonal antibodies and their applications. The invention also
relates to a process for diagnosing brain diseases involving
monoclonal antibodies of the invention, more particularly in
cerebrospinal fluid (CSF) samples. The invention also relates to a
region of the tau molecule modifiable in vivo by the process of
phosphorylation, which is found to be associated with Alzheimer's
disease or with other types of dementia and which is specifically
recognized by the monoclonal antibodies of the invention.
[0002] Alzheimer's disease (AD) is the most common form of
adult-onset dementia. At present, no reliable biochemical test is
available for antemortem diagnosis of AD. The disease is therefore
diagnosed clinically on the basis of exclusion of other forms of
dementia. The diagnosis can be confirmed neuropathologically by the
demonstration of large amounts of neuritic (senile) plaques and
neurofibrillary tangles (NFT) in particular brain regions (McKhann
et al, 1984).
[0003] Neurofibrillary tangles consist of paired helical filaments
(PHFs). Immunocytochemical evidence suggests that the
microtubule-associated protein tau is a major protein component of
PHF and NFT (Brion et al., 1985b; Delacourte and Defossez, 1986;
Grundke-Iqbal et al., 1986; Kosik et al., 1986; Wood et al., 1986).
Definite proof that the tubulin-binding domain of tau is tightly
associated with the core of PHFs was obtained via amino acid
sequencing (Kondo et al., 1988). Nevertheless it has been suggested
that tau peptides may represent only a small portion of the major
component of PHF (Wischik et al., 1988).
[0004] Tau protein exists in different isoforms, of which 4 to 6
are found in adult brain but only 1 isoform is detected in fetal
brain. The diversity of the isoforms is generated from a single
gene on human chromosome 17 by alternative mRNA splicing (Andreadis
et al., 1992). The most striking feature of tau protein, as deduced
from molecular cloning, is a stretch of 31 or 32 amino acids,
occurring in the carboxy-terminal part of the molecule, which can
be repeated either 3 or 4 times. Additional diversity is generated
through 29 or 58 amino acid-long insertions in the
NH.sub.2-terminal part of tau molecules (Goedert et al., 1989). For
simplicity, all numbering in this patent application refers to the
tau variant htau40 containing all exons (441 amino acids long)
according to Goedert et al (1989).
[0005] Under normal circumstances tau promotes microtubule assembly
and stability in the axonal compartment of neurons. The
microtubule-binding domain in tau is localized in the repeat region
of tau (255-381) (Lewis et al, 1990) and is modulated by adjacent
regions: the carboxyterminal tail (382-414) and the proline-rich
region (143-254) (Drubin & Kirschner, 1991). Stability and
bundling of the microtubules is mediated by a short hydrophobic
zipper in the carboxyterminal tail of tau (Lewis et al, 1989). Both
assembly and stability are regulated by alternative mRNA splicing
and phosphorylation.
[0006] In normal circumstances adult brain contains 2 a 3 mol
phosphate per mole of tau (Selden and Pollard, 1983; Ksiezak-Reding
et al, 1992) present amongst others at serine 404 (Poulter et al,
1993), while other results demonstrate that phosphorylation of
different sites in normal tau follows different developmental
profiles (Lee et al, 1991; Bramblett et al, 1993; Goedert et al,
1993a). Abnormal tau variants of 60, 64 and 68 kDa have been
detected exclusively in brain areas showing neurofibrillary changes
and senile plaques (Delacourte et al. 1990). The abnormal
electrophoretical behavior of tau is due to phosphorylation since
alkaline phosphatase treatment of these tau molecules changes their
molecular mass to that of normal tau (Goedert et al., 1992; Flament
et al., 1990b, Greenberg & Davies, 1990). Currently abnormal
phosphorylation sites have been detected in PHF-tau at positions
46, 231, 235, 263 and 396 (Iqbal et al., 1989; Lee et al., 1991;
Hasegawa et al., 1992). In four of these sites, the phosphorylated
residu is followed by a proline residu, indicating that a
proline-directed kinase is involved in some of the abnormal
phosphorylations of tau. In addition to these sites ten others are
present in htau40, two of which are also abnormally phosphorylated,
as indicated by antibody reactivity (Mab tau2: Watanabe et al.,
1992; Mab AT8: Biernat et al., 1992, Goedert et al., 1993).
[0007] The abnormal phosphorylation of tau in Alzheimer's disease
is due to a shift in the phosphatase/kinase equilibrium. In vitro
several kinases can phosphorylate tau: cdc2-kinases (Vulliet et al,
1992; Ledesma et al, 1992), MAP kinases (Drewes et al, 1992, Roder
and Ingram, 1991), glycogen synthase kinases (Mandelkow et al,
1992) and TPKI and TPKII (Ishiguro et al, 1992). The phosphatases
are less well studied in Alzheimer's disease and sofar only one
phosphatase was able in vitro to dephosphorylate the abnormally
phosphorylated sites, namely protein phosphatase 2A.sub.1 (Goedert
et al, 1992).
[0008] Sofar, the detection of PHF-tau in brain extracts, either
via antibodies (Mab Alz50: Ghanbari et al., 1990; Mab Ab423:
Harrington et al., 1991), or via the change in molecular weight
(Flament et al., 1990, Delacourte et al., 1993), or else by
functional assay (Bramblett et al. 1992) has been very useful to
discriminate dementia with altered cytoskeletal properties from
normal aged subjects or from patients with other types of dementia.
Nevertheless the detection of PHF-tau in CSF remained impossible,
even using antibodies directed at one of the abnormally
phosphorylated sites such as serine 202 (Goedert et al., 1993).
This can be ascribed to one or more of the following reasons: 1)
the low concentration of PHF-tau in CSF, 2) non-evenly use of
phosphorylation sites among all the potential phosphorylation
sites, 3) differences in phosphatase sensitivity of these sites,
and, 4) too low affinity constant of the antibodies used.
[0009] The aim of the present invention is therefore to provide
monoclonal antibodies which allow the reliable and sensitive
detection of abnormally phosphorylated tau present in cerebrospinal
fluid.
[0010] The invention also aims at providing the hybridomas which
secrete the above-said monoclonal antibodies.
[0011] The invention furthermore aims at providing the epitopes of
the abnormally phosphorylated tau protein present in brain
homogenates or in body fluids such as cerebrospinal fluid, which
are recognized by said monoclonal antibodies.
[0012] Finally, the invention aims at providing a process for the
detection or diagnosis in vitro of brain diseases involving
abnormally phosphorylated tau proteins.
[0013] The present invention relates more particularly to a
monoclonal antibody which forms an immunological complex with a
phosphorylated epitope of an antigen belonging to abnormally
phosphorylated tau (PHF-tau) residing in the region spanning
positions 143-254 with the following amino acid sequence:
1 143 150 (SEQ ID NO 1) NH.sub.2- Lys Gly Ala Asp Gly Lys Thr Lys
Ile Ala Thr Pro Arg 160 170 Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln
Ala Asn Ala Thr Arg 180 Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr
Pro Pro Ser Ser 190 200 Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly
Tyr Ser Ser Pro 210 Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr
Pro Ser Leu 220 230 Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val Ala
Val Val Arg 240 Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu
Gln Thr 250 Ala Pro Val Pro Met Pro Asp Leu Lys COOH
[0014] and with said monoclonal antibody being characterized by the
the fact that it is capable of specifically detecting abnormally
phosphorylated tau protein (PHF-tau) in cerebrospinal fluid
(CSF).
[0015] The monoclonal antibodies of the invention were selected
from a range of monoclonal antibodies obtained by direct
immunization with PHF-tau, extracted from human brain tissue
derived from Alzheimer patients. More particularly the monoclonal
antibodies of the invention are characterized by the fact that they
specifically bind to naturally occuring abnormally phosphorylated
tau. Further analysis of their epitopes showed that the monoclonal
antibodies of the invention are directed at phosphorylated epitopes
confined to a particular region of the tau molecule, namely the
region between 143 and 254 including several potential
phosphorylation sites such as T153 and S235 used by the SP and TP
directed kinases. The monoclonal antibodies of the invention are
further characterized by the fact that they recognize epitopes
which are different from the epitope of the monoclonal antibody AT8
as defined in Goedert et al. (1993) and upon comparison with the
AT8 antibodies allow the detection of PHF-tau in CSF. They
recognize preferentially PHF-tau either on brain sections,
immunoblots or in ELISA and they are surprisingly able to detect
PHF-tau in CSF, either alone or in combination with other PHF-tau
specific antibodies.
[0016] In conclusion, the monoclonal antibodies of the invention
are characterized in that they specifically bind to naturally
occuring abnormally phosphorylated tau of which the phosphorylation
state is confined to a particular region of the tau molecules as
specified above, or bind to recombinant non-phosphorylated tau
after treatment with proline-directed kinases, which can provoke
the phosphorylation of amongst others Ser-Pro or Thr-Pro sites in
the region as specified. Proline-directed kinases such as MAP
kinases (Sturgill et al., 1991), cdc2 kinases (Labb et al., 1991)
and glycogen synthase kinases (Vandenheede et al., 1980) can be
purified from various tissues or can be present in brain extracts.
The phosphorylation of tau by these kinases is abolished or greatly
diminished when one or more of the following serines/threonines are
mutated to an amino acid such as Ala: T153, T175, T181. S199, S202,
T205, T212, T217, T231, or S235. Consequently, the epitope of these
antibodies can be characterized via such mutant tau, or via
non-phosphorylated tau such as procaryotically expressed
recombinant tau and their phosphorylated homologues, or via
synthetic peptides having the same amino acid sequence as parts of
the region specified above of the human tau 40 protein and with
said peptides being capable of being phosphorylated by said kinases
or being incapable of being phosphorylated upon synthesis of the
peptides. The epitopes of the present invention are thus defined as
the proline rich-region of tau between position 143 and 254 and
which can be abnormally phosphorylated at threonine 153 (T153),
T175, T181, S199, S202, T205, T212, T217, T231 and S235 or a
combination of these sites included in the epitope of these
antibodies, further referred to as a "PHF-tau epitopes".
[0017] The expression "specifically detecting abnormally
phosphorylated tau protein" corresponds to the fact that the
monoclonal antibodies of the invention detect abnormally
phosphorylated tau in CSF without cross-reacting with normal tau
present in CSF.
[0018] The expression "form an immunologically complex with" means
that the monoclonal antibody of the invention binds to the
above-said antigen under conditions as mentioned in one of the
following techniques:
[0019] Light Immunomicroscopy
[0020] Brain tissue samples, of e.g. Alzheimer patients 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.
[0021] Immunoelectron Microscopy in Tissue Sections
[0022] Brain tissue samples e.g. obtained from Alzheimer patients
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., 1985a).
[0023] Immunoblotting Procedures
[0024] For immunoblotting, fractions enriched in PHF-tau are
prepared as described (Greenberg and Davies, 1990). Typically,
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.
[0025] 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).
[0026] 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.
[0027] 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.
[0028] 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.
[0029] The present invention relates more particularly to a
monoclonal antibody as defined above, characterized by the fact it
forms an immunological complex:
[0030] either with a phosphorylated epitope located within the
sequence defined above (SEQ ID NO 1),
[0031] or with any other phosphorylated peptide capable of forming
an immunological complex with a monoclonal antibody, which itself
is capable of forming a complex with a phosphorylated epitope
located in the human tau protein region as shown in SEQ ID NO
1.
[0032] Preferred monoclonal antibodies of the invention, AT180 and
AT270, are produced by hybridomas 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, Porton Down, GB-Salisbury,
Wiltshire SP4 OJG), on Dec. 22, 1992, under No 92122204 or on July
7, 1993 under No 93070774. The above-mentioned monoclonal
antibodies are obtained by a process involving obtention and
isolation of hybridomas which secrete these monoclonal
antibodies.
[0033] The preferred monoclonal antibodies of the invention allow
the detection of at least 1, 5, 10 or 20 pg/ml phosphorylated tau
as determined in an ELISA using these monoclonal antibodies in the
coating phase and incubating them with CSF spiked with different
amounts of phosphorylated and non-phosphorylated tau without
amplification. Phosphorylated tau is prepared by incubating
recombinant non-phosphorylated tau with a rat brain extract capable
of phosphorylation of Ser and Thr amino acids at positions
corresponding to sites of abnormal phosphorylation of tau (Goedert
et al., 1993), as found in tau extracts of brain tissue derived of
patients having died of Alzheimer's disease.
[0034] A process for obtaining the hybridomas of the invention
involves:
[0035] starting from spleen cells of an animal, e.g. mouse or rat,
previously immunized in vivo or from spleen cells of such animals
previously immunized in vitro with an antigen being preferably
abnormally phosphorylated tau (PHF-tau), or a phosphorylated human
tau peptide or immunoaffinity purified abnormally phosphorylated
tau, as disclosed below, recognized by the monoclonal antibodies of
the invention;
[0036] fusing said immunized cells with myeloma cells under
hybridoma-forming conditions; and
[0037] selecting those hybridomas which secrete the monoclonal
antibodies which are capable of specifically recognizing a
phosphorylated epitope of abnormally phosphorylated tau (PHF-tau)
in cerebrospinal fluid (CSF).
[0038] The phosphorylated human tau peptide refers to a peptide
comprising in its amino acid sequence a phosphorylated sequence
comprised in the region spanning amino acids 143 to 254 of human
tau and with said peptide being characterized by the fact that it
can form an immunological complex with the antibodies of the
invention.
[0039] 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 abnormally phosphorylated tau protein is implicated; this
antigen provokes an immunological reaction with the monoclonal
antibody of the invention.
[0040] More particularly, the present invention relates also to
monoclonal antibodies as defined above, obtained by a process such
as as defined above, characterized in that it involves:
[0041] starting from the spleen cells of a mouse previously
immunized with abnormally phosphorylated tau (PHF-tau) extracted
and purified from a human brain sample of a patient suffering from
Alzheimer's disease (as disclosed in the examples section), or a
phosphorylated human tau peptide, or immunoaffinity-purified
abnormally phosphorylated tau capable of reacting with the
monoclonal antibodies of the invention,
[0042] fusing said immunized cells with myeloma cells under
hybridoma-forming conditions,
[0043] selecting those hybridomas which secrete monoclonal
antibodies which specifically recognize PHF-tau and which are
capable of specifically detecting PHF-tau in CSF (as illustrated in
detail in the examples section).
[0044] A process for producing the monoclonal antibodies of the
invention involves:
[0045] culturing the selected hybridomas as indicated above in an
appropriate culture medium; and
[0046] recovering the monoclonal antibodies secreted by said
selected hybridoma; or alternatively
[0047] implanting the selected hybridoma into the peritoneum of a
mouse and, when ascites has been produced in the animal;
[0048] recovering the monoclonal antibodies then formed from said
ascites.
[0049] 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.
[0050] The invention also relates to a peptide capable of forming
an immunological complex with any of the monoclonal antibodies of
the invention, with said peptide being in the phosphorylated form,
and,
[0051] with the sequence of said peptide comprising, or consisting
of phosphorylated parts of the sequence as shown in SEQ ID NO 1,
or,
[0052] with the sequence of said peptide comprising, or consisting
of the sequence of any peptide being capable of forming an
immunological complex with anyone of the monoclonal antibodies
according to the invention.
[0053] Said phosphorylated peptides are preferably from 6 to 100
amino acids long. The peptides according to this embodiment of the
invention can be prepared by classical chemical synthesis. The
synthesis may be carried out in homogenous solution or in solid
phase according to any of the techniques well known in the art.
[0054] Phosphorylated peptides are prepared according to any
technique known in the art, (f.i. de Bont et al., 1990a; de Bont et
al., 1990b; Perich, 1991; Otvos et al., 1989)
[0055] According to a preferred embodiment, the present invention
relates to a phospohorylated peptide as defined above consisting of
or comprising in its amino acid sequence the following sequence:
Val-Arg-Thr-Pro-Pro (amino acid 229-233; human tau 40 numbering,
SEQ ID NO 2), with said Thr(231) being phosphorylated and with said
peptide being characterized in that it is able to form an
immunological complex with the monoclonal antibody AT180 produced
by the hybridoma deposited at the ECACC on Dec. 22, 1992 under No.
92122204.
[0056] According to another preferred embodiment, the present
invention relates also to a phosphorylated peptide as defined above
consisting of or comprising in its amino acid sequence the
following sequence:
[0057] Pro-Lys-Thr-Pro-Pro (amino acid 179-183; human tau 40
numbering; SEQ ID NO 3), with said Thr(181) being phosphorylated
and with said peptide being characterized in that it is able to
form an immunological complex with the monoclonal antibody AT270
produced by the hybridoma deposited at the ECACC on Jul. 7, 1993
under No. 93070774.
[0058] According to yet another embodiment, the present invention
relates to a phosphorylated peptide as defined above, which is
capable of generating a monoclonal antibody according to anyone of
claims 1 to 4 upon immunization.
[0059] The peptides used for immunization are preferentially in the
form in which they are joined to a carrier molecule in order to
achieve a good immunogenic response. Such carrier molecules are
well known in the art and are coupled to the peptide via linker
groups, which are also comprised in the art.
[0060] The invention also relates to a process for the post-mortem
detection or diagnosis in vitro of a brain/neurological disease
involving PHF-tau, such as Alzheimer's disease, which comprises at
least the following steps:
[0061] contacting a monoclonal antibody of the invention 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 abnormally phosphorylated tau protein (PHF-tau)
under conditions suitable for producing an antigen-antibody
complex;
[0062] detecting the immunological binding of said antibody to said
brain homogenate, and possibly separating said complex and
recovering the antigen sought in a purified form.
[0063] Recovering the antigen sought may be done by first washing
the immobilized antibody-antigen complex then formed;
[0064] 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;
[0065] recovering the antigen in a purified form.
[0066] The invention relates also to a process for the detection or
diagnosis in vitro of brain/neurological disease involving
abnormally phosphorylated tau protein, such as in Alzheimer's
disease, which includes:
[0067] bringing a sample of CSF, more preferably unconcentrated
CSF, or a sample of serum from a patient suspected of suffering
from brain disease involving PHF-tau, more particularly Alzheimer's
disease, or proteins or polypeptides as a result of an extraction
procedure starting from brain tissues, cerebrospinal fluid or serum
known to those skilled in the art (Ibqal et al., 1984; Greenberg
& Davies, 1990) into contact under in vitro conditions with a
monoclonal antibody of the invention, with said conditions being
suitable for producing an antigen-antibody complex; and,
[0068] detecting the immunological binding of said antibody to said
sample of brain extract, cerebrospinal fluid or serum, or proteins
or polypeptides.
[0069] Advantageously, the monoclonal antibodies of the invention
are in an immobilized state on a suitable support such as a resin.
Alternatively, the present process may be put into practice by
using any other immunoassay format known to the person skilled in
the art.
[0070] The process for the detection of the antigen can then be
carried out for instance as follows:
[0071] bringing together said antigen-antibody complex formed by
the antigen and the antibodies of the invention with:
[0072] a second antibody
[0073] which can be a monoclonal antibody recognizing an epitope of
abnormally phosphorylated tau protein, or an epitope of any
phosphorylated tau peptide carrying an epitope, with said epitopes
being different from the one of the invention, or
[0074] which can be a polyclonal antibody recognizing abnormally
phosphorylated tau or a polyclonal antibody recognizing a peptide
carrying an epitope of PHF-tau, with said polyclonal antibody being
capable of forming an immunological complex with epitopes which are
different from the epitope of the invention, with said polyclonal
antibody being preferably purified by immunoaffinity chromatography
using immobilized tau protein;
[0075] a marker either for specific tagging or coupling with said
second antibody, with said marker being any possible marker known
to the person skilled in the art;
[0076] 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.
[0077] The detection of the immunologically bound monoclonal
antibody can be achieved by conventional technology comprised in
the art. Advantageously, the second antibody itself carries a
marker or a group for direct or indirect coupling with a
marker.
[0078] The monoclonal antibodies of the invention also enable the
diagnosis of Alzheimer's disease (AD) and of any disease involving
the formation of abnormally phosphorylated tau in the region 143 to
254 on the basis of CSF (i.e. to detect modified forms of tau in
CSF). The problem associated herewith is that this antigen is
present in a very low amount in CSF, so the detection assay must be
very sensitive. This sensitivity problem may be further overcome by
(i) using a combination of the monoclonal antibodies of the
invention, or (ii) a combination of a monoclonal antibody of the
invention with any other normal and/or abnormally phosphorylated
tau monoclonal antibodies known in the art and/or (iii) by using a
monoclonal antibody or a combination of monoclonal antibodies of
the invention in combination with an amplification technique such
as the catalyzed reporter deposition amplification technique (CARD,
Bobrow et al., 1989), allowing a PHF-tau specific ELISA with a
higher sensitivity.
[0079] The results obtained with the monoclonal antibodies of the
invention indicate that elevated PHF-tau levels are found in AD,
but may occur also in other neurological diseases where abnormal
phosphorylation of tau occurs in the region of tau comprised by
amino acids 143 to 254.
[0080] According to another embodiment, the present invention
relates to a kit for the diagnosis in vitro of one of the following
diseases: Alzheimer's disease, Down's syndrome, Pick's disease and
other neurological disorders in which abnormally phosphorylated tau
protein or paired helical filaments are implicated, characterized
in that the kit comprises:
[0081] at least one monoclonal antibody of the invention deposited
on a microplate;
[0082] a preparation containing the sample (CSF, serum or the
proteins extracted therefrom) to be diagnosed in vitro,
[0083] a second antibody
[0084] which can be a monoclonal antibody recognizing an epitope of
abnormally phosphorylated tau protein, or an epitope of any
phosphorylated tau peptide carrying an epitope, with said epitopes
being different from the one of the invention, or
[0085] which can be a polyclonal antibody recognizing abnormally
phosphorylated tau or a polyclonal antibody recognizing a peptide
carrying an epitope of PHF-tau, with said polyclonal antibody being
capable of forming an immunological complex with epitopes which are
different from the epitope of the invention, with said polyclonal
antibody being preferably purified by immunoaffinity
chromatography, using immobilized tau protein;
[0086] a marker either for specific tagging or coupling with said
second antibody;
[0087] 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,
[0088] possibly a peptide carrying an epitope of PHF-tau comprised
in the region spanning amino acids 143 to 254 for standard
purposes, or for competition purposes with respect to the antigen
which is sought.
[0089] A preferred embodiment of the present invention for the
detection or diagnosis in vitro of brain/neurological disease
involving abnormally phosphorylated tau protein, such as
Alzheimer's disease relates to a method or a kit as defined above,
which comprises a mixture (combination) of monoclonal antibodies of
the invention, or a combination of at least one monoclonal antibody
of the invention with other antibodies capable of specifically
recognizing a region of PHF-tau residing in the region spanning
positions 143-254 of human tau 40 (SEQ ID NO 1), with said
monoclonal antibodies being preferably chosen from:
[0090] (1) the monoclonal antibody AT180 produced by the hybridoma
deposited at the ECACC on Dec. 22, 1992 under No. 92122204;
[0091] (2) the monoclonal antibody AT270 produced by the hybridoma
deposited at the ECACC on Jul. 7, 1993 under No. 93070774;
[0092] (3) the monoclonal antibody AT8 produced by the hybridoma
deposited at the ECACC on Oct. 8, 1991 under No. 91100806;
[0093] and with said mixture being preferably chosen from the
following list:
[0094] a mixture of monoclonal antibodies comprsing the monoclonal
antibodies (1) and (2),
[0095] a mixture of the monoclonal antibodies comprising the
monoclonal antibodies (1) and (3),
[0096] a mixture of the monoclonal antibodies comprising the
monoclonal antibodies (2) and (3),
[0097] a mixture of the monoclonal antibodies comprising the
monoclonal antibodies (1), (2) and (3);
[0098] with said method or kit being further characterized as
containing or using:
[0099] a preparation containing the sample to be diagnosed in
vitro;
[0100] a second antibody
[0101] which can be a monoclonal antibody recognizing an epitope of
abnormally phosphorylated tau protein, or an epitope of any
phosphorylated tau peptide carrying an epitope, with said epitopes
being different from the one of the invention, or
[0102] which can be a polyclonal antibody recognising abnormally
phosphorylated tau or a polyclonal antibody recognizing a peptide
carrying an epitope of PHF-tau, with said polyclonal antibody being
capable of forming an immunological complex with epitopes which are
different from the epitope of the invention, with said polyclonal
antibody being preferably purified by immunoaffinity chromatography
using immobilized tau protein;
[0103] a marker either for specific tagging or coupling with said
second antibody;
[0104] 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,
[0105] possibly a peptide carrying an epitope of PHF-tau for
standard purposes, or for competition purposes with respect to the
antigen which is sought.
[0106] According to yet another preferred embodiment, the present
invention relates to a method or kit for detecting or diagnosing in
vitro a brain/neurological disease involving abnormally
phosphorylated tau protein, such as Alzheimer's disease, which
involves a sandwich ELISA detection format comprising coating and
detecting antibodies, with said coating antibodies consisting of at
least one monoclonal antibody of the invention, and with said
detecting antibodies consisting of at least one monoclonal antibody
capable of detecting normal and/or abnormally phosphorylated human
tau of which the epitope is different from any of the epitopes of
the monoclonal antibodies of the invention. Such a preferred
sandwich ELISA format is extensively illustrated in the examples
section of the present invention.
TABLE AND FIGURE LEGENDS
[0107] Table 1
[0108] Detection of PHF-tau and normal tau using PHF-tau specific
monoclonal antibodies AT180 and AT270. Microplates coated with
saturating amounts of a monoclonal antibody specifically
recognizing PHF-tau were incubated with CSF spiked with different
amounts of non-phosphorylated or phosphorylated tau, the latter
prepared by incubating recombinant non-phosphorylated tau with a
rat brain extract capable of phosphorylating Ser and Thr amino
acids at positions corresponding to the sites of abnormal
phosphorylation of tau (Goedert et al., 1993). Bound antigen was
detected as described in the examples section.
[0109] Table 2
[0110] CSF samples from AD patients, control patients and patients
suffering from various non-AD neurological disorders were tested in
ELISA using different combinations of capturing antibodies as
described (example III). All values are expressed as mOD units
except for the determination of total tau which was done using the
Innotest htau (Innogenetics, Belgium) and which are expressed in
pg/ml CSF.
[0111] The different experimental conditions used for each set-up
do allow only intra-lane comparison.
[0112] Table 3
[0113] CSF samples of control patients, AD patients and patients
suffering from various non-AD neurological disorders (OND) were
assayed using the Innotest htau (Innogenetics, Belgium). From the
cohorts of AD patients and OND patients those having high total tau
values were selected for further testing using the PHF-tau specific
ELISA in which AT8, AT180 and AT270 were used as capturing
antibodies and AT120 and HT7 as detecting antibodies as described
(Example IV). Results are expressed in pg/ml tau in CSF for total
tau (Innotest htau) and as mOD units for the PHF-tau specific
ELISA.
[0114] FIG. 1
[0115] Detection of PHF-tau and normal tau using monoclonal
antibody AT180. Microplates coated with saturating amounts of
monoclonal antibody AT180 specifically recognizing PHF-tau were
incubated with CSF spiked with different amounts of
non-phosphorylated or phosphorylated tau, the latter prepared by
incubating recombinant non-phosphorylated tau with a rat brain
extract capable of phosphorylating Ser and Thr amino acids at
positions corresponding to the sites of abnormal phosphorylation of
tau (Goedert et al., 1993). Bound antigen was detected as described
in the examples section.
[0116] FIG. 2
[0117] Detection of PHF-tau and normal tau using monoclonal
antibody AT270. Microplates coated with saturating amounts of
monoclonal antibody AT270 specifically recognizing PHF-tau were
incubated with CSF spiked with different amounts of
non-phosphorylated or phosphorylated tau, the latter prepared by
incubating recombinant non-phosphorylated tau with a rat brain
extract capable of phosphorylating Ser and Thr amino acids at
positions corresponding to the sites of abnormal phosphorylation of
tau (Goedert et al., 1993). Bound antigen was detected as described
in the examples section.
[0118] FIG. 3
[0119] Phosphorylation of wild-type and mutated recombinant tau
(expressed from clone human tau 24; Goedert and lakes, 1990) with
the protein kinase activity from rat brain. Immunoblots with
anti-tau antiserum 134 and monoclonal antibodies AT8 and AT180.
Lanes 1. tau 24; 2, tau 24 plus brain extract; 3, T231 A tau 24; 4,
T231 A tau 24 plus brain extract, 5, S235 A tau 24; 6, S235 A tau
24 plus brain extract.
[0120] FIG. 4
[0121] Phosphorylation of wild-type and mutated recombinant tau
(expressed from clone human tau 24) with the protein kinase
activity from rat brain. Immunoblots with anti-tau antiserum 134
and monoclonal antibodies AT8 and AT270. Lanes 1, tau 24; 2, tau 24
plus brain extract; 3, T175 A tau 24; 4, T175 A tau 24 plus brain
extract; 5, T181 A tau 24; 6, T181A tau 24 plus brain extract.
EXAMPLES
Example I
[0122] Preparation of the Monoclonal Antibodies AT180 and AT270
using PHF-tau as Antigen
[0123] 1. Preparation of the Antigen for Immunization
[0124] 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.
[0125] The antigen preparation was evaluated by a 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, followed by
Western blotting using polyclonal rabbit anti-human normal tau
antiserum (Mercken et al., 1992a).
[0126] 2. Immunization Protocol and Fusion Procedure
[0127] Balb/c mice were primed subcutaneously with 100 .mu.g
PHF-tau preparation 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.
[0128] Mouse spleen cells were fused with SP2/0 myeloma cells,
using a modified procedure of Kohler and Milstein (1975), with PEG
4000.
[0129] 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 of continuous growth for
anti-PHF-tau antibody production by means of a sandwich ELISA as
detailed below.
[0130] Hybridoma growth was performed 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.
[0131] 3. Sandwich ELISA for Anti-PHF-tau Antibody Screening
[0132] The screening ELISA used for the detection of anti-PHF-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
covalent immobilization on 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.
[0133] From such a fusion experiment, using the screening procedure
as described in section 3 above, 28 positive cultures (i.e.
anti-PHF-tau monoclonal antibodies secreting cultures) were
retrieved out of a total of 1440 cultures. These positive cultures
were arbitrarily designated AT1 to AT28 (some of these hybridoma
cultures, i.e. AT1 to AT14 are described by Mercken et al., 1992b).
As in this initial screening round positive cultures were mostly
found to be composed of mixed clones as seen by visual inspection
of the wells (usually between 1 and 4 clones per well). All
hybridoma cultures were further subcloned by limiting dilution, a
technique well known to those skilled in the art, finally resulting
in pure hybridoma clones secreting antibodies with a homogeneous
idiotype. Some of these pure hybridoma clones were further tested
with respect to their reactivity patterns on normal and PHF-tau in
ELISA as described in Example II and the location of their epitopes
by means of Western blot analysis using tau mutants as disclosed in
Example II.
[0134] The latter procedure was carried out as follows: Purified
normal human tau and PHF-tau were applied to 10% SDS-polyacrylamide
gels and run under denaturing conditions according to Laemmli
(1970).
[0135] After SDS-PAGE, the transfer to nitrocellulose (Hybond-C,
Amersham, Brussels, Belgium) was carried out in 10 mM NaHCO3, 3 mM
Na2CO3, 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 the antibody of the respective hybridoma. After three
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 H20.
[0136] As a result of these analyses, 8 hybridomas out of 28
(including AT180 and AT270) were found to be truly PHF-tau
specific. These PHF-tau specific monoclonal antibodies were finally
tested for their detection capacity of PHF-tau in cerebrospinal
fluid using an ELISA (as illustrated in Example IV). As is
illustrated in the further examples, two monoclonal antibodies,
referred to as AT270 and AT180, could be found which allowed the
specific detection of at least than 10 pg/ml phosphorylated tau as
determined in CSF spiked with different amounts of phosphorylated
and non-phosphorylated tau without applying amplification
techniques (phosphorylated tau was prepared by incubating
recombinant non-phosphorylated tau with a rat brain extract capable
of phosphorylating Ser and Thr amino acids at positions
corresponding to the sites of abnormal phosphorylation of tau as
described in Goedert et al., 1993). Moreover, the monoclonal
antibody AT270 was capable to detect PHF-tau in unconcentrated CSF
(see further). An assay based on the use of AT180 allowed to detect
PHF-tau in 5-fold concentrated CSF, while AT8 was not capable to
detect PHF-tau in 10-fold concentrated CSF. Based on these
criteria, the hybridomas AT180 and AT270 were selected for further
characterization of their epitopes and deposited at the ECACC under
numbers 92122104 and 93070774.
[0137] 4. Determination of the Antibody Class and Subclass
[0138] The antibody class and subclass was determined by Inno-LIA
(Innogenetics, Ghent, Belgium). The antibodies AT180 and AT270
appeared to be of the IgG1, kappa subtypes.
EXAMPLE II
Characterization of PHF-tau Specific Antibodies and Their
Epitopes
[0139] 1. Discrimination of Abnormally Phosphorylated tau from
Normal tau in ELISA
[0140] The preparation of affinity purified normal tau is described
in Mercken et al. (1992b) and for PHF-tau is essentially as
described in Greenberg and Davies (1990); Mercken et al. (1992a).
Purity of normal tau and PHF-tau standards was determined by
SDS-PAGE. The samples were also analyzed on 420 A/H amino acid
analyzer (Applied Biosystem B.V., Maarssen, The Netherlands)
according to the manufacturer's instructions. Both normal and
PHF-tau showed the expected amino acid compositions. The exact
protein concentration of both affinity purified normal and PHF-tau
was determined using an internal standard peptide.
[0141] PHF-tau monoclonal antibodies derived from the hybridomas
AT180 or AT270 and 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 100 .mu.l of an appropriately diluted
tau or PHF-tau standards and incubated for 60 min at 37.degree. C.
The plates were washed 5 times with PBS-0.05% Tween 20 (v/v) and
100 .mu.l of two biotinylated antibodies (AT120 and HT7,
Vandermeeren et al., 1993; Mercken, Ph. D. thesis) at a final
concentration of 0.2 .mu.g/ml was added and incubated for 1 hr at
room temperature. After washing horse-radish peroxidase conjugated
streptavidine (Jackson, Innogenetics, Belgium) at a dilution of
{fraction (1/10000)} was added for 30 min at room temperature.
Following a final washing with PBS/Tween 20, 100 .mu.l of 0.42 mM
3,5,3',5'-tetramethylbenz- idine, 0.003% (vol/vol) H.sub.2O.sub.2
in 100 mM citric acid, 100 mM Na.sub.2HPO.sub.4, pH 4.3 were added
as peroxidase substrate for 30 min at room temperature. 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.
[0142] The specificity of AT180 and AT270 for PHF-tau is shown
(Table 1, FIGS. 1 and 2) from which can be seen that even at 1
.mu.g normal tau no reactivity is present.
[0143] 2. Mapping of the Epitope of the Selected PHF-tau Specific
Antibodies Via Recombinant tau Mutants
[0144] A full-length cDNA clone (human tau 24; htau24)
corresponding to a four repeat isoform of tau and with an NdeI site
in the context of the inititiator codon (Goedert and Jakes, 1990)
was subcloned into the EcoRI site of M13mp18. Site-directed
mutagenesis was used to change the codons representing the
following amino acids to an Ala: T153, T175, T181, T199, T205,
T212, T217, T231, S235, further referred to as T153A etc. mutants.
Constructs containing combinations of these sites were also
evaluated. Following cleavage with NdeI and EcoRI the resulting
fragments were subcloned downstream of the T7 RNA polymerase
promoter in the expression plasmid pRK172 (Mc Leod et al., 1987)
and the recombinant plasmids were transformed into E. coli
BL21(DE3) (Studier et al., 1990) cells. The bacterial cultures were
grown, induced and tau proteins purified as described (Goedert and
Jakes, 1990).
[0145] The brain protein kinase activity was prepared by
homogenizing adult rat brain (1 g/2.5 ml) in 10 mM okadaic acid, 1
mM PMSF, 20 .mu.g/ml leupeptin, 20 .mu.g/ml aprotinin and 20
.mu.g/ml pepstatin and centrifuged at 40,000 rpm for 1 h at
4.degree. C. The supernatant was used directly for phosphorylation
(Goedert et al., 1993). Incubations (0.05 ml) were carried out at
37.degree. C. and comprised 40 mM HEPES, pH 7.2, 2 mM ATP, 2 mM
MgCl.sub.2, tau protein (1 .mu.M), rat brain extract (1 .mu.l), 5
mM EGTA, 2 mM DTT, 1 .mu.M okadaic acid, 1 mM PMSF, 20 .mu.g/ml
aprotinin and 20 .mu.g/ml pepstatin. Reactions were initiated by
addition of the brain extract, incubated for 24 h and aliquots used
for SDS-PAGE. Controls were incubated under the same conditions,
except that the brain extract was omitted.
[0146] Results
[0147] The normal htau24 clone or htau24 mutants were
phosphorylated by a protein kinase containing rat brain extract
(Goedert et al, 1993), run on SDS-PAGE and immunoblotted using
AT180, AT270 or a tau antiserum, 134, that is phosphorylation
independent (Goedert et al, 1989). AT270 and AT180 did not stain
wild-type or mutant tau proteins prior to brain extract
phosphorylation. However after a 24 h incubation with brain
extract, AT270 recognized wild-type tau, but not T181A tau (FIG.
3). This establishes that staining by AT270 minimally requires T181
to be phosphorylated. Monoclonal antibody AT180 did equally
recognize the phosphorylated wild-type tau but failed to recognize
the phosphorylated T231A mutant, indicating that the AT180 epitope
needs T231 phosphorylation for recognition (FIG. 4). The rather
weak staining on the S235A mutant is due to the fact that some
factors in the brain extract are limiting in this type of assay and
consequently the S235 site was not always fully converted to its
phosphorylated state, as was confirmed by using activated
recombinant protein kinases as phoshorylating agents (data not
shown). When recombinant tau was treated with activated recombinant
MAP kinase alone or GSK3 kinase alone the AT180 epitope could not
be produced, while the same experiment performed with a mixture of
MAP kinase and GSK3 kinase allowed correct phosphorylation and
immunoreactivity with AT180.
Example III
[0148] Using different combinations of the PHF-tau antibodies to
detect PHF-tau in unconcentrated CSF.
[0149] We have previously shown that an assay to detect abnormally
phosphorylated tau based on the sole use of AT8 antibody as
detector is not capable of detecting the AT8 epitope nor in
unconcentrated nor in concentrated CSF (Vandermeeren et al, 1993).
Continuing experiments with AT180 and AT270 show that the epitope
of AT270 present on abnormally phosphorylated tau can be detected
in most unconcentrated Alzheimer CSF samples, whereas the AT180
epitope only detects abnormally phosphorylated tau in those CSF
samples containing high total tau levels and thus does not detect
PHF-tau in all Alzheimer CSF samples. We subsequently used AT270
alone or in different combinations with other antibodies (AT8,
AT180). Combinations of antibodies were used as solid phase bound
coating antibodies to look for the presence of PHF-tau in CSF of
patients suffering from different neurological disorders in which
abnormally phosphorylated tau has been described, such as Pick's
disease, Creutzfeld-Jacob disease, and Parkinson's disease (see
Table 2).
[0150] A preferred PHF-tau specific assay can be as follows:
coating of the three monoclonal antibodies, AT8, AT180, AT270 at a
final concentration of 5 .mu.g/ml in 10 mM Tris pH 8.6, 10 mM NaCl,
10 mM NaAz overnight at 4.degree. C. on high-binding microtiter
plates (Nunc, GIBCO, Paisley, U.K.). After overcoating for 1 hr
with 150 .mu.l of 10% satured casein in phosphate-buffered saline
(PBS) to reduce non-specific binding, the plates were incubated
with 100 .mu.l of an appropriately diluted recombinant
phosphorylated tau standard, or with unconcentrated CSF samples,
supplemented with 5% Tween 20, overnight at room temperature. The
plates were washed five times with PBS/0.05% Tween 20 (vol/vol),
and 100 .mu.l of two biotinylated antibodies (AT120 and HT7;
Vandermeeren et al., 1993; Mercken, Ph.D. thesis) at a final
concentration of 0.2 .mu.g/ml was added and incubated for 1 hr at
room temperature. After washing, horse-radish peroxidase conjugated
streptavidine (Jackson, Innogenetics, Belgium) at a dilution of
{fraction (1/10000)} was added for 30 min at room temperature.
Following a final washing with PBS/Tween 20, 100 .mu.l of 0.42 mM
3,5,3',5'-tetramethylbenzidine, 0.003% (vol/vol) H.sub.2O.sub.2 in
100 mM citric acid, 100 mM Na.sub.2HPO.sub.4, pH 4.3 were added as
peroxidase substrate for 30 min at room temperature. 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.
Example IV
[0151] Detection of PHF-tau in Cerebrospinal Fluid Samples with the
Selected PHF-tau Specific Monoclonal Antibodies
[0152] Cerebrospinal Fluid Samples
[0153] Antemortem 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
-70.degree. C. until use. Samples were taken from Alzheimer
patients, from patients with no neurological complications and from
patients with various neurological disorders. Those samples were
assayed for total tau concentration using the Innotest htau
(Innogenetics, Belgium) and samples having high total tau values
were retained for further analysis for the presence of PHF-tau with
the preferred PHF-tau assay as specified in Example III.
[0154] Results
[0155] Using this assay and the CSF samples as described, the
results summarized in Table 3 were obtained. From this, it is
obvious that the mean PHF-tau levels remain rather low for controls
and OND patients (controls: 381 mOD; OND, degenerative: 423 mOD;
OND, inflammatory: 392 mOD; OND, vascular: 340 mOD), while the mean
for Alzheimer patients is 814 mOD units. Furthermore, as can be
seen in Table 3, high total tau values in the OND group are not
always reflected by a parallel increase in PHF-tau while in the AD
patients high total tau levels always give rise to increased
concentrations of PHF-tau. The accumulated evidence from the
control group, the OND cohorts and the AD patients therefore
strongly points to the diagnostic specificity of the PHF-tau assay
for AD and AD-related syndromes (such as, multiple infarct
dementia, Parkinson's disease mixed dementia and one unspecified
dementia).
2TABLE 1 tau AT180 AT180 AT270 AT270 pg/ml non-phosphorylated
Phosphorylated non-phosphorylated Phosphorylated 2000 95 449 98
2653 500 92 179 94 1044 100 94 125 96 158 25 85 105 92 113 5 90 106
87 86 1 87 97 90 83 0.2 85 95 91 89 0 89 94 92 94
[0156]
3TABLE 2 tau AT270 AT180, 270 AT8, 270 AT8, 180, 270 Nr Diagnosis
in pg/ml in mOD in mOD in mOD in mOD 3 AD (early onset) 56.5 185
613 126 916 5 AD 62 132 410 105 636 6 AD 71 107 310 87 678 38 AD
68.7 257 685 169 1251 73 AD 27.7 84 96 44 570 88 AD 25.3 124 261 68
705 276 AD 43 106 325 76 612 281 AD 46 184 406 100 199 874 AD 51
128 353 88 777 61 Control 17.3 53 80 87 1435 Hydrocephalus 18 62 38
53 297 401 Control 20.1 77 73 53 242 PNP 20.8 109 169 77 276 1424
Control 21 96 79 61 304 85 Control 22 54 52 43 83 153 CNS lymphoma
22.2 55 111 47 301 1337 Hydrocephalus 29 45 68 47 124 1337
Hydrocephalus 29 43 75 45 124 1381 NPH 32 78 156 75 366 1470 NPH 33
77 152 60 148 641 Control 35 64 135 67 515 349 Meningitis 35.6 76
95 68 367 109 Pick 40.5 89 131 60 398 1467 Pseudotumor 44 111 149
64 373 193 GBS 44.1 158 238 107 295 114 Cerebell Atro. 51.5 90 97
56 203 130 External aftalmoplegia 53.6 102 99 60 229 131 Meningeal
bleeding 66.7 65 82 49 338 214 CJD 92 82 204 70 387 53 PD 57 249
682 137 836 150 Control 79 284 848 207 1557 137 Pick 77.4 423 987
220 1571
[0157]
4TABLE 3 Alzheimer patients tau PHF-tau Nr Sex Age Diagnosis
(pg/ml) (in mOD) 304 F AD (= Alz21) 86 517 3 F ? AD (early onset)
56.5 916 874 F 42 AD 51 777 113 F 44 AD 42.6 627 1085 M 46 AD 543
3049 265 F 47 AD (?) 62 441 161 F 57 AD, Creutzfeld-Jacob (?) 33.8
682 326 F 58 AD 34 394 1499 F 60 AD 147 1425 220 M 60 AD 126 1614 6
M 61 AD 71 678 718 M 62 AD 53 588 335 F 63 AD, probable 83 656 720
M 64 AD 170 1573 174 F 64 AD 66 609 338 F 64 AD 51.2 593 262 F 65
AD 221 2224 254 F 66 AD 80.2 923 73 M 67 AD 27.7 570 209 M 67 AD
74.4 1341 722 M 67 AD 71 433 383 M 67 AD 32.5 691 38 M 67 AD 68.7
1251 1259 F 68 AD (?) 65 698 723 M 69 AD 54 614 721 M 70 AD 99 947
17 F 70 AD 37 582 1 F 72 AD + MS 33 222 229 M 73 AD 70.9 1043 278 F
75 AD 54 1069 719 M 75 AD 70 884 88 F 76 AD 25.3 705 132 F 76 AD
51.9 1187 65 F 77 AD 80.1 1284 287 M 78 AD (?) 58 423 737 F 78 AD
43 330 71 F 78 AD 53.9 741 28 M 78 AD 48.7 476 760 F 78 AD 36 299 5
M 81 AD 62 636 281 F 81 AD 46 199 13 F 81 AD 13 179 289 M 83 AD 29
502 96 F 84 Steele-Richardson (?), AD (?) 41 295 223 M 84 AD 52 487
185 F 85 AD 59 724 276 F 85 AD 43 612 39 F 86 AD 150 2205 343 F 86
AD (?) 44 413 14 F 86 AD 57 825 606 F 86 AD 43 680 724 M 88 AD 31
487 Control patients Nr Sex Age Diagnosis tau PHF-tau 145 F 40 31.9
363 F 68 39 436 F 72 84 718 709 F 56 29 259 1508 M 64 21 155 1100 M
71 31 313 1424 M 64 21 304 F 66 35 515 F 77 43.5 447 544 M 69 17
304 Other Neurological Disorders, Degenerative types Nr Sex Age
Diagnosis tau PHF-tau 196 F 71 Parkinson + dementia 84 421 167 M 61
cerebellum atrophy 14 433 75 M 71 alcohol induced dementia 26 763
53 F 69 Mixed dementia, Parkinson 55 589 53 F 85 Parkinson, mixed
dementia 59.3 1083 137 F 57 Pick's disease 77.4 1571 946 F 75
cortical atrophy, 57 814 periventricular 713 F 48 FLD 39 341 186 M
66 ALS 21.2 208 344 F 68 Parkinson dementia 28 205 22 F 65 Steele
Richardson 13 203 114 M 51 atrophy cerebellum 51.5 203 334 M 57
Parkinson, dyskinesia 22 218 1527 M 61 Parkinson + syphilis 17 277
772 F 70 Steele-Richardson 26 244 169 F 27 Dementia (?) 37.5 337
794 F 61 Steele-Richardson 24 149 214 F 59 Creutzfeld-Jacob 292 387
109 M 63 Pick; ALS 40.5 398 33 M 63 Pick 61 395 230 F 66 Parkinson
21.1 157 Other Neurodegenerative diseases, inflammatory types Nr
Sex Age Diagnosis tau PHF-tau 668 F 79 encephalitis 16 282 673 F 72
encephalitis 16 281 710 F 28 MS 14 186 405 M 29 Guillain-Barr (GBS)
47.6 278 1396 M 67 ALS 150 289 1261 M 28 CIDP 24 293 193 M 68 GBS
44.1 295 279 F 70 M.S. 17 113 314 F 69 GBS 34 292 716 M 54 ALS 15
220 1477 M 67 ALS 150 797 717 M 53 ALS 18 158 163 M 71 Meningitis
150 1891 93 M 85 polyneuropathy 61.9 764 327 F 66 ALS 20 127 708 F
33 Neurocystercosis 20 249 149 M 56 Syphilis 4 176 1493 M 22 SSPE
18 236 207 M 50 Guillain-Barr 70.3 466 706 F 75 GBS 21 309 64 M 58
MS 40.7 512 1447 F 58 ALS 38 340 360 M 54 Lyme disease 34.7 515 532
M 64 TBC 28 470 7 M 17 SSPE 48.1 403 363 F 47 encephalitis 35.9 424
133 M 24 MS 37.1 397 208 M 68 polyneuropathy 28.8 325 398 M 58
meningoencephalitis 117.8 367 715 F 63 ALS 40 333 Other
Neurological diseases, vascular types Nr Sex Age Diagnosis tau
PHF-tau 131 F 59 meningeal bleeding 66.7 338 219 F 58 Pseudobullar
synd. 39.6 231 101 M 55 infarct 52.8 352 228 M 22 ischemic cerebral
infarct 150 392 294 F 71 CVA, diabetes, epilepsia 54 347 409 M 65
multiple vascular pathology 208 222 1492 M 47 infarct, occip. 69
213 712 F 44 Seizure-new onset 14 309 21 M 70 congofilic angiopathy
15 322 457 M 68 infarct 24 280 42 M 78 multi-infarct dementia 38.9
614 320 M 76 diabetes, MID 27 318 714 M 82 MID 29 286 98 F 68 TIA
34.9 577 23 F 82 dementia 31 303 Other neurological disorders, not
defined Nr Sex Age Diagnosis tau PHF-tau 115 M 68 subacute
degeneration 14 561 419 M 1 hydrocephalus 150 2765 275 M 66 PNP 19
448 226 F 54 progress. pyr. affliction 18 445 111 M 67
Polyneuropathy 64.2 549 91 F 81 PNP 31.2 355 1467 M 43 Pseudotumor
44 373 153 F 62 CNS lymphoma 22.2 301 1435 M 65 trauma hydroceph 18
297 150 F 82 mental disturbance 79 1557 274 M 78 cervical
medullopathy 69 160 1454 M 73 hydrocephalus 30 124 268 M 63 PNP,
alcohol 23 500 711 F 31 Pseudotumor cerebri 16 234 170 M 66 Temp. E
24.2 632 195 F 64 E. dialysis 14 239 242 M 72 polyneuropathy 20.8
276 330 M 69 pyr?? 14 246 1478 M 43 Pseudotumor 42 337 1364 M 65
stenosis 32 475 152 F 66 medullar 33 450 438 M 71 polyneuropathy 34
205 1087 F 65 stenosis 24 1086 251 F 77 trauma cerebri commoti 14
209 1576 F 70 korsakoff syndrome 82 863 312 M 72 meta adeno ?? 37
471 1442 F 70 gait disturbance 39 615
REFERENCES
[0158] Andreadis A, Brown W, Kosik K (1992) Structure and novel
exons of the human tau gene. Biochem 31:10626-10633.
[0159] Bramblett G, Trojanowski J, Lee V (1992) Regions with
abundant neurofibrillary pathology in human brain exhibit a
selective reduction in levels of binding-competent tau and
accumulation of abnormal tau isoforms (A68 proteins). Lab Invest
66:212-222.
[0160] Bramblett G, Goedert M, Jakes R, Merrick S, Trojanowski J,
Lee V (1993) The abnoramal phosphorylation of tau at Ser396 in
Alzheimer's disease recapitulates phosphorylation during
development and contributes to reduced microtubule binding. Neuron
10:1089-1099.
[0161] Brion J, Couck A, Passareiro E, Flament-Durand J (1985a)
Neurofibrillary tangles of Alzheimer's disease: an
immunohistochemical study. J Submicrosc Cytol 17:89-96.
[0162] Brion J, Passareiro J, Nunez J and Flament-Durand J (1985)
Mise en evidence immunologique de la proteine tau au niveau des
lesions de degenerescence neurofibrillaire de la maladie
d'Alzheimer. Arch Biol 95:229-235.
[0163] Biernat J, Mandelkow 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.
[0164] 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.
[0165] Butner K, Kirschner (1991) Tau protein binds to microtubules
through a flexible array of distributed weak sites. J Cell Biol
115:717-730.
[0166] de Bont H, van Boom J, Liskamp R (1990a) Automatic synthesis
of phosphopeptides by phosphorylation on the solid phase.
Tetrahedron Letters 31:2497-2500.
[0167] de Bont H, van Boom J, Liskamp R (1990b)
N,N-diiopropyl-bis(4chloro- benzyl)phosphoramidite: A versatile
phosphitylating agent for the phosphorylation of hydroxy amino
acids and preparation of protected phosphopeptides. Recueil des
Travaux Chimiques des Pays-bas 109:27-28.
[0168] 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.
[0169] Delacourte A, Defossez A (1986) Alzheimer's disease: Tau
proteins, the promoting factors of microtubule assembly, are major
components of paired helical filaments. J. Neurol. Sci.
76:173-180.
[0170] Drewes G, Lichtenberg-Kraag B, Doring F, Mandelkow E-M,
Biernat J, Goris J, Doree M, Mandelkow E (1992) Mitogen activated
protein (MAP) kinase transforms tau protein into an Alzheimer-like
state. EMBO J. 11:2131-2138.
[0171] 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.
[0172] Flament S, Delacourte A (1990) Tau Marker? Nature
346:6279.
[0173] 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.
[0174] 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.
[0175] Goedert M, Jakes R (1990) Expression of seperate isoforms of
human tau protein: correlation with the tau protein in brain and
effects on tubulin polymerization. EMBO J. 9:4225-4230.
[0176] Goedert M, lakes R, Crowther R, Six J, Lubke U, Vandermeeren
M, Cras P, Trojanowski J Q, Lee V (1993) The abnormal
phosphorylation of tau protein at serine 202 in Alzheimer's disease
recpitulates phosphorylation during development. Proc Natl Acad Sci
(USA) 90:5066-5070.
[0177] Goedert M, Wishik C, Crowther R, Walker J, Klug A (1988)
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)
85:4051-4055.
[0178] 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.
[0179] Goedert M, Spillantini M, lakes R (1991) Localization of the
Alz-50 epitope in recombinant human microtubule-associated protein
tau. Neurosci Lett. 126:149-154.
[0180] 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.
[0181] 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.
[0182] Greenberg S, Davies P, Schein J, Binder L (1992)
Hydrofluoric acid-treated tauPHF proteins display the same
biochemical properties as normal tau. J Biol Chem 267:564-569.
[0183] Grundke-Iqbal I, Iqbal K, Tung Y, Quinlan M, Wisniewski H
Binder L (1986) Abnormal phosphorylation of the
microtubule-associated protein (tau) in Alzheimer's cytoskeletal
pathology. Proc Natl Acad Sci (USA) 83:4913-4917.
[0184] Grundke-Iqbal I, Iqbal K, Quinlan M, Tung Y, Zaidi M,
Wisniewski H (1986) Microtubule-associated protein tau. J Biol Chem
261:6084-6089.
[0185] Harrington C, Mukaetova E, Hills R, Edwards P, Montejo de
Garcini E, Novak M, Wischik C (1991) Measurement of distinct
immunochemical presentations of tau protein in Alzheimer's disease.
Proc Natl Acad Sci (USA) 88:5842-5846.
[0186] Hasegawa M, Morishima-Kawashima M, Takio K, Suzuki M, Titani
K, Ihara Y (1992) Protein sequence and mass spectrometric analyses
of tau in Alzheimer's disease brain. J Biol Chem
267:17047-17054.
[0187] 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.
[0188] 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.
[0189] Iqbal K, Grundke-Iqbal I, Smith A, George L, Tung Y, Zaidi T
(1989) Identification and localization of a Tau peptide to paired
helical filaments of Alzheimer's disease. Proc Natl Acad Sci (USA)
86:5646-5650.
[0190] Ishiguro K, Takamatsu M, Tomizawa K, Omori A, Takahashi M,
Arioka M, Uchida T, Imahori K (1992) Tau protein kinase I converts
noraml tau protein into AA68-like component of paired helical
filaments. J Biol Chem 267:10897-10901.
[0191] Kanai Y, Chen J, Hirokawa N (1992) Microtubule bundling by
tau proteins in vivo: analysis of functional domains. EMBO J.
11:3953-3961.
[0192] Kondo J, Honda T, Mori H, Hamada Y, Miura R, Ogawara M,
Ihara Y (1988) The carboxyl third of tau is tightly bound to paired
helical filaments. Neuron 1:827-834.
[0193] Kohler G, Milstein C (1975) Continuous cultures of fused
cells secreting antibody of predefined specificity. Nature
256:495-497.
[0194] Kosik K, Joachim C, Selkoe (1986) Microtubule-associated
protein tau is a major antigenic component of paired helical
filaments in Alzheimer's disease. Proc Natl Acad Sci (USA)
83:4044-4048.
[0195] Labb J, Cavadore J and Dore M (1991) M phase-specific cdc2
kinase: preparation from starfish oocytes and properties. Meth
Enzymol 200:291-301.
[0196] Laemmli U (1970) Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 227:680-685.
[0197] Ledesma M, Correas 1, Avila J, Diaz-Nido J (1992)
Implication of brain cdc2 and MAP2 kinases in the phosphorylation
of tau protein in Alzheimer's disease. FEBS Letters
308:218-224.
[0198] 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.
[0199] Lewis S, Wang D, Cowan N (1988) Microtubule-associated
protein MAP2 shares a microtubule binding motif with Tau protein.
Science 242:936-939.
[0200] Mandelkow E-M, Drewes G, Biernat J, Gustke N, Van Lint J,
Vandenheede J, Mandelkow E (1992) Glycogen-synthase kinase-3 and
the Alzheimer's-like state of microtubule-associated protein tau.
FEBS Letters 314:315-321.
[0201] Martin J, Gheuens J, Bruyland M, Cras P, Vandenberghe A,
Masters C, Beyreuther K, Dom R, Ceuterick C, Lubke U, Van
Heuverswijn H, De Winter G, Van Broeckhoven C (1991) Early-onset
Alzheimer's disease in 2 large Belgian families. Neurology
41:62-68.
[0202] McKahn G, Drachman D, Folstein M, Katzman R, Price D,
Stadlan E (1984) Clinical diagnosis of Alzheimer's disease: report
of the NINCDS-ADRA work group under the auspices of department of
health and human services task force on Alzheimer's disease.
Neurology 34:939-944.
[0203] Mc Leod M, Stein M, and Beach D (1987) The product of the
mei3+ gene, expressed under control of the mating-type locus,
induces meiosis and sporulation in fision yeast. EMBO J.
6:729-736.
[0204] Mercken M, Ph. D. thesis: De neurofibrillaire degeneratie
bij de ziekte van Alzheimer: een benadering met monoklonale
antistoffen. Antwerp, 1991.
[0205] 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.
[0206] 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.
[0207] Otvos L, Elekes I, Lee V (1989) Solid phase synthesis of
phosphopeptides. International Journal of Peptide and Protein
Research 34:129-133.
[0208] Perich J (1991) Synthesis of O-phosphoserine and
O-phosphothreonine-containing peptides. Methods in Enzymology
201:225-233.
[0209] Poulter L, Barrat D, Scott C, Caputo C (1983) Localizations
and immunoreactivities of phosphorylation sites on bovine and
porcine tau proteins and a PHF-tau fragment. J Biol Chem
268:9636-9644.
[0210] Roder H, Ingram V (1991) Two novel kinases phosphorylate tau
and the KSP site of heavy neurofilament subunits in high
stoichiometric ratios. J Neurosci 11:3325-3343.
[0211] Sturgill T, Ray L, Anderson N, Erickson A (1991)
Purification of activated protein kinase from epidermal growth
factor treated 3T3-L1 fibroplasts. Meth Enzymol 200:342-351.
[0212] Selden S, Pollard T (1983) Phosphorylation of
microtubule-associated proteins regulates their interaction with
actin filaments. J Biol Chem 258(11):7064-71.
[0213] 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.
[0214] Studier F, Rosenberg A, Dunn J, Dubbendorf J (1990) Use of
T7 RNA polymerase to direct expression of cloned genes. Methods
Enzymol 185:60-89.
[0215] 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.
[0216] Vandenheede J, Yang S, Goris J, Merlevede W (1980)
ATP.times.Mg-dependent protein phosphatase from rabbit skeletal
muscle. Purification of the activating factor and its
characterization as a bifunctional protein also displaying synthase
kinase activity. J Biol Chem 255: 11768-11774.
[0217] Vandermeeren M, Mercken M, Vanmechelen E, Six J, Van de
Voorde A, Martin J, Cras, P (1993) Detection of tau proteins in
normal and Alzheimer's disease fluid with a sensitive sandwich
enzyme linked assay J Neurochem 61:1828-1834.
[0218] Vulliet R, Halloran S, Braun R, Smith A, Lee, G (1992)
Proline-directed phosphorylation of human tau protein. J Biol Chem
267:22570-22574.
[0219] Watanabe N, Takio K, Hasegawa M, Aral T, Titani K, Ihara Y,
(1992) Tau 2: a probe for a ser conformation in the amino terminus
of tau. J Neurochem 58:960-966.
[0220] 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.
[0221] Wolozin B, Davies P (1987) Alzheimer-related neuronal
protein A68: specificity and distribution. Ann Neurol
22:521-526.
[0222] Wolozin B, Pruchnicki A, Dickson D, Davies P (1986) A
neurological antigen in the brains of Alzheimer's patients. Science
232:648-650.
[0223] Wood J, Mirra S, Pollock N, Binder L (1986) Neurofibrillary
tangles of Alzheimer's disease share antigenic determinants with
the axonal mirotubule-associated protein tau. Proc Natl Acad Sci
(USA) 83:4040-4043.
[0224]
Sequence CWU 1
1
* * * * *