U.S. patent application number 12/268177 was filed with the patent office on 2009-05-28 for monoclonal antibodies directed against the microtubule-associated protein tau.
This patent application is currently assigned to N.V. Innogenetics S. A.. Invention is credited to Ev-Maria Mandelkow, Marc Mercken, Marc Vandermeeren, Andre Vandevoorde, Eugeen Vanmechelen.
Application Number | 20090137051 12/268177 |
Document ID | / |
Family ID | 8208630 |
Filed Date | 2009-05-28 |
United States Patent
Application |
20090137051 |
Kind Code |
A1 |
Mercken; Marc ; et
al. |
May 28, 2009 |
Monoclonal Antibodies Directed Against The Microtubule-Associated
Protein Tau
Abstract
A monoclonal antibody which forms an immunological complex with
a phosphorylated epitope of an antigen belonging to human
abnormally phosphorylated tau proteine. The tau protein can be
obtained from a brain homogenate, itself isolated from the cerebral
cortex of a patient having Alzheimer's disease.
Inventors: |
Mercken; Marc; (Turnhout,
BE) ; Mandelkow; Ev-Maria; (Hamburg 52, DE) ;
Vandermeeren; Marc; (Geel, BE) ; Vanmechelen;
Eugeen; (Nazareth-Eke, BE) ; Vandevoorde; Andre;
(Lokeren, BE) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Assignee: |
N.V. Innogenetics S. A.
Ghent
BE
|
Family ID: |
8208630 |
Appl. No.: |
12/268177 |
Filed: |
November 10, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10923323 |
Aug 20, 2004 |
|
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12268177 |
|
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|
09734281 |
Dec 11, 2000 |
6900293 |
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10923323 |
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Current U.S.
Class: |
436/86 ;
530/328 |
Current CPC
Class: |
G01N 2800/28 20130101;
G01N 33/6896 20130101; Y10S 435/975 20130101; G01N 2333/4709
20130101; C07K 14/4711 20130101; C07K 16/18 20130101; G01N
2800/2821 20130101 |
Class at
Publication: |
436/86 ;
530/328 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 7/00 20060101 C07K007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 25, 2001 |
EP |
91402871.7 |
Claims
1. A peptide from 9 to 100 amino acids long, * * comprising the
amino acid sequence YSSPGSPGT (SEQ ID NO:1) * or YSSPGSPGT (SEQ ID
NO:2), phosphorylated at the positions marked with *, and wherein
said peptide is able to form an immunological complex with the
monoclonal antibody AT8 produced by the hybridoma deposited at the
ECACC on Oct. 8, 1991 under No. 91100806.
2. The peptide of claim 1, consisting of the amino * * * acid
sequence YSSPGSPGT (SEQ ID NO:1) or YSSPGSPGT (SEQ ID NO:2),
phosphorylated at the positions marked with *, and wherein said
peptide is able to form an immunological complex with the
monoclonal antibody AT8 produced by the hybridoma deposited at the
ECACC on Oct. 8, 1991 under No. 91100806.
3. A kit for the detection, in a sample, of tau protein
phosphorylated at Ser(199) and/or Ser(202), said kit comprising the
phosphorylated peptide of claim 1 as a standard or competitor.
4. A kit for the detection, in a sample, of tau protein
phosphorylated at Ser(199) and/or Ser(202), said kit comprising the
phosphorylated peptide of claim 2 as a standard or competitor.
Description
[0001] This application is a Divisional of Ser. No. 10/923,323
filed Aug. 20, 2004 which is a Divisional of Ser. No. 09/734,281
filed on Dec. 11, 2000 (U.S. Pat. No. 6,900,293) which is a
Continuation of Ser. No. 08/617,987 filed on Mar. 15, 1996 (U.S.
Pat. No. 6,238,892) which is a Continuation of Ser. No. 08/108,758
filed on Sep. 2, 1993 which claims benefit to EPO No. 91402871.7
filed on Oct. 25, 2001 and which applications are incorporated
herein by reference. To the extent appropriate, a claim of priority
is made to each of the above disclosed applications.
[0002] The invention relates to new monoclonal antibodies directed
against a particular epitope present on the human
microtubule-associated protein tau, to the hybridoma secreting
these monoclonal antibodies, to the process for diagnosing brain
diseases involving the particular epitope of the tau protein, and
to the monoclonal antibodies recognizing said epitope.
[0003] Tau is a microtubule-associated protein which is synthesized
in the neurons (Kosik, K. S. et al., 1989) of several species,
including humans and which is abundantly present in the axonal
compartment of these neurons (Binder, L. I. et al., 1985).
Functionally the tau protein is involved in the polymerization of
tubulin (Weingarten, M. D. et al., 1975) and presumably in reducing
microtubule instability (Bre, M. H. and Karsenti, E. 1990).
[0004] Tau protein is also the major constituent of paired helical
filaments (PHF), characteristic structures found as neurofibrillary
tangles in tissue sections of the brain of Alzheimer patients
(Greenberg, S. and Davies, P., 1990; Lee, V. M.-Y. et al., 1991).
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, M. et al., 1989). The diversity
of the isoforms is generated from a single gene by alternative mRNA
splicing (Himmler, A., 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, M. et al.,
1989).
[0005] Tau variants of 64 and 69 kDa, which are abnormally
phosphorylated as revealed by the decrease in their molecular mass
observed after alkaline phosphatase treatment, have been detected
exclusively in brain areas showing neurofibrillary tangles and
senile plagues (Flament, S. et al., 1989 and 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 a phosphorylation of Ser(405)
which induced a lower electrophoretical mobility (Steiner, B. et
al., 1990).
[0006] Several antibodies are reported that show reactivity to
human tau either because they are directed to nonspecific
phosphorylated epitopes present on neurofilament and subsequently
shown to cross-react with normal and abnormally phosphorylated tau
(Nukina, N. et al., 1987; Ksiezak-Reding et al., 1987) or because
they recognized specific epitopes on normal and abnormally
phosphorylated tau.
[0007] The Alz50 monoclonal antibody (Wolozin, B. L. et al., 1986;
Nukina et al., 1988) recognizing a phosphate-independent epitope
present on tau variants of bovine origin and of normal and
abnormally phosphorylated tau from human origin (Ksiezak-Reding, H.
et al., 1988, Flament, S. and Delacourte, A. 1990) belongs to the
latter class of antibodies. The epitope recognized by this
monoclonal is specifically expressed in the somatodendritic domain
of degenerating cortical neurons during Alzheimer disease
(Delacourte, A. et al., 1990).
[0008] The Alz50 epitope has recently been mapped to the
NH.sub.2-terminal part of the tau molecule (Ksiezak-Reding, H. et
al., 1990; Goedert, M. et al., 1991). Due to its cross-reactivity
with normal tau, this antibody is only able to discriminate normal
from abnormally phosphorylated tau by the use of Western blotting
detection of brain homogenates or by ammonium sulfate-concentrated
CSF, or else by using a sandwich immunoassay on brain homogenates
(Ghanbari et al., 1990; Wolozin, B. and Davies, P. 1987; European
patent publication ("EP") 444 856). A CSF-based assay using
antibodies directed against PHF was first described by Mehta et
al., 1985, but shows considerable overlap between Alzheimer CSF and
CSF from controls. The epitope recognized by this antibody was
identified as part of ubiquitin (Perry et al., 1989).
[0009] Other monoclonal antibodies have been developed to recognize
tau protein. For instance, monoclonal antibody 5E2 was raised by
immunization with human fetal heat-stable microtubule-associated
proteins and recognizes an epitope spanning amino acids 156-175
which is present in normal and abnormally phosphorylated tau
(Kosik, K. S. et al., 1988).
[0010] Other antibodies such as tau 1 and several others were
raised by immunization with bovine tau, bovine heat-stable
microtubule-associated protein, or rat brain extracts (Binder, L.
I. et al., 1985; Kosik, K. S. et al., 1988), and most of the
antibodies recognize the normal and the abnormally phosphorylated
tau (Ksiezak-Reding, H. et al., 1990).
[0011] An antibody named "423", raised against the core of PHF,
reacted specifically with a 9.5 and 12-kDa fragment of the tau
protein, localized in the repetitive elements of tau, but
recognized neither normal human tau nor the abnormally
phosphorylated tau in Alzheimer's brain (Wischik, C. H. et al.,
1988).
[0012] This antibody has been used to discriminate Alzheimer PHF
pathology from normal controls in brain homogenates (Harrington, G.
R. et al., 1990; patent WO89/03993).
[0013] Thus far, none of all the antibodies described heretofore
has had an absolute specificity for the abnormally phosphorylated
tau either by immunohistology, Western blotting, or ELISA.
Quantitative measurements of normal and abnormally phosphorylated
tau have until now only been able to detect tau in brain
homogenates, in brain extracts containing PHF, or in concentrated
CSF samples after Western blotting (Ghanbari H. A. et al., 1990;
Harrington C. R. et al., 1990, Wisniewski, H. M. et al., 1989;
Wolozin, B. and Davies, P. 1987).
[0014] The aim of the present invention is therefore to provide
monoclonal antibodies which are specifically able to detect only
abnormally phosphorylated tau present in brain tissue sections, in
brain extracts, or in body fluids such as cerebrospinal fluid.
[0015] The invention also provides the hybridoma secreting such
monoclonal antibodies.
[0016] The invention further provides the epitope of tau protein
which is expressed in abnormally phosphorylated tau in brain tissue
sections or in brain homogenates or in body fluids, such as
cerebrospinal fluid, and which is recognized by such monoclonal
antibodies.
[0017] The invention still further provides the epitope of tau
protein expressed in the brain of patients affected with
neurological disorders such as Alzheimer's disease and Down
syndrome.
[0018] The invention yet further provides a process for the
detection or diagnosis in vitro of brain diseases involving tau
protein.
[0019] The monoclonal antibodies of the invention are characterized
by the fact that they react with an epitope which is present in
abnormally phosphorylated human tau. The monoclonal antibodies are
furthermore characterized by the fact that they form an
immunological complex with abnormally phosphorylated human tau, and
more specifically with a non-structural epitope present in
abnormally phosphorylated human tau.
[0020] By "non-structural" epitopes is meant epitopes which depend
for their antibody recognition both on their primary structure as
well as on post-translational modifications and conformation in
such a way that particular treatments (e.g. formalin fixation,
detergent treatment, dephosphorylation) may alter or destroy the
epitopes.
[0021] The expression "form an immunologically complex with" means
that a monoclonal antibody of the invention binds to the
aforementioned antigen under the conditions used in any one of the
following techniques:
Light Immunomicroscopy:
[0022] Brain tissue samples, obtained at surgery or autopsy, are
fixed by immersion in 4% formalin or Bouin's fixative and embedded
in paraffin. Four-mm-thick sections are prepared. 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, S. M., et
al., 1981) using 3,3'-diaminobenzidine tetrahydrochloride for
development of color. Sections are counterstained with Harris
haematoxylin stain.
Immunoelectron Microscopy in Tissue Sections:
[0023] Brain tissue sample, obtained at surgery or autopsy, is
fixed in either Bouin's fixative or 10% buffered formalin before
sectioning 60 mm thick 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 following
standard protocols known to those skilled in the art (Brion, J. P.
et al., 1985).
Immunoblotting Procedures:
[0024] For immunoblotting, fractions enriched in PHF are prepared
as described by Iqbal, K. et al. (1984) or Greenberg, S, and
Davies, P. (1990). For the second method, postmortem tissue is
used, consisting mostly of gray matter from the frontal and
temporal cortex, which is obtained from histologically confirmed
Alzheimer patients. This Alzheimer gray matter brain sample (5-10
g) is homogenized with 10 vol. 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 in a 60 Ti MSE
rotor at 27000.times.g for 20 min at 4.degree. C., the pellet is
removed and the supernatant is adjusted to 1% (vol/vol)
N-laurosylsarcosine and 1% (vol/vol) 2-mercaptoethanol and
incubated while rotating on a mixer 820 (Swelab, Sweden) for 2.5
hours at 37.degree. C. The supernatant mixture is centrifuged at
108,000.times.g for 35 min at 20.degree. C. The PHF-tau-containing
pellet is 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 U. K., 1970). After
electrophoresis, the proteins are either fixed and stained with
Coomassie brilliant blue, or transferred (Towbin H. 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 of 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 1/3000 in blocking buffer. After three washes in Tween-PBS,
streptavidin-biotinylated horseradish peroxidase complex (Amersham)
diluted 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] Human abnormally phosphorylated tau is a class of at least
two tau proteins of 64 and 68 kDa (Flament, S. et al., 1989 and
Delacourte, A. et al., 1990) which are specifically expressed in
the somatodendritic domain of the degenerating cortical neurons
during Alzheimer's disease and of which the lower electrophoretic
mobility can be attributed to abnormal phosphorylation. Although
kinase activities have been described that can provoke an
additional phosphorylation of normal tau with the concomitant shift
in electrophoretic mobility, none of these phosphorylations can
induce the formation of the epitope of the invention (Ishiguro, K.
et al., 1988; Steiner, B. et al., 1990).
[0030] According to an advantageous embodiment of the invention,
the monoclonal antibody forms an immunological complex with a
phosphorylated epitope of an antigen belonging to human abnormally
phosphorylated tau protein, with said tau protein being liable to
be obtained from a brain homogenate, itself isolated from the
cerebral cortex obtained from a patient having Alzheimer's disease
or having died from Alzheimer's disease.
[0031] The expression "phosphorylated epitope" means an epitope
that is destroyed when it is treated with a phosphatase enzyme,
such as alkaline phosphatase. In the phosphorylated epitope, the
serines are in the form of phosphoserines.
[0032] A "brain homogenate" and abnormally phosphorylated tau
protein can be obtained by one skilled in the art according to
standard methods such as the method of Iqbal, K. et al. (1984) or
the method of Greenberg, P. G. and Davies, P. (1990).
[0033] Monoclonal antibodies of the invention are characterized by
the fact that they form an immunological complex
[0034] either with the peptide YSSPGSPGT or YSSPGSPGT, preferably
YSSPGSPGT, phosphorylated at the positions marked with *
[0035] or with any other peptide capable of forming an
immunological complex with a monoclonal antibody, which itself is
liable to form a complex with the above-mentioned peptide.
[0036] The peptide YSSPGSPGT or YSSPGSPGT, preferably YSSPGSPGT,
will be hereinafter designated as "the epitope" of the invention.
The serines in positions 199 and 202 using the numbering of human
tau 40 (Goedert, M. et al., 1989) are in the form of
phosphoserines. The epitope spans the amino acids 197-205 using the
above-mentioned numbering.
[0037] 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".
[0038] A peptide phosphorylated at a certain position means that
the serine is in the phosphoserine form.
[0039] The invention also relates to monoclonal antibodies which
are not liable to form an immunological complex with normal tau
protein.
[0040] The monoclonal antibodies of the invention are not able to
form an immunological complex with tau protein present in brain
homogenates derived from human brain, isolated from a patient who
died of non-neurological disorders.
[0041] The monoclonal antibodies of the invention also are not
capable of forming an immunological complex with the above-defined
epitope previously treated with a dephosphorylating agent such as
alkaline phosphatase.
[0042] The monoclonal antibodies of the invention are further not
capable of forming an immunological complex with any variant
peptide above-defined and previously treated with a
dephosphorylating agent.
[0043] Advantageously, the monoclonal antibodies of the invention
are characterized by: [0044] the fact that they form an
immunological complex with the abnormally phosphorylated forms of
tau protein, present in homogenates of human brain obtained from a
patient who died of Alzheimer's disease and [0045] the fact that
these abnormally phosphorylated tau proteins present an apparent
molecular weight which is higher than that of normal tau proteins,
derived from brain homogenate, isolated from a patient who died of
non-neurological disorders and [0046] the fact that the apparent
molecular weight of such abnormally phosphorylated tau proteins can
be decreased to that of normal tau proteins by treatment of such
abnormally phosphorylated tau proteins with a dephosphorylating
agent.
[0047] The invention also relates to monoclonal antibodies which
form an immunological complex with the abnormally phosphorylated 64
and 68 kDa forms of tau protein present in brain homogenate as
defined above.
[0048] 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, Porton Down, GB-Salisbury, Wiltshire SP4
OJG) on Oct. 8, 1991 under No. 91100806. This hybridoma will be
hereinafter referred as "hybridoma ATS8", and the secreted
monoclonal antibody will be referred as "monoclonal antibody
AT8".
[0049] The invention also relates to an hybridoma which secretes a
monoclonal antibody according to the invention, and particularly
the hybridoma filed at ECACC on Oct. 8, 1991 under No.
91100806.
[0050] The monoclonal antibodies of this invention are obtained by
a process involving obtention and isolation of hybridomas which
secrete the monoclonal antibodies.
[0051] A process for obtaining such a hybridoma involves:
[0052] 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 recognized by the
monoclonal antibodies of the invention, such as the monoclonal
antibody secreted by the hybridoma deposited at ECACC on Oct. 8,
1991 under No. 91100806;
[0053] fusing such immunized cells with myeloma cells under
hybridoma-forming conditions; and
[0054] 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 the
abnormally phosphorylated form of tau protein or with the
phosphorylated peptide comprising the epitope of tau protein.
[0055] A process for producing the corresponding monoclonal
antibodies involves:
[0056] culturing the selected hybridoma as indicated above in an
appropriate culture medium; and
[0057] recovering the monoclonal antibodies excreted by the
selected hybridoma, or alternatively
[0058] implanting the selected hybridoma into the peritoneum of a
mouse and, when ascites have been produced in the animal,
recovering the monoclonal antibodies then formed from such
ascites.
[0059] 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
the culturing of cells in homogeneous suspension using, e.g.,
airlift reactors or stirred bioreactors.
[0060] The invention also relates to a peptide (antigen), which can
be obtained from a brain homogenate, itself isolated from the
cerebral cortex obtained from a patient having Alzheimer's disease,
and which forms an immunological complex with the monoclonal
antibody of the invention.
[0061] The invention further relates to peptides (antigens) which
are liable to form an immunological complex with any one of the
monoclonal antibodies of the invention and [0062] which contain or
are constituted by the sequence YSSPGSPGT or YSSPGSPGT,
phosphorylated at the positions marked with *, or [0063] which
contain or are constituted by the sequence of the variant peptides
defined above, i.e., the peptides able to form an immunological
complex with a monoclonal antibody, which itself is liable to form
a complex with the peptide YSSPGSPGT or YSSPGSPGT.
[0064] The invention also relates to peptides (antigens) of about
100 amino acids [0065] which contain the sequence YSSPGSPGT or
YSSPGSPGT, phosphorylated at the positions marked with *, or [0066]
which contain the sequence of the variant peptides defined
above.
[0067] The invention also relates to peptides (antigens) which
contain the sequence [0068] (X.sub.1).sub.p(X.sub.2).sub.qPX.sub.3P
(X.sub.4).sub.r(X.sub.5) or [0069]
(X.sub.1).sub.p(X.sub.2).sub.qSPX.sub.3P(X.sub.4).sub.r(X.sub.5).sub.s,
[0070] preferably
(X.sub.1).sub.p(X.sub.2).sub.qSPX.sub.3P(X.sub.4).sub.r(X.sub.5).sub.s,
[0071] in which X.sub.1, X.sub.2, X.sub.3, X.sub.4, X.sub.5 are any
one of the 20 amino acids and p, q, r, s are 0 or 1, phosphorylated
at places marked by *, provided that such peptides are able to form
an immunological complex with the monoclonal antibodies of the
invention.
[0072] The invention also relates to the above-mentioned peptides
which are liable to generate monoclonal antibodies of the
invention.
[0073] 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 tau protein and which forms an immunological
complex with a monoclonal antibody of the invention.
[0074] A method for preparing the peptides of the invention
preferably involves: 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, care being taken to protect all the reactive
groups carried by these aminoacyls or fragments except for the
amine groups of one and the carboxyl group of the other, 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.
[0075] In this process, it is possible to use previously
phosphorylated amino acids (De Bont H. B. A. et al., 1990) or it is
possible to carry out the phosphorylation after the synthesis of a
non-phosphorylated sequence, as explained hereafter.
[0076] The invention is also related to a process for the
preparation of the antigen according to the invention, starting
from said antigen in non-phosphorylated form which comprises:
[0077] reacting said antigen, which is non-phosphorylated, with a
kinase enzyme capable of recognizing the non-phosphorylated epitope
of the antigen and of modifying the epitope to a phosphorylated
epitope recognized by the monoclonal antibodies of the
invention.
[0078] The kinase used is advantageously extracted from brain
according to methods known to those skilled in the art (Ishiguro K.
et al., 1988; Baudier, J. and Cole, R. D., 1987; Vallee, R. B.,
1980) and is different from the kinases referred to in Steiner et
al. (1990).
[0079] The non-phosphorylated antigen is, for instance, a normal
human tau protein which, by phosphorylation with the
above-mentioned kinase, gives rise to an abnormally phosphorylated
tau protein, which is recognized by the monoclonal antibody of the
invention. Such abnormally phosphorylated tau protein is new.
[0080] The antigen of the invention, which can be prepared by
methods known to those skilled in the art (Iqbal, K. et al., 1984;
Greenberg S. G. and Davies, P., 1990) from the cerebral cortex
obtained from a patient having Alzheimer's disease or having died
of Alzheimer's disease, is characterized by its ability to form an
immunological complex with the monoclonal antibody of the
invention, particularly with the monoclonal antibody secreted by
the hybridoma deposited at the ECACC under No. 91100806.
[0081] 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, SSPE or
other neurological diseases in which the occurrence of PHF and
abnormally phosphorylated tau protein is implicated; this antigen
provokes an immunological reaction with the monoclonal antibody of
the invention.
[0082] The invention also relates to a process for the detection or
the diagnosis in vitro of brain disease involving PHF and tau
protein, i.e. Alzheimer's disease, which involves:
[0083] bringing one of the monoclonal antibodies of the invention
into contact with a preparation of PHF isolated from a patient
having Alzheimer's disease, or preferably from a diseased patient
having had Alzheimer's disease under conditions suitable for
producing an antigen-antibody complex;
[0084] separating the antigen from said complex and recovering the
antigen sought in a purified form.
[0085] The preparation of PHF can be carried out according to Iqbal
K. et al. (1984) or Greenberg et al. (1990).
[0086] Advantageously, the monoclonal antibody used is 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:
[0087] bringing the supernatant containing proteins and
polypeptides, extracted from brain tissues in a known manner
(Iqbal, K. et al., 1989; Greenberg S. S. and Davies, P., 1990),
into contact with the monoclonal antibody under conditions that
allow the formation of an immunological complex;
[0088] washing the immobilized antibody-antigen complex so
formed;
[0089] treating that 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
[0090] recovering the antigen in a purified form.
[0091] The process of the invention for the detection or diagnosis
in vitro of brain disease involving tau protein and PHF, e.g.,
Alzheimer's disease, includes:
[0092] bringing a sample of a brain homogenate, or of cerebrospinal
fluid, or of serum from a patient suspected of suffering of brain
disease involving tau protein and PHF, more particularly
Alzheimer's disease, into contact under in vitro conditions with
the monoclonal antibody of the invention under conditions suitable
for producing an antigen-antibody complex; and
[0093] detecting the immunological binding of said antibody to said
sample of brain homogenate, or of cerebrospinal fluid, or of
serum.
[0094] The detection of the immunologically bound monoclonal
antibody can be achieved in a conventional manner. Advantageously,
the monoclonal antibody of the invention itself carries a marker or
a group for direct or indirect coupling with a marker as
exemplified hereinafter. 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 the polyclonal antibody is
passed over a column to which the antigen is bound and eluting the
polyclonal antibody in a conventional manner. Detection can also be
achieved by competition binding of the antigen with a labeled
peptide comprising the epitope of the invention.
[0095] A particularly advantageous embodiment of the process of the
invention comprises contacting a sample of cerebrospinal fluid
(containing the corresponding antigen) obtained from a patient to
be diagnosed with the monoclonal antibody of the invention.
[0096] 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, SSPE and other neurological disorders in
which abnormally phosphorylated tau protein or paired helical
filaments are implicated. Such a kit would contain:
[0097] at least a microplate for deposition thereon of any
monoclonal antibody of the invention;
[0098] 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 the peptide YSSPGSPGT
or YSSPGSPGT phosphorylated at the positions marked with ;
[0099] a second antibody [0100] which can be a monoclonal antibody
recognizing an epitope of normal tau, or of abnormally
phosphorylated tau protein, or of any peptide of the invention,
such epitope being different from the one of the invention, or
[0101] which can be a polyclonal antibody of normal tau or of
abnormally phosphorylated tau or of a peptide of the invention,
such polyclonal antibody being liable to form an immunological
complex with epitopes which are all different from the epitope of
the invention, and the polyclonal antibody being preferably
purified by immunoaffinity chromatography using immobilized tau
protein, or
[0102] a marker either for specific tagging or coupling with the
second antibody;
[0103] appropriate buffer solutions for carrying out the
immunological reaction between: 1) the monoclonal antibody of the
invention and a test sample and 2) the bonded second antibody and
the marker.
[0104] The labeled peptide mentioned above can be a peptide which
has been labeled by any means known to those skilled in the art.
Likewise, the marker specific for tagging and coupling can be any
marker known to those skilled in the art.
[0105] The invention also relates to a kit, as described above,
also containing the antigen of the invention, the antigen of the
invention being either a standard (for quantitative determination
of an antigen which is sought) or a competitor, with respect to an
antigen which is sought, whereby the kit can be used in a
competition dosage process.
THE FIGURES
[0106] FIG. 1: Reactivity of AT8 to brain homogenates of Alzheimer
patients or normal controls using the AT8 monoclonal antibodies as
binding antibodies and rabbit anti-tau polyclonal antibodies
together with horseradish peroxidase-conjugated donkey anti-rabbit
antiserum for detection. [0107] AD.sub.1-AD.sub.4: Brain
homogenates from 4 different Alzheimer patients diluted 1/200
(solid bars) or 1/1000 (stippled bars). [0108] C.sub.1-C.sub.4:
Brain homogenates from 4 different controls, diluted 1/200 (solid
bars) or 1/1000 (stippled bars). [0109] The ordinate represents the
optical density at 450 nm and the abscissa represents the different
samples.
[0110] FIG. 2: Western blotting detection of normal tau or PHF-tau
using either the monoclonal antibodies Tau-1 or AT8. Lanes 1 and 2:
Coomassie Brilliant Blue staining of PHF-tau (lane 1) or normal tau
(lane 2). Lanes 3 to 6: Western blotting of PHF-tau (lanes 3 and 5)
or normal tau (lanes 4 and 6) using either Tau-1 monoclonal
antibodies (lanes 3 and 4) or AT8 monoclonal antibodies (lanes 5
and 6).
[0111] FIGS. 3A, 3B, 3C and 3D: [0112] Detection of tau protein by
immunochemistry.
[0113] FIG. 3A: Section from hippocampus of a patient with
Alzheimer's disease. Magnification 78.times..
[0114] FIG. 3B: Section from hippocampus of an aged control patient
deceased from non-neurological causes. Magnification 78.times..
[0115] FIG. 3C: Section from hippocampus of a patient with
Alzheimer's disease. Magnification 245.times..
[0116] FIG. 3D: Section from hippocampus of an aged control patient
deceased from non-neurological causes. Magnification
245.times..
[0117] FIG. 4: Immunoblot analysis of Example 5 of a mutated
recombinant tau (clone htau24) phosphorylated with the protein
kinase activity from rat brain. Immunoblots were carried out with
anti-tau antibodies 134 and AT8. Lanes: 1, tau24; 2, tau24+brain
extract; 3, tau24 S199A; 4, tau24 S199A+brain extract; 5, tau24
S202A; 6, tau24 S202A+brain extract; 7, tau24 S199A, S202A; 8,
tau24 S199A, S202A+brain extract.
EXAMPLE I
Preparation of the monoclonal antibodies AT8 IqG1, .lamda.
[0118] 1. Preparation of the Antigen for Immunization:
[0119] 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 vol. 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 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 820 (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.
[0120] The antigen preparation was evaluated by a 10% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis and followed by
Western blotting using immunoblotting with polyclonal rabbit
antihuman normal tau antiserum.
[0121] 2. Immunization Protocol and Fusion Procedure:
[0122] Balb/c mice were primed with 100 .mu.g partially purified
PHF-tau in complete Freund's adjuvant and boosted three times
thereafter at 3-week intervals with 100 .mu.g of the same antigen
in incomplete Freund's. On days 3 and 2 before the fusion, mice
were boosted with 100 .mu.g PHF-tau saline.
[0123] Mouse spleen cells were fused with SP2/0 cells, using a
modified procedure of Kohler and Milstein (Kohler, G. and Milstein,
C., 1975), with PEG 4000.
[0124] Half of the cells were suspended at a density of
4.5.times.104 spleen cells/well on thirty 96-well peritoneal
macrophage feeder layer plates. 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. The other half of the
fusion was grown for three days in tissue culture flasks and stored
frozen in liquid nitrogen. Hybridoma growth was selected by
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 8% CO.sub.2 air incubator.
[0125] 3. Sandwich ELISA for Antibody Screening:
[0126] The screening ELISA used for the detection of anti-tau
monoclonal antibodies was a sandwich ELISA system with polyclonal
rabbit anti-human tau antibodies in the coating phase. For this
purpose polyclonal rabbit anti-human tau serum was
affinity-purified (as described in the passage hereinafter entitled
"Production of polygonal rabbit anti-tau antiserum" in Example IV).
Purified human normal tau (prepared as described in the passage
hereinafter entitled "Production of affinity purified human tau" in
Example IV) 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 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
nonspecific 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 three 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.
[0127] Absorbance was read in a Titertek Multiscan (Flow
Laboratories, Eflab, Oy, Finland) at 450 nm.
[0128] 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
instead of PHF-tau. The hybridoma-secreting antibodies specifically
recognizing the PHF-tau was subcloned by limiting dilutions. Said
hybridoma which secretes the AT8 antibodies will be designated
AT8.
EXAMPLE II
Detection of Pathological Tau and Absence of Detection of Normal
Tau in ELISA by Western Blotting
[0129] 1. Detection of Abnormally Phosphorylated Tau in ELISA and
Absence of Detection of Normal Tau in ELISA:
[0130] According to the protocol outlined in Example I, section 3,
affinity purified polyclonal anti-tau antibodies were coated on
ELISA plates and reacted with different dilutions of either
affinity-purified normal tau as described in the passage titled
"Production of affinity purified human tau" or of PHF-tau, each
prepared in a solution of PBS and 10% casein. After washing, the
plates were incubated with a fixed concentration of the AT8
monoclonal antibodies. All subsequent procedures were as described
(Example I, section 3). The results shown in Table I clearly
indicate that the AT8 monoclonal antibodies react only with
PHF-tau.
TABLE-US-00001 TABLE I Detection of PHF-tau or normal tau in ELISA
using the AT8 monoclonal antibodies for detection A.sub.150 nm
PHF-tau 1/20 1.459 1/200 1.179 1/2000 0.565 Normal tau 1 .mu.g/ml
0.021 100 ng/ml 0.005 10 ng/ml 0.001 Blank 0.000
[0131] The assays were performed as outlined in Example I, section
3. Different dilutions of PHF-tau or normal tau were used, as
indicated in the table.
[0132] The reactivity pattern of the AT8 monoclonal antibodies was
also studied in brain homogenates. To this end, high-binding
microtiter plates (Nunc, Gibco, Paisley, UK) were coated overnight
at 4.degree. C. with 2 .mu.g/ml of purified AT8 monoclonal
antibodies in coating buffer (10 mM Tris, 10 mM NaCl, 10 mM
NaN.sub.3, pH 8.5).
[0133] Overcoating to reduce non-specific binding was performed for
30 min with 120 .mu.l blot buffer (5%, w/v skimmed dried milk and
10% v/v newborn calf serum). After washing 3 times with PBS-0.05%
Tween 20 (v/v), 100 .mu.l sample was added, and the incubation was
carried out for 1 h at 370C. The plates were washed again three
times and incubated with 100 .mu.l of a 1/2000 dilution of rabbit
anti-tau serum. Next, the plates were washed again three times,
after which 100 .mu.l horseradish peroxidase conjugated donkey
anti-rabbit serum diluted 1/2000 in blot buffer was added and the
incubation was continued for 30 min. The plates were washed and 100
.mu.l of a solution consisting of 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 substrate. The reaction was stopped with 50 .mu.l of a 2 M
H.sub.2SO.sub.4 solution. Absorbance was read in Titertek Multiscan
(Flow Laboratories, Eflab, Oy, Finland) at 450 nm.
[0134] As can be seen from FIG. 1, the brain homogenates of the 4
Alzheimer patients (AD.sub.1-AD.sub.4) reacted positively at two
different dilutions, while all extracts prepared from brain derived
from patients who died of non-neurological diseases
(C.sub.1-C.sub.4) were clearly negative at both dilutions.
[0135] 2. Detection of Pathological Tau in Western Blotting and
Absence of Detection of Normal Tau in Western Blotting:
[0136] Purified normal human tau and PHF-tau were applied to 10%
SDS-polyacrylamide gels and run under denaturing conditions
according to Laemmli (1970).
[0137] 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 AT8 as primary antibody. 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 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. 2 indicate
that the AT8 antibody recognizes 64 and 68 kDa tau isoforms but
show that normal tau bands remain unstained.
EXAMPLE III
Detection of Tau by Immunocytochemistry
[0138] 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.
[0139] Cryostat sections from Alzheimer and age-matched control
brain were also prepared. Tissues were immunostained either with
the peroxidase-antiperoxidase (PAP) technique (Steinberger, L. A.
et al., 1970) or with the avidin-biotin complex (ABC) technique
(Hsu, S. M. et al., 1981) using Dakopatts (Denmark) and Amersham
(UK) reagents, respectively. Briefly, after blocking nonspecific
interactions with normal swine serum (Dakopatts X901) diluted 1:25
in Tris-buffered saline (TBS) containing 1% bovine serum albumin
(BSA), sections were incubated overnight with the AT8 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'-diaminobenzidine tetrahydrochloride (Sigma). Sections were
counterstained with Harris' hematoxylin, dehydrated, coverslipped,
and viewed under a light microscope.
[0140] FIG. 3 (A to D) clearly indicates that AT8 does not decorate
any normal structures but only produces abundant staining of NFT,
dystrophic neurites in plaques, and dispersed staining of neuropil
(neuropil threads). Some apparently tangle-free neurons were
diffusely stained, often exhibiting a strong perinuclear
staining.
EXAMPLE IV
Competition ELISAs to Characterize the Epitopes of Known Antibodies
with the Antibody of the Invention
[0141] Affinity-purified rabbit anti-human tau polyclonal antibody
was coated overnight at 4.degree. C. in the wells. Plates were
washed, and 100 .mu.l of purified abnormally phosphorylated tau,
mixed with affinity-purified normal tau, was added per well for 1 h
at 37.degree. C. After washing, 50 .mu.l of the different unlabeled
monoclonal antibodies to be tested were added in several
dilutions.
[0142] Subsequently, 50 .mu.l biotinylated AT8 antibody or
biotinylated BT2 antibody obtained as described in the passage
titled "Production of the monoclonal antibody BT2" was added in an
amount previously determined to provoke 50% of the maximal binding.
After 1 h at 37.degree. C. the plates were washed and a
streptavidin biotin peroxidase complex was added for 30 min to
allow detection of the ELISA with 3,5,3',5'-tetramethylbenzidine.
After stopping the reaction with 2 M H.sub.2SO.sub.4 the plates
were read in a Titertek Multiscan plate reader (Flow) at 450
nm.
[0143] As the results of Table II indicate, the BT2 antibodies
inhibit the binding of biotinylated BT2, while casein or AT8 do not
influence this binding. Conversely, the AT8 antibody completely
blocks the binding of biotinylated AT8, but neither casein or BT2
inhibit this reaction.
TABLE-US-00002 TABLE II Competition ELISA to characterize the
epitopes of known antibodies. BT2-bio AT8-bio Concentration Casein
BT2 AT8 Casein BT2 AT8 9 .mu.g/ml 0.369 0.071 0.369 0.578 0.548
0.056 3 .mu.g/ml 0.390 0.088 0.375 0.573 0.557 0.054 1 .mu.g/ml
0.375 0.122 0.368 0.548 0.550 0.059 333 ng/ml 0.381 0.179 0.369
0.564 0.556 0.093 111 ng/ml 0.385 0.245 0.381 0.576 0.573 0.155 37
ng/ml 0.395 0.318 0.360 0.568 0.549 0.282 BT2 or AT8 were used at
the indicated concentrations in the left column. All antibody
dilutions were carried out in casein which was also used at the
same concentration in the casein controls (column marked
casein).
[0144] Production of Polyclonal Rabbit Anti-Tau Antiserum:
[0145] New Zealand white rabbits were immunized with affinity
purified human tau. Rabbits were injected intradermally with 100
.mu.g affinity purified human tau emulsified in complete Freund's
adjuvant. Two weeks later, this was repeated intramuscularly with
200 .mu.g affinity purified human tau in incomplete Freund's, and a
third intramuscular injection was carried out after one week with
100 .mu.g affinity purified human tau in saline. The rabbits were
bled one week after the third injection, evaluated and again
injected twice, after a month's interval with the same amount of
tau as used for the third injection. The sera were evaluated in a
solid phase ELISA with affinity purified human tau in the coating
phase and in Western blot against affinity purified human tau.
[0146] Production of Affinity Purified Human Tau:
[0147] Typically, 50 grams post-mortem human brain was out into
small pieces with scissors and homogenized 1/1 (W/V) in buffer A
(20 mM w (2-(N-morpholino)ethanesulfonic acid), 80 mM NaCl, 2 mM
EDTA, 0.1 mM EGTA, 1 mM MgCl.sub.2, 1 mM mercaptoethanol, pH 6.75)
with a Potter homogenizer equipped with a teflon plunger. The
homogenate was centrifuged for 1 h at 150000 g at 4.degree. C., and
the supernatant was heated for 5 min in boiling water and chilled
again for 10 min on ice. The slurry was centrifuged for 2 h at
150000 g at 4.degree. C., and the supernatant was collected
thereafter and called the "heat stable cytosolic extract".
[0148] Ten mg BT2 anti-tau monoclonal antibody, purified from
ascites fluid on protein G (Pharmacia, Uppsala, Sweden), was
coupled to 1 gram cyanogen-bromide activated by Sepharose
(Pharmacia) following the method proposed by the manufacturer.
Fifty ml of the heat-stable cytosolic extract were diluted 1/2 in
0.1 M phosphate buffer pH 8.5 and applied to the column. The column
was washed with 0.1 M phosphate and tau was eluted with 0.1 M
citric acid pH 2.5 and neutralized immediately with 1 M NaOH.
Fractions were evaluated in 10% SDS-PAGE in immunoblotting with
anti-tau antibodies.
[0149] Production of the Monoclonal Antibody BT2:
[0150] Antigen Purification:
[0151] Bovine tau: tau was purified from bovine brain by a
modification of the perchloric acid method of Lindwall et al.
(1984). Typically, 50 grams fresh brain was cut into small pieces
with scissors and homogenized 1/1 (w/v) in buffer A (20 mM
w(2-(N-morpholino)ethanesulfonic acid), 80 mM NaCl, 2 mM EDTA, 0.1
M EGTA, 1 mM MgCl.sub.2, 1 mM mercaptoethanol, pH 6.75) with a
Potter homogenizer equipped with a teflon plunger. The homogenate
was centrifuged for 1 h at 150000 g at 4.degree. C., and the
supernatant was heated for 5 min in boiling water and chilled again
for 10 min on ice. The slurry was centrifuged for 2 h at 150000 g
at 4.degree. C., and the supernatant was collected thereafter. The
heat stable cytosolic extract was made to 2.5% perchloric acid and
was centrifuged for 1 h at 150000 g at 4.degree. C. after which the
supernatant was neutralized with 3 M Tris. The supernatant was then
dialyzed and concentrated in water in a centriprep concentrater
(Amicon, Lausanne, Switzerland). The end product, hereinafter
referred to as the "bovine tau", was evaluated in sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) performed
according to the method of Laemmli (1970).
[0152] Monoclonal Anti-Bovine Tau Antibody Production:
[0153] Balb/c mice were primed with 100 .mu.g purified bovine tau
in complete Freund's adjuvant and boosted 3 times with 100 .mu.g
bovine tau in complete Freund's every two weeks. On days 3 and 2
before the fusion, the mice were boosted again with 100 .mu.g
bovine tau in saline. SP2/0 myeloma cells were used as fusion
partner, and the fused cells were seeded on mouse peritoneal
macrophage feeder cells. Half the hybridomas were seeded in 96-well
places and screened after 10 days in a solid-phase ELISA on bovine
tau for anti-tau antibody production, the other half of the fusion
was grown for one day in tissue flasks, and these cells were frozen
and stored in liquid nitrogen. All hybridomas were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%
fetal calf serum, sodium pyruvate (1 mM), L-glutamine (2 mM),
penicillin (100 U/ml), streptomycin (100 .mu.g/ml), and
nonessential amino acids. All products were purchased from Gibco,
Paisley, U. K. Cells were incubated at 37.degree. C. in a
humidified 8% CO.sub.2 air incubator. Positive signals in ELISA
were tested in Western blot against heat stable cytosolic extract
from bovine brain with the anti-tau antibody tau-2 (Sigma, St.
Louis, Mo.) as a control. Positive wells were subcloned and the
clones were frozen in liquid nitrogen.
EXAMPLE V
Expression and Phosphorylation of Mutant Recombinant Tau Proteins
to Determine the Essential Phosphorylation Site(s) of the AT8
Epitope
[0154] A full-length cDNA clone (htau24), corresponding to a four
repeat isoform of tau and with a NdeI site in the context of the
initiator codon, was subcloned into the EcoRI site of M13 mp18. Two
constructs were made in which site-directed mutagenesis was used to
change codon 199 (S to A) or codon 202 (S to A) (using the
numbering of the longest human brain tau isoforms (Goedert, M. et
al., 1989), and a third construct was made, where codons 199 and
202 were both changed to A. Following cleavage with NdeI and EcoRI,
the resulting fragments were subcloned downstream of the T7 RNA
polymerase promoter in the expression plasmid pRK172, and the
recombinant plasmids were transformed into E. coli BL21 (DE3)
cells. The bacterial cultures were grown, induced and harvested as
described (Goedert, M. et al., 1989).
[0155] The pellet from a 500 ml culture was resuspended in 20 ml of
extraction buffer (50 mM PIPES, pH 6.8, 1 mM DTT, 1 mM EDTA, 0.2 mM
PMSF, 0.5 .mu.g/ml leupeptin, 0.5 .mu.g/ml pepstatin) and sonicated
for 2.times.3 min using a Kontes microultrasonic cell disrupter.
Following centrifugation (15,000 rpm for 15 min), the supernatant
was passed through a DE-52 cellulose column. The flow-through was
loaded onto a phosphocellulose column (bed volume 3 ml)
equilibrated in the extraction buffer. After exhaustive washing
with the extraction buffer, protein was eluted batchwise with 3 ml
aliquots of extraction buffer containing 0.5 M NaCl. The fractions
containing the recombinant tau isoforms were pooled and dialyzed
overnight against 50 mM MES, 1 mM DTT, pH 6.25. After
centrifugation, the dialysate was loaded onto a fast flow
carboxymethyl-Sepharose HR 5/5 column. The column was washed with
50 mM MES, 1 mM DTT, 50 mM NaCl, pH 6.25, and the protein was
eluted using 100-300 mM NaCl in 50 mM MES, 1 mM DTT, pH 6.25,
gradient. Column fractions were screened by gel electrophoresis,
and the peak tau fractions were pooled and dialyzed against 40 mM
HEPES, 1 mM DTT, 0.2 mM PMSF, pH 7.2. The protein concentrations
were determined by amino acid composition.
[0156] The resulting recombinant tau proteins were phosphorylated,
using brain protein kinase activity as follows:
[0157] Adult rat brain was homogenized (1 g/2.5 ml) in 10 mM
Tris/HCl, pH7.4, 5 mM EGTA, 2 mM DTT, 1 .mu.M 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. Incubations were
carried out at 37.degree. C. with 40 nM HEPES, pH 7.2, 2 mM AtP, 2
mM MgCl.sub.2, recombinant tau protein (1 .mu.M), and rat brain
extract (0.05 ml) for 24 h, and aliquots were then taken for
immunoblotting. Controls were incubated under the same conditions,
except that the brain extract was omitted.
[0158] The AT8 epitope of the phosphorylated recombinant tau
proteins was analyzed by immunoblots. SDS-PAGE was carried out
using 10% or 10-20% gradient minigels. For immunoblotting,
phosphorylated and non-phosphorylated recombinant tau proteins were
transferred to a polyvinylidene difluoride (PVDF) membrane, and
residual protein binding sites were blocked with 1% gelatin in
phosphate-buffered saline. The blots were then incubated for 5 h at
room temperature with anti-tau antiserum 134 (dilution 1:250)
(Goedert, M. et al., 1989) or with antibody AT8 (dilution 1:500).
Bound antibody was detected by the biotin/peroxidase system
(Vectastain).
[0159] The results of the immunoblots show that phosphorylation of
at least the Ser 202 of the AT8 epitope is required for antibody
recognition.
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