CA2121599C - Monoclonal antibodies directed against the microtubule-associated protein tau - Google Patents
Monoclonal antibodies directed against the microtubule-associated protein tau Download PDFInfo
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- CA2121599C CA2121599C CA002121599A CA2121599A CA2121599C CA 2121599 C CA2121599 C CA 2121599C CA 002121599 A CA002121599 A CA 002121599A CA 2121599 A CA2121599 A CA 2121599A CA 2121599 C CA2121599 C CA 2121599C
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
- G01N2333/4701—Details
- G01N2333/4709—Amyloid plaque core protein
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/28—Neurological disorders
- G01N2800/2814—Dementia; Cognitive disorders
- G01N2800/2821—Alzheimer
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
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.
Description
9~V0 93/0830? ~ ~ ~ ~ ~ ~ ~ ' PC1'/EP92/02392 MONOCLONAL ANTIBODIES DIRECTED AGAINST THE MICROTUBULE-ASSOGIATED
PROTEIN TAU
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.
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 (ire, M. ~. and Karsenti, ~. 1990).
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 l~ilzheimer ~'atients (Greenberg, ~. and Dav~.es, ~. , 19901 ~e, V .M. °~. et al s , 1991) .
The protein exists as a family of different isofor~as of which ~ to 6 isoforms are found in normal adult brain but only 1 isoform is detected in fetal brain (Doedert, M. et al., 1989). The diversity of -the isoforms is generated from a single gene by alternative mRNA
splicing (FIimmler, A., 1989). The most striking feature of tau protein as predicted from molecular cloning is a stretch of 31 or 3~ amino acids occurring in the carboxy-terminal part of the molecule that is repeated 3 or 4 times. Additional diversity is generated through 2 ~ ~. ~.'~ ~. (~ 9 PCT/EP92/02392, , 29 or 58 amino acid long insertions in the NHZ-terminal part of the molecules (Goedert, M. et al., 1989).
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 plaques (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).
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: Xsiezak-Reding et al.~ 1987) or because they recognized specific epitopes on normal and abnormally phosphorylated tau. , The A1x50 monoclonal antibody (idoloxin, 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. , 1.988, Flament, S. and Delacourte, A. 1,990) 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).
The A1x50 epitope has recently been mapped to the NH2-terminal part of the tau molecule (Ksiexak-Reding, H. et al., 1990: Goedert, M, et al., 1991). Due to its WO 93/08302 ~ PCT/EP92/02392 cross-reactivity with normal tau, this antibody is only able to discriminate normal from abnormally phospharylated tau by the use of Western blotting detection of brain homogenates or by ammonium sulfate-concentrated CSF, or alas 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 (ferry et al., 1989).
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).
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.l. et al., 1985: Kosik, K.S.
et al., 1988), and most of the antibodies recognize~the normal and the abnormally phosphoxylated tau (Ksiezak-Redlng, H. et 81., 1990).
An antibody named x423", 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).
This antibody has been used to discriminate Alzheimer PHF pathology from normal controls in brain homogenates (Harrington, G.R. et al., 1990: patent W089/03993).
PROTEIN TAU
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.
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 (ire, M. ~. and Karsenti, ~. 1990).
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 l~ilzheimer ~'atients (Greenberg, ~. and Dav~.es, ~. , 19901 ~e, V .M. °~. et al s , 1991) .
The protein exists as a family of different isofor~as of which ~ to 6 isoforms are found in normal adult brain but only 1 isoform is detected in fetal brain (Doedert, M. et al., 1989). The diversity of -the isoforms is generated from a single gene by alternative mRNA
splicing (FIimmler, A., 1989). The most striking feature of tau protein as predicted from molecular cloning is a stretch of 31 or 3~ amino acids occurring in the carboxy-terminal part of the molecule that is repeated 3 or 4 times. Additional diversity is generated through 2 ~ ~. ~.'~ ~. (~ 9 PCT/EP92/02392, , 29 or 58 amino acid long insertions in the NHZ-terminal part of the molecules (Goedert, M. et al., 1989).
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 plaques (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).
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: Xsiezak-Reding et al.~ 1987) or because they recognized specific epitopes on normal and abnormally phosphorylated tau. , The A1x50 monoclonal antibody (idoloxin, 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. , 1.988, Flament, S. and Delacourte, A. 1,990) 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).
The A1x50 epitope has recently been mapped to the NH2-terminal part of the tau molecule (Ksiexak-Reding, H. et al., 1990: Goedert, M, et al., 1991). Due to its WO 93/08302 ~ PCT/EP92/02392 cross-reactivity with normal tau, this antibody is only able to discriminate normal from abnormally phospharylated tau by the use of Western blotting detection of brain homogenates or by ammonium sulfate-concentrated CSF, or alas 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 (ferry et al., 1989).
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).
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.l. et al., 1985: Kosik, K.S.
et al., 1988), and most of the antibodies recognize~the normal and the abnormally phosphoxylated tau (Ksiezak-Redlng, H. et 81., 1990).
An antibody named x423", 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).
This antibody has been used to discriminate Alzheimer PHF pathology from normal controls in brain homogenates (Harrington, G.R. et al., 1990: patent W089/03993).
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).
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.
Thus, in one aspect, the current. invention provides monoclonal antibody which forms an immunological complex with a phosphorylated epitope of an antigen belonging to human abnormally phosphorylat;ed 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'~~ disease or having died of Alzheimer's disease, wherein said monoclonal antibody is not liable to form an immunological complex with normal tau protein, is not liable to form an immunological complex with tau protein present in brain homogenates derived from human brain, the homogenates beina isolated from a patient having died of non-neurological disorders, is not liable to form an immunological complex with the above defined epitope previously treated with a dephosphorylating agent, such as alkaline phosphatase, is nc>t liable to form.
an immunological complex with any variant peptide defined 4a above and previously treated with a dephosphorylating agent, such as alkaline phosphatase.
According to one aspect of the invention, there is provided a monoclonal antibody capable of forming an immunological complex with a phosphorylated epitope of an antigen belonging to human abnormally phosphorylated tau protein, with said tau protein being obtained from a brain homogenate, said brain homogenate isolated from the cerebral cortex obtained from a patient having Alzheimer's disease or having died of Alzheimer's disease, wherein said monoclonal antibody is not capable of forming an immunological complex with normal tau protein; is not capable of forming an immunological complex with tau protein present in brain homogenates derived from human brain, the homogenates being isolated from a patient having died of non-neurological disorders; and is not capable of forming an immunological complex with the above-defined epitope previously treated with a dephosphorylating agent.
The invention also provides a hybridoma secreting such monoclonal antibodies.
Thus, in another aspect, the current invention further provides monoclonal antibody secreted by the hybridoma deposited at ECACC on October 8, 1991 under No. 91100806.
According to another aspect of the present invention, there is provided a peptide which can be i 4b obtained from a brain homogenate, said brain homogenate isolated from the cerebral cortex obtained from a patient having Alzheimer's disease, wherein said peptide forming an immunological complex with the monoclonal antibody described above, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
According to still another aspect of the present invention, there is provided a peptide capable of forming an immunological complex with the monoclonal antibody described above, said peptide comprising the sequence YSS*PGS*PFT or YSSPGS*PGT, phosphorylated at the positions marked with *, or a sequence capable of forming an immunological complex with a monoclonal antibody capable of forming a complex with the peptide YSS*PGS*PGT or YSSPGS*PGT, phosphorylated at the positions marked with *, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
According to yet another aspect of the present invention, there is provided a peptide (antigen) which contains a sequence selected from the group consisting of:
(Xi) p (X2) qS*PX3S*P (X4) r (Xs) S: and (X1) p (X2) qSPX3S*P (X4) r (X5) s in which X1, X2, X3, X9, XS are selected from the group consisting of naturally occurring amino acids, and p, q, r, s are 0 or 1, phosphorylated at places marked by 4c *, provided that said peptide is capable of forming an immunological complex with the monoclonal antibody described above.
According to a further aspect of the present invention, there is provided a process for obtaining and isolating a hybridoma secreting the monoclonal antibody described above, providing spleen cells of an animal previously immunized in vivo or spleen cells of such animal previously immunized in vitro with an antigen recognized by the monoclonal antibody deposited at ECACC on October 8, 1991 under No. 91100806; fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and selecting those of the hybridomas which secrete the monoclonal antibodies which specifically recognize an epitope of the peptide described above and which form an immunological complex with said epitope.
According to yet a further aspect of the present invention, there is provided a process for producing monoclonal antibodies described above, said process comprising: culturing the selected hybridomas described above, in an appropriate culture medium;
recovering the monoclonal antibodies excreted by said selected hybridomas: or alternatively, implanting the selected hybridomas described above into the peritoneum of a mouse, and when ascites has been produced by the mouse, recovering the monoclonal antibodies then formed from said ascites.
4d According to still a further aspect of the present invention, there is provided a method for the preparation of abnormally phosphorylated tau from brain 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 is implicated, said method comprising: contacting the monoclonal antibody described above, with a preparation of neurofibrillary tangles (NFT) or a detergent-extracted brain homogenate isolated from a patient having had said brain disease under conditions suitable for producing an antigen-antibody complex and separating the antigen from said complex and recovering said antigen in a purified form.
According to another aspect of the present invention, there is provided a kit for the diagnosis in vitro of a disease selected from the group consisting of Alzheimer's disease, Down's syndrome, Pick's disease, subacute spongiform panencephalitis and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated, said kit comprising:
- at least a microplate for deposition thereon of a first antibody, wherein said first antibody is the monoclonal antibody as described above;
- a second antibody, wherein the second antibody is selected from the group consisting of:
i) a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of the peptide as described 4e above, with said epitope being different from an epitope recognized by the first antibody, and ii) a polyclonal antibody recognizing normal tau, or abnormally phosphorylated tau or the peptide described above, with said polyclonal antibody being capable to form an immunological complex with epitopes which are all different from an epitope recognized by the first antibody;
- a marker either for specific tagging or coupling with said second antibody; and - appropriate buffer solutions for carrying out the immunological reaction between the first monoclonal antibody and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
According to a further aspect of the present invention, there is provided a process for the preparation of the peptide described herein, starting from said peptide in non-phosphorylated form which involves:
- reacting said peptide, which is non-phosphorylated, with a kinase enzyme capable of recognizing the non-phosphorylated epitope of the peptide and of modifying the epitope to a phosphorylated epitope recognized by the monoclonal antibodies described herein.
According to still a further aspect of the present invention, there is provided a process for the 4f detection or diagnosis in vitro of brain disease involving paired helical filaments (PHF) and tau protein which involves:
contacting a monoclonal antibody described herein, with a preparation of neurofibrillary tangles (NFT) or a detergent-extracted brain homogenate isolated from a patient having had said brain disease under conditions suitable for producing an antigen-antibody complex; and - separating the antigen from said complex and recovering the antigen sought in a purified form.
The invention further provides a kit comprising the monoclonal antibody described herein together with instructions for the diagnosis in vitro of a disease selected from the group consisting of Alzheimer's disease, Down's syndrome, Pick's Disease, subacute spongiform panencephalitis and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated.
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.
The invention still further provides the epitope of tau protein expressed in the brain of 4g patients affected with neurological disorders such as Alzheimer's disease and Down's syndrome.
The invention yet further provides a process for the detection of diagnosis in vitro of brain diseases involving tau protein.
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.
By "non-structural" epitopes i.s 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.
The expression ''form an immuno~logically 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:
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 pe:roxidase 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 seactions:
Brain tissue sample, obtained at: surgery ar autopsy, is fixed in either Bouin's fixative or 1U%
buffered formalin before sectioning 60 mm thick without embedding (Vibratome). The monoclonaT~.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).
- *Trade-mark f,' 734-2 s Immunoblottina procedures:
For immunoblestting, fractions enriched in PHF are prepared as described by Iqbal, K. et al. (1984) or Greenberg, S. and Davies, P. (199I)). 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 buffet 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 2?000 x g for 20 min at 4'C, the pellet is removed and the supernatant is 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 3?'C. The supernatant mixture is centrifuged at 108 , 000 x g for 35 min at 20' C. The PHF-tau-containing pellet is gently washed with PBS and finally suspended .in 1 ml of the same buffer.
SDS-polyacrylamide electrophoresis is performed under reducing conditions on 12% gels (Laemmli U.K., 19?0) . After electrophoresis, the proteins are eitraer fixed and stained with Coomassie brilliant blue, or transferred (Towbin H. et al., 1979) to nitrocellulose sheets (Hybond-C, Amersham) or Immobilon filters (Millipore).
After transfer the filters are presoaked in PBS
containing 0.05% (v/v) Tween'~ 20 (T4~een-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'C with a monoclonal. antibody of the invention appropriately diluted in blocking buffer.
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
*Trade-mark (Dakopatts, Denmark) diluted 1/3000 in blocking buffer.
After three washes in Tween-PBS, streptavidine-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.
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.
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).
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 2~.21~99 Alzheimer's disease or having died from Alzheimer's disease.
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.
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. (199~j.
Monoclonal antibodies of the invention are characterized by the fact that they form an immunological complex - either with the peptide YS~PG~PGT or YSSPG~PGT, preferably YSSPG~PGT, phosphoxylated at the positions marked with ' - 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.
The peptide YS~PG~PGT or YSSPGSPGT, preferably, YSSPG~PGT, will be hereinafter designated as "the epitope" of the invention. The serines in positions 199 and 20~ using the numbering of human tau 4~ (Goedert, M. et al., 1989) are in the form of phosphoserines. The epitope spans the amino acids 197-205 using the above-mentioned numbering.
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".
A peptide phosphorylated at a certain position means that the serine is in the phosphoserine form.
WO X3/08302 ~ ~ ~ ~ ~~ ~ ~ PCT/EP92/02392 The invention also relates to monoclonal antibodies which are not liable to form an immunological complex with normal tau protein.
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.
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.
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.
Advantageously, the monoclonal antibodies of the invention are characterized by:
- the fact that they form an immunological complex with the abnormally phosphorylated forms of tau protein, present in homogenates of human bra4i.n obtained from a patient who died of Alzheimer's disease and - 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 - 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.
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.
. ...:.; , : ....- . :... ,.v, . . .: .. . - , .. . .. ., .; .:: . .:. . , WO 93/08302 ~ ~ ~ ~ PCT/EP92/02392 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 October 8, 1991 under No. 91100806. This hybridoma will be hereinafter referred as "hybridoma AT8°°, and the secreted monoclonal antibody will be referred as "monoclonal antibody AT8".
The invention also relates to an hybridoma which secretes a monoclonal antibody according to the invention, and particularly the hybridoma filed at ECACC on October 8, 1991 under No. 91100806.
The monoclonal antibodies of this invention are obtained by a process involving obtention and isolation of hybridomas which secrete the monoclonal antibodies.
A process for obtaining such a hybridoma involves:
- 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 October 8, 1991 under I~do. 9110~806;
- fusing such immunized cells with myeloma cells under hybridoma-forming conditions; and - 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.
A process for producing the corresponding monoclonal antibodies involves:
- culturing the selected hybridoma as indicated above in an appropriate culture medium; and - recovering the monoclonal antibodies excreted by the selected hybridoma, or alternat~.vely - 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.
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.
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 manoclonal antibody of the invention.
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 - which contain or are constituted by the sequence YS~PG~PGT or YSSPGSPGT, phosphorylated at the positions marked with *, or Which contain ar 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 liab*e to form a complex with the peptide YSSPGSPGT or YSSPGSPGT, a_ .a_ cz ?-n an embodimer:ar , s~:~:i.~ p~ept.~.cie ~. s nat the 68, 64 or 60 kDa prot~: iri i.sol«ted frar~u AIJ:r~zeimer' s disease brain.
The inventic;n s.iso :e::~e~at:es tea peptides ~ant.ic~ens) of about 100 am~.no aoic~s -- which c:ontairl t: he sec.~uer~c~e YS~SPGSF~GT or YSSPGSPGT, phosp.uory.l.aweci at t.?ue positions marked with * , or WO 93/~302 ~ 121 ~ 9 9 PCT/EP92/02392 which contain the sequence of the variant peptides defined above.
The inventian also relates to peptides (antigens) which contain the sequence (X1)p(X2)q~PX3SP(X,~~r~XS)s ar (X1)p(X2)qSPX3~P(X4) r(X5)s~
preferably (x,)p(x2)qsPX3~P(X~)r(x5)s, in which X~, XZ, X3, X,~, X~ are any one of the 2a amino acids and p, q, r, s are o 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.
The invention also relates to the above-mentioned peptides which are liable to generate monoclonal antibodies of the invention.
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. a 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 noraaally participate in peptide bond formation, in particular after activation of the carboxyl group, Wa 93/08302 2 ~ ~ i C~ (~ g PCT/EP92/02392 according to methods known in peptide synthesis, and so on, proceeding stepwise up to the N-terminal amino acid'.
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.
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:
- r~acting 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.
The kinase used is advantageously extracted from brain according to methods known to those skilled in the art (Isbiguro K. et al., 1988: Gaudier, J. and Cole, R. D. , 1987: Vallee, R. B. , 1980) and i~ different from the kinases referred to in Steiner et al. (1990).
The non-phosphorylated antigen is, for instance, a , normal human tau protein which, by phosphorylation with the above-mentioned kinase, gives rise to an abncirmally phosphorylated tau protein, which is recognized by the monoclonal antibody of the invention. Such abnormally phosphorylated tau protein is new.
The antigen of the invention, which can be prepared. by methods known to those skilled in the art (Iqbal, R. 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 ,. . . ' ,. . ,..._.. ,;'.:, ..: ';.w~. ~. ~-....._.,y.. , , ~ .;::.: ~.
WO 93/0$302 ~~ ~ ~ PCT/EP92/02392 monoclonal antibody secreted by the hybridoma deposited at the ECACC under No. 91100806.
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.
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:
- 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:
separating the antigen from said complex and recovering the antigen sought in a purified form.
The preparation of PHF can be carried outs according to Iqbal K. et al. (1984) or Greenberg et al.
0990) . _ ~ .
Advantageously, the monoclonal antibody used is in an immobilized state on a suitable support such as a resin. The proeess for the detection of the antigen can then be carried out as follows:
- 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;
- washing the immobilized antibody-antigen complex so formed:
W~ 93/0830? 2 ~, ~ ~ ~ ~ ~ fCT/Ef92/02392 treating that complex with a solution (e.g., 3 M
potassium thiocyanate, x.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 - recovering the antigen in a purified form.
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:
- 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 - detecting the immunological binding of said antibody to said sample of brain homogenate, or of cerebrospinal fluid, or of serum.
The detection of the immunologically bound monoclonal antibody can be achieved fn a cpnventional 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.
A particularly advantageous embodiment of the process of the invention comprises contacting a sample of cerebrospinal fluid (containing the corresponding WO 93108302 2121 ~ ~ ~ PGT/EF92/02392 antigen) obtained from a patient to be diagnosed with the monoclonal antibody of the invention.
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:
- at least a microplate for deposition thereon of any monoclonal antibody of the invention;
- 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 YS~PGSPGT or YSSPGSPGT phosphorylated at the positions marked with *:
- a second antibody . which can be a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of .any peptide of the invention, such epitope being different from the one of the invention, or ,.
. 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 wfiich are all different from the epitope of the invention, and the polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein, or - a masker either for specific tagging or coupling with the second antibody;
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.
The labeled peptide mentioned above can be a peptide which has been labeled by any means known to WU 93/08302 ~ Z ~ ~ ~ ~ PCT/EP92/02392 those skilled in the art. Likewise, the marker specific for tagging and coupling can be any marker known to those skilled in the art.
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.
T$e Figures Figure 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.
AD' - AD,~: Brain homogenates from 4 different Alzheimer patients diluted 1/200 (solid bars) or 1/1000 (stippled bars).
C9 - C4: Brain homogenates from .4 different controls, diluted 1/00 (solid bars) or 1/1000 (stippled bars).
The ordinate represents the optical density at 450 nm and the abscissa represents the different samples.
Figure 2: Western blotting detection of normal tau or PHF-tau using either the monoclonal antibodies Tau-1 or ATB. 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 ATS
monoclonal antibodies (lanes 5 and 6).
Figures 3A, 318, 3C and 3D:
Detection of tau protein by immunochemistry.
Figure 3A: Section from hippocampus of a patient with Alzheimer's disease. Magnification 't8 x.
Figure 3B: Section from hippocampus of an aged control patient deceased from non-neurological causes. Magnification 78 x.
Figure 3C: Section from hippocampus of a patient with Alzheimer's disease. Magnification 245 x.
Figure 3D: Section from hippocampus of an aged control patient deceased from non-neurological causes: Magnification 245 x.
Figure 4: Immunoblot analysis of Example 5 of a mutated recombinant tau (clone htau24) phophorylated~
with' the protein kinase activity from rat brain. Immunoblots were carried out with anti-tau antibodies 134 and ATB. panes: 1, tau24; 2, tau24 + brain extract; 3, tau24 S1~9A; 4, tau24 S199A + brain extract; 5, tau24 S202A; 6, tau24 5202.A. + brain extract;
7, tau24 S1~9A, S202A; 8, tau24 S199A, S202A
+ brain extract.
Example I: Preparation of the monoclonal antibodies AT8 ~IQG1, a) 1. Preparation of the antigen for immunization:
Postmortem tissue, consisting mostly of gray matter from the frontal and temporal cortex, was 1:734-2 obtained from histologically confirmed Alzheimer patients. This Alzheimer gray matter brain sample (5-10 g) was homogenized with l0 vol. of cold buffer H (l0 mM
Tris/1 mM EGTA/0.8 M NaCl/10% sucrose, pH 7.4~) in a Teflon%glass Potter' S (Braun, Ger~onany) homogenizer.
After centrifugation in a 60 Ti MSE rotor at 27,000 x g for 20 min at 4'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'C. The supernatant mixture was centrifuged at 108,000 x g for 35 min at 20'C. The PHF-tau containing pellet was gently washed with PBS
and finally suspended in 1 ml of the same buffer.
The antigen preparation was evaluated by a l0%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by Western blotting using immunoblotting with polyclonal rabbit antihuman normal tau antiserum.
2. Immunization protocol and fusion procedure:
Balb/c mice were primed with loo ~g partially purified PHF-tau in complete Freurad's adjuvant and boosted three times thereafter at 3-week intervals with 100 ug of the same antigen in incomplete Freund~s. on days 3 and 2 before the fusion, mice were boasted with 100 ~g PHF-tau saline.
Mouse spleen cells were fused with SP2/o cells, using a modified procedure of Koh.ler and Milatein (Kohler, G. and Milstein, C., 1975), with PEG 4000.
Half of the cells were suspended~at a density of 4.5 x 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 EhISA 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 *Trade-mark WO 93/08302 ~ .~.~, PGT/EP92/02392 selected by Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum, sodium pyruvate (1 ml~i), 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% COZ air incubator.
3. Sandwich ELISA for antibody screening:
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 polyclonal 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 ~cg,/ml) at 4'G on high-binding microtiter plates (Nunc, Gibco, Paisley, UK) in coating buffer (10 mM Tris, 10 mM NaCl, 10 mM NaN~, pH 8.5). After overcoating for 30 min with 125 ~1 10%-saturated casein in P1~S to reduce nonspecific binding, the plates were incubated with 100 u1 of an appropriately diluted PHF-tau preparation and incubated for 60 min at 37'C. The plates were washed three times with PBS-0.05% Tween 20 (v/v); 100 dal hybridoma supernatant was added, and incubation was continued for 1 h at 37'C. After washing, the bound monoclonal antibodies were detected with peroxidase-conjugated rabbit anti-mouse serum (Dakopatts, Glostrup, Denmark). All reagents were w l:'~ 734-2 27.
diluted in PHS with 1o% casein. After final washing, 7.00 ~1 0.42 mM 3,5,3',5'-tetramethylbenzidine, 0.003%
H2p2 v/v in 100 mM citric acid, loo mM disodium hydrogen phosphate, pH 4.3, was added as peroxidase substrate. The reaction was stopped with 50 ~l of a 2 M
H2SO~ solution.
Absorbance was read in a Titeri:ek Multiscan~' (Flow Laboratories, Eflab, Oy, Finland) at 45o nn.
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 P1~F-tau. The hybridoma-secreting antibodies specifically recognizing the PIiF-tau was subcloned by limiting dilutions. Said hybridoma which secretes the AT8 antibodies will be designated AT8:
Example II: Detection of patholoctical tau and absence of detection of normal tau in ELISA by Western blotting 1. Detection of abnormally phosphorylated tau in ELISA and absence of detection of noZSaal tau in ELISA:
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.
*Trade-mark ~Y~ 93/08302 PCT/EP92/02392 ~12~~~~
TABLE I. Detection of PHF-tau or normal tau in ELISA
using the AT8 monoclonal antibodies for detection PHF-tau~~ ~ nm 1/20 1,459 1/200 1,179 1/2000 0,565 Normal tau 1~/ml 0,021 100 n /ml 0,005 n /ml 0,001 Blank 0,000 i 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.
The reactivity pattern of the AT8 monoclonal antibodies was also studied in brain homogenates. To this end, high-binding microtiter plates (Nunc, Gibco,.
Paisley, UIt) were coated overnight at 4 ° C with 2 ~rg/ml of purified AT8 monoclonal antibodies in coating buffer (10 mM Tris, 1.0 mM NaCl, 10 mM NaN3, pH 8. ~) .
~vercoating to reduce non-specific binding was performed for 30 min with 120 u1 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 ~cl sample was added, and the incubation was Carried out for I h at 37°C. The plates were washed again three times and incubated with 100 ~1 of a 1/2000 dilution of rabbit anti-tau serum. Next, the plates were washed again three times, after which 100 ~cl horseradish peroxidase conjugated donkey anti-rabbit serum diluted 1:734-2 1/2000 in blot buffer was added and the incubation was continued for 30 min. The plates were washed and 100 ~cl of a solution consisting of 0.42 mM 3,5,3',5'-tetramethylbenzidine, 0.003% 8202 v/w in 100 mM citric acid, 100 mM disodium hydrogen phosphate, pH 4.3 was added as substrate.. The reaction was stopped with 50 ~.1 of a 2 M H2SO4 solution. Absorbance was read in Titertek Multiscan (Flow haboratories, Eflab, 0y, Finland) at 450 nm.
As can be seen from Fig. 1, the brain homogenates of the 4 Alzheimer patients (AD, - AD4) reacted positively at two different dilutions, while all extracts prepared from brain derived from patients who died of non-neurological diseases (C' - C4) were clearly negative at both dilutions.
2. Detection of pathological tau in Western blotting and absence of detection of normal tau in Western blotting:
Purified normal human tau and PHF-tau were applied to 10% SDS-polyacrylamide gels and run under denaturing conditions according to Laemmli (2970).
After SDS-PAGE, the transfer to nitrocellulose (Hybond-C; Amersham, Brussels, Belgium) was carried out in 10 mM NaHC03, 3 mM Na2C03, 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'e 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, *Trade-mark 1'I734-2 0.05% w/v 3,3'-diaminobenzidine, 0.03% v/v H202) after which the reaction was stopped in HZO. Results, shown in Fig. 2 indicate that the AT8 antibody recognizes 64 and 68 kDa tau isoforms but show tlhat normal tau bands remain unstained.
Example III: Detection of tau bY immunocytochemistry 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.
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.
Figure 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.
*Trade-mark WO 9310830? ~ ~ PCT/EP92/02392 Example IV: Competition ELISAs to characterize the epitopes of known antibodies with the antibody of the invention Affinity-purified rabbit anti-human tau polyclonal antibody was coated overnight at 4'C in the wells.
Plates were washed, and 100 ~cl of purified abnormally phosphorylated tau, mixed with affinity-purified normal tau, was added per well for 1 h at 37'C. After washing, 50 ~l of the different unlabeled monoclonal antibodies to be tested were added in several dilutions.
Subsequently, 50 gel 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'C
the plates were washed and a streptavidine 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 Ii2S0' the plates were read in a Titertek Multiscan plate reader (Flow) at 450 nm. ,, 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 ATB, but neither casein or BT2 inhibit this reaction.
WO 93/08302 PCI'/EP92/02392 ~12~.~~
TABLE II. Competition ELISA to characterize the epitopes of known antibodies.
Concentra- HT2-bio AT8-bio tion Casein HT2 AT8 Casein HT2 AT8 9 ~ /ml 0.369 0.071 0.369 0.578 0.548 0.056 3 ~ /~1 0.390 0.088 0.375 0.573 0.557 0.054 1 a /znl 0.375 0.122 0.368 0.548 0.550 0.059 333 n /ml 0.381 0.179 0.369 0.564 O.~i56 0.093 111 n /ml 0.385 0.245 0.381 0.576 0._i73 0.155 37 n /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). _ Production of polyclonal rabbit anti-tau antiserums New Zealand white rabbits were immunized with affinity purified human tau. ~tabbits were injected intradermally with 100 erg affinity .purified human tau emulsified in complete Freund's adjuvant. Two weeks Later, this was repeated intramuscularly with 200 ~cg affinity purified human tau in incomplete Freund's, and a third intramuscular injection was carried out after one week with 100 ~cg affinity purified human tau in saline. The rabbits were bled one week after the third injection, evaluated and again injected twice, after a 1:? 734-2 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.
- Production of affinity purified human tau:
Typically, 50 grams post-mortem human brain was out into small pieces with scissors and homagenized 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 MgCl2, 1 mM mercaptoethanol, pH 6.75) with a Potter homogenizes equipped with a teflon plunger. The homogenate was centrifuged for 1 h at 150000 g at 4'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'C, and the supernatant was collected thereafter and called the "heat stable cytosolic extract".
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 phi 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.
- Production of the monoclonal antibody BT2:
. Antigen purification:
Bovine tau: tau was purified from bovine brain by a modification of the perchloric acid method of hindwall 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-*Trade-mark 1=1734-2 morpholino)ethanesulfonic acid), .BO mM NaCl, 2 mM EDTA, 0 .1 M EGTA, 1 mM MgCl2, 1 mM merc:aptoethanol , pH 6 . ? 5 ) with a Potter homogenizes equipped with a teflon plunger. The homogenate was centrifuged for 1 h at 150000 g at 4 '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'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'C after which the supernatant was neutralized with 3 M Tris. The supernatant was then dialyzed and concentrated in water in a centriprep~ concentrates (Amicvn; 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).
. Monoclonal anti-bovine tau antibody production:
Balb/c mice were primed with 100 ~g purified bovine tau in complete Freund's adjuvant and boosted 3 times with 100 ~cg bovine tau in complete Freund's every two Weeks. On days 3 and 2 before the fusion, the mice were boosted again with 100 ~cg 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 wioth 10% fetal calf serum, sodium pyruvate (1 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 ~cg/ml), and nonessential amino acids. All products were purchased from Gibco, Paisley, *Trade-mark WO 93/08302 ~ ~ ~ ~, ~ ~ ~ i'CT/EP92/02392 U.K. Cells were incubated at 37°C in a humidifed 8% C02 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 phosphor5~lation of mutant recombinant tau proteins to deteranine the essential phosphorylation sites) of the AT8 epitope A full-length cDNA clone (htau24), corresponding to a four repeat isoform of tau and with a NdeI site in the context of the in~.tiator colon, was subcloned into the EcoRI site of M13mp18. Two constructs were made in which site-directed mutagenesis was used to change colon 199 (S to A) or colon 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 colons 199 and 202 were both changed to A.
Following cleavage with Ndel and EcoRI, the resulting fragments were subcloned downstream of the T7 RNA
polymerase promoter in the expression plasmid pRK172, and the reeomf~inant plasmids were transformed into E.coli BIa21 (DE3) cells. The bacterial cultures were grown, induced and harvested as desribed (Goedert, M.
et al., 1989).
The pellet from a 500 ml culture was resuspended in 20 ml of extraction buffer (50 mM PIPES, pFi 6.8, imM
DTT, 1 mM EDTA, 0.2 mM PMSF, 0.5 ~g/ml leupeptin, 0.5 ~g/ml pepstatin) and sonicated for 2 x 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 m1) equilibrated in the extraction W~ 93/08302 PCT/EP92/02392 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 NaGl, pH 6.25, and the protein was eluted using 100-300 mM NaCl in 50 mM MES, Z mM DTT, pH 6.25, gradient. Column fractions were screened by gel electrophorisis, 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.
The resulting recombinant tau proteins were phosphorylated, using brain protein kinase activity as follows:
Adult rat brain was homogenized (lg/2.5m1) in 10 mM Tris/HCl, pH7.4, 5 mM EGTA, 2 mM DTT, Z EsM okadaic acid, 1 mM PMSF, 20 ~g/ml leupeptin,~ 20 ~g/ml aprotinin and 20 ~g/ml pepstatin and centrifuged at 40,000 rpm for 1 h at 4'C. The supernatant was used directly for phosphorylation. Incubations were earried out at 37 ° C with ~ 0 mM HEPES , pH 7 . 2 , 2 mNt ~lfP, ~ 2 ~ mM
MgCl~, recombinant tau protein (1 ~aM), and rat brain extract (0.05 ml) for 24 h, and aliquots were then taken for immunoblotting. Controls were incubated sander the same conditions, except that the brain extract was omitted.
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 °1734-2 protein binding sites were blocked with 1% gelatin in phosphate-buffered saline. The blots were then incubated far 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).
The results of the immunoblots show that phosphorylation of at least the Ser 202 of the AT8 epitope is required for antibody recognition.
- *Trade-mark WO 93/08382 PCf/EP92/02392 REFERENCES
1. Bawdier, J. and Cole, R.D., J. Biol. Chem. 262, 1?577-17583.
2. Binder, L.I., Frankfurter, A. and Rebhun, L.I., J.
Cell Biol. 101, 1371-1378 (1985).
3. Bre, M.N. and Karsenti., E., Cell Motil.
Cytoskeleton 15, 88-98 (1990).
4. Brion, J.P., Couck, A.M., Passareiro, H, and Flament-Durand, J., J. Submicrosc. Cytol., 17, 89-96 (1985).
Harrington C.R. et al., 1990, Wisniewski, H.M. et al., 1989;
Wolozin, B. and Davies, P. 1987).
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.
Thus, in one aspect, the current. invention provides monoclonal antibody which forms an immunological complex with a phosphorylated epitope of an antigen belonging to human abnormally phosphorylat;ed 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'~~ disease or having died of Alzheimer's disease, wherein said monoclonal antibody is not liable to form an immunological complex with normal tau protein, is not liable to form an immunological complex with tau protein present in brain homogenates derived from human brain, the homogenates beina isolated from a patient having died of non-neurological disorders, is not liable to form an immunological complex with the above defined epitope previously treated with a dephosphorylating agent, such as alkaline phosphatase, is nc>t liable to form.
an immunological complex with any variant peptide defined 4a above and previously treated with a dephosphorylating agent, such as alkaline phosphatase.
According to one aspect of the invention, there is provided a monoclonal antibody capable of forming an immunological complex with a phosphorylated epitope of an antigen belonging to human abnormally phosphorylated tau protein, with said tau protein being obtained from a brain homogenate, said brain homogenate isolated from the cerebral cortex obtained from a patient having Alzheimer's disease or having died of Alzheimer's disease, wherein said monoclonal antibody is not capable of forming an immunological complex with normal tau protein; is not capable of forming an immunological complex with tau protein present in brain homogenates derived from human brain, the homogenates being isolated from a patient having died of non-neurological disorders; and is not capable of forming an immunological complex with the above-defined epitope previously treated with a dephosphorylating agent.
The invention also provides a hybridoma secreting such monoclonal antibodies.
Thus, in another aspect, the current invention further provides monoclonal antibody secreted by the hybridoma deposited at ECACC on October 8, 1991 under No. 91100806.
According to another aspect of the present invention, there is provided a peptide which can be i 4b obtained from a brain homogenate, said brain homogenate isolated from the cerebral cortex obtained from a patient having Alzheimer's disease, wherein said peptide forming an immunological complex with the monoclonal antibody described above, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
According to still another aspect of the present invention, there is provided a peptide capable of forming an immunological complex with the monoclonal antibody described above, said peptide comprising the sequence YSS*PGS*PFT or YSSPGS*PGT, phosphorylated at the positions marked with *, or a sequence capable of forming an immunological complex with a monoclonal antibody capable of forming a complex with the peptide YSS*PGS*PGT or YSSPGS*PGT, phosphorylated at the positions marked with *, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
According to yet another aspect of the present invention, there is provided a peptide (antigen) which contains a sequence selected from the group consisting of:
(Xi) p (X2) qS*PX3S*P (X4) r (Xs) S: and (X1) p (X2) qSPX3S*P (X4) r (X5) s in which X1, X2, X3, X9, XS are selected from the group consisting of naturally occurring amino acids, and p, q, r, s are 0 or 1, phosphorylated at places marked by 4c *, provided that said peptide is capable of forming an immunological complex with the monoclonal antibody described above.
According to a further aspect of the present invention, there is provided a process for obtaining and isolating a hybridoma secreting the monoclonal antibody described above, providing spleen cells of an animal previously immunized in vivo or spleen cells of such animal previously immunized in vitro with an antigen recognized by the monoclonal antibody deposited at ECACC on October 8, 1991 under No. 91100806; fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and selecting those of the hybridomas which secrete the monoclonal antibodies which specifically recognize an epitope of the peptide described above and which form an immunological complex with said epitope.
According to yet a further aspect of the present invention, there is provided a process for producing monoclonal antibodies described above, said process comprising: culturing the selected hybridomas described above, in an appropriate culture medium;
recovering the monoclonal antibodies excreted by said selected hybridomas: or alternatively, implanting the selected hybridomas described above into the peritoneum of a mouse, and when ascites has been produced by the mouse, recovering the monoclonal antibodies then formed from said ascites.
4d According to still a further aspect of the present invention, there is provided a method for the preparation of abnormally phosphorylated tau from brain 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 is implicated, said method comprising: contacting the monoclonal antibody described above, with a preparation of neurofibrillary tangles (NFT) or a detergent-extracted brain homogenate isolated from a patient having had said brain disease under conditions suitable for producing an antigen-antibody complex and separating the antigen from said complex and recovering said antigen in a purified form.
According to another aspect of the present invention, there is provided a kit for the diagnosis in vitro of a disease selected from the group consisting of Alzheimer's disease, Down's syndrome, Pick's disease, subacute spongiform panencephalitis and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated, said kit comprising:
- at least a microplate for deposition thereon of a first antibody, wherein said first antibody is the monoclonal antibody as described above;
- a second antibody, wherein the second antibody is selected from the group consisting of:
i) a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of the peptide as described 4e above, with said epitope being different from an epitope recognized by the first antibody, and ii) a polyclonal antibody recognizing normal tau, or abnormally phosphorylated tau or the peptide described above, with said polyclonal antibody being capable to form an immunological complex with epitopes which are all different from an epitope recognized by the first antibody;
- a marker either for specific tagging or coupling with said second antibody; and - appropriate buffer solutions for carrying out the immunological reaction between the first monoclonal antibody and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
According to a further aspect of the present invention, there is provided a process for the preparation of the peptide described herein, starting from said peptide in non-phosphorylated form which involves:
- reacting said peptide, which is non-phosphorylated, with a kinase enzyme capable of recognizing the non-phosphorylated epitope of the peptide and of modifying the epitope to a phosphorylated epitope recognized by the monoclonal antibodies described herein.
According to still a further aspect of the present invention, there is provided a process for the 4f detection or diagnosis in vitro of brain disease involving paired helical filaments (PHF) and tau protein which involves:
contacting a monoclonal antibody described herein, with a preparation of neurofibrillary tangles (NFT) or a detergent-extracted brain homogenate isolated from a patient having had said brain disease under conditions suitable for producing an antigen-antibody complex; and - separating the antigen from said complex and recovering the antigen sought in a purified form.
The invention further provides a kit comprising the monoclonal antibody described herein together with instructions for the diagnosis in vitro of a disease selected from the group consisting of Alzheimer's disease, Down's syndrome, Pick's Disease, subacute spongiform panencephalitis and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated.
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.
The invention still further provides the epitope of tau protein expressed in the brain of 4g patients affected with neurological disorders such as Alzheimer's disease and Down's syndrome.
The invention yet further provides a process for the detection of diagnosis in vitro of brain diseases involving tau protein.
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.
By "non-structural" epitopes i.s 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.
The expression ''form an immuno~logically 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:
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 pe:roxidase 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 seactions:
Brain tissue sample, obtained at: surgery ar autopsy, is fixed in either Bouin's fixative or 1U%
buffered formalin before sectioning 60 mm thick without embedding (Vibratome). The monoclonaT~.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).
- *Trade-mark f,' 734-2 s Immunoblottina procedures:
For immunoblestting, fractions enriched in PHF are prepared as described by Iqbal, K. et al. (1984) or Greenberg, S. and Davies, P. (199I)). 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 buffet 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 2?000 x g for 20 min at 4'C, the pellet is removed and the supernatant is 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 3?'C. The supernatant mixture is centrifuged at 108 , 000 x g for 35 min at 20' C. The PHF-tau-containing pellet is gently washed with PBS and finally suspended .in 1 ml of the same buffer.
SDS-polyacrylamide electrophoresis is performed under reducing conditions on 12% gels (Laemmli U.K., 19?0) . After electrophoresis, the proteins are eitraer fixed and stained with Coomassie brilliant blue, or transferred (Towbin H. et al., 1979) to nitrocellulose sheets (Hybond-C, Amersham) or Immobilon filters (Millipore).
After transfer the filters are presoaked in PBS
containing 0.05% (v/v) Tween'~ 20 (T4~een-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'C with a monoclonal. antibody of the invention appropriately diluted in blocking buffer.
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
*Trade-mark (Dakopatts, Denmark) diluted 1/3000 in blocking buffer.
After three washes in Tween-PBS, streptavidine-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.
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.
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).
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 2~.21~99 Alzheimer's disease or having died from Alzheimer's disease.
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.
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. (199~j.
Monoclonal antibodies of the invention are characterized by the fact that they form an immunological complex - either with the peptide YS~PG~PGT or YSSPG~PGT, preferably YSSPG~PGT, phosphoxylated at the positions marked with ' - 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.
The peptide YS~PG~PGT or YSSPGSPGT, preferably, YSSPG~PGT, will be hereinafter designated as "the epitope" of the invention. The serines in positions 199 and 20~ using the numbering of human tau 4~ (Goedert, M. et al., 1989) are in the form of phosphoserines. The epitope spans the amino acids 197-205 using the above-mentioned numbering.
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".
A peptide phosphorylated at a certain position means that the serine is in the phosphoserine form.
WO X3/08302 ~ ~ ~ ~ ~~ ~ ~ PCT/EP92/02392 The invention also relates to monoclonal antibodies which are not liable to form an immunological complex with normal tau protein.
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.
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.
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.
Advantageously, the monoclonal antibodies of the invention are characterized by:
- the fact that they form an immunological complex with the abnormally phosphorylated forms of tau protein, present in homogenates of human bra4i.n obtained from a patient who died of Alzheimer's disease and - 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 - 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.
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.
. ...:.; , : ....- . :... ,.v, . . .: .. . - , .. . .. ., .; .:: . .:. . , WO 93/08302 ~ ~ ~ ~ PCT/EP92/02392 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 October 8, 1991 under No. 91100806. This hybridoma will be hereinafter referred as "hybridoma AT8°°, and the secreted monoclonal antibody will be referred as "monoclonal antibody AT8".
The invention also relates to an hybridoma which secretes a monoclonal antibody according to the invention, and particularly the hybridoma filed at ECACC on October 8, 1991 under No. 91100806.
The monoclonal antibodies of this invention are obtained by a process involving obtention and isolation of hybridomas which secrete the monoclonal antibodies.
A process for obtaining such a hybridoma involves:
- 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 October 8, 1991 under I~do. 9110~806;
- fusing such immunized cells with myeloma cells under hybridoma-forming conditions; and - 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.
A process for producing the corresponding monoclonal antibodies involves:
- culturing the selected hybridoma as indicated above in an appropriate culture medium; and - recovering the monoclonal antibodies excreted by the selected hybridoma, or alternat~.vely - 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.
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.
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 manoclonal antibody of the invention.
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 - which contain or are constituted by the sequence YS~PG~PGT or YSSPGSPGT, phosphorylated at the positions marked with *, or Which contain ar 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 liab*e to form a complex with the peptide YSSPGSPGT or YSSPGSPGT, a_ .a_ cz ?-n an embodimer:ar , s~:~:i.~ p~ept.~.cie ~. s nat the 68, 64 or 60 kDa prot~: iri i.sol«ted frar~u AIJ:r~zeimer' s disease brain.
The inventic;n s.iso :e::~e~at:es tea peptides ~ant.ic~ens) of about 100 am~.no aoic~s -- which c:ontairl t: he sec.~uer~c~e YS~SPGSF~GT or YSSPGSPGT, phosp.uory.l.aweci at t.?ue positions marked with * , or WO 93/~302 ~ 121 ~ 9 9 PCT/EP92/02392 which contain the sequence of the variant peptides defined above.
The inventian also relates to peptides (antigens) which contain the sequence (X1)p(X2)q~PX3SP(X,~~r~XS)s ar (X1)p(X2)qSPX3~P(X4) r(X5)s~
preferably (x,)p(x2)qsPX3~P(X~)r(x5)s, in which X~, XZ, X3, X,~, X~ are any one of the 2a amino acids and p, q, r, s are o 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.
The invention also relates to the above-mentioned peptides which are liable to generate monoclonal antibodies of the invention.
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. a 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 noraaally participate in peptide bond formation, in particular after activation of the carboxyl group, Wa 93/08302 2 ~ ~ i C~ (~ g PCT/EP92/02392 according to methods known in peptide synthesis, and so on, proceeding stepwise up to the N-terminal amino acid'.
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.
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:
- r~acting 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.
The kinase used is advantageously extracted from brain according to methods known to those skilled in the art (Isbiguro K. et al., 1988: Gaudier, J. and Cole, R. D. , 1987: Vallee, R. B. , 1980) and i~ different from the kinases referred to in Steiner et al. (1990).
The non-phosphorylated antigen is, for instance, a , normal human tau protein which, by phosphorylation with the above-mentioned kinase, gives rise to an abncirmally phosphorylated tau protein, which is recognized by the monoclonal antibody of the invention. Such abnormally phosphorylated tau protein is new.
The antigen of the invention, which can be prepared. by methods known to those skilled in the art (Iqbal, R. 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 ,. . . ' ,. . ,..._.. ,;'.:, ..: ';.w~. ~. ~-....._.,y.. , , ~ .;::.: ~.
WO 93/0$302 ~~ ~ ~ PCT/EP92/02392 monoclonal antibody secreted by the hybridoma deposited at the ECACC under No. 91100806.
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.
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:
- 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:
separating the antigen from said complex and recovering the antigen sought in a purified form.
The preparation of PHF can be carried outs according to Iqbal K. et al. (1984) or Greenberg et al.
0990) . _ ~ .
Advantageously, the monoclonal antibody used is in an immobilized state on a suitable support such as a resin. The proeess for the detection of the antigen can then be carried out as follows:
- 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;
- washing the immobilized antibody-antigen complex so formed:
W~ 93/0830? 2 ~, ~ ~ ~ ~ ~ fCT/Ef92/02392 treating that complex with a solution (e.g., 3 M
potassium thiocyanate, x.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 - recovering the antigen in a purified form.
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:
- 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 - detecting the immunological binding of said antibody to said sample of brain homogenate, or of cerebrospinal fluid, or of serum.
The detection of the immunologically bound monoclonal antibody can be achieved fn a cpnventional 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.
A particularly advantageous embodiment of the process of the invention comprises contacting a sample of cerebrospinal fluid (containing the corresponding WO 93108302 2121 ~ ~ ~ PGT/EF92/02392 antigen) obtained from a patient to be diagnosed with the monoclonal antibody of the invention.
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:
- at least a microplate for deposition thereon of any monoclonal antibody of the invention;
- 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 YS~PGSPGT or YSSPGSPGT phosphorylated at the positions marked with *:
- a second antibody . which can be a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of .any peptide of the invention, such epitope being different from the one of the invention, or ,.
. 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 wfiich are all different from the epitope of the invention, and the polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein, or - a masker either for specific tagging or coupling with the second antibody;
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.
The labeled peptide mentioned above can be a peptide which has been labeled by any means known to WU 93/08302 ~ Z ~ ~ ~ ~ PCT/EP92/02392 those skilled in the art. Likewise, the marker specific for tagging and coupling can be any marker known to those skilled in the art.
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.
T$e Figures Figure 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.
AD' - AD,~: Brain homogenates from 4 different Alzheimer patients diluted 1/200 (solid bars) or 1/1000 (stippled bars).
C9 - C4: Brain homogenates from .4 different controls, diluted 1/00 (solid bars) or 1/1000 (stippled bars).
The ordinate represents the optical density at 450 nm and the abscissa represents the different samples.
Figure 2: Western blotting detection of normal tau or PHF-tau using either the monoclonal antibodies Tau-1 or ATB. 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 ATS
monoclonal antibodies (lanes 5 and 6).
Figures 3A, 318, 3C and 3D:
Detection of tau protein by immunochemistry.
Figure 3A: Section from hippocampus of a patient with Alzheimer's disease. Magnification 't8 x.
Figure 3B: Section from hippocampus of an aged control patient deceased from non-neurological causes. Magnification 78 x.
Figure 3C: Section from hippocampus of a patient with Alzheimer's disease. Magnification 245 x.
Figure 3D: Section from hippocampus of an aged control patient deceased from non-neurological causes: Magnification 245 x.
Figure 4: Immunoblot analysis of Example 5 of a mutated recombinant tau (clone htau24) phophorylated~
with' the protein kinase activity from rat brain. Immunoblots were carried out with anti-tau antibodies 134 and ATB. panes: 1, tau24; 2, tau24 + brain extract; 3, tau24 S1~9A; 4, tau24 S199A + brain extract; 5, tau24 S202A; 6, tau24 5202.A. + brain extract;
7, tau24 S1~9A, S202A; 8, tau24 S199A, S202A
+ brain extract.
Example I: Preparation of the monoclonal antibodies AT8 ~IQG1, a) 1. Preparation of the antigen for immunization:
Postmortem tissue, consisting mostly of gray matter from the frontal and temporal cortex, was 1:734-2 obtained from histologically confirmed Alzheimer patients. This Alzheimer gray matter brain sample (5-10 g) was homogenized with l0 vol. of cold buffer H (l0 mM
Tris/1 mM EGTA/0.8 M NaCl/10% sucrose, pH 7.4~) in a Teflon%glass Potter' S (Braun, Ger~onany) homogenizer.
After centrifugation in a 60 Ti MSE rotor at 27,000 x g for 20 min at 4'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'C. The supernatant mixture was centrifuged at 108,000 x g for 35 min at 20'C. The PHF-tau containing pellet was gently washed with PBS
and finally suspended in 1 ml of the same buffer.
The antigen preparation was evaluated by a l0%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and followed by Western blotting using immunoblotting with polyclonal rabbit antihuman normal tau antiserum.
2. Immunization protocol and fusion procedure:
Balb/c mice were primed with loo ~g partially purified PHF-tau in complete Freurad's adjuvant and boosted three times thereafter at 3-week intervals with 100 ug of the same antigen in incomplete Freund~s. on days 3 and 2 before the fusion, mice were boasted with 100 ~g PHF-tau saline.
Mouse spleen cells were fused with SP2/o cells, using a modified procedure of Koh.ler and Milatein (Kohler, G. and Milstein, C., 1975), with PEG 4000.
Half of the cells were suspended~at a density of 4.5 x 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 EhISA 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 *Trade-mark WO 93/08302 ~ .~.~, PGT/EP92/02392 selected by Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal calf serum, sodium pyruvate (1 ml~i), 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% COZ air incubator.
3. Sandwich ELISA for antibody screening:
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 polyclonal 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 ~cg,/ml) at 4'G on high-binding microtiter plates (Nunc, Gibco, Paisley, UK) in coating buffer (10 mM Tris, 10 mM NaCl, 10 mM NaN~, pH 8.5). After overcoating for 30 min with 125 ~1 10%-saturated casein in P1~S to reduce nonspecific binding, the plates were incubated with 100 u1 of an appropriately diluted PHF-tau preparation and incubated for 60 min at 37'C. The plates were washed three times with PBS-0.05% Tween 20 (v/v); 100 dal hybridoma supernatant was added, and incubation was continued for 1 h at 37'C. After washing, the bound monoclonal antibodies were detected with peroxidase-conjugated rabbit anti-mouse serum (Dakopatts, Glostrup, Denmark). All reagents were w l:'~ 734-2 27.
diluted in PHS with 1o% casein. After final washing, 7.00 ~1 0.42 mM 3,5,3',5'-tetramethylbenzidine, 0.003%
H2p2 v/v in 100 mM citric acid, loo mM disodium hydrogen phosphate, pH 4.3, was added as peroxidase substrate. The reaction was stopped with 50 ~l of a 2 M
H2SO~ solution.
Absorbance was read in a Titeri:ek Multiscan~' (Flow Laboratories, Eflab, Oy, Finland) at 45o nn.
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 P1~F-tau. The hybridoma-secreting antibodies specifically recognizing the PIiF-tau was subcloned by limiting dilutions. Said hybridoma which secretes the AT8 antibodies will be designated AT8:
Example II: Detection of patholoctical tau and absence of detection of normal tau in ELISA by Western blotting 1. Detection of abnormally phosphorylated tau in ELISA and absence of detection of noZSaal tau in ELISA:
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.
*Trade-mark ~Y~ 93/08302 PCT/EP92/02392 ~12~~~~
TABLE I. Detection of PHF-tau or normal tau in ELISA
using the AT8 monoclonal antibodies for detection PHF-tau~~ ~ nm 1/20 1,459 1/200 1,179 1/2000 0,565 Normal tau 1~/ml 0,021 100 n /ml 0,005 n /ml 0,001 Blank 0,000 i 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.
The reactivity pattern of the AT8 monoclonal antibodies was also studied in brain homogenates. To this end, high-binding microtiter plates (Nunc, Gibco,.
Paisley, UIt) were coated overnight at 4 ° C with 2 ~rg/ml of purified AT8 monoclonal antibodies in coating buffer (10 mM Tris, 1.0 mM NaCl, 10 mM NaN3, pH 8. ~) .
~vercoating to reduce non-specific binding was performed for 30 min with 120 u1 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 ~cl sample was added, and the incubation was Carried out for I h at 37°C. The plates were washed again three times and incubated with 100 ~1 of a 1/2000 dilution of rabbit anti-tau serum. Next, the plates were washed again three times, after which 100 ~cl horseradish peroxidase conjugated donkey anti-rabbit serum diluted 1:734-2 1/2000 in blot buffer was added and the incubation was continued for 30 min. The plates were washed and 100 ~cl of a solution consisting of 0.42 mM 3,5,3',5'-tetramethylbenzidine, 0.003% 8202 v/w in 100 mM citric acid, 100 mM disodium hydrogen phosphate, pH 4.3 was added as substrate.. The reaction was stopped with 50 ~.1 of a 2 M H2SO4 solution. Absorbance was read in Titertek Multiscan (Flow haboratories, Eflab, 0y, Finland) at 450 nm.
As can be seen from Fig. 1, the brain homogenates of the 4 Alzheimer patients (AD, - AD4) reacted positively at two different dilutions, while all extracts prepared from brain derived from patients who died of non-neurological diseases (C' - C4) were clearly negative at both dilutions.
2. Detection of pathological tau in Western blotting and absence of detection of normal tau in Western blotting:
Purified normal human tau and PHF-tau were applied to 10% SDS-polyacrylamide gels and run under denaturing conditions according to Laemmli (2970).
After SDS-PAGE, the transfer to nitrocellulose (Hybond-C; Amersham, Brussels, Belgium) was carried out in 10 mM NaHC03, 3 mM Na2C03, 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'e 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, *Trade-mark 1'I734-2 0.05% w/v 3,3'-diaminobenzidine, 0.03% v/v H202) after which the reaction was stopped in HZO. Results, shown in Fig. 2 indicate that the AT8 antibody recognizes 64 and 68 kDa tau isoforms but show tlhat normal tau bands remain unstained.
Example III: Detection of tau bY immunocytochemistry 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.
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.
Figure 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.
*Trade-mark WO 9310830? ~ ~ PCT/EP92/02392 Example IV: Competition ELISAs to characterize the epitopes of known antibodies with the antibody of the invention Affinity-purified rabbit anti-human tau polyclonal antibody was coated overnight at 4'C in the wells.
Plates were washed, and 100 ~cl of purified abnormally phosphorylated tau, mixed with affinity-purified normal tau, was added per well for 1 h at 37'C. After washing, 50 ~l of the different unlabeled monoclonal antibodies to be tested were added in several dilutions.
Subsequently, 50 gel 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'C
the plates were washed and a streptavidine 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 Ii2S0' the plates were read in a Titertek Multiscan plate reader (Flow) at 450 nm. ,, 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 ATB, but neither casein or BT2 inhibit this reaction.
WO 93/08302 PCI'/EP92/02392 ~12~.~~
TABLE II. Competition ELISA to characterize the epitopes of known antibodies.
Concentra- HT2-bio AT8-bio tion Casein HT2 AT8 Casein HT2 AT8 9 ~ /ml 0.369 0.071 0.369 0.578 0.548 0.056 3 ~ /~1 0.390 0.088 0.375 0.573 0.557 0.054 1 a /znl 0.375 0.122 0.368 0.548 0.550 0.059 333 n /ml 0.381 0.179 0.369 0.564 O.~i56 0.093 111 n /ml 0.385 0.245 0.381 0.576 0._i73 0.155 37 n /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). _ Production of polyclonal rabbit anti-tau antiserums New Zealand white rabbits were immunized with affinity purified human tau. ~tabbits were injected intradermally with 100 erg affinity .purified human tau emulsified in complete Freund's adjuvant. Two weeks Later, this was repeated intramuscularly with 200 ~cg affinity purified human tau in incomplete Freund's, and a third intramuscular injection was carried out after one week with 100 ~cg affinity purified human tau in saline. The rabbits were bled one week after the third injection, evaluated and again injected twice, after a 1:? 734-2 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.
- Production of affinity purified human tau:
Typically, 50 grams post-mortem human brain was out into small pieces with scissors and homagenized 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 MgCl2, 1 mM mercaptoethanol, pH 6.75) with a Potter homogenizes equipped with a teflon plunger. The homogenate was centrifuged for 1 h at 150000 g at 4'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'C, and the supernatant was collected thereafter and called the "heat stable cytosolic extract".
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 phi 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.
- Production of the monoclonal antibody BT2:
. Antigen purification:
Bovine tau: tau was purified from bovine brain by a modification of the perchloric acid method of hindwall 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-*Trade-mark 1=1734-2 morpholino)ethanesulfonic acid), .BO mM NaCl, 2 mM EDTA, 0 .1 M EGTA, 1 mM MgCl2, 1 mM merc:aptoethanol , pH 6 . ? 5 ) with a Potter homogenizes equipped with a teflon plunger. The homogenate was centrifuged for 1 h at 150000 g at 4 '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'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'C after which the supernatant was neutralized with 3 M Tris. The supernatant was then dialyzed and concentrated in water in a centriprep~ concentrates (Amicvn; 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).
. Monoclonal anti-bovine tau antibody production:
Balb/c mice were primed with 100 ~g purified bovine tau in complete Freund's adjuvant and boosted 3 times with 100 ~cg bovine tau in complete Freund's every two Weeks. On days 3 and 2 before the fusion, the mice were boosted again with 100 ~cg 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 wioth 10% fetal calf serum, sodium pyruvate (1 mM), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 ~cg/ml), and nonessential amino acids. All products were purchased from Gibco, Paisley, *Trade-mark WO 93/08302 ~ ~ ~ ~, ~ ~ ~ i'CT/EP92/02392 U.K. Cells were incubated at 37°C in a humidifed 8% C02 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 phosphor5~lation of mutant recombinant tau proteins to deteranine the essential phosphorylation sites) of the AT8 epitope A full-length cDNA clone (htau24), corresponding to a four repeat isoform of tau and with a NdeI site in the context of the in~.tiator colon, was subcloned into the EcoRI site of M13mp18. Two constructs were made in which site-directed mutagenesis was used to change colon 199 (S to A) or colon 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 colons 199 and 202 were both changed to A.
Following cleavage with Ndel and EcoRI, the resulting fragments were subcloned downstream of the T7 RNA
polymerase promoter in the expression plasmid pRK172, and the reeomf~inant plasmids were transformed into E.coli BIa21 (DE3) cells. The bacterial cultures were grown, induced and harvested as desribed (Goedert, M.
et al., 1989).
The pellet from a 500 ml culture was resuspended in 20 ml of extraction buffer (50 mM PIPES, pFi 6.8, imM
DTT, 1 mM EDTA, 0.2 mM PMSF, 0.5 ~g/ml leupeptin, 0.5 ~g/ml pepstatin) and sonicated for 2 x 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 m1) equilibrated in the extraction W~ 93/08302 PCT/EP92/02392 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 NaGl, pH 6.25, and the protein was eluted using 100-300 mM NaCl in 50 mM MES, Z mM DTT, pH 6.25, gradient. Column fractions were screened by gel electrophorisis, 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.
The resulting recombinant tau proteins were phosphorylated, using brain protein kinase activity as follows:
Adult rat brain was homogenized (lg/2.5m1) in 10 mM Tris/HCl, pH7.4, 5 mM EGTA, 2 mM DTT, Z EsM okadaic acid, 1 mM PMSF, 20 ~g/ml leupeptin,~ 20 ~g/ml aprotinin and 20 ~g/ml pepstatin and centrifuged at 40,000 rpm for 1 h at 4'C. The supernatant was used directly for phosphorylation. Incubations were earried out at 37 ° C with ~ 0 mM HEPES , pH 7 . 2 , 2 mNt ~lfP, ~ 2 ~ mM
MgCl~, recombinant tau protein (1 ~aM), and rat brain extract (0.05 ml) for 24 h, and aliquots were then taken for immunoblotting. Controls were incubated sander the same conditions, except that the brain extract was omitted.
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 °1734-2 protein binding sites were blocked with 1% gelatin in phosphate-buffered saline. The blots were then incubated far 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).
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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Claims (27)
1. Monoclonal antibody capable of forming an immunological complex with a phosphorylated epitope of an antigen belonging to human abnormally phosphorylated tau protein, with said tau protein being obtained from a brain homogenate, said brain homogenate isolated from the cerebral cortex obtained from a patient having Alzheimer's disease or having died of Alzheimer's disease, wherein said monoclonal antibody - is not capable of forming an immunological complex with normal tau protein, - is not capable of forming an immunological complex with tau protein present in brain homogenates derived from human brain, the homogenates being isolated from a patient having died of non-neurological disorders, and - is not capable of forming an immunological complex with the above-defined epitope previously treated with a dephosphorylating agent.
2. The monoclonal antibody according to claim 1, wherein the dephosphorylating agent is an alkaline phosphatase.
3. The monoclonal antibody according to claim 1 or 2 wherein said monoclonal antibody is capable of forming an immunological complex - either with a peptide YSS*PGS*PGT or YSSPGS*PGT, phosphorylated at the positions marked with *, - or with any other peptide capable of forming an immunological complex with a monoclonal antibody capable of forming a complex with said peptide YSS*PGS*PGT* or YSSPGS*PGT, phosphorylated at the positions marked with *.
4. ~The monoclonal antibody according to claim 3, wherein the peptide is YSSPGS*PGT, phosphorylated at the position marked with *.
5. ~The monoclonal antibody according to any one of claims 1 to 4, wherein said monoclonal antibody is capable of forming an immunological complex with an abnormally phosphorylated form of tau protein, present in homogenates of human brain obtained from a patient having died of Alzheimer's disease;
wherein said abnormally phosphorylated tau protein presents an apparent molecular weight which is higher than that of a normal tau protein, with said normal tau protein being derived from brain homogenate, said brain homogenate isolated from a patient having died of non-neurological disorders;
and wherein the apparent molecular weight of said abnormally phosphorylated tau protein can be decreased to that of a normal tau protein upon treatment of said abnormally phosphorylated tau protein with a dephosphorylating agent.
wherein said abnormally phosphorylated tau protein presents an apparent molecular weight which is higher than that of a normal tau protein, with said normal tau protein being derived from brain homogenate, said brain homogenate isolated from a patient having died of non-neurological disorders;
and wherein the apparent molecular weight of said abnormally phosphorylated tau protein can be decreased to that of a normal tau protein upon treatment of said abnormally phosphorylated tau protein with a dephosphorylating agent.
6. ~Monoclonal antibody secreted by the hybridoma deposited at ECACC on October 8, 1991 under No.
91100806.
91100806.
7. ~Hybridoma, which secretes a monoclonal antibody according to any one of claims 1 to 6.
8. ~A peptide which can be obtained from a brain homogenate, said brain homogenate isolated from the cerebral cortex obtained from a patient having Alzheimer's disease, wherein said peptide is capable of forming an immunological complex with the monoclonal antibody according to any one of claims 1 to 6, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
9. ~A peptide capable of forming an immunological complex with the monoclonal antibody according to any one of claims 1 to 6, said peptide comprising:
- the sequence YSS*PGS*PFT or YSSPGS*PGT, phosphorylated at the positions marked with *, or - a sequence capable of forming an immunological complex with a monoclonal antibody capable of forming a complex with the peptide YSS*PGS*PGT or YSSPGS*PGT, phosphorylated at the positions marked with *, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
- the sequence YSS*PGS*PFT or YSSPGS*PGT, phosphorylated at the positions marked with *, or - a sequence capable of forming an immunological complex with a monoclonal antibody capable of forming a complex with the peptide YSS*PGS*PGT or YSSPGS*PGT, phosphorylated at the positions marked with *, provided that said peptide is not the 68, 64 or 60 kDa protein isolated from brain obtained from patients having Alzheimer's disease.
10. ~The peptide according to claim 9, wherein the peptide contains or is constituted by the sequence YSSPGS*PGT.
11. ~The peptide according to any one of claims 8 to 10, wherein said peptide is capable of generating the monoclonal antibody according to any one of claims 1 to 6.
12. ~A peptide (antigen) which contains a sequence selected from the group consisting of:
(X1)p (X2) q S*PX3S*P(X4)r(X5)s: and (X1)p (X2) q SPX3S*P (X4)r(X5)s in which X1, X2, X3, X4, X5 are selected from the group consisting of naturally occurring amino acids, and p, q, r, s are 0 or 1, phosphorylated at places marked by *, provided that said peptide is capable of forming an immunological complex with the monoclonal antibody according to any one of claims 1 to 6.
(X1)p (X2) q S*PX3S*P(X4)r(X5)s: and (X1)p (X2) q SPX3S*P (X4)r(X5)s in which X1, X2, X3, X4, X5 are selected from the group consisting of naturally occurring amino acids, and p, q, r, s are 0 or 1, phosphorylated at places marked by *, provided that said peptide is capable of forming an immunological complex with the monoclonal antibody according to any one of claims 1 to 6.
13. ~The peptide according to claim 12, where the sequence is (X1)p(X2)q SPX3S*P(X4)r(X5)s.
14. ~Process for obtaining and isolating a hybridoma secreting the monoclonal antibody according to any one of claims 1 to 6, said process comprising:
- providing spleen cells of an animal previously immunized in vivo or spleen cells of such animal previously immunized in vitro with an antigen recognized by the monoclonal antibody deposited at ECACC on October 8, 1991 under No. 91100806;
- fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and - selecting those of the hybridomas which secrete the monoclonal antibodies which specifically recognize an epitope of the peptide of any one of claims 8 to 13 and which form an immunological complex with said epitope.
- providing spleen cells of an animal previously immunized in vivo or spleen cells of such animal previously immunized in vitro with an antigen recognized by the monoclonal antibody deposited at ECACC on October 8, 1991 under No. 91100806;
- fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and - selecting those of the hybridomas which secrete the monoclonal antibodies which specifically recognize an epitope of the peptide of any one of claims 8 to 13 and which form an immunological complex with said epitope.
15. ~The process of claim 14, wherein the animal is a mouse or a rat.
16. ~Process for producing monoclonal antibodies according to any one of claims 1 to 6, said process comprising:
- culturing the selected hybridomas as defined in claim 7 or as obtained in claim 14, in an appropriate culture medium;
- recovering the monoclonal antibodies excreted by said selected hybridomas;
- or alternatively:
- implanting the selected hybridomas as defined in claim 7 or as obtained in claim 14 into the peritoneum of a mouse, and when ascites has been produced by the mouse, recovering the monoclonal antibodies then formed from said ascites.
- culturing the selected hybridomas as defined in claim 7 or as obtained in claim 14, in an appropriate culture medium;
- recovering the monoclonal antibodies excreted by said selected hybridomas;
- or alternatively:
- implanting the selected hybridomas as defined in claim 7 or as obtained in claim 14 into the peritoneum of a mouse, and when ascites has been produced by the mouse, recovering the monoclonal antibodies then formed from said ascites.
17. ~Process for the preparation of the peptide according to any one of claims 8 to 13, said process comprising:
- providing said peptide in non-phosphorylated form;
- reacting said peptide, which is non-phosphorylated, with a kinase enzyme capable of recognizing the non-phosphorylated epitope of the peptide and of modifying the epitope to a phosphorylated epitope recognized by the monoclonal antibody according to any one of claims 1 to 6.
- providing said peptide in non-phosphorylated form;
- reacting said peptide, which is non-phosphorylated, with a kinase enzyme capable of recognizing the non-phosphorylated epitope of the peptide and of modifying the epitope to a phosphorylated epitope recognized by the monoclonal antibody according to any one of claims 1 to 6.
18. ~Method for the preparation of abnormally phosphorylated tau from brain 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 is implicated, said method comprising:
- contacting the monoclonal antibody according to any one of claims 1 to 6, with a preparation of neurofibrillary tangles (NFT) or a detergent-extracted brain homogenate isolated from a patient having had said brain disease under conditions suitable for producing an antigen-antibody complex;
and - separating the antigen from said complex and recovering said antigen in a purified form.
- contacting the monoclonal antibody according to any one of claims 1 to 6, with a preparation of neurofibrillary tangles (NFT) or a detergent-extracted brain homogenate isolated from a patient having had said brain disease under conditions suitable for producing an antigen-antibody complex;
and - separating the antigen from said complex and recovering said antigen in a purified form.
19. ~Process for the detection or diagnosis in vitro of brain disease involving paired helical filaments (PHF) and tau protein, said process comprising:
- contacting under in vitro conditions a sample of brain homogenate, cerebrospinal fluid, or serum from a patient suspected of suffering from a neurological disorder involving tau protein and PHF, with the monoclonal antibody according to any one of claims 1 to 6, under conditions suitable for producing an antigen-antibody complex and - detecting the immunological binding of said antibody to said sample of brain homogenate, cerebrospinal fluid, or serum.
- contacting under in vitro conditions a sample of brain homogenate, cerebrospinal fluid, or serum from a patient suspected of suffering from a neurological disorder involving tau protein and PHF, with the monoclonal antibody according to any one of claims 1 to 6, under conditions suitable for producing an antigen-antibody complex and - detecting the immunological binding of said antibody to said sample of brain homogenate, cerebrospinal fluid, or serum.
20. ~The process of claim 18 or 19, wherein the brain disease is Alzheimer's disease.
21. ~Kit for the diagnosis in vitro of a disease selected from the group consisting of Alzheimer's disease, Down's syndrome, Pick's disease, subacute spongiform panencephalitis and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated, said kit comprising:
- at least a microplate for deposition thereon of a first antibody, wherein said first antibody is the monoclonal antibody according to any of claims 1 to 6;
- a second antibody, wherein the second antibody is selected from the group consisting of:
i) a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of the peptide according to any one of claims 8 to 13, with said epitope being different from an epitope recognized by the first antibody, and ii) a polyclonal antibody recognizing normal tau, or abnormally phosphorylated tau or the peptide according to any one of claims 8 to 13, with said polyclonal antibody being capable to form an immunological complex with epitopes which are all different from an epitope recognized by the first antibody;
- a marker either for specific tagging or coupling with said second antibody; and - appropriate buffer solutions for carrying out the immunological reaction between the first monoclonal antibody and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
- at least a microplate for deposition thereon of a first antibody, wherein said first antibody is the monoclonal antibody according to any of claims 1 to 6;
- a second antibody, wherein the second antibody is selected from the group consisting of:
i) a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of the peptide according to any one of claims 8 to 13, with said epitope being different from an epitope recognized by the first antibody, and ii) a polyclonal antibody recognizing normal tau, or abnormally phosphorylated tau or the peptide according to any one of claims 8 to 13, with said polyclonal antibody being capable to form an immunological complex with epitopes which are all different from an epitope recognized by the first antibody;
- a marker either for specific tagging or coupling with said second antibody; and - appropriate buffer solutions for carrying out the immunological reaction between the first monoclonal antibody and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
22. ~The kit of claim 21, wherein the second antibody is a polyclonal antibody purified by immunoaffinity chromatography using immobilized tau protein.
23. ~The kit of claim 2I or 22 further comprising the peptide according to any one of claims 8 to 13 for standard purposes, or for competition purposes with respect to the antigen belonging to human abnormally phosphorylated tau protein which is detected.
24. ~The hybridoma of claim 7, wherein said hybridoma is the hybridoma deposited at ECACC on October 8, 1991 under No. 91100860.
25. ~A kit comprising the monoclonal antibody according to any one of claims 1 to 6 together with instructions for the diagnosis in vitro of a disease selected from the group consisting of Alzheimer's disease, Down's syndrome, Pick's Disease, subacute spongiform panencephalitis and other neurological disorders in which abnormally phosphorylated tau protein or paired helical filaments are implicated.
26. ~The kit of claim 25, said kit further comprising a component selected from the group consisting of:
- at least a microplate for deposition thereon of a first antibody, wherein said first antibody is the monoclonal antibody according to any of claims 1 to 6;
- a second antibody, wherein the second antibody is selected from the group consisting of:
i) a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of the peptide according to any one of claims 8 to 13, with said epitope being different from an epitope recognized by the first antibody, and ii) a polyclonal antibody recognizing normal tau, or abnormally phosphorylated tau or the peptide according to any one of claims 8 to 13, with said polyclonal antibody being capable to form an immunological complex with epitopes which are all different from an epitope~
recognized by the first antibody:
- a marker either for specific tagging or coupling with said second antibody; and - appropriate buffer solutions for carrying out the immunological reaction between the first monoclonal antibody and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
- at least a microplate for deposition thereon of a first antibody, wherein said first antibody is the monoclonal antibody according to any of claims 1 to 6;
- a second antibody, wherein the second antibody is selected from the group consisting of:
i) a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of the peptide according to any one of claims 8 to 13, with said epitope being different from an epitope recognized by the first antibody, and ii) a polyclonal antibody recognizing normal tau, or abnormally phosphorylated tau or the peptide according to any one of claims 8 to 13, with said polyclonal antibody being capable to form an immunological complex with epitopes which are all different from an epitope~
recognized by the first antibody:
- a marker either for specific tagging or coupling with said second antibody; and - appropriate buffer solutions for carrying out the immunological reaction between the first monoclonal antibody and a test sample on the one hand, and the bound second antibody and the marker on the other hand.
27. The kit of claim 26, wherein the second antibody is a polyclonal antibody purified by immunoaffinity chromatography using immobilized tau protein.
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-
2000
- 2000-12-11 US US09/734,281 patent/US6900293B2/en not_active Expired - Fee Related
-
2003
- 2003-08-21 JP JP2003297529A patent/JP2004043487A/en active Pending
-
2004
- 2004-08-20 US US10/923,323 patent/US20060008853A1/en not_active Abandoned
-
2008
- 2008-11-10 US US12/268,177 patent/US8163873B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JP2004043487A (en) | 2004-02-12 |
| DE69220503D1 (en) | 1997-07-24 |
| US20090137051A1 (en) | 2009-05-28 |
| US20110143443A9 (en) | 2011-06-16 |
| AU662178B2 (en) | 1995-08-24 |
| EP0610330B1 (en) | 1997-06-18 |
| US20010018191A1 (en) | 2001-08-30 |
| ATE154642T1 (en) | 1997-07-15 |
| EP0610330A1 (en) | 1994-08-17 |
| JP3909084B2 (en) | 2007-04-25 |
| JPH07502888A (en) | 1995-03-30 |
| CA2121599A1 (en) | 1993-04-29 |
| WO1993008302A1 (en) | 1993-04-29 |
| GR3024635T3 (en) | 1997-12-31 |
| DK0610330T3 (en) | 1998-01-26 |
| AU2800292A (en) | 1993-05-21 |
| US6900293B2 (en) | 2005-05-31 |
| US6238892B1 (en) | 2001-05-29 |
| US20060008853A1 (en) | 2006-01-12 |
| DE69220503T2 (en) | 1998-02-05 |
| US8163873B2 (en) | 2012-04-24 |
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| MKEX | Expiry | ||
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Effective date: 20121017 |