Monoclonal antibodies directed against the microtubule-associated protein tau, hybridomas secreting these antibodies, antigen recognition by these monoclonal antibodies and their applications

Abstract

The invention relates to a monoclonal antibody which forms an immunological complex with an epitope of an antigen belonging to normal human tau protein as well as abnormally phosphorylated human tau protein, with said tau protein being liable to be obtained from a brain homogenate, itself isolated from human cerebral cortex. The monoclonal antibodies of the invention can be used to detect tau and abnormally phosphorylated tau in brain extracts and in unconcentrated cerebrospinal fluid.

1 . Monoclonal antibody which forms an immunological complex with an epitope of an antigen belonging to human normal as well as abnormally phosphorylated tau protein, with said tau protein being liable to be obtained from a brain homogenate, itself isolated from the human cerebral cortex, characterized by the fact that: 
 it does not form an immunological complex with other phosphorylated proteins such as MAP-1, MAP-2 and neurofilaments which share part of their sequence with tau protein, as determined by means of an ELISA,    and it is liable to detect human normal as well as abnormally phosphorylated tau protein in cerebrospinal fluid (CSF), with said tau protein being at a concentration as low as 1.0 pg/ml.    and it is liable to detect said tau proteins with 100% recovery upon the addition of a fixed amount of tau proteins in tau-protein-negative CSF.    
     
     
         2 . Monoclonal antibody according to  claim 1 , characterized by the fact that it forms an immunological complex: 
 either with an epitope located within the following amino acid of human tau protein:                          (SEQ ID NO 1)                       155             160                     165         NH 2 -Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln                           170                 175     Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro                   180                 185     Ser Ser Glu Glu Pro Pro Lys Ser Gly Glu Pro Pro           190                 195                 200     Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly                       205                 210     Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro               215                 220     Ser Leu Pro Thr Pro Pro Thr Arg                                               more specifically with an epitope located within the following amino acid sequence:                          (SEQ ID NO 2)                   199 200                 205                 210         Ser Pro Gly Ser Pro Gly Thr Pro Tyr Ser Arg Ser                           215                 220     Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu                    225                230 231     Pro Lys Lys Val Ala Val Val Arg Thr                                      more specifically with an epitope located within the following amino acid sequence:                          (SEQ ID NO 3)                   207         210                 215         Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro               Pro                                          most specifically with an epitope located within the following amino acid sequence:                                          218                     224   (SEQ ID NO 4)             Pro Pro Thr Arg Glu Pro Lys                              or with any other peptide capable of forming an immunological complex with a monoclonal antibody, which is capable of forming a complex with an epitope located in a tau protein regions as shown in any of SEQ ID NO 1 to 4.    
     
     
         3 . Monoclonal antibody according to claims  1  or  2  secreted by the hybridoma deposited at ECACC on Oct. 8, 1992 under No. 92100853.  
     
     
         4 . Hybridoma, which secretes a monoclonal antibody according to anyone of  claims 1  to  3 , more particularly the hybridoma deposited at ECACC on Oct. 8, 1992 under No. 92100853.  
     
     
         5 . Peptides which can be obtained from a brain homogenate, itself isolated from the human cerebral cortex or from cerebral cortex obtained from a patient having Alzheimer's disease and which forms an immunological complex with the monoclonal antibody according to anyone of  claims 1  to  3 .  
     
     
         6 . Peptides liable to form an immunological complex with any of the monoclonal antibodies, according to anyone of  claims 1  to  3 , 
 which are contained in, or are constituted by parts of the sequence as shown in SEQ ID NO 1 to 4,  
 which contain or are constituted by the sequence of the peptides liable to form an immunological complex with a monoclonal antibody according to anyone of  claims 1  to  3 , which itself is liable to form a complex with the epitope located in the tau protein region as shown in SEQ ID NO 1 to 4.  
 
     
     
         7 . Peptides of about 100 amino acids 
 which contain the sequence as shown in SEQ ID NO 1 to 4,    which contain the sequence of the peptides liable to form an immunological complex with a monoclonal antibody according to anyone of  claims 1  to  3 , which itself is liable to form a complex with a peptide within sequence as shown in SEQ ID NO 1 to 4.    
     
     
         8 . Peptides according to anyone of  claims 5  to  7 , which are liable to generate a monoclonal antibody according to anyone of  claims 1  to  3  upon immunization.  
     
     
         9 . Peptides which are contained in the brain, in the cerebrospinal fluid, or the serum of a patient having Alzheimer's disease or any brain disease involving PHF or normal tau protein and which forms an immunological complex with a monoclonal antibody according to anyone of  claims 1  to  3 .  
     
     
         10 . Process for obtaining and isolating a hybridoma according to  claim 4 , secreting a monoclonal antibody according to anyone of  claims 1  to  3 , characterized in that it involves: 
 starting from spleen cells of an animal, e.g. mouse or rat, previously immunized in vivo, or from spleen cells of such cells previously immunized in vitro with an antigen recognized by the monoclonal antibody deposited at ECACC on Oct. 8, 1992 under No. 92100853, or with a peptide containing or which are constituted by parts of any of the sequences as represented in SEQ ID NO 1 to 4;  
 fusing said immunized cells with myeloma cells under hybridoma-forming conditions; and  
 selecting those of the hybridomas which secrete the monoclonal antibodies which specifically recognize an epitope of the antigen according to any of  claims 5  to  8  and which form an immunological complex with said epitope.  
 
     
     
         11 . Process for producing monoclonal antibodies according to anyone of  claims 1  to  3  which involves: 
 culturing the selected hybridomas according to  claim 4 , in an appropriate medium culture; and  
 recovering the monoclonal antibodies excreted by said selected hybridomas; or alternatively: 
 implanting the selected hybridomas of  claim 4  into the peritoneum of a mouse and, when ascites has been produced by the animal, recovering the monoclonal antibodies then formed from said ascites.  
 
 
     
     
         12 . Process for the detection or diagnosis in vitro of brain disease involving either or both PHF-tau or normal tau protein, e.g. Alzheimer's disease, which involves: 
 contacting a monoclonal antibody according to anyone of  claims 1  to  3 , with a preparation of NFT or a detergent-extracted brain homogenate isolated from a patient having had Alzheimer's disease or any other disease involving tau protein or abnormally phosphorylated tau protein under conditions suitable for producing an antigen-antibody complex; and    separating the antigen from said complex and recovering the antigen sought in a purified form.    
     
     
         13 . Process for the detection or diagnosis in vitro of brain disease involving abnormally phosphorylated tau protein, e.g. Alzheimer's disease, which includes: 
 bringing a sample of brain homogenate, or of cerebrospinal fluid, or of serum from a patient suspected of suffering from a neurological disorder involving tau protein or PHF, more particularly Alzheimer's disease, into contact under in vitro conditions with a monoclonal antibody according to anyone of  claims 1  to  3 , with said conditions being suitable for producing an antigen-antibody complex; and    detecting the immunological binding of said antibody to said sample of brain homogenate, or of cerebrospinal fluid, or of serum.    
     
     
         14 . Process for the detection or diagnosis in vitro of brain diseases involving PHF and/or normal tau protein, e.g. Alzheimer's disease, comprising the steps of: 
 bringing a sample of unconcentrated cerebrospinal fluid sample isolated from a patient suspected to suffer from a neurological disorder involving normal or abnormally phosphorylated tau protein, more particulary Alzheimer's disease, into contact under in vitro conditions with a monoclonal antibody according to anyone of  claim 1  to  3 , under conditions suitable for producing an antigen-antibody complex; and,    detecting the immunological binding of said antibody to said sample of cerebrospinal fluid by means of a sandwich ELISA, preferably by applying the catalysed reporter diagnosis enhancement (CARD) procedure.    
     
     
         15 . Kit for the diagnosis in vitro of one of the following diseases: Alzheimer's disease, Down's syndrome, Pick's disease, SSPE and other neurological disorders in which normal tau protein or abnormally phosphorylated tau protein or paired helical filaments are implicated, characterized in that the kit comprises: 
 at least a microplate for deposition thereon of any monoclonal antibody according to anyone of  claims 1  to  3 ;    a preparation containing the sample to be diagnosed in vitro, possibly together with a labeled peptide containing the epitope of the invention and preferably with a peptide lying with as shown in SEQ ID NO 1 to 4;    a second antibody 
 which can be a monoclonal antibody recognizing an epitope of normal tau, or of abnormally phosphorylated tau protein, or of any peptide according to anyone of  claims 5  to  9 , with said epitope being different from the one of the invention, or  
 which can be a polyclonal antibody recognising normal tau or abnormally phosphorylated tau or a peptide according to anyone of  claims 5  to  9 , with said polyclonal antibody being liable to form an immunological complex with epitopes which are all different from the epitope of the invention, with said polyclonal antibody being preferably purified by immunoaffinity chromatography using immobilized tau protein, or  
   a marker either for specific tagging or coupling with said second antibody;    appropriate buffer solutions for carrying out the immunological reaction between the monoclonal antibody of the invention and a test sample on the one hand, and the bound second antibody and the marker on the other hand,    possibly a peptide according to anyone of  claims 5  to  9  for standard purposes, or for competition purposes with respect to the antigen which is sought.