Process for the determination of peptides corresponding to immunologically important epitopes and their use in a process for determination of antibodies or biotinylated peptides corresponding to immunologically important epitopes, a process for preparing them and compositions containing them

Abstract

The technical problem underlying the present invention is to provide peptides corresponding to immunologically important epitopes on bacterial and viral proteins, as well as the use of said peptides in diagnostic or immunogenic compositions. The invention relates to a process for the in vitro determination of antibodies, wherein the peptides used are biotinylated, particularly in the form of complexes of streptavidin-biotinylated peptides or of avidin-biotinylated peptides.

1 . A peptide consisting of an amino acid sequence selected from SEQ ID NO:455 and SEQ ID NO:459:  
       
         
           
                 
                 
                 
               
                     
                 
                   (A)-GHTRVTGGVQGHVTCTLTSLFRPGASQKIQ 
                   (SEQ ID NO 455) 
                     
                 
                   LVNT-(Z) 
                 
                     
                 
                   (A)-AETIVSGGQAARAMSGLVSLFTPGAKQNIQ 
                   (SEQ ID NO 459) 
                 
                   LINT-(Z), 
                 
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
         and, wherein A, when present, represents an amino acid, amino group, or chemically modified amino terminus of the peptide, and wherein Z, when present, represents an amino acid, OH-group, NH 2 -group, or a linkage involving an OH-group or an NH 2 -group or a peptide fragment consisting of at least 5 amino acids of SEQ ID NO: 455 or SEQ ID NO:459 which is immunologically reactive with HCV antisera.  
       
     
     
         2 . A peptide consisting of an amino acid sequence selected from SEQ ID NO:455 and SEQ ID NO:459:  
       
         
           
                 
                 
                 
               
                     
                 
                   (A)-GHTRVTGGVQGHVTCTLTSLFRPGASQKIQ 
                   (SEQ ID NO 455) 
                     
                 
                   LVNT-(Z) 
                 
                     
                 
                   (A)-AETIVSGGQAARAMSGLVSLFTPGAKQNIQ 
                   (SEQ ID NO 459) 
                 
                   LINT-(Z), 
                 
                     
                 
             
                
                
                
                
                
                
                
               
            
           
         
         wherein A, when present, represents an amino acid, amino group, or chemically modified amino terminus of the peptide, and wherein Z, when present, represents an amino acid, OH-group, NH 2 -group, or a linkage involving an OH-group or an NH 2 -group; or a peptide fragment consisting of at least 5 amino acids of SEQ ID NO: 455 or SEQ ID NO:459 which is immunologically reactive with HCV antisera; and said peptide or peptide fragment containing at least one N-terminal biotin group, C-terminal biotin group or biotin group attached to an internal amino acid; said biotin group being attached directly to the peptide or peptide fragment or attached to the peptide or peptide fragment through a linker Y; said linker Y consisting of 1 to 10 chemical entities selected from the group consisting of a glycine residue, beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid and 6-aminohexanoic acid.  
       
     
     
         3 . A peptide or peptide fragment of  claim 1  or  2  wherein at least one of A and Z are not present.  
     
     
         4 . A solid phase comprising a peptide or peptide fragment according to any one of  claims 1  to  3  and a solid support wherein the peptide is anchored to the solid support via at least one covalent or non-covalent bond.  
     
     
         5 . The solid phase according to  claim 3 , wherein said peptide or peptide fragment is anchored via a biotin group to streptavidin present on said solid support.  
     
     
         6 . The solid phase according to  claim 3  or  4  wherein said solid support is a nylon membrane.  
     
     
         7 . An immunological assay kit for detecting antibodies to HCV comprising at least one peptide or peptide fragment according to any one of  claims 1  to  3 .  
     
     
         8 . The immunological assay kit according to  claim 7  which is a line immunoassay kit.  
     
     
         9 . A peptide fragment according to  claim 2  consisting of an amino acid sequence  
       
         
           
                 
                 
                 
               
                     
                 
                   (A)-GHTRVTGGVQGHVTCTLTSLFR-(Z) 
                   (SEQ ID NO 465) 
                     
                 
                     
                 
             
                
                
                
               
            
           
         
         , wherein A, when present, represents an amino acid, amino group, or chemically modified amino terminus of the peptide, and wherein Z, when present, represents an amino acid, OH-group, NH 2 -group, or a linkage involving an OH-group or an NH 2 -group or a peptide fragment consisting of at least 5 amino acids of SEQ ID NO: 465 which is immunologically reactive with HCV antisera.  
       
     
     
         10 . A peptide fragment according to  claim 2  consisting of an amino acid sequence of  
       
         
           
                 
                 
                 
               
                     
                 
                   (A)-GHVTCTLTSLFRPGASQKIQLVNT-(Z) 
                   (SEQ ID NO 466) 
                     
                 
                     
                 
             
                
                
                
               
            
           
         
         , wherein A, when present, represents an amino acid, amino group, or chemically modified amino terminus of the peptide, and wherein Z, when present, represents an amino acid, OH-group, NH 2 -group, or a linkage involving an OH-group or an NH 2 -group; or a peptide fragment consisting of at least 5 amino acids of SEQ ID NO: 466 which is immunologically reactive with HCV antisera; and said peptide or peptide fragment containing at least one N-terminal biotin group, C-terminal biotin group or biotin group attached to an internal amino acid; said biotin group being attached directly to the peptide or peptide fragment or attached to the peptide or peptide fragment through a linker Y; said linker Y consisting of 1 to 10 chemical entities selected from the group consisting of a glycine residue, beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid and 6-aminohexanoic acid.  
       
     
     
         11 . A peptide fragment according to  claim 2  consisting of an amino acid sequence  
       
         
           
                 
                 
                 
               
                     
                 
                   (A)-AETIVSGGQAARAMSGLVSLFT-(Z) 
                   (SEQ ID NO 473) 
                     
                 
                     
                 
             
                
                
                
               
            
           
         
         , wherein A, when present, represents an amino acid, amino group, or chemically modified amino terminus of the peptide, and wherein Z, when present, represents an amino acid, OH-group, NH 2 -group, or a linkage involving an OH-group or an NH 2 -group or a peptide fragment consisting of at least 5 amino acids of SEQ ID NO: 473 which is immunologically reactive with HCV antisera.  
       
     
     
         12 . A peptide fragment according to  claim 2  consisting of an amino acid sequence of  
       
         
           
                 
                 
                 
               
                     
                 
                   (A)-ARAMSGLVSLFTPGAKQNIQLINT-(Z) 
                   (SEQ ID NO 474) 
                     
                 
                     
                 
             
                
                
                
               
            
           
         
         , wherein A, when present, represents an amino acid, amino group, or chemically modified amino terminus of the peptide, and wherein Z, when present, represents an amino acid, OH-group, NH 2 -group, or a linkage involving an OH-group or an NH 2 -group; or a peptide fragment consisting of at least 5 amino acids of SEQ ID NO: 474 which is immunologically reactive with HCV antisera; and said peptide or peptide fragment containing at least one N-terminal biotin group, C-terminal biotin group or biotin group attached to an internal amino acid; said biotin group being attached directly to the peptide or peptide fragment or attached to the peptide or peptide fragment through a linker Y; said linker Y consisting of 1 to 10 chemical entities selected from the group consisting of a glycine residue, beta-alanine, 4-aminobutyric acid, 5-aminovaleric acid and 6-aminohexanoic acid.  
       
     
     
         13 . The peptide fragment according to any one of  claims 9  to  12  wherein at least one of A and Z are not present.  
     
     
         14 . A solid phase comprising the peptide fragment according to any one of  claims 9  to  13  and a solid support wherein the peptide is anchored to the solid support via at least one covalent or non-covalent bond.  
     
     
         15 . The solid phase according to  claim 14 , wherein said peptide or peptide fragment is anchored via a biotin group to streptavidin present on said solid support.  
     
     
         16 . The solid phase according to  claim 14  or  15  wherein said solid support is a nylon membrane.  
     
     
         17 . An immunological assay kit for detecting antibodies to HCV comprising at least one peptide or peptide fragment according to any one of  claims 9  to  13 .  
     
     
         18 . The immunological assay kit according to  claim 17  which is a line immunoassay kit.