Synthetic peptides, antibodies directed against them, and the use thereof

Abstract

The invention relates to synthetic peptides which have amino acid sequences which correspond, in whole or in part, to the amino acid sequence of prothrombin and are antigenic, to the use thereof for the immunization of an animal and for the purification of specific antibodies, to antibodies against these peptides, and to the use of the antibodies for the determination of the fragments F 2 /F 1+2 . The antibodies hitherto used for the determination of the content of the fragments F 2 /F 1+2 have been induced by immunization with natural, highly purified prothrombin fragments F 2 /F 1+2 . The isolation of these prothrombin fragments is elaborate and costly, and the antibodies generated therewith show cross-reactions with intact prothrombin. Used for the immunization according to the invention are synthetic peptides which have amino acid sequences which correspond, in whole or in part, to the amino acid sequence of prothrombin and are antigenic. The resulting antibodies react specifically with the antigen used for the immunization; if the peptides contain the carboxyl-terminal amino acid sequence of the fragments F 2 /F 1+2 resulting after cleavage of the prothrombin molecule with factor Xa, then the antibodies generated react specifically with the fragments F 2 /F 1+2 but not with intact natural prothrombin, so that information can be gained about the degree of activation of prothrombin.

1 . Peptides which have amino acid sequences which partly correspond to the carboxyl-terminal end of the fragments F 2 /F 1+2  resulting from the F Xa cleavage of prothrombin, and which contain the amino acid sequence H-Gly-Asp-Glu-Glu-Gly-Val-Trp-Cys-Tyr-Val-Ala-Gly-Lys-Pro-Gly-Asp-Phe-Gly-Tyr-Cys-Asp-Leu-Asn-Tyr-Cys-Glu-Glu-Ala-Val-Gln-Glu-Glu-Thr-Gly-Asp-Gly-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH, in whole or in part, but at least the four carboxyl-terminal amino acids.  
     
     
         2 . A peptide as claimed in  claim 1 , which has the amino acid sequence H-Leu-Asp-Glu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-ILe-Glu-Gly-Arg-OH, H-Glu-GLu-Arg-Ala-ILe-Glu-Gly-Arg-OH or H-Cys(SH)-Leu-Asp-GLu-Asp-Ser-Asp-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg.  
     
     
         3 . Peptides as claimed in  claim 1 , which are prepared by genetic manipulation or by chemical synthesis.  
     
     
         4 . A peptide as claimed in  claim 1 , which is bound to an insoluble polymeric carrier or keyhole limpet hemocyanin, albumin or ovalbumin.  
     
     
         5 . The use of the peptides as claimed in  claim 1  for the immunization of an animal, preferably of a rabbit, and for the purification of specific antibodies against the said peptide by means of immunoadsorption.  
     
     
         6 . The use of a polypeptide as claimed in  claim 5 , wherein the peptide has the amino acid sequence H-Glu-Glu-Arg-Ala-Ile-Glu-Gly-Arg-OH.  
     
     
         7 . Antibodies against peptides as claimed in  claim 1 , which have been obtained by immunization of an animal with a peptide as claimed in at least one of  claims 1  to  4  and subsequent purification by immunoadsorption as claimed in at least one of claims  5  or  6 .  
     
     
         8 . Antibodies as claimed in  claim 7 , which react specfically with a peptide as claimed in  claim 1  and/or with natural prothrombin or parts thereof.  
     
     
         9 . Antibodies as claimed in  claim 8 , which are specific for binding to the prothrombin fragments F 2 /F 1+2  and are coupled to a carrier.  
     
     
         10 . A method for the determination of prothrombin fragment F 1+2 /F 2  using antibodies as claimed in at least one of  claims 7  to  9 , which comprises the prothrombin fragments F 2 /F 1+2  being bound to the specific antibodies and being detected by a second antibody, and comprises the quantity of the bound second antibody being measurable.  
     
     
         11 . The use of antibodies as claimed in  claim 10 , wherein a marker enzyme, preferably peroxidase, is bound to the second antibody.