Anti-hirudin polyclonal antibodies and their use for the identification, immunopurification and quantitative determination of hirudin

Abstract

A method of producing an immunogen comprising polymerizing a hirudin and/or a hirudin-like protein is provided. A method of producing an antibody specific for a hirudin or a hirudin-like protein is also provided.

1 . A method of producing an immunogen, which method comprises polymerising a hirudin and/or a hirudin-like protein.  
     
     
         2 . A method according to  claim 1  wherein the hirudin and/or the hirudin-like protein are/is polymerized with glutaraldehyde.  
     
     
         3 . A method according to  claim 1  or  2  wherein the hirudin is HV1 (SEQ ID NO: 1), HV2 (SEQ ID NO: 2), HV3 (SEQ ID NO: 3), P1 (SEQ ID NO: 4) or P2 (SEQ ID NO: 5).  
     
     
         4 . A method according to  claim 1  or  2  wherein the hirudin-like protein is a derivative of HV1 (SEQ ID NO: 1), HV2 (SEQ ID NO: 2), HV3 (SEQ ID NO: 3), P1 (SEQ ID NO: 4) or P2 (SEQ ID NO: 5) by way of amino acid substitution, deletion, insertion, extension, functionalisation or chemical modification, said derivative having anti-thrombin activity.  
     
     
         5 . An immunogen comprising a polymerised hirudin and/or hirudin-like protein.  
     
     
         6 . An immunogen according to  claim 5  wherein the hirudin is HV1 (SEQ ID NO: 1), HV2 (SEQ ID NO: 2), HV3 (SEQ ID NO: 3), P1 (SEQ ID NO: 4) or P2 (SEQ ID NO: 5).  
     
     
         7 . An immunogen according to  claim 5  wherein the hirudin-like protein is a derivative of HV1 (SEQ ID NO: 1), HV2 (SEQ ID NO: 2), HV3 (SEQ ID NO: 3), P1 (SEQ ID NO: 4) or P2 (SEQ ID NO: 5) by way of amino acid substitution, deletion, insertion, extension, functionalisation or chemical modification, said derivative having anti-thrombin activity.  
     
     
         8 . An immunogen according to  claim 5 ,  6  or  7  obtainable by polymerising the hirudin and/or the hirudin-like protein with glutaraldehyde.  
     
     
         9 . A method of producing an antibody capable of binding to a hirudin or a hirudin-like protein, said method comprising raising antibody against an immunogen as claimed in  claims 5  to  8 .  
     
     
         10 . A method according to  claim 9 , wherein the said antibody is a polyclonal antibody.  
     
     
         11 . A method according to  claim 9 , wherein the said antibody is a monoclonal antibody.  
     
     
         12 . A method for the quantitative determination, purification or identification of a hirudin or hirudin-like protein, said method comprising raising antibody against an immunogen as claimed in  claims 5  to  8 , and using the antibody thereby obtained in said quantitative determination, purification or identification.