Antibodies specific for a haemostatic protein, their use for isolating protein, haemostatic compositions devoid of proteolytic cleavage products of the protein

Abstract

A method for the generation of Ca++ independent antibodies against blood coagulation factors is described wherein an antibody selection strategy based upon small peptides comprising target sequences for limited proteolysis is employed. These antibodies which distinguish between intact and cleaved species of haemostatic protein provide novel tools for the isolation of intact haemostatic proteins.

We claim:  
     
       1. A method for isolating haemostatic proteins susceptible to specific proteolytic cleavage, from a mixture containing said proteins comprising subjecting said mixture to immunoaffinity chromatography using a Ca 2+ -independent antibody which binds intact but does not bind cleaved species of said haemostatic proteins, and isolating the haemostatic proteins. 
     
     
       2. A method as claimed in  claim 1 , wherein said antibody specifically binds epitopes of said haemostatic proteins which covers an intact proteolytic cleavage sites  site in said haemostatic proteins. 
     
     
       3. A method for isolating a haemostatic protein which is susceptible to specific proteolytic cleavage from a mixture containing said protein, wherein the method comprises 
       (a) subjecting said mixture to affinity chromatography using antibodies capable of binding haemostatic protein wherein the antibodies are not Ca++-dependent and wherein the antibodies are capable of specifically binding activated and are not capable of specifically binding non-activated species of said haemostatic protein, and  
       (b) isolating said haemostatic protein.  
     
     
       4. A method in accordance with  claim 3  wherein said haemostatic protein is a Vitamin K-dependent protein selected from the group consisting of Factor IX, Factor VII, Protein C and Protein S. 
     
     
       5. A method for isolating a protein selected from the group consisting of Factor IX, Factor VII, Protein C and Protein S from a mixture containing said protein which comprises subjecting said mixture to immunoaffinity chromatography using an antibody capable of binding said protein, said antibody being non Ca 2+ -dependent and capable of binding intact but not cleaved species of said protein resulting from specific proteolytic cleavage of the intact form, and isolating the protein. 
     
     
       6. A method as claimed in  claim 3 , wherein said antibody is specific for Factor IX and specifically binds an oligopeptide consisting of the amino acid sequence QTSKLTRAETVFPDVD (SEQ ID NO:1) corresponding to the amino acid residues 139-154 of Factor IX. 
     
     
       7. A method for isolating an uncleaved haemostatic protein susceptible to specific proteolytic cleavage from a mixture containing the uncleaved haemostatic protein and proteolytic cleavage products thereof, the method comprising: 
         immobilizing on a solid support a Ca   2+ - independent antibody having substantially higher affinity for the uncleaved haemostatic protein than for the proteolytic cleavage products;    
       
         contacting the mixture containing the uncleaved haemostatic protein and proteolytic cleavage products with the immobilized antibody to bind the uncleaved haemostatic protein to the antibody;  
       
       
         separating the bound uncleaved haemostatic protein from the proteolytic cleavage products by affinity chromatography; and  
       
       
         isolating the uncleaved haemostatic protein. 
       
     
     
       8. A method according to  claim 7  wherein the antibody is specific for an epitope that covers an intact proteolytic cleavage site in the uncleaved haemostatic protein. 
     
     
       9. A method according to  claim 7  wherein the uncleaved haemostatic protein is a blood coagulation factor. 
     
     
       10. A method according to  claim 9  wherein the blood coagulation factor is Factor IX or Factor VII. 
     
     
       11. A method according to  claim 7  wherein the uncleaved haemostatic protein is an antagonist of a blood coagulation factor. 
     
     
       12. A method according to  claim 11  wherein the antagonist of a blood coagulation factor is Protein C or Protein S. 
     
     
       13. A method for isolating an uncleaved haemostatic protein susceptible to specific proteolytic cleavage from a mixture containing the uncleaved haemostatic protein and proteolytic cleavage products thereof, the method comprising the steps of: 
         immobilizing on a solid support a Ca   2+ - independent antibody having substantially lower affinity for the uncleaved haemostatic protein than for the proteolytic cleavage products;    
       
         contacting the mixture containing the uncleaved haemostatic protein and proteolytic cleavage products with the immobilized antibody to bind the proteolytic cleavage products to the antibody;  
       
       
         separating the bound proteolytic cleavage products from the uncleaved haemostatic protein by affinity chromatography; and  
       
       
         isolating the uncleaved haemostatic protein. 
       
     
     
       14. A method according to  claim 13  wherein the antibody is specific for an epitope that covers an intact proteolytic cleavage site in the uncleaved haemostatic protein. 
     
     
       15. A method according to  claim 13  wherein the uncleaved haemostatic protein is a blood coagulation factor. 
     
     
       16. A method according to  claim 15  wherein the blood coagulation factor is Factor IX or Factor VII. 
     
     
       17. A method according to  claim 13  wherein the uncleaved haemostatic protein is an antagonist of a blood coagulation factor. 
     
     
       18. A method according to  claim 17  wherein the antagonist of a blood coagulation factor is Protein C or Protein S.