Immunoassay for F1.2 prothrombin fragment

Abstract

According to the present invention highly specific-low affinity antibodies are generated which allow for the assay of F1.2 in bodily fluids that also contain prothrombin or other plasma proteins. Antibodies having the necessary properties for this assay are made using synthetic polypeptides which mimic the carboxy terminus of F1.2.

What is claimed is:  
     
         1 . A sandwich assay for the determination of the presence or concentration of a protein comprising: 
 eliciting antibody formation using synthetic peptides which correspond to a portion of said protein;    using said elicited antibodies in a sandwich assay to determine or detect the presence or concentration of said protein.    
     
     
         2 . The assay of  claim 1  wherein the synthetic peptide corresponds to the carboxy terminus region of said protein.  
     
     
         3 . The assay of  claim 1  wherein the synthetic peptide corresponding to a portion of said protein elicits highly specific-low affinity antibodies.  
     
     
         4 . The assay of  claim 1  wherein the elicited antibody has an affinity constant of less than about 1×10 8  L/M.  
     
     
         5 . A sandwich assay for the determination of the presence or concentration of a protein comprising: 
 eliciting antibody formation using synthetic peptides which correspond to a portion of the protein;    immobilizing antibodies on a solid carrier;    contacting said immobilized antibodies with fluid containing said protein;    adding a tag antibody specific for said protein;    determining the concentration or presence of said protein from the presence or concentration of tag detected.    
     
     
         6 . The assay of  claim 5  wherein the synthetic peptide corresponds to the carboxy terminus region of said protein.  
     
     
         7 . The assay of  claim 5  wherein the synthetic peptide corresponding to a portion to said protein elicits highly specific-low affinity antibodies.  
     
     
         8 . The assay of  claim 5  wherein the elicited antibody has an affinity constant of less than about 1×10 8  L/M.  
     
     
         9 . A process according to  claim 5  wherein the labelled antibody is labelled with a member selected from the group consisting of a radioactive isotope, an enzyme or fluorogenic material and said examination is by means selected from the group consisting of radiometric means, enzymatic means or fluorometric means.  
     
     
         10 . A sandwich assay for the determination of the presence or concentration of a F1.2 prothrombin polypeptide fragment comprising: 
 eliciting antibody formation using synthetic peptides which correspond to a portion of said polypeptide fragment;    using said elicited antibodies in a sandwich assay to determine or detect the presence or concentration of said polypeptide fragment.    
     
     
         11 . The assay of  claim 10  wherein the synthetic peptide corresponds to the carboxy terminus region of F1.2 polypeptide fragment.  
     
     
         12 . The assay of  claim 10  wherein the synthetic peptide corresponding to a portion of said polypeptide fragment elicits highly specific-low affinity antibodies.  
     
     
         13 . The assay of  claim 10  wherein the elicited antibody has an affinity constant of less than about 1×10 8  L/M.  
     
     
         14 . A sandwich assay for the determination of the presence or concentration of F1.2 polypeptide fragment in bodily fluids which contain prothrombin comprising: 
 eliciting antibody formation using synthetic peptides which correspond to a portion of the prothrombin F1.2 polypeptide fragment;    immobilizing said antibodies on a solid carrier;    contacting said immobilized antibodies with said bodily fluid;    adding a tag antibody specific for said F1.2 polypeptide fragment;    determining the presence or concentration of said F1.2 polypeptide fragment from the presence or concentration of the tag detected.    
     
     
         15 . The assay of  claim 14  wherein the synthetic peptide corresponds to the carboxy terminus region of F1.2 polypeptide fragment.  
     
     
         16 . The assay of  claim 14  wherein the synthetic peptide amino acid sequence is CYS-GLY-ASP-ARG-ALA-ILE-GLU-GLY-ARG-OH.  
     
     
         17 . The assay of  claim 14  wherein said immobilized antibody has an affinity constant of about 5.3×10 5  L/M.  
     
     
         18 . The assay of  claim 14  wherein the synthetic peptide corresponding to a portion of the F1.2 polypeptide fragment elicits highly specific-low affinity antibodies.  
     
     
         19 . The assay of  claim 14  wherein the synthetic peptide corresponding to a portion of the F1.2 polypeptide elicits an antibody having an affinity constant of less than about 1×10 8  L/M.  
     
     
         20 . A process according to  claim 14  wherein the labelled antibody is labelled with a member selected from the group consisting of a radioactive isotope, an enzyme or fluorogenic material and said examination is by means selected from the group consisting of radiometric means, enzymatic means or fluorometric means.  
     
     
         21 . The process according to  claim 14  wherein the bodily fluid is plasma.  
     
     
         22 . A monoclonal antibody produced by a hybridoma formed by the fusion of cells from a mouse myeloma line and spleen cells from a mouse previously immunized with a synthetic polypeptide which corresponds to a portion of the F1.2 prothrombin polypeptide fragment comprising: 
 an antibody which reacts specifically with F1.2 in the presence of prothrombin, and other plasma proteins.    
     
     
         23 . The monoclonal antibody of  claim 22  wherein said synthetic polypeptide corresponds with carboxy terminus portion of F1.2.  
     
     
         24 . The monoclonal antibody of  claim 22  wherein said synthetic polypeptide has the amino acid sequence: 
 CYS-GLY-ASP-ARG-ALA-ILE-GLU-GLY-ARG-OH.  
 
     
     
         25 . A process to make specific antibodies to proteolytic cleavage fragments arising during zymogen activation, comprising: 
 making a synthetic polypeptide which corresponds to a portion of the carboxy or amino terminus of said proteolytic cleavage fragments.    using said synthetic polypeptide to elicit antibodies specific to said polypeptide.    
     
     
         26 . The process of  claim 25  wherein the proteolytic cleavage fragment corresponds to a portion of F1.2 prothrombin polypeptide fragment.  
     
     
         27 . The process according to  claim 25  wherein the antibody is polyclonal.  
     
     
         28 . The process according to  claim 25  wherein the antibody is monoclonal.