Immunoassays of human prothrombin species which correlate with functional coagulant activities

Specific immunoassays have been developed for forms of human prothrombin that vary in their degree of carboxylation. Human abnormal (des-gamma-carboxyl) prothrombin was isolated in 18% yield from the plasma of a patient treated with warfarin. The purified protein migrated as a single band in electrophoresis and contained an average of three gamma-carboxyglutamic acid residues per molecule. A specific antibody subpopulation was isolated from rabbit anti-abnormal prothrombin antiserum by using affinity chromatography. These antibodies, which bound to abnormal prothrombin but which cross-reacted minimally with prothrombin, were used to establish an immunoassay specific for abnormal prothrombin. In parallel, a specific antibody subpopulation, anti-prothrombin: Ca(II), was isolated from rabbit anti-prothrombin antiserum by conformation perturbation affinity chromatography. This antibody, which bound prothrombin but minimally cross-reacted with abnormal prothrombin, was used to establish a specific immunoassay for native prothrombin. An anti-prethrombin 1 subpopulation bound abnormal prothrombin and prothrombin equivalently and was used for an immunoassay that measured total prothrombin. These assays permit the quantitation of abnormal prothrombin and prothrombin in plasma and serum. The level of native prothrombin antigen correlates precisely with the functional prothrombin activity. These assays provide an example of the use of specific antibodies against functionally important antigenic surfaces to monitor properties of coagulation proteins with the precision and reliability of immunoassy.