Radioimmunoassay of human prothrombin—The quantitation of plasma factor II antigen

Human factor II (prothrombin) was isolated by chromatographic techniques and showed a single band on electrophoresis. 125I‐tagged factor II was prepared by standard chloramine‐T oxidation and sodium metabisulfite reduction. The radioimmunoassay (RIA) method used was a specific double‐antibody radioimmunoassay similar to that described for the pituitary trophic hormones. The method showed high precision and sensitivity. A dose‐response curve was generated by adding known amounts of normal human plasma to human barium sulfate‐absorbed oxalated plasma. A straight‐line relationship existed between 15 to 140% coagulant activity and 7 to 50 μg/ml antigen content. Plasma factor II antigen in 20 normals was 86.1 ± 8.4 μg/ml. Eight patients with induced vitamin K deficiency revealed normal antigen levels and reduced coagulant activity, whereas three patients with severe hepatocellular disease were found to have both antigen and coagulant activity reduced. Although coagulant activity decreased with prolonged storage, it was found that antigen content did not change with up to three months' storage at − 20°C. The use of the RIA for measuring prothrombin protein in conjunction with coagulation factor assays may have clinical application in studying the mechanism of action of vitamin K, the developmental aspects of factor II production and activation, and the understanding of acquired coagulopathies.