Radioimmunoassay for human prothrombin.

Abstract

I describe a radioimmunoassay for human prothrombin, with use of a double-antibody technique. Antiserum raised in rabbits was absorbed with Al(OH)3 and heated to 56 degrees C for 30 min. 125I-labeled prothrombin retaining more than 90% of its biological activity was prepared by the iodine monochloride method. The mean concentration of prothrombin in plasma of 12 normal individuals was 100 +/- 29.4 mg/L (2 SD). Prothrombin values were somewhat lower than those obtained by the Laurell electroimmunoassay or by two-stage biological assay of the same plasma, done the same day. The biological values were converted to protein on the basis of 1960 int. units/mg by comparison with the other two assays. The ability of activation fragments of human prothrombin to inhibit binding of labeled prothrombin to its antibody was evaluated by competitive radioimmunoassay. Although precipitin lines formed with undiluted antiserum against all the fragments tested (F-1, F-1.2, prethrombin-1, and thrombin), none of the fragments competed well with prothrombin, even in 10-fold molar excess. Evidently, the structural integrity of the prothrombin molecule is essential for its maximum binding to the antiserum, and antigenic sites are lost during its activation.