A monoclonal-antibody-based radioimmunoassay for measurement of protein C in plasma

A monoclonal-antibody-based competitive radioimmunoassay for measuring human protein C is reported. With use of a purified protein C standard, the solid-phase assay was sensitive to less than 80 micrograms of protein C per liter. Intraassay CVs ranged from 5% to 8%; the inter-assay CV was 5.4%. Analytical recovery averaged 104% for purified protein C added to 10 samples of normal plasmas. The assay antibody could deplete plasma of all protein C, as determined by immunoaffinity chromatography followed by polyclonal Western blot analysis. Liquid-chromatographic gel permeation of plasma indicated a single immunoreactive species that had an appropriate molecular mass for monomeric protein C. Studies of monoclonal-antibody specificity showed no significant interferences by other vitamin K-dependent proteins. The mean protein C antigen concentration in plasma of 54 healthy subjects was 3.21 (SD 0.56) mg/L and was 1.51 (SD 0.52) mg/L for 22 patients on long-term warfarin therapy. Results of the monoclonal RIA correlated well with those by a polyclonal RIA also developed in our laboratory (r = 0.93) and an amidolytic functional assay (r = 0.88) for both normal plasma and plasma from patients on long-term warfarin therapy.