Quantitative and functional assays compared for determination of zymogen and activated human protein C

Abstract

Abstract We evaluated quantitative and functional assays for protein C, using either purified protein C samples or pooled normal plasma as assay standards. The purified protein C samples were examined as the zymogen form and after activation by thrombin. Mass concentrations of protein C were determined by amino acid analysis and confirmed by enzyme-linked immunosorbent assay (ELISA). Functional activity was assessed in both standard clot inhibition and amidolytic assays. The accuracy and precision of the ELISA was acceptable, with all three preparations of protein C having similar linear curves. The clot inhibition assay demonstrated marked variability when used according to the manufacturer's instructions; however, modifications to the protocol significantly decreased the CV, to less than 10%. Both activated protein C and the zymogen gave linear standard curves. Pooled normal human plasma gave a nonlinear curve, which contributed to inaccurate sample recoveries. We obtained the most nearly accurate recoveries when we used activated protein C as the assay standard. Amidolytic assays provided no insights into the appropriateness of the preparations for that assay format. A uniform, consistent source of protein C, e.g., recombinant activated protein C, would be useful for standardizing all assays of protein C.