Specific measurement of hypocarboxylated prothrombin in plasma or serum and application to the diagnosis of hepatocellular carcinoma

An enzyme-linked immunosorbent assay (ELISA) was developed for measuring human hypocarboxyprothrombin, a protein induced by vitamin K antagonists (PIVKA-II). A specific monoclonal antibody (P-1-2-B9) was prepared and used for coating a micro-ELISA plate, and revelation proceeded with a rabbit polyclonal anti-human prothrombin antibody-peroxidase conjugate. This assay allowed the measurement of PIVKA-II at concentrations ranging from 2 to 200 ng/ml, with an intraassay coefficient of variation of less than 5.2% and an interassay coefficient of variation of less than 7.4%. This assay permits the direct evaluation of PIVKA-II in citrated plasma as well as in serum. The concentration of PIVKA-II is expressed in nanograms per milliliter. It offers specific measurement of PIVKA-II without any cross-reactivity from native prothrombin: the specificity for PIVKA-II respective to prothrombin is >10(5). No reactivity was observed with other vitamin K-dependent proteins, whether fully active or decarboxylated. Complementary studies have demonstrated that the monoclonal antibody used was preferentially directed to 3 to 6 Gla hypocarboxylated prothrombin. PIVKA-II concentration was below 2.4 ng/ml in normal individuals (n = 59). In 61 patients given dicoumarol for more than 90 days (receiving a stable therapy), the measured concentrations of PIVKA-II ranged from 750 to 13,400 ng/ml. Combined with the assay of alpha-fetoprotein (AFP), measurement of PIVKA-II in patients with liver diseases introduces a complementary exploration for hepatocellular carcinoma (HCC). In a study in 59 patients with HCC, the assay sensitivity was 49.1% for PIVKA-II, 47.5% for AFP, and 71% for both markers combined.