Characterization of PIVKA‐II Molecular Forms via Size Exclusion Chromatography and Hydrophobic Interaction Chromatography in HCC Patients

Background Protein induced by the absence of vitamin K or antagonist‐II (PIVKA‐II) is an important serological biomarker for the diagnosis of hepatocellular carcinoma (HCC). The crucial epitope of PIVKA‐II comprises a series of Glu residues located in the Glu‐Gla domain. The other antibody required for the PIVKA‐II sandwich immunoassay recognizes prothrombin fragments 1 and 2. However, the molecular diversity of these regions has not been well documented. Methods PIVKA‐II immuno‐reactivities in blood taken from HCC patients were analyzed by size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC). Obtained fractions were subsequently tested using MU‐3 monoclonal antibody (mAb) and a polyclonal anti‐prothrombin antibody (pAb). Immuno‐reactivity in each fraction was analyzed by an inhibition assay using prothrombin fragments and by a sandwich immunoassay. Results SEC analysis of three paired serum and plasma samples gave a single peak of PIVKA‐II immuno‐reactivity corresponding to the expected molecular mass. However, HIC analysis using three paired sets and 14 HCC specimens revealed immuno‐reactivity present in two distinct peaks of either low (peak‐L: an elution position of 300–500 mM ammonium sulfate) or high (peak‐H: an elution position of 100–300 mM ammonium sulfate) hydrophobicity or both peaks together. Immuno‐reactivity of peak‐L was inhibited by human prothrombin fragment‐1 and detected by mAbs that recognize prothrombin‐1. Immuno‐reactivity of peak‐H was inhibited by human prothrombin fragment‐2 and detected by mAbs for prothrombin‐2. Conclusions These results demonstrate PIVKA‐II exhibits molecular diversity, suggesting its potential as a diagnostic tool. This finding will contribute to further understanding of HCC and improve diagnostic accuracy.