Characterization of PIVKA-II Molecular Forms via Size Exclusion Chromatography and Hydrophobic Interaction...

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.

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.

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.

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.