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

## TL;DR

This study shows that PIVKA-II, a biomarker for liver cancer, exists in different molecular forms, which could improve diagnosis accuracy.

## Contribution

The study reveals the molecular diversity of PIVKA-II in HCC patients using chromatography techniques.

## Key findings

- PIVKA-II exists in two distinct molecular forms with different hydrophobicity levels.
- Each form reacts differently with specific prothrombin fragments and antibodies.
- This molecular diversity could enhance HCC diagnosis accuracy.

## Abstract

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.

PIVKA‐II comprises two heterogeneous molecular forms with different levels regarding hydrophobicity, and PIVKA‐II in serum and plasma exists as distinct molecular forms with varying levels.