# ELISA-Based Detection System for Protein S K196E Mutation, a Genetic Risk Factor for Venous Thromboembolism

## TL;DR

This study developed a reliable ELISA method to detect a genetic mutation in Protein S linked to increased risk of blood clots in Japanese individuals.

## Contribution

A novel ELISA system using mutation-specific antibodies to identify PS K196E carriers with high accuracy.

## Key findings

- Monoclonal antibodies specific to the PS K196E mutation were successfully generated.
- The ELISA system accurately distinguished 11 KE genotype individuals from 122 KK genotype individuals.
- The method provides a rapid and reliable way to identify individuals at higher risk for venous thromboembolism.

## Abstract

Protein S (PS) acts as a cofactor for activated protein C in the plasma anticoagulant system. PS Lys196-to-Glu (K196E) mutation is a genetic risk factor for venous thromboembolism in Japanese individuals. Because of the substantial overlap in PS anticoagulant activity between KK (wild-type) and KE (heterozygous) genotypes, it is difficult to identify PS K196E carriers by measuring PS activity. Here, we generated monoclonal antibodies specific to the PS K196E mutant and developed a simple and reliable method for the identification of PS K196E carriers. We immunized mice with a keyhole limpet hemocyanin-conjugated synthetic peptide with Glu196. The hybridoma cells were screened for the binding ability of the produced antibodies to recombinant mutant EGF-like domains of PS (Ile117–Glu283). We obtained three hybridoma cell lines producing PS K196E mutation-specific antibodies. We established a sandwich enzyme-linked immunosorbent assay (ELISA) system in which the PS K196E mutation-specific monoclonal antibody was used as a detection antibody. We measured human plasma samples by using this system and successfully discriminated 11 individuals with the KE genotype from 122 individuals with the KK genotype. The ELISA system using the PS K196E mutation-specific antibody is a useful tool for the rapid identification of PS K196E carriers, who are at a higher risk for venous thromboembolism.