Development of a sandwich ELISA for the thrombin light chain identified by serum proteome analysis

Abstract

We previously identified novel biomarker candidates in biliary tract cancer (BTC) using serum proteome analysis. Among several candidates, we focused on thrombin light chain which is a 4204 Da peptide as the most promising biomarker for BTC. To move thrombin light chain toward potential diagnostic use, we developed an enzyme immunoassay that enables to measure serum thrombin light chain levels. Both one monoclonal antibody specific to the N-termini and one polyclonal antibody were used to develop a sandwich ELISA for thrombin light chain. The assay was evaluated by comparing the results with those obtained by the ClinProt™ system. Serum samples were obtained from 20 patients with BTC, 20 patients with BBTDs and 20 HVs using the ClinProt™ system and ELISA. The results of the established ELISA showed a positive correlation with the findings by ClinProt™ system (slope=0.3386, intercept=34.901, r²=0.9641). The performance of the ELISA was satisfactory in terms of recovery (97.9-102.5%) and within-run (1.5-4.8%) and between-day (1.9-6.7%) reproducibility. Serum thrombin light chain levels were significantly greater in BTC (176.5±47.2 ng/mL) than in BBTDs (128.6±17.4 ng/mL) and HVs (127.6±16.0 ng/mL) (p<0.001). The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum thrombin light chain levels in various cancers.

Keywords: Biliary tract cancer; Sandwich ELISA; Serum biomarker; Thrombin light chain.

Fig. 1

Western blotting analysis. Immunoreactive bands were observed at thrombin light chain when synthetic and serum thrombin light chain were incubated with anti-thrombin light chain N antibody (A) or thrombin light chain polyclonal antibody (B). Lane1: synthetic thrombin light chain. Lane2: serum thrombin light chain.

Fig. 2

Standard curves for thrombin light chain in the ELISA. (A) The relationship between colorimetric intensity and thrombin light chain concentration in the range of 0.0–200.0 ng/mL. Four concentrations of thrombin light chain were determined by ELISA. (B) Linearity of results from the ELISA, which fitted to an equation: y=1.042x+0.1914 (r²=0.9998, p<0.0001).

Fig. 3

Comparison of the results between ELISA and ClinProt System. The results between ELISA and ClinProt™ System were well-correlated (slope=0.3386, intercept=34.901, r2=0.9641, p<0.0001).

Fig. 4

Serum thrombin light chain levels in HVs, and in patients with benign BTDs and BTC. The levels are significantly greater in BTC (176.5±47.2 ng/mL) compared with the HVs (127.6±16.0 ng/mL) and benign BTDs (128.6±17.4 ng/mL). Serum thrombin light chain levels in patients with BTC were significantly higher than those in HVs and in patients with benign BTDs (P<0.001; Mann–Whitney U-test).