Development of a sandwich ELISA for the 5.9-kDa fibrinogen alpha C chain fragment detected by serum proteome analysis.

Purpose: We previously identified novel biomarker candidates in heavy consumers of alcohol using serum proteome analysis. Among several candidates, a 5.9 kDa peptide identified as a fragment of the fibrinogen alpha C chain (FIC5.9) was the most promising. To move FIC5.9 toward potential diagnostic use, we developed an enzyme immunoassay that enables measurement of serum FIC5.9 levels. Experimental design: Two monoclonal antibodies specific to the N and C-termini of the 5.9-kDa peptide were used to develop a FIC5.9 sandwich ELISA. The assay was evaluated by comparing the results with those obtained by the stable isotope-labeled dilution mass spectrometry (SID-MS) using the ClinProt™ system. Results: The ELISA results correlated with the SID-MS findings (slope=0.795, intercept=−0.011, r2=0.908) and the performance of the ELISA was satisfactory in terms of recovery (98.5–103.0%) and within-run (1.4–4.7%) and between-day (2.8–8.4%) reproducibility. The assay was capable of detecting changes in FIC5.9 during abstinence from drinking in patients with alcohol dependency (p<0.0001). Conclusions and clinical relevance: The sandwich ELISA developed in this study will be useful for validation of the diagnostic significance of serum FIC5.9 levels in various pathological conditions, including alcoholism.