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

Kenta Noda, Kazuyuki Sogawa, Wataru Kikuchi, Iwao Kiyokawa, Toshihide Miura, Ryo Kojima, Katsuhiro Katayama, Yoshio Kodera, Fumio Nomura

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Purpose: We previously identified novel biomarker candidates in heavy consumers of alcohol using serum proteome analysis. Among several candidates, a 5.9kDa 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.

Original languageEnglish
Pages (from-to)141-146
Number of pages6
JournalProteomics - Clinical Applications
Volume5
Issue number3-4
DOIs
Publication statusPublished - 2011 Apr

Keywords

  • Fibrinogen alpha C chain
  • Liver disease
  • Sandwich ELISA
  • Serum biomarker
  • Stable isotope-labeled internal standard dilution-MS (SID-MS)

ASJC Scopus subject areas

  • Clinical Biochemistry

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