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

作者：Noda Kenta; Sogawa Kazuyuki; Kikuchi Wataru; Kiyokawa Iwao; Miura Toshihide; Kojima Ryo; Katayama Katsuhiro; Kodera Yoshio; Nomura Fumio^*

来源：Proteomics - Clinical Applications, 2011, 5(3-4): 141-146.

DOI：10.1002/prca.201000127

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

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 (TM) system.
Results: The ELISA results correlated with the SID-MS findings (slope=0.795, intercept=-0.011, r(2)=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.

• 出版日期2011-4