title: Development of a sandwich ELISA for the 5.9-kDa fibrinogen alpha C chain fragment detected by serum proteome analysis.
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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, 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
  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.
authors.affiliation: R&D Department, Nittobo Medical Co., Ltd., Koriyama, Japan.
authors.forename: Kenta
authors.initials: K
authors.lastname: Noda
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Sogawa
authors.forename: Wataru
authors.initials: W
authors.lastname: Kikuchi
authors.forename: Iwao
authors.initials: I
authors.lastname: Kiyokawa
authors.forename: Toshihide
authors.initials: T
authors.lastname: Miura
authors.forename: Ryo
authors.initials: R
authors.lastname: Kojima
authors.forename: Katsuhiro
authors.initials: K
authors.lastname: Katayama
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kodera
authors.forename: Fumio
authors.initials: F
authors.lastname: Nomura
mesh_terms: D000437:Alcoholism / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005340:Fibrinogen; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008875:Middle Aged; D010446:Peptide Fragments / Q000097:blood*; D020543:Proteome / Q000032:analysis*; D040901:Proteomics; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D010446:Peptide Fragments; D020543:Proteome; C098000:fibrinogen alphaC; D005340:Fibrinogen