title: Bead-based ELISA for validation of ovarian cancer early detection markers.
abstract: OBJECTIVE
  Efforts to validate ovarian cancer early detection biomarkers with immunoassays are challenged by the limited specimen volumes available. We sought to develop a specimen-efficient assay to measure CA125 in serum, assess its reproducibility, validity, and performance, and test its potential for multiplexing and combining with human epididymis protein 4 (HE4), a promising novel ovarian cancer marker.
  
  
  METHODS
  Four pairs of commercially available anti-CA125 antibodies and one pair of anti-HE4 antibodies were evaluated for accuracy in measuring known concentrations of antigen on a bead-based platform. The two best pairs were further assessed for reproducibility, validity, and the ability to discriminate between blinded serum samples obtained from ovarian cancer cases (n = 66) and women without ovarian cancer (n = 125).
  
  
  RESULTS
  Suitability for use in a bead-based assay varied across CA125 antibody pairs. Two CA125 bead-based assays were highly reproducible (overall correlations between replicates >/= 0.95; coefficients of variation < 0.2) and strongly correlated with the research standard CA125II RIA (correlations >/= 0.9). Their ability to distinguish ovarian cancer cases from non-cases based on receiver operating characteristic analyses (area under the curve, AUC, of 0.85 and 0.84) was close to that of the CA125II RIA (AUC, 0.87). The HE4 bead-based assay showed lower reproducibility but yielded an AUC of 0.89 in receiver operating characteristics analysis. Multiplexing was not possible but a composite marker including CA125 and HE4 achieved an AUC of 0.91.
  
  
  CONCLUSIONS
  Optimization procedures yielded two bead-based assays for CA125 that perform comparably to the standard CA125II RIA, which could be combined with an HE4 bead-based assay to improve diagnostic performance, and requires only 15 muL of sample each.
authors.affiliation: Translational Outcomes Research Laboratory, Fred Hutchinson Cancer Research Center, Public Health Sciences, and Harborview Medical Center, University of Washington, Seattle, Washington, USA.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Scholler
authors.forename: Meghan
authors.initials: M
authors.lastname: Crawford
authors.forename: Alicia
authors.initials: A
authors.lastname: Sato
authors.forename: Charles W
authors.initials: CW
authors.lastname: Drescher
authors.forename: Kathy C
authors.initials: KC
authors.lastname: O'Briant
authors.forename: Nancy
authors.initials: N
authors.lastname: Kiviat
authors.forename: Garnet L
authors.initials: GL
authors.lastname: Anderson
authors.forename: Nicole
authors.initials: N
authors.lastname: Urban
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000097:blood*; D018394:CA-125 Antigen / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D030501:Epididymal Secretory Proteins / Q000032:analysis*; D005260:Female; D006801:Humans; D008875:Middle Aged; D010051:Ovarian Neoplasms / Q000097:blood / Q000175:diagnosis*; D011237:Predictive Value of Tests; D012372:ROC Curve; D012680:Sensitivity and Specificity; D023083:beta-Defensins
chemical_list: D014408:Biomarkers, Tumor; D018394:CA-125 Antigen; C426184:DEFB126 protein, human; D030501:Epididymal Secretory Proteins; D023083:beta-Defensins