Bead-Based ELISA for Validation of Ovarian Cancer Early Detection Markers

Abstract

Purpose: 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. Experimental Design: 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. Conclusion: 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 μL of sample each.