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 z 0.95; coefficients of variation < 0.2) and strongly correlated with the research standard CA125II RIA (correlations z 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 AL of sample each.

Method for generation of in vivo biotinylated recombinant antibodies by yeast mating

“…Finally, this method may also be used to generate reagents for emerging technologies such as antibody-based proximity ligation (Fredriksson et al, 2002), antibody-based competition assays using tadpoles (Burbulis et al, 2005), bead-based assays (Vignali, 2000;Gorelik et al, 2005;Scholler et al, 2006) and protein or antibody arrays (Chen et al, 2005;Gao et al, 2005;Utz, 2005;Boozer et al, 2006). In conclusion, the yeast expression system described here allows efficient generation of directly biotinylated high-affinity reagents that are needed for a large range of applications, including evaluation of the products of large-scale proteomics discovery projects.…”

Screening for Ovarian Cancer in Women

“…Various types of tumor markers have been study both early staged and late staged of ovarian cancer (Table 3) (Rein, Gupta et al 2011 Human epididymis protein 4 (HE4) is elevated in ovarian cancer, especially endometrioid adenocarcinoma and serous cystadenocarcinoma (Scholler, Crawford et al 2006). Osteopontin is a glycoprotein and secreted from vascular endothelial cells and osteoblasts.…”

The Role of HE4 in Ovarian Cancer: Inhibiting Tumour Cell Proliferation and Metastasis

“…8 Over the past decade, HE4 has been used widely for the early screening 15,16 and differential diagnosis 17,18 of ovarian cancer, as well as for monitoring disease recurrence 19 and progression. 20 The HE4 promoter is also under investigation in terms of potential ovarian cancer gene therapy.…”