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 muL of sample each.

Figure 1. Determination of optimal coupling concentrations with purified CA125 antigen for five pairs of anti-CA125 antibodies

Five coupling concentrations of capture antibody (0.2, 1, 5, 10 and 20 μg/ml) were incubated with serial dilutions of CA125 antigen in the assay diluent. The captured antigen was detected by 1 μg/ml of biotinylated detection antibody. A: RDI antibodies, X306 capture, X52 detection; B: FDI antibodies, M11 capture, OC-125 detection; C–D: FIII antibodies, C: M002201 capture, M002203 detection; D, orientation 1: M8072320 capture, M8072321 detection; orientation 2: M8072321 capture, M8072320 detection.

Figure 2. Calibration and multiplexing of the bead-based assays

A- Four sera with known CA125 concentration levels were diluted 4 fold and used to compare four CA125 bead-based assays. All detection antibodies were used at 1 μg/ml. The capture antibodies were immobilized on microspheres at 5 μg/ml for RDI mAb; 20 μg/ml for FIII M8072320 mAb; 1 μg/ml for FDI mAb and 1 μg/ml for FIII M002201 mAb. B- Micropheres were coupled with five concentrations of 2H5 anti-HE4 capture mAb as shown, and incubated with two HE4 positive sera (pos1 and pos 2; HE4 concentrations of 5 ng/ml as determined by ELISA) and two HE4 negative sera (neg 1 and neg 2; HE4 concentrations non detectable by ELISA) diluted 4 fold in assay buffer. HE4 serum levels were detected with 6 μg/ml of 3D8 anti-HE4 biotin-conjugated mAb. C- Optimized HE4 bead-based assay was tested in the absence or in the presence of CA125 bead-based assay reagents and/or in presence of serial dilutions of CA125 purified antigen in assay diluent (as shown: 0; 30; 300; 3000 U/ml). D- CA125 bead-based assay using RDI antibodies was tested in the absence or in the presence of HE4 bead-based assay reagents and/or in presence of serial dilutions of HE4-Ig antigen (as shown: 0; 0.5; 1; 2 μg/ml).

Figure 3. CA125 and HE4 bead-based assay performance compared to CA125II using ROC

The ability of the CA125 II RIA to distinguish women with ovarian cancer (cases) from non-cases (A), cases from women who underwent pelvic surgery for reasons unrelated to cancer (B), and cases from a healthy screening population (C) was compared among the two CA125 bead-based assays (RDI and FIII), the HE4 bead-based assay, and the CA125II RIA separately. Total AUC values for each assay are listed in parentheses.

Figure 4. CA125 and HE4 bead-based assay composite marker (CM) performance compared to CA125II using ROC

Each of the two CA125 bead-based assays (RDI and FIII) was combined with the HE4 bead-based assay to form 2 composite markers (CM). The ability of the CA125II RIA to distinguish women with ovarian cancer (cases) from non-cases (A), cases from women who underwent pelvic surgery for reasons unrelated to cancer (B), and cases from a healthy screening population (C) was compared to each of the CM. Total AUC values for each CM and the RIA are listed in parentheses.