Use of Yeast-SecretedIn vivoBiotinylated Recombinant Antibodies (Biobodies) in Bead-Based ELISA

Abstract

Purpose: To measure circulating antigens, sandwich ELISA assays require two complementary affinity reagents. Mouse monoclonal antibodies (mAb) and polyclonal antibodies (pAb) are commonly used, but because their production is lengthy and costly, recombinant antibodies are emerging as an attractive alternative. Experimental Design: We developed a new class of recombinant antibodies called biobodies (Bb) and compared them to mAb for use in serodiagnosis. Bbs were secreted biotinylated in vivo by diploid yeast and used as affinity reagents after Ni purification. Bead-based assays for HE4 and mesothelin were developed using Bbs in combination with pAbs (Bb/pAb assays). To assess precision, reproducibility studies were done using four runs of 16 replicates at six analyte levels for each marker. Pearson correlations and receiver-operator characteristic analyses were done in 214 patient serum samples to directly compare the Bb/pAb assays to mAb assays. Diagnostic performance of the Bb/pAb assay was further assessed in an expanded set of 336 ovarian cancer cases and controls. Results: On average across analyte levels, Bb/pAb assays yielded within-run and between-run coefficients of variations of 11.7 and 23.8, respectively, for HE4 and 14.0 and 14.5, respectively, for mesothelin. In the subset (n = 214), Pearson correlations of 0.95 for HE4 and 0.92 for mesothelin were observed between mAb and Bb/pAb assays. The area under the curves for the mAb and Bb/pAb assays were not significantly different for HE4 (0.88 and 0.84, respectively; P = 0.20) or mesothelin (0.74 and 0.72, respectively; P = 0.38). Conclusion: Yeast-secreted Bbs can be used reliably in cost-effective yet highly sensitive bead^based assays for use in large validation studies.Large-scale screening studies to evaluate candidate ovarian cancer early detection biomarkers with immunoassays are challenged both by limitations in the quantity of patient sample required and by the need for large amounts of expensive and work-intensive affinity reagents. To overcome the first limitation, we adapted double-determinant ELISA assays to a bead-based platform that uses spectrally discrete polystyrene beads or microspheres instead of flat surfaces to immobilize the capture antibody (1, 2). Each antibody-coupled microsphere captures soluble antigens that are then detected with biotinylated antibody and phycoerythrin-conjugated streptavidin. A reading system based on flow cytometry (Bio-Plex Protein Array System, Bio-Rad) measures the fluorescent signals generated by streptavidin-phycoerythrin and the microspheres when they are close to each other after formation of antigen/ antibody complexes. The data are reported as median fluorescence intensity. We developed bead-based assays with available mouse monoclonal antibodies to detect CA125 and HE4 (3) and two biomarkers for ovarian carcinoma (4 -7), and we showed that the CA125 and HE4 assays did comparably with the standard CA125II RIA and the HE4 ELISA, respectively, while requiring only 15 AL of serum.To address t...

Mesothelin as a biomarker for ovarian carcinoma: a meta-analysis

“…In two studies (Bandiera et al 2013, Scholler et al 1999, there was unclear risk of bias in patient selection, and two studies (Moraes 2012, Scholler et al 2008) showed a high risk of bias in patient selection. One study (McIntosh et al 2004) showed high risk of bias in the index test and flow and timing.…”

“…One study (McIntosh et al 2004) showed high risk of bias in the index test and flow and timing. Three studies (McIntosh et al 2004, Moraes 2012, Scholler et al 2008) were classified as having a high risk of bias. The results of the quality assessment were presented in Figure 2 for the 12 included studies.…”

Preventive Strategies in Epithelial Ovarian Cancer

“…Biobodies have been generated against HE4 95 and mesothelin 96 . We have also demonstrated that this technology can be used reliably in a highly-sensitive bead-based ELISA assay for screening large populations of women for ovarian cancer 5,67 and for serum biomarker discovery 6 . These novel approaches to biomarker discovery offers promise for improved ovarian cancer screening and for detection of recurrences.…”

“…In addition to these efforts, we have generated a cost-and time-effective method for generating site-specific in vivo biotinylated recombinant antibodies secreted by yeast (Biobodies 5,95 ). Biobodies have been generated against HE4 95 and mesothelin 96 .…”

Diagnostic Value of Human Epididymis Protein 4 Compared with Mesothelin for Ovarian Cancer: a Systematic Review and Meta-analysis

“…A total of 18 studies (Scholler et al, 1999;Hellstrom et al, 2003;McIntosh et al, Scholler et al, 2008;Huhtinen et al, 2009;Montagnana et al, 2009;Shah et al, 2009;Abdel-Azeez et al, 2010;Nolen et al, 2010;Chang et al, 2011;Holcomb et al, 2011;Jacob et al, 2011;Van Gorp et al, 2011;Park et al, 2012) with 3865 patients who satisfied the inclusion criteria were analyzed. The results are shown in Table 1.…”