Development of a sandwich enzyme-linked immunosorbent assay for the detection of CD44v3 using exon v3- and v6-specific monoclonal antibody pairs.

It has been suggested that soluble CD44 levels in cancer patient sera may be closely associated with tumor progression and metastasis. However, to date, there has been limited methodology for detecting the soluble CD44 variant 3 isoform (CD44v3). Herein, using phage display technology, we isolated monoclonal antibodies specific to exon v3 or v6 of CD44 (CD44-exonv3 or CD44-exonv6) from a human synthetic antibody library. We also confirmed the specificity of antibody binding to CD44-exonv3 or -exonv6. Label-free kinetic analysis using the Octet biolayer interferometry system showed that the Kd values of the anti-CD44-exonv3 and anti-CD44-exonv6 antibodies for CD44v3-10 are approximately 1.1nM and 1.5nM, respectively. Finally, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs. The minimum detection limit of the assay was 6.2ng/ml CD44v3-10 and the linear range was up to 125ng/ml. Intra- and inter-assay coefficients of variation were 2.2% and 2.9%, respectively. The intra- and inter-assay recoveries were 99.3% and 105.3%, respectively. Taken together, these results suggest that this novel sandwich ELISA using the anti-CD44-exonv3 and anti-CD44-exonv6 antibody pairs will be useful for the detection of soluble CD44v3 in cancer patient sera.