Development of a microplate ELISA for circulating E-selectin: Assay characterization, comparison with a commercial kit, and establishment of normal reference values

Abstract

E-selectin, an adhesion molecule, is detectable in the blood. Serum levels of E-selectin can be a prognostic indicator for various malignancies. We developed a microplate enzyme-linked immunosorbent assay (ELISA) for the detection of circulating E-selectin by coating the microwell with monoclonal anti- E-selectin. We incubated the specimen and the biotinylated detecting antibody simultaneously, and used HPR-conjugated streptavidin to generate a signal for quantification. The assay has a sensitivity of 3.8 ng/mL, and the coefficients of variation (CVs) for both within-day and day-to-day precision were all <10%. Our in-house-made kit also compared well with a commercial kit from R&D Systems (r=0.95, slope=0.94). Using our kit we determined a normal reference value for Chinese individuals of different ages (30-80 years). We found no significant difference between females and males; however, age appeared to have an impact on the normal E-selectin value. We found that normal individuals over 60 years old had higher levels of circulating E-selectin (65.8±19.5 ng/mL, N=111) compared to those under 60 years old (59.5±18.1 ng/mL; P=0.002). Our kit appears to have sufficient sensitivity for detecting elevated circulating E-selectin in various carcinomas. We believe that with the established normal reference value, our in-house-developed ELISA kit is well suited for routine clinical laboratory use. © 2003 Wiley-Liss, Inc.