Development of a microplate ELISA for circulating E-selectin: assay characterization, comparison with a commercial kit, wand establishment of normal reference values
✍ Scribed by Kuo-Chien Tsao; Pi-Yueh Chang; Chia-Chi Li; Tsu-Lan Wu; Chien-Feng Sun; James T. Wu
- Publisher
- John Wiley and Sons
- Year
- 2003
- Tongue
- English
- Weight
- 128 KB
- Volume
- 17
- Category
- Article
- ISSN
- 0887-8013
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✦ Synopsis
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. J. Clin. Lab. Anal. 17:97–101, 2003. © 2003 Wiley‐Liss, Inc.