Open AccessDevelopment of a microplate ELISA for circulating E‐selectin: assay characterization, comparison with a commercial kit, wand establishment of normal reference values
Author(s) -
Tsao KuoChien,
Chang PiYueh,
Li ChiaChi,
Wu TsuLan,
Sun ChienFeng,
Wu James T.
Publication year - 2003
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.10077
Subject(s) - biotinylation , monoclonal antibody , microbiology and biotechnology , streptavidin , reference values , chemistry , significant difference , chromatography , antibody , medicine , immunology , biology , biotin , biochemistry
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.