Development of a microplate ELISA for circulating E-selectin: assay characterization, comparison with a commercial kit, wand establishment of normal reference values.
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.
Tsao
Kuo-Chien
KC
Department of Pathology, Chang Gung Memorial Hospital, Taipei, Taiwan.
Chang
Pi-Yueh
PY
Li
Chia-Chi
CC
Wu
Tsu-Lan
TL
Sun
Chien-Feng
CF
Wu
James T
JT
D000328:Adult; D000368:Aged; D000375:Aging; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002138:Calibration / Q000592:standards; D002277:Carcinoma / Q000378:metabolism; D019040:E-Selectin / Q000097:blood* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D011897:Random Allocation; D051381:Rats; D011933:Reagent Kits, Diagnostic; D012016:Reference Values; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D000911:Antibodies, Monoclonal; D019040:E-Selectin; D011933:Reagent Kits, Diagnostic