Development of a microplate ELISA for circulating E‐selectin: assay characterization, comparison with a commercial kit, wand 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.

A panel of multiple markers associated with chronic systemic inflammation and the risk of atherogenesis is detectable in asthma and chronic obstructive pulmonary disease

“…Most markers were measured with in-house developed enzyme linked immunosorbent assay (ELISA) including C-reactive protein (CRP) with a highly sensitive assay (13), serum amyloid A (SAA) (14) adhesion molecules (15,16), uMA (17), 8-hydroxydeoxyguanosine (8-OHdG) (18), interleukin-6 (IL-6) (19), and 3-nitrotyrosine (3NT) (20). All in-house kits have been compared with a commercial kit with good to excellent correlation.…”

Identification of OPN, TNC and E-selectin as potential recognition proteins in cerebral vasospasm after subarachnoid hemorrhage

“…E-selectin is mainly located in vascular endothelial cells, and the expression of which can be obviously induced by inflammatory cytokine especially in cerebrovascular endothelial cells. However, the serum concentration of which will not upregulate at all [11].…”

“…E-selectin is a kind of glycoprotein whose molecular weight is 115KD, which exists in endothelial cells. Pro-inflammatory cytokines such as interleukin, tumor necrosis factor and interferon have stimulatory effects on the synthesis of E-selectin [11]. Studies have revealed that cerebrovascular endothelial cells under normal condition barely have expression of E-selectin.…”

Development of multiple complications in type 2 diabetes is associated with the increase of multiple markers of chronic inflammation

“…All of these in-house kits have been compared with commercial kits with good to excellent correlation, including CRP (20), adhesion molecules (21,22), IL-6 (23), uMA (24), and homocysteine (Hcy) (25). Uric acid (UA) was determined by a Hitachi 7600-210 autoanalyzer (Hitachi, Tokyo, Japan).…”