Quantitation of endothelial cell specific protein E-9 employing a single monoclonal antibody in an indirect sandwich ELISA.

Abstract

An indirect enzyme-linked immunosorbent assay is described for the quantitation of protein E-9 which is specifically expressed on human vascular endothelial cells. The assay capitalizes on the dimeric structure of the E-9 protein by utilizing a single monoclonal antibody as both the capture and detection reagent. Detection is achieved by conjugating the Mab with biotin and is followed by the addition of streptavidin peroxidase to provide high sensitivity. Bound activity is measured by enhanced chemiluminescence utilizing standard Amerlite chemistry. The optimised assay is reproducible and is highly sensitive. Using this assay it was possible to detect the presence of E-9 protein in tissue culture media of endothelial cells and in serum samples--in one case even at 1/100 dilution. In vitro, X irradiation resulted in a greater than two-fold increase (P < or = 0.005) in the level of E-9 protein in culture supernatants of human umbilical vein endothelial cells (HUVEC). There are potential applications for measurements of E-9 protein in body fluids and tissue extracts from patients with a vast variety of diseases characterised by vascular endothelial damage and/or activation.