Sandwich enzyme immunoassay for endothelin with monoclonal antibodies and its application.

The development of a sensitive enzyme immunoassay for endothelin is described. This assay is based on a sandwich method using two different monoclonal antibodies against endothelin-1. A monoclonal antibody, which reacted to the C-terminal region of endothelin, was selected as an immobilized antibody. The Fab' fragment of another monoclonal antibody, which might recognize the N-terminal rigid region of endothelin, was used as a horseradish peroxidase-labeled detector antibody. The assay measures endothelin-1 and endothelin-2 with a sensitivity of 1 fmol/ml. We have determined that cultured endothelial cells actually produced endothelin in significant amounts in a time-dependent manner. The levels of plasma endothelin extracted with Sep-Pak tC18 light cartridges could also be monitored. A basal endothelin level was about 0.3 fmol/ml of plasma, and a transient increase was observed 4 h after starting blood collection under in vivo experimentation in the rat. This enzyme immunoassay will facilitate the investigation of physiological roles of endothelin.