SANDWICH ENZYME-IMMUNOASSAY FOR ENDOTHELIN WITH MONOCLONAL-ANTIBODIESAND ITS APPLICATION

The development of a sensitive enzyme immunoassay for endothelin is de scribed. This assay is based on a sandwich method using two different monoclonal antibodies against endothelin-1. A monoclonal antibody, whi ch 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/m l. We have determined that cultured endothelial cells actually produce d endothelin in significant amounts in a time-dependent manner. The le vels of plasma endothelin extracted with Sep-Pak tC(18) light cartridg es could also be monitored. A basal endothelin level was about 0.3 fmo l/ml of plasma, and a transient increase was observed 4 h after starti ng blood collection under in vivo experimentation in the rat. This enz yme immunoassay will facilitate the investigation of physiological rol es of endothelin. (C) 1995 Academic Press, Inc.