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.
Shima
H
H
Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Company Limited, Osaka, Japan.
Saeki
K
K
Yamanouchi
M
M
Omori
K
K
Sugiura
M
M
D000818:Animals; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D016232:Endothelins / Q000032:analysis* / Q000097:blood / Q000276:immunology*; D004730:Endothelium, Vascular / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D005069:Evaluation Studies as Topic; D006801:Humans; D008297:Male; D051379:Mice; D051381:Rats; D017208:Rats, Wistar; D012680:Sensitivity and Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D016232:Endothelins