A sandwich-type enzyme immunoassay to detect immunoreactive big-endothelin-1 in plasma

A sensitive sandwich-type enzyme immunoassay (sandwich-EIA) has been established for human big endothelin-1 (big-ET-1). A monoclonal antibody AwETN40 which recognizes the N terminal portion of big-ET-1 was used as an immobilized antibody. The Fab' fragment of rabbit antibodies to the human big-ET-1 C terminal peptide (22-38) was used as the enzyme-labeled antibody after being coupled to horseradish peroxidase (HRP). The assay is sensitive enough to detect as little as 0.2 pg/well (5 x 10(-17) mol/well) of big-ET-1 without crossreactivity with ET-1. Immunoreactive big-ET-1 in plasma was determined after being extracted with Sep-pak C-18 cartridges. The average levels were 5.2 +/- 1.0 pg/ml for males and 5.7 +/- 1.6 pg/ml for females (the values were corrected for recovery efficiency). Big-ET-1 was found to be eliminated from the blood stream at a slower rate than ET-1. This may be an important reason for the higher plasma levels of immunoreactive big-ET-1 compared to the levels of immunoreactive ET-1.