SANDWICH-TYPE ENZYME-IMMUNOASSAY FOR BIG ENDOTHELIN-1 IN PLASMA - CONCENTRATIONS IN HEALTHY-HUMAN SUBJECTS UNAFFECTED BY SEX OR POSTURE

A sandwich-type enzyme immunoassay has been developed for measuring hu man big endothelin-l (big ET-1) in human plasma and supernatant fluids from human cell cultures. Big ET-1 is the precursor of endothelin 1 ( ET-1), the most potent vasoconstrictor known. A rabbit antibody raised against the big ET-1 COOH-terminus fragment was used as an immobilize d antibody (anti-P16). The Fab' fragment of a monoclonal antibody (1B3 ) raised against the ET-1 loop fragment was used as the enzyme-labeled antibody, after being coupled to acetylcholinesterase. The lowest det ectable value in the assay was 1.2 pg/mL (0.12 pg/well). The assay was highly specific for big ET-1, demonstrating no cross-reactivity with ET-1, <0.4% cross-reactivity with big endothelin-2 (big ET-2), and <0. 1% with big endothelin-3 (big ET-3). We used this assay to evaluate th e effect of two different postural positions (supine and standing) on plasma big ET-1 concentrations in 11 male and 11 female healthy subjec ts. Data analysis revealed that neither sex nor body position influenc ed plasma big ET-1 concentrations. This assay should thus permit the d etection of possible variations in plasma concentrations of big ET-1 i n certain pathologies and, in association with ET-1 assay, make possib le in vitro study of endothelin-converting enzyme activity in cell mod els. Such studies could clarify the physiological and clinical roles o f this family of peptides.