ABSTRACT
A specific and sensitive radioimmunoassay (RIA) for determination of endothelin-1 (ET-1) in human plasma has been developed. Antibodies were raised in rabbits using synthetic ET-1 conjugated to thyroglobulin as immunogen. The antibodies obtained were used at a final dilution of 1:300,000 yielding maximum binding of 61.7 +/- 3.0% (mean +/- 1 SD, n = 20) of 125I-ET-1. The ID50 (inhibitory dose 50%) was 4.5 +/- 0.6 fmol/100 microliters (mean +/- 1 SD, n = 20). The sensitivity of the RIA was 0.33 fmol/100 microliters standard solution. No cross reactivity was observed with endothelin-3, big-endothelin-1, atrial natriuretic factor, angiotensin I or angiotensin II. The cross-reactivity with endothelin-2 was 100%. Endothelin was extracted from acidified plasma with Sep-pak C18 cartridges and recovery of ET-1 added to normal plasma was 70.9 +/- 10.3% (mean +/- 1 SD, n = 12). The concentration of ET-1 in plasma from normal subjects was 1.5 +/- 0.4 pmol/l (mean +/- 1 SD, n = 11) ranging from 1.0 to 2.2 pmol/1. Extracts of normal human plasma subjected to high performance liquid chromatography on a reverse phase C18 column showed one peak of immunoreactivity co-eluting with the standard for ET-1. From these data it is concluded that the immunoreactive material measured in normal plasma with the present RIA is identical to ET-1.
