Enzyme immunometric assay for endothelin using tandem monoclonal antibodies

Seven distinct mouse monoclonal antibodies (mAbs) directed against human endothelin-1 (ET-1) have been obtained. On the basis of specificity studies performed with competitive immunoassays and of complementary binding studies, these mAbs were classified in two groups. mAbs of group A (Endo-4, -5, -6 and -10) were shown to be directed against the N terminal loop while those of group B (Endo-2, -8 and -18) recognized the C terminal part of the peptide. A pair of monoclonal antibodies with optimal properties for a two-site immunometric assay were selected and the test was performed in 96-well microtiter plates coated with one mAb (Endo-18), while another mAb (Endo-4) covalently labeled with enzyme acetylcholinesterase was used as tracer. Under optimal conditions, the assay appeared to be very sensitive since concentrations as low as 1 pg/ml could be significantly detected. The precision was also very good with a coefficient of variation below 10% from 3 to 250 pg/ml. The assay was specific for mature endothelin presenting no cross-reactivity with the precursor Big ET-1. On the other hand, strong cross-reactivity was observed with other ET-1-related peptides, including ET-2, ET-3, VIC peptide and sarafotoxin 6-b. The assay permitted specific determination of ET-1 in supernatants of cultured endothelial cells and the validity of the test was demonstrated by HPLC fractionation experiments. In addition, the assay also appeared to be suitable for direct determination of ET-1 in plasma. Studies performed with plasma from healthy subjects revealed that circulating levels of ET-1 are below or close to the detection limit of the method (< 8 pg/ml).