DETERMINATION OF ENDOTHELIN BY AN IMMOBILIZED RECEPTOR ASSAY UTILIZING A 96-WELL FORMAT

Bovine cerebellar membranes immobilized on 96-well microtiter plates provide receptors for I-125-labeled endothelin-1 as the basis for a competitive binding assay. Adsorption of the membranes to a surface does not significantly alter the ligand-receptor interaction and reduces non-specific binding to 3-7% of total binding compared to 10-20% for a filtration technique. Considerable savings in reagents are realized since assays can be performed in 100 mul volumes with only 10-20 mug of membrane protein. The 96-well format allows the rapid quantitation of large numbers of samples, and the assay is especially attractive in that it utilizes readily available reagents and equipment without the need for specific antibodies. The endothelin-receptor-based assay may be used to measure conversion of big endothelin-1 to endothelin-1 in aqueous assays. Since the presence of serum does not affect this method, tissue culture medium may be directly analyzed for endothelin production by cultured cells. All three isoforms of endothelin are detected, and the specificity of the receptor is retained since fragments and precursor forms of endothelin are not recognized. In cases where multiple endothelin isoforms may be present or where specificity of binding is in question, this assay may be used in conjunction with high pressure liquid chromatography to distinguish active peptides.