Methodological evaluation of an enzyme-immunoassay for plasma Big-et-1 determination

BACKGROUND: The evaluation of big-endothelin (ET)-1 plasmatic concentrations may serve as another noninvasive marker in patients with cardiovascular diseases, based on the assumption that it may reflect endothelin overproduction more accurately than circulating ET-1. For this reason the analytical performance of an immunoenzimatic assay for plasma Big-ET-1, with or without a preliminary step of extraction, was evaluated. METHODS AND RESULTS: Sensitivity for direct assay was 0.15+/-0.010 fmol/ml (n=27), inter-assay variability, evaluated at 2 different concentrations, resulted 2.5+/-0.062, CV%=5.6 (n=5) and 1.47+/-0.09 fmol/ml, CV%=12.8 (n=4), respectively, while intra-assay variability was 0.89+/-0.022 fmol/ml, CV%=5.6. Sensitivity for the assay with extraction resulted 0.71+/-0.104 (n=6) fmol/ml and intra-assay variability was 3.6+/-0.13 fmol/ml, CV=7.4% (n=4). The comparison between the 2 procedures, performed on 107 plasma samples at different peptide concentrations, showed a close agreement between the results of the 2 procedures for Big-ET-1 values higher than 1 fmol/ml. CONCLUSION: The main limitation of the direct assay is due to possible interference effects while the correct evaluation of extraction yield of the individual samples is the main drawback of the procedure with extraction. These effects become important when assaying samples with low levels of analyte. The direct assay of plasma Big-ET-1, easier to perform, more rapid and less expensive, could be the choice method.