Sensitive Chemiluminescent Enzyme Immunoassay for Determination of Endothelin-1

抄録

A highly sensitive sandwich-type chemiluminescent enzyme immunoassay (CLEIA) for endothelin-1 (ET-1) in plasma which does not involve any extraction steps has been developed using two populations of polyclonal antibodies. One antibody is specific to the C-ter-minal of the endothelin family of peptides and is used as an immobilized antibody. The other antibody is a Fab fragment composed of rabbit antibodies against the N-terminal core region of ET-1 and is coupled with horseradish peroxidase (HRP). Labeled HRP activity was measured using the enhanced chemiluminescent reaction of Luminol/hydrogen peroxide devised by the authors. The assay was sensitive enough to detect 0.05 pg/well (20 amol/well) of ET-1 and had no significant cross-reactivities with other related peptides, including endothelin-3 and the endothelin precursor peptide, big ET-1. Intra-assay CVs were in the range of 8.4%-24.8% for relatively low concentrations of plasma ET-1 (2pg/mI) and 4.8% - 8.1% for higher concentrations of plasma ET-1 (10pg/mI). Inter-assay CVs were 22.6% and 11.1%, respectively. No significant interference caused by bilirubin, neutral fat, hemoglobin and selected anticoagulants was observed. Preliminary investigations indicated that the basal level of ET-1 in normal humanplasma (n=37) was 0.65pg/mI.