Homogeneous immunoassay based on chemiluminescence energy transfer

Abstract

The chemiluminescent compound aminobutylethyl-isoluminol (ABEI) and its isothiocyanate derivative have been coupled to a range of haptens (progesterone, cyclic AMP, cyclic GMP) and protein antigens (IgG, C9). All the derivatives were chemiluminescent, immunologically active, and stable for more than six months. When the ABEI-labeled antigens bind to their respective fluorescein-labeled antibodies, there is a shift in the ratio of chemiluminescence at 460 nm (blue) to that at 525 nm (green). This nonradiative energy transfer was used to establish homogeneous immunoassays, which require no separation step. These assays were at least as sensitive as the conventional radioimmunoassays and could accurately measure substances in serum (i.e., for IgG, results correlated by r = 0.96 relative to those by 125I assay) and tissue extracts (cyclic AMP, r = 0.91 relative to 3H assay), and also were used to evaluate the kinetics of antibody-antigen binding. Chemiluminescence energy transfer provides a new method for quantifying ligand-ligand interactions in the 10-15 to 10-18 mol range without first separating bound and free ligand. This provides a unique opportunity to investigate chemical events in single cells and intact cells.