Direct time-resolved fluorescence immunoassay for serum oestradiol based on the idiotypic anti-idiotypic approach

Abstract

We report a novel competitive type immunoassay for oestradiol based on the idiotypic anti-idiotypic approach. This has been achieved by the production of an anti-idiotypic antibody (anti-Id) which is directed against the oestradiol binding site of the primary idiotypic antibody (Ab 1). In this format the primary Ab 1 was captured onto the surface of microtitre wells and oestradiol standards or serum samples were then allowed to compete with europium labelled anti-Id for the binding sites of Ab 1. Fluorescence was proportional to the concentration of oestradiol over the range 0–8 ng/ml. The sensitivity of the assay was 80 ± 20 pg/ml, whilst the intra-assay variation ranged from 3 to 10%, and the inter-assay variation from 7.3 to 15%. The results obtained by the fluorescence immunoassay correlated well with those obtained by an extraction radioimmunoassay using tritiated antigen and dextran-coated charcoal for separation of bound and free ligand ( n = 60, r = 0.98). The idiotypic anti-idiotypic approach in hapten immunoassays enables antibodies to be labelled instead of haptens, and thus permits the development of robust and sensitive immunoassays.