A highly sensitive microtitre plate enzyme immunoassay for oestradiol-17 beta

A specific and sensitive solid-phase microtitre plate enzyme-linked immunosorbent assay for oestradiol-17 beta (E2) is described. After coating with an IgG anti-E2 fraction, we used E2-6-carboxymethyl-oxime-beta-galactosidase in a competitive binding assay and revealed the bound activity with a fluorogenic substrate. Two methods for the competitive binding assay were tested: (1) a classical one (method A) defined as a 'two-step competition' because the E2 sample was first incubated alone, and then E2-beta-galactosidase conjugate was added; (2) and a new one (method B) also performed in two steps but in which the E2 sample was evaporated to dryness. The detection limit of method A was 100 pg/ml (9 pg/well). Method B was more sensitive since 1 pg/ml (0.3 pg/well) was statistically different from 0 pg/ml. Specificity was equivalent with both methods while precision was better in B. Thus, this new method may be able to measure very low levels of oestradiol-17 beta in, for example, the blood of domestic mammals.