The measurement of oestrone-3-glucuronide in urine by non-competitive idiometric assay

We report a novel non-competitive idiometric assay for the measurement of oestrone-3-glucuronide (EG) in diluted urine. The method is based on the use of two types of anti-idiotypic antibody, the beta-type and alpha-type, that recognize different epitopes within the hypervariable region of the primary anti-EG antibody (Ab1). The beta-type anti-idiotypic antibody is analyte sensitive and competes with the analyte for an epitope of the primary antibody at the binding site. On the other hand, the alpha-type is analyte insensitive, but does not bind the Ab1 in the presence of the beta-type due to epitope proximity. In the present format, reaction mixtures containing the europium labelled Ab1 are reacted sequentially with EG standards or diluted urine samples, with the beta-type anti-idiotypic antibody and biotinylated alpha-type anti-idiotypic antibody on immobilized streptavidin coated microtiter plates. After 1 h incubation, the fluorescence of europium is measured by a time-resolved fluorescence and is proportional to the concentration of EG over a range of 0-10 nmol/l. The method demonstrates good sensitivity, precision and comparability with an alternative competitive fluorescent immunoassay. The idiometric assay for EG may be applied for the monitoring of ovarian function in women and is suitable for dipstick technology.