Monitoring ovarian function by the simultaneous time-resolved fluorescence immunoassay of two urinary steroid metabolites

Abstract

We report the development of a novel time-resolved fluorescence immunoassay utilizing two different assay strategies for the simultaneous measurement of estrone-3-glucuronide (EG) and pregnanediol-3alpha-glucuronide (PG) in samples of early morning urine (EMU). The method for the measurement of EG involves the use of a labeled anti-idiotype as a surrogate antigen, whereas the other method (for the measurement of PG) is a regular competitive immunoassay using a labeled antigen. In addition, the procedure uses different lanthanide chelates as labels to monitor ovarian function in women. After washing the streptavidin-coated plate, we added 10 microL of undiluted urine or mixed standard to the coated wells, followed by the addition of 100 microL of assay buffer containing the labeled reactants (i.e., europium-labeled PG and samarium-labeled anti-idiotype recognizing the binding site of the antibody to EG). Subsequently, we added 100 microL of assay buffer containing the two biotinylated specific monoclonal anti-steroid glucuronide antibodies. After incubation for 1 h on a shaker at room temperature, we washed the plate and added 200 microL of enhancement solution to each well. We measured europium and samarium fluorescence, using a gated plate fluorometer with appropriate emission filters. The method demonstrates appropriate sensitivity and precision (all CVs, 5-8%) across the relevant working ranges for each analyte. The technique has been applied to serial EMUs collected from women with normal and stimulated menstrual cycles.