Multiple immunoassay: the simultaneous measurement of two urinary steroid glucuronides as an index of ovarian function.

Ovarian function in women may be monitored effectively by a simple, solid-phase, multiple immunoassay for the simultaneous measurement of estrone-3-glucuronide and pregnanediol-3 alpha-glucuronide in diluted urine. IgG fractions of the respective antisera are passively absorbed to the walls of polypropylene tubes. The labelled antigens are estrone-3-glucuronyl-5-aminoethyl-ethyl-isoluminol and [6,9-3H]-pregnanediol-3 alpha-glucuronide. Daily samples of early morning urine are diluted in buffer (1:200; v/v), and 200 microliters removed, in duplicate, for assay. After the binding reaction (18 h at 4 degrees C), the solution is removed by aspiration. The antibody-bound fraction is washed twice with buffer (300 microliters) is added and the mixture incubated for 50 min at 22 degrees C. Luminescence is initiated by oxidation of the label with microperoxidase/hydrogen peroxide and the signal integrated for 10 s. Subsequently, liquid scintillation fluid (4 ml) is added to the tube and the radioactivity measured. The unknown values are determined from appropriate calibration curves. The combined method has similar sensitivity, accuracy, precision and clinical utility to the values obtained from the separate measurement of the two analytes.