A nonisotopic enzyme-based immunoassay for assessing human exposure to genistein

Phytoestrogenic isoflavones that are abundant in soybeans may be an important group of natural products that could play a critical role in preventing several chronic human diseases. To facilitate studying the relationship of soya exposure and chronic diseases, we report a simple method for measuring an isoflavone, genistein, in human urine and plasma. The method is a competitive enzyme-linked immunoassay that utilizes a conjugate of horseradish peroxidase (HRP) and genistein as tracer and a monoclonal antibody to genistein (clone 10D8) generated through the 6-position of genistein. Genistein, in diluted hydrolyzed urine or plasma of subjects who ingested soy milk, competes with HRP-genistein conjugate for the binding sites of anti-genistein antibody on rabbit anti-mouse IgG-coated plates. After a one-hour incubation, the wall-bound genistein-HRP activity, after reaction with a chromogen, is measured colorimetrically at 450 nm and is inversely correlated with concentrations of genistein over the range of 0.1-32 ng/well. The sensitivity limit of the method is 0.5 ng of genistein per well or 0.5 ng per 10 microliters of urine and plasma. Urine and plasma levels of genistein measured by this immunoassay correlated well (R2 = 0.92 for urine and 0.77 for plasma) with those determined by chromatographic techniques. This method can be used to assess soya exposure in humans and could facilitate epidemiological studies of the relationship of soya diets and chronic diseases, including cancer.