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

Phytoestrogenic isoflavones that are abundant in soybeans may be an importa nt 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 compe titive enzyme-linked immunoassay that utilizes a conjugate of horseradish p eroxidase (HRP) and genistein as tracer and a monoclonal antibody to genist ein (clone 10D8) generated through the 6-position of genistein. Genistein, in diluted hydrolyzed urine or plasma of subjects who ingested soy milk, co mpetes,vith HRP-genistein conjugate for the binding sites of antigenistein antibody on rabbit anti-mouse IgG-coated plates. After a one-hour incubatio n, the wall-bound genistein-HRP activity, after reaction with a chromogen, is measured colorimetrically at 450 nm and is inversely correlated with con centrations 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 mu l o f urine and plasma. Urine and plasma levels of genistein measured by this i mmunoassay correlated well (R-2 = 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.