Two variants of immunoassay for the determination of biochanin A (5,7-dihydroxy 4'-methoxy isoflavone), i.e., a radioimmunoassay (RIA) and an indirect ELISA, have been developed and evaluated. Both methods employ the same rabbit antiserum to a 7-O-carboxymethyl-5-hydroxy-4'-methoxyisoflavone-bovine serum albumin (BSA) conjugate. A 125I-labeled hapten-tyrosine methyl ester (TME) conjugate was used as a radioligand for the RIA. The indirect ELISA uses immunogen-coated microtitration plates and a peroxidase-labeled antirabbit Ig antibody. Both methods are specific for biochanin A with a comparable sensitivity (3.1 pg/tube for RIA; 5.3 pg/well for ELISA); however, their sensitivity to individual cross-reactants differs. The main cross-reactants are sissotrin (the cross-reactivity 15.7% for RIA; 120% for ELISA), 5-hydroxy, 4',7-dimethoxy isoflavone (51.5% for RIA; 46.5% for ELISA), prunetin (4.5% for RIA; 5.0% for ELISA), genistein (0.8% for RIA; 2.8% for ELISA) and formononetin (0.4% for RIA; 0.3% for ELISA). These methods were used for the analysis of biochanin A in alfalfa and in several nonleguminous plants.

Department of Chemistry of Natural Compounds Institute of Chemical Technology Technická 5 166 28 Praha 6 Czech Republic

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