Enzyme Immunoassay for the Determination of Isoflavones

Abstract

Competitive enzyme-linked immunosorbent assays (ELISA) for the determination of isoflavones daidzein, genistein and biochanin were developed. Isoflavone conjugates coupled at positions C7 and C4' via a carboxymethyl spacer to bovine serum albumin were used as immunogens and also as coating antigens. Assay conditions, including concentrations of antisera and coating antigens, were optimized and the effect of reaction time, various blocking agents and organic solvents was investigated. The developed ELISA assays are highly sensitive, the I₅₀ values of standard curves ranging between 15 60 pg/well, i.e. 0.3 1.2 ng.ml ¹. Cross reactivities to other isoflavones are in an acceptable range and interference of non-isoflavonoid molecules is negligible. The immunoassays have been used in phytochemical research for monitoring isoflavone levels in various plant families. We expect further use of the assays in analysis of food, body fluids, cell culture media and other biological materials.