INDIRECT COMPETITIVE ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE DETECTION OF NATIVE AND HEAT DENATURED BOVINE BETA-LACTOGLOBULIN IN EWES ANDGOATS MILK CHEESE

An indirect competitive enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of native and heat-denatured bovine b eta-lactoglobulin (hd-beta-lg) in goats' and ewes' milk cheese. Polycl onal antibodies raised in chicken against hd-beta-lg were purified by immunadsorption chromatography on native bovine, ovine and caprine bet a-lactoglobulins conjugated to CNBr-activated Sepharose. Although lact oglobulins from ewe, goat and cow have very similar amino acid sequenc es, crossreactivity was eliminated completely by this procedure. A com mercially available goat-anti-chicken alkaline phosphatase conjugate w as used as a secondary antibody to detect the anti-hd-beta-lg antibodi es bound to immobilized hd-beta-lg. The detection limit of the assay w as 100 ng/ml bovine native Ig or hd-beta-lg or 0.1-0.2% of cows' milk equivalent in cheese. Within a method validation study of the European Union, the ELISA test was successfully applied for the detection of n ative and heat-denatured bovine whey proteins in ewes' and goats' milk cheeses.