Detection of cows' milk in ewes' milk and cheese by a sandwich enzyme‐linked immunosorbent assay (ELISA)

A sandwich enzyme‐linked immunosorbent assay (ELISA) has been successfully developed for the detection of defined amounts of cows' milk in ewes' milk and cheese. Polyclonal antibodies were raised in goats against bovine caseins (BC). The resultant antibodies were recovered from the crude antiserum by ammonium sulphate precipitation and further purified by immunoatisorption of the cross‐reacting antibodies onto columns containing immobilised ovine, caprine and bovine caseins, followed by elution of the bovine caseins specific antibodies (anti‐BC) from the column containing the bovine caseins. The anti‐BC bound to the wells of a microtitre plate were used to capture the BC from milk and cheese mixtures. Further immunorecognition of the captured proteins was attained with the same specific antibodies conjugated to biotin. ExtrAvidin‐peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying mixtures of ewes' milk and cheese containing variable amounts of cows' milk.