Sandwich ELISA for detection of goats’ milk in ewes’ milk and cheese

Abstract

A sandwich ELISA has been developed for the detection of defined amounts of goats’ milk (1–100%) in ewes’ milk and cheese. Polyclonal antibodies were raised in rabbits against goat caseins (GC). The resultant antibodies were affinity purified by immunoadsorption of the crude antiserum on to columns containing immobilized cow, ewe and goat caseins, followed by elution of the goats’ milk‐specific antibodies (anti‐GC) from the column containing the goat caseins. The anti‐GC antibodies bound to the wells of a microtiter plate were used to capture the goats’ caseins from milk or cheese mixtures. Further immunorecognition of the captured proteins was achieved with the same antibodies conjugated to biotin. ExtrAvidin‐peroxidase was used to detect biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of the substrate gave clear absorbance differences when assaying mixtures of ewes’ milk and cheese containing variable amounts of goats’ milk.