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Due to the lower prices of cow’s and buffalo’s milk, fraudulent mixing with higher priced milk of other species and sources is economically attractive but illegal and dangerous for allergic consumers. For the rapid detection of cow’s milk, several immunoassays are available but most of them lack the possibility to detect heat-treated cow’s milk due to denaturation of the target protein(s). In the present indirect enzyme-linked immunosorbent assay (ELISA), a mouse monoclonal antibody (Mab) raised against the bovine milk protein ĸ-casein is labelled with the enzyme horseradish peroxidase and the binding to the bovine ĸ-casein-coated wells of a 96-wells microtiter plate is inhibited by free bovine ĸ-casein in the sample or standard. The Mab recognises a 5 amino acids-containing epitope on the glycomacropeptide (GMP) part of bovine ĸ-casein which is absent in ĸ-casein of goat, sheep, horse, donkey, camel, etc, but is present in ĸ-casein of buffalo. Due to the indirect assay format, the cow’s and buffalo’s milk ELISA also recognises heat-treated milk containing denatured ĸ-casein. The measurement range of the bovine ĸ-casein calibration standards is from 0.1 to 2.5 μg/ml, the limit of detection is 0.05 μg/ml and the only milk sample preparation needed is dilution (100 or 1000 times). This new fast and easy-to-apply ELISA is well suited for the detection of the lower priced raw and heated milk of cows and buffalos in the higher priced milk of other species and sources within the range of 0.25 till 50%.