Enzyme linked immunosorbent assay (ELISA) and milk-clotting test (MCT) for determination of the affinity between chymosin and artificial casein micelles

During cheese ripening, the flavour develops partly due to retained milk-clotting enzymes in the curd. The mechanism of incorporation of milk-clotting enzymes into the curd is, however, not known. Two methods, Enzyme Linked Immunosorbent Assay (ELISA) and Milk-Clotting Test (MCT), for determination of the affinity between chymosin and artificial casein micelles were compared. After incubation of chymosin with artificial casein micelles of four different compositions and at four different pH-values, the amount of chymosin in the 'whey' was measured using the two methods. The results showed that there were only small differences between measurements of immunological response (ELISA) and catalytic activity (MCT) of the chymosin not adsorbed onto the artificial casein micelles. This seems to conflict with earlier results, suggesting that rennet enzyme molecules, associated with casein breakdown products, can affect rennet activity determinations. Essentially, the ELISA and MCT methods complement each other and should be used together to determine the affinity between chymosin and artificial casein micelles.