Antipeptide antibodies recognizing plasmin sensitive sites in bovine beta-casein sequence

To investigate plasmin activity in cheese, we produced antibodies to bovine beta -casein with controlled specificity, suitable as markers of the integ rity of the major bonds involved in its initial breakdown. Sixteen rabbits were immunized with synthetic substitutes for six plasmin-sensitive peptide s. Antisera raised to the peptides (f20-39), (f40-56), (f94-113), (f184-202 ), and: (f193-209) recognized beta -casein in ACP-ELISA, Western-blott, and biosensor, assays. Casein in vitro hydrolysis by plasmin or chymosin reduc ed the detection of these determinants in ACP-ELISA, in agreement with the enzymatic sensitivity of bonds included within the binding sites, or in the ir neighborhood. Antiserum to (f20-39) in particular allowed the specific d etection of plasmin cleavage at the bond generating gamma1-CN. Antisera to C-terminus preferentially detected the cleavage by chymosin. Immunoassays u sing these antibodies would allow in situ monitoring of significant proteol ysis events-without bias originated in the secondary degradation Of the rel eased peptides.