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 integrity of the major bonds involved in its initial breakdown. Sixteen rabbits were immunized with synthetic substitutes for six plasmin-sensitive peptides. Antisera raised to the peptides (f20-39), (f40-56), (f94-113), (f184-202), and (f193-209) recognized beta-casein in ACP-ELISA, Western-blot, and biosensor assays. Casein in vitro hydrolysis by plasmin or chymosin reduced the detection of these determinants in ACP-ELISA, in agreement with the enzymatic sensitivity of bonds included within the binding sites, or in their neighborhood. Antiserum to (f20-39) in particular allowed the specific detection of plasmin cleavage at the bond generating gamma1-CN. Antisera to C-terminus preferentially detected the cleavage by chymosin. Immunoassays using these antibodies would allow in situ monitoring of significant proteolysis events without bias originated in the secondary degradation of the released peptides.
Senocq
D
D
INRA-Station de Recherches en Technologie et Analyses Laitières, B.P. 89, F-39801 Poligny, France.
Dupont
D
D
Rolet-Répécaud
O
O
Faurie
F
F
Levieux
D
D
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D015374:Biosensing Techniques; D015153:Blotting, Western; D002364:Caseins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D002611:Cheese; D012085:Chymosin / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005341:Fibrinolysin / Q000378:metabolism*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology; D011817:Rabbits; D013379:Substrate Specificity
D000906:Antibodies; D002364:Caseins; D010446:Peptide Fragments; D005341:Fibrinolysin; D012085:Chymosin