Differential immunochemical characterization of monoclonal antibody directed against the betacasein and its proteolysis products by the plasmin

Milk is extremely complex both by its multiphasic physical nature and by the diversity of itsmolecular constituents whether they are its protein particularly. Caseins (CN), represents ofthe major protein fraction of this dairy undergoes preferential degradation from proteolysis byplasmin activities in Beta-casein (β-CN) with entrainment of gamma casein (γ-CN) andproteose-peptones. In this work aims to study the β-CN antigenicity and its degradationproducts in order to develop a milk quality indicator based on the determination of the degreeof casein proteolysis. A approach consists in producing and characterizing monoclonalantibodies making it possible to differentiate β-CN from its fragments degraded by plasmin.Immunization and fusion in mice with whole caseins followed by pre-selection of hybrids orclones by use of the indirect ELISA technique and finally determination of affinity constants(Ka) of monoclonal antibody. Interaction between the antibody and the target protein that canbe easily monitored by immunoassays such as ELISA. Most of the supernatants studied reactbetter with the degradation products than with the native protein, with each antigen andselected these that recognize strongly the three antigens and those recognizing preferentiallyeither β-CN, either β-5P(f1-105/7), either β-CN(f106-209), (f108-209). Finally, it emerges fromthese results that the SNC46 and 80 have the best discriminating character. They reactexclusively with the C-terminal products of β-CN in immunoblotting as well as by calculationof affinity constants. For the C-terminal fragments against β-CN 1, the SNC25 and 35 alsoexhibit pronounced reactivity, a weak reactivity observed against the N-terminal fragments ofβ-CN, in ELISA and in Immunoblot. Monoclonal antibodies will make an important contributionto the study of functional proteins.