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

Abstract

Milk is extremely complex both by its multiphasic physical nature and by the diversity of its
molecular constituents whether they are its protein particularly. Caseins (CN), represents of
the major protein fraction of this dairy undergoes preferential degradation from proteolysis by
plasmin activities in Beta-casein (β-CN) with entrainment of gamma casein (γ-CN) and
proteose-peptones. In this work aims to study the β-CN antigenicity and its degradation
products in order to develop a milk quality indicator based on the determination of the degree
of casein proteolysis. A approach consists in producing and characterizing monoclonal
antibodies 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 or
clones 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 can
be easily monitored by immunoassays such as ELISA. Most of the supernatants studied react
better with the degradation products than with the native protein, with each antigen and
selected these that recognize strongly the three antigens and those recognizing preferentially
either β-CN, either β-5P(f1-105/7), either β-CN(f106-209), (f108-209). Finally, it emerges from
these results that the SNC46 and 80 have the best discriminating character. They react
exclusively with the C-terminal products of β-CN in immunoblotting as well as by calculation
of affinity constants. For the C-terminal fragments against β-CN 1, the SNC25 and 35 also
exhibit pronounced reactivity, a weak reactivity observed against the N-terminal fragments of
β-CN, in ELISA and in Immunoblot. Monoclonal antibodies will make an important contribution
to the study of functional proteins.