An ELISA to detect proteolysis of UHT milk upon storage

International audienc

ELISA to detect proteolysis of ultrahight-temperature milk upon storage

International audienceCasein proteolysis can occur in milk during storage leading to its gelation. The two main proteolytic systems suspected to be involved are the plasmin and the proteases produced by psychrotrophic bacteria. The latter have been shown to cleave κ-casein at the Phe105−Met106 bond. Although several techniques allow the determination of plasmin in milk, few rapid and easy-to-perform analytical techniques are available to check for bacterial proteolytic activity. This study presents the development of an inhibition ELISA allowing for the quantification of the κ-casein intact at the Phe105−Met106 bond. It uses a monoclonal antibody specifically directed against this peptide bond that binds to the protein as long as the molecule's cleavage site is intact but not when it is cleaved. This simple technique allows for the rapid analysis of more than 20 samples within 3 h. Applied to commercial milks, this assay allowed for the detection of unstable milk

An ELISA to detect proteolysis of UHT milk upon storage

International audienc

Epitope characterization of a supramolecular protein assembly with a collection of monoclonal antibodies : the case of casein micelle

In milk, κ-, β-, αs1- and αs2-casein (CN) are associated into a supramolecular assembly, the micelle. In this work, CN micelles contained in fresh skim milk were used to produce over 100 monoclonal antibodies. The specificity of these probes was determined using libraries of synthetic peptides and peptides fractionated from tryptic hydrolysis of purified CNs. Although κ-CN and αs2-CN are minor proteins in the micelle (ratio 1:1:4:4 for κ, αs2, αs1, β) a proportionally high number of clones were produced towards these two proteins (32 for each), compared to 9 and 29 for αs1-CN and β-CN, respectively. Most of the β-CN and κ-CN epitopes were identified, while about 50% of αs1-CN and αs2-CN antibodies were suspected to react to conformational linear or discontinuous epitopes, since no peptide binding could be identified. Antibody binding to the phosphoserine rich regions of the three calcium sensitive CNs was weak or non-existing, suggesting them to be hidden in the micelle structure together with αs1-CN. The C-terminal glycomacropeptide of κ-CN and the C-terminal moiety of β-CN were well exposed generating the majority of the antibodies specific for these two proteins. The two major antigenic sites of αs2 were αs2-CN (f96–114) and (f16–35). Cross-reaction between αs2-CN specific antibodies with αs1-CN illustrated the tangled structure between the two proteins. Immuno-dominant epitopes identified in the present study totally differ from those known for the purified caseins suggesting they were specific for the micelle supramolecular structure