Evaluation of the Allergenicity and Antigenicity of Bovine‐Milk αs1‐Casein Using Extensively Purified Synthetic Peptides

Abstract

Alphas1-Casein (CAS1_BOVIN), the major allergen of cow's milk (CM), is widely used as hydrolysates in infant diet formulae and additive to other processed food items. To date, most of the reported B-cell epitope mapping were performed on polyethylene pins or cellulose-derivative membrane. We sought to locate the motifs critical for human-specific IgE and rabbit polyclonal IgG binding using extensively purified CAS1_BOVIN, synthetic peptides and derivatives. Thirteen overlapping peptides covering the whole CAS1_BOVIN encompassing 17 : 20 amino acid (AA) were synthesized by f-moc AA solid-phase polyamide peptide synthesis. In addition, six cyanogen bromide (CNBr) cleavage fragments were prepared. Limited hydrolysis, oxidized and reduced/alkylated derivatives were also produced. The preparations were purified by ion exchange, gel filtration chromatography, reversed phase and high-performance liquid chromatography. The homogeneity was visualized by sodium dodecyl sulfate (SDS) and poly acryl amide gel electrophoresis (PAGE) followed by IgE and IgG immunoblotting. IgE binding was measured by Biotin Streptavidin (Bio/strep) fluoro enzyme immuno assay (FEIA) or ELISA-inhibition. Eighteen CM allergy (CMA) sera from 45 clinically examined children (Melbourne) and five adults (Bergen) were selected. Individual sera and pools were used for mapping IgE-binding epitopes. Rabbit IgG sera and pools were used for locating the antigenic sites of the molecule. Results indicated that all the individual CMA sera and pools recognized the intact molecule and three of the CNBr fragments as major antibody-binding allergens. The N- and C-terminal peptides (CAS 16-35; CAS 136-155) showed high IgE-binding affinity. CAS 1-18 and CAS 181-199 showed high IgG bindings. Considering the diversity of the antibody specificities, a reasonable agreement between IgE and IgG epitopes were found at the N- and C-terminals of CAS1_BOVIN. Mapping IgE B-cell epitopes by direct Bio/strep FEIA allowed the development of a sensitive modified technique for detecting unlabelled, casein immune dominant peptides in food products.