Allergenic epitopes of bovine α_s1‐casein recognized by human IgE and IiG

Abstract

B-cell epitopes of bovine alpha S1-casein, one of the major allergens of cow's milk, were identified by a screening method based on synthetic peptides. According to the known amino acid sequence of alpha S1-casein, a set of 188 overlapping sequential decapeptides shifted by one amino acid was manually synthesized on polyethylene pins by the 9-fluorenyl-methoxycarbonyl (Fmoc) method. Peptides were screened by an enzyme-linked immunosorbent assay (ELISA) specific for human IgE and IgG. Bound antibodies were detected by successive incubation with up to three polyclonal antibodies, the last one conjugated to horseradish peroxidase. Tested sera were from 15 patients with acute clinical reactions to cow's milk and IgE-specific reactions to bovine alpha-casein in the ELISA and immunoblot. Sera from 10 healthy subjects without remarkable reactions to cow's milk proteins were used as controls. All sera from allergic subjects showed reactions with three regions of alpha S1-casein, corresponding to amino acids 19-30, 93-98, and 141-150. Furthermore, individual sera showed reactions with other parts of the protein. No essential differences in the epitope specificity of IgE and IgG were found. Inhibition of IgE binding to alpha S1-casein with soluble synthetic peptides confirmed the results and revealed peptide CN-2 as the most inhibiting one.

The Recognition Pattern of Sequential B Cell Epitopes of Beta–Lactoglobulin Does Not Vary with the Clinical Manifestations of Cow’s Milk Allergy

“…The previously described Pin–ELISA method had been improved for these studies by introducing a monoclonal antibody in order to reduce the background [26]. Using this Pin–ELISA method, we were able to pick up the protein region amino acids 95–113 which was recognized by all BLG sensitized individuals tested.…”

Specificity of the Human IgE Response to the Different Purified Caseins in Allergy to Cow’s Milk Proteins

“…The study of the specific antibody response, and particularly the IgE response after oral sensitization, first to whole casein fraction, and then to each of its isolated purified constituents, may provide information valuable in elucidating the structure-allergenicity relationships of milk proteins. Whereas the characterization of certain epitopes has been reported for some caseins [20, 21, 22], little is known about their respective allergenicity. Some studies involving skin tests [13] or the determination of the specific IgE response to different caseins have been reported.…”

Analysis of casein alpha S1 & S2 proteins from different mammalian species

“…Cow's milk contains more than 25 different proteins [19] but the casein fraction composed of alpha S1­, alpha S2­, beta­, and k­casein particularly alpha S1­casein seems to be a major allergen [20–23]. The chemical property and biological function of a protein is a direct consequence of its primary structure[27].…”

“…Cow's milk contains more than 25 different proteins, but only the whey proteins alpha­lactalbumin, beta­lactoglobulin, BSA, and lactoferrin, as well as the four caseins, have been identified as allergens [19]. The casein fraction is composed of alpha S1­, alpha S2­ , beta­, and k­casein, of which alpha S1­casein seems to be a major allergen according to IgE and T cell recognition data [20–23]. In developed countries there is increasing interest in goat milk and its derivates, the quality of which is considered of special importance in the light of current tendencies favouring healthy eating.…”