The allergic potential of alpha-caseins from bovine, ovine, and goat's milk sharing more than 85% identical amino acids was compared. Caseins were purified by anion-exchange chromatography and used for a specific IgE and IgG ELISA with diluted human sera. Sera were from 17 children with immediate-type allergy to cow's milk, from 59 children with atopy but without food allergy, and from 27 healthy children without atopy disease. The sera of cow's milk-allergic children showed a significantly higher IgE and IgG binding to alpha-caseins from all three species than the sera of the other groups. All groups showed an increased antibody binding to bovine alpha-casein compared to the sheep and goat proteins, but the differences were significant only in the groups of atopic children and of healthy controls. Furthermore, inhibition of the IgE binding to bovine alpha-casein with alpha-casein from cow, goat, and sheep revealed that the alpha-casein from these species are highly cross-reactive; on the basis of the small differences in their primary structure. In conclusion, the milk of goat and sheep harbor an allergic potential and is not suitable for the nutrition of milk-allergic patients.

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Background: β–Lactoglobulin (BLG) represents one of the major allergens causing cow’s milk allergy (CMA) – a disease with a wide spectrum of clinical symptoms. The aim of this study was to evaluate sequential B cell epitopes of BLG by the Pin–ELISA method. Furthermore, we wanted to investigate a possible association of the IgE recognition patterns in sera of patients with BLG sensitization and the type of clinical reactions following contact with cow’s milk. Methods: Overlapping sequential decapeptides corresponding to the amino acid sequence of BLG were used in Pin–ELISAs specific for human IgE. Tested sera were from 14 individuals with CMA, 8 of them with a history of immediate systemic reactions and 6 with delayed skin reactions following contact with cow’s milk. All of them showed specific IgE antibodies to BLG in the CAP–RAST. Control sera were from 5 healthy nonallergic individuals. Results: All sera from BLG–sensitized individuals showed IgE binding with one region of BLG corresponding to amino acids 95–113. Furthermore, individual sera showed reactions with two further regions, 12–27 and 124–135. Inhibition of IgE binding to BLG with one soluble synthetic peptide confirmed the major epitope. No differences were found in the B cell epitope recognition pattern to BLG in the two groups of patients with CMA, characterized by acute systemic or delayed skin reactions. Conclusions: Using IgE Pin–ELISAs we were able to confirm previously described sequential B cell epitopes of BLG. However, the recognition pattern of one of the major cow’s milk allergens is not predictive of the clinical type of reaction.

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.

Sequence/structure relationships have been explored by site-directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermodynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X-ray structure analysis. Heat-denaturation experiments yielded melting temperatures Tm and melting enthalpy changes delta Hm. The heat capacity change delta Cp of the wild-type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and delta Hm. Using the wild-type delta Cp value, Gibbs free energy changes delta G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced-fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X-ray analysis at 223-pm resolution which showed the structural changes. The enzyme was stabilized by adding a -Pro-His-His tail to the C-terminal alpha-helix for nickel-chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.