Identification of the human β-casein C-terminal fragments that specifically bind to purified antibodies to bovine β-lactoglobulin

The presence of foreign proteins in human milk after the ingestion of bovine dairy products is thought to be one of the possible causes of allergic sensitization in exclusively breast-fed predisposed infants. The immunologic determination of bovine β-lactoglobulin (LG) concentration in human milk has been reported by several researchers, but the results are conflicting. Moreover, a strong cross-reactivity between antibodies to bovine β-LG and human milk proteins and peptides was reported, throwing doubt on the reliability of radioimmunoassay and enzyme-linked immunosorbent assay detection and quantification assays for bovine β-LG in human milk. Thus, the goal of this study was to isolate human milk peptides with a molecular mass ≥ 1,000 Da cross-reactive with antibodies to bovine β-LG in order to identify possible common epitopes between human and bovine milk proteins. The proteins were first isolated by affinity chromatography with purified polyclonal antibodies to bovine β-LG, followed by gel filtration fast phase liquid chromatography and reverse phase-high performance liquid chromatography purification of the components specifically bound in the affinity separation step. Affinity-bound peptides were identified by determining their amino acid sequence. All the sequenced peptides belonged to the C-terminal part of human β-casein, which confirms the cross-reactivity of human milk proteins and peptides with antibodies to bovine β-LG and allows the identification of possible common epitopes between the two proteins. No bovine β-LG peptides with a molecular mass ≥ 1,000 Da were found in our milk samples from healthy mothers on a diet rich in bovine milk and dairy products.