Evaluation of cross-reactivity between casein components using inhibition assay and in silico analysis

We previously reported that the specific IgE levels to αs1-casein (CN) and β-CN in patients with cow's milk allergy decreased with similar dynamics during oral immunotherapy. Therefore, we hypothesized that αs1- and β-CN have strong cross-reactivity among CN components, despite the low similarity in the full-length amino acid sequences.

The αs1-, β-, and κ-CN were purified from commercial cow's milk. We recruited 39 patients with cow's milk allergy, and the serum IgE levels for each CN component were measured by enzyme-linked immunosorbent assay (ELISA). Cross-reactivity between CN components was investigated by competitive ELISA against αs1-CN. Sequence homology between CN components at the peptide level was calculated using in silico analysis and quantified by the property distance (PD) value.

The αs1-CN-specific IgE levels exhibited a strong positive correlation with the β-CN-specific IgE (r = 0.945, P < .001). Complete competition was observed by β-CN against αs1-CN, suggesting the presence of common epitopes between them. In silico analysis detected 24 peptide sets with PD values lower than 10 between αs1- and β-CN, and 14 sets between αs1- and κ-CN. The amino acid sequences of αs1-CN (E61-E70) and β-CN (I12-E21) that showed the lowest PD value (5.30) were present in the characteristic sequence known as casein phosphopeptide (CPP).

We detected strong cross-reactivity between CN components. Furthermore, we found highly homologous sequences in the CPP region, which contains a core sequence of "SSSEE" with phosphorylated serine residues.