Evaluation of cross-reactivity between casein components using
inhibition assay and in silico analysis
Background: 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. Methods: 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.
Results: The αs1-CN-specific IgE levels exhibited a strong positive
correlation with the β-CN-specific IgE (r = 0.945, P < 0.001).
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- (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).
Conclusion: 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