3.2 Cross-reactivity of αs1-casein with casein components
To confirm whether a cross-reactive epitope exists between the αs1- and β-CN components, we performed a competitive ELISA of β-CN against αs1-CN-specific IgE (Figure 2). Complete inhibition was observed at high concentrations of β-CN (10–100 µg/mL, Figure 2A). The IC50 of the β-CN (0.160 µg/mL) was about 10 times higher than that of αs1-CN itself (0.0139 µg/mL), suggesting differences in the number or the affinity of IgE epitopes. On the other hand, the IC50 of κ-CN (13.6 µg/mL) was about 1,000 times higher than that of αs1-CN, and the maximum inhibition rate remained 54.5% at the highest concentration (Figure 2B). These results suggested the possibility that a cross-reactive epitope exists between αs1- and β-CN.
To exclude the possibility that αs1-CN was contaminated with the purified β-CN, we performed SDS-PAGE and immunoblotting using α-casein-specific monoclonal antibody. Even in the ten times more protein supplementation of β-CN by SDS-PAGE, no bands detected by the αs1-CN-specific immunoblotting (Figure S2). Therefore, the possibility of protein contamination was unlikely.