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.