Abstract: The aim of this study was to explore a method for screening for proteases specifically hydrolyzing the epitopes of αs1-casein. First, the epitope aa 83–105 of αs1-casein was synthesized by solid-phase synthesis method. After purification and identification, the peptide was coupled to bovine serum albumin (BSA) to prepare a complete antigen, and then BALB/c mice were immunized to prepare monoclonal antibodies. In addition, an indirect competitive enzyme linked immunosorbent assay (ELISA) was established. The monoclonal antibody prepared with αs1-casein and the established method were regarded as controls. The results showed that the purity of the synthetic epitope was over 90%, and the coupling rate with BSA was 6.31. The monoclonal antibody belonged to IgG1, with a titer of 1:320 000, and it could react specifically with αs1-casein, but did not cross-react with soybean protein. The linear regression equation of the competitive inhibition curve was y = ? 9.22x + 100.78 (R2 = 0.989 1). The indirect competitive ELISA method showed good repeatability and accuracy, and its detection limit was lower than that of the monoclonal antibody method. The amounts of residual antigen in papain and alcalase hydrolysates were relatively small, which needs to be further verified by in vivo test. The successful preparation of G1 monoclonal antibody against αS1-casein epitope aa 83–105 provides a specialized tool for ELISA detection of antigen residues and for the development of hypoallergenic formula.

Key words: αs1-casein; epitope; monoclonal antibody; antigen residue; protease