Identification of the linear immunodominant epitopes in the β subunit of β-conglycinin and preparation of epitope antibodies

Abstract

β-conglycinin is one of the major allergens in soybean protein. The purpose of this study was to predict and to identify the major linear epitopes of the β subunit of β-conglycinin. Potential linear epitopes were predicted and confirmed by three immunoinformatics tools combined with the Immune Epitope Database (IEDB). Ten potential epitope peptides were synthesized by Fmoc (9-fluorenylmethoxycarbonyl) solid phase peptide synthesis and were validated by the indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) using sera from soybean allergic patients. Polyclonal antibodies, which were prepared by immunizing rabbits with synthesized peptides, were used to confirm their binding ability with β-conglycinin through western blot and dot blot assays. The results showed that 10 peptides were screened as the main epitopes for the β subunit of β-conglycinin. All 10 peptides (P1-P10) presented IgG binding activity, and P2 and P6 were also validated as IgE binding peptides. Moreover, the results of dot blot showed that P5 and P8 might be located inside the protein molecule. Western blot indicated that most of polyclonal antibodies were bound effectively to the β subunit of β-conglycinin. In addition, few polyclonal antibodies exhibited an immune cross-reaction with the α and α' subunits.

Keywords: Epitope peptides; Immunoinformatics prediction; β subunit of β-conglycinin.