The alpha subunit of β-conglycinin is one of the main allergens found in soybeans. For the preparation of a specific monoclonal antibody (mAb) against the α subunit, potential epitopes were predicted using Protean and evaluated by Wu's Antigenic Index. The specific epitope 85EQDERQFPFPR95 was synthesized, and then conjugated to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) for use as an immunogen and the plate-coating antigen, respectively. The resulting mAb, termed mAb-60K, was characterized as belonging to the IgG1 isotype and containing the κ light chain; it exhibited high specificity for the α subunit and did not cross react with the α′ and β subunits of β-conglycinin or other proteins found within soybeans. A competitive enzyme-linked immunosorbent assay (cELISA) with high accuracy and reproducibility was developed based on mAb-60K and the plate-bound peptide. Co-incubation with the full-length α subunit showed a 50% mAb binding inhibition concentration (IC50) value of 4.42 ng/mL, with a linear inhibition curve observed α subunit concentrations between 0.65 and 29.84 ng/mL. The newly developed mAb-60K and the companion cELISA could provide a valuable tool for determining sensitivity towards the α subunit of soybean β-conglycinin and for future studies on food allergies resulting from this protein.
Highlights
A highly specific mAb-60K directed against the α subunit of β-conglycinin was prepared based on predicted B cell epitope detection.
The monoclonal mAb-60K could identify the β-conglycinin α subunit specifically but did not cross-react with either the α′ or β subunit or other soybean meal proteins despite of a high homology between the α, α′ and β subunits.
The resulting mAb-60K-based cELISA using BSA-12 as the coating antigen exhibited a lower for β-conglycinin α subunit detection limit of 0.65 ng/mL.
The present study broadens the current body of methods for detecting the α subunit of β-conglycinin in soybean and soybean meals. The cELISA based on mAb-60K is very useful for quantitative detection of the α subunit of β-conglycinin in soybeans and soybean meal.
