Development of a competitive ELISA for the detection of soybean α subunit of β-conglycinin

The alpha subunit of beta-conglycinin is one of the main allergens found in soybeans. For the preparation of a specific monoclonal antibody (mAb) against the a subunit, potential epitopes were predicted using Protean and evaluated by Wu's Antigenic Index. The specific epitope (85)EQDERQFPFPR(95) 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 IgC1 isotype and containing the kappa light chain; it exhibited high specificity for the alpha subunit and did not cross react with the alpha' and beta subunits of beta-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 alpha subunit showed a 50% mAb binding inhibition concentration (IC50) value of 4.42 ng/mL, with a linear inhibition curve observed alpha 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 alpha subunit of soybean beta-conglycinin and for future studies on food allergies resulting from this protein. (C) 2011 Elsevier Ltd. All rights reserved.