DETERMINATION OF THE MAJOR FOOD ALLERGEN, OVOMUCOID, WITH AN OLIGOCLONAL ENZYME-LINKED IMMUNOSORBENT ASSAY Oligoclonal sandwich ELISA for ovomucoid

Ovomuciod (OM), a glycoprotein of a molecular weight of 28 kDa, is thought to be the major allergen in egg white. We have established three monoclonal antibodies (mAbs) against OM. MAb 5C is specific to native OM, mAb 6H is specific to heated OM, and mAb 7D is specific to carbohydrate moiety of OM, which recognizes native and heated OM equally. Then, an enzyme-linked immunosorbent assay (ELISA) capable to determine the total OM content, irrespective of the degree of heat denaturation of OM, was constructed by using mAb 7D. Furthermore, two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAbs 7D, 5C, and 6H as a second antibody. Second, it was shown that usage of denaturing reagents such as SDS and β-mercaptoethanol for extraction of allergens from foods was acceptable for ELISA within a range of stability of a first antibody on a solid phase. Properties of the oligoclonal sandwich ELISA system thus constructed will provide important information for establishing a common international method for allergen determination.