Immunoassay for wheat processing quality: Utilization of a sandwich assay incorporating an immobilized single-chain fragment

A single-chain fragment (scFv) was engineered from a monoclonal antibody to high molecular weight glutenin subunits (HMW-GS), wheat flour polypeptides that play a major role in determining the mixing- and extension strength-r elated properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows d isulfide bond formation in the cytoplasm and incorporated into a diagnostic test for wheat quality. Although the scFv lacks the more highly conserved antibody constant regions usually involved with immobilization, it was able to be directly immobilized to a polystyrene microwell solid phase without chemical or covalent modification of the protein or solid phase and utilize d as a capture antibody in a double-antibody (two-site) immunoassay. In the sandwich assay, increasing HMW-GS concentrations produced increasing assay color, and highly significant correlations were obtained between optical d ensities obtained in the ELISA using the scFv and the content-of large glut enin polymers in flours as well as measures of dough strength as measured b y resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract-dilutions th an that required for a similar assay utilizing a monoclonal capture antibod y. This research shows that engineered antibody fragments can be utilized t o provide superior assay performance in two-site ELISAs over monoclonal ant ibodies and is the first application of an engineered antibody to the analy sis of food processing quality.