title: Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment.
abstract: 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-related properties of dough and its subsequent baking performance. The scFv was expressed in a thioredoxin mutant Escherichia coli strain that allows disulfide 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 utilized 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 densities obtained in the ELISA using the scFv and the content of large glutenin polymers in flours as well as measures of dough strength as measured by resistance to dough extension in rheological testing. The assay using the scFv was able to be carried out at lower flour sample extract dilutions than that required for a similar assay utilizing a monoclonal capture antibody. This research shows that engineered antibody fragments can be utilized to provide superior assay performance in two-site ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality.
authors.affiliation: CSIRO Plant Industry and Quality Wheat Cooperative Research Centre Ltd., GPO Box 1600, Canberra, ACT, Australia. A.Hill@pi.csiro.au
authors.forename: A S
authors.initials: AS
authors.lastname: Hill
authors.forename: T M
authors.initials: TM
authors.lastname: Giersch
authors.forename: C S
authors.initials: CS
authors.lastname: Loh
authors.forename: J H
authors.initials: JH
authors.lastname: Skerritt
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005983:Glutens / Q000031:analogs & derivatives* / Q000276:immunology; D006801:Humans; D007118:Immunoassay; D011786:Quality Control; D014908:Triticum / Q000737:chemistry* / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D005983:Glutens; C037302:glutenin