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GrainGenes Reference Report: JFC-47-4484

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Reference	JFC-47-4484
Title	Immunoassay for wheat processing quality: utilization of a sandwich assay incorporating an immobilized single-chain fragment
Journal	Journal of Agricultural and Food Chemistry
Year	1999
Volume	47
Pages	4484-4490
Author	Hill AS Giersch TM Loh CS Skerritt JH
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 twosite ELISAs over monoclonal antibodies and is the first application of an engineered antibody to the analysis of food processing quality
Keyword	[ Hide all but 1 of 43 ] analogs and derivatives antibodies,monoclonal antibody assay baking baking performance color cytoplasm density disulfide bond dough elisa enzyme-linked immunosorbent assay escherichia coli flour food food processing quality gluten glutenin glutenin subunit hmw glutenin hmw-gs human immunoassay immunology its methods mixing molecular weight monoclonal antibodies mutant performance polymer polypeptide processing quality quality control recombinant proteins resistance thioredoxin weight wheat flour wheat quality

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