Publication no. C-1999-0416-03R |  VIEW ARTICLE

Development of a Panel of Specific Monoclonal Antibodies to High Molecular Weight Glutenin Subunits and Their Application in Genetic Screening.

Thomas M. Giersch (1), Amanda S. Hill (1), and John H. Skerritt (1,2). (1) CSIRO Plant Industry and Quality Wheat Cooperative Research Centre Ltd., GPO Box 1600, Canberra, ACT, Australia (2) Corresponding author. E-mail: <J.Skerritt@pi.csiro.au> Phone: +61 2 6246 5350. Fax: + 61 2 6246 5351. Cereal Chem. 76(3):380-388. Accepted January 28, 1999. Copyright 1999 American Association of Cereal Chemists, Inc.

High molecular weight glutenin subunits (HMW-GS) encoded by different chromosomal loci and alleles (1, 2, 5, 7, 10, and 12) were purified using reversed-phase HPLC from reduced, aqueous propanol extracts of flour from aneuploid or null wheat lines. Unlike previous libraries of monoclonal antibodies developed in our laboratory to SDS-extracted or alkylated HMW-GS, several of the monoclonal antibodies (mAb) developed in this study had a range of specificity patterns for HMW-GS in enzyme-linked immunosorbent assay (ELISA) and on immunoblots. A subset of the mAb bound either x- or y-type HMW-GS but not other gluten proteins, while a few antibodies bound one (mAb 110622, 110421, 140820), or two (mAb 101319, 110804, 140705, 1410460) HMW-GS expressed in each cultivar tested. In most cases, antibodies bound equally to the subunits encoded by different HMW-GS alleles. The more specific antibodies should be useful in research on the quantitative variation of HMW-GS expression and in studies of the role of particular HMW-GS in dough structure. The mAb 101319, which was prepared to subunit 1, bound to HMW-GS 1Bx subunits in ELISA and on immunoblots. This antibody also provided a higher absorbance value in ELISA with extracts of wheat lines expressing the Glu-B1e allele (HMW-GS 20) compared with the Glu-B1i allele (HMW-GS 17+18). Another mAb (110622) detected subunit 2 more strongly than subunit 5 in ELISA and produced a higher signal in immunoblots with subunit 2 even though these subunits are >98.7% homologous in amino acid sequence. An ELISA assay using this antibody was optimized for discrimination of wheat lines with the allelic pairs of subunits 1Dx5-1Dy10 from those with 1Dx2-1Dy12, with the former lines providing stronger dough properties and superior breadmaking quality. The performance of this assay was unaffected by other variations at HMW-GS loci and was demonstrated in sets of biotypes, doubled haploid, and cross-bred breeder's lines.