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Development of a Panel of Specific Monoclonal Antibodies to High Molecular Weight Glutenin Subunits and Their Application in Genetic Screening
Author(s) -
Giersch Thomas M.,
Hill Amanda S.,
Skerritt John H.
Publication year - 1999
Publication title -
cereal chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.558
H-Index - 100
eISSN - 1943-3638
pISSN - 0009-0352
DOI - 10.1094/cchem.1999.76.3.380
Subject(s) - glutenin , monoclonal antibody , microbiology and biotechnology , antibody , protein subunit , chemistry , biochemistry , biology , gene , genetics
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‐Ble allele (HMW‐GS 20) compared with the Glu‐Bli 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.