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Two-Site Sandwich ELISA for Discriminating Different Gli-1 (gliadin)/Glu-3 (LMW-glutenin subunit) Alleles in Hexaploid Wheat

¹CSIRO Plant Industry and Quality Wheat Cooperative Research Centre Ltd. GPO Box 1600, Canberra, ACT, Australia. ²Department of Pathology, Columbia University, New York, NY 10032. ³Australia New Zealand Food Authority, PO Box 7186, Canberra MC 2610 ACT Australia. ⁴Australian Centre for International Agricultural Research, GPO Box 1571, Canberra, ACT 2601, Australia. ⁵Corresponding author. Phone: +61 2 6217 0559. E-mail: Skerritt@aciar.gov.au

A panel of monoclonal antibodies was assessed in a two-site sandwich ELISA format, using both reduced glutenin subunit and gliadin-rich antigen preparations, to develop assays that could potentially discriminate between Gli-1/Glu-3 allelic variants in hexaploid wheat. Each antibody was assessed as the immobilized and the enzyme-labeled antibody in the sandwich ELISA. A number of antibody combination were identified which could discriminate different Gli-1/Glu-3 allelic variants in a population of doubled haploid lines derived from a cross between parents that differed at each of these loci. Certain labeled antibodies consistently detected allelic variation at a particular locus when used in conjunction with any of several immobilized antibodies. However, the level of discrimination was largely dependent on the choice of immobilized antibody. Two antibody combinations were identified that provided twofold differences in ELISA absorbances in flour extracts from different allelic variants at the Gli-A1/Glu-A3 and Gli-B1/Glu-B3 loci. By analyzing the prolamin composition of the antigen preparations, and the performance of the assays with flour extracts from a set of Gli-1/Glu-3 biotypes and a range of diverse cultivars, the biochemical basis for the discrimination was determined. The assays may have potential for use in high-throughput screening in wheat breeding programs.