Development of a simple, antibody-based test for granule-bound starch synthase Wx-B1b (Null-4A) wheat varieties

Alignment of the mature protein sequences of the three isoforms of granule-bound starch synthase 1 (GBSS1) of hexaploid wheat Triticum aestivum was used to identify regions showing sequence divergence. Two synthetic peptides based on the Wx-B 1 sequence and differing from the corresponding Wx-A1 and Wx-D1 sequences by two or three amino acids were used to immunise rabbits. One peptide elicited an antibody response that was highly selective for the Wx-B1 isoform when used in an indirect enzyme-linked immunosorbent assay (ELISA) format to assay starch granule protein (SGP) preparations prepared from a series of GBSS1 double null lines. A monoclonal antibody specific for GBSS, but not for any one isoform of the enzyme, was subsequently used as the capture antibody in a sandwich ELISA format, with the Wx-B1-selective rabbit polyclonal serum as the detection antibody. The sandwich ELISA format enabled good resolution of Wx-B1b (protein absent) from Wx-B1a (protein present) varieties using direct extraction of antigen from wholemeal flour. A simple method for the solubilisation of SGP using 8 M urea at room temperature was developed. This assay represents a simple, high-throughput means for the identification of wheat lines carrying the Wx-B1b allele that is suitable for testing small samples of starch, flour or crushed grain with a turnaround time of 1 day. (C) 2004 Elsevier Ltd. All rights reserved.