Detection of mutations in the 7A allele of wheat ( Triticum aestivum) granule-bound starch synthase (Wx-7A) with a monoclonal antibody produced by targeted peptide immunisation

Abstract

A peptide sequence unique for the granule-bound starch synthase (GBSS1) of hexaploid wheat ( Triticum aestivum) was identified by sequence alignment of the three isoforms. Multiple antigenic peptides (MAP) of different length (16 and 20 AA) were synthesised against the selected sequence. The assessment of the immune response in mice against differently presented forms (resin-bound, free) and two types of adjuvants indicated that the free MAP with the longer peptide is more immunogenic. A 20AA MAP that elicited a strong immune response was used to produce a monoclonal antibody for the 7A isoform of GBSS 1. Epitope mapping of the selected Mab (F5-1F2) revealed that a 12mer partial sequence of the immunising peptide was specifically detected. Based on this Mab, a simple high throughput ELISA was developed that allows the quick identification of wheat lines carrying the 7A allele of GBSS 1 with minute amounts of sample.