Annual Meeting, American Association of Cereal Chemists (AACC) location:San Francisco, USA date:17 - 20 September 2006
Proteinaceous xylanase inhibitors in cereals are of high technological relevance, as they inhibit the majority of microbial xylanases often used in cereal based biotechnological processes. They are equally believed to be involved in the defense mechanism of wheat. An important challenge is the separate and accurate quantification of the three known types of cereal xylanase inhibitors. Here we report on the development of an indirect sandwich enzyme-linked immunosorbent assay (ELISA) for TAXI (Triticum aestivum xylanase inhibitor)- and XIP (xylanase inhibiting protein)-type xylanase inhibitor quantification. Polyclonal antibodies were raised against wheat TAXI and XIP xylanase inhibitors by rabbit immunization, and subsequently purified by affinity chromatography. The purified antibodies were then used for indirect ELISA. The sandwich format of the essay consisted of a layer of Bacillus subtilis and Aspergillus oryzae xylanases coated into the wells of a multiwell plate and able to catch the TAXI and XIP proteins, respectively, from sample extracts. The purified antibodies were then allowed to bind to captured antigen and served as a tag recognised by anti rabbit IgG labeled with horseradish peroxidase. The activity of the latter enzyme was quantified. The procedure was optimised for reproducibility and accuracy. It allows high throughput analysis and permits the routine detection and quantification of TAXI and XIP levels as low as 40 and 3 ng by ml, respectively. As the antibodies against the wheat xylanase inhibitors cross-reacted with their homologous targets from other cereals, the technique could also be used for quantification of these proteins.