Antibodies against wheat xylanase inhibitors as tools for the selective identification of their homologues in other cereals

Polyclonal antibodies (PAbs) were raised against wheat (Triticum aestivum L.) TAXI- and XIP-type xylanase inhibitors by rabbit immunization. A small contaminant in both antigens, not detected by SDS-PAGE and later identified through Western blot as a recently discovered third type of xylanase inhibitor from wheat, i.e. thaumatin-like xylanase inhibitor (TL-XI), led to the coproduction of PAbs against this protein in the rabbits. To obtain inhibitor-specific PAbs, the PAbs against TAXI, XIP and TL-XI were separated by affinity chromatography using immobilised recombinant and native xylanase inhibitors. The purified PAbs allowed the immunoquantification of each type of wheat xylanase inhibitor using Western blot and densitometric analysis against purified inhibitor standards. The method allowed the detection of the purified inhibitors at the 20 ng level. As the PAbs against the wheat xylanase inhibitors cross-reacted with their homologous targets from other cereals, immunoprobing allowed identification of XIP homologues in oats (Avena sativa L.) and TL-XI homologues in durum wheat (Triticum durum Desf.) and rye (Secale cereale L.).