Antibodies against wheat xylanase inhibitors as tools for the selective identification of their homologues in other cereals |
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| Institution: | 1. Laboratory of Food Chemistry, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium;2. Laboratory of Livestock Physiology, Immunology and Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 30, 3001 Leuven, Belgium;1. Functional Biotechnology Unit, Advanced Biotechnology and Breeding Centre, Malaysian Palm Oil Board (MPOB), PO Box 10620, 50720 Kuala Lumpur, Malaysia;2. Institute of Biological Sciences, Faculty of Science, Universiti Malaya, 50603 Kuala Lumpur, Malaysia;1. Diyarbakir Plant Protection Research Station, Diyarbakir, Turkey;2. Department of Plant Protection, Faculty of Agriculture, Insect Molecular Biology Laboratory, Ankara University, 06110, Diskapi, Ankara, Turkey;3. Department of Plant Protection, Faculty of Agriculture, Mustafa Kemal University, 31030, Hatay, Turkey;1. Department of Microbiology, Science Laboratory Building, Faculty of Science, King Mongkut''s University of Technology, Thonburi, Thailand;2. Theoretical and Computational Physics Group, Department of Physics, KMUTT, Thailand, Faculty of Science, King Mongkut''s University of Technology, Thonburi, Thailand;3. Theoretical and Computational Science Center (TaCS), Science Laboratory Building, Faculty of Science, King Mongkut''s University of Technology Thonburi (KMUTT), 126 Pracha-Uthit Road, Bang Mod, Thrung Khru, Bangkok, 10140, Thailand;4. Department of Physics, Faculty of Science, Kano University of Science and Technology, Wudil, Kano, Nigeria;1. School of Forest Sciences, University of Eastern Finland, P.O. Box 111, FI80101 Joensuu, Finland;2. College of Forestry, Northwest A&F University, 3 Taicheng Road, Yangling 712100, China;3. Department of Biology, University of Eastern Finland, P.O. Box 111, FI80101 Joensuu, Finland;1. Laboratory of Neural and Neuroendocrine Regulations, Institute of Developmental Biology, Russian Academy of Sciences, 26 Vavilov St., Moscow 119334, Russia;2. Laboratoire de Pathophysiologie des maladies du système nervoux central, UMR5 INSERM 952, IFR 83, Université P. et M. Curie, 7 quai St. Bernard, 75252 Paris Cedex 05, France;3. IINS, UMR CNRS 5297, Université Bordeaux Segalen, 146 rue Léo Saignat, 33076 Bordeaux-Cedex, France |
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| Abstract: | 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.). |
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