Towards a new gliadin reference material–isolation and characterisation R. van Eckert a,b,c , E. Berghofer d , P.J. Ciclitira e , F. Chirdo f , S. Denery-Papini g , H.J. Ellis e , P. Ferranti h , P. Goodwin i,2 , U. Immer j , G. Mamone h , E. Me ´ndez k , T. Mothes l , S. Novalin d , A. Osman l,3 , M. Rumbo f , M. Stern m , L. Thorell n , A. Whim i , H. Wieser o, * a School of Biological Sciences, Victoria University of Wellington, Kelburn Parade, New Kirk Building, Wellington, New Zealand b Department of Biological Sciences, School of Science, University of Waikato, private Bag 3105, Hamilton, New Zealand c Department of Food Chemistry and Technology, University of Technology of Graz, Petersgasse 12/II, 8010 Graz, Austria d Universita ¨t fu ¨r Bodenkultur, Muthgasse 18, 1190 Wien, Austria e Gastroenterology Unit, The Rayne Institute (KCL), St Thomas’ Hospital, Lambeth Palace Road, London SE1 7EH, UK f Ca ´tedra de Inmunologia, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina g Laboratoire de Biochimie et Technologie des Prote ´ines, INRA, BP 71627, 44316 Nantes Cedex 03, France h Istituto di Scienze dell’ Alimentazione, CNR, Via Roma 52 A/C, 83100 Avellino, Italy i Tepnel BioSystems Limited, One Newtech Square, Deeside Industrial Park, Deeside, Flintshire CH5 2NT, UK j R-Biopharm AG, Landwehrstr. 54, 64293 Darmstadt, Germany k Centro Nacional de Biotecnologia, 28049 Cantoblanco Madrid, Spain l Institut fu ¨r Laboratoriumsmedizin, Klinische Chemie und Molekulare Diagnostik, Universita ¨tsklinikum Leipzig, Liebigstrasse 27, 04103 Leipzig, Germany m University Children’s Hospital, Hoppe-Seyler-Str. 1, 72076 Tu ¨bingen, Germany n Arla Foods, Torsgatan 14, 105 46 Stockholm, Sweden o Deutsche Forschungsanstalt fu ¨r Lebensmittelchemie, Lichtenbergstr. 4, 85748 Garching, Germany Received 30 June 2005; received in revised form 9 December 2005; accepted 14 December 2005 Abstract Twenty-eight wheat cultivars representative of the three main European wheat producing countries, France, UK and Germany, were selected as a source for the preparation of a reference gliadin. One kilogram of kernels from each cultivar were mixed and milled. The resulting white flour was defatted and vacuum dried. Albumins and globulins were eliminated by extraction using 0.4 M NaCl solution and gliadins were extracted with 60% ethanol. The gliadin extracts were concentrated, desalted by ultrafiltration, freeze-dried, and homogenised. After tests had shown good solubility and homogeneity, aliquots of the reference gliadin were sent to 16 different laboratories for further investigations: The material was analysed by various methods including RP-HPLC, SE-HPLC, RP-HPLC-ESI-MS, MALDI-TOF, capillary electrophoresis, acid-PAGE, 2D- PAGE, SDS-PAGE and immunoblotting and ELISA-tests with different monoclonal and polyclonal antibodies. The results showed that the gliadin composition of the source flour and the reference gliadin matched perfectly, demonstrating that no major gliadin components had been lost during the isolation procedure. The reference gliadin showed good immunochemical sensitivity with different gliadin antibodies in enzyme immunoassays. Because of its high protein and gliadin content, good solubility, homogeneity, stability and representative character, the product is regarded as a suitable universal reference material. q 2006 Elsevier Ltd. All rights reserved. Keywords: Gliadin; Reference material; Gluten determination; Gluten-free food; Coeliac disease Journal of Cereal Science 43 (2006) 331–341 www.elsevier.com/locate/jnlabr/yjcrs 0733-5210/$ - see front matter q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2005.12.009 Abbreviations: acid-PAGE, acidic polyacrylamide gel electrophoresis; CE, capillary electrophoresis; ELISA, enzyme linked immunosorbent assay; ESI-MS, electrospray mass spectrometry; IRMM, Institute for Reference Materials and Measurements; LC, liquid chromatography; MALDI-TOF, matrix-assisted laser desorption ionisation time-of-flight mass spectrometry; PWG, Working Group on Prolamin Analysis and Toxicity; RP-HPLC, reversed phase high-performance liquid chromatography; SE-HPLC, size exclusion chromatography; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; 2D-PAGE, two- dimensional polyacrylamide gel electrophoresis.. * Corresponding author. Tel.: C49 89 289 13260; fax: C49 89 289 14183. E-mail address: h.wieser@lrz.tum.de (H. Wieser). 2 Present address: Hallmark Analytical Ventures Limited, 98B High Street (Cwrt Erwain), Saltney, Chester, CH4 8SQ, UK. 3 Present address: Roboscreen GmgH, Delitzscher Strasse 135, 04129 Leipzig, Germany.