Towards a new gliadin reference material–isolation and characterisation

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.