Deutsche Forschungsanstalt für Lebensmittelchemie, Lise-Meitner-Strasse 34, 85354 Freising, Germany.
Anal Bioanal Chem. 2009 Nov;395(6):1721-8. doi: 10.1007/s00216-009-3080-6.
Celiac disease (CD) is a permanent gastrointestinal disorder characterized by the intolerance to a group of proteins called gluten present in wheat, rye, barley, and possibly oats. The only therapy is a strict lifelong gluten-free diet. The standard method for gluten determination in foods produced for CD patients is the R5-enzyme-linked immunosorbent assay (ELISA) as proposed by the recent Codex Alimentarius Draft Revised Standard. This test is based on the determination of prolamins, the alcohol-soluble proteins of gluten, and is available as a sandwich ELISA for intact proteins and as a competitive ELISA for gluten-derived peptides. While the suitability of the sandwich ELISA including a wheat prolamin (gliadin) reference for calibration has been shown by various studies and a ring test, the competitive ELISA still lacks a convenient reference for the quantitation of gluten peptides in fermented cereal foods (e.g., sourdough products, starch syrup, malt extracts, beer). Therefore, the aim of the present study was to prepare a suitable reference for the quantitation of partially hydrolyzed gluten in fermented wheat, rye, and barley products. The prolamin fractions from barley (hordein) and rye (secalin) were isolated from corresponding flours by means of a modified preparative Osborne fractionation. The prolamin fraction from wheat was obtained as reference gliadin from the Prolamin Working Group. The prolamin fractions were successively digested by pepsin and trypsin or pepsin and chymotrypsin procedures, which have been used for CD-specific toxicity tests on cereal storage proteins for many years. The protein/peptide content (N x 5.7) of the prolamin fractions and digests, which was the basis for the calculation of the gluten content by means of ELISA, varied between 67.1% and 96.0%. The prolamin fractions and enzymatic digests were then tested for their response in both sandwich and competitive assays. Intact prolamins responded similarly in both ELISA showing no important differences between the cereals. In the case of digested proteins, however, the sandwich ELISA was considerably less sensitive than the competitive ELISA. The former provided approximately 40% and the latter 70% of the signal intensity obtained with the intact prolamins. Thus, the combination of the competitive ELISA and the enzymatic digests of prolamin fractions as reference was considered to be an adequate system for the analysis of partially hydrolyzed gluten. The limit of detection using a peptic-tryptic hordein digest as reference was 2.3 microg prolamin equivalent per milliliter, and the limit of quantitation was 6.7 microg prolamin equivalent per milliliter. This system was applied for the determination of gluten equivalents in five commercial beverages based on fermented cereals.
乳糜泻（CD）是一种永久性的胃肠道疾病，其特征是对一组称为麸质的蛋白质不耐受，这些蛋白质存在于小麦、黑麦、大麦中，可能也存在于燕麦中。唯一的治疗方法是严格的终生无麸质饮食。为 CD 患者生产的食品中麸质测定的标准方法是最近的食品法典标准修订草案中提出的 R5-酶联免疫吸附测定（ELISA）。该测试基于醇溶蛋白的测定，即麸质的可溶蛋白，可作为完整蛋白的夹心 ELISA 和源自谷蛋白的肽的竞争 ELISA。虽然夹心 ELISA 包括用于校准的小麦醇溶蛋白（麦醇溶蛋白）参考物的适用性已被各种研究和环试验证明，但竞争 ELISA 仍然缺乏用于发酵谷物食品（例如，酸面团产品、淀粉糖浆、麦芽提取物、啤酒）中部分水解谷蛋白定量的便捷参考物。因此，本研究的目的是制备一种适用于定量发酵小麦、黑麦和大麦产品中部分水解谷蛋白的参考物。大麦（醇溶蛋白）和黑麦（麦醇蛋白）的醇溶蛋白部分通过改良的 Osborne 分级分离从相应的面粉中分离出来。小麦醇溶蛋白部分是从醇溶蛋白工作组获得的参考麦醇溶蛋白。醇溶蛋白部分依次用胃蛋白酶和胰蛋白酶或胃蛋白酶和糜蛋白酶进行消化，这些方法多年来一直用于谷物储存蛋白的 CD 特异性毒性测试。通过 ELISA 计算谷蛋白含量的基础是醇溶蛋白部分和消化物的蛋白质/肽含量（N x 5.7），其在 67.1%至 96.0%之间变化。然后，对醇溶蛋白部分和酶解物进行夹心和竞争测定的反应性测试。完整的醇溶蛋白在两种 ELISA 中反应相似，显示出谷物之间没有重要差异。然而，对于消化的蛋白质，夹心 ELISA 的灵敏度明显低于竞争 ELISA。前者提供了与完整醇溶蛋白获得的信号强度的大约 40%，而后者提供了 70%。因此，竞争 ELISA 与醇溶蛋白部分的酶解产物作为参考的组合被认为是分析部分水解谷蛋白的合适系统。使用肽酶消化醇溶蛋白作为参考时的检测限为 2.3μg 醇溶蛋白当量/毫升，定量限为 6.7μg 醇溶蛋白当量/毫升。该系统用于测定基于发酵谷物的五种商业饮料中的谷朊粉当量。
