Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol
Innovative approach to low-level gluten determination in foods using a novel sandwich enzyme-linked immunosorbent assay protocol
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DOI：
10.1097/01.meg.0000059119.41030.df
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发表时间：
2003-05-01
影响因子：
2.1
通讯作者：
Méndez, E
中科院分区：
文献类型：
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作者：
Valdés, I;García, E;Méndez, E
目前有很多人呼吁建立一种可靠的酶联免疫吸附试验（ELISA）方案来测定食品中的麸质，以作为食品法典委员会进一步法规的基础。鉴于其识别潜在的乳糜泻毒性表位QQPFP的能力，这种表位在α -， γ -和ω -麦胶蛋白，蜂群蛋白和西卡林蛋白中反复出现，针对西卡林提取物提出的单克隆抗体R5可能被证明是谷蛋白分析的重要工具。本研究旨在开发一种高灵敏度和特异性的夹心ELISA法，用于量化乳糜泻患者食物中低水平的小麦、大麦和黑麦蛋白。方法采用单克隆抗体（R5）作为包被和检测的夹心ELISA法。对热加工食品的定量鸡尾酒谷蛋白提取程序也进行了测试。结果R5-ELISA法检测出麦胶蛋白、果苷蛋白和花青素，检测灵敏度分别为0.78、0.39和0.39 ng/ml。该方法的检出限为1.5 ng麦胶蛋白/ml （1.56 ppm麦胶蛋白，3.2 ppm谷蛋白）。该系统对非乳糜中毒谷物玉米、水稻和燕麦不敏感，并且不依赖于品种。它还能够检测未加工和热处理的小麦和大麦产品中的麦胶蛋白和蜂胶蛋白，并估计水解食品中的麸质含量。结论建立了新一代的夹心r5酶联免疫吸附测定试剂盒，具有良好的重现性（8.7%）和重复性（7.7%）。其麸质检测限为3.2 ppm，低于现有的20-200 ppm的阈值。ELISA对大麦、小麦和黑麦蛋白同样敏感，适用于热加工食品的定量鸡尾酒提取程序。除鸡尾酒程序外，Prolamin分析和毒性工作组目前正在评估食品法典委员会提议的R5-ELISA系统。[j] .中华胃肠病杂志，2003,15 (3):465-474 (C)。
Objectives There is currently much call for a reliable enzyme-linked immunosorbent assay (ELISA) protocol for determining gluten in foods to serve as a basis for further Codex Alimentarius regulations. Given its ability to recognize the potential coeliac-toxic epitope QQPFP, which occurs repeatedly in alpha-, gamma- and omega-gliadins, hordeins and secalins, the monoclonal antibody R5 raised against a secalin extract may prove to be an essential tool for gluten analysis. This study was designed to develop a highly sensitive and specific sandwich ELISA to quantify low levels of wheat, barley and rye prolamins in foods for coeliacs.Methods Simple sandwich ELISA based on the use of a single monoclonal antibody (R5) as both the coating and detection was developed. A quantitative cocktail gluten-extraction procedure for heat-processed foods was also tested.Results R5-ELISA was able to identify gliadins, hordeins and secalins with assay sensitivities of 0.78, 0.39 and 0.39 ng/ml, respectively. The assay's detection limit was 1.5 ng gliadins/ml (1.56 ppm gliadins, 3.2 ppm gluten). The system proved insensitive to the non-coeliac-toxic cereals maize, rice and oats, and was non-cultivar-dependent. It was also able to detect gliadins and hordeins in unprocessed and heat-processed wheat- and barley-based products, and to estimate the gluten content of hydrolysed foods.Conclusion We present a new generation of a robust sandwich R5-ELISA with good reproducibility (8.7%) and repeatability (7.7%). Its gluten-detection limit of 3.2 ppm is lower than the existing threshold of 20-200 ppm. The ELISA, which is equally sensitive to barley, wheat and rye prolamins, is compatible with the quantitative cocktail extraction procedure for heat-processed foods. Along with the cocktail procedure, the Working Group on Prolamin Analysis and Toxicity is currently evaluating an R5-ELISA system as proposed by the Codex Alimentarius Commission. EurJ Gastroenterol Hepatol 15:465-474 (C) 2003 Lippincott Williams Wilkins.