Rallabhandi Prasad, Sharma Girdhari M, Pereira Marion, Williams Kristina M
Immunobiology Branch, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration , 8301 Muirkirk Road, Laurel, Maryland 20708, United States.
J Agric Food Chem. 2015 Feb 18;63(6):1825-32. doi: 10.1021/jf505716p. Epub 2015 Feb 5.
Gluten proteins in wheat, rye and barley cause celiac disease, an autoimmune disorder of the small intestine, which affects approximately 1% of the world population. Gluten is comprised of prolamin and glutelin. Since avoidance of dietary gluten is the only option for celiac patients, a sensitive gluten detection and quantitation method is warranted. Most regulatory agencies have set a threshold of 20 ppm gluten in foods labeled gluten-free, based on the currently available ELISA methods. However, these methods may exhibit differences in gluten quantitation from different gluten-containing grains. In this study, prolamin and glutelin fractions were isolated from wheat, rye, barley, oats and corn. Intact and pepsin-trypsin (PT)-digested prolamin and glutelin fractions were used to assess their immunoreactivity and gluten recovery by three sandwich and two competitive ELISA kits. The Western blots revealed varied affinity of ELISA antibodies to gluten-containing grain proteins and no reactivity to oat and corn proteins. ELISA results showed considerable variation in gluten recoveries from both intact and PT-digested gluten fractions among different kits. Prolamin fractions showed higher gluten recovery compared to their respective glutelin fractions. Among prolamins, barley exhibited higher recovery compared to wheat and rye with most of the ELISA kits used. Hydrolysis resulted in reduced gluten recovery of most gluten fractions. These results suggest that the suitability of ELISA for accurate gluten quantitation is dependent upon various factors, such as grain source, antibody specificity, gluten proteins and the level of their hydrolysis in foods.
小麦、黑麦和大麦中的麸质蛋白会引发乳糜泻，这是一种小肠自身免疫性疾病，全球约1%的人口受其影响。麸质由醇溶蛋白和谷蛋白组成。由于避免食用含麸质食物是乳糜泻患者的唯一选择，因此需要一种灵敏的麸质检测和定量方法。基于目前可用的酶联免疫吸附测定（ELISA）方法，大多数监管机构设定了无麸质标签食品中麸质的阈值为20 ppm。然而，这些方法在对不同含麸质谷物的麸质定量上可能存在差异。在本研究中，从小麦、黑麦、大麦、燕麦和玉米中分离出醇溶蛋白和谷蛋白组分。完整的以及经胃蛋白酶-胰蛋白酶（PT）消化的醇溶蛋白和谷蛋白组分被用于通过三种夹心ELISA试剂盒和两种竞争ELISA试剂盒评估其免疫反应性和麸质回收率。蛋白质印迹法显示ELISA抗体对含麸质谷物蛋白的亲和力各不相同，且对燕麦和玉米蛋白无反应性。ELISA结果表明，不同试剂盒对完整和PT消化的麸质组分的麸质回收率存在显著差异。醇溶蛋白组分的麸质回收率高于各自对应的谷蛋白组分。在醇溶蛋白中，使用的大多数ELISA试剂盒显示，大麦的回收率高于小麦和黑麦。水解导致大多数麸质组分的麸质回收率降低。这些结果表明，ELISA用于准确麸质定量的适用性取决于多种因素，如谷物来源、抗体特异性、麸质蛋白及其在食物中的水解程度。
