Panda Rakhi, Boyer Marc, Garber Eric A E
Division of Bioanalytical Chemistry, Office of Regulatory Science, Center for Food Safety and Applied Nutrition (CFSAN), FDA, 5001 Campus Drive, College Park, MD, 20740, USA.
Office of Analytics and Outreach, Center for Food Safety and Applied Nutrition (CFSAN), FDA, 5100 Paint Branch Parkway, College Park, MD, 20740, USA.
Anal Bioanal Chem. 2017 Dec;409(30):6959-6973. doi: 10.1007/s00216-017-0677-z. Epub 2017 Nov 7.
A novel competitive ELISA was developed utilizing the G12, R5, 2D4, MIoBS, and Skerritt antibody-HRP conjugates employed in nine commercial ELISA test kits that are routinely used for gluten detection. This novel multiplex competitive ELISA simultaneously measures gliadin-, deamidated gliadin-, and glutenin-specific epitopes. The assay was used to evaluate 20 wheat beers, 20 barley beers, 6 barley beers processed to reduce gluten, 15 soy sauces, 6 teriyaki sauces, 6 Worcestershire sauces, 6 vinegars, and 8 sourdough breads. For wheat beers, the apparent gluten concentration values obtained by the G12 and Skerritt antibodies were typically higher than those obtained using the R5 antibodies. The sourdough bread samples resulted in higher apparent gluten concentration values with the Skerritt antibody, while the values generated by the G12 and R5 antibodies were comparable. Although the soy-based sauces showed non-specific inhibition with the multiple R5 and G12 antibodies, their overall profile was distinguishable from the other categories of fermented foods. Cluster analysis of the apparent gluten concentration values obtained by the multiplex competitive ELISA, as well as the relative response of the nine gluten-specific antibodies used in the assay to different gluten proteins/peptides, distinguishes among the different categories of fermented-hydrolyzed foods by recognizing the differences in the protein/peptide profiles characteristic of each product. This novel gluten-based multiplex competitive ELISA provides insight into the extent of proteolysis resulting from various fermentation processes, which is essential for accurate gluten quantification in fermented-hydrolyzed foods. Graphical abstract A novel multiplex competitive ELISA for the detection and characterization of gluten in fermented-hydrolyzed foods.
利用九种常用于麸质检测的商业ELISA检测试剂盒中使用的G12、R5、2D4、MIoBS和Skerritt抗体-辣根过氧化物酶（HRP）偶联物，开发了一种新型竞争性ELISA。这种新型多重竞争性ELISA可同时检测麦醇溶蛋白、脱酰胺麦醇溶蛋白和麦谷蛋白特异性表位。该检测方法用于评估20种小麦啤酒、20种大麦啤酒、6种经过处理以降低麸质含量的大麦啤酒、15种酱油、6种照烧酱、6种伍斯特沙司、6种醋和8种酸面团面包。对于小麦啤酒，G12和Skerritt抗体获得的表观麸质浓度值通常高于使用R5抗体获得的值。酸面团面包样品使用Skerritt抗体时产生的表观麸质浓度值更高，而G12和R5抗体产生的值相当。尽管基于大豆的酱油对多种R5和G12抗体表现出非特异性抑制，但它们的总体特征与其他发酵食品类别不同。通过对多重竞争性ELISA获得的表观麸质浓度值以及该检测中使用的九种麸质特异性抗体对不同麸质蛋白/肽的相对反应进行聚类分析，通过识别每种产品特有的蛋白质/肽谱差异，区分不同类别的发酵水解食品。这种基于麸质的新型多重竞争性ELISA有助于深入了解各种发酵过程导致的蛋白水解程度，这对于准确量化发酵水解食品中的麸质至关重要。图形摘要一种用于检测和表征发酵水解食品中麸质的新型多重竞争性ELISA。
