Enzyme-linked immunosorbent assay (ELISA) is commonly used to determine food allergens in food products. However, a significant number of ELISAs give an erroneous result, especially when applied to highly processed food. Accordingly, an improved ELISA, which utilizes an extraction solution comprising the surfactant sodium lauryl sulfate (SDS) and reductant 2-mercaptoethanol (2-ME), has been specially developed to analyze food allergens in highly processed food by enhancing analyte protein extraction. Recently, however, the use of 2-ME has become undesirable. In the present study, a new extraction solution containing a human- and eco-friendly reductant, which is convenient to use at the food manufacturing site, has been established. Among three chemicals with different reducing properties, sodium sulfite, tris(3-hydroxypropyl)phosphine, and mercaptoethylamine sodium sulfite was selected as a 2-ME substitute. The protein extraction ability of SDS/0.1 M sodium sulfite solution was comparable to that of SDS/2-ME solution. Next, the ELISA performance for egg, milk, wheat, peanut, and buckwheat was evaluated by using model-processed foods and commercially available food products. The data showed that the SDS/0.1 M sulfite ELISA significantly correlated with the SDS/2-ME ELISA for all food allergens examined (p< 0.01), thereby establishing the validity of the SDS/0.1 M sulfite ELISA performance. Furthermore, the new SDS/0.1 M sulfite solution was investigated for its applicability to the lateral-flow (LF) test. The result demonstrated the successful analysis of food allergens in processed food, showing consistency with the SDS/0.1 M sulfite ELISA results. Accordingly, a harmonized analysis system for processed food comprising a screening LF test and a quantitative ELISA with identical extraction solution has been established. The ELISA based on the SDS/0.1 M sulfite extraction solution has now been authorized as the revised official method for food allergen analysis in Japan.
Background: It is important to analyze the presence of wheat/gluten in food to avoid wheat allergy or celiac disease. Objective: The Wheat/Gluten ELISA kit was developed to measure total wheat protein or gluten content in wheat, barley, and rye cereals as raw materials, and processed foods. Validation as to whether this kit is suitable for quantifying total wheat protein/gluten was carried out. Methods: The Wheat/Gluten ELISA kit was designed as a sandwich ELISA based on antigliadin polyclonal antibody. Selectivity, interference study, matrix study including incurred food, robustness, stability, and lot-to-lot consistency studies were conducted for the Wheat/Gluten ELISA kit. Incurred matrix studies were also conducted in an independent laboratory. Results: The analysis of 38 different substances revealed no cross-reactivity above the LOQ except for oats. Recoveries of the spiked samples were mostly in the range of 75–140%, including an independent laboratory result. The LOD of the ELISA was found to be 0.02–0.16 mg/kg. Robustness testing proved that extraction time and incubation time of first reaction and enzyme reaction had no significant influence on quantified value. The stability at 2–8°C was found to exceed 12 months. Good lot-to-lot consistency was observed. Conclusions: The Wheat/Gluten ELISA kit showed good analytical performance in the quantitative analysis of total wheat protein/gluten in the identified food products using the AOAC Performance Tested Method(s)SM program. Highlights: The Wheat/Gluten ELISA kit was validated and showed good analytical performance in the quantitative analysis of total wheat protein/gluten in food.
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