Food allergen analysis for processed food using a novel extraction method to eliminate harmful reagents for both ELISA and lateral-flow tests.

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.

A chimeric epidermal growth factor with fibrin affinity promotes repair of injured keratinocyte sheets.

The aim of the present study is to create a novel chimeric protein of epidermal growth factor (EGF) with fibrin affinity and demonstrate its potential for repairing injured tissues by immobilization to fibrin. The chimeric protein (FBD-EGF) was produced by the fusion of the fibronectin fibrin-binding domain (FBD) to EGF. It showed dose-dependent binding to fibrin and its binding was stable for at least 7days, while native EGF showed little affinity. FBD-EGF promoted the growth of fibroblasts and keratinocytes in the fibrin-bound state as well as in the soluble state. Its activity was further studied in a keratinocyte culture system in which fibrin was exposed upon injury of cell sheets. Fibrin-bound FBD-EGF promoted growth of the sheets over the injured area at a significantly faster rate (approximately eightfold) than native EGF (p<0.01). Wounds 2mm wide were closed in 7-9days. This repair process was inhibited by anti-EGF. Keratinocytes proliferated more extensively in the leading edges of sheets contacting fibrin with FBD-EGF, approximately 1.7-fold more than in the adjacent regions. These results imply that the stable binding of chimeric EGF to fibrin is effective for the repair of injured keratinocyte sheets, suggesting a potential use in tissue engineering.