The Validation of the Wheat Gluten ELISA Kit.

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.

Reliable enzyme-linked immunosorbent assay for the determination of coconut milk proteins in processed foods.

This study was designed to develop a novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of coconut milk proteins in processed foods. The developed sandwich ELISA was able to detect coconut milk proteins from various coconut milk products and did not show any cross-reactivity with 41 of 42 kinds of popularly used food ingredients, thus reflecting great specificity for coconut milk proteins. In addition, the established ELISA is highly sensitive and allowed the detection of 0.31 µg/g of coconut milk protein in complex food matrices. This proposed assay could serve as a useful tool for the detection of the presence of hidden coconut milk proteins in processed foods.

Determination of walnut protein in processed foods by enzyme-linked immunosorbent assay: interlaboratory study.

Because food allergens from tree nuts, including walnuts, are a frequent cause of adverse food reactions for allergic patients, the labeling of foods containing ingredients derived from tree nuts is required in numerous countries. According to Japanese regulations, the labeling of food products containing walnuts is recommended. To ensure proper labeling, a novel sandwich ELISA kit for the determination of walnut protein in processed foods (Walnut Protein [2S-Albumin] Kit; Morinaga Institute of Biological Science, Inc.; "walnut kit") has been developed. We prepared seven types of incurred samples (model processed foods: biscuits, bread, sponge cake, orange juice, jelly, chicken meatballs, and rice gruel) containing 10 microg walnut soluble protein/g of food for use in interlaboratory evaluations of the walnut kit. The walnut kit displayed sufficient reproducibility relative standard deviations (interlaboratory precision: 5.8-9.9% RSDR) and a high level of recovery (81-119%) for all the incurred samples. All the repeatability relative standard deviation (RSDr) values for the incurred samples that were examined were less than 6.0%. The results of this interlaboratory evaluation suggested that the walnut kit could be used as a precise and reliable tool for determination of walnut protein in processed foods.

A reliable enzyme linked immunosorbent assay for the determination of bovine and porcine gelatin in processed foods.

Since gelatin-containing foods pose a risk for eliciting allergic reactions in sensitized individuals, a novel sandwich enzyme linked-immunosorbent assay (ELISA) for the detection and quantification of bovine and porcine gelatin in processed foods was developed. Rabbits and goats were immunized with bovine gelatin, and three antisera (pAb1 and pAb2 from rabbits, and pAb3 from goats) were obtained. We established a sandwich ELISA method based on a combination of these antibodies. In this study, two sandwich ELISA methods, rabbit pAb2-pAb1 and goat pAb3-pAb3, were evaluated for sensitivity, specificity, cross-reactivity, and applicability. Both ELISA methods were highly specific for bovine and porcine gelatin but had little reactivity with fish gelatin. The detection and quantification limits for porcine gelatin were found to be 0.78 ng/mL and 1.56 ng/mL, respectively. The established sandwich ELISA methods produced no false-positives, except for heated meat products or false negatives when various commercial foods were analyzed for their gelatin content. The rabbit pAb2-pAb1 ELISA cross-reacted with boiled squid, while the goat pAb3-pAb3 ELISA did not. Thus, the proposed goat pAb3-pAb3 ELISA method is a reliable tool for the detection of gelatin contaminants present in processed foods.

Reliable enzyme-linked immunosorbent assay for the determination of walnut proteins in processed foods.

Among food allergens of tree nuts, walnuts are a frequent cause of adverse food reactions in allergic patients. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of walnut soluble proteins in processed foods was developed. The sandwich ELISA is highly specific for walnut soluble proteins. The recovery ranged from 83.4 to 123%, whereas the intra- and interassay coefficients of variation were less than 8.8 and 7.2%, respectively. This study showed that the proposed method is a reliable tool for detection in the presence of hidden walnut proteins in processed foods.

Functional activation of arylhydrocarbon receptor (AhR) in primary T cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exerts diverse adverse health effects by activating the transcription factor arylhydrocarbon receptor (AhR). The activated AhR induces the expression of various genes having xenobiotic responsive elements (XREs) in their enhancer regions, such as the gene for cytochrome P-450 1A1 (CYP1A1). The immune system is sensitively affected by TCDD, while the precise mechanism of how TCDD acts in each immune cell type remains to be determined. The results of previous studies on AhR activation in B cell lines, T cell clones, and thymocytes, which mainly consist of immature T cells, suggested that AhR in mature T cells is inactive, whereas that in B cells and immature T cells act functionally. In the present study, we investigated whether or not TCDD induces the CYP1A1 gene by functionally activating AhR in primary mature T cells in mice. When the splenocytes that contain mature T and B lymphocytes as their predominant cell types or the thymocytes were cultured in the presence of TCDD, each of them showed a similar magnitude of CYP1A1 induction with a peak induction at 4 h. Both mature T cells and B cells that had been separated from total splenocytes also showed CYP1A1 induction at the same magnitude with a peak induction at 4 h. Gene expression of CYP1A1 was observed at 0.1 nM or greater concentrations of TCDD in splenocytes and separated T cells. The induction of CYP1A1 in T cells was confirmed in mice exposed to TCDD. These results indicate that TCDD induces the functional activation of AhR in primary mature T cells in mice.