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Pistachios (Pistacia vera) are popular snacks and consumption of pistachios is notably increasing due to their usage as ingredients for confections, ice cream, and baked goods. The increased consumption of pistachios may lead to a higher frequency of allergic sensitization and an increased prevalence of allergic reactions to pistachios. Trace amounts of undeclared pistachio allergens can pose serious health risks for food-allergic consumers, including severe anaphylactic reactions. A highly sensitive analytical method, sandwich-type enzyme-linked immunosorbent assay (ELISA) is applied as a critical tool for food manufacturers and food scientists to detect minute amounts of allergenic food residues in processed foods. The aim of the study was to develop an ELISA for the detection and quantification of pistachio residues in processed foods. California raw pistachios and a mixture of Californian, Iranian, and Turkish roasted pistachios were used as immunogens to immunize one sheep, one goat, and three rabbits. Both raw and roasted pistachio ELISAs were developed by using pooled sheep antisera as the capture reagent and pooled rabbit antisera as the detector reagent. Binding of antigen-antibody complex was visualized through a colorimetric reaction involving goat anti-rabbit IgG antibody (labeled with alkaline phosphatase) and substrate (p-nitrophenyl phosphate). A total of 102 food ingredients were evaluated using the developed pistachio ELISA for potential cross-reactivity. Vanilla ice cream and sugar cookies with known amounts of pistachio were prepared as manufactured model foods. The sensitivity, specificity, and robustness of the assay were determined by the percent of recovery from both of the model foods.
The optimized ELISA had a limit of quantification (LOQ) of <1 ppm>(1μg/g). Minor cross-reactivity was observed with cashew at a level equivalent to 4 ppm of roasted pistachio. The performance of the ELISA was not affected in the presence of the ice cream and cookie matrices. The mean percent recovery of pistachio from the vanilla ice cream, cookie dough, and baked sugar cookie are 115 ± 3.0, 131 ± 20.8, and 53.9 ± 3.0 respectively. The pistachio-ELISA developed in this study is sufficiently specific and sensitive to be used to help food manufacturers in complying with the FDA labeling guidelines and to safeguard allergic consumers from undeclared pistachio residues.