A Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Almonds in Foods†

Abstract

An enzyme-linked immunosorbent assay was developed to detect almonds as potential allergenic contaminants in food. Polyclonal antibodies directed against roasted almonds were partially purified from immunized sheep and rabbits and used as capture and secondary antibodies, respectively, in a sandwich-type, 96-well plate format. Food samples and almond-spiked samples were extracted 1:10 in phosphate-buffered saline at 60°C for 2 h, centrifuged, and applied to wells coated with sheep anti-almond antibody. After incubation, washing, and the addition of rabbit anti-almond antibody, the amount of almond present was detected with the subsequent addition of goat anti-rabbit immunoglobulin G–alkaline phosphatase conjugate and p-nitrophenyl phosphate substrate. Plate absorbances were read at 410 nm, and standard curves were developed in all matrices to quantify unknowns. Antibodies developed were specific for almond; however, some cross-reactivity was observed with extracts of some tree nuts and sesame seeds. Sodium dodecylsulfate–polyacrylamide gel electrophoresis and Western immunoblotting indicated that sheep anti-almond antibody recognized proteins extracted from black walnuts, Brazil nuts, cashews, hazelnuts, macadamia nuts, pistachios, and sesame seeds in addition to those from almond. The assay was optimized to detect less than 1 ppm of almond and was used successfully to determine almond residues in cereal and chocolate without cross-reacting interferences. A retail survey of 20 brands of cereal demonstrated that the assay produced statistically consistent results. This assay provides a useful quality control tool for the food industry for the protection of consumers allergic to almonds.