A sandwich enzyme-linked immunosorbent assay for the detection of almonds in foods

An enzyme-linked immunosorbent assay was developed to detect almonds as pot ential allergenic contaminants in food. Polyclonal antibodies directed agai nst roasted almonds were partially purified from immunized sheep and rabbit s and used as capture and secondary antibodies, respectively, in a sandwich -type, 96-well plate format. Food samples and almond-spiked samples were ex tracted 1:10 in phosphate-buffered saline at 60 degrees C for 2 h, centrifu ged, and applied to wells coated with sheep anti-almond antibody. After inc ubation, washing, and the addition of rabbit anti-almond antibody, the amou nt of almond present was detected with the subsequent addition of goat anti -rabbit immunoglobulin G-alkaline phosphatase conjugate and p-nitrophenyl p hosphate substrate. Plate absorbances were read at 410 nm, and standard cur ves were developed in all matrices to quantify unknowns. Antibodies develop ed were specific for almond; however, some cross-reactivity was observed wi th extracts of some tree nuts and sesame seeds. Sodium dodecylsulfate-polya crylamide gel electrophoresis and Western immunoblotting indicated that she ep anti-almond antibody recognized proteins extracted from black walnuts, B razil nuts, cashews, hazelnuts, macadamia nuts, pistachios, and sesame seed s 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 sur vey of 20 brands of cereal demonstrated that the assay produced statistical ly consistent results. This assay provides a useful quality control tool fo r the food industry for the protection of consumers allergic to almonds.