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This research project focused on macadamia nut detection. Two specific aims were proposed: (i) develop a competitive inhibition ELISA with a sensitivity of 10 ppm (ii) assess the assay specificity and robustness. The assay specificity of the rabbit anti-macadamia pAb-based ELISA was evaluated by determining assay interference by several select food matrices. The assay robustness was assessed by determining its ability to detect the presence of proteins in variously processed nut seeds. The optimized ELISA has a detection range of 3.2 to 400 ng of macadamia nut protein/mL of BSB. The protein G-purified rabbit anti-macadamia nut protein polyclonal antibodies did not exhibit detectable cross-reactivity with tested protein extracts prepared from several seed and food matrices. The assay was able to detect the seed proteins extracted from macadamia nut seeds subjected to select thermal processing methods.