Effects of processing on the immunochemical detection of peanut residues and the development of a sensitive and Ara h 2 specific competitive elisa for the detection of peanut

In the first phase of this study, six commercial ELISA kits were compared for their specificity in detecting peanut allergens. Five kits predominantly detected the heat labile peanut allergen, Ara h 3. The other kit was most sensitive to the heat stable and more potent allergens, Ara h 2 and Ara h 6. The kits that showed higher sensitivity to Ara h 3 underestimated the protein content in medium and dark roasted peanut flours. The Ara h 2 sensitive kit was the only kit that showed sufficient sensitivity in detecting peanuts at concentrations of ≤ 25 ppm of dark roast peanut flours, although the overall recovery of peanut with this kit was low. During the second phase of this study, several buffers were evaluated for their efficacy in recovering peanut from a model solid food matrix (pastry squares) incurred with peanut and subjected to different processing conditions using two commercial ELISA kits (Veratox and Morinaga peanut kits). Compared to the kit supplied buffer of the Veratox kit, sodium carbonate at pH 9.6 and PBS containing 1 M GuHCl recovered significantly higher amounts of peanut from fried, high pressure processed (HPP) and baked samples. More peanut was recovered from boiled and fried pastry squares when the Morinaga buffer was replaced with PBS containing β-ME and SDS indicating that selected buffers are significantly more effective than the kit supplied buffers in recovering peanut from certain processed solid food matrices. In the final phase of this study, a competitive inhibition ELISA targeting peanut Ara h 2 was developed. Polyclonal IgG antibodies raised against purified Ara h 2 were used to develop a sensitive ELISA with a range of quantification of 40-400 ng/mL of Ara h 2. The LOD and IC50 were 27± 8 ng/mL and 134± 32 ng/mL of Ara h 2 respectively. Among 45 food ingredients evaluated for cross reactivity with the polyclonal antibodies only cloves, cocoa powder, mustard and pistachio showed some matrix interference. The competitive ELISA showed better performance in recovering peanut from a differentially processed solid food matrix when compared to two commercial ELISA kits