Development of a sensitive sandwich ELISA specific to 2S albumin (Ana o 3) as a stable protein marker for cashew nut residue detection in pre-packaged food products

In this study, a cashew allergen, Ana o 3 (2S albumin), was used as a protein marker to develop two sensitive and specific sandwich ELISAs (denoted as CAS-ELISA-2 and Ano3-ELISA-1) with the limits of detection (LOD, determined as a concentration that yield 20% of maximum colour development) at 0.04 and 0.06 mg protein kg−1, respectively. The ELISAs were highly specific to cashew nut with very low cross-reactivity with pistachio, pecan, almond, peanut, and hazelnut. Assay validation on the model cookies observed lower cashew protein recovery of 40.2-44.9% with the CAS-ELISA-2 assay and 25.2-79.8% with the Ano3-ELISA-1 assay. Both assays exhibited a limit of quantitation (LOQ) of 40 mg protein kg−1 cookies. While for the model chocolate, the acceptable protein recoveries were obtained with both CAS-ELISA-2 (74.0-87.7%) and Ano3-ELISA-1 (62.8-136.2%). The LOQ of 4 mg protein kg−1 chocolate were achieved.