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

摘要

Cashew nut (Anacardium occidentale L.) as a nutritious and popular snack is also a potent allergenic food which could elicit severe or even life-threatening allergic reactions in sensitive individuals. The only recommended management option is the strict avoidance of offending cashew nut, and to achieve this, reliable and accurate food labelling is essential.Cashew allergen Ana o 3 (2S albumin), due to its compact and stable structure, is more stable after thermal processing and is potentially a good biomarker for ELISA development. This study aims to develop a sensitive sandwich ELISA specific to Ana o 3, which is less influenced by food processing conditions or protein composition of cashew nut cultivars so to detect traces of cashew residues in pre-packed food products more reliably. Ana o 3 was purified from raw and roasted cashew nut kernels and used as an immunogen to raise polyclonal antibodies. Four sandwich ELISAs differing in their capture-detection antibody pairs were developed and compared for their performance. The CAS–ELISA–1 developed for crude protein presented a sensitivity of 0.58 mg protein kg-1 (57.1 ± 10.8 g L-1). The other three ELISAs, CAS–ELISA–2, Ano3–ELISA–1 and Ano3–ELISA–2, by using anti-Ana o 3 antibody to detect thermally stable cashew nut allergen, offered LODs of 0.04 mg protein kg-1 (4.1 ± 0.02 μg L-1), 0.06 mg protein kg-1 (0.6 ± 0.1 μg L-1) and 0.13 mg protein kg-1 (1.3 ± 0.4 μg L-1), respectively. The assay provided less variation in detecting cashew nut (protein) in different cultivars in raw and roasted form, indicating Ana o 3 was an ideal biomarker for ELISA. The ELISAs were highly specific to cashew nut only showing very low cross-reactivity with pistachio, pecan, almond, peanut and hazelnut. Two cashew nut ELISAs were validated by spiking cashew nut into model biscuits and chocolate. The biscuit extracts significantly interfered with assay performance, resulting in lower recovery of 40.2–44.9% with CAS–ELISA–2 and 25.2–79.8% with Ano3–ELISA–1 in the model biscuits for CAS–ELISA–2 and Ano3–ELISA–1 and a limit of quantitation (LOQ) of 40 mg protein kg-1 for both assays. Chocolate provided less matrix interference than biscuit. CAS–ELISA–2 and Ano3–ELISA–1 provided LOQs of 4 mg protein kg-1 and 0.4 mg protein kg-1 in chocolate with an acceptable recovery of 74.0–87.7% (CAS–ELISA–2) and 43.8–136.2% (Ano3–ELISA–1). Therefore, they are valuable tools for detecting cashew nut residues under the recommended VITAL action level.