Development Of Enzvme-Linked Immunoassay And Effects Of Processing For Almond Allergen, Amandin

Almonds are one of tree nuts and one of most popular allergens, which can induce severe allergic reactions mediated by IgE. So far, the best defense against food allergen is to avoid the protein, so developing a sensitive detect method and researching the decrease-allergenicity ways are the technology supports to control food allergen.In the article, amandin, the allergen proteins were extracted from Chinese almonds (sweet almond and bitter almond), and the polyclonal antibodies against amandin were obtained by immunizing rabbits and mice with the proteins, respectively. Then sensitive sandwich-antibody enzyme linked immunosorbent assays based on polyclonal antibodies were developed and could detect amandin of Chinese almonds as low as 10.16±1.27 ng/mL and 6.36±1.02 ng/mL respectively. The assays didn’t show any cross-reactivity with 14 common food proteins, showing a high specificity. From the results of detecting other kinds of almonds by two assays, the ELISA based sweet almond could detect Chinese bitter almonds and Amygdingus comnnis better, with the limit detection of 12.11±1.70 μg/L and 18.95±1.52 μg/L respectively, so the assay could be used to detect almonds in food. The sandwich ELISA were used to detect the amandin in bread, biscuit and Skim milk with recoveries ranging from 78.94%±2.00% to 125.15%±2.73%, and the assay showed high accuracy and repeatability from the experiments of variance in-plate and between-plate.The article focused on the effects of amandin by the processings include dry-heating (100℃,200℃,250℃,400℃), moist-heating (60℃, boiling), autoclave sterilization and high pressure treatment (400 MPa,500 MPa,580 MPa). The results showed that amandin were relative stable at the condition of dry heating, however, synergistic effects of moist with temperature and pressure on amandin destroyed the protein structure obviously, so that the solubility and immunoreactivity of amandin decreased significantly. The 22-24 KDa polypeptide of amandin disappered after moist-heating 100℃ and autoclaving with PBS, and both peptides between of 20-22 KDa and 38-41 KDa were depolymerized into small molecule peptide after high pressured with PBS, which decreased the immunoreactivity of amandin greatly.In general, water combined with heat and high pressure treatment might be a new strategy to decrease or eliminate the antigenicity of allergen during food processing.