Establishment And Application Of Immunoassay For Detecting Allergen Of Macadamia Nut

Macadamia tree nuts,with high nutritional value,but its allergen protein can cause allergic reactions in some people.Currently,an effective method to prevent allergy is to avoid allergen exposure in allergic patients.Therefore,it is of great significance to establish an effective detection method for macadamia protein.In this thesis,the ELISA method for the determination of whole protein of macadamia was optimized and applied to the detection of the target protein in heat-processed foods.Ammonium sulfate precipitation method was used to extract total macadamia nut protein,which was used as immunogen to immunize rabbits and BALB/c mice to prepare rabbit anti-polyclonal antibody and mouse anti-polyclonal antibody.Cross-reaction experiments showed that the antibodies could not identify proteins in soybean,walnut,almond,peanut,cashew and wheat except for some proteins from pistachio and hazelnut,indicating that the prepared antibodies had better specificity.The rabbit polyclonal antibody was used to optimize indirect competitive enzyme-linked immunosorbent assay(ELISA).The volume of antigen coated was 0.025μg/well,the blocking solution was 1%skimmed milk powder,the dilution of rabbit polyclonal antibody was 1:16000,Standard secondary antibody dilution factor 1:20000.The detection limit of this method was 5.12±0.57 ng/mL and IC50 was 63.94 ± 0.85 ng/mL.The rabbit polyclonal antibody was used as the capture antibody and the mouse polyclonal antibody was used as the detection antibody.The double antibody sandwich enzyme-linked immunosorbent assay(ELISA)was established.The capture antibody coating amount was 0.05 μg/well and the blocking solution was 0.5%skimmed milk powder.The dilution of detection antibody was 1:2000,the dilution of goat anti-mouse HRP-ELISA secondary antibody was 1:20000,the linear range of the method was 3.13 ng/mL to 30 ng/mL.The limit of detection was 0.95 ± 0.17 ng/mL.The limit of quantification was 3.13 ± 0.57 ng/mL.The intra-plate and inter-plate variation of this method are in the range of 1.76%-7.20%and 3.83%-12.05%,respectively,indicating that the method has good reproducibility.The established double-antibody sandwich ELISA method was applied to the determination of macadamia in baked macadamia nuts and crackers,and the applicability of the method to detect the target in heat-processed food was evaluated.