| Walnuts are a common leisure and puzzle food in daily life.While having extremely high economic benefits,it also has extremely high nutritional levels,with a walnut protein content of up to 26.38% per 100 grams,which is favored by consumers.But it is also one of the eight major food allergens,including nuts.Some people may experience allergic symptoms after consuming products containing walnuts,which can be life-threatening in severe cases.Therefore,strengthening its detection methods has become a trend.This experiment uses fresh walnuts sold on the market as raw materials,and uses physical and chemical methods for preliminary protein extraction and purification.Subsequently,animal immunological tests are conducted to evaluate the antibody titer.Based on the immunological characteristics of antigen,antibody,and enzyme-linked secondary antibody specific binding,a double antibody sandwich ELISA method is established to detect walnut allergic proteins,and its related experimental links are optimized.Finally,assemble the reagent kit.This study is a convenient detection method for walnut protein,with precise detection limits and quantification limits,less susceptible to interference,and lower prices,making it easier to promote new methods.The research content and results of this article are as follows:(1)The fat in walnut was removed by organic reagent,the walnut protein extract was salted out with saturated ammonium sulfate,dialyzed with a dialysis bag with a molecular weight of 8000 KD,and purified with dextran gel G-100 column chromatography.The molecular weight of allergen protein in the purified protein solution is about 55 k D,43 k D,34-30 k D and 15-10 k D respectively.(2)Immunize the experimental animals to prepare antibodies for biological detection,and use the chessboard method to optimize the dilution and action time of the antibody.The antiserum titer is 1:2.4×105 μ g/L,and the inhibition rate of walnut protein is 52.67%.The antibody was purified by affinity chromatography column,and the concentration of antibody was 2.58g/L.After optimizing the reaction conditions of double antibody sandwich ELISA,the antibody coating amount is 2 μ g per well,the dilution ratio of enzyme-labeled second antibody is 1:1800,and the optimal p H value of PBS buffer is 5.7.Use 1% BSA solution as the blocking solution,dilute the concentration of walnut protein standard sample by 6concentrations,and then construct a quantitative detection method of walnut protein by plotting the standard curve,which has high sensitivity,and the detection limit is 30 ± 5 μ G/L,with good stability,the variation range within the plate is 0.84~7.82%,and the variation range between plates is 1.33~0.10%.Compared with the detection method of liquid chromatography-mass spectrometry,the R2 is 0.9977,indicating that this experimental analysis method can obtain accurate quantitative results.(3)The kit was assembled,the standard curve was evaluated,three products were selected,and five repeated tests were conducted to observe the working concentration range of the standard curve and the IC50 variation range.The linear performance evaluation of three batches of reagent kits showed that the linear range of reagent detection was 0.1~8.1 ppb,and the IC50 range was 0.303~0.434,which met the design requirements. |
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