Establishment Of A DAS-ELISA Method For Detectimg Antigenicity Changes Of Peanut Allergens-Ara H 3

As a common food in life,peanut contains a lot of fat and protein.Peanut protein,as a good quality and price-advantage plant protein,can improve quality and strengthen nutrition whether it is added to animal or plant ingredients.However,as one of the eight classes of sensitized foods,the main sensitizing allergen protein,Ara h 3,has stable structure and high similarity with legume protein,which will affect the physical health of allergic patients.In this study,by exploring the influence of damp-heat treatment on Ara h 3 antigenicity and protein structure,and establish the detection method of double-antibody-sandwich（double-antibody-sandwich Enzyme Linked Immunosorbent Assay,ELISA）,detect the change of antigenicity of peanut after damp-heat treatment,correctly guide the production of the food containing Ara h 3 processing,so as to develop Ara h 3-containing foods with low sensitization or no sensitization,is significant to peanut allergy population.The main working methods of this test are:first,Ara h 3 was extracted and purified from degreted peanut powder by graded precipitation of ammonium sulfate to study the effect of damp-heat treatment on the antigenicity and protein structure of Ara h 3,and to determine the optimal damp-heat treatment conditions to reduce its antigenicity;secondly,the digestion stability of Ara h 3 after damp-heat treatment was evaluated by in vitro simulated digestion test;finally,a double-antibody-sandwich ELISA detection method for the antigenic change of Ara h 3 after damp-heat treatment was established to explore the antigenic change of the food containing Ara h 3 after damp-heat treatment.The study results are as follows:The purity of Ara h 3 was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis（Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis,SDS-PAGE）:electrophoresis showed that the extracted Ara h 3 showed high purity after purification.The gray scale value of the extracted by Band Scan analysis software showed that the purity of Ara h 3 was above 75%and met the requirements of the test.Effect of damp-heat treatment on the antigenicity of Ara h 3:The effect of damp-heat treatment on the antigenicity of Ara h 3 was measured by indirect ELISA（enzyme-linked immunosorbent method）.The optimal damp-heat treatment condition of Ara h 3 was:the temperature of damp-heat treatment was 90℃,and the time was 30 min.Effect of damp-heat treatment on the structural characteristics of Ara h 3:The analysis of the prototype electrophoresis results showed that Ara h 3 depolymerization took place after 100℃,30 min of damp-heat treatment,resulting in smaller molecular mass protein bands and the appearance of insoluble macromolecular mass protein aggregates.The exogenous fluorescence of Ara h 3 by fluorescence spectroscopy showed that at 90℃and 20 min increased the fluorescence intensity,and the tertiary structure of Ara h 3 changes.The results of UV-visible spectroscopy showed that the content of free sulfhydryl group increased significantly when the temperature of damp-heat treatment was 80 to 90℃and the time was20 to 30 min.The effect of circular dichroism spectroscopy（CD）on the secondary structure changes of Ara h 3 after damp-heat treatment showed that the ratio ofβ-fold andα-helix in Ara h 3 spatial structure and the proportion of random coil significantly increased after 90℃damp-heat treatment.Evaluation by in vitro simulated digestion:damp-heat treatment can significantly reduce the antigenicity of Ara h 3.After in vitro simulated digestion,the untreated Ara h 3 bands were gradually digested into small molecules,and the antigenicity was significantly reduced.Compared with the untreated Ara h 3,the damp-heat treated Ara h3 bands were further digested and even completely digested,and the antigenicity was lower.Establish double-antibody-sandwich ELISA detection method for Ara h 3,the optimal damp-heat treatment conditions of Ara h 3 and anti-Ara h 3 rabbit source polyclonal antibody mixed after temperature,prepared the damp-heat treatment destroy Ara h 3 antigen epitope specific antibody as coating antibody,with horseradish peroxide enzyme（HRP）antibody,the analysis of antigenicity after damp-heat treatment containing Ara h 3 food:the specificity determination results show that this method has a relatively good specificity,its critical value is 0.2431,beyond the critical value is positive.By sensitivity analysis,the limit of detection obtained was 39.06 ng/m L.The coefficient of variation in the present method is lower than 3.05%and lower than 6.79%.The addition recovery test showed that the addition recovery rate of Ara h 3 ranged from 78.48%to 90.93%.For the detection of damp-heat-treated foods containing Ara h 3 using the established double-antibody-sandwich ELISA method,The results showed that the OD₄₅₀values of peanut butter,peanut cheese,peanut milk and boiled peanut were lower than the OD₄₅₀values of skimmed peanut powder.The results show that the above several kinds of food containing Ara h 3 are caused by damp-heat processing.It may be that during the process of wet heat treatment,part of the epitopes contained in Ara h 3 in food are destroyed or embedded,which reduces the ability to bind to specific antibodies that destroy the Ara h 3 epitopes by damp-heat treatment,resulting in a decrease in the OD₄₅₀detection value.However,the Ara h 3 in degreasing peanut powder is not destroyed or embedded,and the allergenic protein can specifically bind specifically to the antibody,so the OD ₄₅₀detection value is high.This method can be used to detect the changes in the antigenicity of Ara h 3-containing foods after damp-heat treatment,so as to better guide the safe production of damp-heat processed food containing Ara h 3,and then reduce its effects on patients with Ara h 3 sensitization.