Establishment Of DAS-ELISA For Detecting Antigenicity Changes Of Glycinin After Heat Processing

Soybean is high in nutrients,but it also carries a potential risk of food allergies.Effective processing can reduce the antigenicity of soybean globulin.This test to explore the antigenicity of hot working glycinin by means of heat treatment and the relationship between protein structure,and establish the double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA)detection products containing soybean globulin hot working class,the antigenic change after heat treatment,containing soybean globulin correct guidance to product safety in production,thereby reducing the potential risk of glycinin allergic patients.The main contents of this project are as follows:in the first step,three New Zealand rabbits were immunized with the extracted and purified soybean globulin to prepare the soybean globulin polyclonal antiserum.The polyclonal antiserum was purified by ammonium sulfate precipitation and further purified by immunoaffinity chromatography.The second step is to explore the effect of different heat treatment temperature and time on the structure and antigenicity of soybean globulin.The best heat treatment condition is the corresponding heat treatment parameters when the antigenicity of soybean globulin is the lowest.The third step is to establish a DAS-ELISA method to explore the antigenicity changes of heat-treated products containing soy globulin.In this process,the heat-treated antigen-absorbing antiserum is used as coating antibody,and the horseradish peroxidase(HRP)labeled rabbit polyclonal antibody is used as detection antibody.Finally,the DAS-ELISA method is optimized and established.The results are as follows:The extracted and purified soy globulin was analyzed by gel analysis imaging system.Its purity was 81%,and 3 New Zealand rabbits were immunized to prepare the polyclonal serum.Then the immunological characteristics of the prepared serum were identified.The results showed that the titer of rabbit 1 was 1:6×10⁴,and the highest.The indirect competitive ELISA inhibition curve shows that the inhibition rate of IC₅₀ is 527 ng/m L,that is,the sensitivity of rabbit 1 is the highest.The results showed that the cross reaction rate of the antiserum with?-conglycinin and peanut protein was 8.6%and 4.6%,and the cross reaction rate with other proteins(CR<0.2%).The rabbit polyclonal antiserum with high titer,high sensitivity and specificity was prepared.The serum was purified with CNBr activated seprose-4B to obtain high purity antibody.The results of indirect ELISA showed that the optimal heat treatment conditions of soybean globulin were protein concentration 10 mg/m L,temperature 110?and time 50 min,and the antigenicity was the lowest at this time.The results of SDS-PAGE showed that the primary structure of soybean globulin was not changed by heat treatment.Non-reduction SDS-PAGE results showed that soybean globulin was depolymerized by heat treatment to produce lower molecular weight protein,and large molecular weight protein aggregates were formed.Under the conditions of 90??120?and 40?50 min of heat treatment,the UV-Vis spectra showed that the content of free sulfhydryl group increased significantly after heat treatment.The results of fluorescence spectrum showed that the maximum red shift occurred when heated for 50 minutes,indicating that the change of tryptophan microenvironment changed the tertiary structure of soybean globulin,and the change of antigenic epitope of soybean globulin made the antigenicity of soybean globulin shown by indirect ELISA lowest.The results of circular dichroism(CD)spectrum analysis showed that the content of?-helix and?-fold decreased after heat treatment,while the content of irregular curled structure increased.Therefore,it is speculated that the antigenic epitopes of soybean globulin may mainly exist in?-helix and?-fold.The specificity of the established DAS-ELISA showed that the specificity of the method was good.The cut off value was 0.388,which was higher than that,it could be judged as positive.The sensitivity test results showed that the detection limit was 19.53 ng/m L.The variation coefficient of the method was less than 5.25%(between plates)and 9.50%(Inside plates).In the milk powder addition test,the recovery of soybean globulin was between83.65%and 90.13%.DAS-ELISA method was used to detect the hot processed foods containing soy globulin.The results showed that the OD₄₅₀ values of soy sauce,steamed fish soy sauce,soybean paste,beef paste,soybean milk powder and tofu were lower than those of defatted soybean powder.The results showed that the soybean globulin products had undergone different degrees of thermal processing,most of the epitopes of soybean globulin in the products were destroyed by thermal processing,and could not bind to the thermal treated antigen-absorbing antiserum,which reduced the detection value of OD₄₅₀.The method can be used to analyze the antigenicity of soybean globulin in food,and further guide the safety production of heat-processing products containing soybean globulin,so as to reduce the impact on soybean sensitized population.