Development Of Sandwich ELISA Detection Method For Three Major Allergens In Milk

Milk,one of the relatively common sources of protein in people's daily life,is also recognized by WHO as one of the eight categories of food allergies to humans.Casein(CN),?-lactoglobulin(?-LG),and?-lactalbumin(?-LA)are considered to be the main allergens in milk.At present,there is no effective treatment for milk allergy.Therefore,the detection for main allergens of milk including CN,?-LG and?-LA,not only can provide theoretical and technical supports for the development of allergen identification standard of milk and dairy products,but also provide protection for the health of susceptible special population.In this paper,the preparation and performance identification of rabbit polyclonal antibodies to three main allergens including casein,?-LA and?-LG,the preparation and performance identification of mouse monoclonal antibodies to three main allergens,and the development and methodological assessment of three main allergens for sandwich ELISA detection methods were studied.The specific research contents and results were as follows:1.Preparation and identification of casein,?-LA and?-LG rabbit polyclonal antibodiesAfter mixing equal amounts of CN,?-LG and?-LA as the immune antigens with Freund's adjuvant,the healthy New Zealand white rabbits were immunized by subcutaneous injection in the back of the neck,then rabbit serum was prepared and purified by caprylic acid-saturated ammonium sulfate precipitation method.Subsequently,SDS-PAGE gel electrophoresis was used to determine the purity and the titer was determined by indirect ELISA.The results showed that high-purity rabbit polyclonal antibodies against CN,?-LG and?-LA could be successfully prepared;the three proteins were immunized four times,and the dose of each immunization was500?g.After four times,the titers of the three multi-antibodies were all up to 1:729000.2.Preparation of casein,?-LA and?-LG monoclonal antibodiesThe same immunization method and immune antigens as the rabbit polyclonal antibody were used to immunize healthy,age-appropriate female BALB/c mice.All three proteins were immunized seven times,the three proteins were immunized six times,the first dose fot the first immunization was 100?g/every mouse,the second to sixth dose for booster immunization was 50?g/every mouse,and the sprint immunization was 25?g/every mouse.Then indirect ELISA was used to detect the immune serum of mice,and the mice with the highest serum titer(casein titer for1:81000,?-LA and?-LG titers for 1:243000)were selected after the seventh immunization.Next,positive fusion wells were detected by cell fusion and indirect ELISA.After three times of subcloning and screening by the limiting dilution method,two hybridoma cell lines(2A8,8B6)stably secreting casein McAb,eight hybridoma cell lines(1C3,2F8,2H11,3A5,3A10,4A2,4A3,4B10)stably secreting?-LA McAb,and one hybridoma cell line(2A8)stably secreting?-LG McAb were obtained by limited dilution method.Finally,the in vivo ascites inducing method was used for mass production of monoclonal antibodies.3.Identification of casein,?-LA and?-LG monoclonal antibodiesThe ascites monoclonal antibody obtained was purified by caprylic acid-saturated ammonium sulfate precipitation method,and then its purity was identified by SDS-PAGE gel electrophoresis,titer was determined by indirect ELISA method,specificity by Westen-blotting,non-competitive indirect ELISA The results showed that the titers of 2 monoclonal antibodies 2H11 and 3A10 of?-LA were not too high(1:27000),and that of 2(2A8,8B6)monoclonal antibodies and?-LG of casein 1(2A8)monoclonal antibody and the remaining 6(1C3,2F8,3A5,4A2,4A3,4B10)monoclonal antibodies can reach 1:243000;the casein monoclonal antibody affinity constant was 3.1×10~7 L/mol,and the?-LA monoclonal antibody affinity constant was 3.5×10~7L/mol,?-LG monoclonal antibody affinity constant is 3.27×10~7L/mol;identification by commercial monoclonal antibody subtype kits shows that except that the monoclonal antibody subtype secreted by 3A10 of?-LA is IgG2b Monoclonal antibody subtypes secreted by protein,?-LG and other?-LA cell lines IgG1.4.Development of Sandwich ELISA for Detection of CNBy screening the optimal capture antibody,and optimizing the working concentration of rabbit polyclonal antibody and enzyme-labeled secondary antibody,a double-antibody sandwich ELISA method for casein was established.The optimal capture antibody is 2A8,and the optimal working concentration of rabbit polyclonal antibody is 1:10000,the optimal working concentration of HRP-labeled goat anti-rabbit IgG antibody(enzyme-labeled secondary antibody)is 1:10000.The linear range of this method is 40~1280 ng/mL,and the limit of detetion(LOD)is 40 ng/mL.The intra-assay variation coefficient CV of this method for casein detection is between 2.72%and 5.97%,and the inter-assay variation coefficient CV is between7.99%and 12.33%,indicating a high degree of precision;this method only cross-reacts with casein,and does not cross-react with several other major proteins in cow's milk,indicating good specificity;the The recovery rate of casein from the method was 80.4%~92.25%,which could meet the detection requirements of casein allergens in cow milk.5.Development of Sandwich ELISA for Detection of?-LABy screening the optimal capture antibody,and optimizing the working concentration of rabbit polyclonal antibody and enzyme-labeled secondary antibody,a dual-antibody sandwich ELISA detection method for?-LA was established.The optimal capture antibody was 4A2,and the optimal working concentration of rabbit polyclonal antibody It is 1:15000.The optimal working concentration of HRP-labeled goat anti-rabbit IgG antibody(enzyme-labeled secondary antibody)is 1:10,000.The linear range of this method is 2.5~40 ng/mL,and the LOD is 2.5 ng/mL.The intra-assay coefficient of variation(CV)of this method for?-LA detection is between2.69%and 6.27%.The coefficient of variation CV is between 8.06%and 12.41%,indicating a high degree of precision;this method only cross-reacts with?-LA and does not cross-react with several other major proteins in cow's milk,indicating a better Specificity;The recovery of?-LA from this method is 84.6%~93.6%,which can meet the detection requirements of?-LA allergens in cow's milk.6.Development of Sandwich ELISA for Detection of?-LGBy optimizing the working concentration of rabbit polyclonal antibody and enzyme-labeled secondary antibody,a dual-antibody sandwich ELISA method for?-LG was established.The capture antibody was 2A8,and the optimal working concentration of rabbit polyclonal antibody was 1:10,000.The optimal working concentration of goat anti-rabbit IgG antibody(enzyme-labeled secondary antibody)is 1:10000.The linear range of this method is 12.5~200 ng/mL,and the LOD is 12.5ng/mL.The intra-assay coefficient of variation CV of this method for casein detection is between 3.33%and 5.96%,and the inter-assay coefficient of variation CV is between 7.81%and 12.36%,indicating a high degree of precision;this method only cross-reacts with casein and does not cross-react with several other major proteins in milk,indicating good specificity;The srecovery rate of?-LG from this method is86.8%~92.3%,which can meet the detection requirements of?-LG allergens in cow's milk.