Study On Gold Magnetic Enzyme-linked Immunoassay For Detecting Allergen Casein, α-lactalbumin In Bovine Milk

Milk products are rich in nutrients, bovine milk and its dairy products are considered to cause human one of the eight kind of food allergies by FAO and WHO. Casein(CN) and alpha lactalbumin(α-LA) of bovine milk are regarded as the main allergens. Therefore, there is a great theoretical and realistic significance for the strong specificity, high sensitivity of detection of CN and α-LA in food products.In this paper, we mainly studied including preparation and appraisal of monoclonal antibody against CN and α-LA, preparation of gold magnetic composite nanoparticles and the enzyme-labeled antigen, and development of gold magnetic enzyme-linked immunoassay for detecting allergens CN and α-LA in bovine milk.The concrete research content is as follows:1. Preparation of monoclonal antibody against CN and α-LA4 BALB/c mice were immuned with the high pure CN and α-LA respectively, and indirect ELISA method was used to test the titer of antiserum of mice to obtain the highest antiserum titer of mice. Cell fusion was carried out by immunized mice spleen cells and Sp2/0 myeloma cell, so as to obtain hybridoma cells. The indirect ELISA method was used to screen the positive hole, and through 3 subcloning, Secreting homogeneous hybridoma cell lines were obtained by limited dilution method. Finally, one hybridoma cell line specific to CN was obtained and named as 3D3. Three hybridoma cell lines specific to α-LA were obtained and named as 11G10 and 11F8. A large number of monoclonal antibodies were prepared by the method of induced ascites in mice.2. Appraisal of monoclonal antibody against CN and α-LAMonoclonal antibody was purified using caprylic acid-ammonium sulfate precipitation joint. The purity of the obtained monoclonal antibody was characterized by SDS-PAGE electrophoretic, and the indirect competitive ELISA, Western blotting and non competitive ELISA were used to detect the antibody titer and specificity and affinity. The results showed the concentration of purified CN and α-LA m Ab were 8.425 mg/m L and 8.591 mg/m L respectively. The titer of anti-CN 3D3 monoclonal antibody was 1:128000, and the titer of anti- α-LA 11G10, 11F8 monoclonal antibody was respectively 1:256000 and 1:64000, which was to ensure that the monoclonal antibodies better activity and specificity. The results showed that the two monoclonal antibodies were type Ig G1. Anti-CN, α-LA m Ab affinity constants were 0.32 × 108 L/mol and 0.37 × 108 L/mol.3. Synthesis of gold magnetic composite and the enzyme-labeled antigenAmino-functionalized Fe3O4-PEI composites, which were synthesized by the one-pot solvothermal method. After two layers of Au particles were coated on the surface, of preparing gold magnetic( Fe3O4/Au) nanoparticles. Shapes of gold magnetic composite were round, uniform, whose particle size were about 170-185 nm. CN and α-LA were labeled with horseradish peroxidase( HRP) by the sodium periodate method. Also protein concentration of CN-HRP and α-LA-HRP was 1.991 mg/m L, 2.816 mg/m L respectively; the enzyme loss rate of CN-HRP and α-LA-HRP were 8.7 %, 7.63 %.4. Development of gold magnetic enzyme-linked immunoassay for detecting allergens CN and α-LA in bovine milk.After the gold immunomagnetic probe and CN/α-LA enzyme labeled antigen and HRP-CAP were modified as the optimum conditions described above, they were mixed with CN/α-LA standard solution to establish gold magnetic enzyme linked immunosorbent assay system. And the feasibility of the system in the detection of CN/α-LA in bovine milk was investigated preliminarily. The results showed that there was an excellent linear tendency between logarithm of CN concentration and the inhibition rate in the measured range of 4 ng/m L ~256 ng/m L, the standard curve was y=32.654x-5.17143, R2 = 0.993, IC50 was 50ng/m L and LOD was 2.9 ng/ m L. There was an excellent linear tendency between logarithm of α-LA concentration and the inhibition rate in the measured range of 2 ng/m L ~256 ng/m L, the standard curve was y=35.718x-2.8429, R2=0.99545, IC50 was 30.2 ng/m L and LOD was 2.3 ng/ m L. In addition, the detection system was evaluated, including precision, specificity and stability. The results showed that there was a good precision with intra-assay coefficient of variation of 3.7% ~ 6.9%, inter-assay coefficient of variation of 3.6% ~ 8.7% in detecting CN and with intra-assay coefficient of variation of 3.9% ~ 6.8%, inter-assay coefficient of variation of 5.5% ~ 9.8% in detecting α-LA. The cross-reactivity rates of the system with other major proteins in milk was less than 2 %, which had a good specificity. The detection system was finally applied in the detection of CN/ α-LA in milk samples. The result, with the recovery rate of CN 82% ~105% and the recovery rate of α-LA 87.1% ~ 111%, meets the requirements of detecting CN/ α-LA. This method can improve the sensitivity and specificity, and has an important practical application value in food safety detection.