| Human health is closely related with the food safety. It is important to detect residues of pesticide and veterinary drugs in the field of food safety. In this study, monoclonal antibodies (McAb) of atrazine and ampicillin were prepared and were identified. These work laid the foundation for the further testing. Aim for preparing high specificity monoclonal antibodies of atrazine and ampicillin, our study mainly includes transformation and identification of hapten, synthesis and identification of complete antigen, the animals immunization, screening of the hybridoma cell strain, preparation of monoclonal antibody and purification and characterization of monoclonal antibody, and so on.Atrazine was carboxylated and its Cl atom was replaced by 3 - mercaptopropionic acid. After identified by infrared spectroscopy (IR), nuclear magnetic resonance(NMR), and mass spectrum (MS), the carboxylated atrazine was then coupled to BSA and OVA by DCC to prepare the complete antigen, which was identified by ultraviolet spectrum(UV) and SDS-PAGE. The efficiency of the coupling reaction of was 20:1. The ampicillin was also coupled to BSA and OVA by Biosynthesis method and the complete antigen was identified by ultraviolet spectrum(UV) and SDS-PAGE. The efficiency of the coupling reaction of was 12.1:1. 8-week-old Balb / C mice were selected as laboratory animals. A small dose of medium-range immunity were carried out with the mixture of complete antigen with equal volume Freund adjuvant(FA) and immunization interval was about two weeks. At the first immunization, every mouse was injected with 40μg of AT-BSA or 50μg of Amp-OVA intraperitoneally; at the second immunization the dose was 50μg of AT-BSA or 60μg of Amp-OVA; and at the third immunization was 70μg of AT-BSA or 70μg of Amp-OVA. On the 20th day after the third immunization, the animals were injected with 70μg of AT-BSA or 80μg of Amp-OVA. 20 days later, the mouse used for cell fusion was injected with 160μg of complete antigen without adjuvant. Respectively, after the second and the forth immunization, the antiserum was collected by cutting tails and the titers were determined by indirect ELISA. The specificity of antiserum for the target antigen was validated by competitive ELISA. To prepare fusion cells, Splenocytes from immunized mouse was fused with mouse myelomas cells SP2/0 by 50% PEG4000, followed by HAT medium culture. Hybridoma clones were screened by indirect ELISA and antibody-positive cell lines were subcloned twice by limited dilution. Four monoclonal antibodies cell strains against atrazine were obtained and named C7B1A12,C7B1C9,C7B1F3,C12A4D10 and three cell lines against ampicillin were named H3D1H12,H3D1B2,H3D1C9. After determinated reproducibility (batch to batch) and stability by indirect ELISA and analyzed chromosomes by Giemsa staining, the cell lines were injected into mice intraperitoneally used paraffin oil as inductor to prepare the monoclonal antibody ascites. The monoclonal antibody was evaluated as follow: 1) the titer of the monoclonal antibody; 2) the specificity of the monoclonal antibody; 3) the affinity of the monoclonal antibody; 4) the immunoglobulin subclass; 5) the establishment of regression equation.Characterized the McAb against atrazine as follow: the titer of the antibody was 1:240000 or more. The cross-reaction rates between the four McAbs and durshan,monocrotophos,parathion were all lower than 0.01%. The rate between C7B1A12 and melamine was 0.043%, that of other antibodies were not higher than 0.01%. The rates of the four antibodies with simazine were 21.29%,80.33%,17.23%,84.77%, respectively. the antibody affinity constants were 2.76×1011,2.97×1011,3.04×1011,2.13×1011 L?mol. By Sigma monoclonal antibody immunoglobulin subclass identification kits, antibodies immunoglobulin subclasses were IgG1,IgG1,IgG1,IgG2b, respectively. Thereby, the antibodies secreted by C7B1A12,C7B1C9,C7B1F3 were better than that of C12A4D10 , and C7B1C9 better than that of C7B1A12,C7B1F3. The establishment of regression equation: after validated the most suitable coating amount of complete antigen and the dilute strength of antibody by the chessboard titration, the standard curve of the four antibodies were obtained by direct competitive ELISA. The regression equations were: I=37.28logC+120.39, R2=0.9847; I=36.26logC+120.77, R2=0.9856; I=36.07logC +119.13, R2=0.9857; I=33.29logC+124.9, R2=0.9926. The detection range were 0.9～0.001μg/mL; 0.9～0.001μg/mL; 0.9～0.001μg/mL; 0.9～0.004μg/mL, respectively, and the lowest detection limit were IC20=0.0020μg/mL;IC20=0.0017μg/mL;IC20=0.0018μg/mL;IC20=0.007μg/mL. The sensitivity were IC50=0.013μg/mL;IC50=0.011μg/mL;IC50=0.012μg/mL;IC50=0.056μg/mL. All of the lowest detection limit were on the ng level and the specificity of the antibodies secreted by C7B1A12,C7B1C9,C7B1F3 were higher than that secreted by C12A4D10.Characterized the McAb against ampicillin as follow: the titer of the antibody was 1:64000 or more. The Cross-Reactivity rates between the three McAbs and penicillin, phytomycin,kanamycin,sulfadiazine were all lower than 0.01%. The antibody affinity constants were 12.37×109,8.96×109,9.03×109 L?mol. By Sigma monoclonal antibody immunoglobulin subclass identification kits, antibodies immunoglobulin subclasses were IgG1, respectively. thereby, the antibodies secreted by H3D1C9 were better than that of H3D1B2,H3D1H1. The establishment of regression equation: after validated the most suitable coating amount of complete antigen and the dilute strength of antibody by the chessboard titration, the standard curve of the three antibodies were obtained by direct competitive ELISA. The regression equations were: I=35.163logC+116.05, R2=0.9829; I=36.065logC+121.12, R2=0.9768; I=33.865logC+113.67, R2=0.9941. The detection range were 0.094～0.0018μg/mL; 0.072～0.0015μg/m; 0.101～0.0017μg/mL, respectively, and the lowest detection limit were IC20=0.00185μg/mL; IC20=0.0015μg/mL; IC20=0.0017μg/mL, the sensitivity were IC50=0.013μg/mL; IC50=0.0106μg/mL; IC50=0.013μg/mL. All of the lowest detection limit were on the ng level and the specificity of the antibodies secreted by H3D1C9 were higher than that secreted by H3D1H1,H3D1B2.In this study, we prepared McAb against atrazine or ampicillin with high specificity,sensibility and affinity. The purified monoclonal antibodies could be applied to the rapid detection of atrazine or ampicillin residues. For example, the antibody can be used in a rapid and sensitive ELISA, or combination with the preparation of colloidal gold technique which can obtained test strip. In a word, this study laid the foundation for the monitoring of pesticide and veterinary drug residue. We believe the antibodies, such as that described above, could play an important role in targeting official surveillance activities as well as providing a tool for the agriculture and food industries. |
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