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Tetracyclines (tetracyclines, TCs) broad spectrum antimicrobial efficacy, has been widely used in the treatment of various infections. But in recent years, its residual hazards in animal tissues has attracted people's attention, while detection methods are constantly improved, domestic also did not see the use of tetracycline monoclonal antibody to the establishment of the official reports of the tetracycline residue detection method. Therefore, this study was prepared in a lot of information on the basis of tetracycline hydrochloride monoclonal antibody immunological detection of tetracycline residues, which laid the foundation for the development and application of the kit. The test results are as follows: 1. Diazonium synthesis of TC-BSA, TC-OVA, artificial antigens were identified by UV scanning. The UV scanning Spectrum shows TC-BSA and TC-OVA has a maximum absorption peak at a wavelength of 276.6nm and 278nm, respectively, in addition to the TC characteristic peaks, further described hapten TC respectively with the carrier BSA and OVA crosslinked. 2. TC-BSA as immune immunize 8-week-old BALB / C strain mice. Three free mouse tail blood serum was separated, TC-OVA coating antigen screening serum high titer and specificity by indirect ELISA and indirect competitive ELISA positive rats. Take positive mouse spleen cells with SP2 / 0 cells were fused with 50% PEG-1500. Testing, cloning, screening and other steps, the final filter out the two cell lines produced anti-TC mAb mAb subtypes identified, and cell culture supernatant, indicating that the two cells producing monoclonal antibody of the IgG2b type antibodies . A hybridoma cell mice by intraperitoneal injection to produce ascites, and the ascites was purified by caprylic acid - sulfuric acid amine method, the protein content of purified ascites 1.326mg/mL then identified by SDS-PAGE purity, the results of electrophoresis for two clear bands, a heavy chain, a light chain, the purity is higher. 3. Application of purified monoclonal initially established indirect competitive ELISA, the antigen best package determined by titration concentration (5μg/mL), the concentration of monoclonal antibody purified 1:8000, and its whole reaction system The reaction conditions are optimized in order to establish a TC residual ELISA method; draw the TC pairs E 10 cell lines produced by the monoclonal antibody of the inhibition curve of the standard curve y =-20.439x 81.03 (R 2 = 0.9498), the limit of detection (LOD) 0.68ng/mL the the the IC 50 for 31.62ng/mL; TC monoclonal antibody with ciprofloxacin ofloxacin, gentamicin, penicillin potassium sulfadiazine, streptomycin of six had no cross-reactivity. The results show that the test established TC residue detection ELISA method with high sensitivity and specificity. 4. Series of concentrations of drugs of the tetracycline hydrochloride is added to the milk, and then for the standard curve, and the establishment of the regression equation; were taken again high, medium and low the three tetracycline hydrochloride drug concentration recovery test the same sample with the standard curve approach Add recoveries of tetracycline hydrochloride in milk between 61.3% -76.5%. The results show that: the application TC monoclonal antibody establish indirect competitive ELISA method can be used to detect tetracycline residues in milk.
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