Study Of Chlortetracyline Immunological Rapid Detection Method

Chlortetracyline are extracted from the streptomyces mediua alkaline broad-spectrum antibiotics. It inhibits Gram-positive bacteria （G+） and Gram negativebacteria （G-）, would prevention Animal diseases and growth.But because it abuses, thebacteria resistance increasely. So, Established a rapid, sensitive Chlortetracylineresidues detection methods, it is of great significance for monitor system and humansecurity.Based on the analysis of molecular structure and physical and chemical propertiesof Chlortetracyline, successffully, gains artificial antigens which obtains its micepolyclonal antiserum by immunizing with artificial antigens; obtais monoclonalantibody by hybridoma technology, We gets cell lines which high titer and specificityof the mAb anti Chlortetracyline by competitive and indirect ELISA, adapts for anexpanded system of ascites by mice, then it was identifide for immunologicalcharacterization, development of rapid determination of Chlortetracyline residuemethod.The main contents and results were as follows:1. Synthesis and identification of the artificial antigen for ChlortetracylineChlortetracyline was conjugated to carrier protein BSA and OVA by the methodof Glutaraldehyde and EDC to form the artificial antigens BSA-CTC and OVA-CTC.The artificial antigens were identified by ultra violet scanning spectrum and SDS-PAGE. The titre and specificity of polyclonal antibody（pAb） was detected bycompetitive and indirect ELISA. The results showed that the hapten Chlortetracylinewas successffully linked to carrier proteins. Five BALB/c mice indirect ELISA titeragainst CTC were above1×10^-3 and the IC₅₀ of No.3mice was71.47ng/mL. The rateof cross reaction of Chlortetracyline monoclonal antibody with tetracycline was1.662%and with oxytetracycline was0.714%, there were less0.08%cross-reactivityto other compounds. The high titer and specific anti CTCpAb has been produced. 2. Development of monoclonal antibody against Chlortetracyline andestablishment of immunology ELISANo.3mouse which high titer and specificity was selected for cell fusing. Thethree hybridoma lines of6H5-H4,6H5-B2,3G9that fused with NS0myeloma cellsand lymphocytes by using monoclonal antibody hybridoma technology. Adapts for anexpanded system of ascites by mice were used for Chlortetracyline monoclonalantibody. The isotypes of the Chlortetracyline monoclonal antibody was IgG1. Theindirect ELISA titer of the mAb were1:1.2×10²,1:1.0×10²,1:0.8×10²in supernatant;1∶2.5×10⁴in ascites, the affinity constant （Ka） was4.05×10¹⁰L/moL, the mAb of6H5-H4showed good sensitivity with an IC50of29.86ng/mL to CTC1.48%cross-reactivity to Tetracycline and little or no cress-reactivity to other compounds. The hightiter and specific anti CTCmAb has been produced, which used for establishimmunoassay of Chlortetracyline residues in food.3. Development of rapid determination of Chlortetracyline residue methodBased on the enzyme linked immunosorbent assay method, the CTC-Kit wassuccessffully developed. The calibration curve of CTC-Kit was typical sigmoid curvefitted with logistic equation, the detection limit of Chlortetracyline IC50was29.86ng/mL. The recoveries from milk and pig pork were83.69%and82.93%whenChlortetracyline were spiked. The validity of CTC-Kit of hightiter and specificity,which useful for the quantitative or qualitative detection of Chlortetracyline residues.