Preparation Of Clenbuterol Monoclonal Antibody With Subtractive Immunization And Identification Of Its Characterization

Clenbuterol (CL) is a kind ofβ2-adrenoceptor agonist,commonly used as bronchodilators for treatment of bronchial spasms caused by asthma, emphysema and other respiratory diseases. It can enhance the animal's muscle growth and reduce the fat deposition when 5~10 times beyond it's treatment dose caplendused. Because of its unique biological properties, often illegally used as feet additives resulting in considerable residues in livestock products,People will be poisoned accompanies with such symptom as dizziness, cardiopalmus and finger thrillingness etc. after taking the meat products with CL residues, In recent years, Poisoning incidents have occurred frequently after taking the meat products with CL residues. So it is necessary to establish simple, rapid, sensitive detection method based on immunology to tacho-detect for CL residue in meat, in order to prevent food-poisoning efficiently.To establish the detection method based on immunology, the specific antibody must be prepared at first. CL is a micromolecule organics without immunogenicity. So it is unable to be used of immunization of animal experiment directly. To stimulate zoic immunologic mechanism producing specific antibody, CL must be coupled with macromolecule carrier protein. The antigen of interest which is used to immune animal has to be absolutely pure in order to obtain high specific antibody which direct at the antigen. It is fairly difficult to prepare the high purity conjugate of CL and the macromolecule protein . To eliminate or weaken the immunogenicity of other antigen out of interest, decrease the production of the irrespective antibody utility, and increase the probability of produce more desired antibody which stimulated by low abundance antigen of interest, subtractive immunization was used in this experiment. Thus subtractive immunization could eliminate or weaken the humoral immunization reaction to the antigen out of interest by altering the immunologic mechanism of the experimental animal. And then, the immunologic reaction system could direct to the antigen determinant on the antigen of interest as well as the high specific CL antibody was obtained.1 Synthesis the complete antigen and immune BALB/c mice with subtractive immunizationImmunogen CL-BSA was prepared by diazotization as well as characterized by ultra-violet analysis detection method in the experiments. And the result of binding ratio is 1:15.8, which could stimulate animal organism to producing more antibody. We can regard BSA and CL-BSA as tolerance and target antigen separately ,which exist in the original immunogen.After the immune tolerance procedure, ELISA experiment with the experimental animal's tail blood confirmed that the experimental mice had obtained immune tolerance to BSA. And then, the immunization after immune tolerance was done. After the third injection of the immunogen, ELISA experiment was repeated with the experimental animal's tail blood, and high level antibody to CL-BSA was obtained. The antibody titer of each was 106. Among the five experimental mice which were immuned with subtractive immunization, mouse No.2 and mouse No.5 were much more successful subtractive immuned. Took the spleen of mouse No.2, and subsequently prepared the unicell suspension to carry out the next step—cell fusion. 2 Cell fusion and hybridoma cell line establishedPEG4000 was used as the fusogenic agent, took SP2/0 myeloma cell and the spleen cell immuned confluenced. Add the confluent cells to the well of 96-well containing plates which were added the breeding cells-mice'abdominal cavity exfiltration cells in advance. Selective culture the cells after cell fusion with HAT and HT cell culture fluid. 12-14d after cell fusion ,screening the desired specificity and affinity hybridoma cells ELISA experiment which had been established in advance. The confluence rate was 97%. The rate of the hybridoma cell with positive reaction which had obtained was 8.2%. Freezed the hybridoma cells of positive reaction in time. A method of limiting dilution assay was used for cloning the hybridoma cells with positive reaction. Screeninged the positive reaction hybridoma cell with ELISA experiment after cloning every time. After four times clone, five hybridoma cell lines which stable product CL monoclonal antibody had been screeninged out with ELISA experiment, which named: 1A2,1B1,1B4,2B12 and 2C3. Chromosome analysis was done to the hybridoma cells. The chromosome analysis was done abiding the following steps: fixed the hybridoma cells at exponential phase of growth by fixation fluid, dyed with Giemsa, and then observed what was showed under micro. Chromosome, the result was that the chromosome number was between 99 and 107, which was roughly corresponding to the total of the chromosome number of mice' spleen cells and SP2/0 myeloma cells.3 Produce CL monoclonal antibody and identify its characterizationAscites monoclonal antibody and supernatant of hybridoma cell monoclonal antibody produced by hybridoma cell line 1A2 with the maximum antibody activity. The antibody titer of the ascites and the hybridoma cell culture fluid supernatant were determined by ELISA experiment which had been established in this paper, and the data were 106 and104, respectively. The mice'IgG standard curve was obtained by Sandwich ELISA method. Worked out the utility antibody concentration of supernatant monoclonal antibody and ascites monoclonal antibody could be calculated based on the standard curve, and they were 32.80ug/mL and 4.48mg/mL, respectively. The result of the indirect competition ELISA confirmed that the monoclonal antibody had no cross-reaction with BSA and a few kind of CL analogue. Western-blot experiment showed that the monoclonal antibody could react with CL-BSA, but it could not react with BSA, which further proved that the monoclonal antibody was specific to CL. The ELISA experiment showed that the monoclonal antibody affinity constants is 2.90×1010 L/mol, which proved the antibody had high affinity to CL. At first the ascites was salting-out with different concentration of (NH4)2SO4. After that, the utility antibody was found in the sediment which was salted-out by 50% (NH4)2SO4 , which was corroborated by SDS-PAGE electrophoresis and ELISA experiment. The sediment which was salting-out by 50% (NH4)2SO4 was further purification by affinity column, and highly purified monoclonal antibody to CL was obtained, which was corroborated by SDS-PAGE electrophoresis and the indirect competition ELISA, either. The molecular corer.-weight of the monoclonal antibody was 83kd, which showed by the SDS-PAGE protein electrophoresis film. The ascites antibody titer after purification was 105. the detection standard curve of the monoclonal antibody was established with indirect competition ELISA,The IC50 was 14.554ng/mL and the lowest detectable limit was 1.0ng/mL.CL monoclonal antibody was Prepared using subtractive immunization, which weakened the immunogenicity of the interferent BSA in the immunogen, increased the probability of obtaining the CL monoclonal antibody secreting type hybridoma cell, and then, lightened the workload of screening the monoclonal antibody. High specific CL monoclonal antibody was obtained at last. This had established the foundation to the advanced development of the ELISA kit.