Preliminary Preparation And Application Study Of Anti-TB Monoclonal Antibody Target To CT Contrast

Part OneProkaryotic Expression and Purification of Tuberculosis Specific Proteins and Preparation of Anti-TB Specific Monoclonal AntibodiesObjective The purpose of this study was to prokaryotic expression recombinant plasmid with the construction genes. To expres and purify of secreted protein Ag85B and ESAT-6of Mycobacterium tuberculosis. To prepare and purify anti-TB specific murine monoclonal antibodies with the isolated hybridoma cells, which against the85B and ESAT-6antigen of Mycobacterium tuberculosis.Materials and Methods1. The bacterium was amplified and cultured with expression of containing the recombinant plasmid, collection and lysis bacterium. Extract the recombinant plasmid with DNA polymerase chain reaction (PCR) and endonuclease appraisal. To induced the expression of Ag85B and ESAT-6protein and analyzed by SDS-PAGE. Protein was Purified to obtain antigen and quantitative to reserve.2. The strains of hybridoma cell (2H4and1B103) were prepared by routine procedure with hybridoma technology, against the85B and ESAT-6antigen of Mycobacterium tuberculosis, and identified with which immune blotting, confirmed by Western blot analysis. The anti-85B and ESAT-6specific monoclonal antibodies (MAb) were prepared by hybridoma cell, and purified the ascite fluids with silicon dioxide adsorption.3. Identification the binding activity between the protein Ag85B and ESAT-6of Mycobacterium tuberculosis by Enzyme-Linked Immuno Sorbent Assay (ELISA), which was induced to expression of recombinant plasmid, and the monoclonal antibodies against the85B and ESAT-6antigen of Mycobacterium tuberculosis, which was obtained from the hybridoma technique.Results 1. The results of PCR amplification and recombinant plasmid (pET-30a-85B and pET-30a-ESAT6) restriction enzyme digestion confirmed that the Ag85B and ESAT-6protein was successfully expressed.The inclusion body was induced at37degree, and the soluble expression protein was obtained by reducing the induction temperature. The concentration of antigen85B and ESAT-6protein were8.31mg/mL and2.5mg/mL separately after purification. The results of SDS-PAGE showed that the purity was more than95%, and it could be used for the determination of the antibody titer.2. Hybridoma cell lines producing ascite fluids monoclonal antibodies were given2mg, and the results of SDS-PAGE showed that the purity was more than95%, which was met the desired amount of follow-up mark the contrast agent.3. The anti-85B and ESAT-6monoclonal antibodies could bind specifically with85B and ESAT-6protein of Mycobacterium tuberculosis and appearance strong bands of immune reaction, as confirmed by ELISA. The specific antibodies titer in the sera was more than1:625000, and the purity were high.Conclusion1. Secreted protein Ag85B and ESAT-6of Mycobacterium tuberculosis was successfully expressed and purified with a good immunogenicity as antigen for further evaluation of monoclonal antibodies titer.2. The specific anti-85B and ESAT-6murine monoclonal antibodies were prepared, which have been built and lay a foundation for preliminary studying the targeted contrast agents. Part TwoThe Preparing of Anti-TB Monoclonal Antibody Targeted Contrast Agent of Computed TomographyObjective To prepare the anti-85B and ESAT-6murine monoclonal antibody targeted contrast agent of computed tomography, conjugating iodine atoms and to explore the feasibility of preparation and the biological activity in vitro. Materials and Methods1. The study of the amplification effect of the biotin-avidin system (BAS)Label the bovine serum albumin instead of antibodies by Iodogen method, as well as indirectly radio iodine label by biotin-avidin system. To initially establish the measurement method of the label efficiency of the iodine, comparing the label efficiency of iodine between the Iodogen method and the biotin-avidin system indirect label method, to provide the standard for evaluation, to optimize the conditions for preparing the targeted contrast agent of computed tomography.2. The preparation of the*I-anti-murine monoclonal antibody(1) Label the anti-85B and ESAT-6murine monoclonal antibodies with radioactive iodine Na125I tracing non-radioactive iodine Na17I by chlorine glycoluril (brand name Iodogen) label method, and measurement the iodine label efficiency rate by silica gel chromatography method, calculation the iodine binding capacity, purification and determination the radiochemical purity, and investigate the stability.(2) For example as*I-anti-85B antibody, to observe the influence of label rate by different Na127I feeding amount, different Iodogen feeding amount and different Iodogen reaction tube, to improve label efficiency through optimize the preparation conditions of iodine labeled.(3) To explore the binding capacity of iodine labeled anti-murine monoclonal antibodies with mycobacterium tuberculosis antigen by indirect ELISA, and the positive was optical density value greater than or equal to2.1times compared to the negative control.3. The preparation of anti-murine monoclonal antibodies targeted contrast agent of computed tomographyLabel the anti-85B and ESAT-6murine monoclonal antibodies with the non radioactive iodine (Na127I) of the Iodogen method. Using sample tags, purification, and calculate the quality of antibody and iodine content in the contrast agent solutionResults1. The amplification effect of the BASThe experiments showed that direct label bovine serum albumin, the molar ratio (mol/mol) of iodine and protein was up to6.372, while the molar ratio of indirect label method was only0.617.2. The preparation of the*I-anti-murine monoclonal antibody (1) The iodine label rate of anti-85B monoclonal antibody was (86.25±6.34)%, the radiochemical purity was more than98%, the protein recovery rate was up to (82.95±4.17)%after purification. The radiochemical purity was more than93%in vitro when storing a week at4degree.(2) The iodine label rate of anti-ESAT-6monoclonal antibody was (83.07±2.05)%, the radiochemical purity was more than98%, the protein recovery rate was up to (36.75±12.80) after purification. The radiochemical purity was more than95%in vitro after1day and7days.(3)*I-anti-85B antibodies as an example. The label rate were89%,95%,70%when the Na127I (0.4μg/μL) feeding amount were5μL,10μL,20μL respectively, and the127I of anti-85B antibody was64.3μg; The label rate were70%,95%,93%when the amount of Iodogen were200μg,100μ,50μg. The label rate were95%,86%,80%,84%of the different Iodogen reaction tube, which prepared in2010-2-25,2010-3-23,2010-3-29,2010-3-31respectively.(4) The results of indirect ELISA show that the anti-85B and ESAT-6antibody of non-radioactive iodine (Na127I) labeled remained a good binding capacity.3. The preparation of anti-murine monoclonal antibodies targeted contrast agent of computed tomography(1) The volume of127I-anti-mouse monoclonal antibody85B after purified was2.52mL, the quality of anti-85B antibody and127I were range from210to255μg and range from10.5to16.6μg respectively in the collection fluid of antibody.(2) The volume of127I-anti-mouse monoclonal antibody ESAT-6after purified was2.93mL, the quality of anti-ESAT-6antibody and127I were147μg and20.58μg in the collection fluid antibody respectivelyConclusion1. As an example of*I-anti-85B antibodies, the ideal label conditions were the Iodogen100μg, Na125I150μCi, Na127I4μg, and the quality of antibody was50μg, the reaction time was five minutes indoor temperature. The label rate was more than85%, the radiochemical was more than98%, and stability well.2. Preliminary prepared the127I-anti-85B and ESAT-6murine monoclonal antibody targeted contrast agent. The stability and biologic activity of the contrast agent in vitro were well. Part ThreePreliminary Study of Anti-TB Specific Monoclonal Antibody Targeted Contrast Agent of Computed TomographyOneEstablishment and Comprehensive Evaluation of Acute Tuberculosis Animal ModelObjective The study of the research is to establish the acute murine tuberculosis model and make comprehensive evaluation of it, selection and application of animal model in the advanced research of tuberculosis.Materials and Methods1. The C57BL/6J mice were infected with standard strains of mycobacterium tuberculosis H37Rv by intravenous injection and intranasal administration, which were randomly divided into the high dose moderate dose and low dose group of intravenous, as well as intranasal infection group, there were twenty mice in each group, and five ones in each control group of intravenous and intranasal. The two kinds of tuberculosis mouse models developed by different infection routes were compared by observing the lesion of the tissues and distribution of bacteria.2. At the4th,6th and8th weeks after infection, the infected mice were scanned by Micro-CT respectively and their lungs were sterile separated after the scanning. The histopathologic changes and the bacterium fixed in the murine lung tissue were investigated by observation, Gross findings, histological staining (HE, PAS, and acid-fast) and colony counting. CD68Immune histochemistry analysis was also performed.3. Evaluation the murine chest Micro-CT scanning images from the pulmonary manifestations of the animal model, the indexes such as artifact from CT apparatus, the sharpness and contrast of the thoracic lesions, and also make an overall subjective sensory score.Results1. The deterioration of lung infection in the murine models was indicated by visual observation and Micro-CT scanning and the histopathology results. The murine infection gradually increased with the infection time, and the pulmonary lesions showed obvious and diffused throughout the whole lungs after infected six and eight weeks. The scan-positive rate and Micro-CT score of the lesion display were both high in the intranasal infection group.2. Meanwhile, the HE staining results of the lung lesion showed that each of the infection group with varying degrees of inflammatory changes. The organ pathological changes were serious with the amount of inoculants increase. The lesions of the intranasal group were more obvious than the intravenous infecton. And the acid-fast staining showed that red dyed short mycobacterium.3. Both of the two different infection routes can cause pathological changes of the lung tissues of the mice, Standard strains of mycobacterium tuberculosis H37Rv was diluted to1×106cfu is the best concentration. Colony counts show no significant difference between the infected group (P>0.05), the lung pathological changes were more significant in the intranasal infection group than the intravenous with the same amount of inoculation bacterium.Conclusion1. The acute tuberculosis mouse models were successfully established in the intravenous and intranasal infection methods. Intranasal administration of bacteria is the most efficient method to develop mouse tuberculosis model, because it was simple and convenient, and tolerate the experimental operation, with a high successful rate of the model. Although the model could not absolutely represent the same situation of tuberculosis in human but the comprehensive evaluation of this research proved that it is an ideal animal model for radiology research.2. The Micro-CT scan sensitivity of the animal model acutely infected tuberculosis and the correlation with histopathology findings were high. Met the basic requirements of the chest imaging of mice, can be used as one of the evaluations of iive animal model. TwoPreliminary Observation of Targeted Contrast Agent of Computed Tomography by the Murine Acute Tuberculosis Animal Model Objective To explore the feasibility of anti-85B and ESAT-6monoclonal antibodies targeted contrast agent of computed tomography by the murine acute tuberculosis animal model.Materials and Methods1. Preparation the targeted contrast agent of computed tomography by iodine atoms coupled with anti-85B and ESAT-6murine monoclonal antibodies after purified. Calculate the label rate and the quality of127I of the targeted contrast agent solution, and dilute the contrast agent solution to the required concentration (5μgI/ml) to spare.2. There were twenty mice of acute tuberculosis animal model, which were divided into three groups of targeted contrast agent, common contrast agent and blank control, the targeted contrast agent group was divided into85B group and ESAT-6group according to the different antibody, and each group was five animals. The common contrast agent group was injected with diluents of iohexol, which was diluted into the same concentration with the targeted contrast agent. The control group was injected with antibody diluents PBS. All the animals were scanned before and after injection the contrast agent in different time, such as immediate, six hours, twelve hours and twenty-four hours. Observe the display and changes of the murine tuberculosis lesions, and measurement the computed tomography value.Results1. The volume of anti-85B contrast agent solution was2.52mL, and the quality of antibody and127I were range from210ug to255ug and10.5μg to16.6μg respectively. The volume of anti-ESAT-6contrast agent solution was2.93mL, and the quality of antibody and I27I were147ug and20.58μg respectively.2. The lesions of the control group showed no visible density changes before and after injection of PBS. The CT value of the lung lesions in the targeted contrast agent group were gradually increased with time, and the lesion showed visible enhancement after the contrast injection twelve hours, and also remained visible enhancement after injection twenty-four hours, which is significant difference compared to the common contrast agent (P<0.05).Conclusion1. The targeted property of anti-85B and ESAT-6murine monoclonal antibody contrast agent of computed tomography had been partly proved by the acute tuberculosis animal model. 2. The injection dosage of the contrast agent and the scan time of Micro-CT provide an experimental basis for further study of tuberculosis targeted contrast agent.The anti-85B mice monoclonal antibody targeted CT contrast agent has targeted properties in these pulmonary tuberculosis mice models, which make the early specific diagnosis of tuberculosis become possible.