Preliminary Study Of Using Anti-ESAT-6Monoclonal Antibody Targeted Probe For The Imaging Diagnosis Of Tuberculosis

Part OneThe preparing of targeted probe of computed tomography and fluorescence and testing of their immunocompetenceObjectiveThe purpose of this study was to prepare the anti-ESAT-6mouse monoclonal antibody (mAb), to develop the monoclonal antibody targeted probes of computed tomography and fluorescence by being labeled with a certain number of iodine atoms or fluorescein and evaluate their immunocompetence.Methods1. The mice were immunized with the purified ESAT-6antigen expressed in E. Coli. Four stably secreting anti-ESAT-6specific mAbs cell strains were obtained with the hybridoma cell fusion technique. The four strains of above cell strains were identified and used to prepare the ascitic fluids. The purity and titer of antibody was detected.2. The anti-ESAT-6mouse monoclonal antibody targeted probe of computed tomography was prepared through Iodogen method directly radioactive iodine mark, radioactive iodine Na125I tracer the non-radioactive iodine Na127I, and calculation of the purification and determination of radiochemical purity.3. IR783and Rho were labeled on the antibody by a N-hydroxysuccinimide ester (NHS). The purity of mAb-IR783-Rho was determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).4. The immunocompetence of the targeted probes of computed tomography and fluorescence was tested by indirect ELISA method.Results1. The ELISA titers in the ascite fluids of the four mAbs reached1:128000. The ascite mAbs were purified up to90%purity. IgG isotypes showed that all the mAbs secreted by the four stains belong to IgGl/k subclass. ELISA method proved that the mAbs reacted specifically with ESAT-6antigen of Mycobacterium tuberculosis, but did not react specifically with his-85b.2. Adopted the Iodogen method for the iodine mark, the radiochemical purity of the probe was higher than98%, and the probes kept in vitro at4℃conditions for a week later, the radiochemical purity>95%, proving good stability markers.3. The ELISA method proved that the targeted probes of computed tomography and fluorescence reacted specifically with the ESAT-6antigen of Mycobacterium tuberculosis and had high immunocompetence.Conclusion1. The mAbs with high purity and high titer to ESAT-6protein of Mycobacterium tuberculosis were obtained, which may react specifically with ESAT-6antigen.2. The anti-ESAT-6mouse monoclonal antibody directly iodine labled by the Iodogen method has been successfully prepared, which has good stability in vitro. The targeted probe labeled with fluorescein has high purity. The two targeted probes reacted specifically with ESAT-6antigen and proved good immunocompetence. These results provide the basis for the further experimental imaging in animals. Part TwoEstablishment of murine tuberculosis model and expression of ESAT-6antigen in tuberculous tissues of murine lungsObjectiveTo explore the feasibility of establishment of the murine tuberculosis model for mycobacterium tuberculosis infection in intranasal. To analyze the expression and significance of ESAT-6antigen in the tuberculous tissues of murine lungs.Methods1. Thirty C57BL/6J mice divided into the infection group (n=24) and the control group (n=6). The mice of the infection group were infected with standard strain of tubercle bacillus H37Rv by intranasal administration while the mice of the control group were given the same dose saline.2.4,8and12weeks after infection, the infected mice were scanned by Micro-CT and their lungs were separated. The pathologic changes were investigated by observation of histological staining (H&E and acid-fast staining). The tissue homogenate of murine lungs were cultured to calculate the colony counting of Mycobacterium tuberculosis.3. Immunehistochemistry and immunofluoresence staining were performed to observe the expression of ESAT-6antigen in the lung tuberculosis lesions.Results1.4,8and12weeks after infection, the bacteria colony count from lung tissue was about106CFU, which had a slight upward trend with time. The positive rate of pulmonary lesions of the infected mice on Micro-CT was respectively50.0%,83.3%,100%, the volume of pulmonary lesions of the infected mice increased with the time extension (P<0.05). The positive rate of murine lungs by pathological examination was respectively83.3%,100%and100%at three monitoring time points. The positive rate of Micro-CT scans and pathological examination of the mice at8and12weeks after infection was similar, the pathological manifestations of lesions appeared major the fertile pathological changes with consolidation and granuloma, but rare caseous necrosis.4,8and12weeks after infection, scattered or a large number of mycobacterium tuberculosis were found in the lung lesions of mice by acid-fast staining.2.4,8and12weeks after infection, the sheet brown material deposition were observed in the exudation and consolidation lesions of murine lungs and the strip, the granular brown positive material deposition at the mycobacterium tuberculosis and the macrophage distribution areas by immunohistochemistry. The fluorescence signal increased obviously at the consolidation and granuloma areas was confirmed by immunofluorescence staining.Conclusion1. The tuberculosis mice model was successfully established according to the comprehensive analysis by the Micro-CT dynamic observation and pathology results.2.8weeks after infection is an ideal time point to establish the tuberculosis mouse model for the experimental imaging. 3. The expression of ESAT-6antigen in lung tuberculous tissues of mice is confirmed by immunehistochemistry analysis and immunofluoresence staining, which lay a foundation for the further imaging tuberculosis by targeted probes. Part ThereImaging of experimental pulmonary tuberculosis in mice by Micro-CT and near-infrared fluorescenceObjectiveTo explore the feasibility of detecting pulmonary tuberculosis in mice by Micro-CT and near-infrared fluorescence using anti-ESAT-6targeted probes.Methods1. Twenty-four C57BL/6J mice were divided into the CT group and the fluorescence group. Each group had12mice and divided into the experimental group (n=9) and the control group (n=3). All the mice infected with standard strain of tubercle bacillus H37Rv by intranasal administration and were used for imaging after8weeks. The mice of the experimental group were administrated via tail vein of the probe while the control group were given non-specific IgG antibody labeled with*I and fluorescein.2. Micro-CT scanning were performed in6mice before injection of targeted probe and6hours,24hours,48hours after injection of targeted probe, then the CT value of the ROI were measured at each time point and the mean data were calculated. The mice of control group were observed at the same time.24hours after injection of probe, the radioactive iodine distribution of pulmonary lesions and surrounding normal tissues were detected by the activity detection instrument.3. Ex vivo NIRF was performed at24hours after tail vein iniection of the probe, then the signal intensities (SI) of lung lesions, normal lung tissues and background noise were measured. The contrast to noise ratios (CNR) were calculated as CNR=(SIlesions-SIlung)/SInosie. The frozen section of lung tissues was observed by fluorescence microscope and compared with the results of HE staining. The mice of control group were observed by the same method. Results1. In the CT experimental group, the pulmonary lesions gradually enhanced after probe injection, and the highest CT value of the ROI was occurred24hours after injection.In the control group, the pulmonary lesions didn’t enhance. The radioactive iodine distribution of pulmonary lesions of mice in the experimental group was higher than the surrounding normal tissues (P<0.05).2. In the fluorescence experimental group, the significant fluorochrome accumulated in the pulmonary lesions, and the fluorescence signal was much higher than the control group24hours after injection of targeted probe. The CNR of the pulmonary lesions of the experimental group was60.33±3.44and much higher than the control group10.49±2.03(P<0.05). The tuberculosis lesions accumulated fluorescence by fluorescence microscope observation, which was confirmed by HE staining.Conclusion1. The anti-ESAT-6mice monoclonal antibody targeted CT probe has targeted properties in these pulmonary tuberculosis mice models. The biology distribution of radioactive iodine consists with in vivo Micro-CT imaging.2. The anti-ESAT-6mice monoclonal antibody targeted fluorescence probe can reach the area of pulmonary tuberculosis in mice according to the ex vivo NIFR imaging and fluorescence microscope observation.3. The two kinds anti-ESAT-6mice monoclonal antibody targeted probes can be used for targeted and specific imaging of the pulmonary tuberculosis in mice, which provide the basis for further multi-mode imaging of tuberculosis.