抗ESAT-6单克隆抗体靶向探针对结核影像诊断的初步研究

发布时间：2018-05-11 19:00

本文选题：结核分枝杆菌 + 杂交瘤　；参考：《复旦大学》2013年博士论文

【摘要】：第一部分碘与荧光标记抗ESAT-6单克隆抗体靶向探针的制备及免疫活性评价目的制备抗结核分枝杆菌ESAT-6蛋白的单克隆抗体,以碘原子和荧光标记单克隆抗体研制CT和荧光靶向探针,并评价两种探针的免疫活性。方法 1.以重组大肠杆菌表达纯化的ESAT-6蛋白为免疫原免疫小鼠,采用淋巴细胞杂交瘤技术,获得了4株针对结核分枝杆菌ESAT-6抗原的单克隆抗体杂交瘤细胞株,进行小鼠腹水制备、纯化,并测定抗体纯度和效价。 2.采用Iodogen法,以放射性碘Na125I示踪非放射性碘Na127I标记抗ESAT-6单克隆抗体,纸层析法测定放化纯度和稳定性。 3.将抗ESAT-6单克隆抗体偶联两种荧光基团IR783和罗丹明制备荧光探针,SDS-PAGE凝胶电泳确定荧光探针表征。 4.间接ELSIA法测定碘原子与荧光标记探针的抗体效价,评价标记后探针免疫活性。结果 1.4株单克隆抗体的腹水效价达到1：128000,纯化后抗体纯度高于90%,抗体亚型均为IgGl/k型。ELISA结果显示制备的单克隆抗体与结核分枝杆菌ESAT-6抗原可发生特异反应,而不与非相关性蛋白His-85b发生反应。 2.纯化后的碘原子标记抗ESAT-6抗体的放化纯度大于98%。稳定性试验测定结果显示,放射性碘标记抗ESAT-6抗体在体外4℃条件下存放一周后放化纯度大于95%。 3.荧光探针每个抗体分子上标记2个IR783和1.5个罗丹明。采用不同色调分析方式进行处理,显示探针无杂条带。 4.标记后单克隆抗体仍能与ESAT-6抗原发生特异性反应,抗体效价大于1：128000,碘原子和荧光标记探针具有较强免疫活性。结论 1.制备了高纯度、高效价抗结核分枝杆菌ESAT-6单克隆抗体,可与ESAT-6蛋白产生特异性反应。 2.碘原子标记抗ESAT-6单克隆抗体具有较好的体外稳定性；荧光探针纯度优良。碘与荧光标记抗ESAT-6单克隆抗体能与ESAT-6抗原特异性结合,较好保持其免疫活性。为进一步应用于动物体内成像实验奠定了基础。第二部分小鼠肺结核模型建立及ESAT-6抗原在小鼠肺结核组织中的表达目的探讨滴鼻途径建立C57BL/6J小鼠肺部结核分枝杆菌感染模型的可行性,分析ESAT-6抗原在小鼠肺结核组织中的表达和意义。方法 1. C57BL/6J小鼠30只,分为感染组24只,对照组6只。结核分枝杆菌H37Rv标准株滴鼻法接种感染组小鼠,对照组小鼠给予同等剂量生理盐水。 2.感染后4周、8周、12周,Micro-CT扫描小鼠肺部评价肺部病灶变化,并与HE染色及抗酸染色病理对照分析,小鼠肺组织匀浆培养计算肺部结核分枝杆菌菌落数。 3.应用免疫组化及免疫荧光染色法检测ESAT-6抗原在小鼠肺结核组织中的表达。结果 1.感染后4周、8周、12周,小鼠肺组织匀浆培养结核分枝杆菌菌落计数在106CFU左右,呈轻度上升趋势；小鼠肺部Micro-CT扫描显示肺部病灶阳性率分别为50.0%、83.3%、100%,肺部病灶体积增加与时间递增相关(P0.05)；3个监测时间点小鼠肺部病理学检查阳性率分别为83.3%、100%、100%。感染后8周、12周两个时间点小鼠肺部Micro-CT扫描阳性率及病理阳性率相似,病理改变均以增殖性实变和肉芽肿为主,干酪样坏死少见。感染后4周、8周、12周,抗酸杆菌染色均可见病变区散在或大量结核分枝杆菌聚集。 2.免疫组化结果显示感染后4周、8周、12周可见在小鼠肺结核渗出及实变区域片状褐色阳性物质沉积,在结核分枝杆菌及巨噬细胞分布区域见条状、颗粒状、结节状褐色阳性物质沉积。免疫荧光染色在实变区及肉芽肿区域荧光信号明显增强。结论 1. Micro-CT动态观察与病理学对照分析表明经滴鼻感染途径成功建立了小鼠肺结核感染模型。 2.感染后8周是成像实验模型建立的理想时间点。. 3.免疫组化及免疫荧光观察表明ESAT-6抗原在小鼠肺结核组织中的表达明显增强,为进一步研究单克隆抗体探针靶向诊断小鼠肺结核提供了依据。第三部分小鼠肺结核Micro-CT和近红外荧光成像的实验研究目的探讨抗ESAT-6单克隆抗体靶向探针Micro-CT和近红外荧光成像诊断小鼠肺结核的可行性。方法 1.24只C57BL/6J小鼠分为CT组和荧光组,每组12只。CT组和荧光组又分为实验组和对照组,实验组各9只小鼠,对照组各3只小鼠。小鼠经滴鼻法感染结核后8周用于成像实验。实验组经尾静脉注射碘和荧光标记单克隆抗体探针,对照组注射等量碘和荧光标记非特异IgG抗体。 2.CT组6只小鼠注射探针前、注射探针后6h、24h、48h活体Micro-CT动态成像,测量不同时间点肺部病灶CT值并计算平均值；对照组小鼠相同时间点观察。CT组3只小鼠在注射探针24h后,放射性活度仪检测肺部病灶及周围正常组织放射性碘分布。 3.荧光组9只小鼠注射荧光探针后24h,取小鼠肺、心、肝、脾、肾、肌肉组织离体近红外荧光成像。测量肺部病灶、正常肺组织、背景噪声信号强度(SI),计算对比噪声比(CNR), CNR=(SI病灶-SI正常肺)／SI背景噪声。肺组织冰冻切片荧光显微镜观察并与HE染色对照分析,评价荧光探针到达结核病灶的能力。对照组小鼠相同方法观察。结果 1.注射碘标记抗ESAT-6靶向探针后,Micro-CT小鼠肺部动态增强显示肺部结核病灶强化,且24h时CT值最高。对照组小鼠肺部病灶未见强化。放射性活度仪检测实验组小鼠肺部病灶放射性碘分布高于正常肺组织(P0.05)。 2.离体近红外荧光成像显示实验组小鼠肺部结核病灶区域荧光信号明显增强,对照组结核病灶区域微弱荧光或无荧光信号。单克隆抗体诱导的肺部病灶信号明显高于同型对照抗体,对比噪声比分别为60.33±3.44(n=9)和10.49±2.03(n＝3),两者存在统计学差异(P0.05)。荧光显微镜显示荧光积聚区域与HE染色肺结核组织部位一致。结论 1.活体Micro-CT成像显示碘原子标记探针能够靶向性聚集于结核病灶,放射性碘体内生物学分布与活体成像一致。 2.离体近红外荧光成像及荧光显微镜观察证明了抗ESAT-6单克隆抗体荧光靶向探针可特异性到达小鼠肺结核组织。 3.应用以上两种抗ESAT-6单克隆抗体探针可靶向、特异成像小鼠肺结核,为进一步多模式成像诊断肺结核奠定了基础。
[Abstract]:Part one
Preparation of iodine and fluorescent labeled anti ESAT-6 monoclonal antibody targeting probes and evaluation of their immune activity
The monoclonal antibodies against Mycobacterium tuberculosis ESAT-6 protein were prepared. The CT and fluorescent target probe were developed with iodine atom and fluorescence labeled monoclonal antibody, and the immunological activity of the two probes was evaluated.
Method
1. the ESAT-6 protein expressed and purified by recombinant Escherichia coli was immunized with immunogenic mice. By using lymphocyte hybridoma technique, 4 monoclonal antibody hybridoma cell lines were obtained for ESAT-6 antigen of Mycobacterium tuberculosis. The mouse ascites was prepared, purified, and the antibody purity and titer were measured.
2. using Iodogen method, radioiodine Na125I was used to trace the non radioactive iodine Na127I labeled monoclonal antibody against ESAT-6. The radiochemical purity and stability were determined by paper chromatography.
3. the monoclonal antibody against ESAT-6 was coupled with two fluorescent groups IR783 and Luo Danming to prepare fluorescent probes. SDS-PAGE gel electrophoresis was used to determine the fluorescence probe characterization.
4. indirect ELSIA method was used to determine the antibody titer of iodine atom and fluorescent labeled probe, and the immune activity of labeled probe was evaluated.
Result
The ascitic titer of 1.4 monoclonal antibodies reached 1:128000, and the purity of the purified antibody was higher than 90%. The antibody subtypes were all IgGl/k.ELISA results showed that the monoclonal antibody was specific to the ESAT-6 antigen of Mycobacterium tuberculosis and did not react against the unrelated protein His-85b.
The purity of anti ESAT-6 antibody after 2. purified iodine was greater than that of 98%. stability test, and the radioiodine labeled anti ESAT-6 antibody was stored for one week at 4 centigrade, and the purity was more than 95%.
3. fluorescent probes labeled 2 IR783 and 1.5 rhodamine on each antibody molecule. Different hue analysis methods were used to show that there were no heterozygous bands in the probe.
After 4. labeling, the monoclonal antibody can still react with ESAT-6 antigen specifically, the titer of the antibody is greater than 1:128000, and the iodine atom and the fluorescent labeled probe have strong immune activity.
conclusion
1. a highly purified and highly effective anti Mycobacterium tuberculosis ESAT-6 monoclonal antibody was prepared, which could react specifically with ESAT-6 protein.
2. iodide labeled anti ESAT-6 monoclonal antibody has good in vitro stability, and the purity of fluorescence probe is excellent. Iodine and fluorescent labeling anti ESAT-6 monoclonal antibody can be specifically combined with ESAT-6 antigen, and better maintain its immune activity. It lays a foundation for further application of imaging experiments in animal body.
The second part
Establishment of a mouse model of pulmonary tuberculosis and expression of ESAT-6 antigen in mice with pulmonary tuberculosis
objective
To explore the feasibility of establishing a model of Mycobacterium tuberculosis infection in C57BL/6J mice by means of nasal drip, and to analyze the expression and significance of ESAT-6 antigen in the tuberculosis of mice.
Method
1. C57BL/6J mice were divided into infection group (24 mice) and control group (6 rats). The H37Rv standard strain of Mycobacterium tuberculosis was inoculated with nosed mice in infection group, and the same dose of saline was given to the control group.
2. after 4 weeks of infection, 8 weeks and 12 weeks, the lung lesions were evaluated by Micro-CT scan in the lungs, and the lung tissue homogenate culture was used to calculate the number of Mycobacterium tuberculosis in the lung tissue of mice.
3. immunohistochemistry and immunofluorescence staining were used to detect the expression of ESAT-6 antigen in mouse pulmonary tuberculosis.
Result
1. after 4 weeks of infection, 8 weeks and 12 weeks, the colony count of Mycobacterium tuberculosis in lung tissue homogenate in mice was about 106CFU, showing a slight increase. The positive rates of lung lesions were 50%, 83.3%, 100% in lung Micro-CT scan in mice, respectively, and the increase of lung lesion volume was associated with time increasing (P0.05), and 3 monitoring time points of lung pathology in mice. The positive rates of the examination were 83.3%, 100%, 8 weeks after 100%. infection and two time at 12 weeks in 12 weeks, and the pathological positive rates were similar. The pathological changes were mainly proliferative and granulomatous, and the caseous necrosis was rare. 4 weeks, 8 weeks and 12 weeks after infection, the pathological area was scattered in the lesion area or a large number of tuberculosis branches. Bacteria gather.
2. the results of immunofluorescence showed that 4 weeks, 8 weeks, and 12 weeks after infection, the nodular Brown positive substance in the tubercular exudation and real region of the mice was deposited. In the distribution area of Mycobacterium tuberculosis and macrophage, a strip, granular and nodular Brown positive substance was deposited. The fluorescence staining of immunofluorescence in the real and granulomatous regions was significantly increased. Strong.
conclusion
1. Micro-CT dynamic observation and pathology analysis showed that a mouse model of pulmonary tuberculosis infection was successfully established through nasal infection.
2. the 8 week after infection is the ideal time to establish the imaging experimental model.
3. immunofluorescence and immunofluorescence showed that the expression of ESAT-6 antigen was obviously enhanced in the tuberculosis tissues of mice, which provided a basis for the further study of the monoclonal antibody probe targeted to the diagnosis of tuberculosis in mice.
The third part
Experimental study on Micro-CT and near infrared fluorescence imaging of pulmonary tuberculosis in mice
objective
Objective to investigate the feasibility of using ESAT-6 monoclonal antibody targeted probe Micro-CT and near infrared fluorescence imaging in the diagnosis of pulmonary tuberculosis in mice.
Method
1.24 C57BL/6J mice were divided into CT group and fluorescence group, each group of 12.CT groups and fluorescent groups were divided into experimental group and control group, 9 mice in the experimental group and 3 mice in the control group. The mice were infected by the nose drip method for 8 weeks after tuberculosis and were used for imaging experiments. The experimental group was injected with iodine and fluorescence labeled monoclonal antibody probes by the tail vein, and the control group was injected with equal amount of iodine. Non specific IgG antibodies were labeled with fluorescence and fluorescence.
Before injection of probe into 6 mice in group 2.CT, the 6h, 24h, 48h living body Micro-CT dynamic imaging after injection of probe was used to measure the CT value of lung lesions at different time points and to calculate the average value. In the control group, 3 mice in the.CT group were observed at the same time point, and the radioactivity meter was used to detect the radioactive iodine distribution in the lung lesions and surrounding normal tissues after the injection probe 24h.
3. fluorescent group 9 mice were injected with 24h after injection of fluorescent probe. The lung, heart, liver, spleen, kidney, and muscle tissue were taken near infrared fluorescence imaging in mice. The lung lesions, normal lung tissues, background noise signal intensity (SI) were measured, contrast noise ratio (CNR), CNR= (SI lesion -SI normal lung) / SI background noise. HE staining was used to evaluate the ability of fluorescent probes to reach tuberculosis foci.
Result
After 1. injection of iodine labeled anti ESAT-6 target probe, the lung dynamic enhancement showed that the pulmonary tuberculosis focus was enhanced in the Micro-CT mice, and the CT value was the highest at 24h. The lung lesions in the control group did not strengthen. The radioiodine distribution in the lung lesions of the mice was higher than that of the normal lung tissue (P0.05).
2. in vitro near infrared fluorescence imaging showed that the fluorescence signal of the pulmonary tuberculosis focus in the experimental group was obviously enhanced and the tuberculosis focus area of the control group was weak fluorescent or no fluorescence signal. The lung focal signal induced by the monoclonal antibody was significantly higher than that of the same type control antibody. The contrast noise ratio was 60.33 + 3.44 (n=9) and 10.49 + 2.03 (n = 3), respectively. There was statistical difference (P0.05). Fluorescence microscopy showed that the area of fluorescence accumulation was consistent with that of HE stained tuberculosis tissue.
conclusion
1. in vivo Micro-CT imaging showed that the iodine labeled probe could target aggregation in the tuberculosis foci. The biodistribution of radioiodine in vivo coincide with in vivo imaging.
2. in vitro near infrared fluorescence imaging and fluorescence microscopy confirmed that the fluorescent targeting probe against ESAT-6 monoclonal antibody could specifically reach the pulmonary tuberculosis tissue in mice.
3. the above two anti ESAT-6 monoclonal antibody probes can be targeted and specifically imaging pulmonary tuberculosis in mice, thus laying the foundation for further multi-mode imaging diagnosis of pulmonary tuberculosis.

【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R52;R816.4

【参考文献】