抗人CD105抗体的制备及其在卵巢癌显像和检测中的初步应用
发布时间：2018-08-19 16:13
【摘要】：研究背景： 卵巢癌是妇科癌症死亡的首要原因，晚期卵巢癌的治疗主要包括肿瘤细胞减灭术和紫杉醇/卡铂化疗。但是大部分患者死于肿瘤复发。血管生成是卵巢癌进展和转移的基础，抗血管生成治疗是控制和治愈卵巢癌的希望所在。两个随机研究表明通过添加抗VEGF单克隆抗体贝伐单抗，可延长无进展生存期（PFS），因此导致FDA批准贝伐单抗可用卵巢癌的一线治疗，也表明靶向血管生成是有前途的抗肿瘤治疗策略。 CD105又名Endoglin（EDG），是一种细胞膜糖蛋白，是内皮细胞增殖的标志之一，还是转化生长因子-β (TGF-β)受体复合物的组分之一。CD105在肿瘤血管和淋巴管内皮细胞有较高的表达，而在成熟的血管内皮细胞中表达较弱或基本不表达。体外研究表明，内皮细胞对TGF的反应需CD105调节，而CD105尚可促内皮细胞增殖，抑制其凋亡，并且有促新生血管生成等功能。大量研究表明，CD105可作为肿瘤显像及治疗的理想靶点。以CD105为标记的肿瘤微血管密度（MVD）可判断多种实体肿瘤的恶性度及预后，由MMP-14剪切产生的可溶性的sCD105可用于多种转移性肿瘤，如乳腺癌、肠癌患者的预后判断，而多项报道认为子痫前期的孕妇血清中增加sEndoglin水平升高。 卵巢癌是妇科恶性度最高的肿瘤之一，也是血管化程度较高的恶性肿瘤，以贝伐单抗为代表的抗血管生成药物已显示出较好的抗肿瘤效应，但也存在持续时间短、副作用较大等缺点，CD105在卵巢癌微血管内皮高表达，抗人CD105抗体在其诊断及治疗中有可能发挥重要作用。目前有较多有关CD105单克隆抗体（McAb）或人鼠嵌合抗体及其偶联物抗肿瘤的报道。国外已有CD105-McAb商品，动物试验表明CD105-McAb有较好的抗肿瘤作用，目前人鼠嵌合抗体I期临床试验正在进行，国内尚无抗人CD105功能性抗体的报道。研制具有自主知识产权的CD105-McAb并用于临床诊断及治疗具有重大的意义。 制备抗人Endoglin抗体不仅能将其应用于抗肿瘤药物的筛选，亦可将其构建免疫检测技术应用于卵巢癌转移及预后的评测。但是抗体药物的制备难度大，无论在特异性、亲和力、中和阻断活性及生物反应活性等个方面均有很高要求。但在诊断应用上相对要求较低，因此在成功制备抗人CD105抗体后，可以充分利用所制备的抗体，建立Endoglin体内及体外检测技术。目前检测sCD105仍多采用酶联免疫吸附法或放射免疫法，均存在检测费用高，需多人份血清同时检测，等候时间长的缺点。电化学免疫分析法成功的将免疫反应的高选择性和电化学测定的高灵敏度相结合而受到人们的青睐，其测定灵敏度与放射免疫法相近，而又不需要使用放射性同位素，充分显示了电化学免疫法在临床检测中的优越性和发展前途。电化学免疫分析法自上世纪70年代提出研究理论至今，已成功的将电化学检测到高灵敏性，高稳定性等与免疫反应的高选择性相结合而研发出适用于医疗，，生物等多领域的电化学免疫传感器，并越来越受到人们的青睐，其测定灵敏度相当或优于目前高灵敏检测所依赖的放射免疫法，而又不需要使用放射性同位素，不仅减少了医护人员及病患的放射损伤，同时也避免了放射性标记对抗体或抗原等于有标记所造成的化学损伤，而最大程度地保持了抗体或抗原的免疫反应活性，提高了检测效率。这些都充分显示了电化学免疫法在临床检测应用中的优越性和发展前景。 主要的研究内容和结果： 1、人CD105分子胞外区基因的克隆及在原核系统中的表达与纯化： 通过PCR技术克隆了CD105分子胞外段基因，经酶切鉴定及序列分析表明其序列与文献报道完全一致。将该片段亚克隆到表达载体pET32a(+)，转化BL21，经IPTG诱导，SD-SAPGE分析，在80kd处有一明显的诱导表达带，其分子量大小与预期相符。Western-blot分析表明，该表达蛋白带可被商业化CD105-McAb特异识别。表达产物以包含涵体形式存在，通过镍离子柱亲和纯化得到纯化蛋白并复性，最终获得纯度可达90%的目的重组蛋白。该蛋白为后续研究中抗体制备及检测技术建立奠定基础。 2、人CD105单克隆抗体的制备及鉴定： 2.1CD105表位特异性线性多肽多克隆抗体的制备： 为了能获得更多不同表位的CD105抗体，达到建立双抗体夹心配对检测CD105的目的，采用表位在线分析及文献报道CD105抗体的表位，我们设计并合成了CD105分子B细胞线性表位多肽，并与BSA交联。将偶联BSA的多肽免疫兔子，得到高滴度的抗体，纯化后抗体经ELISA、SDS-PAGE等检测具有抗CD105特异性。 2.2CD105单克隆抗体的制备及鉴定： 利用纯化的CD105胞外段蛋白作为免疫原，我们采用改良的免疫程序，采用快速的杂交瘤技术，经历4细胞融合，成功制备了数十株阳性克隆，使用从北京义翘购买的CHO细胞表达的重组CD105蛋白筛选出6株抗糖基化CD105的特异性单克隆抗体，经酶联免疫吸附法（ELISA）、Western Blot、细胞及组织免疫荧光等多种手段对所得单抗进行特异性分析，显示我们制备的单克隆抗体具有抗CD105特异。 3、131碘(~(131)I)标记的CD105单克隆抗体在荷人卵巢癌裸鼠体内显像和分布： 3.1用Iodogen法合成~(131)I-CD105单抗及对照单抗（鼠抗人单克隆抗体），SephadexG50柱纯化，利用纸层析法测定其标记率及放化纯度，结果表明Iodogen法标记CD105单抗及对照单抗，标记率分别为92.2%和83.6%，放化纯度超过97%，放射比活性分别为147.52μci/μg和129.58μci/μg，放射性浓度达737.6μci/ml和732.09μc。 3.2经鼠尾静脉注射~(131)I标记单抗入荷人卵巢癌裸鼠体内，SPECT仪下观察肿瘤显像情况，同时，在注射~(131)I-CD105单抗后24h，48h，72h脱颈椎处死一组小鼠，取各重要脏器及肿瘤组织，滤纸吸干，γ放射免疫计数器测定各组织的每分钟放射性计数，计算每克组织每分钟放射性计数，计算放射比活性，计算肿瘤组织和非肿瘤组织放射性比值（T/NT比值）。SPECT观察注射后24h，48h，96h放射自显影成像情况。 实验组心、肝、肺、肾、胃等组织在24小时时点均有较高的每克组织摄取注射量的百分比（%ID/g），随着时间的延长，肿瘤组织的放射性摄取率明显增高，而其他重要脏器摄取率逐渐下降，至96小时肿瘤组织每克组织摄取注射量的百分比高达29.10±1.61%ID/g，而肝、肾等脏器均在明显下降。相应的T/NT比值在96小时时点均3，证明~(131)I-CD105单抗能够在肿瘤组织内浓聚并在96小时达到高点。而对照组未发现肿瘤部位的放射性浓聚。 ~(131)I-CD105单抗组在尾静脉注射~(131)I标记物后，移植瘤部位和肾脏，肝脏，胃开始出现放射性浓聚，随着时间的延长，胸腹部逐渐变淡，肿瘤区域逐渐浓聚,48小时组肿瘤区域清晰可见，96小时本底背景明显减弱，而肿瘤显像明显增强。 4、人CD105免疫传感器检测的建立及评测 在抗原（重组蛋白）及高效抗体制备的基础上，采用电沉积纳米金技术在电极表面构建生物兼容性良好的纳米金表面，并进一步修饰半胱氨酸用于吸附修饰小粒径的纳米金增加工作面对比表面积，提高抗体的固定量，将筛选的单克隆抗体固定于制备电极表面，通过抗原/抗体特异性反应，以及蛋白在电极表面的结合对电化学响应信号的影响，初步建立了应用于CD105检测的电化学免疫传感器。在此基础上，为了进一步提高免疫检测的特异性及灵敏性，采用纳米铂及硫堇（电子媒介体）标记的配对抗CD105抗体作为检测抗体，通过硫堇实现电化学响应，及纳米铂的催化性能进一步提高研究的电化学免疫传感器的灵敏性，并通过检测抗体与所检测抗原的特异性反应进一步提高免疫检测体系的特异性，实现CD105的高特异，高灵敏性检测。电化学免疫传感器的研究及制备过程，表征鉴定我们采用循环伏安法（CV）考察了电极表面的电化学特性，充分地研究了免疫传感器的性能。采用CV对人CD105进行检测，初步研究获得了良好的检测效果，基本建立了应用于人CD105检测的电化学免疫传感器，进一步验证了所研究的电化学免疫传感技术的良好实用性，并充分证明了所研究制备的重组蛋白及高效抗体的良好检测应用性能及可能的临床检测应用前景。同时，此研究为进一步改良并研发适用于临床应用的CD105检测技术奠定基础。
[Abstract]:Research background:
Ovarian cancer is the leading cause of death in gynecologic cancer. Treatment for advanced ovarian cancer includes cytoreductive surgery and chemotherapy with paclitaxel/carboplatin. But most patients die of recurrence. Angiogenesis is the basis of ovarian cancer progression and metastasis. Antiangiogenic therapy is the hope of controlling and curing ovarian cancer. Two randomized studies It has been shown that bevacizumab can prolong progression-free survival (PFS) by adding anti-VEGF monoclonal antibody, which leads to FDA approval of bevacizumab as a first-line treatment for ovarian cancer. Targeted angiogenesis is also a promising anti-tumor strategy.
CD105, also known as Endoglin (EDG), is a membrane glycoprotein, a marker of endothelial cell proliferation, or a component of transforming growth factor-beta (TGF-beta) receptor complex. CD105 is highly expressed in tumor blood vessels and lymphatic endothelial cells, but weakly or basically not expressed in mature vascular endothelial cells. These results suggest that the endothelial cell response to TGF is regulated by CD105, which can promote the proliferation of endothelial cells, inhibit their apoptosis and promote angiogenesis. Many studies have shown that CD105 can be used as an ideal target for tumor imaging and treatment. Prognosis, soluble sCD105 produced by MMP-14 shearing can be used to predict the prognosis of many metastatic tumors, such as breast cancer and bowel cancer, and many reports suggest that increased serum sEndoglin levels in preeclampsia pregnant women are elevated.
Ovarian cancer is one of the most malignant tumors in gynecology. It is also a highly vascularized malignant tumor. Bevacizumab as a representative of anti-angiogenesis drugs has shown good anti-tumor effect, but there are also shortcomings such as short duration and side effects. CD105 is highly expressed in the microvascular endothelium of ovarian cancer, and anti-human CD105 antibody in its diagnosis. There are many reports about the anti-tumor effect of CD105 monoclonal antibody (McAb) or human-mouse chimeric antibody and its conjugates. There are CD105-McAb products abroad. Animal tests show that CD105-McAb has a good anti-tumor effect. At present, phase I clinical trials of human-mouse chimeric antibody are under way, but there is no anti-tumor effect in China. It is of great significance to develop CD105-McAb with independent intellectual property rights for clinical diagnosis and treatment.
Preparation of anti-human Endoglin antibody can be used not only in screening of anti-tumor drugs, but also in the detection of metastasis and prognosis of ovarian cancer by constructed immunoassay. Therefore, after the successful preparation of anti-human CD105 antibody, we can make full use of the prepared antibody to establish Endoglin in vivo and in vitro detection technology. Point. Electrochemical immunoassay (EIA) successfully combines the high selectivity of immune reaction with the high sensitivity of electrochemical assay. Its sensitivity is similar to that of radioimmunoassay without the use of radioisotopes. It fully demonstrates the superiority and development prospects of EIA in clinical detection. Since the theory of chemical immunoassay was put forward in the 1970s, electrochemical immunosensors have been successfully developed for medical, biological and other fields by combining electrochemical detection with high sensitivity, high stability and high selectivity of immune reaction, and have attracted more and more attention. It is superior to the radioimmunoassay, which is currently used in highly sensitive detection, and does not require the use of radioisotopes. It not only reduces the radiation damage of medical staff and patients, but also avoids the chemical damage caused by the radiolabeling of antibodies or antigens equal to the labeling, and maintains the immune response of antibodies or antigens to the greatest extent. All these fully demonstrate the superiority and development prospect of electrochemical immunoassay in clinical detection.
Main research contents and results:
1, cloning and expression and purification of the extracellular domain of human CD105 molecule in the prokaryotic system.
The extracellular segment of CD105 gene was cloned by PCR and its sequence was identical with that reported in literatures. The subcloned fragment was transformed into BL21 by subcloning into expression vector pET32a (+). After induction by IPTG and SD-SAPGE analysis, an obvious induction expression band was found at 80 kd. The molecular weight of the fragment was in agreement with the expected value. The results showed that the expressed protein band could be specifically recognized by commercial CD105-McAb. The expressed product was in the form of inclusion body. The purified protein was purified by Ni-ion affinity purification and renatured, and the purity of the recombinant protein was up to 90%. This protein lays a foundation for antibody preparation and detection technology in the follow-up study.
2, preparation and characterization of human CD105 monoclonal antibody.
Preparation of 2.1CD105 epitope specific linear polypeptide polyclonal antibody:
In order to obtain more CD105 antibodies with different epitopes and to establish a double antibody sandwich matching method for detection of CD105, we designed and synthesized a linear epitope polypeptide of CD105 molecule B cells by on-line epitope analysis and reported CD105 antibody epitopes. The polypeptides conjugated with BSA were immunized to rabbits to obtain high titer antibodies. After purification, the antibody was tested for anti CD105 specificity by ELISA and SDS-PAGE.
Preparation and characterization of monoclonal antibodies against 2.2CD105:
Using purified CD105 extracellular segment protein as immunogen, we have successfully prepared dozens of positive clones by rapid hybridoma technique and 4-cell fusion. Six specific monoclonal antibodies against glycosylated CD105 have been screened by using recombinant CD105 protein expressed by CHO cells purchased from Beijing Yiqiao. Enzyme-linked immunosorbent assay (ELISA), Western Blot, cell and tissue immunofluorescence were used to analyze the specificity of the monoclonal antibody. The results showed that the monoclonal antibody prepared by us was specific to CD105.
Imaging and distribution of 3131 iodine (~ (131) I) labeled CD105 monoclonal antibody in nude mice bearing human ovarian cancer:
3.1 The ~ (131) I-CD105 monoclonal antibody and the control monoclonal antibody (mouse anti-Human Monoclonal antibody) were synthesized by Iodogen method and purified by Sephadex G50 column. The labeling rate and radiochemical purity of the monoclonal antibody and the control monoclonal antibody were determined by paper chromatography. The results showed that the labeling rates of the monoclonal antibody and the control monoclonal antibody were 92.2% and 83.6%, respectively. The radiochemical purity of the monoclonal antibody and the control monoclonal antibody were over 97% and 147 / g and 129.58 mu ci/ g, the radioactive concentration is 737.6 ci/ml and 732.09 C.
3.2 Nude mice bearing human ovarian cancer were injected with ~ (131) I-labeled monoclonal antibody via tail vein of mice. The tumor imaging was observed by SPECT. At the same time, a group of mice were sacrificed 24 hours, 48 hours and 72 hours after injection of ~ (131) I-CD105 monoclonal antibody. The vital organs and tumor tissues were taken out, filtered paper was dried, and the minute radioimmunoassay was performed by gamma-ray counter. Radioactive counts per gram of tissue per minute were calculated, radioactivity was calculated, and radioactivity ratios (T/NT ratios) between tumor and non-tumor tissues were calculated. SPECT was used to observe autoradiography 24, 48 and 96 hours after injection.
The heart, liver, lung, kidney, stomach and other tissues in the experimental group all had a higher percentage of uptake of injection per gram of tissue at 24 hours (% ID / g). With the prolongation of time, the uptake rate of radioactivity in tumor tissue increased significantly, while the uptake rate of other important organs gradually decreased, and the percentage of uptake of injection per gram of tumor tissue reached 29.1% at 96 hours. The corresponding T/NT ratios were 3 at 96 hours, indicating that ~ (131) I-CD105 monoclonal antibody could accumulate in tumor tissues and reach a high level at 96 hours.
After injection of ~ (131) I marker into tail vein, the tumor site, kidney, liver and stomach began to show radioactivity concentration in the 131 I-CD105 McAb group. With the time prolonged, the tumor area became thinner in the thorax and abdomen, and gradually thickened. In the 48-hour group, the tumor area was clearly visible, the background was significantly weakened in 96 hours, and the tumor imaging was significantly enhanced.
4, the establishment and evaluation of human CD105 immunosensor detection.
On the basis of preparation of antigen (recombinant protein) and high-performance antibody, electrodeposited gold nanoparticles were used to construct biocompatible gold nanoparticles on the electrode surface, and cysteine was further modified to adsorb and modify small-sized gold nanoparticles to increase the specific surface area of working face and increase the amount of antibody immobilization. An electrochemical immunosensor for the detection of CD105 was established by immobilizing the body on the prepared electrode surface. Nano-platinum and thionine were used to improve the specificity and sensitivity of immunoassay. The electrochemical response of CD105 antibody labeled with electronic mediator is realized by thionine, and the catalytic performance of nano-platinum further improves the sensitivity of the electrochemical immunosensor. The specificity of the immunoassay system is further enhanced by detecting the specific reaction between the antibody and the antigen detected. The electrochemical characteristics of the electrode surface were investigated by cyclic voltammetry (CV), and the performance of the immunosensor was fully studied. The detection of human CD105 by CV was carried out, and good results were obtained. The electrochemical immunosensor applied to the detection of human CD105 further validates the good practicability of the electrochemical immunosensor technology, and fully proves the good detection performance and potential clinical application prospect of the recombinant protein and high-efficiency antibody. It lays the foundation for clinical application of CD105 detection technology.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R737.31
本文编号：2192171
[Abstract]:Research background:
Ovarian cancer is the leading cause of death in gynecologic cancer. Treatment for advanced ovarian cancer includes cytoreductive surgery and chemotherapy with paclitaxel/carboplatin. But most patients die of recurrence. Angiogenesis is the basis of ovarian cancer progression and metastasis. Antiangiogenic therapy is the hope of controlling and curing ovarian cancer. Two randomized studies It has been shown that bevacizumab can prolong progression-free survival (PFS) by adding anti-VEGF monoclonal antibody, which leads to FDA approval of bevacizumab as a first-line treatment for ovarian cancer. Targeted angiogenesis is also a promising anti-tumor strategy.
CD105, also known as Endoglin (EDG), is a membrane glycoprotein, a marker of endothelial cell proliferation, or a component of transforming growth factor-beta (TGF-beta) receptor complex. CD105 is highly expressed in tumor blood vessels and lymphatic endothelial cells, but weakly or basically not expressed in mature vascular endothelial cells. These results suggest that the endothelial cell response to TGF is regulated by CD105, which can promote the proliferation of endothelial cells, inhibit their apoptosis and promote angiogenesis. Many studies have shown that CD105 can be used as an ideal target for tumor imaging and treatment. Prognosis, soluble sCD105 produced by MMP-14 shearing can be used to predict the prognosis of many metastatic tumors, such as breast cancer and bowel cancer, and many reports suggest that increased serum sEndoglin levels in preeclampsia pregnant women are elevated.
Ovarian cancer is one of the most malignant tumors in gynecology. It is also a highly vascularized malignant tumor. Bevacizumab as a representative of anti-angiogenesis drugs has shown good anti-tumor effect, but there are also shortcomings such as short duration and side effects. CD105 is highly expressed in the microvascular endothelium of ovarian cancer, and anti-human CD105 antibody in its diagnosis. There are many reports about the anti-tumor effect of CD105 monoclonal antibody (McAb) or human-mouse chimeric antibody and its conjugates. There are CD105-McAb products abroad. Animal tests show that CD105-McAb has a good anti-tumor effect. At present, phase I clinical trials of human-mouse chimeric antibody are under way, but there is no anti-tumor effect in China. It is of great significance to develop CD105-McAb with independent intellectual property rights for clinical diagnosis and treatment.
Preparation of anti-human Endoglin antibody can be used not only in screening of anti-tumor drugs, but also in the detection of metastasis and prognosis of ovarian cancer by constructed immunoassay. Therefore, after the successful preparation of anti-human CD105 antibody, we can make full use of the prepared antibody to establish Endoglin in vivo and in vitro detection technology. Point. Electrochemical immunoassay (EIA) successfully combines the high selectivity of immune reaction with the high sensitivity of electrochemical assay. Its sensitivity is similar to that of radioimmunoassay without the use of radioisotopes. It fully demonstrates the superiority and development prospects of EIA in clinical detection. Since the theory of chemical immunoassay was put forward in the 1970s, electrochemical immunosensors have been successfully developed for medical, biological and other fields by combining electrochemical detection with high sensitivity, high stability and high selectivity of immune reaction, and have attracted more and more attention. It is superior to the radioimmunoassay, which is currently used in highly sensitive detection, and does not require the use of radioisotopes. It not only reduces the radiation damage of medical staff and patients, but also avoids the chemical damage caused by the radiolabeling of antibodies or antigens equal to the labeling, and maintains the immune response of antibodies or antigens to the greatest extent. All these fully demonstrate the superiority and development prospect of electrochemical immunoassay in clinical detection.
Main research contents and results:
1, cloning and expression and purification of the extracellular domain of human CD105 molecule in the prokaryotic system.
The extracellular segment of CD105 gene was cloned by PCR and its sequence was identical with that reported in literatures. The subcloned fragment was transformed into BL21 by subcloning into expression vector pET32a (+). After induction by IPTG and SD-SAPGE analysis, an obvious induction expression band was found at 80 kd. The molecular weight of the fragment was in agreement with the expected value. The results showed that the expressed protein band could be specifically recognized by commercial CD105-McAb. The expressed product was in the form of inclusion body. The purified protein was purified by Ni-ion affinity purification and renatured, and the purity of the recombinant protein was up to 90%. This protein lays a foundation for antibody preparation and detection technology in the follow-up study.
2, preparation and characterization of human CD105 monoclonal antibody.
Preparation of 2.1CD105 epitope specific linear polypeptide polyclonal antibody:
In order to obtain more CD105 antibodies with different epitopes and to establish a double antibody sandwich matching method for detection of CD105, we designed and synthesized a linear epitope polypeptide of CD105 molecule B cells by on-line epitope analysis and reported CD105 antibody epitopes. The polypeptides conjugated with BSA were immunized to rabbits to obtain high titer antibodies. After purification, the antibody was tested for anti CD105 specificity by ELISA and SDS-PAGE.
Preparation and characterization of monoclonal antibodies against 2.2CD105:
Using purified CD105 extracellular segment protein as immunogen, we have successfully prepared dozens of positive clones by rapid hybridoma technique and 4-cell fusion. Six specific monoclonal antibodies against glycosylated CD105 have been screened by using recombinant CD105 protein expressed by CHO cells purchased from Beijing Yiqiao. Enzyme-linked immunosorbent assay (ELISA), Western Blot, cell and tissue immunofluorescence were used to analyze the specificity of the monoclonal antibody. The results showed that the monoclonal antibody prepared by us was specific to CD105.
Imaging and distribution of 3131 iodine (~ (131) I) labeled CD105 monoclonal antibody in nude mice bearing human ovarian cancer:
3.1 The ~ (131) I-CD105 monoclonal antibody and the control monoclonal antibody (mouse anti-Human Monoclonal antibody) were synthesized by Iodogen method and purified by Sephadex G50 column. The labeling rate and radiochemical purity of the monoclonal antibody and the control monoclonal antibody were determined by paper chromatography. The results showed that the labeling rates of the monoclonal antibody and the control monoclonal antibody were 92.2% and 83.6%, respectively. The radiochemical purity of the monoclonal antibody and the control monoclonal antibody were over 97% and 147 / g and 129.58 mu ci/ g, the radioactive concentration is 737.6 ci/ml and 732.09 C.
3.2 Nude mice bearing human ovarian cancer were injected with ~ (131) I-labeled monoclonal antibody via tail vein of mice. The tumor imaging was observed by SPECT. At the same time, a group of mice were sacrificed 24 hours, 48 hours and 72 hours after injection of ~ (131) I-CD105 monoclonal antibody. The vital organs and tumor tissues were taken out, filtered paper was dried, and the minute radioimmunoassay was performed by gamma-ray counter. Radioactive counts per gram of tissue per minute were calculated, radioactivity was calculated, and radioactivity ratios (T/NT ratios) between tumor and non-tumor tissues were calculated. SPECT was used to observe autoradiography 24, 48 and 96 hours after injection.
The heart, liver, lung, kidney, stomach and other tissues in the experimental group all had a higher percentage of uptake of injection per gram of tissue at 24 hours (% ID / g). With the prolongation of time, the uptake rate of radioactivity in tumor tissue increased significantly, while the uptake rate of other important organs gradually decreased, and the percentage of uptake of injection per gram of tumor tissue reached 29.1% at 96 hours. The corresponding T/NT ratios were 3 at 96 hours, indicating that ~ (131) I-CD105 monoclonal antibody could accumulate in tumor tissues and reach a high level at 96 hours.
After injection of ~ (131) I marker into tail vein, the tumor site, kidney, liver and stomach began to show radioactivity concentration in the 131 I-CD105 McAb group. With the time prolonged, the tumor area became thinner in the thorax and abdomen, and gradually thickened. In the 48-hour group, the tumor area was clearly visible, the background was significantly weakened in 96 hours, and the tumor imaging was significantly enhanced.
4, the establishment and evaluation of human CD105 immunosensor detection.
On the basis of preparation of antigen (recombinant protein) and high-performance antibody, electrodeposited gold nanoparticles were used to construct biocompatible gold nanoparticles on the electrode surface, and cysteine was further modified to adsorb and modify small-sized gold nanoparticles to increase the specific surface area of working face and increase the amount of antibody immobilization. An electrochemical immunosensor for the detection of CD105 was established by immobilizing the body on the prepared electrode surface. Nano-platinum and thionine were used to improve the specificity and sensitivity of immunoassay. The electrochemical response of CD105 antibody labeled with electronic mediator is realized by thionine, and the catalytic performance of nano-platinum further improves the sensitivity of the electrochemical immunosensor. The specificity of the immunoassay system is further enhanced by detecting the specific reaction between the antibody and the antigen detected. The electrochemical characteristics of the electrode surface were investigated by cyclic voltammetry (CV), and the performance of the immunosensor was fully studied. The detection of human CD105 by CV was carried out, and good results were obtained. The electrochemical immunosensor applied to the detection of human CD105 further validates the good practicability of the electrochemical immunosensor technology, and fully proves the good detection performance and potential clinical application prospect of the recombinant protein and high-efficiency antibody. It lays the foundation for clinical application of CD105 detection technology.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R737.31
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