基于单克隆抗体技术的贾第虫胶体金试纸条诊断方法的建立及其初步应用

发布时间：2018-01-07 03:18

本文关键词：基于单克隆抗体技术的贾第虫胶体金试纸条诊断方法的建立及其初步应用　出处：《中国疾病预防控制中心》2017年硕士论文　论文类型：学位论文

【摘要】：蓝氏贾第鞭毛虫(贾第虫)是一种引起人和其他哺乳动物腹泻的机会性致病寄生虫,其主要寄生在人和某些哺乳动物的小肠,引起人的腹泻、腹痛和消化不良等胃肠道症状。贾第虫滋养体引发的贾第虫病,目前已被列为全世界危害人类健康的十种主要寄生虫病之一,该病的严重性和危害性日益受到重视。目前,利用显微镜对样本中的包囊和滋养体进行镜检的病原学检测方法,被认为是诊断贾第虫感染的"金标准"。该法现场应用时费时费力,劳动强度大,检出率低,在感染率/度低时容易出现漏检。免疫学检测方法具有敏感性强、特异性高、操作简单且易于现场推广等优点,贾第虫的免疫学检测引起研究人员更多的关注。随着单克隆抗体技术的应用,进一步加强了贾第虫免疫学检测的特异性。本研究拟以贾第虫滋养体免疫小鼠制备单克隆抗体,建立诊断贾第虫病的免疫学诊断方法。1、贾第虫单克隆抗体制备以贾第虫滋养体免疫BALB/c小鼠,利用细胞融合技术建立抗贾第虫抗原杂交瘤细胞株并制备单克隆抗体(Monoclonal antibody,McAb)。采用免疫球蛋白亚类(型)检测试剂盒鉴定McAb的类型及亚型;蛋白质印迹鉴定McAb对贾第虫滋养体可溶性抗原和排泄分泌抗原的识别;酶联免疫吸附试验检测McAb与贾第虫滋养体反应性,并检测与其他虫种的交叉反应。结果获得12株分泌贾第虫单克隆抗体细胞株,检测后均为IgG1亚型。其中EB2株能识别贾第虫滋养体可溶性抗原和排泄分泌抗原中相对分子量分别为175KDa和191KDa的2条蛋白条带,与大肠杆菌、猪蛔虫、人芽囊原虫、日本血吸虫虫卵、日本血吸虫成虫和卫氏并殖吸虫抗原均无交叉反应。2、建立双抗体夹心ELISA法检测贾第虫抗原将制备的贾第虫单抗和多抗进行纯化后,建立双抗体夹心ELISA方法检测贾第虫粪抗原。结果初步建立了单抗-抗原-多抗-二抗的双抗体夹心ELISA方法检测贾第虫粪抗原,并对其条件进行了优化。该方法的灵敏度为1:10倍稀释,批内变异系数CV5%,且与其他虫种无交叉反应,是一个快速、特异、灵敏的检测方法,为进一步的现场应用奠定了基础。3、胶体金试纸条试制检测贾第虫抗原成功制备胶体金试纸条。该试纸条具有较好的敏感性、特异性和稳定性。检测贾第虫滋养体抗原的检测限为0.25μg。该试条与其他虫种模拟粪样无交叉反应;通过对10份临床腹泻患者粪样检测,该试纸条与市售试纸条(德国拜发公司)符合率一致,检测阳性率低于市售ELISA试剂盒(德国拜发公司(R-Biopharm)产品)。结论:1、成功进行贾第虫滋养体的体外培养,并获得滋养体可溶性抗原和排泄分泌抗原。2、以贾第虫滋养体抗原免疫家兔成功获得贾第虫兔多克隆抗体(Polyclonal antibody,PcAb)。3、以贾第虫滋养体免疫小鼠并进行杂交瘤融合和筛选,成功获得十二株单克隆抗体细胞株。其中EB2株分泌上清效价较高且稳定分泌抗体,能特异识别贾第虫滋养体可溶性抗原和排泄分泌抗原中相对分子量分别大约为175 kDa和191 kDa的2条蛋白条带,与大肠杆菌、猪蛔虫、人芽囊原虫、日本血吸虫虫卵、日本血吸虫成虫和卫氏并殖吸虫抗原均无交叉反应。4、利用贾第虫McAb和PcAb成功建立双抗体夹心ELISA法体系,用于粪样贾第虫抗原的检测。5、基于贾第虫McAb和PcAb成功建立胶体金试纸条用于粪样贾第虫抗原的快速检测,并具有较高的敏感性、特异性和稳定性。
[Abstract]:Giardia lamblia (Giardia) is an opportunistic pathogenic parasite causing diarrhea in human and other mammals, the main intestinal parasite of humans and some mammals, cause diarrhea, abdominal pain and indigestion and other gastrointestinal symptoms. Giardia trophozoites caused by Giardia, has been listed as one of the ten the main parasitic disease in the world of serious harm to human health, and harmfulness of the disease has attracted more and more attention. At present, the methods for assessing the microscopic examination of cysts and trophozoites in the sample with microscope pathogen, is considered to be the diagnosis of Giardia infection "gold standard". The application of the site time-consuming. High labor intensity, low detection rate, the infection rate is low when / prone to leak. Immunological detection method has high sensitivity, high specificity, simple operation and easy site promotion, the insect immunology inspection Jia The researchers measured caused more attention. With the application of monoclonal antibody technology, to further strengthen the specificity of Jia Di worm immunological detection. In this study, preparation of monoclonal antibodies to Jia Di worm trophozoites of mice were immunized, establishment of immunological diagnostic methods in diagnosis of Jia Di disease.1, Jia Di. Preparation of monoclonal antibodies to Jia Di worm immune trophozoite BALB/c mice, using technology to establish the anti Jia Di worm antigen hybridoma cell lines and cell fusion to prepare monoclonal antibody (Monoclonal antibody, McAb). The immunoglobulin subclass (type) type detection kit and identification of McAb subtype identification; Western blot McAb secretory antigen of Jia Di worm trophozoites and excretion of soluble antigen; enzyme linked immunosorbent assay to detect McAb and Jia Di worm trophozoites reactivity, and detection and other insect species cross reaction. Results 12 strains secreted Jia Di worm clones Antibody cell lines were detected after IgG1 subtype. The strain EB2 can identify the trophozoites of Giardia lamblia soluble antigens and excretory secretory antigen molecular weight were 175KDa and 191KDa 2 protein bands, and Escherichia coli, Ascaris suum, Blastocystis hominis, Schistosoma japonicum and Schistosoma japonicum and Wei Paragonimus antigen had no cross reaction with.2, detection of Giardia antigen double antibody sandwich ELISA method for the preparation of Giardia monoclonal and polyclonal antibody was purified. Double antibody sandwich ELISA method detecting Giardia fecal antigen is established. Results the monoclonal antibody - antigen - antibody - two anti double antibody sandwich ELISA method detection of Giardia fecal antigen, and the conditions were optimized. The sensitivity of this method is 1:10 times of dilution, the intraassay coefficient of variation of CV5%, and no cross reaction with other species, is a rapid, specific and sensitive method for detection. Laid the foundation for the further application in the field of.3, colloidal gold test paper Giardia antigen successfully prepared colloidal gold strip. The strip has good sensitivity, specificity and stability. Detection of trophozoites of Giardia lamblia antigen limit was 0.25 g. the test strip and other insect feces without cross simulation based on the reaction; 10 clinical patients with diarrhea feces detection, the test strip with the commercially available test strip (German R-biopharm company) coincidence rate, the positive detection rate lower than the commercially available ELISA Kit (R-biopharm German company (R-Biopharm) products). Conclusion: 1. Successfully cultured by trophozoites of Giardia lamblia in vitro, and get the trophozoites of soluble antigens and excretory secretory antigen.2 in trophozoites of Giardia lamblia antigen immunized rabbits successfully Giardia rabbit polyclonal antibody (Polyclonal, antibody,.3, PcAb) in trophozoites of Giardia lamblia in immunized mice and hybridoma fusion And screening, successfully obtained Twelve Strains of monoclonal antibodies. The strain EB2 secreted supernatant titer high and stable secretion of antibody can specifically recognize the trophozoites of Giardia lamblia soluble antigens and excretory secretory antigen respectively. The relative molecular weight of approximately 175 kDa and 191 kDa 2 protein bands, and Escherichia coli, Ascaris suum, man B.H, Schistosoma japonicum and Schistosoma japonicum and Paragonimus antigen had no cross reaction with.4, successfully established the method of double antibody sandwich ELISA system using McAb and Giardia PcAb,.5 for the detection of fecal Giardia antigen, Giardia McAb and PcAb successfully established colloidal gold strip for rapid detection of fecal based on the sample of Giardia antigen, and has high sensitivity, specificity and stability.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R446.6;R532.1

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