消减免疫法制备大肠杆菌O157：H7单克隆抗体及初步应用

发布时间：2018-02-25 23:13

本文关键词： O157:H7 消减免疫法单克隆抗体胶体金　出处：《中国人民解放军军事医学科学院》2008年硕士论文　论文类型：学位论文

【摘要】： 大肠杆菌O157:H7已成为威胁人类健康的重要致病菌之一。由于其致病力强、感染剂量低、潜伏期长,使得对传染源的控制、监测及追踪成为难题。因此发展快速、敏感、特异的检测方法是确保饮水和食品安全,预防大肠杆菌O157:H7感染发生的重要手段。目前,在大肠杆菌O157:H7的免疫学检测方法中,所用抗体多为多克隆抗体。抗O157:H7的多克隆抗体与抗其它肠道细菌的抗体存在着严重交叉反应,通常的纯化不能满足精确的血清学检验需求。而用单克隆抗体进行检测的研究国内外报道不多,已有的报道多为使用菌体抗原直接免疫。制备针对细菌抗原的单克隆抗体存在主要的问题是筛选困难,细菌抗原的单克隆抗体易与其它型别肠杆菌发生交叉反应,不能定向地获得有价值的抗体。为解决筛选困难的问题,本实验利用消减免疫法制备特异针对大肠杆菌O157:H7的单克隆抗体,从而为其检测和防治提供物质基础。 1.消减免疫法制备大肠杆菌O157:H7单克隆抗体:以甲醛固定法制备抗原,消减免疫法免疫BALB/c小鼠,间接ELISA筛选阳性克隆,有限稀释法对阳性孔细胞进行三次克隆化,最终获得19株能稳定分泌抗大肠杆菌O157:H7抗体的杂交瘤细胞株,分别为1C6、1D8、1H6、3F5、3G12、4A7、4D7、5A2、5A5、5A9、5C12、5D8、5F10、5F11、5H4、6C11、6D12、7F1、7F8。杂交瘤细胞染色体分析:经Gimsa染色,显微镜下观察,杂交瘤细胞株的染色体数目均在为96-105条之间,基本上是小鼠脾细胞与SP2/0骨髓瘤细胞两者染色体数目之和。 2.单克隆抗体特性鉴定:采用间接ELISA检测19株单克隆抗体与81株细菌交叉反应情况,发现1C6、1D8、1H6、4A7、5A2、5D8等6株杂交瘤细胞株分泌的单克隆抗体特异性较好,且与4株分离株的大肠杆菌O157:H7(E11、E22、北医、天防)有很好的抗体反应谱。酶联免疫斑点实验结果与间接ELISA相似。对1C6、1D8、4A7、5A2、5D8 5株单克隆抗体进行了亚类、亲和力、免疫印迹等特性鉴定。5株杂交瘤细胞均能稳定分泌抗体,细胞培养液上清及腹水的ELISA效价分别为1:103和1:106。其中1D8、4A7免疫球蛋白类型为IgG,1C6、5A2、5D8为IgM。5株单克隆抗体均具有很高的亲和力,5A2亲和力较高,达1.47×1011(mol/L)-1。大肠杆菌O157:H7抗原的免疫印迹图谱显示5A2和5D8单克隆抗体在25kDa相对分子质量的位置都有一抗原决定簇;而1C6、1D8和4A7单克隆抗体对应抗原表位的相对分子质量由2或3个相近的抗原蛋白构成。 3.单克隆抗体的初步应用:制备4A7、5A2两种免疫胶体金探针,用于免疫渗滤实验。5A2株免疫胶体金探针对O157免疫渗滤实验特异性良好,与74株非O157菌株均无交叉反应。5A2株免疫胶体金探针免疫渗滤实验检测大肠杆菌O157:H7的灵敏度5.5×106cfu/mL,4A7株免疫胶体金探针相加实验和银加强实验均能将检测的灵敏度提高一个数量级。免疫纳米磁颗粒浓集与免疫胶体金渗滤实验结合检测大肠杆菌O157:H7,经银加强实验,可检测大肠杆菌O157:H7菌悬液浓度为5.5×104cfu/mL。通过制备和初步应用抗大肠杆菌O157:H7特异性单克隆抗体,为进一步临床应用打下基础,为大肠杆菌O157:H7的检测和疾病诊断提供一种较好的试剂和快速、准确、实用的诊断方法。
[Abstract]:Escherichia coli O157:H7 has become a threat to human health is one of the important pathogens. Because of its strong pathogenicity, infection of low dose, long incubation period, the control of the source of infection, monitoring and tracking becomes a problem. So the development of rapid, sensitive and specific method for the detection of drinking water and is to ensure food safety, prevention of Escherichia coli O157:H7 infection is an important means of happen.
At present, the immunological detection of E. coli O157:H7, the antibody is a polyclonal antibody. The polyclonal antibody of anti O157:H7 antibody and anti other intestinal bacteria exist serious cross reaction, purification can not meet the precision demand of serological tests. But usually single clone antibody of domestic detection reports not much has been reported for the use of direct somatic antigen immunization. Preparation of monoclonal antibodies against bacterial antigens are the main problem is the difficult screening of monoclonal antibody, bacterial antigens react with other types of Enterobacteriaceae, not directed to obtain antibody value.
In order to solve the problem of screening difficulty, the monoclonal antibody against Escherichia coli O157:H7 was prepared by subtractive immunization in order to provide a material basis for its detection and prevention.
1. subtractive immunization prepared Escherichia coli O157:H7 monoclonal antibody in formalin fixed preparation of antigen, subtractive immunization immunization of BALB/c mice. Positive clones were screened by indirect ELISA, limited dilution of positive hole cells were cloned three times, obtained 19 strains of hybridoma cell lines secreting anti Escherichia coli O157:H7 antibody, respectively. Analysis of 1C6,1D8,1H6,3F5,3G12,4A7,4D7,5A2,5A5,5A9,5C12,5D8,5F10,5F11,5H4,6C11,6D12,7F1,7F8. chromosome of hybridoma cells by Gimsa staining, observed under the microscope, the chromosome number of hybridoma cell lines were in 96-105, basically is the spleen cells of mice with SP2/0 myeloma cell chromosome number of both.
2. characterization of monoclonal antibody by indirect ELISA detection of 19 monoclonal antibodies and 81 strains of bacteria cross reaction, 1C6,1D8,1H6,4A7,5A2,5D8 found that 6 strains of hybridoma cell lines secreting monoclonal antibody specific for the better, and 4 strains of Escherichia coli O157:H7 isolates (E11, E22, Taipei, day -) have a good antibody response the spectrum of ELISPOT results with indirect ELISA. Similar to 1C6,1D8,4A7,5A2,5D8 5 monoclonal antibodies of subclass, affinity, Western blotting characterization of.5 hybridoma cells stably secreting antibody, cell culture supernatant and ascites titers of ELISA were 1:103 and 1:106. which 1D8,4A7 type of immunoglobulin for IgG, 1C6,5A2,5D8 for IgM.5 monoclonal antibodies have high affinity, high affinity 5A2, 1.47 * 1011 (mol/L) -1. of Escherichia coli O157:H7 antigen blotting map display 5A2 and 5D8 monoclonal antibodies have an antigenic determinant in the relative molecular mass of 25kDa, while the relative molecular mass of 1C6,1D8 and 4A7 monoclonal antibodies corresponding to epitopes is composed of 2 or 3 similar antigen proteins.
Preliminary application of 3. monoclonal antibodies: preparation of 4A7,5A2 two colloidal gold probe for immuno infiltration assay.5A2 strains of immune colloidal gold probe of O157 immuno infiltration assay showed good specificity, and 74 strains of non O157 strains had no cross reaction with.5A2 strain of immune colloidal gold immunofiltration test probe detection sensitivity of Escherichia coli O157:H7 * 5.5 106cfu/mL, 4A7 strains of immune colloidal gold probe experiments and experiments were added silver enhanced detection can improve the sensitivity of an order of magnitude. Detection of Escherichia coli O157:H7 with immune magnetic nano particles concentration and immunogold filtration experiments by silver enhancement experiment, detection of E. coli O157:H7 suspension concentration is 5.5 * 104cfu/mL.
Through preparation and preliminary application of specific monoclonal antibodies against Escherichia coli O157:H7, we will lay a foundation for further clinical application, and provide a better reagent and a fast, accurate and practical diagnostic method for detection and diagnosis of O157:H7.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【引证文献】