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  本文關鍵詞： O157:H7 消減免疫法 單克隆抗體 膠體金　出處：《中國人民解放軍軍事醫(yī)學科學院》2008年碩士論文　論文類型：學位論文

【摘要】： 大腸桿菌O157:H7已成為威脅人類健康的重要致病菌之一。由于其致病力強、感染劑量低、潛伏期長,使得對傳染源的控制、監(jiān)測及追蹤成為難題。因此發(fā)展快速、敏感、特異的檢測方法是確保飲水和食品安全,預防大腸桿菌O157:H7感染發(fā)生的重要手段。 目前,在大腸桿菌O157:H7的免疫學檢測方法中,所用抗體多為多克隆抗體。抗O157:H7的多克隆抗體與抗其它腸道細菌的抗體存在著嚴重交叉反應,通常的純化不能滿足精確的血清學檢驗需求。而用單克隆抗體進行檢測的研究國內(nèi)外報道不多,已有的報道多為使用菌體抗原直接免疫。制備針對細菌抗原的單克隆抗體存在主要的問題是篩選困難,細菌抗原的單克隆抗體易與其它型別腸桿菌發(fā)生交叉反應,不能定向地獲得有價值的抗體。 為解決篩選困難的問題,本實驗利用消減免疫法制備特異針對大腸桿菌O157:H7的單克隆抗體,從而為其檢測和防治提供物質(zhì)基礎。 1.消減免疫法制備大腸桿菌O157:H7單克隆抗體:以甲醛固定法制備抗原,消減免疫法免疫BALB/c小鼠,間接ELISA篩選陽性克隆,有限稀釋法對陽性孔細胞進行三次克隆化,最終獲得19株能穩(wěn)定分泌抗大腸桿菌O157:H7抗體的雜交瘤細胞株,分別為1C6、1D8、1H6、3F5、3G12、4A7、4D7、5A2、5A5、5A9、5C12、5D8、5F10、5F11、5H4、6C11、6D12、7F1、7F8。雜交瘤細胞染色體分析:經(jīng)Gimsa染色,顯微鏡下觀察,雜交瘤細胞株的染色體數(shù)目均在為96-105條之間,基本上是小鼠脾細胞與SP2/0骨髓瘤細胞兩者染色體數(shù)目之和。 2.單克隆抗體特性鑒定:采用間接ELISA檢測19株單克隆抗體與81株細菌交叉反應情況,發(fā)現(xiàn)1C6、1D8、1H6、4A7、5A2、5D8等6株雜交瘤細胞株分泌的單克隆抗體特異性較好,且與4株分離株的大腸桿菌O157:H7(E11、E22、北醫(yī)、天防)有很好的抗體反應譜。酶聯(lián)免疫斑點實驗結(jié)果與間接ELISA相似。對1C6、1D8、4A7、5A2、5D8 5株單克隆抗體進行了亞類、親和力、免疫印跡等特性鑒定。5株雜交瘤細胞均能穩(wěn)定分泌抗體,細胞培養(yǎng)液上清及腹水的ELISA效價分別為1:103和1:106。其中1D8、4A7免疫球蛋白類型為IgG,1C6、5A2、5D8為IgM。5株單克隆抗體均具有很高的親和力,5A2親和力較高,達1.47×1011(mol/L)-1。大腸桿菌O157:H7抗原的免疫印跡圖譜顯示5A2和5D8單克隆抗體在25kDa相對分子質(zhì)量的位置都有一抗原決定簇;而1C6、1D8和4A7單克隆抗體對應抗原表位的相對分子質(zhì)量由2或3個相近的抗原蛋白構(gòu)成。 3.單克隆抗體的初步應用:制備4A7、5A2兩種免疫膠體金探針,用于免疫滲濾實驗。5A2株免疫膠體金探針對O157免疫滲濾實驗特異性良好,與74株非O157菌株均無交叉反應。5A2株免疫膠體金探針免疫滲濾實驗檢測大腸桿菌O157:H7的靈敏度5.5×106cfu/mL,4A7株免疫膠體金探針相加實驗和銀加強實驗均能將檢測的靈敏度提高一個數(shù)量級。免疫納米磁顆粒濃集與免疫膠體金滲濾實驗結(jié)合檢測大腸桿菌O157:H7,經(jīng)銀加強實驗,可檢測大腸桿菌O157:H7菌懸液濃度為5.5×104cfu/mL。 通過制備和初步應用抗大腸桿菌O157:H7特異性單克隆抗體,為進一步臨床應用打下基礎,為大腸桿菌O157:H7的檢測和疾病診斷提供一種較好的試劑和快速、準確、實用的診斷方法。
[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.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392

【引證文獻】

相關碩士學位論文 前1條

1 才琳;抗黃曲霉毒素B1單克隆抗體和單鏈抗體的制備及活性研究[D];黑龍江八一農(nóng)墾大學;2010年

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本文編號：1535551