本文關(guān)鍵詞：基于單克隆抗體技術(shù)的賈第蟲膠體金試紙條診斷方法的建立及其初步應(yīng)用　出處：《中國(guó)疾病預(yù)防控制中心》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 藍(lán)氏賈第鞭毛蟲 單克隆抗體 酶聯(lián)免疫吸附試驗(yàn) 膠體金試紙條

【摘要】：藍(lán)氏賈第鞭毛蟲(賈第蟲)是一種引起人和其他哺乳動(dòng)物腹瀉的機(jī)會(huì)性致病寄生蟲,其主要寄生在人和某些哺乳動(dòng)物的小腸,引起人的腹瀉、腹痛和消化不良等胃腸道癥狀。賈第蟲滋養(yǎng)體引發(fā)的賈第蟲病,目前已被列為全世界危害人類健康的十種主要寄生蟲病之一,該病的嚴(yán)重性和危害性日益受到重視。目前,利用顯微鏡對(duì)樣本中的包囊和滋養(yǎng)體進(jìn)行鏡檢的病原學(xué)檢測(cè)方法,被認(rèn)為是診斷賈第蟲感染的"金標(biāo)準(zhǔn)"。該法現(xiàn)場(chǎng)應(yīng)用時(shí)費(fèi)時(shí)費(fèi)力,勞動(dòng)強(qiáng)度大,檢出率低,在感染率/度低時(shí)容易出現(xiàn)漏檢。免疫學(xué)檢測(cè)方法具有敏感性強(qiáng)、特異性高、操作簡(jiǎn)單且易于現(xiàn)場(chǎng)推廣等優(yōu)點(diǎn),賈第蟲的免疫學(xué)檢測(cè)引起研究人員更多的關(guān)注。隨著單克隆抗體技術(shù)的應(yīng)用,進(jìn)一步加強(qiáng)了賈第蟲免疫學(xué)檢測(cè)的特異性。本研究擬以賈第蟲滋養(yǎng)體免疫小鼠制備單克隆抗體,建立診斷賈第蟲病的免疫學(xué)診斷方法。1、賈第蟲單克隆抗體制備以賈第蟲滋養(yǎng)體免疫BALB/c小鼠,利用細(xì)胞融合技術(shù)建立抗賈第蟲抗原雜交瘤細(xì)胞株并制備單克隆抗體(Monoclonal antibody,McAb)。采用免疫球蛋白亞類(型)檢測(cè)試劑盒鑒定McAb的類型及亞型;蛋白質(zhì)印跡鑒定McAb對(duì)賈第蟲滋養(yǎng)體可溶性抗原和排泄分泌抗原的識(shí)別;酶聯(lián)免疫吸附試驗(yàn)檢測(cè)McAb與賈第蟲滋養(yǎng)體反應(yīng)性,并檢測(cè)與其他蟲種的交叉反應(yīng)。結(jié)果獲得12株分泌賈第蟲單克隆抗體細(xì)胞株,檢測(cè)后均為IgG1亞型。其中EB2株能識(shí)別賈第蟲滋養(yǎng)體可溶性抗原和排泄分泌抗原中相對(duì)分子量分別為175KDa和191KDa的2條蛋白條帶,與大腸桿菌、豬蛔蟲、人芽囊原蟲、日本血吸蟲蟲卵、日本血吸蟲成蟲和衛(wèi)氏并殖吸蟲抗原均無交叉反應(yīng)。2、建立雙抗體夾心ELISA法檢測(cè)賈第蟲抗原將制備的賈第蟲單抗和多抗進(jìn)行純化后,建立雙抗體夾心ELISA方法檢測(cè)賈第蟲糞抗原。結(jié)果初步建立了單抗-抗原-多抗-二抗的雙抗體夾心ELISA方法檢測(cè)賈第蟲糞抗原,并對(duì)其條件進(jìn)行了優(yōu)化。該方法的靈敏度為1:10倍稀釋,批內(nèi)變異系數(shù)CV5%,且與其他蟲種無交叉反應(yīng),是一個(gè)快速、特異、靈敏的檢測(cè)方法,為進(jìn)一步的現(xiàn)場(chǎng)應(yīng)用奠定了基礎(chǔ)。3、膠體金試紙條試制檢測(cè)賈第蟲抗原成功制備膠體金試紙條。該試紙條具有較好的敏感性、特異性和穩(wěn)定性。檢測(cè)賈第蟲滋養(yǎng)體抗原的檢測(cè)限為0.25μg。該試條與其他蟲種模擬糞樣無交叉反應(yīng);通過對(duì)10份臨床腹瀉患者糞樣檢測(cè),該試紙條與市售試紙條(德國(guó)拜發(fā)公司)符合率一致,檢測(cè)陽性率低于市售ELISA試劑盒(德國(guó)拜發(fā)公司(R-Biopharm)產(chǎn)品)。結(jié)論:1、成功進(jìn)行賈第蟲滋養(yǎng)體的體外培養(yǎng),并獲得滋養(yǎng)體可溶性抗原和排泄分泌抗原。2、以賈第蟲滋養(yǎng)體抗原免疫家兔成功獲得賈第蟲兔多克隆抗體(Polyclonal antibody,PcAb)。3、以賈第蟲滋養(yǎng)體免疫小鼠并進(jìn)行雜交瘤融合和篩選,成功獲得十二株單克隆抗體細(xì)胞株。其中EB2株分泌上清效價(jià)較高且穩(wěn)定分泌抗體,能特異識(shí)別賈第蟲滋養(yǎng)體可溶性抗原和排泄分泌抗原中相對(duì)分子量分別大約為175 kDa和191 kDa的2條蛋白條帶,與大腸桿菌、豬蛔蟲、人芽囊原蟲、日本血吸蟲蟲卵、日本血吸蟲成蟲和衛(wèi)氏并殖吸蟲抗原均無交叉反應(yīng)。4、利用賈第蟲McAb和PcAb成功建立雙抗體夾心ELISA法體系,用于糞樣賈第蟲抗原的檢測(cè)。5、基于賈第蟲McAb和PcAb成功建立膠體金試紙條用于糞樣賈第蟲抗原的快速檢測(cè),并具有較高的敏感性、特異性和穩(wěn)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.

【學(xué)位授予單位】：中國(guó)疾病預(yù)防控制中心
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R446.6;R532.1

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