抗胆汁螺杆菌单克隆抗体的研制及其初步应用
发布时间：2018-07-28 14:35
【摘要】： 以胆汁螺杆菌等为主的啮齿动物螺杆菌在实验啮齿类动物的感染已引起人们的极大关注,使之成为各国实验动物微生物学质量标准中必须排除的病原菌。但我国尚缺乏有效的检测方法,至今未将其列入国家标准。血清学检测方法以其灵敏度高、简便快捷及费用相对低廉等各种优点已经广泛用于病原微生物感染的实验室诊断,但其检测结果准确性的关键是检测试剂的特异性和敏感性。本研究利用B细胞杂交瘤技术制备抗胆汁螺杆菌的特异性单克隆抗体(以下简称单抗),进而建立以检测抗原为目的的双抗体夹心ELISA法及间接免疫荧光法,以期发展起一种特异、敏感和快速的实验动物胆汁螺杆菌检测方法。为此,我们从以下几个方面进行了研究。 首先,单抗的制备与鉴定。使用自行分离自实验小鼠的胆汁螺杆菌B2m株全菌抗原免疫BABL/c小鼠,经细胞融合,用ELISA法筛选获得A～K共11个阳性杂交瘤细胞株,其分泌抗体的效价最高达1:4×10~5以上,并与实验动物常见的15种病原菌呈阴性反应;IgG亚类为IgG_(2a)和IgG_(2b);免疫印迹试验显示,6株(A～F)与胆汁螺杆菌大约相对分子质量(17、20、21、30、52、66)×10~3的抗原特异结合,5株(G～K)皆与胆汁螺杆菌、幽门螺杆菌等三种螺杆菌大约相对分子质量(52、82)×10~3的抗原呈阳性反应,表明A～F株针对的是胆汁螺杆菌特异性抗原,G～K株可能具有属特异性。 其次,我们建立了以单抗为诊断抗体的双抗体夹心ELISA法及间接免疫荧光法(IFA),并初步探索其用于实验动物胆汁螺杆菌隐性感染检测的可行性。 在双抗体夹心ELISA法,采用最佳配对实验,筛选出以单抗D、E作为包被抗体、C-辣根过氧化物酶标记(C-HRP)作为检测抗体的配对,对胆汁螺杆菌抗原的检测限均可达到ng级水平;用于胆汁螺杆菌感染阳性鼠群小鼠盲肠内容物的检测显示5/10、4/10的阳性。 在IFA,通过6种单抗的比较,确定了单抗C作为诊断抗体。该单抗与胆汁螺杆菌感染阳性鼠群小鼠盲肠内容物反应,荧光显微镜下能清晰地观察到螺旋状、周质纤毛缠绕的典型胆汁螺杆菌菌体形态,与免疫血清比较,背景更清晰,很容易排除假阳性反应,并克服了双抗体夹心ELISA的不足。其用于胆汁螺杆菌感染阳性鼠群小鼠盲肠内容物的检测显示6/10的阳性。 最后,通过PCR法对上述阳性动物群进行检测,阳性数为8/10。由此可见,双抗体夹心ELISA法与之符合率为50～60%,IFA符合率为75%。因此,可以认为所建立的两种血清学方法基本能满足实验啮齿类胆汁螺杆菌隐性感染的检测。 综上所述,本研究采用B淋巴细胞杂交瘤细胞技术成功地制备出了11株较高特异性和敏感性的抗胆汁螺杆菌单抗,并以其为诊断抗体建立起以检测抗原为目的的双抗体夹心ELISA法和间接免疫荧光法。在实验小鼠胆汁螺杆菌自然感染的检测中初步显示基本能满足实验动物质量检测需求。其中抗多种螺杆菌单抗在进一步的检测方法学研究中有重要意义。本研究为胆汁螺杆菌血清学常规检测方法的完善、试剂盒的研制提供了保障,也为其他啮齿动物螺杆菌血清学检测方法的建立奠定了基础。
[Abstract]:The infection of Helicobacter pylori, which is the main bile screw bacteria, has aroused great concern in the experimental rodents, making it the pathogenic bacteria that must be eliminated in the standard of the microbiology of experimental animals in various countries. However, our country still lacks effective detection methods and has not been included in the national standard. Many advantages, such as high sensitivity, simple, quick and low cost, have been widely used in the laboratory diagnosis of pathogenic microorganism infection, but the key to the accuracy of the detection results is the specificity and sensitivity of the detection reagents. This study uses B cell hybridoma technology to prepare the specific monoclonal antibodies against the bile spirilli (hereinafter referred to as the following abbreviation) In order to develop a specific, sensitive and rapid test method for the detection of Helicobacter pylori in experimental animals, we have studied the following aspects in the following aspects: the double antibody sandwich ELISA method and indirect immunofluorescence method for detecting antigen.
First, the preparation and identification of the monoclonal antibody. The BABL/c mice were immunized with the total bacteria antigen of the B2m strain of Helicobacter pylori isolated from the experimental mice. After cell fusion, 11 positive hybridoma cells from A to K were screened by ELISA method. The titer of the secretory antibody was up to 1:4 * 10~5 above, and was negative against the 15 common pathogenic bacteria in the experimental animals. IgG subclasses were IgG_ (2a) and IgG_ (2b); immunoblotting showed that 6 strains (A to F) were specifically associated with the relative molecular mass (17,20,21,30,52,66) * 10~3 of Helicobacter pylori, and 5 (G to K) were all positive for the relative molecular mass of the three kinds of Helicobacter pylori, Helicobacter pylori, etc., about the relative molecular mass (52,82). The F strain is targeted to the specific antigens of bile bacteria, and G to K strains may be of specific genus.
Secondly, we established a double antibody sandwich ELISA method and indirect immunofluorescence (IFA) with monoclonal antibody as diagnostic antibody, and preliminarily explored its feasibility for detection of recessive infection of Helicobacter pylori in experimental animals.
In the double antibody sandwich ELISA method, the best paired experiment was used to screen out the monoclonal antibody D, E as a clad antibody and the C- horseradish peroxidase labeling (C-HRP) as a detection antibody. The detection limit for the antigen of the Helicobacter beliscum could reach the level of ng. The detection of the content of the cecum in the mice with positive BBA infection mice showed 5/10,4/1 0 of the positive.
In IFA, the monoclonal antibody C was determined as a diagnostic antibody by comparison of 6 McAbs. The monoclonal antibody was reacted with the cecum content of the mice infected by the infection of the Helicobacter pylori, and the morphology of the typical spiral and periplasmic cilia was clearly observed under the fluorescence microscope. Compared with the immune sera, the background was clearer and easy to be excluded. The false positive reaction and overcoming the deficiency of double antibody sandwich ELISA showed that 6/10 was positive for cecal contents in mice infected with positive bile duct infection.
Finally, the positive fauna was detected by PCR, the positive number was 8/10., the coincidence rate of the double antibody sandwich ELISA method was 50 ~ 60%, and the coincidence rate of IFA was 75%., so the two serological methods established could basically meet the detection of the recessive infection of the experimental rodent Helicobacter.
To sum up, 11 highly specific and sensitive anti bile screw bacteria monoclonal antibodies were successfully prepared by B lymphocyte hybridoma cell technique, and the double antibody sandwich ELISA and indirect immunofluorescence were established for the detection of antigen as the diagnostic antibody. The preliminary results showed that it was basically able to meet the needs of testing the quality of experimental animals. Among them, it is of great significance in the further study of the detection methodology. This study provides a guarantee for the improvement of the routine testing methods for the serology of the Helicobacter pylori, the development of the kit and the serological test for other rodent Helicobacter The establishment of the law laid the foundation.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
本文编号：2150548
[Abstract]:The infection of Helicobacter pylori, which is the main bile screw bacteria, has aroused great concern in the experimental rodents, making it the pathogenic bacteria that must be eliminated in the standard of the microbiology of experimental animals in various countries. However, our country still lacks effective detection methods and has not been included in the national standard. Many advantages, such as high sensitivity, simple, quick and low cost, have been widely used in the laboratory diagnosis of pathogenic microorganism infection, but the key to the accuracy of the detection results is the specificity and sensitivity of the detection reagents. This study uses B cell hybridoma technology to prepare the specific monoclonal antibodies against the bile spirilli (hereinafter referred to as the following abbreviation) In order to develop a specific, sensitive and rapid test method for the detection of Helicobacter pylori in experimental animals, we have studied the following aspects in the following aspects: the double antibody sandwich ELISA method and indirect immunofluorescence method for detecting antigen.
First, the preparation and identification of the monoclonal antibody. The BABL/c mice were immunized with the total bacteria antigen of the B2m strain of Helicobacter pylori isolated from the experimental mice. After cell fusion, 11 positive hybridoma cells from A to K were screened by ELISA method. The titer of the secretory antibody was up to 1:4 * 10~5 above, and was negative against the 15 common pathogenic bacteria in the experimental animals. IgG subclasses were IgG_ (2a) and IgG_ (2b); immunoblotting showed that 6 strains (A to F) were specifically associated with the relative molecular mass (17,20,21,30,52,66) * 10~3 of Helicobacter pylori, and 5 (G to K) were all positive for the relative molecular mass of the three kinds of Helicobacter pylori, Helicobacter pylori, etc., about the relative molecular mass (52,82). The F strain is targeted to the specific antigens of bile bacteria, and G to K strains may be of specific genus.
Secondly, we established a double antibody sandwich ELISA method and indirect immunofluorescence (IFA) with monoclonal antibody as diagnostic antibody, and preliminarily explored its feasibility for detection of recessive infection of Helicobacter pylori in experimental animals.
In the double antibody sandwich ELISA method, the best paired experiment was used to screen out the monoclonal antibody D, E as a clad antibody and the C- horseradish peroxidase labeling (C-HRP) as a detection antibody. The detection limit for the antigen of the Helicobacter beliscum could reach the level of ng. The detection of the content of the cecum in the mice with positive BBA infection mice showed 5/10,4/1 0 of the positive.
In IFA, the monoclonal antibody C was determined as a diagnostic antibody by comparison of 6 McAbs. The monoclonal antibody was reacted with the cecum content of the mice infected by the infection of the Helicobacter pylori, and the morphology of the typical spiral and periplasmic cilia was clearly observed under the fluorescence microscope. Compared with the immune sera, the background was clearer and easy to be excluded. The false positive reaction and overcoming the deficiency of double antibody sandwich ELISA showed that 6/10 was positive for cecal contents in mice infected with positive bile duct infection.
Finally, the positive fauna was detected by PCR, the positive number was 8/10., the coincidence rate of the double antibody sandwich ELISA method was 50 ~ 60%, and the coincidence rate of IFA was 75%., so the two serological methods established could basically meet the detection of the recessive infection of the experimental rodent Helicobacter.
To sum up, 11 highly specific and sensitive anti bile screw bacteria monoclonal antibodies were successfully prepared by B lymphocyte hybridoma cell technique, and the double antibody sandwich ELISA and indirect immunofluorescence were established for the detection of antigen as the diagnostic antibody. The preliminary results showed that it was basically able to meet the needs of testing the quality of experimental animals. Among them, it is of great significance in the further study of the detection methodology. This study provides a guarantee for the improvement of the routine testing methods for the serology of the Helicobacter pylori, the development of the kit and the serological test for other rodent Helicobacter The establishment of the law laid the foundation.
【学位授予单位】：中国协和医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392
【引证文献】
相关期刊论文 前1条
1 丁聪;冯洁;谢建云;陈庆庆;;应用PCR方法调查实验大小鼠螺杆菌感染情况[J];扬州大学学报(农业与生命科学版);2012年02期
相关硕士学位论文 前2条
1 丁聪;实验大小鼠螺杆菌流行病学调查及肝螺杆菌flaB基因的表达[D];扬州大学;2011年
2 李瑞娇;啮齿动物螺杆菌多重PCR检测方法的建立及流行病学调查[D];东华大学;2013年
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