抗膽汁螺桿菌單克隆抗體的研制及其初步應用
發(fā)布時間：2018-07-28 14:35
【摘要】： 以膽汁螺桿菌等為主的嚙齒動物螺桿菌在實驗嚙齒類動物的感染已引起人們的極大關注,使之成為各國實驗動物微生物學質量標準中必須排除的病原菌。但我國尚缺乏有效的檢測方法,至今未將其列入國家標準。血清學檢測方法以其靈敏度高、簡便快捷及費用相對低廉等各種優(yōu)點已經廣泛用于病原微生物感染的實驗室診斷,但其檢測結果準確性的關鍵是檢測試劑的特異性和敏感性。本研究利用B細胞雜交瘤技術制備抗膽汁螺桿菌的特異性單克隆抗體(以下簡稱單抗),進而建立以檢測抗原為目的的雙抗體夾心ELISA法及間接免疫熒光法,以期發(fā)展起一種特異、敏感和快速的實驗動物膽汁螺桿菌檢測方法。為此,我們從以下幾個方面進行了研究。 首先,單抗的制備與鑒定。使用自行分離自實驗小鼠的膽汁螺桿菌B2m株全菌抗原免疫BABL/c小鼠,經細胞融合,用ELISA法篩選獲得A～K共11個陽性雜交瘤細胞株,其分泌抗體的效價最高達1:4×10~5以上,并與實驗動物常見的15種病原菌呈陰性反應;IgG亞類為IgG_(2a)和IgG_(2b);免疫印跡試驗顯示,6株(A～F)與膽汁螺桿菌大約相對分子質量(17、20、21、30、52、66)×10~3的抗原特異結合,5株(G～K)皆與膽汁螺桿菌、幽門螺桿菌等三種螺桿菌大約相對分子質量(52、82)×10~3的抗原呈陽性反應,表明A～F株針對的是膽汁螺桿菌特異性抗原,G～K株可能具有屬特異性。 其次,我們建立了以單抗為診斷抗體的雙抗體夾心ELISA法及間接免疫熒光法(IFA),并初步探索其用于實驗動物膽汁螺桿菌隱性感染檢測的可行性。 在雙抗體夾心ELISA法,采用最佳配對實驗,篩選出以單抗D、E作為包被抗體、C-辣根過氧化物酶標記(C-HRP)作為檢測抗體的配對,對膽汁螺桿菌抗原的檢測限均可達到ng級水平;用于膽汁螺桿菌感染陽性鼠群小鼠盲腸內容物的檢測顯示5/10、4/10的陽性。 在IFA,通過6種單抗的比較,確定了單抗C作為診斷抗體。該單抗與膽汁螺桿菌感染陽性鼠群小鼠盲腸內容物反應,熒光顯微鏡下能清晰地觀察到螺旋狀、周質纖毛纏繞的典型膽汁螺桿菌菌體形態(tài),與免疫血清比較,背景更清晰,很容易排除假陽性反應,并克服了雙抗體夾心ELISA的不足。其用于膽汁螺桿菌感染陽性鼠群小鼠盲腸內容物的檢測顯示6/10的陽性。 最后,通過PCR法對上述陽性動物群進行檢測,陽性數(shù)為8/10。由此可見,雙抗體夾心ELISA法與之符合率為50～60%,IFA符合率為75%。因此,可以認為所建立的兩種血清學方法基本能滿足實驗嚙齒類膽汁螺桿菌隱性感染的檢測。 綜上所述,本研究采用B淋巴細胞雜交瘤細胞技術成功地制備出了11株較高特異性和敏感性的抗膽汁螺桿菌單抗,并以其為診斷抗體建立起以檢測抗原為目的的雙抗體夾心ELISA法和間接免疫熒光法。在實驗小鼠膽汁螺桿菌自然感染的檢測中初步顯示基本能滿足實驗動物質量檢測需求。其中抗多種螺桿菌單抗在進一步的檢測方法學研究中有重要意義。本研究為膽汁螺桿菌血清學常規(guī)檢測方法的完善、試劑盒的研制提供了保障,也為其他嚙齒動物螺桿菌血清學檢測方法的建立奠定了基礎。
[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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
本文編號：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.
【學位授予單位】：中國協(xié)和醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2008
【分類號】：R392
【引證文獻】
相關期刊論文 前1條
1 丁聰;馮潔;謝建云;陳慶慶;;應用PCR方法調查實驗大小鼠螺桿菌感染情況[J];揚州大學學報(農業(yè)與生命科學版);2012年02期
相關碩士學位論文 前2條
1 丁聰;實驗大小鼠螺桿菌流行病學調查及肝螺桿菌flaB基因的表達[D];揚州大學;2011年
2 李瑞嬌;嚙齒動物螺桿菌多重PCR檢測方法的建立及流行病學調查[D];東華大學;2013年
，本文編號：2150548
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