抗S. paratyphi A O2抗原单克隆抗体制备及ELISA检测方法的建立

发布时间：2018-03-23 15:32

本文选题：甲型副伤寒沙门氏菌　切入点：快速检测　出处：《华中农业大学》2008年硕士论文

【摘要】： 伤寒副伤寒是由伤寒杆菌(Salmonella typhi)和副伤寒杆菌甲、乙、丙(S.paratyphiA,B,C)引起的急性肠道传染病,在全球分布很广,估计每年伤寒发病1600万～1700万,死亡60万,主要在发展中国家。以前我国伤寒发病占伤寒副伤寒总发病的90%以上,后来由于伤寒Vi疫苗的广泛使用,20世纪90年代中期,流行菌株发生转变,我国部分省份伤寒病例逐渐下降,而由甲型副伤寒沙门氏菌引起的副伤寒病例却大幅上升,并呈现由散发到局部暴发,由沿海省份向内地省份推进的流行趋势,近几年来成为我国一些地区传染病突发事件的主要病种,常发展成重大疫情,急需建立快速、准确的诊断方法。实验室常规检测、诊断甲型副伤寒的主要方法为细菌培养、肥达氏凝集反应和伤寒血球快速诊断。这些方法费时费力,不利于早期诊断,给流行病学专家在追踪传染源、早期控制与临床医生早期诊断工作带来了一定的难度。以酶联免疫吸附试验为原理建立起来的各种免疫学检测方法,特异性好、灵敏度高、操作简便、样品处理量大,已经广泛应用于临床和食品中病原菌的检测。但是目前国内外还很少有用ELISA方法检测甲型副伤寒沙门氏菌的报道,本研究中,我们先制备抗甲型副伤寒沙门氏菌O2抗原的单克隆抗体和多克隆抗体,然后以此为基础建立夹心ELISA方法,快速检测该菌。 1.甲型副伤寒沙门氏菌单克隆抗体的制备与鉴定甲型副伤寒沙门氏菌的菌体抗原包括O1、O2和O12三种,其中O2抗原为该菌所特有。我们选用甲型副伤寒沙门氏菌模式菌株50001,经沸水浴2h后制备菌体抗原,免疫5只6-8周龄雌性Balb/C小鼠,经过11次细胞融合筛选出两株分泌特异性抗体的杂交瘤细胞株,2C6和4F10,其腹水效价分别为1:25600和1:12800,通过SDS-PAGE分析,两株杂交瘤细胞所产生的单克隆抗体分子量均在180KD左右。2C6和4F10分泌的单克隆抗体不与伤寒等其它细菌发生交叉反应,为特异性针对甲型副伤寒沙门氏菌02抗原的抗体。 2.夹心ELISA检测方法的建立用相同抗原免疫兔子,制备多克隆抗体,选用单抗2C6和HRP标记的羊抗鼠酶标二抗,建立检测甲型副伤寒沙门氏菌双抗夹心ELISA方法。实验结果表明,此ELISA方法不与属内外常见致病菌发生交叉反应,特异性较好;检测限达到10~5CFU/mL,灵敏度高;整个检测过程4-5h即可完成,为食品检验以及临床医生早期诊断提供一种快速、灵敏、有效的方法。通过以上研究工作,初步确定了甲型副伤寒沙门氏菌的夹心ELISA检测程序并优化了反应条件。其方法的稳定性和实际应用情况还有待进一步完善,以便为试剂盒的研制打下基础。
[Abstract]:Typhoid paratyphoid is an acute enteric disease caused by Salmonella typhimurium (Salmonella typhimurium) and Bacillus paratyphiae A, B, C). It is widely distributed in the world. It is estimated that typhoid fever has an incidence of 16 million ~ 17 million per year and causes 600000 deaths. Mainly in developing countries. Previously, typhoid accounted for more than 90% of the total typhoid and paratyphoid fever in China. Later, due to the widespread use of typhoid Vi vaccine, epidemic strains changed in the mid-1990s. The number of typhoid cases in some provinces of China has gradually decreased, while the cases of paratyphoid fever caused by salmonella paratyphoid A have increased significantly, showing a trend of epidemic from sporadic to local outbreaks and from coastal provinces to inland provinces. In recent years, it has become the main type of infectious disease emergency in some regions of our country, and it often develops into a major epidemic situation. It is urgent to establish a rapid and accurate diagnostic method. Routine laboratory tests show that the main methods for diagnosis of paratyphoid A are bacterial culture, Widal's agglutination and rapid diagnosis of typhoid blood cells. These methods are time-consuming and laborious, not conducive to early diagnosis, giving epidemiologists the ability to track the source of infection. All kinds of immunological detection methods based on enzyme linked immunosorbent assay (Elisa) are characterized by good specificity, high sensitivity, simple operation and large amount of sample handling. It has been widely used in the detection of pathogenic bacteria in clinic and food. However, there are few reports about the detection of salmonella paratyphoid A by ELISA method at home and abroad. We first prepared monoclonal antibody and polyclonal antibody against O _ 2 antigen of Salmonella paratyphi A and then developed a sandwich ELISA method for rapid detection of the bacterium. 1. Preparation and identification of monoclonal antibody against salmonella paratyphoid A. The bacterial antigens of Salmonella paratyphi A include O _ 1O _ 2 and O _ 12, among which O _ 2 antigen is unique to this strain. We selected the model strain 50001 of Salmonella paratyphoid A and prepared the antigen after 2 hours of boiling water bath. Five 6-8 week-old female Balb/C mice were immunized. Two hybridoma cell lines 2C6 and 4F10 secreting specific antibodies were screened by cell fusion for 11 times. The ascites titers were 1: 25600 and 1: 12800, respectively. The ascites titers were analyzed by SDS-PAGE. The molecular weight of monoclonal antibodies produced by the two hybridoma cells were about .2C6 of 180KD and the monoclonal antibodies secreted by 4F10 did not cross react with other bacteria such as typhoid fever, so they were specific antibodies against the antigen of Salmonella paratyphi A 02. 2. Establishment of sandwich ELISA detection method. Rabbits were immunized with the same antigen and polyclonal antibodies were prepared. The double antibody sandwich ELISA method for detection of Salmonella paratyphi A was established by using the second antibody labeled by monoclonal antibody 2C6 and HRP. This ELISA method does not cross react with common pathogenic bacteria inside and outside of the family, and has good specificity, the detection limit is up to 105 CFU / mL, the sensitivity is high, the whole detection process can be completed 4-5 hours, which provides a fast and sensitive method for food testing and early diagnosis by clinicians. An effective method. Through the above research work, the sandwich ELISA detection procedure of Salmonella paratyphi A was preliminarily determined and the reaction conditions were optimized. The stability and practical application of the method need to be further improved. In order to lay a foundation for the development of the kit.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392;R446.6;R516.3

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