基于人血清中抗伤寒Vi多糖特异IgG抗体含量测定ELISA方法建立的研究

发布时间：2018-07-03 07:32

本文选题：伤寒沙门菌 + IgG抗体　；参考：《兰州大学》2017年硕士论文

【摘要】：伤寒是一种肠道感染的侵袭性疾病,伤寒沙门菌是引起伤寒的病原体,常通过摄入污染的水和食物传播,每年引起21,000,000例新增病例,216,000人死亡。在一些基础设施不完善的发展中国家是一种很常见的疾病。目前,伤寒的预防制剂主要是口服免疫的Ty21a减毒活疫苗和肌肉注射的伤寒Vi多糖疫苗。Ty21a减毒活疫苗存在基因背景不清楚、免疫效果不佳等缺陷,而尼泊尔和南非以及我国江苏、广西的临床试验证明,伤寒Vi多糖疫苗可提供70%左右的保护力,随着伤寒Vi多糖疫苗在世界范围内的广泛使用,其良好的安全性和70%左右的保护效果为预防伤寒疾病的传播做出了巨大的贡献,但由于Vi多糖是T细胞非依赖型抗原,普遍认为对5岁以下儿童免疫效果不理想,尤其是2岁以下儿童基本不产生免疫应答。近年来,随着结合疫苗技术的发展,国际多家科研机构先后开展了伤寒Vi结合疫苗的临床研究工作,美国国立卫生研究院(NIH)率先完成了在越南的Ⅲ期临床研究工作,结果显示,伤寒Vi结合疫苗是一种安全有效的疫苗,且在越南高发病区2-5岁儿童中可产生91.1%的保护力,并可持续4年【6-9】。并且确定3.52 ELISA单位(EU)为伤寒Vi IgG最低保护性抗体水平,并得到WHO的认可。我国兰州生物制品研究所有限责任公司生产的伤寒Vi多糖蛋白结合疫苗临床研究也得出了优效于伤寒Vi多糖疫苗的结果。国际多家科研机构对伤寒Vi结合疫苗的研制,预示着消灭伤寒这个古老疾病的利器即将诞生。相应的,评价伤寒结合疫苗免疫效果的检测方法的建立也成为该疫苗发展的重要环节。本研究旨在建立一种准确、快捷、易操作的检测人血清中抗伤寒Vi多糖IgG抗体含量的方法。通常酶联免疫吸附测定法(ELISA)是检测伤寒Vi IgG抗体水平最常用的方法。本研究在已有文献报道的间接ELISA方法基础上,以美国国立卫生研究院(NIH)测定Vi抗体的酶联免疫吸附测定法(Enzyme-linked immunosorbent assay,ELISA)为主要参考,对两种包被抗原(伤寒Vi多糖及枸橼酸杆菌Vi多糖)、不同吸附力酶标板、抗原包被浓度、样品反应温度和时间、酶标IgG反应时间、显色时间、终止反应后放置时间等逐一进行了考察及优化,建立了测定人血清中抗伤寒Vi多糖特异IgG抗体含量ELISA方法。并依据方法学验证要求,对建立的ELISA方法进行了标准曲线一致性、特异性、线性、准确性、精密度以及最低检测值的验证。研究结果表明,6次试验各自所得标准曲线方程中A、B、C、D 4个参数无差异,且6次试验r2均大于0.99,因此可以认为本实验建立的ELISA方法标准曲线具有良好的一致性;质控(Quality Control,QC)血清经不同剂量多糖吸收后,抗体含量与多糖吸收剂量具有明显的剂量依赖关系,多糖抑制率最高可达92%左右,说明建立的ELISA方法具有良好的特异性;血清稀释度与抗体含量存在负相关的线性关系(R2=0.999),说明建立的ELISA方法具有良好的线性;本试验检测的QC血清抗体浓度76.80EU/mL,CV=8.55%,与美国NIH检测的数据(75EU/mL,CV≤15%)保持一致,证明此方法准确性良好;重复性(CV≤15%)及中间精密度(CV≤20%)均符合要求,说明此ELISA方法具有良好的精密度;同时确定0.00078EU/mL为本方法最低检测值。
[Abstract]:Typhoid is an invasive disease of intestinal infection. Salmonella typhi is a pathogen that causes typhoid fever, often transmitted through the intake of contaminated water and food. It causes 21000000 new cases and 216000 deaths each year. In some developing countries with imperfect infrastructure, it is a common disease. At present, the main preventive preparations of typhoid are mainly The Ty21a attenuated live vaccine of oral immunization and the.Ty21a attenuated Vi vaccine of the intramuscular injection of typhoid Vi polysaccharide vaccine have the defects of unclear genetic background and poor immune effect, while the clinical trials of Nepal and South Africa and Jiangsu, China, and Jiangsu, China, have proved that the typhoid Vi polysaccharide vaccine can provide about 70% of the protective power and with the typhoid Vi polysaccharide vaccine Extensive use in the world, its good safety and about 70% of the protection effect to prevent the spread of typhoid disease has made great contributions. But because Vi polysaccharide is a T cell non dependent antigen, it is generally believed that the immune effect of children under the age of 5 is not ideal, especially in children under 2 years of age. In recent years, With the development of combined vaccine technology, a number of international research institutes have carried out clinical research on typhoid Vi binding vaccine. The National Institutes of Health (NIH) took the lead in completing the stage III clinical study in Vietnam. The results showed that the typhoid Vi conjugate vaccine was a safe and effective vaccine and 2-5 year old children in the high incidence area of Vietnam. It could produce 91.1% protection and sustainable 4 years [6-9]. And 3.52 ELISA unit (EU) was the lowest protective antibody level of typhoid Vi IgG and approved by WHO. The clinical study of typhoid Vi polysaccharide conjugate vaccine produced by the Lanzhou Institute of Biological Products Research Institute of China also obtained the excellent effect on the typhoid Vi polysaccharide vaccine. The development of a number of international scientific research institutions on typhoid Vi binding vaccine indicates that the weapon of eradication of this old disease is about to be born. Accordingly, the establishment of a method for evaluating the immune effect of typhoid vaccine is also an important link in the development of the vaccine. This study aims to establish an accurate, quick, and easy to operate test. The method of anti typhi Vi polysaccharide IgG antibody content in human serum. The commonly used method of enzyme linked immunosorbent assay (ELISA) is the most common method for detecting the level of Vi IgG antibody in typhoid fever. On the basis of the existing indirect ELISA method reported in the literature, the enzyme linked immunosorbent assay (Enzyme-linked) for determining Vi antibody was determined by the National Institutes of Health (NIH) of the United States (Enzyme-linked). Immunosorbent assay, ELISA) for the main reference, two kinds of envelope antigen (typhoid Vi polysaccharide and citric acid bacilli Vi polysaccharide), different adsorbability enzyme plate, antigen inclusion concentration, sample reaction temperature and time, enzyme reaction time, color time, termination time after the termination of reaction were investigated and optimized, and the determination of human serum was established. ELISA method of anti typhoid Vi polysaccharide specific IgG antibody content. According to the verification requirements of methodology, the standard curve consistency, specificity, linearity, accuracy, precision and minimum detection value of the established ELISA method are verified. The results show that the 6 tests have no difference between the 4 parameters of A, B, C and D in the standard curve equation, and 6, and 6 The secondary test R2 is more than 0.99, so it is considered that the standard curve of ELISA method established in this experiment has good consistency. After the absorption of different doses of polysaccharide in the plasma (Quality Control, QC), the antibody content has a significant dose dependence with the absorbed dose of polysaccharide, and the maximum inhibition rate of polysaccharide can reach about 92%, indicating the established ELISA square. The method had a good specificity, the linear relationship between the serum dilution and the antibody content was negative (R2=0.999), indicating that the established ELISA method had good linearity; the serum antibody concentration of QC, CV=8.55%, was consistent with the data of NIH detection in the United States (75EU/ mL, CV < 15%). The renaturation (CV < 15%) and intermediate precision (CV < 20%) all meet the requirements, indicating that the ELISA method has good precision and 0.00078EU/mL is the lowest detection value of this method at the same time.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R516.3

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