本文選題：傷寒沙門菌 + IgG抗體　； 參考：《蘭州大學(xué)》2017年碩士論文

【摘要】：傷寒是一種腸道感染的侵襲性疾病,傷寒沙門菌是引起傷寒的病原體,常通過攝入污染的水和食物傳播,每年引起21,000,000例新增病例,216,000人死亡。在一些基礎(chǔ)設(shè)施不完善的發(fā)展中國家是一種很常見的疾病。目前,傷寒的預(yù)防制劑主要是口服免疫的Ty21a減毒活疫苗和肌肉注射的傷寒Vi多糖疫苗。Ty21a減毒活疫苗存在基因背景不清楚、免疫效果不佳等缺陷,而尼泊爾和南非以及我國江蘇、廣西的臨床試驗(yàn)證明,傷寒Vi多糖疫苗可提供70%左右的保護(hù)力,隨著傷寒Vi多糖疫苗在世界范圍內(nèi)的廣泛使用,其良好的安全性和70%左右的保護(hù)效果為預(yù)防傷寒疾病的傳播做出了巨大的貢獻(xiàn),但由于Vi多糖是T細(xì)胞非依賴型抗原,普遍認(rèn)為對5歲以下兒童免疫效果不理想,尤其是2歲以下兒童基本不產(chǎn)生免疫應(yīng)答。近年來,隨著結(jié)合疫苗技術(shù)的發(fā)展,國際多家科研機(jī)構(gòu)先后開展了傷寒Vi結(jié)合疫苗的臨床研究工作,美國國立衛(wèi)生研究院(NIH)率先完成了在越南的Ⅲ期臨床研究工作,結(jié)果顯示,傷寒Vi結(jié)合疫苗是一種安全有效的疫苗,且在越南高發(fā)病區(qū)2-5歲兒童中可產(chǎn)生91.1%的保護(hù)力,并可持續(xù)4年【6-9】。并且確定3.52 ELISA單位(EU)為傷寒Vi IgG最低保護(hù)性抗體水平,并得到WHO的認(rèn)可。我國蘭州生物制品研究所有限責(zé)任公司生產(chǎn)的傷寒Vi多糖蛋白結(jié)合疫苗臨床研究也得出了優(yōu)效于傷寒Vi多糖疫苗的結(jié)果。國際多家科研機(jī)構(gòu)對傷寒Vi結(jié)合疫苗的研制,預(yù)示著消滅傷寒這個(gè)古老疾病的利器即將誕生。相應(yīng)的,評價(jià)傷寒結(jié)合疫苗免疫效果的檢測方法的建立也成為該疫苗發(fā)展的重要環(huán)節(jié)。本研究旨在建立一種準(zhǔn)確、快捷、易操作的檢測人血清中抗傷寒Vi多糖IgG抗體含量的方法。通常酶聯(lián)免疫吸附測定法(ELISA)是檢測傷寒Vi IgG抗體水平最常用的方法。本研究在已有文獻(xiàn)報(bào)道的間接ELISA方法基礎(chǔ)上,以美國國立衛(wèi)生研究院(NIH)測定Vi抗體的酶聯(lián)免疫吸附測定法(Enzyme-linked immunosorbent assay,ELISA)為主要參考,對兩種包被抗原(傷寒Vi多糖及枸櫞酸桿菌Vi多糖)、不同吸附力酶標(biāo)板、抗原包被濃度、樣品反應(yīng)溫度和時(shí)間、酶標(biāo)IgG反應(yīng)時(shí)間、顯色時(shí)間、終止反應(yīng)后放置時(shí)間等逐一進(jìn)行了考察及優(yōu)化,建立了測定人血清中抗傷寒Vi多糖特異IgG抗體含量ELISA方法。并依據(jù)方法學(xué)驗(yàn)證要求,對建立的ELISA方法進(jìn)行了標(biāo)準(zhǔn)曲線一致性、特異性、線性、準(zhǔn)確性、精密度以及最低檢測值的驗(yàn)證。研究結(jié)果表明,6次試驗(yàn)各自所得標(biāo)準(zhǔn)曲線方程中A、B、C、D 4個(gè)參數(shù)無差異,且6次試驗(yàn)r2均大于0.99,因此可以認(rèn)為本實(shí)驗(yàn)建立的ELISA方法標(biāo)準(zhǔn)曲線具有良好的一致性;質(zhì)控(Quality Control,QC)血清經(jīng)不同劑量多糖吸收后,抗體含量與多糖吸收劑量具有明顯的劑量依賴關(guān)系,多糖抑制率最高可達(dá)92%左右,說明建立的ELISA方法具有良好的特異性;血清稀釋度與抗體含量存在負(fù)相關(guān)的線性關(guān)系(R2=0.999),說明建立的ELISA方法具有良好的線性;本試驗(yàn)檢測的QC血清抗體濃度76.80EU/mL,CV=8.55%,與美國NIH檢測的數(shù)據(jù)(75EU/mL,CV≤15%)保持一致,證明此方法準(zhǔn)確性良好;重復(fù)性(CV≤15%)及中間精密度(CV≤20%)均符合要求,說明此ELISA方法具有良好的精密度;同時(shí)確定0.00078EU/mL為本方法最低檢測值。
[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.
【學(xué)位授予單位】：蘭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R516.3

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王志高,周偉忠,史鑒寶,王同來,謝廣中,辜清吾,王秉瑞,王立亞,王洪飛,丁兆松,楊躍,沈玉霖,王薇媛,王祥初;傷寒Vi多糖菌苗流行病學(xué)效果[J];江蘇預(yù)防醫(yī)學(xué);1996年03期

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本文編號：2092886