流式荧光微球技术高通量筛查血液中多种病毒模型的建立及检测血液感染HBV、HCV的应用研究

发布时间：2018-05-25 04:04

本文选题：荧光微球 + 血液筛查　；参考：《西北大学》2005年硕士论文

【摘要】：因输血导致感染乙肝、丙肝、艾滋、梅毒等疾病的现象在当今社会已经越来越普遍,引起国家和社会的高度重视。目前最主要的任务就是严格控制对外来血源的筛查,而传统的ELISA方法检测血液传播疾病的方法仍在不同方面存在局限性,如检测通量低、需时长、灵敏度较低等。本研究采用流式荧光微球技术,应用ELISA双抗体夹心法和间接法原理以及生物素-亲和素系统,目的在于建立一种高通量、快速、灵敏检测HBV、HCV等病毒或病菌血清标志物的方法。具体研究工作可分为三部分: 1.用Hb-PcAb和羊抗人IgG优化抗体生物素化条件。 2.用Hb和人IgG建立抗原(抗体)与荧光微球的偶联条件。 3.以聚苯乙烯羧基荧光微球为固相载体,将HBsAg-McAb-S6和HCV Core Ag与不同编号的聚苯乙烯羧基荧光微球偶联;应用ELISA双抗体夹心法和间接法原理在Luminex-100~(TM)流式荧光微球检测系统下检测人血清中的HBsAg和HCV-Ab。结果表明抗体在以PBS作为第一次透析液、DMF作为BNHS溶剂的条件下生物素化效果较好;抗体生物素化后活性保持较好,生物素化效率较高。抗原/抗体分别在100mM MES(PH6.0)和50mM MES(PH5.0)中,浓度为100μg/ml条件下与荧光微球的偶联效率最高。荧光微球检测128例HBV患者血清中HBsAg的特异性和灵敏度均为100%,大大高于ELISA检测的特异性(71.43%)和灵敏度(66.67%),显示了荧光微球技术检测的优越性;荧光微球检测74例HCV患者血清中HCV-Ab的灵敏度为89.19%,检测方法还有待于提高。
[Abstract]:Infection with hepatitis B, hepatitis C, AIDS, syphilis and other diseases caused by blood transfusion has become more and more common in today's society. At present, the most important task is to strictly control the screening of foreign blood sources. However, the traditional ELISA method for detecting blood-borne diseases still has some limitations in different aspects, such as low detection flux, long detection time, low sensitivity and so on. In this study, ELISA double antibody sandwich method and indirect method and biotin avidin system were used to establish a high throughput, rapid and sensitive method for the detection of HBV virus or virus markers. The specific research can be divided into three parts: 1. Hb-PcAb and sheep anti-human IgG were used to optimize the biotinylation conditions. 2. The coupling conditions between antigens (antibodies) and fluorescent microspheres were established by HB and human IgG. 3. Using polystyrene carboxyl fluorescent microspheres as solid phase carrier, HBsAg-McAb-S6 and HCV Core Ag were coupled with polystyrene carboxyl fluorescent microspheres with different numbers. ELISA double antibody sandwich method and indirect method were used to detect HBsAg and HCV-Abin in human serum under the Luminex-100T TM flow fluorescence microsphere detection system. The results showed that the biotinylation effect of antibody was better when PBS was used as the first dialysate and the biotinylation efficiency was higher. When the concentration of antigen / antibody was 100 渭 g/ml, the coupling efficiency with fluorescent microspheres was the highest. The specificity and sensitivity of fluorescent microspheres in the detection of HBsAg in serum of 128 patients with HBV were 100, which was much higher than that of ELISA (71.43) and the sensitivity was 66.67%, which showed the superiority of fluorescent microspheres in the detection of HBsAg. The sensitivity of fluorescent microspheres in the detection of HCV-Ab in 74 patients with HCV was 89.19, and the detection method needed to be improved.
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R373

【引证文献】