ELISA法检测H3N2亚型流感病毒Vero细胞适应株  

Detection of a Vero Cell-adapted H3N2 Influenza Virus Strain by ELISA Method

作　　者：卢勇[1] 宋绍辉[1] 蔡玮[1] 马磊[1] 吴雅楠[1] 李卫东[1] 廖国阳[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118

出　　处：《现代生物医学进展》2013年第10期1811-1815,共5页Progress in Modern Biomedicine

摘　　要：目的:流感病毒Vero细胞适应株A/Yunnan/1/2005va(H3N2)是一株以Vero细胞为培养基质能高产的病毒株,可用于制备以Vero细胞为基质的流感病毒裂解灭活疫苗;通过在低温下连续传代培养,可选育出流感病毒Vero细胞冷适应株,用于制备Vero细胞流感病毒减毒活疫苗。为了便于对Vero细胞冷适应株的进一步研究,建立一个检测该病毒株的ELISA方法。方法:以流感病毒Vero细胞适应株A/Yunnan/1/2005Va(H3N2)的纯化抗原为免疫原制备羊抗A/Yunnan/1/2005Va(H3N2)和鸡抗A/Yunnan/1/2005Va(H3N2)的抗血清。将抗血清先后经硫酸铵沉淀法和Protein G亲和层析柱纯化后,以纯化的羊抗A/Yunnan/1/2005Va(H3N2)IgG为包被抗体,鸡抗A/Yunnan/1/2005Va(H3N2)IgG为第二抗体,测定包被抗体、第二抗体和酶标抗体最佳工作浓度,建立双抗体夹心ELISA检测方法。结果:羊抗A/Yunnan/1/2005Va(H3N2)IgG的最佳工作浓度为5μg/mL,鸡抗A/Yunnan/1/2005Va(H3N2)IgG的最适浓度为10μg/mL,酶标抗体最佳稀释倍数为1:4000。对已知的阳性样品,经双抗体夹心ELISA法测定的病毒滴度比血凝方法测定病毒滴度灵敏度高。结论:通过测定包被抗体、第二抗体和酶标抗体最佳工作浓度,建立了双抗体夹心ELISA检测流感病毒Vero细胞适应株病毒含量的方法。该方法操作简单、方便快速、敏感性高,可应用于Vero细胞冷适应株选育时对流感病毒Vero细胞适应株A/Yunnan/1/2005va(H3N2)的检测,对于研制Vero细胞流感减毒活疫苗有重要意义。Objective： The Vero cell-adapted Influenza Virus strain A/Yurman/1/2005Va （H3N2） was a high-yield virus strain on Vero cell which can be applied to the production of Vero cell-based inactivated split influenza vaccine. By successive passage at low temperature, a Vero cell based cold-adapted influenza virus strain can be produced which can be used as a vaccine candidate strain for live attenuated cold-adapted influenza virus vaccine. The aim of this study was to establish a ELISA method for detection of a Vero cell-adapted Influenza Virus strain A/Yunnan/1/2005Va （H3N2） so as to do further research on Vero cell based cold-adapted influenza virus strain. Methods： Goats and chickens were immunized with the purified strain. And then antiserum was purified by methods of am- monium sulfate precipitation and Protein G affinity chromatography. Goat anti-A/Yunnan/1/2005Va （H3N2） IgG was coated on the ELISA plate and chicken anti-A/Yunnardl/2005Va （H3N2） IgG was the second antibody. The optimal concentration of the two kinds of antibodies and enzyme-labe!ed antibody were determined in this study. Results： The optimal concentration of two kinds of antibodies were 5 ~g/mL（goat） and 10 ixg/mL（chicken） respectively, and the optimal dilution ratio of the enzyme-labeled antibody was 1：4000. The sensitivity of the ELISA was higher than that of hemagglutination assay. Conclusion： A ELISA method for detection of a Vero cell-adapted influenza virus strain A/Yunnan/1/2005Va （H3N2） was initially established in this study by determining the optimal concentration of the two kinds of antibodies and enzyme-labeled antibody. The ELISA method, which was simple, convenient, quick and sensitive, was ap- plied to detection of a Vero cell-adapted influenza virus strain in the process of the development of a Vero cell based cold-adapted in- fluenza virus strain. Thus, the ELISA method was of profound significance for the research on live attenuated cold-adapted influenza virus vaccine.

关 键 词：流感病毒 H3N2亚型 VERO细胞适应株 ELISA 

分 类 号：Q75[生物学—分子生物学] R373[医药卫生—病原生物学]