百日咳丝状血凝素（FHA）的纯化、抗-FHA单克隆抗体的研制及初步应用
发布时间：2018-12-14 04:07
【摘要】： 目的从大罐发酵培养的百日咳杆菌的菌液中纯化得到百日咳丝状血凝素(Filamentous Hemagglutinin:FHA),制备抗-FHA单克隆抗体,并初步建立特异、准确的FHA定量检测方法。方法采用硫酸铵沉淀结合离子交换层析纯化百日咳菌液获得百日咳丝状血凝素蛋白,以此蛋白免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠SP2／0细胞融合制备单克隆抗体,间接ELISA法筛选稳定分泌抗-FHA单克隆抗体的杂交瘤细胞,制备小鼠腹水单抗并纯化,对抗体进行全面鉴定；过碘酸钠标记法制备辣根过氧化物酶(horseradish peroxidase:HRP)标记的兔抗-FHA多克隆抗体,棋盘滴定法建立夹心ELISA定量检测方法。结果从百日咳菌液中纯化得到三批FHA,纯度分别为：80.2%,86.2%,92.4%；获得四株稳定分泌抗-FHA单克隆抗体的杂交瘤细胞株,分别命名为2C11、8D1、8G11、5B9,抗体类别为IgG1和IgG2b,腹水单抗ELISA效价达1：105～1：106,并能特异的与FHA反应,与百日咳毒素和流感病毒血凝素不发生交叉反应；ELISA定量检测方法的线性检测范围为1.56-100ng／ml,板内变异系数为2.54%-8.13%,板间变异系数为3.42%-5.85%,高、中、低三个浓度的回收率分别为：94.73%,108.67%,116.37%。结论已成功从百日咳菌液中纯化得到FHA并获得稳定分泌抗-FHA单克隆抗体的杂交瘤细胞株,初步建立一种特异、准确的定量检测FHA的ELISA方法,可用于无细胞百日咳疫苗生产中间产品中FHA的定量检测,作为无细胞百日咳疫苗生产的质量监测措施之一。
[Abstract]:Objective to purify pertussis filamentous hemagglutinin (Filamentous Hemagglutinin:FHA) from the fermenting liquid of Bacillus pertussis and to prepare monoclonal antibody against FHA and to establish a specific and accurate FHA quantitative detection method. Methods pertussis filamentous hemagglutinin protein was purified by ammonium sulfate precipitation and ion exchange chromatography. Balb/c mice were immunized with this protein. The spleen cells of immunized mice were fused with SP2/0 cells to prepare monoclonal antibodies. The hybridoma cells secreting monoclonal antibody against FHA were screened by indirect ELISA method. The mouse ascites monoclonal antibody was prepared and purified, and the antibody was fully identified. Horseradish peroxidase (horseradish peroxidase:HRP) labeled rabbit polyclonal antibody against FHA was prepared by sodium periodate labeling method. The sandwich ELISA quantitative detection method was established by chessboard titration. Results the purity of three batches of FHA, purified from pertussis was 80.2% and 86.2%, respectively. Four hybridoma cell lines stably secreting monoclonal antibodies against FHA were obtained. They were named 2C11O8D1O8G115B9. The antibodies were classified as IgG1 and IgG2b, ascites monoclonal antibody ELISA with the titer of 1: 105 and 1: 106, and could react specifically with FHA. There was no cross reaction with pertussis toxin and influenza virus hemagglutinin. The linear detection range of ELISA quantitative detection was 1.56-100ng / ml, the coefficient of variation was 2.54-8.13g / ml, the coefficient of variation between plates was 3.42-5.85g / ml, the coefficient of variation was 3.42- 5.85g / ml, and the coefficient of variation was 3.42- 5.85g / ml, respectively. The recoveries of the three low concentrations were 94.73 and 108.67 and 116.37, respectively. Conclusion FHA was successfully purified from pertussis and hybridoma cell lines secreting monoclonal antibodies against FHA were obtained. A specific and accurate ELISA method for the quantitative detection of FHA was established. It can be used for the quantitative detection of FHA in the intermediate products of acellular pertussis vaccine production and as one of the quality monitoring measures in the production of acellular pertussis vaccine.
【学位授予单位】：武汉生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
[Abstract]:Objective to purify pertussis filamentous hemagglutinin (Filamentous Hemagglutinin:FHA) from the fermenting liquid of Bacillus pertussis and to prepare monoclonal antibody against FHA and to establish a specific and accurate FHA quantitative detection method. Methods pertussis filamentous hemagglutinin protein was purified by ammonium sulfate precipitation and ion exchange chromatography. Balb/c mice were immunized with this protein. The spleen cells of immunized mice were fused with SP2/0 cells to prepare monoclonal antibodies. The hybridoma cells secreting monoclonal antibody against FHA were screened by indirect ELISA method. The mouse ascites monoclonal antibody was prepared and purified, and the antibody was fully identified. Horseradish peroxidase (horseradish peroxidase:HRP) labeled rabbit polyclonal antibody against FHA was prepared by sodium periodate labeling method. The sandwich ELISA quantitative detection method was established by chessboard titration. Results the purity of three batches of FHA, purified from pertussis was 80.2% and 86.2%, respectively. Four hybridoma cell lines stably secreting monoclonal antibodies against FHA were obtained. They were named 2C11O8D1O8G115B9. The antibodies were classified as IgG1 and IgG2b, ascites monoclonal antibody ELISA with the titer of 1: 105 and 1: 106, and could react specifically with FHA. There was no cross reaction with pertussis toxin and influenza virus hemagglutinin. The linear detection range of ELISA quantitative detection was 1.56-100ng / ml, the coefficient of variation was 2.54-8.13g / ml, the coefficient of variation between plates was 3.42-5.85g / ml, the coefficient of variation was 3.42- 5.85g / ml, and the coefficient of variation was 3.42- 5.85g / ml, respectively. The recoveries of the three low concentrations were 94.73 and 108.67 and 116.37, respectively. Conclusion FHA was successfully purified from pertussis and hybridoma cell lines secreting monoclonal antibodies against FHA were obtained. A specific and accurate ELISA method for the quantitative detection of FHA was established. It can be used for the quantitative detection of FHA in the intermediate products of acellular pertussis vaccine production and as one of the quality monitoring measures in the production of acellular pertussis vaccine.
【学位授予单位】：武汉生物制品研究所
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392
【参考文献】
相关期刊论文 前10条
1 郭杰标;郭锐;刘艳华;;以抗体竞争结合抗原测定单抗亲和力常数的研究[J];南方医科大学学报;2006年07期
2 单璞 ,苏锦锋 ,刘保奎;无细胞百日咳疫苗的质量控制[J];国外医学(预防、诊断、治疗用生物制品分册);2005年01期
3 谢广中;;无细胞百日咳疫苗的发展及应用[J];上海预防医学杂志;2006年01期
4 侯启明,张路民,梁雅文,秦进才;应用CHO细胞法检测百日咳疫苗中的残余毒性[J];中国生物制品学杂志;1997年02期
5 孙述学,宋继萍,陈晓航,王剑虹,梁玉珍,杨淼,陈怀恭,王师真,罗广,孙晓琳,武毅;百日咳疫苗血清学效力试验与小鼠保护力试验结果的相关性[J];中国生物制品学杂志;2001年02期
6 杨晓明;百日咳疫苗现状和发展方向[J];中国生物制品学杂志;2003年02期
7 徐颖华,孟淑芳,张庶民,侯启明,雷殿良;抗百日咳丝状血凝素单克隆抗体的制备、鉴定及应用[J];中国生物制品学杂志;2005年06期
8 崔文禹;徐静;李树香;刘敬;王欣怡;赵玉秀;陈蕾;沈心亮;;抗肠道病毒71型单克隆抗体的制备及鉴定[J];中国生物制品学杂志;2009年12期
9 赵伟栋;韩文瑜;Q，
本文编号：2377893
本文链接：https://www.wllwen.com/yixuelunwen/shiyanyixue/2377893.html
最近更新
教材专著
