【摘要】： 目的從大罐發(fā)酵培養(yǎng)的百日咳桿菌的菌液中純化得到百日咳絲狀血凝素(Filamentous Hemagglutinin:FHA),制備抗-FHA單克隆抗體,并初步建立特異、準(zhǔn)確的FHA定量檢測方法。方法采用硫酸銨沉淀結(jié)合離子交換層析純化百日咳菌液獲得百日咳絲狀血凝素蛋白,以此蛋白免疫Balb/c小鼠,取免疫小鼠脾細(xì)胞與小鼠SP2／0細(xì)胞融合制備單克隆抗體,間接ELISA法篩選穩(wěn)定分泌抗-FHA單克隆抗體的雜交瘤細(xì)胞,制備小鼠腹水單抗并純化,對抗體進(jìn)行全面鑒定；過碘酸鈉標(biāo)記法制備辣根過氧化物酶(horseradish peroxidase:HRP)標(biāo)記的兔抗-FHA多克隆抗體,棋盤滴定法建立夾心ELISA定量檢測方法。結(jié)果從百日咳菌液中純化得到三批FHA,純度分別為：80.2%,86.2%,92.4%；獲得四株穩(wěn)定分泌抗-FHA單克隆抗體的雜交瘤細(xì)胞株,分別命名為2C11、8D1、8G11、5B9,抗體類別為IgG1和IgG2b,腹水單抗ELISA效價達(dá)1：105～1：106,并能特異的與FHA反應(yīng),與百日咳毒素和流感病毒血凝素不發(fā)生交叉反應(yīng)；ELISA定量檢測方法的線性檢測范圍為1.56-100ng／ml,板內(nèi)變異系數(shù)為2.54%-8.13%,板間變異系數(shù)為3.42%-5.85%,高、中、低三個濃度的回收率分別為：94.73%,108.67%,116.37%。結(jié)論已成功從百日咳菌液中純化得到FHA并獲得穩(wěn)定分泌抗-FHA單克隆抗體的雜交瘤細(xì)胞株,初步建立一種特異、準(zhǔn)確的定量檢測FHA的ELISA方法,可用于無細(xì)胞百日咳疫苗生產(chǎn)中間產(chǎn)品中FHA的定量檢測,作為無細(xì)胞百日咳疫苗生產(chǎn)的質(zhì)量監(jiān)測措施之一。
[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.
【學(xué)位授予單位】：武漢生物制品研究所
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

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