14型肺炎球菌多糖单克隆抗体的制备与初步应用

发布时间：2018-07-23 08:31

【摘要】：目的制备14型肺炎球菌多糖单克隆抗体,并进行鉴定及初步应用。方法制备免疫原,免疫BALB/c小鼠,制备单克隆抗体,经3次亚克隆筛选单抗细胞株,并制备腹水。对筛选的单抗进行ELISA效价、特异性、亚型、中和效价、浊度法特异性等鉴定。采用制备的单抗腹水检测市售23价肺炎球菌多糖疫苗、7价肺炎球菌结合疫苗中14型肺炎球菌多糖含量,计算回收率;并与丹麦血清研究所(State Serum Institute,SSI)14型肺炎球菌多糖兔多抗血清检测结果进行比较,计算两者之间的变异系数(CV)。结果共获得7株14型肺炎球菌多糖单抗细胞株:PP-14-101~107,效价分别为105、104、104、104、105、105、105;7株单抗与23价肺炎疫苗中包含的除14型以外的其他所有血清型的多糖均不发生交叉反应;PP-14-101、104、105、106这4株单抗具有免疫浊度趋势,另3株单抗无浊度信号;PP-14-101和PP-14-104株单抗与6A、33F型肺炎球菌多糖具有一定的交叉反应,而PP-14-105和PP-14-106株单抗与23型多糖均无交叉反应;PP-14-101、PP-14-104株单抗为Ig M亚型,PP-14-105、PP-14-106株单抗为Ig G1亚型,PP-14-106比PP-14-105株单抗稀释倍数高,反应性更强,因此选择PP-14-106株单抗为目标单抗;PP-14-106株单抗腹水对14型肺炎球菌的中和效价远高于1∶17 496,中和效价较高;采用PP-14-106株单抗腹水检测市售23价肺炎球菌多糖疫苗和7价肺炎球菌结合疫苗,回收率为90.2%~120.0%,该单抗与SSI血清检测上述疫苗成品14型肺炎球菌多糖含量的CV值在2.5%~11.9%之间。结论成功获得1株效价高、具有杀菌活性、特异性好的单抗,该单抗具有免疫浊度趋势,能准确测定肺炎球菌疫苗中的多糖含量,可替代SSI血清用于多糖疫苗和结合疫苗中14型肺炎球菌多糖含量的定量检测。
[Abstract]:Objective to prepare monoclonal antibody against type 14 pneumococcal polysaccharide, and to identify and apply it. Methods Immunogen was prepared and BALB / r / c mice were immunized. Monoclonal antibodies were prepared. The McAb cell lines were subcloned for 3 times and ascites were prepared. Elisa titer, specificity, subtype, neutralization titer and turbidity specificity of the selected McAbs were identified. The concentration of type 14 pneumococcal polysaccharides in 23 valence pneumococcal polysaccharide vaccine and 7 valent pneumococcal conjugate vaccine was determined by using the prepared monoclonal antibody in ascites, and the recovery rate was calculated. The results were compared with the polyantisera of pneumococcal polysaccharide type 14 from the State Serum Institute (SSI), and the coefficient of variation (CV) was calculated. Results A total of 7 strains of pneumococcus pneumoniae polysaccharide monoclonal antibody cell line: PP-14-101H107 were obtained, and the titers were 105104104104105105105105107 and none of the polysaccharides of all the serotypes except type 14 contained in the 23-valent pneumonia vaccine had any cross-reaction with PP-14-101104105106. Antigens have the tendency of immune turbidity, The other three McAbs, PP-14-101 and PP-14-104, had a certain cross reaction with 6AMA-33F pneumococcal polysaccharides, and PP-14-101 and PP-14-104 McAbs had some cross-reactions with 6A3F pneumococcal polysaccharides. However, PP-14-105 and PP-14-106 McAbs had no cross reaction with 23 polysaccharides. PP-14-101 PP-14-104 McAbs were Ig M subtype PP-14-106 McAbs, which were Ig G 1 subtype, PP-14-106 and PP-14-106 McAbs had higher dilution times and stronger reactivity than PP-14-105 McAbs, and PP-14-104 McAbs were Ig M subtype and PP-14-106 McAbs were Ig G 1 subtype and PP-14-106 McAbs had higher dilution times than PP-14-105 strains. Therefore, the neutralization titer of PP-14-106 strain monoclonal antibody against pneumococcus 14 was much higher than that of 1:17 496, and the neutralization titer of PP-14-106 McAb was higher than that of PP-14-106 strain. PP-14-106 strains of monoclonal antibody were used to detect 23 valent pneumococcal polysaccharide vaccine and 7 valent pneumococcal conjugate vaccine on the market. The recovery rate was 90. 2% 120. 0. The CV value of the monoclonal antibody and SSI serum for detecting the polysaccharide content of pneumococcus type 14 was between 2.5% and 11. 9%. Conclusion A monoclonal antibody with high titer, bactericidal activity and specificity was successfully obtained. The monoclonal antibody has the tendency of immune turbidity and can accurately determine the polysaccharide content in pneumococcal vaccine. It can be used for quantitative detection of pneumococcal polysaccharide type 14 in polysaccharide vaccine and conjugate vaccine instead of SSI serum.
【作者单位】：清华大学化学工程系;北京科兴生物制品有限公司;
【分类号】：R378.14;R392-33

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