【摘要】：目的制備14型肺炎球菌多糖單克隆抗體,并進(jìn)行鑒定及初步應(yīng)用。方法制備免疫原,免疫BALB/c小鼠,制備單克隆抗體,經(jīng)3次亞克隆篩選單抗細(xì)胞株,并制備腹水。對篩選的單抗進(jìn)行ELISA效價、特異性、亞型、中和效價、濁度法特異性等鑒定。采用制備的單抗腹水檢測市售23價肺炎球菌多糖疫苗、7價肺炎球菌結(jié)合疫苗中14型肺炎球菌多糖含量,計算回收率;并與丹麥血清研究所(State Serum Institute,SSI)14型肺炎球菌多糖兔多抗血清檢測結(jié)果進(jìn)行比較,計算兩者之間的變異系數(shù)(CV)。結(jié)果共獲得7株14型肺炎球菌多糖單抗細(xì)胞株:PP-14-101~107,效價分別為105、104、104、104、105、105、105;7株單抗與23價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發(fā)生交叉反應(yīng);PP-14-101、104、105、106這4株單抗具有免疫濁度趨勢,另3株單抗無濁度信號;PP-14-101和PP-14-104株單抗與6A、33F型肺炎球菌多糖具有一定的交叉反應(yīng),而PP-14-105和PP-14-106株單抗與23型多糖均無交叉反應(yīng);PP-14-101、PP-14-104株單抗為Ig M亞型,PP-14-105、PP-14-106株單抗為Ig G1亞型,PP-14-106比PP-14-105株單抗稀釋倍數(shù)高,反應(yīng)性更強(qiáng),因此選擇PP-14-106株單抗為目標(biāo)單抗;PP-14-106株單抗腹水對14型肺炎球菌的中和效價遠(yuǎn)高于1∶17 496,中和效價較高;采用PP-14-106株單抗腹水檢測市售23價肺炎球菌多糖疫苗和7價肺炎球菌結(jié)合疫苗,回收率為90.2%~120.0%,該單抗與SSI血清檢測上述疫苗成品14型肺炎球菌多糖含量的CV值在2.5%~11.9%之間。結(jié)論成功獲得1株效價高、具有殺菌活性、特異性好的單抗,該單抗具有免疫濁度趨勢,能準(zhǔn)確測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結(jié)合疫苗中14型肺炎球菌多糖含量的定量檢測。
[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.
【作者單位】： 清華大學(xué)化學(xué)工程系;北京科興生物制品有限公司;
【分類號】：R378.14;R392-33

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本文編號：2138814