毛细管电泳法检测Y群和W135群脑膜炎球菌多糖结合物原液中游离载体蛋白含量  

Determination of free carrier protein content in bulks of groups Y and W135 meningococcal polysaccharide conjugates by capillary electrophoresis

作　　者：李茂光[1] 武琳琳 米健秋[3] 毛琦琦[1] 李亚南[1] 杨英[4] 李健峰 叶强[1] 

机构地区：[1]中国食品药品检定研究院卫生部生物技术产品检定方法及其标准化重点实验室,北京100050 [2]江苏省(泰州)疫苗工程技术研究中心,江苏泰州225312 [3]安捷伦科技(中国)有限公司,北京100102 [4]北京放射与辐射医学研究所,北京100850

出　　处：《中国生物制品学杂志》2016年第7期740-743,747,共5页Chinese Journal of Biologicals

基　　金：国家863计划"疫苗效果和质量评价新技术研究"(2012AA02A402)

摘　　要：目的建立毛细管电泳法检测Y群和W135群脑膜炎球菌多糖结合物原液中游离载体蛋白破伤风类毒素(tetanus toxoid,TT)含量,并对方法进行验证及初步应用。方法采用胶束电动毛细管电泳法测定TT含量,使用内径50μm未涂层熔融石英毛细管柱(有效柱长为104 cm),以40 mmol/L硼酸盐-SDS缓冲液(p H 9.30)为电泳介质,电压为30 k V,在200 nm紫外波长条件下进行检测。对方法的专属性、线性、精密度、重复性、稳定性、准确性、定量限进行验证,并用建立的方法检测Y群和W135群脑膜炎球菌结合物原液各3批样品中的游离蛋白TT含量。结果该方法专属性、精密度、重复性、稳定性、准确性良好,TT在10~35μg/ml范围内与色谱峰相对内标峰面积比值呈良好的线性关系,r^2=0.995,游离蛋白的定量限为7μg/ml。用建立的方法检测Y群和W135群脑膜炎球菌结合物原液各3批样品中的游离蛋白TT含量均小于10μg/ml。结论建立的毛细管电泳法操作简便、准确,可用于检测Y群和W135群脑膜炎球菌多糖结合物原液中的游离蛋白含量。Objective To develop,verify and preliminarily apply a method for determination of the content of free carrier protein,tetanus toxoid（TT）,in the bulks of groups Y and W135 meningococcal polysaccharide conjugates by capillary electrophoresis（CE）.Methods Micellar electrokinetic chromatography（MEKC） was used for the quantitative determination of tetanus toxoid（TT）.A uncoated fused-silica capillarie（50 μm in inside diameter,104 cm from inlet to detector）was used,serving 40 mmol/L sodium borate-SDS buffer（p H 9.30）as a separation buffer at a voltage of 30 k V and a UV detection wavelength of 200 nm.The method was verified for specificity,linearity,precision,reproducibility,stability,accuracy and quantitative detection limit,and used for determination of free TT contents in bulks of groups Y and W135 meningococcal polysaccharide conjugate,three batches for each.Results The method showed high specificity,precision,reproducibility,stability and accuracy.TT at a concentration of 10 ~ 35 μg/ml showed a considerable linear relationship to the relative peak area of internal standard（r2 = 0.995）.The quantitative detection limit of free protein was7 μg/ml.All the free TT contents in the bulks of three batches of group Y and three batches of group W135 meningococcal polysaccharide conjugate were less than 10 μg/ml.Conclusion The developed method was simple and accurate,which might be used for the determination of free carrier protein content in bulks of groups Y and W135 meningococcal polysaccharide conjugates.

关 键 词：胶束电动毛细管电泳法 Y群脑膜炎球菌结合物 W135群脑膜炎球菌结合物 游离蛋白 

分 类 号：O657.8[理学—分析化学] R392-33[理学—化学]