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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 establish a capillary electrophoresis method for the determination of tetracycline toxoid (TT), a free carrier protein of the meningococcal polysaccharide conjugate of group Y and group W135, and to validate the method and its preliminary application. Methods The content of TT was determined by micellar electrokinetic capillary electrophoresis. Electrophoresis was carried out using 40 mmol / L borate-SDS buffer (p H 9.30) with an inner diameter of 50 μm uncoated fused silica capillary column (effective column length of 104 cm) Medium, at 30 kV, is tested at 200 nm UV wavelength. The specificity, linearity, precision, repeatability, stability, accuracy and limit of quantification of the method were validated. The free protein TT in three batches of samples of group Y and group W135 meningococcal conjugate was detected by the established method content. Results The specificity, precision, repeatability, stability and accuracy of the method were good. The TT showed a good linear relationship with the internal standard peak area ratio of chromatographic peak in the range of 10 ~ 35μg / ml, r ~ 2 = 0.995, The limit of quantitation of protein is 7μg / ml. The content of free protein TT in three batches of samples of Y group and W135 meningococcal conjugate stock solution was less than 10μg / ml. Conclusion The established capillary electrophoresis method is simple and accurate, and can be used to detect free protein content of the meningococcal polysaccharide conjugate stock solution of Y group and W135 group.