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目的建立差异火箭免疫电泳(differential rocket immunoelectrophoresis,DRIEP)定量检测四价脑膜炎球菌结合疫苗游离多糖的方法,并对该方法进行验证。方法制备非均一性抗体琼脂糖凝胶,建立脑膜炎A群、C群、Y群、W135群游离多糖的DRIEP定量检测法,并对该方法的专属性、分离效果、线性、重复性及稳定性进行验证。分别采用DRIEP法及DOC沉淀化学法对11批各群脑膜炎球菌单价结合物进行检测。结果各群多糖抗原只与其相应特异性抗体发生结合,无交叉反应;各群结合物或混合物中的TT可完全与多糖分离;A群、W135群游离多糖定量对照品在2.5~20μg/ml范围内,C群、Y群游离多糖定量对照品在2~10μg/ml范围内,与沉淀峰高度呈良好线性关系,R2均>0.99;各群单价结合物的9次检测结果的CV值均<15%;多价脑膜炎球菌结合疫苗中各群游离多糖含量均比较接近。DRIEP法检测值较DOC沉淀化学法高约1/2倍,但两种方法检测结果的高低趋势具有相似性。结论DRIEP法可用于四价脑膜炎球菌结合疫苗中游离多糖的定量检测,提高了该疫苗的质控水平。
Objective To establish a method for the quantitative determination of free polysaccharides of tetravalent meningococcal conjugate vaccine by differential rocket immunoelectrophoresis (DRIEP), and to validate this method. Methods The non-uniform antibody agarose gel was prepared to establish the quantitative detection method for DRIEP of meningitis group A, C group, Y group and W135 group free polysaccharide. The specificity, separation, linearity, repeatability and stability Sex to verify. The 11 batches of meningococcal monovalent conjugates were detected by DRIEP and DOC precipitation chemistries, respectively. Results The polysaccharide antigen of each group only bound to the corresponding specific antibody without cross-reaction. The TT in each conjugate or mixture could be completely separated from the polysaccharide. The quantitative control of group A and W135 free polysaccharide was in the range of 2.5 ~ 20μg / ml The quantitative control of free polysaccharides in group C and group Y was in the range of 2 ~ 10μg / ml with a good linear relationship with the height of precipitation peak, with R2> 0.99. The CV values of 9 tests of each group of monovalent conjugates were < 15%; polyvalent meningococcal conjugate vaccine groups in each free polysaccharide content are relatively close. The DRIEP method is about one-and-a-half times higher than the DOC precipitation method, but the two methods show similarities in the trend of the test results. Conclusion The DRIEP method can be used for the quantitative detection of free polysaccharides in the tetravalent meningococcal conjugate vaccine and improve the quality control of the vaccine.