差异火箭免疫电泳定量检测四价脑膜炎球菌结合疫苗游离多糖方法的建立及验证  

Development and verification of differential rocket immunoelectrophoresis for quantitative determination of free polysaccharide content in tetravalent meningococcal conjugate vaccine

作　　者：侯亚莉[1] 于旭博 刘方蕾[1] 杨英英[1] 吴兵[1] 刘佳[1] 王晶[1] 毛睿[1] 谢贵林 

机构地区：[1]兰州生物制品研究所有限责任公司甘肃省疫苗工程技术研究中心,甘肃兰州730046 [2]中国生物技术股份有限公司,北京100029

出　　处：《中国生物制品学杂志》2015年第11期1193-1197,1201,共6页Chinese Journal of Biologicals

基　　金：国家"重大新药创制"专项(2013ZX09402-302-215);甘肃省科技重大专项(0801NKDA003);甘肃省"十二五陇药产业发展"专项

摘　　要：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 develop and verify the differential rocket immunoelectrophoresis（DRIEP） for quantitative determination of free polysaccharide content in tetravalent meningococcal conjugate vaccine（MCV）.Methods Nonhomogeneous antibody agarose gel was prepared,based on which a DRIEP method for quantitative determination of free polysaccharide content of groups A,C,Y and W135 was developed and verified for specificity,separation efficacy,linearity,reproducibility and stability.Eleven batches of monovalent meningococcal conjugates of various groups were determined by DRIEP and DOC methods respectively.Results The polysaccharide antigen of various groups showed only binding to the corresponding antibodies,while no cross reactions were observed.The TT in conjugates and mixtures of various groups were completely separated from polysaccharide.The quantitative references for free polysaccharides of groups A and W135 at concentrations of 2.5 ~ 20 μg / ml as well as those of groups C and Y at 2 ~ 10 μg / ml showed good linear relationship to the height of precipitation peak,with R2 values of more than 0.99.All the CVs of nine test results of monovalent conjugates of various groups were less than 15%.The contents of free polysaccharide of various groups in polyvalent meningococcal conjugate vaccine were similar.Though the test value by DRIEP was about 1 / 2times higher than that by DOC,the trends of test values by the two methods were in consistent.Conclusion DRIEP may be used for the quantitative determination of free polysaccharide content in tetravalent meningococcal conjugate vaccine,by which the quality control of vaccine may be improved.

关 键 词：差异火箭免疫电泳 四价脑膜炎球菌结合疫苗 游离多糖 

分 类 号：R392-33[医药卫生—免疫学]