冻干ACYW135群脑膜炎球菌多糖结合疫苗中各群多糖含量检测方法的建立及验证

Establishment and verification of a determination method for contents of polysaccharide of various groups in freeze-dried groups A,C,Y and W135 meningococcal polysaccharide conjugate vaccine

目的 建立冻干ACYW135群脑膜炎球菌多糖结合疫苗中多糖含量的测定方法。方法 将10支ACYW135群脑膜炎球菌多糖结合疫苗用注射用水复溶后,分为10份,70μl/份,依次加入20 mg/ml蛋白酶K溶液0、1、2、3、4、5、6、7、8、9μl,降解结合物中载体蛋白,采用火箭免疫电泳法进行多糖含量的测定,确定蛋白酶K最适加量。对建立的方法进行线性范围、定量限度确定以及准确度、精密性及专属性验证。结果20 mg/ml蛋白酶K的最适加量为5μl;该方法的线性范围为2.5-40μg/ml,定量限度为2.5μg/ml;3个不同浓度的供试品重复测定3次,A、C、Y、W135群多糖含量的平均回收率分别为99.1%、96.5%、96.5%和97.4%;不同试验人员在不同时间对同一供试品重复测定9次,A、C、Y、W135群多糖含量的CV值分别为3.40%、4.11%、3.35%和4.88%;各群多糖抗原均与其对应的抗血清产生免疫沉淀,而与其他群抗血清不产生免疫沉淀,具有较强的特异性。结论 建立了冻干ACYW135群脑膜炎球菌多糖结合疫苗中多糖含量的检测方法,该方法在确定的限度范围内具有较好的准确度、精密性及专属性。

Objective To establish a method for determination of polysaccharide content in freeze-dried groups A,C,Y and W135 meningococcal polysaccharide conjugate vaccine. Methods Ten containers of freeze-dried groups A,C,Y and W135 meningococcal polysaccharide conjugate vaccine were reconstituted with water for injection,and divided into ten aliquots,70 μl for each,then added with 0,1,2,3,4,5,6,7,8 and 9 μl of 20 mg / ml protease K respectively to degrade the carrier protein,and determined for polysaccharide content by rocket immunoelectrophoresis,based on which the dosage of protease K was optimized. The developed method was determined for linear range,quantitative limit and verified for accuracy,precision and specificity. Results The optimal dosage of protease K was 5 μl. The linear range and quantitative limit of the developed method were 2. 5 ~ 40 μg / ml and 2. 5 μg / ml respectively. The mean recovery rates of groups A,C,Y and W135 polysaccharide in test samples at three concentrations in three repeat tests were 99. 1%,96. 5%,96. 5% and 97. 4% respectively. The CVs of groups A,C,Y and W135 polysaccharide contents in the same test sample determined for 9 times by various personnel at various time points were 3. 40%,4. 11%,3. 35% and 4. 88%respectively. The polysaccharide antigens of various groups showed immunoprecipitation with the antisera of the corresponding groups,while showed no immunoprecipitation with those of other groups,indicating a high specificity of the method. Conclusion A method for determination of polysaccharide content in freeze-dried groups A,C,Y and W135 meningococcal polysaccharide conjugate vaccine was established,which showed high accuracy,precision and specificity within the defined linear range.