朱涛;朱婉玉;宇学峰;毛慧华;邵忠琦;
ZHU Tao;ZHU Wan-yu;YU Xue-feng;MAO Hui-hua;SHAO Zhong-qi;Tianjin University of Science and Technology;

• 1:天津科技大学
• 2:天津康希诺生物技术有限公司天津市呼吸道细菌重组及结合疫苗企业重点实验室

摘要(Abstract)：

目的建立一种脑膜炎球菌多糖-白喉类毒素(CRM197)结合物中游离多糖含量的检测方法,并进行验证。方法根据脱氧胆酸钠(sodium deoxycholate,Na DC)在酸性条件下可沉淀蛋白的原理,分别对标准蛋白溶液和CRM197蛋白溶液进行酸沉淀处理,考察其蛋白沉淀效果;向脑膜炎球菌多糖-白喉类毒素结合物中标加多糖衍生物,计算加标回收率,验证方法的准确性;分别采用Na DC沉淀法处理脑膜炎球菌多糖-白喉类毒素(A、C、Y、W135群)结合物,检测其游离多糖含量,试验重复6次,验证方法的重复性;向标准蛋白溶液中标加不同浓度的Na DC溶液,验证Na DC对蛋白检测的干扰,考察方法的耐用性。结果不同浓度的标准蛋白溶液经Na DC沉淀法处理后,蛋白浓度在100³00μg/ml范围内,沉淀中蛋白回收率在90%300μg/ml范围内,沉淀中蛋白回收率在90%¹10%之间,蛋白浓度大于300μg/ml的样品回收率显著降低;4种脑膜炎球菌多糖-CRM197结合物标加多糖衍生物后,再经Na DC沉淀法处理后的游离多糖含量回收率均在80%110%之间,蛋白浓度大于300μg/ml的样品回收率显著降低;4种脑膜炎球菌多糖-CRM197结合物标加多糖衍生物后,再经Na DC沉淀法处理后的游离多糖含量回收率均在80%¹10%之间;4种脑膜炎球菌多糖-CRM197结合物6次检测结果的相对标准偏差(relative standard deviation,RSD)均<15%;当溶液中Na DC的浓度不高于2%时,对蛋白检测无干扰。结论 Na DC沉淀法准确性、重复性和耐用性良好,可用于脑膜炎球菌多糖-白喉类毒素结合物中游离多糖含量的测定。
Objective To develop and verify a method for determination of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid(CRM197) conjugate. Methods Based on the principle that sodium deoxycholate(Na DC) may precipitate protein under acidic condition, standard BSA and CRM197 solutions were precipitated with Na DC, and the precipitation efficacy was evaluated. Polysaccharide derivatives were added to meningococcal polysa-ccharide-CRM197 conjugates to calculate the spike recovery rate and verify the accuracy of the method. Meningococcal polysaccharide-CRM197 conjugates of groups A, C, Y and W135 were precipitated with Na DC respectively, of which the polysaccharide contents were determined for 6 times to verify the reproducibility of the method. Na DC at various concentrations were added to the standard BSA solution to verify the interference of Na DC to determination of protein and evaluate the durability of the method. Results The concentrations of standard BSA solutions at various concentrations ranged from 100 to 300 μg / ml after precipitation with Na DC, and the recovery rate of protein in precipitate was 90% 110%之间;4种脑膜炎球菌多糖-CRM197结合物6次检测结果的相对标准偏差(relative standard deviation,RSD)均<15%;当溶液中Na DC的浓度不高于2%时,对蛋白检测无干扰。结论 Na DC沉淀法准确性、重复性和耐用性良好,可用于脑膜炎球菌多糖-白喉类毒素结合物中游离多糖含量的测定。
Objective To develop and verify a method for determination of free polysaccharide in meningococcal polysaccharide-diphtheria toxoid(CRM197) conjugate. Methods Based on the principle that sodium deoxycholate(Na DC) may precipitate protein under acidic condition, standard BSA and CRM197 solutions were precipitated with Na DC, and the precipitation efficacy was evaluated. Polysaccharide derivatives were added to meningococcal polysa-ccharide-CRM197 conjugates to calculate the spike recovery rate and verify the accuracy of the method. Meningococcal polysaccharide-CRM197 conjugates of groups A, C, Y and W135 were precipitated with Na DC respectively, of which the polysaccharide contents were determined for 6 times to verify the reproducibility of the method. Na DC at various concentrations were added to the standard BSA solution to verify the interference of Na DC to determination of protein and evaluate the durability of the method. Results The concentrations of standard BSA solutions at various concentrations ranged from 100 to 300 μg / ml after precipitation with Na DC, and the recovery rate of protein in precipitate was 90% ¹10%. The recovery rate of sample at protein concentration of more than 300 μg / ml decreased remarkably. The recovery rates of free polysaccharide contents in meningococcal polysaccharide-CRM197 conjugates of four groups were 80% 110%. The recovery rate of sample at protein concentration of more than 300 μg / ml decreased remarkably. The recovery rates of free polysaccharide contents in meningococcal polysaccharide-CRM197 conjugates of four groups were 80% ¹10%. The relative standard deviation(RSD) of six determination results of conjugate of four groups was less than 15%.The Na DC at a concentration of not more than 2% showed no interference to the protein assay. Conclusion Na DC precipitation method showed high accuracy, reproducibility and durability, which might be used for determination of free polysaccharide content in meningococcal polysaccharide-CRM197 conjugate.

关键词(KeyWords)： 脑膜炎球菌多糖;白喉类毒素;结合物;游离多糖;脱氧胆酸钠
Meningoccocal polysaccharide;Diphtheria toxoid;Conjugate;Free polysaccharide;Sodium deoxycholate

Abstract:

Keywords:

基金项目(Foundation): 863计划“基因重配多价轮状病毒等新型疫苗研发技术及产品研发”(2012AA02A401)

作者(Authors): 朱涛;朱婉玉;宇学峰;毛慧华;邵忠琦;
ZHU Tao;ZHU Wan-yu;YU Xue-feng;MAO Hui-hua;SHAO Zhong-qi;Tianjin University of Science and Technology;

DOI: 10.13200/j.cnki.cjb.000857

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