离子色谱法测定ACYW135群脑膜炎球菌结合物中Y群及W135群多糖含量  

Quantification of polysaccharide in serogroup W135 and Y in ACYW135 polysaccharide-protein conjugates by HPAEC-PAD

作　　者：王晶[1] 于旭博 赵阳[1] 孔素娟[1] 刘爱萍[1] 袁菲[1] 吴兵[1] 谭小梅[1] 谢贵林[1] 

机构地区：[1]兰州生物制品研究所有限责任公司第一研究室甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2016年第5期7-14,共8页Progress In Microbiology and Immunology

基　　金：国家科技支撑计划课题(2008BAI66B01)

摘　　要：目的：利用离子色谱法即高效阴离子交换柱层析—脉冲安培检测法（ HPAEC-PAD）,测定ACYW135脑膜炎球菌多糖蛋白结合物中Y群和W135群多糖含量的方法,并加以验证。方法将ACYW135脑膜炎球菌多糖蛋白结合物中Y群和W135群多糖用三氟乙酸（ TFA）水解为特异性单糖（葡萄糖、半乳糖）,并去除水解液中残留的TFA,用HPAEC-PAD分析检测,用PA10糖分析柱分离单糖,电化学检测器测定葡萄糖及半乳糖含量,用Chrome-leon色谱工作站记录并分析数据。对上述方法进行专属性、准确性、重复性验证,确定该方法的检出限和定量限。结果 Y群和W135群多糖在TFA2.5 mol/L（终浓度）、90℃、4 h可完全水解为葡萄糖、半乳糖。对照品葡萄糖及半乳糖在0.10-60.00μg/mL 范围内,质量浓度和色谱峰面积呈较好的线性关系, r 均大于0.99,回收率为87.94%-109.80%,相对标准偏差（RSD）为1.00%-2.00%,检出限为0.05μg/mL（信噪比3∶1）,定量限为0.10μg /mL（信噪比10∶1）。结论离子色谱法可同时检测脑膜炎球菌多糖蛋白结合物中Y群和W135群糖含量,该方法操作简便、灵敏、快速,干扰小,重现性好,适用于对相关疫苗生产过程中的质量控制。Objective To set up a determination and verification of meningococcal polysaccharides （ PS） in serogroup W135 and Y in ACYW135 tetravalent polysaccharide-protein conjugates by means of HPAEC-PAD （ High performance ani-on exchange chromatography-pulsed amperometric detection）. Methods The polysacchrides from the tetravalent conju-gates was hydrolyzed by TFA into monosacharide such as, glucose （ Gal）/galactose （ Glc） , which is characteristic of PSY/PSW respectively, and then analyzed by HPAEC-PAD followed with residual TFA removing from hydrolysis solution. Fur-thermore, Gal/Glc was determined by separation of PA10 anion exchange column, followed by detection with electrochemi-cal detector and Chromeleon workshop analysis. The specificity, accuracy and repeatability was verified for the test , re-spectively, and detection limit and quantitative limit were determined. Results The Gal/Glc was completely released un-der an optimized hydrolysis including, TFA with a final concentration 2. 5 mol/L, 90℃, and 4 hs. A good linear relation-ship was obtained from the concentration and peak’s area, with r〉0. 99, within the range of 0. 10-60. 00 μg/mL for con-trols Gal and Glc. The average recovery rate was in 87. 94% ~109. 80%, and RSD （Relative standard deviation） was in 1. 00%-2. 00%. The detection limit was 0. 05μg/mL（Signal to noise ratio, SNR 3 ∶ 1） , and quantitative limit was 0. 10μg/mL （SNR 3 ∶ 1）. Conclusion The content of polysaccharide in serogroup W135 and Y could be determined in the same time in the tetravalent conjugates by means of HPAEC-PAD, and the method was sensitive with a good repeatability associated with less interference, simple and fast in performance. It should be suitable to quality control of the related vaccine production.

关 键 词：脑膜炎球菌多糖蛋白结合物 高效阴离子交换柱层析-脉冲安培检测法( HPAEC-PAD) 多糖含量 

分 类 号：R392-33[医药卫生—免疫学]