摘要
目的建立a型流感嗜血杆菌（Haemophilus influenzae type a,Hia）结合物游离多糖含量测定方法,并对方法进行验证。方法利用脱氧胆酸钠（Na DC）盐酸法和蒽酮硫酸法测定Hia结合物的游离多糖含量。通过检测Na DC盐酸法沉淀破伤风类毒素（tetanus toxoid,TT）、TT ADH衍生物（ADH derivative of TT,TTAH）及TT琥珀酰化衍生物（succinic anhydride derivative of TT,TTSA）的最大能力以及沉淀多糖降解物（depolymerized polysaccharide,de-PS）或降解多糖ADH衍生物（ADH derivative of de-PS,de-PSAH）与相应载体蛋白质混合物的最佳离子强度,并利用最佳离子强度检测混合物中多糖与蛋白质质量的最小比例,以确定Hia结合物游离多糖含量的测定条件。通过添加回收试验验证方法的准确度,多次测定结合物的游离多糖含量验证方法的精密度。结果 Na DC盐酸法对蒽酮硫酸法测定多糖含量几乎无影响;Na DC盐酸法沉淀TT、TTAH和TTSA的最大能力分别为402.8、352.5和691.0μg/ml;在0.6 mol/L NaCl的离子强度下,Na DC盐酸法沉淀多糖与蛋白质混合物,当de-PS与TT或TTAH的质量比≥1∶2、dePSAH与TT的质量比≥1∶4、de-PSAH与TTSA的质量比≥1∶5时,多糖回收率均在95%以上。向结合物添加6、12、35和56μg/ml的de-PS或de-PS_（AH）时,多糖回收率均在80%-100%之间;游离多糖含量检测结果的CV值均在10%以内。结论建立的方法适用于不同结合方法制备的Hia结合物游离多糖含量的测定。
Objective To develop and verify a method for determination of free polysaccharide content in Haemophilus influenzae type a（Hia）conjugate. Methods Na DC-HCl method and Anthrone-sulfuric acid method were used to measure the content of free polysaccharide in Hia conjugate. The maximum capacities of Na DC-HCl method in precipitation of tetanus toxoid（TT）, ADH derivative of TT（TTAH） and succinic anhydride derivative of TT（TTSA）, as well as the optimal ionic strength for precipitation of depolymerized polysaccharide（de-PS） or ADH derivative of de-PS（de-PS_（AH））and the mixture of the corresponding carrier protein, were determined, based on which the minimum ratio of polysaccha-ride to protein in mixture was measured at the optimal ionic strength to determine the condition for determination of free polysaccharide content in Hia conjugate. The method was verified for accuracy by spiking experiment, and for precision by multiple determination of free polysaccharide content in Hia conjugate. Results Na DC-HCl method showed little effect on determination of polysaccharide content by Anthrone-sulfuric acid method. The maximum capacities of Na DC-HCl method in precipitation of TT, TTAHand TTSAwere 402. 8, 352. 5 and 691. 0 μg / ml respectively. At an ionic strength of 0. 6 mol / L sodium chloride, the recovery rates of polysaccharide in mixture of polysaccharide and protein were more than 95% when the mass ratio of de-PS to TT or TTAHwas not less than 1 ∶ 2, while that of de-PS_（AH）to TT was not less than1 ∶ 4, and that of de-PS_（AH）to TTSAwas not less than 1 ∶ 5. However, all the recovery rates of polysaccharide were 80% ~100% when 6, 12, 35 and 56 μg / ml de-PS or de-PS_（AH）was added into the conjugate. All the CVs of determination results of free polysaccharide content were less than 10%. Conclusion The developed method was suitable for the determination of contents of free polysaccharide in Hia conjugates prepared by various conjugate methods.
出处
《中国生物制品学杂志》
CAS
CSCD
2016年第6期623-627,631,共6页
Chinese Journal of Biologicals
关键词
a型流感嗜血杆菌
多糖蛋白质结合物
脱氧胆酸钠
游离多糖
Haemophilus influenzae type a
Polysaccharide-protein conjugate
Sodium deoxycholate
Free polysaccharide
