a型流感嗜血杆菌结合物游离多糖含量测定方法的建立及验证
发布时间：2018-10-21 10:53
【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-PS_(AH))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、dePS_(AH)与TT的质量比≥1∶4、de-PS_(AH)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。
[Abstract]:Objective to establish a method for the determination of free polysaccharides of Haemophilus influenzae type a (Haemophilus influenzae type ahia) conjugate and to verify the method. Methods the content of free polysaccharide of Hia conjugate was determined by deoxycholate (Na DC) hydrochloric acid method and anthrone sulfuric acid method. The maximum ability to precipitate tetanus toxoid (tetanus toxoid,TT), TT ADH derivative (ADH derivative of TT,TTAH) and TT succinylated derivative (succinic anhydride derivative of TT,TTSA by Na DC hydrochloric acid method and to precipitate the polysaccharide degradable substance (depolymerized polysaccharide,de-PS) or the degradation polysaccharide ADH derivative (ADH derivative of de-PS,de-PS_ (AH) were determined. Optimum ionic strength of the mixture with the corresponding carrier protein, The optimum ionic strength was used to determine the minimum ratio of polysaccharides to protein in the mixture to determine the conditions for the determination of free polysaccharides in Hia conjugates. The accuracy of the method was verified by adding the recovery test, and the precision of the verification method for the free polysaccharide content of the conjugate was determined several times. Results the maximum ability of Na DC hydrochloric acid method to determine the content of polysaccharide by; Na DC hydrochloric acid method was 402.8352.5 渭 g / ml and 691.0 渭 g / ml, respectively, and the content of polysaccharides and protein was precipitated by, Na DC hydrochloric acid method at 0.6 mol/L NaCl. When the mass ratio of de-PS to TT or TTAH 鈮，
本文编号：2284880
[Abstract]:Objective to establish a method for the determination of free polysaccharides of Haemophilus influenzae type a (Haemophilus influenzae type ahia) conjugate and to verify the method. Methods the content of free polysaccharide of Hia conjugate was determined by deoxycholate (Na DC) hydrochloric acid method and anthrone sulfuric acid method. The maximum ability to precipitate tetanus toxoid (tetanus toxoid,TT), TT ADH derivative (ADH derivative of TT,TTAH) and TT succinylated derivative (succinic anhydride derivative of TT,TTSA by Na DC hydrochloric acid method and to precipitate the polysaccharide degradable substance (depolymerized polysaccharide,de-PS) or the degradation polysaccharide ADH derivative (ADH derivative of de-PS,de-PS_ (AH) were determined. Optimum ionic strength of the mixture with the corresponding carrier protein, The optimum ionic strength was used to determine the minimum ratio of polysaccharides to protein in the mixture to determine the conditions for the determination of free polysaccharides in Hia conjugates. The accuracy of the method was verified by adding the recovery test, and the precision of the verification method for the free polysaccharide content of the conjugate was determined several times. Results the maximum ability of Na DC hydrochloric acid method to determine the content of polysaccharide by; Na DC hydrochloric acid method was 402.8352.5 渭 g / ml and 691.0 渭 g / ml, respectively, and the content of polysaccharides and protein was precipitated by, Na DC hydrochloric acid method at 0.6 mol/L NaCl. When the mass ratio of de-PS to TT or TTAH 鈮，
本文编号：2284880
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