發(fā)布時間：2018-10-21 10:53

【摘要】：目的建立a型流感嗜血桿菌(Haemophilus influenzae type a,Hia)結(jié)合物游離多糖含量測定方法,并對方法進(jìn)行驗證。方法利用脫氧膽酸鈉(Na DC)鹽酸法和蒽酮硫酸法測定Hia結(jié)合物的游離多糖含量。通過檢測Na DC鹽酸法沉淀破傷風(fēng)類毒素(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))與相應(yīng)載體蛋白質(zhì)混合物的最佳離子強(qiáng)度,并利用最佳離子強(qiáng)度檢測混合物中多糖與蛋白質(zhì)質(zhì)量的最小比例,以確定Hia結(jié)合物游離多糖含量的測定條件。通過添加回收試驗驗證方法的準(zhǔn)確度,多次測定結(jié)合物的游離多糖含量驗證方法的精密度。結(jié)果 Na DC鹽酸法對蒽酮硫酸法測定多糖含量幾乎無影響;Na DC鹽酸法沉淀TT、TTAH和TTSA的最大能力分別為402.8、352.5和691.0μg/ml;在0.6 mol/L NaCl的離子強(qiáng)度下,Na DC鹽酸法沉淀多糖與蛋白質(zhì)混合物,當(dāng)de-PS與TT或TTAH的質(zhì)量比≥1∶2、dePS_(AH)與TT的質(zhì)量比≥1∶4、de-PS_(AH)與TTSA的質(zhì)量比≥1∶5時,多糖回收率均在95%以上。向結(jié)合物添加6、12、35和56μg/ml的de-PS或de-PS_(AH)時,多糖回收率均在80%~100%之間;游離多糖含量檢測結(jié)果的CV值均在10%以內(nèi)。結(jié)論建立的方法適用于不同結(jié)合方法制備的Hia結(jié)合物游離多糖含量的測定。
[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，

本文編號：2284880