摘要
为建立一种准确可靠的高效液相色谱-紫外(HPLC-UV)方法,检测EV71-CA16二价手足口病(HFMD)灭活疫苗原液中的相关抗原,肠道病毒71型(EV71)和柯萨奇病毒A组16型(CA16)的纯度。本文采用Waters体积排阻色谱柱(SEC,UltrahydrogelTM 2507.8×300 mm,6μm),流动相为0.01 mol/L的PBS中再加入0.1 mol/L氯化钠溶液,等度洗脱,流速0.3 mL/min,检测波长280 nm,进样量50μL,柱温21℃,进行EV71与CA16病毒原液的纯度检测,并对该方法的检测限、拖尾因子、专属性、重复性及检测范围进行验证。结果显示,2种抗原的灵敏度溶液的信噪比(S/N)分别为37.053和47.710,均远大于3,各试样的拖尾因子均小于3;CA16原液和EV71原液经HPLC分离纯化后的目标抗原峰馏分经SDS-PAGE和Western-Blot特异性鉴定,结果显示,色谱峰样品组成分别为CA16和EV71的2种病毒衣壳的结构蛋白,表明本检测方法专属性高。不同浓度的EV71与CA16病毒抗原经多次检测后,各浓度条件下的峰面积百分比与保留时间的RSD均小于1%,重复性良好。不同抗原浓度批样品检测结果显示,CA16原液抗原浓度在89 U/mL~26398 U/mL之间,EV71原液抗原浓度在170 U/mL~9074 U/mL之间时,2种抗原溶液经多次检测统计显示峰面积百分比与保留时间的RSD均小于1%,本方法在该抗原浓度范围内准确可靠,适用于EV71-CA16二价手足口病灭活疫苗原液中病毒抗原纯度的检测。
To establish a relatively accurate and reliable HPLC-UV method for the purity detection of relevant antigens in EV71-CA16 bivalent inactivated vaccine of hand-foot-mouth disease(HFMD):Enterovirus 71(EV71)and Coxsackievirus group a type 16(CA16).HPLC analysis was performed on a Waters volume exclusion chromatography column(SEC,UltrahydrogelTM 2507.8×300 mm,6μm).The mobile phase consisted of 0.01 mol/L PBS and 0.1 mol/L NaCl with equivalent elution.The flow rate was 0.3 mL/min,the detection wavelength was 280 nm,the injection volume was 50μL and the column temperature was 21℃,were used to detect antigen purity of EV71 and CA16 virus drug substances,and the detection limit,tailing factor,specificity,repeatability and detection range of the method were verified.The results showed that the signal-to-noise ratios(S/N)of the sensitivity solutions for the two antigens were 37.053 and 47.710,respectively,those were both greater than 3,and the tailing factor of each specimen was less than 3.The peak fractions of the CA16 stock and EV71 stock target antigen after purification by HPLC were specifically examined by SDS-PAGE and Western-blot,those results showed that the chromatographic peak sample composition were identified as the structural proteins of the capsids of the two viruses,CA16 and EV71,respectively,indicating a reliable specificity.After multiple testing with different concentrations of EV71 and CA16 viral antigens,the coefficient of variation RSD between the peak area percentage and retention time at each concentration condition was less than 1%,indicating acceptable repeatability.The test results of samples of different antigen concentration batches showed that,the antigen concentration of CA16 stock solution was between 89 U/mLand 26398 U/mL and the antigen concentration of EV71 stock solution was between 170 U/mLand 9074 U/mL,both antigen solutions statistically showed less than 1%RSD of peak area and retention time after multiple testing,indicating this assay was accurate and reliable at such antigen co
作者
曹明翔
徐华
张浩然
刘鹏
毛正睿
姜莉
杨二霞
CAO Mingxiang;XU Hua;ZHANG Haoran;LIU Peng;MAO Zhengrui;JINAG Li;YANG Erxia(Aimei Action Biopharmaceutical Co.,Ltd.,Taizhou Jiangsu,225300,China)
出处
《质量安全与检验检测》
2024年第2期21-26,共6页
QUALITY SAFETY INSPECTION AND TESTING
基金
艾美疫苗股份有限公司(2014ZX09102042-001)。
