重组肠道病毒71型疫苗(汉逊酵母)抗原含量测定方法的建立
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摘要
目的建立重组肠道病毒71型疫苗(EV-A71)(汉逊酵母)抗原含量测定方法,用于重组EV-A71(汉逊酵母)疫苗的质量控制。方法以抗EV-A71 (汉逊酵母)多克隆抗体为包被抗体,经辣根过氧化物酶(horseradish peroxidase,HRP)标记的抗EV-A71单克隆抗体为检测抗体,建立双抗体夹心酶联免疫法(enzyme-linked immunosorbent assay,ELISA),确定定量限度及最佳定量范围,并对该方法的准确度、精密度和特异性进行方法学验证。结果线性范围为5～80 U/ml,线性和平行性良好。定量下限为5U/ml,检测不同浓度的EV-A71国家抗原标准品,回收率在88%～115%,准确度良好;重复性和中间精密度测定,其变异系数均小于15%;与重组柯萨奇病毒A16、甲型肝炎灭活疫苗(HAV)、冻干水痘减毒活疫苗(VZV)、Sabin株脊髓灰质炎灭活疫苗(IPV)、五价口服轮状病毒减毒活疫苗(RV)等疫苗、Tris缓冲液、PBS缓冲液、破碎液、汉逊酵母宿主蛋白(HCP)均无交叉反应,其专属性良好。结论建立了重组EV-A71疫苗(汉逊酵母)抗原含量测定方法并进行了初步验证,为疫苗研制中抗原含量的质控提供了可靠的检测手段。
Objective To establish and verify a method for the detection of recombinant enterovirus 71(EVA71)vaccine(Hansenula polymorpha)antigen content for the quality control. Methods The anti-recombinant EV-A71(Hansonella)polyclonal antibody was used as the coating antibody,and the HRP-labeled antiEV71 monoclonal antibody as the detection antibody to establish a double antibody sandwich ELISA.The quantitative limit and the optimal quantitative range were determined,and the accuracy,precision and specificity were verified. Results The linear range was 5～80 U/ml,and the linearity and parallelism were good.The lower limit of quantification was 5 U/ml.The recovery rate was 88%～115% when testing the EV-A71 national antigen standard at different concentrations,and the accuracy was good.The reproducibility of the three batches of the original solution was more than 15% after tested for 6 times and measured the intermediate precision.It showed no cross-reactivity with recombinant coxsackievirus A16(CV-A16),inactivated hepatitis A vaccine(HAV),live attenuated varicella vaccine(VZV),Sabin polio inactivated vaccine(IPV),pentavalent oral rotavirus live vaccines(RV),Tris buffer,PBS buffer,disrupted solution,and Hansenula host protein(HCP),and the specificity was good.Repeated determinations of intermediate products during the process of one batch were done,and the results indicated a good applicability. Conclusions A double antibody sandwich ELISA for detecting the antigen content of recombinant EV-A71(Hansonella)was established and verified.This assay can provide a simple and rapid detection method for the quality control of the key index-antigen content in the research and development of vaccine.
Objective To establish and verify a method for the detection of recombinant enterovirus 71(EVA71)vaccine(Hansenula polymorpha)antigen content for the quality control. Methods The anti-recombinant EV-A71(Hansonella)polyclonal antibody was used as the coating antibody,and the HRP-labeled antiEV71 monoclonal antibody as the detection antibody to establish a double antibody sandwich ELISA.The quantitative limit and the optimal quantitative range were determined,and the accuracy,precision and specificity were verified. Results The linear range was 5～80 U/ml,and the linearity and parallelism were good.The lower limit of quantification was 5 U/ml.The recovery rate was 88%～115% when testing the EV-A71 national antigen standard at different concentrations,and the accuracy was good.The reproducibility of the three batches of the original solution was more than 15% after tested for 6 times and measured the intermediate precision.It showed no cross-reactivity with recombinant coxsackievirus A16(CV-A16),inactivated hepatitis A vaccine(HAV),live attenuated varicella vaccine(VZV),Sabin polio inactivated vaccine(IPV),pentavalent oral rotavirus live vaccines(RV),Tris buffer,PBS buffer,disrupted solution,and Hansenula host protein(HCP),and the specificity was good.Repeated determinations of intermediate products during the process of one batch were done,and the results indicated a good applicability. Conclusions A double antibody sandwich ELISA for detecting the antigen content of recombinant EV-A71(Hansonella)was established and verified.This assay can provide a simple and rapid detection method for the quality control of the key index-antigen content in the research and development of vaccine.
引文
[1]Gao X,Lou JR.Research progress of enterovirus 71viruslike particle vaccines[J].Guoji Shengwu Zhipinxue Zazhi,2016,39(4):171-175,201.(in Chinese)高雪,楼觉人.肠道病毒71型病毒样颗粒疫苗研究进展[J].国际生物制品学杂志,2016,39(4):171-175,201.
[2]Mao Q,Wang Y,Bian L,et al.EV71vaccine,a new tool to control outbreaks of hand,foot and mouth disease(HFMD)[J].Expert Rev Vaccines,2016,15(5):599-606.
[3]Chung YC,Ho MS,Wu JC,et al.Immunization with viruslike particles of enterovirus 71elicits potent immune responses and protects mice against lethal challenge[J].Vaccine,2008,26(15):1855-1862.
[4]Liu CC,Chang HW,Yang G,et al.Development of a quantitative enzyme linked immunosorbent assay for monitoring the enterovirus 71 vaccine manufacturing process[J].JVirol Methods,2011,176(1/2):60-68.
[5]Jia H,Cai F,Gao Q,et al.Development of a quantitative ELISA method for EV71antigen[J].Zhongguo Shengwu Zhipinxue Zazhi,2010,23(1):87-90.(in Chinese)贾卉,蔡芳,高强,等.EV71抗原ELISA定量检测方法的建立[J].中国生物制品学杂志,2010,23(1):87-90.
[6]Wang YP,Yao X,Mao QY,et al.Progress in quality control in developing enterovirus 71vaccine[J].Weishengwuxue Mianyixue Jinzhan,2015,43(5):69-72.(in Chinese)王一平,姚昕,毛群颖,等.肠道病毒71型疫苗质量控制研究进展[J].微生物学免疫学进展,2015,43(5):69-72.
[7]Liang Z,Mao Q,Gao Q,et al.Establishing China′s national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71(EV71)vaccines[J].Vaccine,2011,29(52):9668-9674.
[8]National Pharmacopoeia Commission.Chinese pharmacopoeia(IV)[M].Beijing:China Medical Science and Technology Press,2015:9000Guiding Principles 374-377.(in Chinese)国家药典委员会.中华人民共和国药典(四部)[M].北京:中国医药科技出版社,2015:9000指导原则374-377.
[2]Mao Q,Wang Y,Bian L,et al.EV71vaccine,a new tool to control outbreaks of hand,foot and mouth disease(HFMD)[J].Expert Rev Vaccines,2016,15(5):599-606.
[3]Chung YC,Ho MS,Wu JC,et al.Immunization with viruslike particles of enterovirus 71elicits potent immune responses and protects mice against lethal challenge[J].Vaccine,2008,26(15):1855-1862.
[4]Liu CC,Chang HW,Yang G,et al.Development of a quantitative enzyme linked immunosorbent assay for monitoring the enterovirus 71 vaccine manufacturing process[J].JVirol Methods,2011,176(1/2):60-68.
[5]Jia H,Cai F,Gao Q,et al.Development of a quantitative ELISA method for EV71antigen[J].Zhongguo Shengwu Zhipinxue Zazhi,2010,23(1):87-90.(in Chinese)贾卉,蔡芳,高强,等.EV71抗原ELISA定量检测方法的建立[J].中国生物制品学杂志,2010,23(1):87-90.
[6]Wang YP,Yao X,Mao QY,et al.Progress in quality control in developing enterovirus 71vaccine[J].Weishengwuxue Mianyixue Jinzhan,2015,43(5):69-72.(in Chinese)王一平,姚昕,毛群颖,等.肠道病毒71型疫苗质量控制研究进展[J].微生物学免疫学进展,2015,43(5):69-72.
[7]Liang Z,Mao Q,Gao Q,et al.Establishing China′s national standards of antigen content and neutralizing antibody responses for evaluation of enterovirus 71(EV71)vaccines[J].Vaccine,2011,29(52):9668-9674.
[8]National Pharmacopoeia Commission.Chinese pharmacopoeia(IV)[M].Beijing:China Medical Science and Technology Press,2015:9000Guiding Principles 374-377.(in Chinese)国家药典委员会.中华人民共和国药典(四部)[M].北京:中国医药科技出版社,2015:9000指导原则374-377.