摘要
目的建立柯萨奇病毒A组6型(Coxsackievirus A6,CV-A6)疫苗抗原定量检测试剂盒,用于CV-A6疫苗以及含有CV-A6的手足口病(hand,foot and mouth disease,HFMD)多价疫苗的研发及质量控制。方法分别采用多个CV-A6毒株与多家企业的CV-A6抗原,通过结合能力和中和能力比较,从6个单克隆抗体(9H1、3D9、10F4、5A6、6D12和10G8)中筛选出具有广谱高结合能力及高中和能力的单克隆抗体,建立ELISA法CV-A6抗原定量检测试剂盒。对试剂盒进行线性、专属性、准确度、精密度、耐用性等验证。将试剂盒分发国内主要CV-A6疫苗研发企业,由各企业分别采用该试剂盒对各自制备的CV-A6疫苗原液以及生产工艺各中间环节制品进行CV-A6抗原含量检测,考核试剂盒适用性。结果以多克隆抗体CV-A6-3I作为包被抗体,最佳浓度为1∶8 000,以5A6单克隆抗体作为酶标抗体,最佳浓度为1∶8 000;包被条件为4℃过夜;封闭液为10%FBS+PBST+5%蔗糖,37℃封闭2 h;抗体稀释液为ED-33,酶标抗体作用条件为37℃1 h;显色条件为37℃10 min。建立的CV-A6抗原检测试剂盒线性范围为11~178 ng/mL,线性良好,定量检出限为11 ng/mL;与同为小RNA病毒科的CV-A16、甲型肝炎病毒(hepatitis A virus,HAV)、CV-A10、EV-A71以及MEM、DMEM和PBS缓冲液等均无交叉反应,专属性良好;检测回收率在83%~114%之间,单个实验员和2个实验员重复检测CV均小于15%,准确度、重复性和中间精密度均良好;抗原或酶标抗体孵育时间上下浮动5 min或反应温度上下浮动2℃,回收率在83%~107%之间,耐用性良好。该试剂盒检测4个企业制备的CV-A6疫苗原液和疫苗制备过程中的中间制品均具有良好的线性和平行性,提示该试剂盒对国内主要CVA6疫苗及中间制品均具有良好的适用性。结论本研究采用具有高效价、广谱中和活性的单克隆抗体和多克隆抗体,建立了双抗体夹心ELISA法CV-A6疫苗抗原定量检测试剂盒。经方法验证和适用性研究,证明该试剂盒各项指标良好,可作为CV-A6疫苗通用试剂盒用于CV-A6抗原含量检测,为CV-A6疫苗和HFMD多价疫苗研发及质量控制提供了可靠的检测手段。
Objective To develop a universal quantitative detection kit for Coxsackievirus A6(CV-A6) vaccine antigen and apply to the research,development and quality control of CV-A6 vaccine and CV-A6-containing polyvalent hand-footmouth disease(HFMD) vaccine. Methods By comparing the binding and neutralizing abilities of six monoclonal antibodies(mAbs),9 H1,3 D9,10 F4,5 A6,6 D12 and 10 G8,with different CV-A6 strains or antigens,the mAb with widely binding and potently neutralizing abilities was screened and used for developing an ELISA kit for the quantitative detection of CV-A6 antigen. The developed kit was verified for linearity,specificity,accuracy,precision and durability.The kit was dispensed to the main manufacturers developing CV-A6 vaccines and used for the quantitative detection of CVA6 antigen in bulks and intermediate products of vaccine. Results Polyclonal antibody CV-A6-3I was used as coating antibody,while mAb 5A6 as enzyme-labeled antibody,of which the optimal dilutions were both 1∶8 000. The microtiter plate was incubated at 4 ℃ overnight for coating,then blocked with 10% FBS + PBST + 5% sucrose at 37 ℃ for 2 h. The antibody dilution solution was ED-33,while the temperature and time for treatment with enzyme-labeled antibody were 37 ℃and 1 h respectively. However,the optimal temperature and time for color development were 37 ℃ and 10 min respectively. The linear range of the developed kit was 11 ~ 178 ng/mL,while the limit of quantitation was 11 ng/mL. No cross reactions with CV-A16,hepatitis A virus(HAV),CV-A10,EV-A71 as well as MEM,DMEM and PBS buffer were observed,indicating high specificity of the kit. The recovery rate was 83% ~ 114%,and both the CVs of repeat test results by one and two persons were less than 15%,indicating high accuracy,reproducibility and intermediate precision.After the reaction time increased or decreased within a range of 5 min,or temperature within a range of 2 ℃,the recovery rates were 83% ~ 107%,indicating good durability. The kit showed good linearity and parallelism in determination of CV-A6 antigen contents in bulks and intermediate products of CV-A6 vaccine from four manufacturers,indicating good suitability to the main domestic CV-A6 vaccines and their intermediate products. Conclusion A double antibody sandwich ELISA kit for quantitative detection of CV-A6 vaccine antigen was successfully developed by using the mAb and pAb with high titers and broad spectrum neutralization capabilities. The validation and applicability research indicated satisfactory indexes of the kit which may be used as a universal kit for detection of antigen content. It provided a reliable tool for development and quality control of CV-A6 and polyvalent HFMD vaccine.
作者
刘思远
卞莲莲
高帆
刘佩
霍雅倩
王泽鋆
张中洋
李雅静
戈小琴
顾美荣
安超强
李克雷
毛群颖
姜崴
LIU Si-yuan;BIAN Lian-lian;GAO Fan;LIU Pei;HUO Ya-qian;WANG Ze-jun;ZHANG Zhong-yang;LI Ya-jing;GE Xiao-qin;GU Mei-rong;AN Chao-qiang;LI Ke-lei;MAO Qun-ying;JIANG Wei(National Institutes for Food and Drug Control,Beijing 102600,China;不详)
出处
《中国生物制品学杂志》
CAS
CSCD
北大核心
2021年第3期300-306,共7页
Chinese Journal of Biologicals
基金
国家十三五重大新药创制(2018ZX09737-011)。
