柯萨奇病毒A组16型抗原定量检测ELISA方法的建立及初步应用  

Development and preliminary application of ELISA method for quantitative detection of Coxsackievirus group A type 16 antigen

作　　者：马淑花[1] 郭会杰 吴蕴怡[1,2] 郝春生[1,2] 宋冬梅[1,2] 王潇潇[1,2] 杨永娟[1,2] 李秀玲[1,2] 

机构地区：[1]北京生物制品研究所有限责任公司,北京100024 [2]北京微谷生物医药有限公司,北京100029

出　　处：《中国生物制品学杂志》2014年第10期1288-1294,共7页Chinese Journal of Biologicals

基　　金：十二五"重大新药创制"科技重大专项(2013ZX09402302);北京市科委基金资助(Z131100006513007);国药新产品基金(2013SW03)

摘　　要：目的 建立柯萨奇病毒A组16型（Coxsackievirus group A type 16,CA16）抗原定量检测的ELISA方法,用于CA16疫苗中抗原的定量检测。方法 以纯化的CA16病毒颗粒作为免疫原,分别免疫家兔和BALB/c小鼠,制备抗CA16多克隆抗体和单克隆抗体。以抗CA16多抗作为包被抗体,经HRP酶标记的单抗作为酶标抗体,建立CA16抗原定量检测的双抗体夹心ELISA方法,确定该方法的线性范围,并进行准确度、精密度、稳定性及特异性验证。用建立的方法检测CA16疫苗原液制备过程样品中CA16抗原含量。结果 以抗CA16多抗为包被抗体,针对74株不同基因型的CA16的ELISA检测结果均为阳性,该抗CA16单抗的广谱性较好。该方法的线性范围为23.4～750 ng/ml,R2跃0.99,最小检出量为23.4 ng/ml;样品回收率在89%～108%之间,CV约15%,准确度和精密度较好;抗体包被板干燥后于37℃放置6 d,CV约20%,稳定性较好;PV纯化样品、EV71收获液、Vero细胞样品中抗原的A值均小于cutoff值,特异性较好。随着CA16疫苗制备过程的不断进行,中间品1-4比活分别为0.44、0.64、92.13和1 239.03,呈逐渐上升趋势。结论 建立了CA16抗原定量检测的双抗体夹心ELISA方法,该方法准确度、精密度高,稳定性及特异性较好,为CA16疫苗的研制及工艺开发奠定了基础。Objective To develop an ELISA method for quantitative determination of Coxsackievirus group A type 16（CA16）antigen and use for CA16 vaccine. Methods Polyclonal and monoclonal antibodies against CA16 antigen were prepared from the serum and ascites of rabbits and BALB / c mice immunized with purified CA16 respectively. A double antibody sandwich ELISA method was developed using polyclonal antibody as coating antibody and HRP-labeled monoclonal antibody as enzyme-labeled antibody,determined for linear range,verified for accuracy,precision,stability and specificity. The CA16 antigen content in test samples during preparation of bulk of CA16 vaccine was determined by the developed method. Results All the determination results of 74 strains of CA16 virus of various genotypes by ELISA using polyclonal antibody against CA16 as coating antibody were positive. The monoclonal antibody against CA16 showed a broad spectrum. The linear range of the developed ELISA method was 23. 4 - 750 ng / ml,with a R2 value of more than 0. 99. The recovery rate of samples was 89% - 108%,with a CV of less than 15%,indicating high accuracy and precision. After the antibody-coated ELISA microplate was dried and stored at 37 ℃ for 6 d,the CV of determination results was less than 20%,indicating high stability. All the A values of antigens in purified PV,harvest of EV71 and Vero cells were less than the cutoff value,indicating a high specificity. The specific activities of intermediates 1 - 4 were 0. 44,0. 64,92. 13 and 1 239. 03 respectively,which showed a gradually increasing tendency. Conclusion A double antibody sandwich ELISA method for quantitative determination of CA16 antigen was developed,which showed high accuracy,precision,stability and specificity,and laid a foundation of development of CA16 vaccine and process.

关 键 词：柯萨奇病毒A组16型 抗原 酶联免疫吸附法 

分 类 号：R373.23[医药卫生—病原生物学]