O型口蹄疫病毒血清抗体竞争ELISA检测方法的建立及验证  

Development and verification of competitive ELISA for serum antibody against foot-and-mouth disease virus type O

作　　者：杨志元[1] 白满元 张韵[2] 闻晓波[1] 孙世琪[2] 郭慧琛[2] 冉旭华[1] YANG Zhi-yuan;BAI Man-yuan;ZHANG Yun;WEN Xiao-bo;SUN Shi-qi;GUO Hui-chen;RAN Xu-hua(College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongiang Province,China)

机构地区：[1]黑龙江八一农垦大学动物科技学院,黑龙江大庆163319 [2]中国农业科学院兰州兽医研究所家畜疾病病原生物学国家重点实验室OIE/国家口蹄疫参考实验室,甘肃兰州730046

出　　处：《中国生物制品学杂志》2020年第4期438-444,共7页Chinese Journal of Biologicals

基　　金：国家重点研发计划(2016YFD0500700、2016YFE0204100、2017YFD0500900、2017YFD0501100);黑龙江八一农垦大学研究生创新科研项目(YJSCX2017-Y43)。

摘　　要：目的建立O型口蹄疫病毒(foot-and-mouth disease virus,FMDV)血清抗体的竞争ELISA检测方法,并进行验证。方法采用O型FMDV病毒样颗粒(virus-like particle,VLP)作为包被抗原,建立用于检测FMDV血清抗体的竞争ELISA方法。对方法的抗原包被浓度(0.1、0.2、0.3、0.4、0.5、0.6、0.7、0.8、0.9、1.0μg/mL)、抗原包被温度及时间(4℃过夜、37℃1 h、37℃1.5 h、37℃2 h、37℃2.5 h、37℃3 h)、血清稀释度(1∶2、1∶4、1∶8、1∶16稀释)、HRP-IgG稀释度(1∶10000~1∶20000稀释)、封闭液种类(不封闭、1%BSA封闭、2%BSA封闭、5%脱脂乳封闭)、封闭液作用时间(30、60、90、120 min)、血清与HRP-IgG作用时间(30、60、90、120、150 min)、底物TMB作用时间(10、15、20、25、30 min)进行优化。同时验证方法的交叉反应、特异性及敏感性、精密性。采用建立的方法及O型FMDV抗体液相阻断ELISA检测试剂盒分别检测未知血清背景的138份猪和牛血清,计算两者的符合率。结果方法最适检测条件为:抗原包被浓度0.5μg/mL,抗原包被温度及时间为4℃过夜,血清稀释度为1∶4;HRP-IgG稀释度为1∶18000;封闭液为1%BSA,封闭液作用时间为30 min;HRP-IgG与血清作用时间为30 min;底物作用时间为10 min。21份不同猪病的阳性血清中,除3份O型FMD阳性血清为阳性外,其他血清样品为阴性,该方法无交叉反应;敏感性为90.4%,特异性为95.7%;批内和批间精密性试验变异系数(CV)均<10%。该方法与口蹄疫O型抗体液相阻断ELISA检测试剂盒检测结果的符合率为97%。结论建立的方法具有良好的特异性及精密性,且操作简便,可用于O型FMDV血清抗体的检测。Objective To develop and verify a competitive ELISA for serum antibody against foot-and-mouth disease virus(FMDV)type O. Methods The virus-like particle(VLP)of FMDV type O was used as coating antigen to develop a competitive ELISA for serum antibody against FMDV type O. The antigen concentration(0. 1,0. 2,0. 3,0. 4,0. 5,0. 6,0. 7,0. 8,0. 9 and 1. 0 μg/mL)as well as temperature and time(4 ℃ overnight,37 ℃ 1 h,37 ℃ 1. 5 h,37 ℃ 2 h,37 ℃2. 5 h and 37 ℃ 3 h)for coating,serum dilution(1 ∶ 2,1 ∶ 4,1 ∶ 8 and 1 ∶ 16),HRP-IgG(1 ∶ 10 000 ~ 1 ∶ 20 000),kinds of blocking reagent(non-blocking,blocking with 1% BSA,blocking with 2% BSA and blocking with 5% skimmed milk),blocking time(30,60,90 and 120 min),reaction time of serum with HRP-IgG(30,60,90,120 and 150 min)and treatment time with substrate TMB(10,15,20,25 and 30 min)were optimized. The developed method was verified for cross reaction,specificity,sensitivity and precision. A total of 138 porcine and bovine serum samples with unknown backgrounds were determined by the developed kit and liquid phase blocking ELISA kit for antibody against FMDV type O respectively, and the coincidence rate of test results was calculated. Results The antigen was coated at a concentration of 0. 5 μg/mL and a temperature of 4 ℃ overnight. The optimal dilutions of serum and HRP-IgG were 1 ∶ 4 and 1 ∶ 18 000 respectively. The 1% BSA was used as blocking reagent,while the optimal time for blocking was 30 min.However,the optimal reaction time of HRP-IgG with serum was 30 min,while the optimal treatment time with substrate was 10 min. Twenty-one porcine serum samples positive for various viruses were tested by the developed method,of which the results of three samples positive for FMDV type O were positive,while those of the rest samples were negative,indicating no cross reactions. The sensitivity and specificity of the method were 90. 4% and 95. 7% respectively,while the coefficients of variation(CVs)in intra-and inter-assays were less than 10%. The coincidence rate

关 键 词：O型口蹄疫病毒 病毒样颗粒 血清抗体 竞争ELISA法 

分 类 号：S855.3[农业科学—临床兽医学]