牛呼吸道合胞体病毒双抗体夹心ELISA检测方法的建立

发布时间：2018-04-11 16:13

本文选题：牛呼吸道合胞体病毒 + G蛋白　；参考：《中国人兽共患病学报》2017年07期

【摘要】：目的建立牛呼吸道合胞体病毒(BRSV)双抗体夹心ELISA(DAS-ELISA)检测方法。方法以重组纯化的BRSV-G蛋白免疫家兔和小鼠,制备抗G蛋白的多克隆抗体(PcAb)和单克隆抗体(MAb),方阵滴定法优化DAS-ELISA方法抗体工作浓度及反应条件,并对其敏感性、特异性、符合率进行验证。结果获得5株稳定分泌抗G蛋白MAb的杂交瘤细胞株,分泌抗体亚类均为IgG1型κ链,Western blot、IFA试验表明PcAb和MAb均与G蛋白和BRSV发生特异性反应,MAb作为捕获抗体的最佳包被浓度为2.5μg/mL,PcAb作为检测抗体最佳工作浓度为5μg/mL,判定临界值为0.22,最低检测限为1.43μg/mL,批内和批间重复性试验变异系数均小于10%,与常引起牛呼吸道疾病的几种病原均无交叉反应,与RT-PCR的敏感性、特异性、符合率分别为92.0%、100%、95.6%。结论建立的检测BRSV的DAS-ELISA方法可用于临床样品的大规模检测,为BRSV疫情的监测和快速诊断奠定了基础。
[Abstract]:Objective to establish a double antibody sandwich ELISAV DAS-ELISAA method for detection of bovine respiratory syncytial virus (BRSVV).Methods Rabbits and mice were immunized with recombinant purified BRSV-G protein to prepare anti-G protein polyclonal antibody (PCAB) and monoclonal antibody (McAb). The working concentration and reaction conditions of DAS-ELISA antibody were optimized by square array titration, and the antibody was sensitive and specific.The coincidence rate is verified.Results five hybridoma cell lines secreting anti-G protein MAb stably were obtained.The specific reaction of PcAb and MAb with G protein and BRSV showed that the best concentration of antibody was 2.5 渭 g / mLPcAb as the best working concentration of 5 渭 g / mL. the critical value was determined.The lowest detection limit was 1.43 渭 g 路mL ~ (-1). The coefficient of variation of repeated tests was less than 100.There was no cross reaction with several pathogens that often caused bovine respiratory diseases.The sensitivity, specificity and coincidence rate with RT-PCR were 92. 0% and 95. 6%, respectively.Conclusion the established DAS-ELISA method for the detection of BRSV can be used for the large-scale detection of clinical samples, which lays a foundation for the monitoring and rapid diagnosis of BRSV.
【作者单位】：黑龙江八一农垦大学动物科技学院;
【基金】：黑龙江八一农垦大学研究生创新科研项目(No.YJSCX2016-Y18) 黑龙江省教育厅资助项目(No.12521372)联合资助~~
【分类号】：S852.653

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