Establishment of double-antibody sandwich ELISA for detection of respiratory syncytial virus titer
王婉 1唐悦 1赵忆宁 1陈玥如 1傅生芳 1程亚慧 1李雄雄1
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作者信息
1. 兰州生物制品研究所有限责任公司第二研究室 甘肃省疫苗工程技术研究中心，兰州 730046
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摘要
目的 建立双抗体夹心酶联免疫吸附试验(double antibody sandwich enzyme-linked immunosorbent assay，DAS-ELISA)检测呼吸道合胞病毒(respiratory syncytial virus，RSV)滴度。 方法 以RSV融合前F蛋白的两种抗体AM14与D25作为捕获抗体和检测抗体，通过对其抗体浓度的优化来确定DAS-ELISA较适反应条件，并对该方法的特异性、重复性、灵敏度及适用性进行验证。 结果 选择用AM14抗体作为捕获抗体，D25作为检测抗体。捕获抗体AM14较适工作浓度为20 μg/mL，检测抗体D25的稀释比例为1∶3 000。建立的DAS-ELISA与轮状病毒(rotavirus，RV)和人3型副流感病毒(human parainfluenza virus type 3，HPIV-3)无交叉反应，且不同稀释倍数的RSV病毒进行批内与批间重复试验的变异系数(coefficient of variation，CV)均<10%；不同亚型的RSV及蛋白进行试验，均能检测到病毒滴度；检测结果与CCID50比较，可检测的稀释倍数略高。 结论 建立了检测RSV滴度的DAS-ELISA，该方法可为实验室RSV病毒滴度检测提供依据。 Objective In order to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detecting respiratory syncytial virus(RSV) titer. Methods Two antibodies AM14 and D25 of RSV pre-fusion protein were used as capture antibody and detection antibody separately. The optimal reaction condition of DAS-ELISA was determined through antibody concentration optimization, and the specificity, repeatability, sensitivity and applicability were validated. Results Choosing AM14 antibody as capture antibody and D25 as detection antibody, the working concentration of AM14 was determined to be 20 μg/mL, and the working concentration of D25 was determined to be 1∶3 000 dilution. The established DAS-ELISA had no cross-reaction with rotavirus(RV) and human parainfluenza virus type 3(HPIV-3). The coefficient variation ( CV) of RSV viruses with different dilution ratios in both intra and inter batch repeated experiments were less than 10%. The virus titer of different subtype RSV and protein were detected by this method. Compared with CCID50, the detectable dilution ratio is slightly higher. Conclusion DAS-ELISA in detection of RSV titer was successfully developed, which provided a basis for RSV titer detection in laboratory.
Abstract
Objective In order to develop a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) for detecting respiratory syncytial virus(RSV) titer. Methods Two antibodies AM14 and D25 of RSV pre-fusion protein were used as capture antibody and detection antibody separately. The optimal reaction condition of DAS-ELISA was determined through antibody concentration optimization, and the specificity, repeatability, sensitivity and applicability were validated. Results Choosing AM14 antibody as capture antibody and D25 as detection antibody, the working concentration of AM14 was determined to be 20 μg/mL, and the working concentration of D25 was determined to be 1∶3 000 dilution. The established DAS-ELISA had no cross-reaction with rotavirus(RV) and human parainfluenza virus type 3(HPIV-3). The coefficient variation ( CV) of RSV viruses with different dilution ratios in both intra and inter batch repeated experiments were less than 10%. The virus titer of different subtype RSV and protein were detected by this method. Compared with CCID50, the detectable dilution ratio is slightly higher. Conclusion DAS-ELISA in detection of RSV titer was successfully developed, which provided a basis for RSV titer detection in laboratory.
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