猪流行性乙型脑炎病毒prM-E抗原捕获ELISA检测方法的建立

Development of an antigen capture ELISA for detecting the prM-E protein of porcine Japanese encephalitis virus

为建立猪流行性乙型脑炎病毒(JEV)的检测方法,本研究以pr M-E结构蛋白特异性单克隆抗体(MAb)5B10和5E7分别作为捕获抗体和辣根过氧化酶标记的检测抗体,对反应条件进行优化,建立了抗原捕获ELISA(AC-ELISA)方法。该方法与其它常见猪病病原无交叉反应,特异性强。该方法可以检出1.25×105 pfu/m L的病毒和48.44 ng/m L pr M-E重组蛋白,最佳线性检测范围为0.05μg/m L^1.00μg/m L,线性相关系数(R2)为0.996。组装的试剂盒批内和批间重复性试验的变异系数均小于10%,具有良好的重复性。试剂盒4℃保存12个月稳定。该研究为JEV亚单位疫苗的研制与生产提供了技术支持。

To develop a method for detecting porcine Japanese encephalitis virus （JEV）, an antigen capture ELISA （AC-ELISA） was established with monoclonal antibody （MAb） 5B10 as capture antibody and horseradish peroxidase conjugated MAb 5E7 as detection antibody. The AC-ELISA was specificity which had no cross-reactivity with other related pathogens of swine diseases. The detection limit was 1.25 ×105 pfu/mL for JEV and 48.44 ng/mL for viral prM-E protein expressed by mammalian cell lines. The linear quantitative range was 0.05 μg/mL to 1.00 μg/mL, and the liner correlation （R2） was 0.996. The coefficients of variation intra-assay and inter-assay were less than 10%. The AC-ELISA kit was validity for 12 months stored at 4 ℃. The established AC-ELISA kit provided technical support to the development and production of JEV subunit vaccine.