裂谷热病毒NP蛋白双抗体夹心ELISA检测方法的建立  

Development of a double-antibody sandwich ELISA detection method for Rift Valley fever virus ribonucleoprotein

作　　者：郭瑶 单逢缘 陆阳 陶丽红[1] 王欣慧 蒋永伦 步志高[1] 钟功勋[1] GUO Yao;SHAN Feng-yuan;LU Yang;TAO Li-hong;WANG Xin-hui;JIANG Yong-lun;BU Zhi-gao;ZHONG Gong-xun(Harbin Veterinary Research Institute,State Key Laboratory of Animal Disease Control and Prevention,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所动物疫病防控全国重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第10期1043-1048,1056,共7页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发项目(2022YFC2604200)。

摘　　要：为建立裂谷热病毒(RVFV)的快速检测方法,本研究选取RVFV高度保守的核蛋白(NP)基因,采用哺乳动物真核表达系统表达并纯化重组NP蛋白(r NP),经SDS-PAGE检测无误后将其分别免疫小鼠和大白兔,按常规实验方法制备RVFV鼠源NP单克隆抗体(MAb)5D8和兔源NP多克隆抗体(PAb)rab NP-1,经ELISA检测二者效价均为1:51200。以rab NP-1 PAb作为捕获抗体,5D8 MAb经HRP标记后作为检测抗体,经条件优化后结果显示,该检测方法捕获抗体浓度为1.56 mg/mL,0.2μg/孔;检测抗体浓度为1.17 mg/mL,2.24 ng/孔,初步建立RVFV双抗体夹心ELISA抗原检测方法。利用该方法分别对克里米亚-刚果出血热病毒(CCHFV)、拉沙病毒(LSV)、新冠病毒(SARS-CoV-2)和RVFV的NP以及RVFV Gn蛋白样品进行检测,结果显示,仅RVFV NP蛋白检测结果呈阳性,其余几种病毒蛋白均为阴性,无交叉反应;利用该方法检测2倍倍比稀释(2^(1)~2^(16))的RVFV复制缺陷型灭活病毒样品,结果显示经214倍稀释后的RVFV检测结果仍为阳性,该方法对RVFV检测限为6.1×10^(-1)FFU/mL;利用同一批次和不同批次包被的ELISA板进行批内和批间重复性检测,结果显示批内和批间变异系数均小于8%。使用RVFV灭活病毒对建立的双抗体夹心ELISA检测方法进行验证,结果符合预期。本研究建立的RVFV双抗体夹心ELISA检测方法特异性强、灵敏度高、重复性好,可用于RVFV的快速检测,也可为NP蛋白功能的研究提供技术支撑。To establish a rapid detection method for Rift Valley fever virus(RVFV),this study selected the highly conserved ribonucleo protein(NP)in RVFV as the antigen.The NP was expressed and purified using a mammalian eukaryotic expression system.Mice and rabbits were immunized to prepare RVFV NP monoclonal antibodies(MAb)and RVFV NP polyclonal antibodies(PcAb)with ELISA titer of 1:51200.By utilizing the prepared anti-RVFV NP PcAb as the capture antibody and 5D8-HRP as the detected antibody,a double-antibody sandwich ELISA method for detecting RVFV NP was established after optimizing the conditions.The results show that the optimal concentration of the capture antibody in this detection method is 1.56mg/mL(0.2μg/well),and the detected antibody concentration is 1.17mg/mL(2.24ng/well).Moreover,positive samples tested positive even after being diluted 214 times.This double-antibody sandwich ELISA method exhibits highly specificity and good repeatability,with intra-batch and inter-batch repeatability coefficients of less than 8%.Inactivated RVFV was used to verify the established doubleantibody sandwich ELISA detection method,and the S/P value of the sample was 1.03,indicating that the dual-antibody sandwich ELISA method was successfully established.The method established in this study can be utilized for the rapid diagnosis of Rift Valley fever and also provide technical support for research on the NP.

关 键 词：裂谷热病毒 核蛋白 双抗体夹心ELISA 

分 类 号：S852.65[农业科学—基础兽医学]