小反刍兽疫病毒竞争ELISA抗体检测方法的建立  

Establishment of a Competitive ELISA for Antibody Detection of PPRV

作　　者：许芳 蔡杰 薛华平 罗均[1] 蒋永青 郭霄峰[1] XU Fang;CAI Jie;XUE Hua-ping;LUO Jun;JIANG Yong-qing;GUO Xiao-feng(College of Veterinary Medicine,South China Agricultural University,Guangzhou,Guangdong 510642,China;Shenzhen Lvshiyuan Biotechnology Co.,Ltd,Shenzhen,Guangdong 518120,China)

机构地区：[1]华南农业大学兽医学院,广东广州510642 [2]深圳市绿诗源生物技术有限公司,广东深圳518120

出　　处：《动物医学进展》2024年第4期1-7,共7页Progress In Veterinary Medicine

基　　金：岭南现代农业科学与技术广东省实验室肇庆分中心“十四五”项目(P20211154-030303)。

摘　　要：为建立一种能准确、灵敏地检测小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)抗体的ELISA方法,通过诱导BL21(DE3)-pET-30a-PPRV N表达菌种获得PPRV N蛋白,将纯化的PPRV N蛋白免疫Balb/c小鼠,通过杂交瘤技术获得1株能够高效表达、稳定分泌和具有较好竞争效果的抗PPRV单克隆抗体的杂交瘤细胞株(命名为1G2)。以纯化的PPRV N蛋白为包被抗原,HRP标记的1G2单克隆抗体(1G2-HRP)作为竞争抗体,建立了小反刍兽疫病毒竞争ELISA(competitive ELISA,cELISA)抗体检测方法。经优化反应条件确定最佳抗原包被浓度为1.0μg/mL,待检血清最佳稀释条件为4倍稀释,1G2-HRP酶标抗体最佳工作浓度为250 ng/mL;当阻断率(PI值)小于42%,判定为抗体阴性;阻断率大于或等于42%,判定为抗体阳性。该方法最低可检测128倍稀释的PPRV抗体阳性血清,具有较高的敏感性;仅能检测PPRV抗体,与羊源常见病原和pET-30a-BL21(DE3)的抗体阳性血清无交叉反应,具有较好的特异性;批内、批间变异系数均小于15%,具有良好的重复性。该方法与血清中和试验的阳性符合率为87.80%(95%CI:73.80,95.92),阴性符合率为94.95%(95%CI:88.61,98.34),总符合率为92.86%(95%CI:84.11,97.64),Kappa值为0.83(95%CI:53.10,112.50),具有较高的符合率。表明建立的ELISA方法在我国PPRV的流行病学调查、疫苗免疫效果评估方面具有良好的应用前景。In order to establish an accurate and sensitive ELISA method for detecting antibodies of peste des petits ruminants virus(PPRV),the PPRV N protein was obtained by inducing BL21(DE3)-pET-30a-PPRV N expression strain,and the purified PPRV N protein was immunized to Balb/C mice.A hybridoma cell line(designated as mAb 1G2)with high expression,stable secretion and good competitive effect of anti-PPRV monoclonal antibody was obtained by hybridoma technique.Using purified PPRV N protein as the coating antigen and HRP-labeled 1G2 monoclonal antibody(1G2-HRP)as the competitive antibody,a competitive ELISA(cELISA)method for the detection of PPRV antibodies was established.After optimizing the reaction conditions,it is determined that the optimal antigen coating concentration is 1.0μg/mL;the optimal dilution of the serum to be tested is 4 times;the optimal working concentration of the 1G2-HRP enzyme-labeled antibody is 250 ng/mL;when the blocking rate(PI value)is less than 42%,it is determined to be antibody negative;when the blocking rate is greater than or equal to 42%,the antibody is determined to be positive.This method can detect the PPRV antibody positive serum diluted at least 128 times and has high sensitivity.This method can only detect PPRV antibody and has good specificity without cross-reaction with sheep common pathogens and pET-30a-BL21(DE3)antibody positive serum.The intra-and inter-batch coefficients of variation of the method are less than 15%,and have good repeatability.The positive coincidence rate and negative coincidence rate with serum neutralization test were 87.80%(95%CI:73.80,95.92)and 94.95%(95%CI:88.61,98.34),respectively.The total coincidence rate was 92.86%(95%CI:84.11,97.64),with a high Cohen’s Kappa value of 0.83(95%CI:53.10,112.50).The ELISA method established in this study has a good application prospect in the epidemiological investigation and the evaluation of vaccine immune effect of PPRV in China.

关 键 词：小反刍兽疫病毒 小反刍兽疫病毒N蛋白 竞争酶联免疫吸附试验 

分 类 号：S852.65[农业科学—基础兽医学] S858.2[农业科学—兽医学]