摘要
为了建立检测猪繁殖与呼吸综合征病毒(PRRSV)抗体的ELISA方法,本研究以纯化的PRRSV全病毒为包被抗原,利用PRRSV N蛋白单克隆抗体(MAb)为阻断抗体,经棋盘法优化各反应条件,建立了检测PRRSV抗体的阻断ELISA(B-ELISA)方法。结果显示,B-ELISA方法的最佳抗原包被浓度为1μg/mL,血清样品稀释度为1:4,酶标PRRSV N蛋白MAb的最佳稀释度为1:3000。ELISA结果判定标准为血清样品抑制率PI≥40%时判定为阳性,PI<40%判定为阴性。利用该方法检测PRRSV-1、PRRSV-2、猪伪狂犬病病毒(PRV)、猪圆环病毒2型(PCV2)、猪瘟病毒(CSFV)、猪细小病毒(PPV)、猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)等猪病毒阳性血清,结果显示该方法仅检测PRRSV-1和PRRSV-2为阳性,与其他病毒阳性血清均无交叉反应,特异性强。利用该方法和IDEXX试剂盒分别检测2倍倍比(1:2~1:2048)稀释的PRRSV 71#标准阳性血清,结果显示,该方法能检测出256倍稀释的71#标准阳性血清,而IDEXX试剂盒能检测到128倍稀释的71#标准阳性血清,本实验建立的方法敏感性更高;从同一批次和不同批次包被的ELISA板中各取3块,分别检测5份猪血清样品,每份样品各重复3次,进行批内和批间重复性试验,结果显示批内重复性试验的变异系数小于10%,批间重复性试验的变异系数小于15%。利用该方法和商品化IDEXX试剂盒对黑龙江地区采集的1012份临床血清样品进行检测,结果显示,本研究建立的B-ELISA对样品的阳性检测率为73.42%(743/1012),阴性率为26.58%(269/1012),IDEXX试剂盒对样品的阳性检测率为71.84%(727/1012),阴性率为28.16%(285/1012),二者的总符合率达97.43%,Kappa值为0.957,二者检测结果高度符合。本研究建立的B-ELISA方法为PRRSV的流行病学调查及其抗体水平的监测与检测提供了新的技术手段。
In order to establish an enzyme-linked immunosorbent assay(ELISA)method for detection of N protein antibodies against porcine reproductive and respiratory syndrome virus,a blocking-ELISA(B-ELISA)method was developed in this study using purified PRRSV whole virus as coating antigen and a monoclonal antibody against the N protein.The results showed that the optimal antigen coating concentration was 1μg/mL,the best dilution ratio of serum was 14,and the optimal dilution of the HRP-conjugated antibody was 13000.The ELISA result criteria:if the serum sample percentage inhibition(PI)was not less than 40%,it was considered to be positive or it was negative.The specificity test showed that this method can only detect PRRSV-1 and PRRSV-2 and had no cross-reaction with those positive serums against PRV,PCV2,CSFV,PPV,PEDV,and TGEV,with good specificity.The sensitivity test showed that this method had a relatively higher sensitivity than IDEXX PRRSV kit.This method can detect the 71#standard positive serum diluted 256 times.The detection results of the IDEXX kit showed that it can detect the 71#standard positive serum diluted 128 times,indicating this method has high sensitivity.Three ELISA plates coated in the same batch and three ELISA plates coated in different batches were respectively selected.Five serum samples were detected respectively,and each sample was repeated three times for the intra-batch and inter-batch repeatability tests.The results showed the coefficient of variation(CV)of the intra-batch repeatability test was less than 10%.The CV of the inter-batch repeatability test was less than 15%.1012 clinical serum samples collected in Heilongjiang region were tested by B-ELISA and IDEXX PRRSV kit.The established B-ELISA detected 743 positive serum samples with a positive rate of 73.42%,and 269 negative serum samples with a negative rate of 26.58%.The IDEXX kit detected 727 positive serum samples with a positive rate of 71.84%,and 285 negative serum samples with a negative rate of 28.16%.The coincidence rate with the commercial IDEXX PRRSV kit calculated by using EXCEL 2017 software was 97.43%,and the Kappa value was 0.957,indicating that these two detection results exhibited high consistency.Therefore,the B-ELISA method established in this study provides a novel detection method for the epidemiological investigation of PRRSV and the monitoring antibody levels.
作者
冯子萱
李宛生
张洪亮
许浒
李超
郭镇洋
彭金美
周国辉
王倩
田志军
FENG Zi-xuan;LI Wan-sheng;ZHANG Hong-liang;XU Hu;LI Chao;GUO Zhen-yang;PENG Jin-mei;ZHOU Guo-hui;WANG Qian;TIAN Zhi-jun(State Key Laboratory For Animal Disease Control and Prevention,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)
出处
《中国预防兽医学报》
北大核心
2025年第6期585-592,613,共9页
Chinese Journal of Preventive Veterinary Medicine
基金
中央级公益性科研院所基本科研业务费课题(1610302022008)
国家自然科学基金项目(32402920)。
关键词
猪繁殖与呼吸综合征病毒
N蛋白
抗体
阻断ELISA
porcine reproductive and respiratory syndrome virus
N protein
antibody
blocking ELISA
作者简介
共同第一作者:冯子萱(1999-),男,北京人,硕士研究生,主要从事PRRS的诊断研究;共同第一作者:李宛生(1994-),男,河南南阳人,博士研究生,主要从事PRRS的诊断研究.通信作者:王倩,E-mail:wangqian@caas.cn;通信作者:田志军,tianzhijun@caas.cn。
