猪繁殖与呼吸综合征病毒直接免疫荧光检测方法的建立

DEVELOPMENT OF DIRECT IMMUNOFLUORESCENT METHOD USING FITC-LABELED MONOCLONALANTIBODY AGAINST THE NUCLEOPROTEIN OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS

为了建立一种快速、准确的猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)直接免疫荧光检测方法,对前期制备的抗猪繁殖与呼吸综合征病毒核蛋白单克隆抗体进行亲和层析法纯化,经异硫氰酸荧光素标记;通过对荧光染色的固定试剂、固定时间、染色时间和荧光抗体工作浓度的测定,确定荧光抗体染色法在PRRSV感染细胞和病料中的最佳工作条件。利用荧光抗体染色法对本实验室收集的62份病料进行检测,同时以RT-PCR进行验证,确定荧光染色法的检测符合率。结果显示,FITC标记的荧光抗体检测细胞培养物最佳工作条件:80%丙酮4℃固定20 min,荧光抗体按1:400稀释,感作时间为1 h;检测组织病料最佳工作条件:病料组织进行涂片后,用80%丙酮4℃固定30 min,荧光抗体按1:200稀释,感作时间为2 h;病料组织荧光检测与RT-PCR的检测一致率为74.2%。本研究建立的快速、准确的猪繁殖与呼吸综合征病毒直接免疫荧光检测方法为今后该疾病诊断奠定了基础。

To establish a direct immunofluorescent method for diagnosis of Porcine reproductive and respiratory syndrome virus, monoclonal lgG antibody against the nucleocapsid protein was purified by affinity chromatography and labeled with FITC. The immunofluorescent antibody was optimized regarding cell fixing agents and conditions, antibody reaction time and working concentration. Then, 140 clinical samples were tested using the immunofluorescent antibody and RT-PCR. If cell culture samples were examined by immunofluoresece method, cell cultures were fixed with 80% acetone at 4 ℃ for 20 min and stained with 1：400 immunofluorescent antibodies following by incubation for 1 h. When clinical tissue samples were examined, cell cultures were fixed with 80% acetone at 4℃ for 30 min and stained 1：200 immunofluorescent antibodies following by incubation for 2 h. Agreement between immunoflorescent antibody and RT-PCR was 74.2%. The results suggested that the immunofluorescent antibody method could be used as an alternative for detecting PRRSV.