猪传染性胃肠炎病毒免疫过氧化物酶单层细胞试验原位检测方法的建立

Development of immunoperoxidase monolayer assay detecting transmissible gastroenteritis virus in situ

为建立猪传染性胃肠炎病毒(TGEV)的原位检测方法,本研究以TGEV N蛋白的单克隆抗体为一抗,羊抗小鼠IgG-HRP为二抗,AEC过氧化物酶底物为显色液,建立了TGEV的免疫过氧化物酶单层细胞试验(IPMA)检测方法。检测结果显示,一抗的最佳使用浓度为0.5 ng/μL,二抗的最佳稀释倍数为1∶2 000倍,经AEC显色,TGEV感染的阳性细胞呈红棕色;对常见的猪流行性腹泻病毒、猪轮状病毒和猪圆环病毒2型等猪病病原检测均为阴性,具有良好特异性;同时该方法能够在普通显微镜下观察到病毒在细胞中的分布情况。本研究建立的方法为TGEV的实验室鉴定及TGEV在感染细胞中的定位和动态分布提供有效的检测手段。

In order to develop an method for detection of transmissible gastroenteritis virus （TGEV） in situ, the immuno- peroxidase monolayer assay （IPMA） was established with a monoclonal antibody against TGEV N protein as the primary antibody and the HRP labeled goat anti-mouse IgG as the second antibody with the AEC as substrate-chromogen. Then, the optimized reaction conditions included using 0.5 ng/μL of the primary antibody and the 1：2,000 diluted second antibody. The detection results showed that TGEV IPMA-positive staining reactions were characterized by red-brown staining in the TGEV-infected PK-15 cell monolayer. The assay was specific for detecting TGEV, and had no cross-reactions with other related pathogenic porcine viruses, such as porcine epidemic diarrhea virus, porcine rotavirus and porcine cirovirus type 2. Moreover, this assay only need light microscopy, which was more convenient to examine the distribution of TGEV in PK15 cells in situs.