IPMA筛选猪繁殖与呼吸综合征病毒单克隆抗体方法研究

Development of a IPMA Method Applicable for Screening mAb Targeting PRRSV

为了得到一种能够高通量检测细胞培养物的方法,并将其用于筛选特异性强、敏感度高的抗猪繁殖与呼吸综合征病毒(PRRSV)单克隆抗体。用每孔能感染约100个细胞的病毒量接种单层覆盖96孔板的Marc-145细胞,12h后用含3%H2O2的甲醇固定细胞,以制备免疫过氧化物酶单层细胞试验(IPMA)反应板。以100μL/孔的量将融合后继续培养10d的杂交瘤细胞的培养上清加入至IPMA反应板,以辣根过氧化物酶标记的羊抗鼠IgG-HRP作为二抗,以3-氨基-9-乙基咔唑(AEC)作为显色底物,于倒置显微镜下进行观察。结果表明,共筛选出1D1、7G8等39份PRRSV单克隆抗体,这39份单抗能够使PRRSV中高致病毒株HN07-1和经典毒株BJ-4感染的Marc-145细胞被特异性染色,而对猪瘟病毒、猪伪狂犬病毒、猪流行性腹泻病毒感染的Marc-145细胞无交叉染色。因此,构建的IPMA方法能够敏感、准确地捕捉到PRRSV单克隆抗体。

To develop a high-throughput cell culture detection method for screening specific and sensitive monoclonal antibodies against Porcine reproductive and respiratory syndrome virus (PRRSV). A single layer of Marc-145 cells covered with 96-well plates was inoculated with a virus amount of about100 cells infected per well. After12 h,cells were fixed with 3% H 2 O 2 in methanol to prepare an immunoperoxidase monolayer cell assay (Immunoperoxidase monolayer assay,IPMA)reaction plate. The culture supernatant of the hybridoma cells cultured for10 days after the fusion was added to the IPMA reaction plate in an amount of100 μL/well,and horseradish peroxidase-labeled goat anti- mouse IgG-HRP was used as the secondary antibody and 3-amino-9- ethylcarbazole(AEC)was used as a chromogenic substrate,then observed under an inverted microscope. The results showed that 39 PRRSV monoclonal antibodies,such as1D1 and 7G8,were screened, and these 39 monoclonal antibodies were able to specifically stain Marc-145 cells infected with high-viral strain HN07-1 and classical strain BJ-4 PRRSV. There was no cross-staining of Marc- 145 cells infected with Classical swine fever virus,Pseudorabies virus, and Porcine epidemic diarrhea virus. Therefore,this IPMA method constructed by the study can capture PRRSV monoclonal antibodies sensitively and accurately.