摘要
应用制备的牛病毒性腹泻病毒(BVDV) CC13B毒株E2蛋白的单克隆抗体与多克隆抗体,建立了基于单抗捕获牛病毒性腹泻-黏膜病病毒(Bovine viral diarrhea-mucosal disease virus,BVD-MDV)抗原的双抗体夹心ELISA方法。方阵试验确定的捕获BVDV抗原的单克隆抗体最佳抗体包被量为200 ng/孔,酶标多克隆抗体的最佳稀释浓度为1 000倍稀释。对100份BVDV阴性粪便样品进行检测与统计学处理,确定了夹心ELISA检测BVDV抗原的判定标准为待检样品D_(490)值≥0.156 7时,检测结果判定为阳性。特异性、敏感性和重复性试验结果显示,建立的检测BVDV抗原的方法具有特异、敏感、快速及重复性好等特点。以建立的单抗捕获BVDV抗原的夹心ELISA方法,调查了山东、河南和吉林3省部分地区牛群BVDV感染情况,发现上述地区牛群的BVDV感染率低至6%。本研究结果为BVDV感染的诊断、检疫、净化及防制提供了有效的技术手段及流行病学理论依据。
A sandwich ELISA for capture of BVD-MDV antigen based on monoclonal antibody was developed using antibodies raised against the recombinant protein E2 derived from BVDV CC13 B strain.The concentrations of antigen-capture mcAb and HRP-conjugated polyclonal antibody againsti-E2 were optimized using square matrix titrimetry.The criterion for the double antibody sandwich ELISA was determined and sample was assessed as positive when its D_(490)≥0.156 7.The established double antibody sandwich ELISA had high specificity and sensitivity with a well repeatability and simplicity.Investigation on BVDV infections in cattle herds from Shandong Province,Henan Province and Jilin Province revealed a low to 6% BVDV infection in different regions.This study provides an effective means and epidemiological theoretical basis for the diagnosis,quarantine and prevention of BVDV infection in the future.
引文
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