摘要
旨在制备牛肠道病毒2型(BEV-2)单克隆抗体,建立检测BEV-2抗原的ELISA方法。将纯化的BEV-2VP1+重组蛋白质接种BALB/c小鼠,取其脾淋巴细胞与骨髓瘤细胞SP2/0用PEG4000进行融合,对杂交瘤培养上清进行筛选,获得2株稳定分泌抗BEV-2VP1+蛋白的单克隆抗体的杂交瘤细胞株(1G1、5G6)。用制备的单克隆抗体包被酶标板,通过一系列的优化,建立了BEV-2双抗夹心ELISA检测方法。建立的ELISA方法检测BEV-2病毒量的最低限为100TCID50·0.1mL-1,与BVDV、IBRV、BLV等病毒无交叉反应。本研究首次制备了牛肠道病毒2型单克隆抗体,建立了检测BEV-2抗原的双抗体夹心ELISA方法,该方法特异性强、敏感性高、重复性好,为进一步研究和开发快速诊断牛肠道病毒试剂盒奠定了基础。
The study was aimed to prepare monoclonal antibody against BEV-2 and develop ELISA for the detection of antigen of bovine enterovirus type 2(BEV-2)based on the monoclonal antibody.The purified BEV-2VP1+recombinant protein was used to immunize BALB/c mice,of which splenocytes were fused with SP2/0cells by PEG4000.The selection of positive hybridomas was conducted.Two hybridomas(designated as 1G1 and 5G6)secreting monoclonal antibodies(mAb)against BEV-2 VP1+protein were selected.Based on the monoclonal antibodies against BEV-2,a sandwich ELISA was established to detect BEV-2through a series of optimization.The sensitivity of the sandwich ELISA was 100TCID50·0.1mL-1,and this method had no cross-reaction with BVDV,IBRV and BLV.The results showed that the sandwich ELISA was specific,sensitive and reproducible.This is the first domestic report of establishment of sandwich ELISA for the detection of BEV-2based on the preparation of the monoclonal antibodies,and it provided a basis for development of kit for rapid diagnosing BEV-2.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2015年第11期2025-2031,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家质检总局科研项目(2013IK026)
