赤羽病病毒单克隆抗体的制备及c-ELISA抗体检测方法建立

Preparation of Monoclonal Antibody for Akabane Disease and Establishment of c-ELISA Antibody Detection Method

为建立一种用于快速检测赤羽病病毒抗体的竞争ELISA法。用AKV病毒免疫Balb/C小鼠,将其脾细胞与SP2/0细胞进行免疫融合,以获得抗AKV的单克隆抗体;利用Bac-to-Bac杆状病毒表达的SBV N蛋白作为诊断特异性抗原,山羊抗鼠HRP-IgG为二抗,建立并优化AKV抗体检测的竞争ELISA方法。得到一株持续稳定繁殖的能够分泌单抗AKV核蛋白抗体的杂交瘤细胞株,单抗亚型鉴定为:重链IgG1,轻链kappa,仅能与AKV病毒呈阳性反应,与BTV、FAMD、EHDV病毒等病毒抗原不发生特异性反应;建立的检测AKV抗体ELISA检测方法,诊断抗原最佳包被浓度为0.5μg/mL,1∶1000抗体稀释比,1∶50血清稀释比,1∶2000二抗稀释比,封闭条件为5%BSA,37℃封闭2 h,确定了血清抑制率大于等于44%时为阳性,小于44%为阴性;所建立的ELISA方法敏感性和特异性鉴定结果与ID Screen AKV Competition检测试剂盒一致。本试验成功制备出一株分泌针对AKV N蛋白的杂交瘤细胞系,建立的ELISA检测方法能够用于检测动物AKV抗体,为进一步开展AKV抗体诊断试剂工艺研究、产业化奠定了基础。

To establish a competitive ELISA method for the rapid detection of antibodies to Akabane disease virus.Balb/C mice were immunized with AKV virus,and their splenocytes were immunofused with SP2/0 cells to obtain monoclonal antibodies against AKV.Using the SBV N protein expressed by Bac-to-Bac baculovirus as a diagnostic specific antigen,and goat anti-murine HRP-IgG as a secondary antibody,a competitive ELISA method for AKV antibody detection was established and optimized.A hybridoma cell line that could secrete monoclonal antibody AKV nucleoprotein antibody was obtained with continuous and stable reproduction,and the monoclonal antibody subtype was identified as:heavy chain IgG1 and light chain kappa,which could only react positively with AKV virus,and did not react specifically with viral antigens such as BTV,FAMD,and EHDV virus.The established detection method for AKV antibody ELISA showed that the optimal coating concentration of diagnostic antigen was 0.5μg/mL,1∶1000 antibody dilution ratio,1∶50 serum dilution ratio,1∶2000 secondary antibody dilution ratio,5%BSA,37℃blocking for 2 h,and it was determined that the serum suppression rate was positive when it was greater than or equal to 44%,and it was negative when it was less than or equal to 44%.The established ELISA method sensitivity and specificity identification results are consistent with the ID Screen AKV Competition assay.A hybridoma cell line secreting AKV N protein was successfully prepared,and the established ELISA detection method can be used to detect AKV antibodies in animals,which lays a foundation for further research and industrialization of AKV antibody diagnostic reagents.