抗鹿源牛病毒性腹泻-粘膜病病毒特异性单克隆抗体的制备与鉴定

Preparation and identification of specific monoclonal antibody against Sika bovine viral diarrhea virus

为建立鹿源牛病毒性腹泻-粘膜病病毒(BVDV)检测方法,本研究制备了抗鹿源BVDV特异性单克隆抗体(MAb)。用纯化的BVDV免疫BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,经3次有限稀释法克隆和间接ELISA法筛选,获得2株稳定分泌MAb的杂交瘤细胞株2A3和4B12。通过间接ELISA检测,MAb效价为:上清液及腹水分别为1∶512、1∶640和1∶12800、1∶16000;MAb的亚类鉴定结果表明,2株杂交瘤细胞分泌的抗体亚类均为IgGl亚类;经ELISA测定,2A3和4B12与BVDVC24V株、HCV、BDV、PRV均不发生交叉反应,但与BVDVCCSYD株发生交叉反应;IFA试验结果显示,4B12和2A3与接种BVDVN71株病毒液的细胞反应产生较强的特异荧光;抗原识别位点分析结果表明,2A3和4B12两株MAb所识别的抗原位点相同。这两株MAb的获得为鹿BVDV的检测方法的建立奠定良好的基础。

The monoclonal antibodies （MAbs） against bovine viral diarrhea virus （BVDV） was developed for the detection of BVDV infection in deer. Two hybridoma cell lines designated as 2A3 and 4B12 were produced by fusing mouse mycloma cells （SP2/0） with spleen cells from BALB/c immunized with BVDV N71 strain. The antibody titres of hybridoma supernatant and ascetic fluids of 2A3 and 4B12 were 1∶512, 1∶640 and 1∶12 800, 1∶16 000, respectively when detected by indirect ELISA. Indirect immunofluorescent assay （IFA） showed that both MAbs reacted specifically with BVDV N71 strain prepared in MDBK cells. The 2A3 and 4B12 could also react with BVDV CCSYD strain, but showed no cross reactions with BVDV C24V strain, classical swine fever virus, sheep border virus, and pseudorabies virus. The additive ELISA showed that 2A3 and 4B12 reacted with the same antigen determinant.