副猪嗜血杆菌OMP5单克隆抗体的制备与鉴定  

Preparation and identification of monoclonal antibodies against OMP5 of Haemophilus parasuis

作　　者：秦翠丽[1,2] 高祥辉[1,2,3] 李春玲[2,3] 

机构地区：[1]河南科技大学,河南洛阳471003 [2]广东省农业科学院兽医研究所,广东广州510640 [3]广东省兽医公共卫生公共实验室,广东广州510640

出　　处：《中国兽医学报》2012年第10期1468-1472,共5页Chinese Journal of Veterinary Science

基　　金：广东省农业科技重大专项资助项目(2009A0201006);广州市农业科技重大专项资助项目(2009A1-E04)

摘　　要：用副猪嗜血杆菌(HPS)血清5型灭活菌液免疫BALB/c小鼠,取其脾细胞与Sp2/0骨髓瘤细胞融合,以HPS外膜蛋白5(OMP5)为抗原,经间接ELISA法筛选阳性融合孔,并用有限稀释法对筛选的阳性孔进行亚克隆,获得2株分泌抗HPS OMP5单克隆抗体的杂交瘤细胞株1E2和8D9;间接ELISA法效价检测,1E2和8D9细胞培养上清抗体效价均为1∶2 560,纯化后腹水抗体效价分别为1∶204 800和1∶51 200;抗体型别鉴定,1E2和8D9分别分泌IgG2b、IgG2a型抗体;特异性检测表明,2株单抗均能特异识别OMP5。将2株杂交瘤细胞反复冻融3次复苏后仍能稳定分泌抗体。2株抗HPS OMP5单克隆抗体的成功制备,将为HPS感染快速检测方法的建立以及检测试剂的研制奠定基础。Two monoclonal antibody hybridomases（McAbs） named 1E2 and 8D9 were produced,respectively.In the procedure,SP2/0 and spleen cells from BALB/c mice immunized with Formalin-killed whole bacteria cells of serotype 5 of Haemophilus parasuis were fused.The positive hybridomases against OMP5 were screened by indirect enzyme-linked immunosorbent assay,and then subcloned by limiting dilution assay.The ELISA titers of culture supernatant were both 1∶2 560,and those of ascites were 1∶204 800 and 1∶51 200,respectively.The McAbs isotypes were detected as 1E2：IgG2b and 8D9：IgG2a with McAB isotypes kits.Both of the McAbs can recognize OMP5 with indirect ELISA assay.The two hybridomases can stably secrete antibody after three times of repeated freezing and thawing.Two McAbs against HPS OMP5 were successfully prepared,which laid a foundation of developing a rapid determination for HPS.

关 键 词：副猪嗜血杆菌 OMP5 单克隆抗体 杂交瘤技术 

分 类 号：S852.612[农业科学—基础兽医学]