群特异性蓝舌病病毒单克隆抗体的制备和鉴定

目的：制备群特异性抗蓝舌病病毒（BTV）单克隆抗体,并对其特性进行鉴定,为建立检测BTV抗原及抗体的ELISA方法奠定基础。方法：用纯化的BTV颗粒为免疫抗原免疫BALB/c鼠,以大肠杆菌表达的VP7蛋白作为筛选抗原,用间接ELISA法筛选杂交瘤细胞株;选取抗体效价最高的一株制备BTV单克隆抗体,以该抗体为捕获抗体与8种不同血清型BTV进行ELISA反应,结果与细胞病变反应进行比对;以该抗体为竞争抗体,与12种不同血清型绵羊BTV抗血清进行竞争ELISA反应,并将结果与参比c-ELISA试剂盒结果进行比对。结果：筛选出5株稳定分泌BTV单克隆抗体的杂交瘤细胞株,并选其中一株（3E2）制备了高纯度的单克隆抗体;该单抗用于检测不同血清型BTV,与细胞病变反应结果完全相符;用于检测不同血清型绵羊BTV抗血清,其结果与参比c-ELISA试剂盒符合率为100%,与鹿流行性出血热病毒抗原和抗体均无交叉反应。结论：制备的BTV单克隆抗体具有良好的群特异性,可用于检测不同血清型BTV抗原及BTV抗体。

Preparation and Characterization of Group Specific Monoclonal Antibody Against Bluetongue Virus

Objective： To prepare and characterize group specific monoclonal antibody （McAb） against bluetongue virus （BTV） for establishing ELISA detecting BTV antigen and antibody. Method： Purified BTV visions were used to immunize BALB/c mice. The hybridoma culture supernatants were screened by indirect ELISA with bacterial expressed VP7 protein as coating antigen. The hybridoma clones with the highest antibody titer were selected and employed for preparation of McAb. Antigen capture ELISA was performed with this McAb to detect BTV of eight different groups. Competition ELISA was performed with this MeAb to detect BTV antiserum of twelve different groups. Results： Five hybridoma clones that constitutively secrete McAb against BTV were screened. High purified McAb was prepared with 3E2 hybridoma clone. The result of antigen capture ELISA was consistent with cell pathological effect. The level of agreement between the MeAb and the reference c-ELISA test was 100%. There is no cross-reaction with antibody to EHDV in sera. Conclusion： Group specific McAb against BTV has been prepared successfully. The MeAb represents a convenient tool for detecting BTV antigen as well as antibody of different groups.