牛支原体单克隆抗体的制备与鉴定

Preparation and Identification of Monoclonal Antibodies against Mycoplasma bovis

以牛支原体(Mycoplasma bovis)湖北分离株HB0801作为抗原免疫8周龄BALB/c小鼠,利用杂交瘤技术筛选出了6株能稳定分泌抗牛支原体的单克隆抗体细胞株,分别生产腹水并对单抗进行了纯化和特性鉴定。经亚型测定,这些单抗都属IgG类。腹水ELISA效价在1×105～1.6×106。ELISA特异性分析结果表明,6株单抗与临床分离的牛支原体菌株以及ATCC标准株PG45都显阳性反应,但与牛的其他常见病原菌如多杀性巴氏杆菌、化脓隐秘杆菌等都显阴性反应。所有制备的单抗都与无乳支原体有交叉反应,其中两株单抗1A5和1C11只与无乳支原体有交叉反应,与其他支原体无交叉反应。经Western blotting验证,6株单抗分别识别牛支原体全菌蛋白中的不同条带,说明分别针对不同的蛋白抗原。这些牛支原体单克隆抗体为后期建立牛支原体检测方法及致病机理研究奠定了良好基础。

8-week-old female BALB/c mice were immunized with Mycoplasrna boris HB0801. By using the hybridoma technique, six hybridoma cell lines secreting monoclonal antibodies （MAbs） were identified. The ascite for the six MAbs were prepared and the characteristics of them were further studied. All of the six MAbs belonged to IgG category. The antibody ti- ters of these MAbs ranged from 1 × 10^5 to 1.6 × 10^6. The specificity of these MAbs was detected by ELISA. The result sugges- ted that all these MAbs can react with Mycoplasma boris strains clinically isolated from China and the ATCC reference strain PG45, but they did not react with other bovine pathogenic bacteria tested such as Pasteurella multocida, Arcanobacterium pyogenes and so on. All MAbs investigated cross-reacted with Mycoplasma agalactiae which was known to be closely related to Mycoplasma boris, prevalent in goats but not in cattle. Among them, two MAbs 1A5 and 1Cll did not exhibit further cross reactions to other Mycoplasma species tested. In addition, Western blotting assay demonstrated that these six antibodies recognized various Mycoplasma boris proteins. In conclusion, the MAbs prepared in this study layed the foundation for the es- tablishment of novel detection methods for Mycoplasma boris and studying its pathogenesis.