免疫磁珠分离技术纯化和检测泰泽氏病原体的研究

Isolation and Detection of Clostridium piliformis by Immunomagnetic Bead Assays

目的建立泰泽氏病原体(Ty)纯化方法,获得纯的菌体供抗原研究,为ELISA诊断抗原的制备提供依据;并尝试建立Ty隐性感染血清学抗原检查方法。方法选择特异性抗体包被磁珠,从感染大鼠肝脏中富集和纯化Ty;用SDS-PAGE和Western blot技术考察纯化Ty的蛋白和抗原图谱;同时用免疫磁珠分离技术(IMS)直接检查隐性感染大鼠肠道上皮细胞内的Ty。结果用辛酸-硫酸铵纯化的Ty单克隆抗体M5以0·5μg/107beads以上浓度包被抗IgG抗体预结合的磁珠4h,可最大效率地富集Ty;分离反应进行1h,敏感性达到103菌体/mL;吖啶黄染色镜检法可以直接、快速地观察到结合于磁珠上的细菌;抗原分析表明,IMS较好地去除了肝组织和真核细胞成分,纯化的Ty RJ株具有3个免疫优势的抗原成分,相对分子质量(Mr)分别为160×103、116×103、55×103;此外,IMS法可直接从隐性感染大鼠盲肠上皮细胞中检测到少量寄生的Ty。结论用IMS技术可有效地富集和纯化Ty,并可以作为Ty隐性感染血清学抗原检查的候选方法。

Objective To establish a method for purification of Clostridium piliformis （Ty）, and take advantage of this method to prepare serodiagnostic antigen for specific ELISA test. Method To concentrate and purify Ty from infected liver homogenate suspension using immunomagnetic beads coated with monoclonal antibody specific for Ty. To analyze the protein and antigen components of purified Ty with SDS-PAGE and Western blotting. Meanwhile, to directly isolate parasitized Ty in the cecum epithelium of subclinical infected animals by using immunomagnetic separation method （IMS）. Result The sheep antimouse IgG pre-combined beads coated with purified monoclonal antibody M5 specific for Ty in a concentration of 1.0 μg Ab/10^7 beads or more for 4 hours can most efficiently concentrate the pure Ty from heterogeneous suspension and remove most of the matrix-associated components. The whole detection process required 1 h and the sensitivity was lOs bacteria/mL. Captured bacteria were easily identified by acridine yellow staining. The protein and antigen profiles of pure RJ isolate showed that the main immunodominant antigens located at molecular weight 160 × 10^3 , 116 × 10^3 and 55 × 10^3 . The properly coated immunomagnetic beads can sensitively detect low level intracellular Ty in the cecum epithelialium which is the major tissue site of Tysubclinical infection. Conclusion Ty can be successfully purified by using IMS techniques. IMS is also an ideal method for direct antigen examination of Ty in cecum epithelium.