Srinivasan Sangeetha, Baszler Timothy, Vonlaufen Nathalie, Leepin Angela, Sanderson Sanya J, Wastling Jonathan M, Hemphill Andrew
Institute of Parasitology, University of Bern, Switzerland.
J Parasitol. 2006 Dec;92(6):1235-43. doi: 10.1645/GE-889R.1.
Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.
以经超声处理的犬新孢子虫速殖子全虫作为免疫原制备单克隆抗体（mabs）。使用相同抗原以及在抗体结合前用过碘酸钠处理的抗原，对杂交瘤培养上清液的反应性进行初步酶联免疫吸附测定（ELISA）筛选，结果鉴定出8种对假定碳水化合物表位有反应性的上清液。免疫印迹后，选择结合52/48-kDa双峰的单克隆抗体6D12（IgG1）和结合190/180-kDa双峰的单克隆抗体6C6（IgM）进行进一步研究。速殖子感染培养物的免疫荧光显示，相应表位不在犬新孢子虫速殖子的表面，而是位于顶端部分的内部表位。在体外速殖子向缓殖子阶段转化过程中，单克隆抗体6C6的标记向包囊周边移位，而单克隆抗体6D12的定位未见变化。用非离子去污剂Triton-X-100提取速殖子后，单克隆抗体6D12识别的52-kDa条带仅存在于犬新孢子虫和刚地弓形虫速殖子的不溶性细胞骨架部分。串联质谱分析鉴定该蛋白为犬新孢子虫β微管蛋白。单克隆抗体6D12标记的48-kDa条带是Vero细胞蛋白污染。与单克隆抗体6C6反应的蛋白无法通过质谱法明确鉴定。免疫荧光始终未能标记刚地弓形虫速殖子，表明刚地弓形虫和犬新孢子虫中的β微管蛋白可能有不同修饰，或者刚地弓形虫中的反应性表位被掩盖。对分离的顶端细胞骨架制剂进行免疫金透射电镜分析以及用抗微管蛋白抗体进行双重免疫荧光分析，证实单克隆抗体6D12与顶端复合体相关微管的前部结合。通过免疫印迹和ELISA证实了与β微管蛋白相关表位的高碘酸钠敏感性，并且在单克隆抗体6D12标记前用唾液酸酶处理犬新孢子虫细胞骨架蛋白导致抗体结合显著丧失，表明单克隆抗体6D12与唾液酸化的β微管蛋白反应。
Zotero 插件