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首页> 外文期刊>Journal of Parasitology >MONOCLONAL ANTIBODY DIRECTED AGAINST NEOSPORA CANINUM TACHYZOITE CARBOHYDRATE EPITOPE REACTS SPECIFICALLY WITH APICAL COMPLEX–ASSOCIATED SIALYLATED BETA TUBULIN
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MONOCLONAL ANTIBODY DIRECTED AGAINST NEOSPORA CANINUM TACHYZOITE CARBOHYDRATE EPITOPE REACTS SPECIFICALLY WITH APICAL COMPLEX–ASSOCIATED SIALYLATED BETA TUBULIN

机译：针对新新犬齿犬齿T碳酸盐表位的单链抗体，特别是与APIAPI缔合的唾液酸化β-微管蛋白反应

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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 β 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 β 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 β 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 β tubulin.
机译：针对整个超声处理的新孢子虫速殖子产生了单克隆抗体（单克隆抗体）作为免疫原。使用相同的抗原和在抗体结合前用高碘酸钠处理过的抗原对杂交瘤培养上清液的反应性进行了ELISA初步筛选，结果鉴定出8种上清液具有对假定的碳水化合物表位的反应性。免疫印迹后，选择结合52 / 48-kDa双峰的mab6D12（IgG1）和结合190 / 180-kDa双峰的mab6C6（IgM）进行进一步研究。速殖子感染的培养物的免疫荧光定位将相应的抗原决定簇定位在犬新孢子虫速殖子的顶端而不是表面，而是定位在内部抗原决定簇上。在体外速殖子向缓殖子阶段的转化过程中，mab6C6标记移向囊肿外围，而对于mab6D12，未发现定位变化。用非离子型去污剂Triton-X-100提取速殖子后，被mab6D12识别的52 kDa条带仅存在于犬新孢子虫和弓形虫速殖子的不溶性细胞骨架部分中。串联质谱分析鉴定该蛋白为犬新孢子虫β微管蛋白。 mab6D12标记的48 kDa条带是Vero细胞蛋白质污染。与mab6C6反应的蛋白质无法通过质谱法最终确定。免疫荧光法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的β微管蛋白可能被差异修饰或刚地弓形虫的反应性表位被掩盖。分离的根尖细胞骨架制剂的免疫金相TEM和具有微管蛋白抗体的双重免疫荧光证实了mab6D12与根尖复合体相关的微管的前部结合。通过免疫印迹和ELISA证实了β微管蛋白相关表位的高碘酸钠敏感性，并且在mab6D12标记之前用唾液酸酶处理犬新孢子虫细胞骨架蛋白会导致抗体结合的大量丧失，这表明mab6D12与唾液酸化的β微管蛋白反应。

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