Balczon R, West K
Department of Structural and Cellular Biology, University of South Alabama, Mobile 36688.
Cell Motil Cytoskeleton. 1991;20(2):121-35. doi: 10.1002/cm.970200205.
Human autoimmune sera were screened for the presence of anticentrosome autoantibodies. Two high titer sera were identified that reacted with HeLa, CHO, and PtK2 centrosomes by immunofluorescence, although the fluorescent patterns that were obtained using the two antisera were separate and distinct. Serum obtained from patient IJ contained antibodies that reacted with epitopes present only in mitotic centrosomes; staining of interphase centrosomes was never detected uing IJ antiserum. Immunoblot analysis demonstrated that antibodies present in IJ antiserum reacted with a 190 kD spindle pole antigen. Immunofluorescent staining of cultured mammalian cells demonstrated that antibodies present in serum obtained from patient SPJ reacted with both interphase and mitotic centrosomes. Characterization of SPJ antiserum by immunoblotting demonstrated that antibodies present in the SPJ serum recognized proteins of Mrs of 39, 185, and 220 kD, although the possibility that the 185 kD polypeptide was a proteolytic breakdown product of the 220 kD protein has not been eliminated. Neither antiserum was able to inhibit microtubule nucleation from centrosomes in a lysed cell system in which pure 6S tubulin was added to permeabilized cells following pretreatment of the cells with either SPJ or IJ antiserum. These antisera should be useful probes for studying the biochemistry of the mammalian centrosome.
对人自身免疫血清进行筛选，以检测抗中心体自身抗体的存在。鉴定出两份高滴度血清，通过免疫荧光法可与HeLa、CHO和PtK2中心体发生反应，不过使用这两种抗血清获得的荧光模式是分开且不同的。从患者IJ获得的血清含有仅与有丝分裂中心体中存在的表位发生反应的抗体；使用IJ抗血清从未检测到间期中心体的染色。免疫印迹分析表明，IJ抗血清中存在的抗体与一种190 kD的纺锤体极抗原发生反应。对培养的哺乳动物细胞进行免疫荧光染色表明，从患者SPJ获得的血清中存在的抗体与间期和有丝分裂中心体均发生反应。通过免疫印迹对SPJ抗血清进行鉴定表明，SPJ血清中存在的抗体识别分子量为39、185和220 kD的蛋白质，不过185 kD多肽是220 kD蛋白质的蛋白水解降解产物这种可能性尚未排除。在一个裂解细胞系统中，在用SPJ或IJ抗血清对细胞进行预处理后，向通透细胞中加入纯6S微管蛋白，这两种抗血清均无法抑制中心体的微管成核。这些抗血清应是研究哺乳动物中心体生物化学的有用探针。
