MONOCLONAL-ANTIBODIES AGAINST MYOFIBRILLAR COMPONENTS OF RAT SKELETAL-MUSCLE DECORATE THE INTERMEDIATE FILAMENTS OF CULTURED-CELLS
MONOCLONAL-ANTIBODIES AGAINST MYOFIBRILLAR COMPONENTS OF RAT SKELETAL-MUSCLE DECORATE THE INTERMEDIATE FILAMENTS OF CULTURED-CELLS
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DOI：
10.1073/pnas.78.4.2335
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
LIN, JJC
中科院分区：
文献类型：
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作者：
LIN, JJC
用大鼠骨骼肌原纤维免疫BALB/c小鼠，取脾细胞与骨髓瘤细胞融合，体外制备单特异性抗体。在琼脂糖上克隆3次后，获得两个稳定的克隆并选择用于进一步表征。第一个克隆JLB 1产生的抗体识别分布在肌原纤维的M线区域和Z线的任一位点上的抗原。第二个克隆JLB 7产生的抗体仅与位于肌原纤维M线区域的抗原反应。JLB 1和JLB 7抗体都修饰各种培养细胞的典型中间丝。在与两种抗体反应之前对细胞进行秋水仙胺处理导致细胞核周围的纤维卷曲或加帽（或两者）。用细胞松弛素B对细胞进行短暂处理并不影响两种抗体染色的纤维的完整性，而在相同条件下，另一种抗肌动蛋白单克隆抗体（JLA 20）观察到的微丝束在细胞质中被分解成许多聚集体。通过对用这些单克隆抗体和兔自身免疫血清（已显示与中间丝的组分反应）染色的相同细胞进行双标记免疫荧光显微镜检查，获得中间丝的相同染色模式。用免疫沉淀法鉴定了鸡胚成纤维细胞中分子量为210，000和95，000道尔顿的蛋白质条带，分别为JLB 1和JLB 7单克隆抗体识别的潜在抗原。这些抗原决定簇在不同培养细胞中的广泛存在表明这些中间丝组分的高度保守性。
Monospecific antibodies were produced in vitro by fusing mouse myeloma cells with spleen cells from a BALB/c mouse immunized with rat skeletal myofibrils. After cloning 3 times on agarose, two stable clones were obtained and chosen for further characterization. The first clone, JLB1, produced an antibody that recognizing an antigen distributed in the M-line region and on either site of the Z line of myofibrils. The second clone, JLB7, produced an antibody reacting only with an antigen located at the M-line region of myofibrils. Both JLB1 and JLB7 antibodies decorate the typical intermediate filaments of a variety of cultured cells. Colcemid treatment of cells before reaction with both antibodies resulted in the coiling or capping (or both) of the fibers around the nucleus. Brief treatment of cells with cytochalasin B did not affect the integrity of the fibers stained by both antibodies whereas, under the same conditions, microfilament bundles visualized by another monoclonal antibody (JLA20) against actin were disassembled into many aggregates in the cytoplasm. Identical staining patterns of the intermediate filaments are obtained by double-label immunofluorescence microscopy of the same cell stained with these monoclonal antibodies and rabbit autoimmune serum (which has been shown to react with the components of the intermediate filaments). By using immunoprecipitation, protein bands at 210,000 and 95,000 daltons from chicken embryo fibroblasts were identified as the potential antigens recognized by JLB1 and JLB7 monoclonal antibodies, respectively. The widespread occurrence of these antigenic determinants in different cultured cells suggests the highly conservative property of these intermediate-filament components.