MONOCLONAL-ANTIBODIES TO MITOTIC CELLS
MONOCLONAL-ANTIBODIES TO MITOTIC CELLS
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DOI：
10.1073/pnas.80.10.2926
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
RAO, PN
中科院分区：
文献类型：
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作者：
DAVIS, FM;TSAO, TY;RAO, PN
某些蛋白质或活性存在于有丝分裂细胞中，但不存在于间期细胞中。这些蛋白质可以在有丝分裂前合成或激活，或两者兼而有之，并负责核膜的破裂和染色体的浓缩。为了了解更多关于这些蛋白质的性质，提出了针对有丝分裂细胞的单克隆抗体。用同步有丝分裂的HeLa [人宫颈癌]细胞的0.15 M NaCl提取物免疫的小鼠的脾细胞与SP2/0-Ag 14小鼠骨髓瘤细胞融合，并在含有次黄嘌呤、甲氨蝶呤、胸苷和甘氨酸的培养基中选择杂交体。分离了两种不同的杂交瘤克隆，其分泌与来自每个试验物种[人、小鼠、仓鼠、鸡、青蛙、线虫、蚊子、大鼠]的有丝分裂和减数分裂细胞反应的抗体。有丝分裂细胞的染色体和细胞质与抗体反应，通过间接免疫荧光检测。通过在NaDodSO 4 [十二烷基硫酸钠]/聚丙烯酰胺平板凝胶中电泳分离有丝分裂细胞的蛋白质，转移到硝酸纤维素片上，并进行免疫化学染色。这2种抗体被命名为MPM-1和MPM-2，识别表观分子量为0.40至> 200千道尔顿（kDA）的多肽家族。两种抗体均与182 kDa、118 kDA和70 kDA的3条多肽带反应。只有有丝分裂细胞显示出被抗体识别的蛋白条带。经~（32）P标记、放射自显影及碱性磷酸酶处理后，这些条带均为磷蛋白。
Certain proteins or activities are present in mitotic cells but not in interphase cells. These proteins may be synthesized or activated, or both, just prior to mitosis and are responsible for the breakdown of the nuclear envelope and the condensation of chromosomes. To learn more about the nature of these proteins, monoclonal antibodies to mitotic cells were raised. Spleen cells from mice immunized with a 0.15 M NaCl extract of synchronized mitotic HeLa [human cervical carcinoma] cells were fused with SP2/0-Ag14 mouse myeloma cells, and hybrids were selected in medium containing hypoxanthine, methotrexate, thymidine and glycine. Two different hybridoma clones secreting antibodies reactive with mitotic and meiotic cells from every species [human, mouse, hamster, chicken, frog, nematode, mosquito, rat] tested were isolated. Chromosomes and cytoplasm in mitotic cells reacted with the antibodies, as detected by indirect immunofluorescence. The proteins for mitotic cells were separated by electrophoresis in NaDodSO4[sodium dodecyl sulfate]/polyacrylamide slab gels, transferred to nitrocellulose sheets, and stained immunochemically. The 2 antibodies, designated MPM-1 and MPM-2, recognize a family of polytpeptides with apparent molecular masses of 0.40 to > 200 kilodaltons (kDA). Both antibodies reacted strongly with 3 polypeptide bands of 182 kDa, 118 kDA and 70kDA. Only mitotic cells exhibited the protein bands that were recognized by the antibodies. All these bands were phosphoproteins as shown by 32P labeling and autoradiography and their removal by alkaline phosphatase treatment.