Preparation and characterization of a human aurora-A kinase monoclonal antibody
Preparation and characterization of a human aurora-A kinase monoclonal antibody
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DOI：
10.1023/a:1021608012253
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发表时间：
2003-01-01
影响因子：
4.3
通讯作者：
Prigent, C
中科院分区：
文献类型：
--
作者：
Cremet, JY;Descamps, S;Prigent, C
我们已经开发了针对人极光-A丝氨酸/苏氨酸激酶的单克隆抗体。在对小鼠进行免疫后，进行融合以获得杂交瘤，所述杂交瘤被选择，因为它们产生对用于免疫的蛋白质呈阳性反应的免疫球蛋白。我们使用一系列选择性测定分离出一种特定的单克隆，我们将其命名为35 C1。筛选单克隆抗体的第一个标准是在96孔板中针对纯化的重组组氨酸标记的aurora-A进行的ELISA（酶联免疫吸附测定）测定。第二个是针对相同重组蛋白的阳性Western印迹。第三个标准是针对HeLa细胞提取物的阳性蛋白质印迹，所选单克隆抗体应仅检测到一种在SDS（十二烷基硫酸钠）-聚丙烯酰胺凝胶电泳上迁移至46 kDa（千道尔顿）的蛋白质。最后，通过间接免疫荧光，单克隆抗体必须与人MCF 7培养细胞中的重复中心体和纺锤体极结合。在此阶段，几个单克隆抗体仍呈阳性。然后，我们通过寻找能够与小鼠极光-A激酶交叉反应的抗体，通过蛋白质印迹和间接免疫荧光来增加选择性。我们选择并克隆了35 C1杂交瘤以产生抗体。35 C1抗体的进一步表征揭示，它能够免疫沉淀激酶，它不抑制极光-A激酶活性，因此可以用于测量免疫沉淀后体内极光-A激酶活性。
We have developed monoclonal antibodies against the human aurora-A serine/threonine kinase. After immunization of a mouse, a fusion was performed to obtain hybridomas that were selected because they produced immunoglobulin positively reacting against the protein used for immunization. We isolated one particular monoclonal that we named 35C1 using a series of selective assays. The first criteria of the screen for monoclonals was an ELISA ( Enzyme Linked Immunosorbant Assay) assay performed in 96-well plates against the purified recombinant histidine-tagged aurora-A. The second was a positive Western blot against the same recombinant protein. The third criteria was a positive western blot against an HeLa cell extract, the selected monoclonal should detect only one protein migrating at 46 kDa (kiloDalton) on SDS ( Sodium Dodecyl Sulfate)-polyacrylamide gel electrophoresis. Finally, the monoclonal had to bind to duplicated centrosomes and spindle poles in human MCF7 cultured cells by indirect immunofluorescence. At this stage several monoclonals were still positive. We then increased the selectivity by searching for antibodies that were able to cross-react with the mouse aurora-A kinase both by western blot and indirect immunofluorescence. We selected and cloned the 35C1 hybridoma to produce the antibody. Further characterization of the 35C1 antibody revealed that it was able to immunoprecipitate the kinase, that it did not inhibit the aurora-A kinase activity and consequently could be used to measure the aurora-A kinase activity in vivo after immunoprecipitation.