Key G, Becker M H, Baron B, Duchrow M, Schlüter C, Flad H D, Gerdes J
Forschungsinstitut Borstel, Div. Molecular Immunology, Federal Republic of Germany.
Lab Invest. 1993 Jun;68(6):629-36.
The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all cells that are not in G0. Recently, we could demonstrate that Ki-67 detects a double band in Western blots of proliferating cells with apparent molecular weights of 345 kilodaltons and 395 kilodaltons, respectively. Furthermore, initial molecular biologic data favored the view that the epitope detected by Ki-67 might be encoded by a repetitive 66 bp element.
In order to verify this assumption, parts of the Ki-67 cDNA were bacterially expressed, and the fusion proteins obtained were used to elicit new monoclonal antibodies. The specificities of the new reagents were tested by immunohistochemistry, Western blot, and enzyme-linked immunosorbent assay techniques.
The somatic cell fusions revealed a number of antibodies with immunoreactivities comparable to Ki-67. Three antibodies, designated MIB 1-3, were further characterized. Besides the fact that their immunostaining reactivity is identical with that of Ki-67, all new antibodies react in Western blots with native Ki-67 antigen. Furthermore, Western blot and competitive binding assays by enzyme-linked immunosorbent assay clearly demonstrate that MIB 1 and MIB 3, like the original Ki-67 antibody, react with an epitope that is encoded by the 66 bp repetitive element mentioned above. MIB 2, however, reacts with an epitope distinct from this latter structure. In addition, after antigen unmasking by microwave treatment, MIB 1 and MIB 3 detect the Ki-67 antigen in paraffin sections.
Our results demonstrate that it is possible to use bacterially expressed parts of the Ki-67 antigen as immunogen to elicit antibodies that react with the native antigen. While MIB 1 and MIB 3 detect the same or a very similar epitope as the original antibody Ki-67, MIB 2 clearly differs in its fine specificity. Our results provide a circle of evidence that the cDNA sequence thus far determined encodes for the Ki-67 antigen. Furthermore, the new antibodies may become powerful tools for routine histopathology and for further functional characterization of the Ki-67 antigen.
单克隆抗体Ki-67与一种人类核细胞增殖相关抗原发生反应，该抗原在所有非G0期的细胞中表达。最近，我们能够证明，在增殖细胞的蛋白质免疫印迹中，Ki-67检测到两条明显分子量分别为345千道尔顿和395千道尔顿的条带。此外，初步的分子生物学数据支持这样一种观点，即Ki-67检测到的表位可能由一个66碱基对的重复元件编码。
为了验证这一假设，部分Ki-67 cDNA在细菌中表达，并将获得的融合蛋白用于制备新的单克隆抗体。通过免疫组织化学、蛋白质免疫印迹和酶联免疫吸附测定技术检测新试剂的特异性。
体细胞融合产生了许多免疫反应性与Ki-67相当的抗体。对三种命名为MIB 1-3的抗体进行了进一步的特性分析。除了它们的免疫染色反应性与Ki-67相同这一事实外，所有新抗体在蛋白质免疫印迹中均与天然Ki-67抗原发生反应。此外，蛋白质免疫印迹和酶联免疫吸附测定的竞争性结合试验清楚地表明，MIB 1和MIB 3与原始的Ki-67抗体一样，与上述66碱基对重复元件编码的表位发生反应。然而，MIB 2与后一种结构不同的表位发生反应。此外，经微波处理进行抗原修复后，MIB 1和MIB 3在石蜡切片中检测到Ki-67抗原。
我们的结果表明，有可能将细菌表达的Ki-67抗原部分用作免疫原，以制备与天然抗原发生反应的抗体。虽然MIB 1和MIB 3检测到的表位与原始抗体Ki-67相同或非常相似，但MIB 2在精细特异性方面明显不同。我们的结果提供了一系列证据，证明迄今确定的cDNA序列编码Ki-67抗原。此外，这些新抗体可能成为常规组织病理学以及Ki-67抗原进一步功能特性研究的有力工具。
