New Ki-67-equivalent murine monoclonal antibodies (MIB 1-3) generated against bacterially expressed parts of the Ki-67 cDNA containing three 62 base pair repetitive elements encoding for the Ki-67 epitope.

Background: 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.

Experimental Design: 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.

Results: 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.

Conclusions: 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.