NEW KI-67-EQUIVALENT MURINE MONOCLONAL-ANTIBODIES (MIB 1-3) GENERATEDAGAINST BACTERIALLY EXPRESSED PARTS OF THE KI-67 CDNA CONTAINING 3 62BASE-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 t hat are not in G0. Recently, we could demonstrate that Ki-67 detects a double band in Western blots of proliferating cells with apparent mol ecular weights of 345 kilodaltons and 395 kilodaltons, respectively. F urthermore, initial molecular biologic data favored the view that the epitope detected by Ki-67 might be encoded by a repetitive 66 bp eleme nt. EXPERIMENTAL DESIGN: In order to verify this assumption, parts of the Ki-67 cDNA were bacterially expressed, and the fusion proteins obt ained 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 somat ic cell fusions revealed a number of antibodies with immunoreactivitie s comparable to Ki-67. Three antibodies, designated MIB 1-3, were furt her characterized. Besides the fact that their immunostaining reactivi ty is identical with that of Ki-67, all new antibodies react in Wester n blots with native Ki-67 antigen. Furthermore, Western blot and compe titive binding assays by enzyme-linked immunosorbent assay clearly dem onstrate 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 menti oned above. MIB 2, however, reacts with an epitope distinct from this latter structure. In addition, after antigen unmasking by microwave tr eatment, MIB 1 and MIB 3 detect the Ki-67 antigen in paraffin sections . CONCLUSIONS: Our results demonstrate that it is possible to use bact erially expressed parts of the Ki-67 antigen as immunogen to elicit an tibodies that react with the native antigen. While MIB 1 and MIB 3 det ect 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 powe rful tools for routine histopathology and for further functional chara cterization of the Ki-67 antigen.