Production and characterization of mouse monoclonal antibodies to wild-type and oncogenic FLI-1 proteins.
Mouse monoclonal antibodies were raised against the C-terminal domain of human FLI-1, a member of the ETS family of transcription factors which is involved in various murine and human malignancies. This FLI-1 specific domain is included in the fusion product EWS-FLI-1, an oncogenic variant of FLI-1 expressed in Ewing tumors. Antibodies were screened first by enzyme-linked immunosorbent assay onto recombinant FLI-1-coated plates. Positive clones were then tested for their ability to immunoprecipitate over-expressed EWS-FLI-1 protein. Three monoclonal antibodies were selected and further characterized. One of them, termed 7.3 MoAb, was shown to react with FLI-1 and EWS-FLI-1 in immunoblotting, immunoprecipitation, and immunofluorescence experiments. With all three methods, this antibody not only enabled the detection of overexpressed proteins but also more interestingly, that of endogenously expressed proteins. Furthermore, the 7.3 MoAb can specifically inhibit the interaction of FLI-1 with its DNA-binding site as shown by electrophoretic mobility shift assay. The 7.3 MoAb appears to be specific for FLI-1 because it does not react with ERG, the ETS family member most closely related to FLI-1. This antibody should be a useful tool in the diagnostic evaluation of Ewing tumors and should permit biochemical analyses to study the function of the wild-type FLI-1 protein and of the EWS-FLI-1 fusion protein.
Melot
T
T
Laboratoire de Génétique des Tumeurs, INSERM U 434, Paris.
Gruel
N
N
Doubeikovski
A
A
Sevenet
N
N
Teillaud
J L
JL
Delattre
O
O
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D051379:Mice; D015514:Oncogene Proteins, Fusion / Q000276:immunology / Q000378:metabolism; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D034802:RNA-Binding Protein EWS; D015534:Trans-Activators / Q000276:immunology* / Q000378:metabolism; D014157:Transcription Factors / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C106031:EWS-FLI fusion protein; C497426:Fli1 protein, mouse; D015514:Oncogene Proteins, Fusion; D051789:Proto-Oncogene Protein c-fli-1; D011518:Proto-Oncogene Proteins; D034802:RNA-Binding Protein EWS; D015534:Trans-Activators; D014157:Transcription Factors; D004247:DNA