Production and Characterization of Mouse Monoclonal Antibodies to Wild-Type and Oncogenic FLI-1 Proteins

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Abstract
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 over-expressed 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.