Production and characterization of mouse monoclonal antibodies to wild-type and oncogenic FLI-1 proteins

Area of Science:

• Oncology
• Molecular Biology
• Immunology

Background:

• FLI-1 is an ETS family transcription factor implicated in various malignancies.
• The EWS-FLI-1 fusion protein, an oncogenic variant of FLI-1, is characteristic of Ewing tumors.
• Developing specific tools to detect and study FLI-1 and EWS-FLI-1 is crucial for cancer research and diagnostics.

Purpose of the Study:

• To generate and characterize monoclonal antibodies against the C-terminal domain of human FLI-1.
• To identify an antibody capable of detecting both wild-type FLI-1 and the EWS-FLI-1 fusion protein.
• To assess the utility of the antibody in diagnosing Ewing tumors and studying FLI-1 protein function.

Main Methods:

• Monoclonal antibodies were raised against the C-terminal domain of human FLI-1.
• Antibodies were screened using ELISA, immunoprecipitation, and immunoblotting.
• Functional activity was assessed via electrophoretic mobility shift assay (EMSA) to evaluate DNA-binding inhibition.
• Specificity was confirmed by testing against ERG, a related ETS family member.

Main Results:

• Three monoclonal antibodies were selected, with 7.3 MoAb showing significant reactivity.
• The 7.3 MoAb detected both overexpressed and endogenously expressed FLI-1 and EWS-FLI-1 proteins via multiple methods.
• Electrophoretic mobility shift assays demonstrated that 7.3 MoAb inhibits FLI-1 DNA-binding.
• The antibody exhibited specificity for FLI-1, with no cross-reactivity to ERG.

Conclusions:

• The 7.3 MoAb is a specific and effective tool for detecting FLI-1 and the oncogenic EWS-FLI-1 fusion protein.
• This antibody is valuable for the diagnostic evaluation of Ewing tumors.
• The 7.3 MoAb facilitates biochemical studies on the function of wild-type FLI-1 and the EWS-FLI-1 fusion protein.