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Production of anti-fibroblast growth factor receptor monoclonal antibodies by in vitro immunization
S Venkateswaran1, V Blanckaert, M Schelling
1Department of Genetics and Cell Biology, Washington State University, Pullman 99164-4234.
Abstract:
Monoclonal antibodies against the high affinity tyrosine kinase Fibroblast Growth Factor (FGF) receptor may help define receptor epitopes involved in FGF binding and signal transduction which mediate coronary and tumor angiogenesis (the development of blood vessels). Monoclonal antibodies against the FGF receptor were made by in vitro immunization. Receptor was PAGE-purified and transblotted to nitrocellulose. The nitrocellulose was dissolved with acetonitrile, and the receptor used as antigen in an in vitro immunization system. Screening for FGF receptor positive clones was done using membrane preparations from Coronary Venular Endothelial Cells (CVEC) expressing the FGF receptor, both by Enzyme Linked Immunosorbent Assay (ELISA) and Western blot. Culture supernatants of several clones tested positive for IgM or IgG monoclonal antibodies against the FGF receptor. Antibodies were affinity purified. Ascites fluid was produced in Balb/c mice primed with Incomplete Freund's adjuvant (IFA). The monoclonal antibodies were also found to be suitable for receptor immunoprecipitation. Immunocytochemistry was done on sections from a variety of species. Further characterization of these antibodies, as well as the production of the remainder of a panel of anti-FGF receptor monoclonal antibodies, is underway.
Insights
Researchers developed novel monoclonal antibodies targeting the Fibroblast Growth Factor (FGF) receptor to study its role in blood vessel development. These antibodies are crucial tools for understanding angiogenesis in both cardiac and tumor tissues.
Area of Science:
- Molecular Biology
- Immunology
- Cell Biology
Background:
- The Fibroblast Growth Factor (FGF) receptor is a high-affinity tyrosine kinase.
- FGF receptor signaling is critical for both coronary and tumor angiogenesis.
- Defining receptor epitopes is key to understanding FGF binding and signal transduction.
Purpose of the Study:
- To generate monoclonal antibodies against the FGF receptor.
- To characterize these antibodies for their utility in studying FGF receptor function.
- To facilitate research into angiogenesis mediated by FGF signaling.
Main Methods:
- In vitro immunization using PAGE-purified and transblotted FGF receptor.
- Screening of antibody-producing clones via ELISA and Western blot using Coronary Venular Endothelial Cells (CVEC).
- Affinity purification of antibodies, ascites production, and testing for immunoprecipitation and immunocytochemistry.
Main Results:
- Successfully produced and identified IgM and IgG monoclonal antibodies against the FGF receptor.
- Demonstrated the antibodies' suitability for immunoprecipitation and immunocytochemistry across species.
- Established a panel of anti-FGF receptor monoclonal antibodies for further characterization.
Conclusions:
- Generated a valuable panel of monoclonal antibodies targeting the FGF receptor.
- These antibodies serve as essential tools for investigating FGF receptor-mediated angiogenesis.
- Further characterization will enhance their application in biomedical research.