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Establishment of epitope-defined monoclonal antibodies with specificity for fibroblast growth factor receptor types 1
D Larocca1, A Witte, A M Gonzalez
1PRIZM Pharmaceuticals, San Diego, CA 92121, USA.
Abstract:
The development of specific antibody probes for characterizing the expression of the family of 4 fibroblast growth factor receptor (FGFR) types has been difficult because of their close homology to each other and high degree of evolutionary conservation. Of the existing anti-FGFR monoclonal antibodies (MAbs), there are few that are useful for staining paraffin-embedded tissues. We have raised MAbs against human FGFR1 and FGFR2 in both rats and mice using bacterial recombinant receptor fusion proteins as immunogens. We used peptide epitope mapping to characterize the immune sera and the selected MAbs. Immunized animals were selected that displayed the broadest reactivity against epitopes unique to the immunizing receptor type. We produced FGFR1 specific MAbs that bind epitopes in immunoglobulin domain I (Ig-I) and FGFR2 specific MAbs that bind epitopes in Ig-I, Ig-II, and the acid box. The specificity of the antibodies was demonstrated by ELISA and immunoblot analysis of purified recombinant FGFR1 and FGFR2 extracellular domains produced both in E. coli and in eucaryotic cells. Based on the lack of epitope homology, these MAbs would not be expected to cross-react with FGFR3 or FGFR4. We isolated MAbs that bound to paraffin embedded tissue and immunoblots of recombinant receptor. These epitope-defined MAbs can distinguish between members of the FGF receptor family and should be useful as tools for assessing FGF receptor expression in a variety of normal and diseased tissues.
Insights
Developing specific antibody probes for fibroblast growth factor receptors (FGFRs) is challenging. New monoclonal antibodies (MAbs) targeting FGFR1 and FGFR2 have been created, offering precise tools for assessing receptor expression in tissues.
Area of Science:
- Biotechnology
- Immunology
- Molecular Biology
Background:
- Characterizing fibroblast growth factor receptor (FGFR) expression is hindered by high homology among the four FGFR types.
- Existing anti-FGFR monoclonal antibodies (MAbs) often lack utility for paraffin-embedded tissue staining.
Purpose of the Study:
- To develop specific antibody probes for distinguishing between the four fibroblast growth factor receptor (FGFR) types.
- To generate monoclonal antibodies (MAbs) that are effective for staining paraffin-embedded tissues.
Main Methods:
- Raised MAbs against human FGFR1 and FGFR2 using bacterial recombinant receptor fusion proteins.
- Employed peptide epitope mapping to characterize immune sera and selected MAbs.
- Validated antibody specificity using ELISA and immunoblot analysis of purified recombinant FGFR extracellular domains.
Main Results:
- Generated FGFR1-specific MAbs binding to epitopes in immunoglobulin domain I (Ig-I).
- Produced FGFR2-specific MAbs binding to epitopes in Ig-I, Ig-II, and the acid box.
- Isolated MAbs demonstrated specificity for FGFR1 and FGFR2, with no expected cross-reactivity to FGFR3 or FGFR4, and were effective in paraffin-embedded tissue staining.
Conclusions:
- Developed epitope-defined MAbs capable of distinguishing between FGFR family members.
- These novel MAbs are valuable tools for assessing FGFR expression in various normal and diseased tissues.
- The antibodies overcome limitations of existing probes, enabling more accurate FGFR characterization.