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Monoclonal antibodies that recognize the native human thyrotropin receptor
A P Johnstone1, J C Cridland, C R DaCosta
1Department of Cellular and Molecular Sciences, St George's Hospital Medical School, London, UK.
Molecular and Cellular Endocrinology
|November 1, 1994
Summary
Researchers developed a new method to create monoclonal antibodies targeting the native human thyrotropin receptor. This technique successfully identified antibodies that recognize the functional receptor, aiding in understanding its structure-function relationship.
Area of Science:
- Immunology
- Endocrinology
- Molecular Biology
Background:
- The human thyrotropin receptor (TSHR) is crucial for thyroid hormone regulation.
- Developing antibodies that recognize the native, functional TSHR is essential for studying its structure-function relationship.
- Existing screening methods often favor antibodies that bind denatured receptors.
Purpose of the Study:
- To generate monoclonal antibodies that specifically recognize the native human thyrotropin receptor.
- To compare the efficacy of a novel screening protocol with standard methods.
- To characterize the binding properties and epitope specificities of the generated antibodies.
Main Methods:
- Production of recombinant full-length functional human TSHR in eukaryotic cells.
- Immunization of mice with TSHR produced in eukaryotic or prokaryotic systems.
- Screening of antibodies using flow cytofluorimetry and ELISA.
- Characterization of antibody binding to native and denatured receptor via flow cytofluorimetry, ELISA, immunoprecipitation, and immunoblotting.
- Epitope mapping using different recombinant TSHR constructs.
Main Results:
- A novel flow cytofluorimetry-based screening protocol successfully identified antibodies against the native TSHR.
- An IgM antibody (7B5) recognized only the native receptor, indicating a conformational epitope.
- Two IgG1 antibodies (2C11 and 3B12) recognized both native and denatured receptor, with epitopes mapped to region 125-369.
- One IgG1 antibody (2C11) inhibited thyrotropin binding to the receptor, but none stimulated cAMP production.
Conclusions:
- The developed screening protocol is effective for generating antibodies against native receptors.
- The characterized antibodies provide valuable tools for investigating TSHR structure and function.
- These antibodies can help elucidate the mechanisms of TSHR activation and regulation.