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Hapten-mediated identification of cell membrane antigens using an anti-FITC monoclonal antibody
P Balogh1, G Szekeres, P Németh
1Immunological and Biotechnological Laboratory, University Medical School of Pécs, Hungary.
Insights
A new anti-fluorescein antibody (F4/1) detects fluorescein in various forms. This antibody enables detection of fluorescein-conjugated molecules, like carboxyfluorescein succinimidyl ester, on cell membranes and in immunoprecipitated proteins.
Area of Science:
- Immunology
- Biochemistry
- Cell Biology
Background:
- Fluorescein isothiocyanate (FITC) is a widely used fluorescent dye for labeling biomolecules.
- Accurate detection of FITC-conjugated molecules, especially on cell surfaces or within protein complexes, is crucial for various biological assays.
- Characterizing novel labeling reagents and their specificities is essential for advancing molecular detection techniques.
Purpose of the Study:
- To produce and characterize a monoclonal antibody specific for fluorescein and its derivatives.
- To evaluate the utility of this antibody in detecting cell-bound fluorescein labeling.
- To assess the antibody's performance in identifying protein-conjugated fluorescein in immunoprecipitated samples.
Main Methods:
- Production and characterization of a monoclonal anti-FITC antibody (F4/1).
- Immunohistochemical staining of mouse thymocytes labeled with 5(6)-carboxyfluorescein succinimidyl ester (CFl-NSE).
- Immunoblotting of detergent-solubilized cell extracts.
- Immunoprecipitation of Thy-1.2 antigen from labeled thymocyte lysates followed by antibody detection.
Main Results:
- The F4/1 antibody demonstrated specificity for free and protein-conjugated fluorescein, including carboxyfluorescein.
- CFl-NSE labeling of mouse thymocytes resulted in predominantly membrane-bound fluorescein incorporation.
- Immunoblotting revealed a random distribution pattern of CFl-NSE coupling to cells.
- The anti-FITC antibody successfully detected Thy-1.2 antigen immunoprecipitated from labeled thymocytes.
Conclusions:
- The developed monoclonal anti-FITC antibody (F4/1) is a versatile tool for detecting fluorescein conjugates in various biological contexts.
- This antibody facilitates the study of cell surface labeling and protein interactions involving fluorescein-tagged molecules.
- The antibody aids in molecular weight estimation of FITC-labeled immunoprecipitated proteins.
Abstract:
A monoclonal anti-FITC antibody (F4/1) was produced and demonstrated to be specific for both the free and protein-conjugated (either soluble or cell-bound) form of fluorescein, or carboxyfluorescein. When mouse thymocytes were labelled with a novel fluorescein derivative 5(6)-carboxyfluorescein succinimidyl ester (CFl-NSE), the incorporation of fluorescein was predominantly membrane-bound as demonstrated immunohistochemically. The coupling of CFl-NSE to cells displays a random distribution pattern as shown by immunoblotting of cell extracts prepared by detergent solubilization of CFl-NSE-labelled thymocytes. In addition, the Thy-1.2 antigen immunoprecipitated from a CFl-NSE-labelled thymocyte lysate with a rat monoclonal antibody (Mab) could be detected using the anti-FITC Mab. The molecular weight of the immunoprecipitated material could be estimated immediately by reference to the FITC-labelled molecular weight markers electrophoresed simultaneously.
