Monitoring Antibody Binding Events in Homogeneous Solution by Single-Molecule Fluorescence Spectroscopy
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Silvia Scheffler
Abstract
We demonstrate the potential of modern confocal fluorescence microscopy in combination with quenched peptide-based fluorescence probes for sensitive detection of p53 antibodies directly in homogeneous solution. Single tryptophan (Trp) residues in the sequences of short, synthetic peptide epitopes of the human p53 protein efficiently quench the fluorescence of an oxazine fluorophore attached to the amino terminal ends of the peptides. The fluorescence quenching mechanism is thought to be a photoinduced electron transfer reaction from Trp to the dye enabled by the formation of intramolecular complexes between dye and Trp. Specific recognition of the epitope by the antibody confines the conformational flexibility of the peptide. Consequently, complex formation between dye and Trp is abolished and fluorescence is recovered. Using fluorescence correlation spectroscopy (FCS), antibody binding can be monitored observing simultaneously two parameters: the diffusional mobility of the peptide as well as the quenching amplitude induced by the conformational flexibility of the peptide change significantly upon antibody binding. Furthermore, we demonstrate that the strong fluorescence increase upon binding can also be used to directly detect p53 autoantibodies from human blood serum samples in fluorescence intensity time traces. Our data demonstrate that new refined single-molecule fluorescence techniques in combination with quenched peptide epitopes open new possibilities for the reliable detection of antibody binding events in homogeneous solution.
© by Oldenbourg Wissenschaftsverlag, München
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Artikel in diesem Heft
- Preface
- Advanced Laser Imaging Diagnostics in Combustion
- Carbon Dioxide Thermal Decomposition: Observation of Incubation
- Action Spectroscopy and Predissociation of Vibrationally Excited C2HD
- Combination of Laser- and Mass-Spectroscopic Techniques for the Investigation of Fuel-Rich Flames
- Rovibrational Energy Transfer in the 4νCH Manifold of Acetylene, Viewed by IR-UV Double Resonance Spectroscopy. 3. State-to-State J-Resolved Kinetics
- Conditional Velocity Measurements by Simultaneously Applied Laser Doppler Velocimetry and Planar Laser-Induced Fluorescence in a Swirling Natural Gas/Air Flame
- High-Growth-Rate Chemical Vapor Deposition of Silicon: an Experimental and Modeling Approach
- Monitoring Antibody Binding Events in Homogeneous Solution by Single-Molecule Fluorescence Spectroscopy
- NO Reburn and Formation Chemistry in Methane Diffusion Flames
- SACM/CT Study of Product Energy Distributions in the Dissociation of n-Propylbenzene Cations
- Classical Trajectory and Statistical Adiabatic Channel Study of the Dynamics of Capture and Unimolecular Bond Fission. VII. Thermal Capture and Specific Rate Constants k(E,J) for the Dissociation of Molecular Ions
