PUB - Publikationen an der Universität Bielefeld

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.