%0 Journal Article %1 Scheffler2005 %A Scheffler, S %A Sauer, M %A Neuweiler, H %C LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY %D 2005 %I R OLDENBOURG VERLAG %J ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY & CHEMICAL PHYSICS %K hannes sauer %N 5 %P 665-678 %T Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy %U http://dx.doi.org/10.1524/zpch.219.5.665.64323 %V 219 %X 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.

@article{Scheffler2005, 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.}, added-at = {2011-03-01T10:57:13.000+0100}, address = {LEKTORAT MINT, POSTFACH 80 13 60, D-81613 MUNICH, GERMANY}, affiliation = {Sauer, M (Reprint Author), Univ Bielefeld, Fac Phys, Univ Str 25, D-33615 Bielefeld, Germany. Univ Bielefeld, Fac Phys, D-33615 Bielefeld, Germany.}, author = {Scheffler, S and Sauer, M and Neuweiler, H}, author-email = {sauer@physik.uni-bielefeld.de neuweiler@physik.uni-bielefeld.de}, biburl = {https://www.bibsonomy.org/bibtex/23d4cc64d1d4570d119c61397c14730df/reichert}, cited-references = {BANKS L, 1986, EUR J BIOCHEM, V159, P529. BARTEK J, 1993, J PATHOL, V169, P27. HUDGINS RR, 2002, J AM CHEM SOC, V124, P556. KRICHEVSKY O, 2002, REP PROG PHYS, V65, P251. KRIEGER F, 2003, J MOL BIOL, V332, P265, DOI 10.1016/S0022-2836(03)00892-1. KUBBUTAT MHG, 1997, NATURE, V387, P299. LANE DP, 1992, NATURE, V358, P15. LUBIN R, 1993, CANCER RES, V53, P5872. LUBIN R, 1995, NAT MED, V1, P701. MARME N, 2004, ANGEW CHEM INT EDIT, V43, P3798, DOI 10.1002/anie.200453835. MEDINA MA, 2002, BIOESSAYS, V24, P758. NEUWEILER H, 2002, ANGEW CHEM INT EDIT, V41, P4769. NEUWEILER H, 2003, J AM CHEM SOC, V125, P5324, DOI 10.1021/ja034040p. NIE SM, 1994, SCIENCE, V266, P1018. PORTEFAIX JM, 2002, J IMMUNOL METHODS, V259, P65. SAKURAI K, 2004, J AM CHEM SOC, V126, P16288, DOI 10.1021/ja044883w. SAUER M, 1997, APPL PHYS B-LASERS O, V65, P427. SAUER M, 1998, CHEM PHYS LETT, V284, P153. SOUSSI T, 1996, IMMUNOL TODAY, V17, P354. TYAGI S, 1996, NAT BIOTECHNOL, V14, P303. VAIANA AC, 2003, J AM CHEM SOC, V125, P14564, DOI 10.1021/ja036082j.}, doc-delivery-number = {925TY}, groups = {public}, interhash = {bca8f6bbd65ffc27a3d372dbb83457dc}, intrahash = {3d4cc64d1d4570d119c61397c14730df}, issn = {0942-9352}, journal = {ZEITSCHRIFT FUR PHYSIKALISCHE CHEMIE-INTERNATIONAL JOURNAL OF RESEARCH IN PHYSICAL CHEMISTRY \& CHEMICAL PHYSICS}, journal-iso = {Z. Phys. Chemie-Int. J. Res. Phys. Chem. Chem. Phys.}, keywords = {hannes sauer}, keywords-plus = {INTRAMOLECULAR CONTACT FORMATION; MONOCLONAL-ANTIBODIES; ELECTRON-TRANSFER; P53 ANTIBODIES; PEPTIDE; CANCER; PROBES; DYNAMICS; LEVEL; SERUM}, language = {English}, number = 5, number-of-cited-references = {21}, pages = {665-678}, publisher = {R OLDENBOURG VERLAG}, subject-category = {Chemistry, Physical}, times-cited = {6}, timestamp = {2011-03-10T12:36:33.000+0100}, title = {Monitoring antibody binding events in homogeneous solution by single-molecule fluorescence spectroscopy}, type = {Article}, unique-id = {ISI:000229077400009}, url = {http://dx.doi.org/10.1524/zpch.219.5.665.64323}, username = {reichert}, volume = 219, year = 2005 }