(19) & (11) EP 2 084 519 B1 (12) EUROPEAN PATENT SPECIFICATION (45) Date of publication and mention (51) Int Cl.: of the grant of the patent: G01N 23/223 (2006.01) G01T 1/36 (2006.01) 01.08.2012 Bulletin 2012/31 C12Q 1/00 (2006.01) (21) Application number: 07874491.9 (86) International application number: PCT/US2007/021888 (22) Date of filing: 10.10.2007 (87) International publication number: WO 2008/127291 (23.10.2008 Gazette 2008/43) (54) X-RAY FLUORESCENCE ANALYSIS METHOD RÖNTGENFLUORESZENZ-ANALYSEVERFAHREN PROCÉDÉ D’ANALYSE PAR RAYONS X FLUORESCENTS (84) Designated Contracting States: • BURRELL, Anthony, K. AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Los Alamos, NM 87544 (US) HU IE IS IT LI LT LU LV MC MT NL PL PT RO SE SI SK TR (74) Representative: Albrecht, Thomas Kraus & Weisert (30) Priority: 10.10.2006 US 850594 P Patent- und Rechtsanwälte Thomas-Wimmer-Ring 15 (43) Date of publication of application: 80539 München (DE) 05.08.2009 Bulletin 2009/32 (56) References cited: (60) Divisional application: JP-A- 2001 289 802 US-A1- 2003 027 129 12164870.3 US-A1- 2003 027 129 US-A1- 2004 004 183 US-A1- 2004 017 884 US-A1- 2004 017 884 (73) Proprietors: US-A1- 2004 093 526 US-A1- 2004 235 059 • Los Alamos National Security, LLC US-A1- 2004 235 059 US-A1- 2005 011 818 Los Alamos, NM 87545 (US) US-A1- 2005 011 818 US-B1- 6 329 209 • Caldera Pharmaceuticals, INC. US-B2- 6 719 147 Los Alamos, NM 87544 (US) • GOLDIN E M ET AL: "Quantitation of antibody (72) Inventors: binding to cell surface antigens by X-ray • BIRNBAUM, Eva, R. fluorescence spectrometry" BIOCHIMICA ET Los Alamos, NM 87544 (US) BIOPHYSICA ACTA. BIOMEMBRANES, • KOPPISCH, Andrew, T. AMSTERDAM, NL, vol. 552, no. 1, 23 March 1979 Los Alamos, NM 87544 (US) (1979-03-23), pages 120-128, XP023505472 ISSN: • BALDWIN, Sharon, M. 0005-2736 [retrieved on 1979-03-23] Santa Fe, NM 87507 (US) • POTTS PHILIP J ET AL: "Atomic spectrometry • WARNER, Benjamin, P. update - X-ray fluorescence spectrometry" Los Alamos, NM 87544 (US) JOURNAL OF ANALYTICAL ATOMIC • MCCLESKEY, Mark, T. SPECTROMETRY, vol. 21, no. 10, 11 September Los Alamos, NM 87544 (US) 2006 (2006-09-11), pages 1076-1107, • BERGER, Jennifer, A. XP002543563 ISSN: 0267-9477 Los Alamos, NM 87544 (US) • STEWART, Jeffrey, J. Los Alamos, NM 87544 (US) • HARRIS, Michael, N. Los Alamos, NM 87544 (US) Note: Within nine months of the publication of the mention of the grant of the European patent in the European Patent Bulletin, any person may give notice to the European Patent Office of opposition to that patent, in accordance with the Implementing Regulations. Notice of opposition shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention). EP 2 084 519 B1 Printed by Jouve, 75001 PARIS (FR) (Cont. next page) EP 2 084 519 B1 • MERTENS ET AL.: ’Evaluation of the protein • NAGATA ET AL.: ’Multivariate calibrations for the concentration in enzymes via determination of SR-TXRF determination of trace concentrations sulfur by total reflection X-ray fluorescence of lead and arsenic in the presence of bromine.’ spectrometry-? limitations of the method.’ X-RAYSPECTROMETER vol. 35, no. 1, 24 October SPECTROCHIMICA ACTA PART B: ATOMIC 2005, pages 79 - 84, XP008112758 SPECTROSCOPY vol. 56, no. 11, 30 November 2001, pages 2157 - 2164, XP008112772 Remarks: • RICCI E. ET AL.: ’A versatile, compact system for Thefile contains technical information submitted after X-ray fluorescence analysis.’ X-RAY the application was filed and not included in this SPECTROMETRY vol. 10, no. 1, 1981, pages 12 - specification 16, XP008112759 • RINDERS ET AL.: ’The use of a scanning X-ray microprobefor simultaneous XRF/XRD studies of fly-ashparticles.’ JSYNCHROTRON RADIAT. vol. 4, no. 4, 01 July 1997, pages 228 - 235, XP008112757 2 EP 2 084 519 B1 Description FIELD OF THE INVENTION 5 [0001] The present invention relates generally to detecting binding events and more particularly to estimating binding selectivities for chemicals, analogs, and drugs being tested with receptors. BACKGROUND OF THE INVENTION 10 [0002] The desire to hasten the identification of potentially important drugs, catalysts, chemical and biological sensors, medical diagnostics, and other materials is a constant challenge that has prompted the use of combinatorial synthetic and screening strategies for synthesizing these materials and screening them for desirable properties. Combinatorial synthesis involves assembling a "library", i.e. a very large number of chemically related compounds and mixtures, usually in the form of an array on a substrate surface. High throughput screening of an array involves identifying which members 15 of the array, if any, have the desirable property or properties. The array form facilitates the identification of a particular material on the substrate. Combinatorial arrays and high-throughput screening techniques have been used to solve a variety of problems related to the development of biological materials such as proteins and DNA because the screening techniques can be used to rapidly assay many biological materials. [0003] The binding properties of a protein largely depend on the exposed surface amino acid residues of its polypeptide 20 chain (see, for example, Bruce Alberts et al., "Molecular Biology of the Cell", 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., "Molecular Cell Biology", 4th edition, W. H. Freeman and Company, 2000). These amino acid residues can form weak noncovalent bonds with ions and molecules. Effective binding generally requires the formation of many weak bonds at a "binding site," which is usually a cavity in the protein formed by a specific arrangement of amino acids. There should be a precise fit with the binding site for effective binding to occur. 25 [0004] The chemical properties and in particular, the binding properties of a protein depend almost entirely on the exposed surface amino acid residues of the polypeptide chain. These residues can form weak noncovalent bonds with other molecules. An effective binding between the protein, one example of a group of materials herein referred to as "receptors", and the material that binds to the receptor, referred to herein as "chemical", generally requires that many weak bonds form between the protein receptor and the chemical. Chemicals include organic molecules, inorganic mol- 30 ecules, salts, metal ions, and the like. The bonds between the protein and the chemical form at the "binding site" of the protein. The binding site is usually a cavity in the protein that is formed by a specific arrangement of amino acids that often belong to widely separated regions of the polypeptide chain and represent only a minor fraction of the total number of amino acids present in the chain. Chemicals should fit precisely into the binding site for effective binding to occur. The shape of these binding sites can differ greatly among different proteins, and even among different conformations 35 of the same protein. Even slightly different conformations of the same protein may differ greatly in their binding abilities. For further discussion of the structure and function of proteins, see: Bruce Alberts et al., "Molecular Biology of the Cell", 2nd edition, Garland Publishing, Inc., New York, 1989; and H. Lodish et al., "Molecular Cell Biology", 4th edition, W. H. Freeman and Company, 2000. [0005] After a receptor array is prepared, it is screened to determine which members have the desirable property or 40 properties. U. S. Patent 5,143,854 to M. C. Pirrung et al. entitled "Large Scale Photolithographic Solid Phase Synthesis of Polypeptides and Receptor Binding Screening Thereof", which issued September 1, 1992, hereby incorporated by reference, describes one such screening method. A polypeptide array is exposed to a ligand (an example of a chemical) to determine which members of the array bind to the ligand. The ligands described are radioactive, or are "tagged", i.e. attached via one or more chemical bonds to a chemical portion that fluoresces when exposed to non-ionizing, ultraviolet 45 radiation. Thus, the attached portion, i.e. the tag, makes the chemical visible by interrogation with ultraviolet radiation. Tagged molecules have also been used to aid in sequencing immobilized polypeptides as described, for example, in U. S. Patent 5,902,723 to W. J. Dower et al. entitled "Analysis of Surface Immobilized Polymers Utilizing Microfluorescence Detection," which issued May 11, 1999. Immobilized polypeptides are exposed to molecules labeled with fluorescent tags. The tagged molecules bind to the terminal monomer of a polypeptide, which is then cleaved and its identity 50 determined. The process is repeated to determine the complete sequence of the polypeptide. [0006] It is generally assumed that the attachment of a fluorescent tag to a chemical only serves to make visible the otherwise invisible chemical, and does not alter its binding properties. Since it is well known that even small changes to the structure of a molecule could affect its function, this assumption that a tagged chemical, i.e. a "surrogate", has the same binding affinity as the untagged chemical may not be a valid one. Small structural changes that accompany even 55 a conformational change of a receptor have been known to affect the binding affinity of the receptor. The tagged surrogates are structurally different from their untagged counterparts, and these structural differences could affect their binding affinities. Since binding affinities derived using tagged surrogates are suspect, the binding properties of receptors and chemicals should be evaluated using the untagged chemical or receptor and not with a tagged surrogate. 3 EP 2 084 519 B1 [0007] Pharmaceutical chemicals are the active ingredients in drugs, and it is believed that their therapeutic properties are linked to their ability to bind to one or more binding sites.