# US 8,785,201 B2 · Assay buffer, compositions containing the same, and methods of using the same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,785,201 B2 - Document ID: US8785201B2 - Status: Granted - Kind: B2 - Application number: 12/368,742 - Filed: 2009-02-10 - Published: 2009-07-02 - Granted: 2014-07-22 - Earliest claimed: 2001-09-10 - Assignees: Meso Scale Technologies, LLC. - Applicants: Michael Tsionsky; Eli N. Glezer; Selen Altunata; George Sigal; Jonathan K. Leland; Mark A. Billadeau; Svetlana Leytner; Mark Martin; Larry Helms - Inventors: Michael Tsionsky; Eli N. Glezer; Selen Altunata; George Sigal; Jonathan K. Leland; Mark A. Billadeau; Svetlana Leytner; Mark Martin; Larry Helms - Web page: https://patentgrep.com/patent/US8785201B2 ## CPC - G01N 33/573: for enzymes or isoenzymes - C12Q 1/485: {involving kinase} - G01N 21/76: Chemiluminescence; Bioluminescence - G01N 33/5306: {Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding} - G01N 33/58: involving labelled substances (G01N33/53 takes precedence) - G01N 33/582: {with fluorescent label} - G01N 2333/91205: Phosphotransferases in general - G01N 2458/30: Electrochemically active labels - G01N 2458/40: Rare earth chelates - Y10S 435/967: Standards, controls, materials, e.g. validation studies, buffer systems - Y10S 435/968: High energy substrates, e.g. fluorescent, chemiluminescent, radioactive - Y10T 436/10: Composition for standardization, calibration, simulation, stabilization, preparation or preservation; processes of use in preparation for chemical testing - Y10T 436/108331: Preservative, buffer, anticoagulant or diluent - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - Y10S: TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS - Y10T: TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION ## Abstract The invention relates to improved electrochemiluminescence assay methods for phosphorylated peptides or proteins employing phospho-specific antibodies and buffer compositions that are substantially free of inorganic phosphate. ## Claims ### Claim 1 A method of conducting an electrochemiluminescence (ECL) assay for detecting a phosphorylated peptide or protein or phosphorylation of a peptide or protein in a sample, said method comprising: (a) contacting an assay electrode with said sample and a phospho-specific antibody comprising an ECL label, said assay electrode including a binding domain comprising a binding reagent immobilized thereto, wherein said binding reagent is specific for said phosphorylated peptide or protein, said contacting step thereby forming a complex immobilized to said electrode, said complex comprising said binding reagent, said phosphorylated peptide or protein, and said phospho-specific antibody; (b) contacting said electrode with an ECL assay buffer, wherein said ECL assay buffer has a concentration of less than 5 mM inorganic phosphate and comprises an ECL coreactant, glycylglycine and an organic co-solvent; (c) applying electrochemical energy to said electrode under conditions suitable to induce ECL labels on said antibody to emit electrochemiluminescence; and (d) measuring emitted electrochemiluminescence; wherein a phosphorylated peptide or protein, or phosphorylation of a peptide or protein, is detected by an increase in electrochemiluminescence. ### Claim 2 (depends on 1) The method of claim 1, wherein said ECL assay buffer further comprises an additional component selected from the group consisting of a detergent, preservative, anti-foaming agent, salt, a chelator, a base, and combinations thereof. ### Claim 3 (depends on 1) The method of claim 1, wherein said ECL assay buffer comprises at least one additional assay component selected from the group consisting of a non-immobilized binding reagent, a cofactor, a kinase, a phosphorylase, a phosphatase or an inhibitor or substrate thereof, and combinations thereof. ### Claim 4 (depends on 1) The method of claim 1, wherein said ECL assay buffer is substantially aqueous. ### Claim 5 (depends on 1) The method of claim 1, wherein said organic co-solvent is selected from the group consisting of DMSO, DMF, methanol, ethanol, and combinations thereof. ### Claim 6 (depends on 1) The method of claim 1, wherein said ECL assay buffer is selected from the group consisting of glycylglycine, tris[hydroxymethyl]aminomethane and combinations thereof. ### Claim 7 (depends on 2) The method of claim 2, wherein said base is potassium hydroxide. ### Claim 8 (depends on 1) The method of claim 1, wherein said ECL assay buffer has a concentration of said ECL coreactant ranging from 50 to 1000 mM. ### Claim 9 (depends on 2) The method of claim 2 wherein said chelator is ethylenediaminetetraacetic acid (EDTA). ### Claim 10 (depends on 1) The method of claim 1 wherein the concentration of inorganic phosphate in said ECL assay buffer is less than 1 mM. ### Claim 11 (depends on 1) The method of claim 1 wherein said ECL assay buffer is free of inorganic phosphate. ### Claim 12 A method of measuring a binding interaction between a phospho-peptide or protein and a phospho-specific antibody, said method comprising: (a) combining said phospho-peptide or protein and said phospho-specific antibody in a pH buffer, wherein said pH buffer has a concentration of less than 5 mM inorganic phosphate and comprises glycylglycine, wherein said combining step is conducted under conditions suitable to form said binding interaction; and (b) detecting said binding interaction. ### Claim 13 (depends on 12) The method of claim 12 wherein the concentration of inorganic phosphate in said buffer is less than 1 mM. ### Claim 14 (depends on 12) The method of claim 12 wherein said buffer is free of inorganic phosphate. ### Claim 15 (depends on 12) The method of claim 12 wherein said pH buffer comprises an organic co-solvent. ### Claim 16 (depends on 15) The method of claim 15, wherein said organic co-solvent is selected from the group consisting of DMSO, DMF, methanol, ethanol, and combinations thereof. ### Claim 17 (depends on 3) The method of claim 3, wherein one of said at least one additional assay components comprises an ECL label.