# US 8,541,179 B2 · Immunodetection assay for Mycobacterium tuberculosis complex > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 8,541,179 B2 - Document ID: US8541179B2 - Status: Granted - Kind: B2 - Application number: 12/810,217 - Filed: 2008-12-19 - Published: 2010-11-11 - Granted: 2013-09-24 - Earliest claimed: 2007-12-28 - Assignees: Bl Incorporated - Applicants: Yasuharu Namba - Inventors: Yasuharu Namba - Web page: https://patentgrep.com/patent/US8541179B2 ## CPC - C07K 16/1289: Mycobacteriaceae (F) - G01N 33/5695: {Mycobacteria} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - 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) ## Abstract The present invention provides a method for specifically detecting a Mycobacterium tuberculosis complex-specific secretory protein MPT64 antigen in a biological sample, whereby diagnosis of infection with Mycobacterium tuberculosis is carried out rapidly and safely with higher accuracy than before. An antibody that recognizes an epitope for MPB64 located in any one of amino acid sequences of SEQ ID NOS: 2 to 4, particularly a monoclonal antibody was obtained. Thus, an immunoassay using the antibody, particularly a sandwich immunoassay using first and second antibodies to MPB64, particularly an immunochromatographic assay and an immunochromatographic test strip are provided. A biological sample can be rapidly subjected to the immunoassay without culturing or after culturing for a time before Mycobacterium tuberculosis complex bacteria in the sample substantially start to grow. The biological sample may be pretreated by treatment for inactivation of Mycobacterium tuberculosis, or treatment by dispersion or solubilization. ## Claims ### Claim 1 A method for detecting Mycobacterium tuberculosis complex, which comprises an immunoassay using an antibody to a Mycobacterium tuberculosis complex-specific secretory protein MPB64, in which said antibody comprises a monoclonal antibody to an epitope of MPB64 located in any one of amino acid sequences of SEQ ID NOS: 2, 3 or 4. ### Claim 2 A method for detecting Mycobacterium tuberculosis complex, which comprises a sandwich immunoassay using first and second antibodies to a Mycobacterium tuberculosis complex-specific secretory protein MPB64, in which at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in any one of amino acid sequences of SEQ ID NOS: 2, 3 or 4. ### Claim 3 (depends on 2) The detection method according to claim 2, wherein either of the first and second antibodies is immobilized to a carrier. ### Claim 4 (depends on 2) The detection method according to claim 2, wherein a biological sample is subjected to said immunoassay without culturing or after culturing for a time sufficient for secreting a detectable amount of MPB64 before the Mycobacterium tuberculosis complex bacteria in the sample start to grow. ### Claim 5 (depends on 4) The detection method according to claim 4, wherein said biological sample is subjected to a treatment for inactivation of Mycobacterium tuberculosis or a pretreatment by dispersion or solubilization before subjected to the immunoassay. ### Claim 6 (depends on 5) The detection method according to claim 5, wherein said treatment by dispersion or solubilization is carried out by stirring operation, or by addition to the biological sample of at least one member selected from the group consisting of an alkali substance, a reducing substance, a protease and a surfactant. ### Claim 7 (depends on 4, 5, 6) The detection method according to any one of claims 4 to 6, wherein said biological sample is sputum. ### Claim 8 An immunochromatographic assay for detecting Mycobacterium tuberculosis complex, which comprises: providing a membrane carrier having a capturing zone which is formed in a predetermined position thereof by immobilizing a first antibody to a Mycobacterium tuberculosis complex-specific secretory protein MPB64, chromatographically developing a mixed solution in the membrane carrier toward the capturing zone, the mixed solution containing a second antibody to the MPB64 and a predetermined amount of a test sample, whereby a complex of an antigen contained in the test sample and the second antibody is captured by the capturing zone, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in any one of amino acid sequences of SEQ ID NOS: 2, 3 or 4. ### Claim 9 (depends on 8) The immunochromatographic assay according to claim 8, wherein said test sample contains a biological sample which has not been cultured or has been cultured for a time sufficient for secreting a detectable amount of MPB64 before the Mycobacterium tuberculosis complex bacteria in the sample start to grow. ### Claim 10 (depends on 9) The immunochromatographic assay according to claim 9, wherein said biological sample is pretreated by treatment for inactivation of Mycobacterium tuberculosis, or treatment by dispersion or solubilization. ### Claim 11 (depends on 10) The immunochromatographic assay according to claim 10, wherein said treatment by dispersion or solubilization of the biological sample is carried out by stirring operation, or by addition to the biological sample of at least one member selected from the group consisting of an alkali substance, a reducing substance, a protease and a surfactant. ### Claim 12 (depends on 9, 10, 11) The immunochromatographic assay according to any one of claims 9 to 11, wherein said biological sample is sputum. ### Claim 13 (depends on 8) The immunochromatographic assay according to claim 8, wherein the second antibody is labeled with a metallic colloidal particle or a latex particle. ### Claim 14 (depends on 13) The immunochromatographic assay according to claim 13, wherein the membrane carrier is a nitro cellulose membrane. ### Claim 15 An immunochromatographic test strip for detecting a Mycobacterium tuberculosis complex, which at least comprises first and second antibodies to a Mycobacterium tuberculosis complex-specific secretory protein MPB64, and a membrane carrier, wherein the first antibody is previously immobilized in a predetermined position of the membrane carrier so as to form a capturing zone, the second antibody is labeled with a suitable labeling agent and is prepared at a position remote from the capturing zone for being chromatographically developed in the membrane carrier, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in any one of amino acid sequences of SEQ ID NOS: 2, 3 or 4. ### Claim 16 (depends on 15) The immunochromatographic test strip according to claim 15, wherein the second antibody is labeled with a metallic colloidal particle or a latex particle. ### Claim 17 (depends on 16) The immunochromatographic test strip according to claim 16, wherein the membrane carrier is a nitro cellulose membrane. ### Claim 18 A monoclonal antibody selected from the group consisting of: (a) a monoclonal antibody which recognizes an epitope of MPB64 located in SEQ ID NO:2; (b) a monoclonal antibody which recognizes an epitope of MPB64 located in SEQ ID NO:3; and (c) a monoclonal antibody which recognizes an epitope of MPB64 located in SEQ ID NO:4. ### Claim 19 (depends on 18) A monoclonal antibody according to claim 18, wherein said monoclonal antibody recognizes an epitope of MPB64 located in SEQ ID NO:2. ### Claim 20 (depends on 18) A monoclonal antibody according to claim 18, wherein said monoclonal antibody recognizes an epitope of MPB64 located in SEQ ID NO:3. ### Claim 21 (depends on 18) A monoclonal antibody according to claim 18, wherein said monoclonal antibody recognizes an epitope of MPB64 located in SEQ ID NO:4. ### Claim 22 (depends on 1) The detection method according to claim 1, wherein said antibody comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:2. ### Claim 23 (depends on 1) The detection method according to claim 1, wherein said antibody comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:3. ### Claim 24 (depends on 1) The detection method according to claim 1, wherein said antibody comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:4. ### Claim 25 (depends on 2) The detection method according to claim 2, wherein said antibody comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:2. ### Claim 26 (depends on 2) The detection method according to claim 2, wherein said antibody comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:3. ### Claim 27 (depends on 2) The detection method according to claim 2, wherein said antibody comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:4. ### Claim 28 (depends on 8) The immunochromatographic assay according to claim 8, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in amino acid sequence of SEQ ID NO: 2. ### Claim 29 (depends on 8) The immunochromatographic assay according to claim 8, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:3. ### Claim 30 (depends on 8) The immunochromatographic assay according to claim 8, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in the amino acid sequence of SEQ ID NO:4. ### Claim 31 (depends on 15) The immunochromatographic test strip according to claim 15, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in amino acid sequence of SEQ ID NO: 2. ### Claim 32 (depends on 15) The immunochromatographic test strip according to claim 15, wherein at least one of the first and second antibodies comprises a monoclonal antibody to an epitope of MPB64 located in amino acid sequence of SEQ ID NO: 3. ### Claim 33 (depends on 15) The immunochromatographic test strip according to claim 15, wherein at least one of the first and second antibodies comprises to a monoclonal antibody epitope of MPB64 located in amino acid sequence of SEQ ID NO: 4. ### Claim 34 (depends on 6) The detection method according to claim 6, wherein said treatment by dispersion or solubilization of the biological sample is carried out by addition to the biological sample of at least one member selected from the group consisting of an alkali substance, a reducing substance and a protease. ### Claim 35 (depends on 11) The immunochromatographic assay according to claim 11, wherein said treatment by dispersion or solubilization of the biological sample is carried out by addition to the biological sample of at least one member selected from the group consisting of an alkali substance, a reducing substance and a protease. ### Claim 36 (depends on 6) The detection method according to claim 6, wherein said treatment by dispersion or solubilization of the biological sample is carried out by addition to the biological sample of an alkali substance and a reducing substance. ### Claim 37 (depends on 11) The detection method according to claim 11, wherein said treatment by dispersion or solubilization of the biological sample is carried out by addition to the biological sample of an alkali substance and a reducing substance.