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EP-3845903-B1 - IMMUNOCHROMATOGRAPHY KIT, AND METHOD FOR DETECTING MYCOBACTERIUM TUBERCULOSIS

EP3845903B1EP 3845903 B1EP3845903 B1EP 3845903B1EP-3845903-B1

Inventors

  • OHARA TOMOYA
  • OYABU NATSUKI
  • WADA ATSUHIKO
  • KATADA JUNICHI
  • UJIHARA DAI

Dates

Publication Date
20260506
Application Date
20190830

Claims (12)

  1. An immunochromatographic kit for detecting Mycobacterium tuberculosis, the kit comprising: a label substance modified with a first antibody against lipoarabinomannan; a porous carrier having a reaction site holding a second antibody against lipoarabinomannan; a compound containing silver; and a reducing agent reducing silver ions, wherein both of the first antibody and the second antibody are monoclonal antibodies, and the second antibody is a monoclonal antibody that recognizes a 5-deoxy-5-methylthio-xylofuranose structure of lipoarabinomannan; and wherein the second antibody comprises: (a) Heavy chain CDR1 consisting of an amino acid sequence set forth in SEQ ID NO: 11; (b) Heavy chain CDR2 consisting of an amino acid sequence set forth in SEQ ID NO: 12; (c) Heavy chain CDR3 consisting of an amino acid sequence set forth in SEQ ID NO: 13; (d) Light chain CDR1 consisting of an amino acid sequence set forth in SEQ ID NO: 14; (e) Light chain CDR2 consisting of an amino acid sequence set forth in SEQ ID NO: 15; and (f) Light chain CDR3 consisting of an amino acid sequence set forth in SEQ ID NO: 16; wherein the kit detects one or more of M. tuberculosis, M. bovis, M. africanum, BCG Pasteur Strain, BCG Connaught Strain, BCG Tokyo Strain and M. microti and does not detect M. abscessus subsp. Abscessus, M. abscessus subsp. Bolletii, M. abscessus subsp. Massiliense, M. chelonae and M. fortuitum.
  2. The immunochromatographic kit according to claim 1, wherein the label substance is a metal particle.
  3. The immunochromatographic kit according to any one of claims 1 to 2, wherein the label substance is gold, silver, platinum, or a compound thereof.
  4. The immunochromatographic kit according to any one of claims 1 to 3, wherein an average particle size of the label substance 1 nm to 500 nm.
  5. The immunochromatographic kit according to any one of claims 1 to 4, wherein the porous carrier is a nitrocellulose carrier.
  6. The immunochromatographic kit according to any one of claims 1 to 5, further comprising a coloring reagent for detecting the reducing agent reducing silver ions.
  7. The immunochromatographic kit according to claim 6, wherein the coloring reagent is a compound that reacts with ions and develops color.
  8. The immunochromatographic kit according to claim 6, wherein the coloring reagent is carried by the porous carrier.
  9. The immunochromatographic kit according to any one of claims 1 to 8, further comprising a housing case including the porous carrier having the reaction site, the compound containing silver, and the reducing agent reducing silver ions.
  10. The immunochromatographic kit according to any one of claims 1 to 9, further comprising: pots each including a tearable member, wherein the compound containing silver and the reducing agent reducing silver ions are respectively sealed in the pots.
  11. A method for detecting Mycobacterium tuberculosis, the method comprising: a step of spreading a complex body of lipoarabinomannan in a specimen and a label substance modified with a first antibody against lipoarabinomannan on a porous carrier having a reaction site holding a second antibody against lipoarabinomannan; a step of trapping the complex body at the reaction site; and a step of amplifying the label substance of the complex body trapped at the reaction site using a compound containing silver and a reducing agent reducing silver ions, wherein both of the first antibody and the second antibody are monoclonal antibodies, and the second antibody is a monoclonal antibody that recognizes a 5-deoxy-5-methylthio-xylofuranose structure of lipoarabinomannan; and wherein the second antibody comprises: (a) Heavy chain CDR1 consisting of an amino acid sequence set forth in SEQ ID NO: 11; (b) Heavy chain CDR2 consisting of an amino acid sequence set forth in SEQ ID NO: 12; (c) Heavy chain CDR3 consisting of an amino acid sequence set forth in SEQ ID NO: 13; (d) Light chain CDR1 consisting of an amino acid sequence set forth in SEQ ID NO: 14; (e) Light chain CDR2 consisting of an amino acid sequence set forth in SEQ ID NO: 15; and (f) Light chain CDR3 consisting of an amino acid sequence set forth in SEQ ID NO: 16; wherein the method detects one or more of M. tuberculosis, M. bovis, M. africanum, BCG Pasteur Strain, BCG Connaught Strain, BCG Tokyo Strain and M. microti and does not detect M. abscessus subsp. Abscessus, M. abscessus subsp. Bolletii, M. abscessus subsp. Massiliense, M. chelonae and M. fortuitum.
  12. The method for detecting Mycobacterium tuberculosis according to claim 11, the method further comprising detecting a label substance having an average particle size of equal to or more than 1 µm and equal to or less than 20 µm in a case of detection.

Description

Field of the Invention The present invention relates to an immunochromatographic kit for detecting Mycobacterium tuberculosis, the kit including a label substance, a porous carrier, a compound containing silver, a reducing agent capable of reducing silver ions and monoclonal antibodies against lipoarabinomannan. The present invention further relates to a method for detecting Mycobacterium tuberculosis using a label substance, a porous carrier, a compound containing silver, and a reducing agent capable of reducing silver ions ions and monoclonal antibodies against lipoarabinomannan. Description of the Related Art Among immunoassay methods, an immunochromatographic method is generally utilized, because operation is easy and measurement can be performed within a short period of time. Competitive responses or sandwich-based responses are widely used as an immune response used in the immunochromatographic method. Among them, sandwich-based responses are the mainstream in the immunochromatographic method, and in a typical example thereof, the following operation is performed in order to detect a test substance composed of antigens in a specimen. First, fine particles sensitized with an antibody against an antigen which is a test substance are immobilized as solid-phase fine particles on a chromatographic carrier, or an antibody itself is directly immobilized on a chromatographic carrier, and thereby a chromatographic carrier having a reaction site is prepared. Meanwhile, labeled fine particles are sensitized with an antibody against a test substance to prepare sensitized labeled fine particles. The sensitized labeled fine particles are then chromatographically moved together with a specimen on a chromatographic carrier. By the above operation, the immobilized antibody serves as an immobilization reagent at a reaction site formed on the chromatographic carrier, and the sensitized labeled fine particles specifically bind to this immobilized antibody via an antigen which is a test substance. As a result, by visually determining the presence or absence or a degree of signals generated by the sensitized labeled fine particles trapped at the reaction site, it is possible to measure the presence or absence or an amount of a test substance in a specimen. In the immunochromatographic method, detection signals may be amplified in some cases in order to avoid a problem of false negatives indicating that an antigen is not detected due to a low sensitivity. As a method of signal amplification, there are known method such as a method in which an enzyme such as alkaline phosphatase or peroxidase is used as a label, and a method of amplification (silver amplification) in which a compound containing silver and a reducing agent capable of reducing silver ions are used for a label selected from the group consisting of a metal colloid label and a metal sulfide label. Major tuberculosis antigens are glycolipids, which are major constituents of cell membranes and cell walls. Among glycolipids, it is known that lipoarabinomannan (LAM) is detected for diagnosis of tuberculosis, which is an infectious disease. For example, Patent Document 1 discloses an immunoassay for mycobacterial diseases by using a monoclonal antibody that specifically binds to lipoarabinomannan, and the above antibody. Patent Document 2 discloses an immunoassay using a monoclonal antibody that specifically binds to lipoarabinomannan. Patent Document 3 discloses a method and a kit which are for detecting Mycobacterium tuberculosis by using a polyclonal antibody that specifically binds to lipoarabinomannan. Patent Document 4 discloses a method and a kit which are for detecting Mycobacterium tuberculosis by using a monoclonal antibody that specifically binds to lipoarabinomannan. Non-Patent Document 1 discloses detection of lipoarabinomannan by using a monoclonal antibody that recognizes an epitope having a 5-deoxy-5-methylthio-xylofuranose structure (also referred to as an MTX structure) of lipoarabinomannan. Non-Patent Document 2 discloses a method and a kit which are for detecting Mycobacterium tuberculosis by using a polyclonal antibody that specifically binds to lipoarabinomannan. Prior Art Documents Patent Documents Patent Document 1: WO2013/129634APatent Document 2: WO2017/139153APatent Document 3: JP2008-507544APatent Document 4: WO2012/102679A Non-Patent Documents Non-Patent Document 1: BMC Infectious Diseases 2012, 12: 103Non-Patent Document 2: J Immunol. 2018 May 1; 200(9): 3053-3066 SUMMARY OF THE INVENTION Lack of diagnostic sensitivity, lack of specificity for Mycobacterium tuberculosis caused by cross-reaction with a nontuberculous mycobacterial group due to use of polyclonal antibodies, and the like have been pointed out in existing kits and methods for detecting lipoarabinomannan (LAM) and diagnosing tuberculosis, which is an infectious disease. Because lack of diagnostic sensitivity leads to delayed treatment of tuberculosis, and lack of specificity l