Immunodetection assay for
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.
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.
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.
3. The detection method according to claim 2 , wherein either of the first and second antibodies is immobilized to a carrier.
4. 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.
5. 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.
6. 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.
7. The detection method according to any one of claims 4 to 6 , wherein said biological sample is sputum.
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.
9. 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.
10. 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.
11. 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.
12. The immunochromatographic assay according to any one of claims 9 to 11 , wherein said biological sample is sputum.
13. The immunochromatographic assay according to claim 8 , wherein the second antibody is labeled with a metallic colloidal particle or a latex particle.
14. The immunochromatographic assay according to claim 13 , wherein the membrane carrier is a nitro cellulose membrane.
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.
16. The immunochromatographic test strip according to claim 15 , wherein the second antibody is labeled with a metallic colloidal particle or a latex particle.
17. The immunochromatographic test strip according to claim 16 , wherein the membrane carrier is a nitro cellulose membrane.
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.
19. A monoclonal antibody according to claim 18 , wherein said monoclonal antibody recognizes an epitope of MPB64 located in SEQ ID NO:2.
20. A monoclonal antibody according to claim 18 , wherein said monoclonal antibody recognizes an epitope of MPB64 located in SEQ ID NO:3.
21. A monoclonal antibody according to claim 18 , wherein said monoclonal antibody recognizes an epitope of MPB64 located in SEQ ID NO:4.
22. 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.
23. 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.
24. 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.
25. 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.
26. 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.
27. 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.
28. 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.
29. 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.
30. 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.
31. 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.
32. 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.
33. 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.
34. 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.
35. 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.
36. 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.
37. 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.