Method and kit for immunological detection of
Definitive diagnosis and early start of treatment cannot be made for tuberculosis since conventional methods for detecting a Mycobacterium tuberculosis complex require a long time plus enormous labor and expense. Because detection is difficult to perform directly from a biological sample, if the biological sample contains no or a very small amount of MPT64, there is a risk infection with the Mycobacterium tuberculosis complex will be missed. The present method and a kit address these long-standing needs by more rapidly and conveniently detecting a Mycobacterium tuberculosis complex, without culturing a biological sample containing the Mycobacterium tuberculosis complex, in which a biological sample is subjected to a heat-treatment so as to extracellularly secrete a Mycobacterium tuberculosis complex-specific secretory protein, particularly, MPB64, and subjecting the resulting treated sample to an immunological measurement/assay.
1. A method for detecting the presence of a Mycobacterium tuberculosis complex-specific protein in a heat-treated biological sample, the method comprising
subjecting a heat-treated biological sample to an immunological assay for the presence of the Mycobacterium tuberculosis complex-specific protein, wherein the Mycobacterium tuberculosis complex-specific protein is MPB64/MPT64,
wherein the heat-treated biological sample to be immunologically assayed is obtained by subjecting a biological sample that may contain Mycobacterium tuberculosis complex to a heat-treatment carried out at 45° C. to 60° C. for a time sufficient to promote extracellular secretion of the Mycobacterium tuberculosis complex-specific protein MPB64/MPT64, and
wherein the biological sample is lysed with a sample treating agent before being subjected to the heat-treatment.
2. A method according to claim 1 , wherein the heat-treatment of the biological sample is carried out at 45° C. to 55° C.
3. A method according to claim 2 , wherein the heat-treatment of the biological sample is carried out at 45° C. to 50° C.
4. A method according to claim 1 , wherein the biological sample is selected from the group consisting of sputum, bronchial secretion, pleural effusion, gastric juice, blood, spinal fluid, urine, feces, bronchial lavage fluid, and a tissue collected from the bronchus or the lung.
5. A method according to claim 4 , wherein the biological sample is sputum.
6. A method for according to claim 1 , wherein the biological sample lysed with a sample treating agent is dispersed or dissolved in a solvent before being subjected to the heat-treatment.
7. A method according to claim 1 , wherein the immunological assay is a sandwich immunoassay using a first antibody and a second antibody against the Mycobacterium tuberculosis complex-specific protein.
8. A method according to claim 7 , wherein the immunological assay is an immunochromatographic assay.
9. A method according to claim 7 , wherein the immunological assay is ELISA.
10. A method for extracellularly secreting a Mycobacterium tuberculosis complex-specific protein MPB64/MPT64, comprising subjecting a biological sample containing a Mycobacterium tuberculosis complex that secretes a Mycobacterium tuberculosis complex-specific protein MPB64/MPT64 to a heat-treatment that is carried out at 45° C. to 60° C. for 15 to 60 minutes whereby said Mycobacterium tuberculosis complex-specific protein MPB64/MPT64 is extracellularly secreted, wherein the biological sample is lysed with a sample treating agent before being subjected to the heat-treatment.
11. A method according to claim 1 , said method comprising:
subjecting the lysed biological sample to the heat-treatment at 45° C. to 60° C. for 15 to 60 minutes to promote extracellular secretion of the Mycobacterium tuberculosis complex-specific protein MPB64/MPT64;
preparing the heat-treated sample for immunological assaying; and
subjecting the heat-treated sample to the immunological assay for the presence of Mycobacterium tuberculosis complex-specific protein MPB64/MPT64.
12. A method for detecting the presence of a Mycobacterium tuberculosis complex-specific protein in a heat-treated biological sample, the method comprising
subjecting a heat-treated biological sample to an immunological assay for the presence of the Mycobacterium tuberculosis complex-specific protein, wherein the Mycobacterium tuberculosis complex-specific protein is MPB64/MPT64,
wherein the heat-treated biological sample to be immunologically assayed for the presence of the Mycobacterium tuberculosis complex-specific protein is obtained from a biological sample, which was obtained from a subject, that has been subjected to a heat-treatment carried out at 45° C. to 60° C. for a time sufficient for promoting Mycobacterium tuberculosis complex that may be present in the biological sample to extracellularly secrete a detectable amount of the Mycobacterium tuberculosis complex-specific protein MPB64/MPT64, and wherein said biological sample has been lysed with a sample treating agent before being subjected to the heat-treatment.
13. A method for detecting for the presence of Mycobacterium tuberculosis complex-specific protein MPB64/MPT64 in a heat-treated biological sample, the method comprising:
subjecting an aliquot of a heat-treated biological sample to an immunological assay to detect for the presence of Mycobacterium tuberculosis complex-specific protein MPB64/MPT64,
wherein the aliquot of the heat-treated biological sample to be immunologically assayed for the presence of said Mycobacterium tuberculosis complex-specific protein MPB64/MPT64 is obtained by
collecting a biological sample from a subject, wherein said biological sample may contain Mycobacterium tuberculosis complex and said biological sample is selected from the group consisting of sputum, bronchial secretion, pleural effusion, gastric juice, blood, spinal fluid, urine, feces, bronchial lavage fluid, and a tissue collected from the bronchus or the lung,
lysing the biological sample with a sample treating agent,
subjecting the lysed biological sample to a heat-treatment carried out at 45° C. to 60° C. for a time sufficient for inducing Mycobacterium tuberculosis complex that may be present in the biological sample to extracellularly secrete Mycobacterium tuberculosis complex-specific protein MPB64/MPT64, and
collecting and preparing the aliquot from the heat-treated biological sample.
14. A method for detecting the presence of Mycobacterium tuberculosis complex-specific protein MPB64/MPT64 in a biological sample, the method comprising:
collecting a biological sample from a subject, wherein said biological sample may contain Mycobacterium tuberculosis complex and said biological sample is selected from the group consisting of sputum, bronchial secretion, pleural effusion, gastric juice, blood, spinal fluid, urine, feces, bronchial lavage fluid, and a tissue collected from the bronchus or the lung,
lysing the biological sample with a sample treating agent,
subjecting the lysed biological sample to a heat-treatment carried out at 45° C. to 60° C. for 15 to 60 minutes to induce Mycobacterium tuberculosis complex that may be present in the biological sample to extracellularly secrete the Mycobacterium tuberculosis complex-specific protein,
collecting and preparing an aliquot from the heat-treated biological sample for immunological assay, and
subjecting the aliquot of the heat-treated biological sample to an immunological assay to detect for the presence of the Mycobacterium tuberculosis complex-specific protein MPB64/MPT64.