JP2002062299A - Method and reagent for detecting tubercle bacillus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To eliminate an obstacle against an identification diagnosis and early treatment of tuberculosis in a conventional tubercle bacillus detecting method costing long hours, labor, and costs and to solve public health problems such as peripheral infection caused by discharged bacteria. SOLUTION: In this method and reagent for quickly and easily detecting a tubercle bacillus, a highly sensitive immunoassay is used so as to detect a tubercle bacillus-specific secretory protein existing in a biological sample without cultivating any tubercle bacillus in the biological sample.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method and a reagent for detecting Mycobacterium tuberculosis by measuring a secretion protein specific for Mycobacterium tuberculosis in a biological sample by an immunoassay. More specifically, without culturing Mycobacterium tuberculosis in a biological sample,
The present invention relates to a method and a reagent for detecting Mycobacterium tuberculosis by measuring a Mycobacterium tuberculosis group-specific secreted protein in a biological sample by an immunoassay using an anti-M. Tuberculosis group-specific secreted protein antibody.

[0002]

BACKGROUND OF THE INVENTION Tuberculosis in humans is a respiratory and other infections caused by infection with Mycobacterium tuberculosis. Pulmonary tuberculosis, which accounts for the majority of tuberculosis, is difficult to clinically distinguish from lung cancer, pulmonary mycosis, respiratory worm and the like. For the definitive diagnosis of Mycobacterium tuberculosis, a method of detecting Mycobacterium tuberculosis by culturing a specimen is used, but it takes a long time to detect the Mycobacterium tuberculosis, and identification of the detected bacterium is essential. The PCR method has been applied as a method for rapidly detecting M. tuberculosis, but this method requires a great deal of labor and cost, and the detection sensitivity is not always satisfactory.

It is said that the group of Mycobacterium tuberculosis secretes as many as 300 kinds of various proteins into a medium. Among them, MPT64 is known as a Mycobacterium tuberculosis group-specific secreted protein [Infec
tionand Immunity, 62 (5) , 2058 (1994)]. As a specific protein produced by BCG bacteria, MPB64 [Infection and Im
munity, 57 (1) , 283 (1989)]. The amino acid sequences of both are identical.

[0004] A method of detecting Mycobacterium tuberculosis by an immunological method using an antibody against MPT64 (hereinafter abbreviated as anti-MPT64 antibody) is known (Japanese Patent Application Laid-Open No. Hei 7-110332). As an immunological method, a method using immunochromatography is known [JP-A-11-1089].
No. 31, Medical Examination, 48 (6), 976 (1999)]. However, in these methods, since the tuberculosis bacterium in the biological sample is cultured and the culture solution is used as the sample, it also takes about one week before detection, and requires culturing and identification operations. Therefore, a lot of labor and cost are required.

[0005]

As described above, the conventional method requires a long time to detect M. tuberculosis and requires a great deal of labor and cost. Not only is it a major obstacle, but it also poses a serious public health problem, such as infection of surrounding people by bacterial germination. An object of the present invention is to provide a method, a reagent and a kit for quickly and easily detecting Mycobacterium tuberculosis.

[0006]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and using a highly sensitive immunological assay method, without culturing Mycobacterium tuberculosis in a biological sample, The present inventors have found that a Mycobacterium tuberculosis bacterium can be diagnosed quickly and easily by detecting a Mycobacterium tuberculosis group-specific secreted protein from a biological sample, thereby completing the present invention. That is, the present invention relates to the following (1) to (20). (1) A method for detecting Mycobacterium tuberculosis in a biological sample, wherein the Mycobacterium tuberculosis group-specific secreted protein is detected by an immunological assay without culturing the Mycobacterium tuberculosis in the biological sample. (2) Detecting a Mycobacterium tuberculosis group-specific secreted protein by an immunoassay using an anti-tuberculosis group-specific secreted protein antibody after pretreating the biological sample without culturing the M. tuberculosis in the biological sample. A method for detecting Mycobacterium tuberculosis in a biological sample. (3) An immunological method using a tuberculosis group-specific secreted protein antibody and a labeled body sensitizing reagent after pretreatment of the biological sample without culturing the tuberculosis bacterium in the biological sample; Characterized by detecting by a measuring method,
A method for detecting Mycobacterium tuberculosis in a biological sample. (4) The biological sample is sputum or pleural effusion as described in (1) to above.
The method according to any one of (3). (5) The method according to (2) or (3), wherein the pretreatment includes a step of inactivating tuberculosis bacteria in the biological sample.

(6) The above (2) or (3), wherein the pretreatment is performed with at least one selected from an alkaline substance, a reducing substance, a protease, a sugar, a surfactant, and a protein denaturant. the method of. (7) The method according to any one of (1) to (3) above, wherein the Mycobacterium tuberculosis group-specific secretory protein is MPT64. (8) Anti-M. Tuberculosis group-specific secreted protein antibody is anti-MPT64
The method according to the above (2) or (3), which is an antibody. (9) The method according to the above (8), wherein the anti-MPT64 antibody is a monoclonal antibody. (10) The method according to any one of the above (1) to (3), wherein the immunological assay is an enzyme immunoassay.

(11) The method according to any one of the above (1) to (3), wherein the immunological assay is a luminescent immunoassay. (12) The method according to any one of the above (1) to (3), wherein the immunological assay is a fluorescent immunoassay. (13) The immunoassay according to any one of (10) to (12), wherein the immunoassay is a sandwich method.
-The method according to any one of (12). (14) containing an alkaline substance as an active ingredient,
A reagent for pretreating a Mycobacterium tuberculosis complex-specific protein in a biological sample. (15) The above-mentioned (14), wherein the biological sample is sputum or pleural effusion.
The reagent as described.

(16) A kit for detecting Mycobacterium tuberculosis, comprising the reagent according to (14) or (15) and a reagent containing an anti-M. Tuberculosis group-specific secreted protein antibody. (17) The reagent according to the above (14) or (15),
A kit for detecting Mycobacterium tuberculosis, comprising a reagent containing an anti-M. Tuberculosis group-specific protein antibody and a reagent for detecting a label of the antibody. (18) The reagent according to the above (14) or (15),
A kit for detecting Mycobacterium tuberculosis, comprising a reagent containing an anti-Mycobacterium tuberculosis group-specific secretory protein antibody, a reagent for detecting the label of the antibody, and a labeled sensitizing reagent. (19) the Mycobacterium tuberculosis group-specific secreted protein is MPT64;
The kit according to any one of the above (16) to (18). (20) The kit according to any one of (16) to (18) above, wherein the anti-tuberculosis group-specific protein antibody is an anti-MPT64 antibody.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, detecting a Mycobacterium tuberculosis group-specific secreted protein by an immunoassay without culturing the M. tuberculosis in a biological sample involves culturing the M. tuberculosis in the biological sample. Any method can be used as long as the method can measure the Mycobacterium tuberculosis group-specific secreted protein by an immunological assay without performing the method. However, a method in which the Mycobacterium tuberculosis in a biological sample is pretreated and then subjected to the immunological assay is preferable.

As the biological sample used in the present invention, any biological sample in which M. tuberculosis can be present can be used. For example, sputum, pleural effusion and the like can be used. Preferably, sputum is used because of its high bacterial concentration. The pretreatment of the present invention means a step including a step of treating a Mycobacterium tuberculosis group-specific secreted protein so as to increase sensitivity to an antibody against the protein without impairing antigenic activity. It is preferable to include a step of inactivating Mycobacterium tuberculosis for safety in measurement. The step of inactivating M. tuberculosis may be before or after the step of pretreating the M. tuberculosis group-specific secretory protein.

The reagent for pretreating a Mycobacterium tuberculosis group-specific secretory protein from a biological sample according to the present invention includes increasing the sensitivity of a Mycobacterium tuberculosis group-specific secretory protein to an antibody to the protein without impairing antigenic activity. Can be anything if possible. Preferably, a reagent having a function of lowering the viscosity of a biological sample, particularly sputum, and more preferably containing a substance capable of solubilizing proteins in the biological sample is used.

Examples of the substance that reduces the viscosity of sputum include an alkaline substance, a reducing substance, a protease, a sugar, a surfactant, a protein denaturant, and the like, and preferably, an alkaline substance and a reducing substance. Examples of the alkaline substance include sodium hydroxide, and the concentration thereof is not particularly limited. However, considering the effect on the antigen activity of the M. tuberculosis secretory protein, it is preferably used in an amount of 0.5% to 4%.

Examples of the reducing substance include N-acetyl-L-cysteine and dithiothreitol, and the concentration thereof is not particularly limited, but is preferably used at 0.05% to 1%. As protease, proteinase K (Proteina
se K), ficin (FICIN) and the like, and the concentration thereof is not particularly limited, but is preferably used at 0.05% to 1%.

Examples of the sugar include fucose, trehalose and the like, and the concentration thereof is not particularly limited, but it is preferable to use 5% to 50%. Examples of the surfactant include nonylphenoxy-polyethoxy-ethanol (NP-40) and 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS). Although not limited, it is preferable to use 1% to 50%.

Examples of the protein denaturing agent include urea and guanidine hydrochloride, and the concentration thereof is not particularly limited.
Preferably it is used at ~ 10M. By reacting the above reagent on a biological sample and then performing a neutralization reaction as needed, the sensitivity of the Mycobacterium tuberculosis group-specific secreted protein to an antibody to the protein can be increased without impairing the antigen activity. The neutralization reaction can be performed by, for example, adding an acidic substance when an alkaline substance is used as a sputum pretreatment agent, and adding a protease inhibitor when using a protease as a sputum pretreatment agent. Alternatively, the heat treatment can be performed.

The step of inactivating M. tuberculosis includes heat treatment, filtration and the like. Preferably, a heat treatment is used. The heating temperature is not particularly limited, but is 50 ° C to 140 ° C, preferably 100 ° C. The heating time is not particularly limited, but is 1 to 60 minutes, preferably 15 to 30 minutes.

When performing the heat treatment, it is preferable to add a substance that stabilizes the antigen activity in order to prevent a decrease in the antigen activity of the M. tuberculosis group-specific secretory protein. Examples of substances that stabilize antigen activity include bovine serum albumin, gelatin and the like. The concentration of the substance that stabilizes the antigen activity is not particularly limited, but is preferably 0.01 to 10%,
More preferably, it is 0.1%.

By performing the method for inactivating M. tuberculosis, the subsequent operations described later do not need to be performed in a safety cabinet, and the test can be easily carried out. Therefore, the method is suitable for clinical examination. The M. tuberculosis group-specific secreted protein of the present invention may be any protein as long as it is specifically secreted by the M. tuberculosis group. Preferably, MPT64 is used.

The antibody against the Mycobacterium tuberculosis group-specific secretory protein in the present invention may be either a polyclonal antibody or a monoclonal antibody. As these antibodies, antibodies prepared by known methods can be used. As a method for preparing an antigen for preparing an antibody used in the present invention, DNA encoding the MPB64 protein (Japanese Patent No. 26247)
No. 59), a method of preparing MPT64 protein by separating and purifying MPT64 protein from a culture supernatant of M. tuberculosis, or selecting a partial amino acid sequence of the amino acid sequence of MPT64 protein And a method for preparing a synthetic peptide.

The antibody used in the present invention can be produced according to a known method for producing a monoclonal antibody using the aforementioned antigen as an immunogen. The monoclonal antibody can be produced by a method described in, for example, "Sakuji Toyama, Monoclonal Antibody Experimental Manual, Kodansha". Animals immunized with the immunogen include mice, rats, hamsters, rabbits, guinea pigs, goats, sheep and chickens, and it is preferable to use mice and rats for monoclonal antibody production.

Examples of the immunization method include [Nishimichi, Teshima,
New Chemistry Experiment Course, 1 , 389 (1990), Tokyo Kagaku Doujin] and the like. For example, the immunogen is emulsified in Freund's complete or incomplete adjuvant and administered intraperitoneally, subcutaneously, or intramuscularly. For example, 7 to 30 days, preferably 12
The administration can be carried out two or more times, preferably two to four times at regular intervals of from 16 days to complete immunity.

In the case of preparing a monoclonal antibody, the source of the antibody-producing cells may be the spleen, lymph node, peripheral blood, etc. of the immunized animal. Also, so-called in vitro immunization (Arai, Ota, Experimental Medicine, etc.), in which antibody-producing cells are extracted from the spleen, lymph nodes, peripheral blood, etc. of non-immunized animals, and these cells are directly immunized to produce antibody-producing cells. 6 , 43 (1988)].

The myeloma cells used for cell fusion between the antibody-producing cells and myeloma cells are not particularly limited, but it is preferable to use animal-derived cell lines of the same species as the antibody-producing cells. In order to efficiently select only cells that have been appropriately subjected to cell fusion, those having a specific drug marker are preferable. For example, 8-azaguanine-resistant myeloma cells cannot grow in a medium containing hypoxanthine, aminopterin, and thyminedin (hereinafter referred to as HAT medium), but cells in which these cells are fused with normal cells are HA.
It is preferably used because it can grow in T medium and can be distinguished from unfused myeloma cells. Specifically, P3
× 63-Ag.8.653 [Journal of Immunology (J.
Immunol.), 123 , 1548 (1979)] and P3 × 63-Ag.8.U1 [Cur
r. Topics. Microbiol. Immunol., 81 , 1 (1978)] (hereinafter simply abbreviated as P3U1), Sp / O-Ag14 [Nature, 276 , 269 (1978)] and the like.

Cell fusion was invented by Koehler and Milstein [Nature, 256 , 495 (1975)], and rapidly developed and variously improved methods can be applied. As a frequently used method, antibody-producing cells and myeloma cells are mixed at a ratio of 10 to 3: 1 and mixed with 30 to 50% of polyethylene glycol (average molecular weight of 1,500 to 6,6).
000) as a fusion agent. It can also be fused by electric pulses [Kawachi et al., Experimental Medicine, 6 , 50 (1988)].

The cells after cell fusion are suspended in a selection medium, and only the fused cells are grown using a culture vessel such as a 96-well culture plate, which is advantageous for the subsequent selection of target cells.
At the stage where only the fused cells have been selectively grown, only cells producing antibodies to the MPT64 protein are selected. In this selection, the presence or absence of the target antibody in the culture supernatant of the fused cells is examined using a method such as enzyme immunoassay or radioimmunoassay. The selected cells are cloned using, for example, a limiting dilution method or a soft agar medium method to establish a hybridoma cell line producing an MPT64 protein-specific monoclonal antibody.

The monoclonal antibody is obtained by culturing the established hybridoma cell line in an appropriate medium and collecting the culture, or by transplanting the cell line into the peritoneal cavity of an animal and growing it in ascites to collect ascites. It can be obtained from the obtained culture solution or ascites. The antibody in the culture solution or ascites can be purified and used as needed. Purification methods include, for example, salting-out fractionation using ammonium sulfate, ion-exchange chromatography, gel filtration column chromatography, affinity column chromatography using protein A or protein G, and a gel on which an antigen is immobilized. Various methods such as an affinity column chromatography method to be used or a method combining these methods can be mentioned. Further, as the antibody used in the present invention, Fab, Fab ′, F (a) obtained by treating the antibody obtained by the above-mentioned method with an enzyme such as pepsin and / or a reduction treatment.
b) Antibody fragments such as ₂ may be used.

The immunological assay for the secretory protein specific to the Mycobacterium tuberculosis group used in the present invention includes any known immunological assay. For example, radioimmunoassay (RIA), enzyme immunoassay (EIA or ELIS)
A), fluorescence immunoassay (FIA), luminescence immunoassay (lumine
scent immunoassay), physicochemical detection method (TIA, LAPI
A, PCIA) and the like, preferably an enzyme immunoassay.

The label used in the enzyme immunoassay includes:
Any known enzyme label (edited by Eiji Ishikawa et al., Enzyme immunoassay, Medical Shoin) can be used. For example, alkaline phosphatase label, peroxidase label, luciferase label and the like can be used. As the label used in the luminescence immunoassay, any known [Imai Kazuhiro,
Bioluminescence and chemiluminescence, Hirokawa Shoten; Clinical Laboratory 42 (1998)]
Can be used. For example, an acridinium ester label, a roffin label, or the like can be used.

The label used in the immunofluorescence assay includes:
Any known fluorescent label (Akira Kawao, fluorescent antibody method, Soft Science) can be used. For example, FITC labels, RITC labels, and the like can be used. The immunological measurement method is a method for measuring the amount of an antibody or the amount of an antigen using the above-described variously labeled antigens or antibodies. As the immunological measurement method of the present invention, any method may be used as long as it is a method for detecting or measuring an antigen. For example, competitive method, sandwich method [Immunology Illustrated
Plate (Nankodo)], but the sandwich method is preferred.

The sandwich method is a method in which after binding a first antibody to a solid phase, an antigen to be measured is trapped, and a labeled second antibody is reacted. As the antibody used for the sandwich method, any of a polyclonal antibody and a monoclonal antibody may be used.
Antibody fragments such as b 'and F (ab) ₂ may be used. The combination of the two types of antibodies used in the sandwich method may be a combination of a monoclonal antibody or an antibody fragment that recognizes a different epitope, or a combination of a polyclonal antibody and a monoclonal antibody or an antibody fragment.

Hereinafter, the immunoassay will be described in detail. First, an antibody against the Mycobacterium tuberculosis group-specific secreted protein obtained above is adsorbed and fixed on the surface of a suitable solid carrier. The antibody is brought into contact with a pretreated biological sample containing a Mycobacterium tuberculosis complex-specific protein. Thereby, the Mycobacterium tuberculosis group-specific secreted protein in the biological sample specifically binds to the antibody against the Mycobacterium tuberculosis group-specific secreted protein immobilized on the solid support. As a result, the Mycobacterium tuberculosis group-specific secretory protein in the biological sample is immobilized on the immobilization carrier via an antibody against the Mycobacterium tuberculosis group-specific secretory protein which has been solidified in advance.

Next, the carrier on which the Mycobacterium tuberculosis group-specific secretory protein is immobilized is brought into contact with a solution containing a labeled antibody against the Mycobacterium tuberculosis group-specific secretory protein as a secondary antibody. Thereby, the secondary antibody binds to the solid-phase carrier via the antibody against the Mycobacterium tuberculosis group-specific secretory protein and the Mycobacterium tuberculosis group-specific secretory protein, which are fixed in advance, and in this case, the immobilized amount of the secondary antibody is reduced. It reflects the amount of Mycobacterium tuberculosis group-specific secreted proteins in biological samples.

The amount of the secondary antibody immobilized as described above can be measured according to the label of the secondary antibody. In addition, a labeled sensitizing reagent can be used to measure a trace amount of antigen. By measuring the amount of the immobilized secondary antibody as described above, the amount of the M. tuberculosis group-specific secreted protein in the biological sample can be determined.

Examples of the solid carrier include a polyvinyl chloride microtiter plate and a polystyrene microtiter plate. For immobilization of the antibody, for example, after diluting the antibody in an appropriate buffer, for example, a phosphate buffer, a borate buffer, a carbonate buffer, or the like, the diluted antibody is brought into contact with the surface of the immobilization carrier, and then heated to 4 to 37 ° C. For 30 minutes or more.

In order to remove the antibody against the unadsorbed Mycobacterium tuberculosis group-specific secreted protein, a buffer containing a surfactant such as Tween 20, for example, a phosphate buffer, a borate buffer, a carbonate buffer, or the like is used. And wash several times. Examples of the blocking buffer for blocking free binding groups on the surface of the solid carrier include a buffer containing 1 to 10% bovine serum albumin and 10 to 30% Block Ace (manufactured by Snow Brand Milk Products), for example, phosphate buffer. Liquid, borate buffer, carbonate buffer and the like.
The blocking treatment can be performed by incubation at 4 to 37 ° C. for 30 minutes or more.

The biological sample may be one diluted with a buffer containing a protein such as bovine serum albumin of 0.01 to 1%, a phosphate buffer, a borate buffer, a carbonate buffer or the like. Contact between the antibody and the biological sample should be performed at
It can be carried out by incubation for 0 minutes or more. After the contact, the substrate is washed several times with a buffer containing a surfactant such as Tween 20, for example, a phosphate buffer, a borate buffer, a carbonate buffer, or the like.

Detection or measurement of the bound and immobilized secondary antibody can be performed according to any known method. For example, as described above, the secondary antibody itself is labeled with an enzyme, a luminescent material, a fluorescent material, or the like, and can be directly detected or measured, or a tertiary antibody specific to the secondary antibody , The tertiary antibody can be labeled by various methods, and the label of the tertiary antibody can be detected or measured.

As a labeled sensitizing reagent, for example, when an alkaline phosphatase label is used in enzyme immunoassay, an enzyme cycling sensitizing reagent, Amplifier (DAK
In the case of using peroxidase labeling, a Radical-EIA method using electron spin resonance (JP-A-9-28396) can be used. The present invention provides a kit for detecting Mycobacterium tuberculosis, the kit including at least a pretreatment liquid for a biological sample and a reagent containing an antibody against a Mycobacterium tuberculosis group-specific protein. Antibodies to Mycobacterium tuberculosis group-specific secreted proteins include:
The antibodies described above are used. When the immunological assay to be used is a sandwich, two kinds of antibodies are used, but a combination of a monoclonal antibody or an antibody fragment that recognizes a different epitope may be used, or a combination of a polyclonal antibody and a monoclonal antibody or an antibody fragment may be used.

In addition, any one of the two types of antibodies is labeled with the above-described label. Therefore, a reagent for detecting a labeled substance is also included in the kit for detecting Mycobacterium tuberculosis. Also, if necessary, a biological sample dilution buffer, various reagent dilution buffers,
A washing solution and the like are also included in the kit of the present invention. Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited by the Examples and the like.

[0041]

EXAMPLES Example 1 Preparation of Monoclonal Antibody Against MPT64 (1) Immunization of Animals and Preparation of Antibody-Producing Cells Known Methods [Jpn. J. Med. Sci. Biol., 49 , 15 (1996)]
, MPT64 was obtained from the M. tuberculosis culture supernatant.

For the purpose of enhancing the immunogenicity of MPT64, a conjugate with methylated BSA (Sigma) was prepared by the following method and used as an immunogen. That is, methylated BSA dissolved in double distilled water was mixed at 4 ° C. so that BSA: HVC002 = 1: 4 (weight ratio), and stirred with a vortex mixer for 10 seconds. Then, after diluting with a 0.4 M NaCl solution so that the HVC002 concentration becomes 200 μg / ml, the mixture was mixed with complete Freund's adjuvant or incomplete Freund's adjuvant at a volume ratio of 1: 1 using a syringe with a connecting needle, and mixed with the immunogen. did.

To a 5-week-old female mouse (Balb / c), 30 μg of the immunogen MPT64 prepared as described above using complete Freund's adjuvant was administered. Two weeks later, the same procedure was performed using incomplete Freund's adjuvant. The adjusted immunogen was administered once a week for a total of four times. Blood was collected from the fundus venous plexus, and its serum antibody titer was examined by the binding ELISA described in Example 1 (4). From the mouse showing a sufficient antibody titer, the spleen was extracted 3 days after the last immunization.

The spleen was shredded in a MEM medium (manufactured by Nissui Pharmaceutical Co., Ltd.), loosened with forceps, and centrifuged (1200 rpm,
After 5 minutes), the supernatant was discarded and treated with a Tris-ammonium chloride buffer (pH 7.65) for 1 to 2 minutes to remove red blood cells, washed three times with MEM medium, and used for cell fusion. (2) Preparation of mouse myeloma cells An 8-azaguanine-resistant mouse myeloma cell line P3-U1 was cultured in a normal medium, and 2 × 10 ⁷ or more cells were secured at the time of cell fusion and used as a parent strain for cell fusion.

(3) Preparation of hybridoma The mouse spleen cells obtained in Example 1 (1) and the myeloma cells obtained in (2) were mixed at a ratio of 10: 1, and centrifuged (1,200 rpm). 5 minutes), discard the supernatant,
After loosening the precipitated cell group, the mixture was stirred for 37
At room temperature, polyethylene glycol-1,000 (PEG
-1,000) 2 g, MEM medium 2 ml and dimethyl sulfoxide 0.7 ml mixture 0.2-1 ml / 10 8
Mouse spleen cells were added, and MEM medium 1
After adding 2 ml several times, a total amount of 50 ml was added by adding MEM medium.
ml. After centrifugation (900 rpm, 5 minutes), the supernatant was discarded, and the cells were loosened gently.
The cells were suspended in 100 ml of AT medium.

This suspension was dispensed at 100 μl / well into a 96-well culture plate and cultured in a 5% CO ₂ incubator at 37 ° C. for 10 to 14 days under 5% CO ₂ . The culture supernatant was examined by a binding ELISA described in Example 1 (4), a well that did not react with an antigen not containing MPT64 in response to MPT64 was selected, and further replaced with an HT medium and a normal medium, and cloning was repeated twice. , Anti-MPT6
A four protein monoclonal antibody producing hybridoma was established.

(4) Selection of Monoclonal Antibody As an antigen for binding ELISA assay, MPT64 conjugated with methylated BSA by the method described in Example 1 (1) was used. BSA used for preparation of the antigen conjugate was used as a control antigen. The conjugate prepared as described above was added to a 96-well EIA plate (manufactured by Greiner) at an MPT64 or IL-1β concentration of 1 μm.
The solution was dispensed at 50 μl / well at g / ml and allowed to stand at 4 ° C. overnight for adsorption. After washing, 1% BSA-PBS was added at 100 μl / well and allowed to react at room temperature for 1 hour to block the remaining active groups. Discard 1% BSA-PBS, immunized mouse antiserum, anti-MPT
64 protein monoclonal antibody culture supernatant or purified antibody
The MPT64 protein monoclonal antibody was dispensed at 50 μl / well and allowed to react for 2 hours. After washing with tween-PBS, diluted peroxidase-labeled rabbit anti-mouse immunoglobulin (manufactured by Dako) was added at 50 μl / well and reacted at room temperature for 1 hour. -Ethylbenzothiazole-6-sulfonic acid) ammonium], and the absorbance at OD 415 nm was measured with a plate reader (Emax; manufactured by Wako Pure Chemical Industries, Ltd.).

(5) Purification of Monoclonal Antibody 5 to 20 weeks of the hybridoma strain obtained in Example 1 (3) was added to pristane-treated 8-week-old nude female mice (Balb / c).
× 10 ⁶ cells / animal were injected intraperitoneally. 10-21
One day later, the hybridoma developed ascites cancer. Ascites was collected (1-8 ml / animal) from the mice with accumulated ascites, and the solid content was removed by centrifugation (3,000 rpm, 5 minutes). When the monoclonal antibody is IgG, caprylic acid precipitation method [Antibodies-A Laboratory Manual, Cold Sprin
g Harbor Laboratory, 1988] to obtain a purified monoclonal antibody.

The antibody subclass was determined by enzyme immunoassay using a subcluster typing kit.
Was determined.

Example 2 MPT64 Measurement Using Enzyme Sandwich Immunoassay 100 μL of the anti-MPT64 monoclonal antibody (10 μg / mL) -containing PBS solution obtained in Example 1 was added to a 96-well microplate and incubated at room temperature for 4 hours. This was left as an immobilized antibody. Next, in order to suppress nonspecific adsorption, the wells were washed three times with a PBS solution, and then 250 μL of a PBS solution containing 1% bovine serum albumin was added, and the mixture was allowed to stand at room temperature for 4 hours. PB Well
After washing three times with S, 100 µL of a culture solution of Mycobacterium tuberculosis (diluted from 1 to 80,000) diluted with a PBS solution containing 0.1% bovine serum albumin was added, and the mixture was incubated at 37 ° C for 2 hours. Amp Well
After washing three times with a liQ washing solution (manufactured by Dako), 100 μL of an alkaline phosphatase-labeled anti-MPT64 monoclonal antibody was added, and the mixture was incubated at 37 ° C. for 2 hours. After washing the wells 5 times with AmpliQ washing solution (manufactured by Dako), 100 μL of AmpliQ Am
plifierA (manufactured by Dako) and 100 μL AmpliQ AmplifierB
(Manufactured by Dako) and stirred at room temperature for 40 minutes.
The color reaction was stopped by adding AmpliQ stop solution (DAKO). Finally, the absorbance at 492 nm was measured with a microtiter plate reader. The result is shown in FIG. As a result, it was shown that detection of M. tuberculosis was possible even when the culture solution of M. tuberculosis was diluted 10,000 times.

Example 3 Pretreatment of sputum 0.5 mL of sputum pretreatment agent NALC-NaOH solution (BBL mycoprep; manufactured by Nippon Becton Dickinson Company) was added to 0.5 mL of sputum, and mixed for several seconds with a vortex mixer. At room temperature
Left for 5 minutes. 0.3 mL of 2M NaH ₂ PO ₄ to neutralize pH
The solution was added and mixed on a vortex mixer for several seconds. Then, in order to prevent a decrease in MPT64 antigen activity due to the heat treatment, bovine serum albumin was added to a concentration of 0.1%, and then to inactivate Mycobacterium tuberculosis in sputum, 10
Heat treatment was performed at 0 ° C. for 30 minutes.

Example 4 Detection of Mycobacterium tuberculosis by Measurement of MPT64 in Sputum from a Tuberculosis Patient Sputum from a tuberculosis patient which had been pretreated by the same method as in Example 3 was used as a measurement sample. The MPT64 measurement of each sample was performed in the same manner as in Example 2. The determination of the result was performed based on the measured value (absorbance 492 nm), and the case where the measured value exceeded 0.180 was defined as positive. As a control, the cells were cultured for 8 weeks using an Ogawa medium by a conventional method, and the growth of Mycobacterium tuberculosis was visually confirmed. Table 1 shows the results.

[0053]

[Table 1]

As a result, only the tuberculosis patient specimen became positive, and the same results were obtained with the Ogawa medium.

[0055]

According to the reagent and method for detecting Mycobacterium tuberculosis of the present invention, Mycobacterium tuberculosis can be detected without culturing Mycobacterium tuberculosis in a biological sample. As a result, the test period, which conventionally required 1 to 8 weeks, can be reduced to several hours, and a definite diagnosis of tuberculosis can be made quickly and easily.

[Brief description of the drawings]

FIG. 1 shows the results obtained in Example 2 by the method of Example 2.
FIG. 4 shows the results of measuring MPT64 in a culture solution.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Shuji Mori 600-1 Kamiyamachi, Minami-Ishiki, Nagaizumi-cho, Sunto-gun, Shizuoka Pref. -2-2

Claims (20)

[Claims] 1. A method for detecting Mycobacterium tuberculosis in a biological sample, comprising detecting a Mycobacterium tuberculosis group-specific secreted protein by an immunoassay without culturing the Mycobacterium tuberculosis in the biological sample. 2. A method for detecting a Mycobacterium tuberculosis group-specific secreted protein by an immunological assay using an anti-tuberculosis group-specific secreted protein antibody after pretreating the biological sample without culturing the Mycobacterium tuberculosis in the biological sample. A method for detecting Mycobacterium tuberculosis in a biological sample. 3. A method of pretreating a biological sample without culturing the M. tuberculosis in the biological sample, and then immunizing the M. tuberculosis group-specific secretory protein with an anti-M. Tuberculosis group-specific secretory protein antibody and a labeled sensitizing reagent. A method for detecting Mycobacterium tuberculosis in a biological sample, wherein the method is performed by a biological measurement method. 4. The biological sample is sputum or pleural effusion.
The method according to any one of claims 1 to 3. 5. The method according to claim 2, wherein the pretreatment includes a step of inactivating tuberculosis bacteria in the biological sample. 6. The method according to claim 2, wherein the pretreatment is performed with at least one selected from an alkaline substance, a reducing substance, a protease, a sugar, a surfactant, and a protein denaturant.
Or the method of 3. 7. The method according to any one of claims 1 to 3, wherein the Mycobacterium tuberculosis group-specific secreted protein is MPT64. 8. An anti-tuberculosis group-specific secreted protein antibody,
The method according to claim 2 or 3, which is an MPT64 antibody. 9. The method according to claim 8, wherein the anti-MPT64 antibody is a monoclonal antibody. 10. The method according to claim 1, wherein the immunoassay is an enzyme immunoassay. 11. The method according to claim 1, wherein the immunoassay is a luminescence immunoassay. 12. The method according to claim 1, wherein the immunological assay is a fluorescent immunoassay. 13. The immunoassay according to any one of claims 10 to 12, wherein the immunoassay is a sandwich method.
13. The method according to any one of 12 above. 14. A reagent for pretreating a Mycobacterium tuberculosis group-specific protein in a biological sample, comprising an alkaline substance as an active ingredient. 15. The reagent according to claim 14, wherein the biological sample is sputum or pleural effusion. 16. A kit for detecting Mycobacterium tuberculosis, comprising the reagent according to claim 14 or 15 and a reagent comprising an anti-M. Tuberculosis group-specific secreted protein antibody. 17. The reagent according to claim 14 or 15,
A kit for detecting Mycobacterium tuberculosis, comprising a reagent containing an anti-M. Tuberculosis group-specific protein antibody and a reagent for detecting a label of the antibody. 18. The reagent according to claim 14 or 15,
A kit for detecting Mycobacterium tuberculosis, comprising a reagent containing an anti-Mycobacterium tuberculosis group-specific secretory protein antibody, a reagent for detecting the label of the antibody, and a labeled sensitizing reagent. 19. The kit according to claim 16, wherein the Mycobacterium tuberculosis group-specific secreted protein is MPT64. 20. The anti-M. Tuberculosis group-specific protein antibody is anti-MPT6.
The kit according to any one of claims 16 to 18, which is a 4 antibody.