JPH11108931A - Method and kit for detection of tubercle bacillus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To detect a tubercle bacillus in a short period, with high sensitivity and safely by a method wherein human tubercle bacillus intermingled MPT 64 protein or bovine tubercle bacillus intermingled MPB 64 protein in which a sample is cultured by a solid substrate so as to be proliferated and a color labeled anti-MPB 64 protein antibody are coupled by an immunoreaction and their coupled substance is captured by a second anti-MPB 64 protein antibody. SOLUTION: A member 5 for sample addition, a member 2 which is impregnated with a color labeled antibody, a membrane carrier 3 for chromatography development and a member 4 for adsorption are pasted on the adhesive face of an adhesive sheet 1. The member 2 is impregnated wholly with a color-labeled anti-MPB protein antibody solution, and the carrier 3 is impregnated with a second anti-MPB protein antibody solution so as to be a spot shape. A sample such as the cultured substance of a sample to be inspected is irrigated to a member 5. When MPT 64 protein or MPB 64 protein which is produced from a tubercle bacillus is contained in the sample, a color-coupled substance is generated by the immunoreaction of it with the color-labeled anti-MPB 64 protein antibody. The sample is developed toward the downstream of the membrane carrier 3 for chromatography development, and the color-coupled substance is captured and integrated by a capture part 31 so as to be colored.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for detecting Mycobacterium tuberculosis and a kit for detecting Mycobacterium tuberculosis. More specifically, the present invention relates to a method for detecting the presence or absence of M. tuberculosis or M. bovis in a subject such as sputum. The present invention relates to a detection method for testing and a detection kit therefor.

[0002]

2. Description of the Related Art Mycobacterium tuberculosis is a taxonomic group of mycobacteria. Mycobacterium tuberculosis includes Mycobacterium tuberclos
i), Mycobacterium bovis Mycob
acterium bovis), Mycobacterium microti, Mycobacterium aviu
m) and cold-blooded Mycobacterium tuberculosis are known. Thus, of these, almost all of M. tuberculosis is involved in M. tuberculosis, but rarely there is infection with M. bovis. Microorganisms belonging to the genus Mycobacterium other than Mycobacterium tuberculosis and Mycobacterium bovis are called atypical acid-fast bacteria.

On the other hand, as a conventional diagnostic method for human tuberculosis, a so-called "niacin production test" is widely performed. This niacin production test was performed on patients with suspected tuberculosis.
For example, tuberculosis in which sputum, warmed 1% by weight sterile saline is deeply inhaled as an aerosol with a nebulizer, and sputum, sputum, gastric lavage, feces and urine, etc., which are excreted with cough, are cultured in a medium and propagated in tuberculosis This is a method for extracting the presence or absence of Mycobacterium tuberculosis in a subject by extracting the bacterium from the bacteria with boiling distilled water and knowing the presence or absence of niacin in the extract.

[0004] However, in the niacin production test, 93% or more of Mycobacterium tuberculosis are positive, but certain other acid-fast bacteria or, rarely, atypical acid-fast bacteria are also positive. Even if the provisional instruction and the niacin production test are positive, it is not always possible to conclude that the strain is Mycobacterium tuberculosis. Therefore, generally, in addition to the niacin production test, the growth rate of the bacteria, pigmentation, and the properties of the colonies are simultaneously examined to discriminate between Mycobacterium tuberculosis and other acid-fast bacteria.

[0005] Niacin of Mycobacterium tuberculosis grown by culturing a specimen is detected by qualitative chemical analysis means. However, this qualitative chemical analysis has low sensitivity, and a large amount of niacin is required to obtain accurate test results. Needed. Further, in this qualitative chemical analysis, analytical operations over several steps are performed, and such analytical operations have a high risk of Mycobacterium tuberculosis infection due to scattering of cells. Therefore, it is said that these analysis operations must be performed in an extremely safe environment, using extremely safe equipment, and with great care. However, in order to carry out the niacin production test, not only does qualitative chemical analysis itself take some time, but it also requires culturing of the specimen and this culturing takes a long time. Long hours will be spent.

[0006] On the other hand, the niacin production test may be performed by subjecting the subject to liquid culture or solid culture. The former method is carried out, for example, in the United States and the like, and the culturing period of the specimen is reduced, but the risk of M. tuberculosis infection during the operation is great, and highly safe facilities and equipment are required. In Japan, the latter is performed and the risk of M. tuberculosis infection is reduced, but it requires a long period of about two months before the results of the analysis become clear. Thus, in the latter case, the risk of M. tuberculosis infection during operation is reduced, but still, use a highly safe environment and highly safe equipment, and pay close attention. It must be done.

[0007] The protein MPT64 (hereinafter referred to as MPT64 protein) is a mycobacterial protein specifically produced by Mycobacterium tuberclosis and secreted out of the cells. Also, protein MP
It is known that it is the same substance as B64 (hereinafter referred to as MPB64 protein). On the other hand, MPB64 protein
BCG strain Mycobacterium bovis BCG (hereinafter referred to as B
CG bacterium) and secreted extracellularly. M. Harboe (M. Harboe) reports that this MPB64 protein has been isolated and purified from BCG bacteria and studied for its properties (Infection and Inmmunity 1986, Vol. 52, No. 1,
293-302).

In Japanese Patent Application Laid-Open No. 1-247094, the MPB64 protein is not produced by atypical mycobacteria which infects humans and exhibits symptoms very similar to tuberculosis. It is a protein specific to BCG bacteria and Mycobacterium tuberculosis bacteria, which are the same substance as MPT64 protein.

Therefore, this means that the anti-MPB64 protein antibody is an antibody whose antigen is the MPB64 protein,
It means that it is also an antibody of PT64 protein. Therefore,
A pathogenic harmless BCG bacterium is cultured, the MPB64 protein produced by BCG is extracted and purified from the culture solution, an anti-MPB64 protein antibody is prepared using the MPB64 protein as an antigen, and the antigen using the antibody is used. By detecting Mycobacterium tuberculosis in a subject by an antibody reaction (immune reaction),
It is possible to establish a simple method for diagnosing tuberculosis, which can clearly and quickly determine human tuberculosis.

[0010] As a test drug and a method for detecting Mycobacterium tuberculosis using an anti-MPB64 protein antibody, see, for example,
The method described in Japanese Patent No. 10332 is known. As an immunological method in this method, reverse passive hemagglutination (R
PHA), reverse passive latex agglutination (RPLA) and enzyme-linked immunosorbent assay (ELISA). The former two methods have the disadvantages that the aggregation time is long, for example, about 3 hours, the sensitivity is low, and there is no quantitative property. The latter has the drawback that the test operation is more complicated than the former two, and it is not easy to carry out in a safety cabinet due to the use of special machines.

[0011]

SUMMARY OF THE INVENTION The present inventors have solved the above-mentioned drawbacks in the prior art, and have a short period from the collection of a specimen to the determination of a test result, high sensitivity, and semi-quantitative properties. In addition, as a result of intensive studies to develop a highly safe detection method for M. tuberculosis, for example, anti-MPB
Color-labeled anti-MPB obtained by binding a color-labeling substance such as colloidal gold particles, which are dispersed particles of gold sol, to antibody 64 protein.
The present inventors have obtained new knowledge that the above-mentioned drawbacks in the prior art can be solved by preparing a 64 protein antibody and using the same, and based on this finding, have reached the present invention.

[0012]

That is, the first aspect of the present invention is to provide a method of culturing a test sample in a solid medium and growing the same, wherein the mixed M. tuberculosis mixed MPT64 protein or M. bovis mixed M.
The PB64 protein and the color-labeled anti-MPB64 protein antibody are bound by an immunological reaction,
A method for detecting Mycobacterium tuberculosis characterized by capturing with a 64-protein antibody.

[0013] The second invention is to mix a culture obtained by culturing a subject with a color-labeled anti-MPB64 protein antibody,
Next, the mixed solution was brought into contact with a second anti-MPB64 protein antibody, and the presence or absence of coloration due to capture of the conjugate of the MPT64 protein or MTB64 protein with the color-labeled anti-MPB64 protein antibody by the second anti-MPB64 antibody was determined. And determining the presence or absence of M. tuberculosis or M. bovis.

The third aspect of the present invention is to provide at least a chromatographic test strip or a chromatographic test strip lacking a color-marked antibody member contained in a case and a color-labeled anti-MPB64 protein antibody contained in a container. It is a kit for detecting Mycobacterium tuberculosis.

[0015]

The specimens are mainly sputum of patients with suspected tuberculosis and test patients. For example, sputum and gastric lavage which are expectorated with cough by inhaling sterile saline aerosol. , Feces and urine, respectively. The subject is subjected to solid culture or liquid culture by a conventional method.

In the case of solid culture, for example, the cells are cultured at 37 ° C. using a test tube Kudo PD solid slope medium (a product of Kyowa Pharmaceutical Co., Ltd.). Distilled water is added to the Mycobacterium tuberculosis cultured on the slope to extract a culture secreted outside the cells during the culture period, and the Mycobacterium tuberculosis mixed solution is used as a sample for inspection. In the case of liquid culture, for example, a test solution and an aqueous sodium hydroxide solution are mixed, and the cells are separated from this mixed solution by centrifugation. The separated cells are, for example, Dubos' liquid medium (Becton).・ A culture solution cultured at 37 ° C. under the trade name of Dickinson is used as a sample for the test.

The MPB64 protein is described in, for example, M. It is prepared by a method known per se such as a method of separating and purifying Harbo. That is, for example, salting out, dialysis, and column chromatography are performed on a culture obtained by culturing a strain having the ability to produce MPB64 protein, such as the BCG bacterium Tokyo strain belonging to Mycobacterium bovis, in a liquid medium such as Sorton's medium. Purified MPB64 protein can be obtained by successively identifying proteins by chromatography, electrophoresis, or the like.

The anti-MPB64 protein antibody may be either a monoclonal antibody or a polyclonal antibody obtained by a conventional method using the purified MPB64 protein thus obtained as an antigen. Monoclonal antibodies are obtained, for example, by the method of Keller-Milstein. The polyclonal antibody is preferably obtained by immunizing a goat.

The anti-MPB64 protein antibody thus obtained is conjugated with a color-labeling substance to form a color-labeled anti-M
PB64 protein antibody. Colored labeling substances include, for example, colloidal metals and colored latex. Representative examples of the colloidal metal include metal colloidal particles which are dispersed particles such as gold sol, silver sol, selenium sol, tellurium sol, and platinum sol.

The size of the colloidal metal particles is usually
The diameter is about 3 to 60 nm. As a typical example of the colored latex, a synthetic latex such as polystyrene latex colored with respective pigments such as red and blue can be given. Natural latex such as natural rubber latex can be used as the latex. The size of the colored latex ranges from a few nanometers to several hundreds in diameter.
It can be selected from about nm. Commercially available products can be used for these color labeling substances as they are, but they may be further processed in some cases or manufactured by a method known per se.

The binding between the anti-MPB64 protein antibody and the color labeling substance is carried out by a conventional method. That is, for example,
When the color labeling substance is colloidal gold particles that are dispersed particles of gold sol, usually, the anti-MPB64 protein antibody and gold sol are mixed at room temperature to room temperature for several minutes, and at most 10 minutes. Both can be physically combined.

The antigen MPB64 protein or MPT64 protein which is the same substance as the protein (hereinafter, MPT64 protein is referred to as antigen similarly to MPB64 protein) and the color-labeled anti-MPB64 protein antibody are subjected to a normal antigen-antibody reaction. It is combined. That is, for example, a test sample which may contain MPB64 protein or MPT64 protein which is an antigen produced by M. tuberculosis and a color-labeled anti-MPB64.
The antigen-antibody reaction is carried out by allowing the mixture to stand at room temperature to room temperature in a buffer solution in which both the protein and the antibody are mixed, and a conjugate of the antigen MPB64 protein or MPT64 protein with the color-labeled anti-MPB64 protein antibody (Hereinafter, simply referred to as a color combination). This colored conjugate is charged, for example, on a band-shaped solid support on which a second anti-MPB64 protein antibody is immobilized and chromatographed, and all this is captured by the second anti-MPB64 protein antibody.
A color peculiar to the used color labeling substance is presented, whereby tuberculosis bacteria in the subject are detected.

In the second anti-MPB64 protein antibody, the binding site of the antigen-antibody reaction to the antigen MPB64 protein or MPT64 protein is determined by the anti-MPB64 protein antibody used for the labeled anti-MPB64 protein antibody. It must be different from the binding site.

The known number of M. tuberculosis and the second anti-MPB
By using the criterion derived from the relationship with the color concentration of the color conjugate captured by the 64 protein antibody, the approximate number of tuberculosis bacteria contained in the subject can be known. In this case, all of the test conditions of the subject must be substantially the same as those used when creating the criteria.

The capture of the colored conjugate with the second anti-MPB64 protein antibody is preferably carried out using a chromatographic test strip. A chromatographic test strip is a test strip that is irrigated with one end of a strip-shaped solid carrier test strip from a sample inlet of a case lid to start chromatographic development and to infiltrate into a colored label on the test strip. When the antibody reaches the antibody-impregnated member and immunoreacts with the color-labeled anti-MPB64 protein antibody of the member, a color-conjugated product is formed. M
This is a method in which the capture site is captured by an immune reaction with a PB64 protein antibody, and the capture site is colored.

For the preparation of a test strip for the chromatography method, for example, the method described in JP-B-7-13460 is suitably applied. In the method for detecting Mycobacterium tuberculosis of the present invention using a chromatographic test strip, a test mixture obtained by mixing a test sample or a test sample obtained from the test sample and a color-labeled anti-MPB64 protein antibody in advance is chromatographed by a chromatographic test strip. Alternatively, a sample addition member and a color-labeled antibody-impregnated member may be provided in advance on a chromatographic test strip, and a predetermined amount of the subject or a test sample obtained from the subject may be irrigated with the sample addition member. Alternatively, the above-described method of performing chromatographic development may be used.

In order to perform the method for detecting Mycobacterium tuberculosis of the present invention using the above-described chromatographic test strip, at least
A kit can be made by combining a chromatographic test strip or a chromatographic test strip lacking a colored labeled antibody impregnated member and a colored labeled anti-MPB64 protein antibody contained in a container.

This kit can further include a relational table between the protein concentration and the degree of coloration, which is prepared in advance as the criterion. In the method for detecting Mycobacterium tuberculosis of the present invention, an immune reaction between MPT64 protein or MPB64 protein and a color-labeled anti-MPB64 protein antibody, and a color-conjugated product produced by this immune reaction and a second anti-MPB64
Since the immune reaction with the protein antibody is completed within a few minutes, the test itself is completed in about 5 minutes at the longest.

In the method for detecting Mycobacterium tuberculosis of the present invention, as in the other methods for detecting Mycobacterium tuberculosis, sufficient care must be taken to prevent Mycobacterium tuberculosis infection when treating the subject. However, in the detection method of the present invention, only a small amount of a test sample obtained from a subject or a test sample which may contain M. tuberculosis is added to a chromatographic test strip, and Since the time required for itself is at most about 15 minutes, the risk of infection with M. tuberculosis
The test operation is complicated and the number of tests is small compared to the conventional method for detecting Mycobacterium tuberculosis, which requires a long time for the test.

[0030]

EXAMPLES The present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

1. Isolation of MPB64 protein (1) Culture of BCG bacterium Tokyo strain The BCG bacterium Tokyo strain was obtained from the Tuberculosis Research Institute, Japan Tuberculosis Prevention Society, and inoculated into 20 x 500 ml Sorton's medium having the following composition and incubated at 37 ° C for 5 weeks. After culturing, 10 l of a culture solution was obtained.

Composition of Sorton's medium Asparagine 0.4% Citric acid 0.2% Sodium citrate 0.28% Potassium phosphate 0.05% Magnesium sulfate 0.05% Ferrous ammonium citrate 0.005% Glycerin 6 % (“%” Indicates “weight / volume%”; the same applies hereinafter)

(2) Separation and Purification of MPB64 Protein Ammonium sulfate (hereinafter referred to as ammonium sulfate) was added to the culture solution obtained in the above (1) to form an 80% saturated solution, and the precipitated protein fraction was centrifuged. The obtained protein fraction was dissolved in 30 mM Tris-HCl buffer (pH 8.7) and the solution was dissolved at 10 ° C. in 30 mM Tris-HCl buffer (pH
8.7) was dialyzed to obtain about 30 ml of a dialysate containing about 9 g of protein. About 30 ml of the above solution containing about 9 g of this protein was added to D
EAE Sephadex A.50 (a product of Pharmacia Biotech Co., Ltd.) was charged into a column (57 cm × 5.5 cm), and a 30 mM Tris-HCl buffer (pH 8.0) was added.
7) The above column was washed with 100 ml to obtain an eluate.

Next, 30 mM Tris-HCl buffer / 200 mM
Chromatographic development was performed with a sodium chloride solution (pH 8.7). Ammonium sulfate was added to the eluate of this chromatographic development to obtain a 70% saturated solution, and the precipitated protein fraction was obtained by centrifugation. This protein fraction was dissolved in 30 mM Tris-HCl buffer (pH 8.7) and dialyzed at 10 ° C. against 30 mM Tris-HCl buffer (pH 8.7) to obtain about 3.5 proteins.
About 35 ml of dialysate containing g were obtained.

About 35 ml of the above solution containing about 3.5 g of this protein was applied to a DEAE Sepharose CL-6B (a product of Pharmacia Biotech) column (64 cm).
× 3.6 cm) and the column was washed with about 700 ml of 30 mM Tris-HCl buffer (pH 8.7). Then, 30 mM Tris-HCl buffer (pH 8.7)
Then, using a 30 mM Tris-HCl buffer / 200 mM sodium chloride solution (pH 8.7), the solution was chromatographed by a concentration gradient method from a sodium chloride concentration of 0 to 200 mM, and the developed solution was collected as a 20 ml fraction. The developing solutions of fraction numbers 105 to 121 were collected. Ammonium sulfate was added to this developing solution to obtain a 70% saturated solution, and the precipitated protein fraction was obtained by centrifugation. This protein fraction was dissolved in 30 mM Tris-HCl buffer (pH 8.7), and the solution was dissolved at 10 ° C. in 30 mM Tris-HCl buffer (pH 8.7).
8.7) was dialyzed to obtain about 7 ml of a dialysate containing about 0.7 g of protein.

Approximately 360 ml of this developing solution was used to prepare a fractionated molecular weight of 1
Concentrate to about 6 ml at 5 ° C. by suction ultrafiltration using an ultrafiltration membrane with a pore size of 000, and then at 10 ° C. 30 mM Tris-HCl buffer (pH
8.7) was dialyzed to obtain about 7 ml of dialysate. About 7 ml of this solution is freeze-dried by a conventional method to obtain about 0.
A freeze-dried product containing 7 g was obtained.

A freeze-dried product containing 0.7 g of this protein was dissolved in 7 ml of purified water, and the whole amount was charged into a column (45 cm × 3.0 cm) of DEAE Sepharose CL-6B (a product of Pharmacia Biotech Co., Ltd.). 30 mM Tris-HCl buffer, 3 M urea, 40 mM sodium chloride solution (pH 7.5) and 30 mM Tris-HCl buffer, 3 mM
Chromatographic development was carried out by using a concentration gradient method from 40 mM to 90 mM sodium chloride concentration using a 90 mM sodium urea / 90 mM sodium chloride solution (pH 7.5), and 5 ml of the developing solution was collected as a fraction.

After spotting a small amount of each fraction on a solid support of polyacrylamide gel and performing electrophoresis, a band of a protein having a molecular weight of about 23,000 was recognized in an electrophoresis image obtained by Coomassie brilliant blue staining. Each fraction was collected. This solution was concentrated to about 2 ml at 5 ° C. by suction ultrafiltration using an ultrafiltration membrane having a pore size of 10,000 and a 30 mM Tris-HCl buffer (pH 8). .7) and dialysate about 2.
5 ml were obtained. About 2.5 ml of this solution was freeze-dried by a conventional method to obtain a freeze-dried product containing about 60 mg of protein.

A freeze-dried product containing about 60 mg of this protein was dissolved in 2 ml of purified water, and the whole amount was charged into a DEAE Sepharose CL-6B (a product of Pharmacia Biotech) column (34 cm × 2.7 cm). Next, the column was washed with a 30 mM Tris-HCl buffer / 60 mM sodium chloride solution (pH 8.7).

Next, using a 30 mM Tris-HCl buffer / 60 mM sodium chloride solution (pH 8.7) and a 30 mM Tris-HCl buffer / 110 mM sodium chloride solution (pH 8.7), a concentration gradient method using a sodium chloride concentration of 60 mM to 110 mM. , And 1 ml of the developing solution was collected as a fraction. After spotting a small amount of each fraction on a solid support of polyacrylamide gel and performing electrophoresis, the electrophoresis image obtained by Coomassie brilliant blue staining showed a molecular weight of about 23,
Fractions in which 000 protein bands were observed were collected.

This solution was concentrated to about 1 ml at 5 ° C. by suction ultrafiltration using an ultrafiltration membrane having a pore size of 10,000 and a molecular weight cutoff of 10,000. Then 1
Dialysis was performed at 0 ° C. against 30 mM Tris-HCl buffer (pH 8.7) to obtain about 1.3 ml of dialysate. About 1.3 ml of this solution was freeze-dried by a conventional method to obtain a freeze-dried product containing about 6 mg of protein.

Finally, the freeze-dried product containing about 6 mg of this protein was dissolved in 1 ml of purified water, and the whole amount was charged into a Sephacryl S-200 (a product of Pharmacia Biotech Co., Ltd.) column (30 cm × 2 cm). The solution was chromatographed using a 30 mM Tris-HCl buffer / 0.5 mM sodium chloride solution (pH 7.5), and the developed solution was collected as a 0.5 ml fraction. After spotting a small amount of each fraction on a solid support of polyacrylamide gel and performing electrophoresis, each fraction in which a protein band having a molecular weight of about 23,000 was recognized in an electrophoresis image obtained by Coomassie brilliant blue staining Collected. This solution was concentrated to about 1 ml at 5 ° C. by suction ultrafiltration using an ultrafiltration membrane having a pore size of 10,000 and a 30 mM Tris-HCl buffer (pH 10).
8.7) was dialyzed to obtain about 1.3 ml of a dialysate. About 1.3 ml of this solution was freeze-dried by a conventional method to obtain a freeze-dried product containing about 5 mg of protein. The thus obtained MPB64 protein having a molecular weight of about 23,000 showed a single peak electrophoretic image in desk electrophoresis (7.7% acrylamide gel), and thus was judged to be of high purity. Was.

2. Preparation of Anti-MPB64 Protein Monoclonal Antibody (Mouse) (1) Cell fusion between spleen cells of MPB64 protein-administered mouse (BALB / C system) and mouse myeloma cells (P3U1) Using the purified MPB64 protein obtained by the above method, the method of Keller-Milstein (G. Kohker and C. Milstein) was used.
n; Eur. J. Immunol. 6 : 511-519, 1976) to perform cell fusion to prepare a hybridoma.

That is, the above 1. Purified MPB obtained in
500 μl of a 64 protein solution (200 μg / ml) and 500 μl of Freund's incomplete adjuvant are mixed by a conventional method until emulsification, and 500 μl of this mixed emulsion is mixed with BALB / C
Immunization was performed by intraperitoneal administration of strain mice (female, 4 weeks old). Two weeks after the previous administration, 40 μl of the mixed emulsion prepared in the same manner as above was again administered to the above-mentioned mouse intraperitoneal cavity. Two weeks later, purified MPB64
A final immunization was performed by intraperitoneally administering a saline solution of the protein (200 μg / ml) to the above mice.

Three days after the final immunization, the mice were sacrificed and their spleens were immediately aseptically removed. The spleen was placed in a Petri dish having a diameter of 60 mm containing about 10 ml of a sterile RPMI-1640 medium, cut along scissors along the long axis of the spleen, and cells were released while pressing the spleen with forceps quickly. The cells were passed through a nylon mesh and suspended in fresh sterile RPMI-1640 medium to obtain a spleen cell suspension.

The above spleen cell suspension was subjected to RPMI-16.
The spleen cells obtained by mixing with 40 medium and removing erythrocytes by specific gravity centrifugation using Ficoll Pake were suspended in sterile RPMI-1640 serum-free medium.

On the other hand, mouse myeloma cells P3U1 (hereinafter, referred to as P3U1 cells) were expressed in 10% by volume of fetal bovine serum.
After the cells were cultured and grown in a sterile RPMI-1640 medium containing, the P3U1 cells were separated by centrifugation (250 × G), and the cells were washed twice with a sterile RPMI-1640 medium to obtain a sterile RPMI-1640 medium. The P3U1 cell suspension was suspended in 1640 medium.

The above spleen cell suspension and the above P3
The U1 cell suspension was mixed and both cells were separated from this mixture by centrifugation (250 × G). The separated cells are combined with a sterile RPMI-164 containing 40% of polyethylene glycol having a molecular weight of 4,000.
Suspension cells were suspended in 1 ml of medium 0 (pH 7.4) and allowed to stand at 37 ° C. for 6 minutes to fuse spleen cells and P3U1 cells to obtain hybridomas.

Next, sterile RPMI-1 was added to these cells.
640 medium was added to make a total volume of 10 ml, and the cell suspension was separated by centrifugation (250 × G).
To these cells, 10 ml of a sterile RPMI-1640 medium containing 10% by volume of fetal bovine serum was added to suspend the cells. This cell suspension is centrifuged (2
50 × G), and the liquid was removed to obtain cells.

HAT medium was added to the cells and 1 × 10
^As a cell suspension of ⁶ cells / ml, 100 μl of this cell suspension
Each well was dispensed into each well of a 96-well (well) plastic microtiter plate (a product of Sumitomo Beclite Co., Ltd.), and carbon dioxide was 5% by volume in the incubator.
The cells were cultured at 37 ° C. in the presence of the contained air.

On the first day after the start of the culture, 100 μl of HAT medium was further added to each well.
On each of day 8, day 11, and day 11, 100 μl of the culture solution was removed, and the culture was continued while supplementing 100 μl of HAT medium instead. 14 after culture
On the day, an average of 2-3 hybridoma colonies per well was found in the wells where the hybridomas were present. In these wells, the hybridomas proliferated sufficiently about 2 weeks after the start of the culture. At this time, the antibody titer of the culture supernatant was determined by enzyme-linked immunosorbent assay (ELISA).
Method).

In the enzyme-linked immunosorbent assay, 50 μl of the culture in which these hybridomas were cultured was added to wells of a plastic microtiter plate for ELISA in which the MPB64 protein as an antigen had been solid-phased in advance. For 1 hour, and after removing this culture solution,
Wash with saline containing 0.05% Tween20.
Then, 50 μl of a peroxidase-labeled goat anti-mouse immunoglobulin solution was added thereto, and the mixture was allowed to stand at 37 ° C. for 1 hour to carry out immunoreaction. The peroxidase-labeled goat anti-mouse immunoglobulin solution was removed, and 0.05% Tween 20 was removed.
Wash with saline solution. In addition, 4-
A normal peroxidase color developing reagent consisting of aminoantipyrine, hydrogen peroxide and phenol was added to develop color, and the antibody titer was determined according to a conventional method. on the other hand,
For the hybridomas in the wells producing the antibody, the cloning operation by the limiting dilution method was repeated three times. As a result, 5 cloned cell lines stably producing anti-MPB64 protein antibodies were obtained.

(2) Purification of monoclonal antibody The BALB / C mouse, which had been intraperitoneally injected with 0.5 ml of 2,6,10,14-tetramethylpentanedecane per mouse one week before, Of anti-MP among cloned cell lines
0.5 ml of the cloned cell suspension (10 ⁷ cells / ml), which produced a particularly large amount of B64 protein antibody, was injected. One week later, ascites containing the anti-MPB64 protein antibody was added to the mouse at a rate of 5 ml / mouse.
〜１０10 ml were obtained.

This ascites was added to 50 mM Tris-HCl buffer · 15
The column was charged with a Protein A (a product of Pharmacia Biotech) equilibrated with a 0 mM sodium chloride solution (pH 8.5). Next, the column was washed with 50 mM Tris-HCl buffer / 150 mM sodium chloride solution (pH 8.5), and then washed with 100 mM citrate buffer / 150 mM.
The above-mentioned anti-MPB6 with sodium chloride solution (pH 5.0)
The four protein antibodies were eluted.

The anti-MPB64 protein antibody eluate was used for 10
After dialyzing against 20 mM phosphate buffer and 150 mM sodium chloride solution (pH 7.4) at 20 ° C., sodium azide (N) was used as a preservative so that the final concentration was 0.1%.
aN ₃ ) was added.

3. Preparation of colloidal gold-labeled antibody and blue latex-labeled antibody (1) Preparation of gold sol Boil 99 ml of ultrapure water, and add chloroauric acid (a product of Katayama Chemical Industry Co., Ltd.) %)
One ml was added, and one minute later, 1.5 ml of a sodium citrate solution (concentration of sodium citrate: 1%) was added. The mixture was boiled for 5 minutes, and then left to cool at room temperature. Then, the solution was adjusted to pH 9.0 with a 200 mM potassium carbonate solution.
, And ultrapure water was added to make a total volume of 100 ml to obtain a gold sol.

(2) Preparation of colloidal gold-labeled antibody 1 μg in terms of protein of the anti-MPB64 protein mouse monoclonal antibody obtained in the above (hereinafter, when expressing the protein equivalent weight of the antibody, only the numerical value of the weight is shown) and 1 ml of the above gold sol are mixed, and the mixture is mixed at room temperature. After standing for 2 minutes, all of this antibody was bound to colloidal particles, which are dispersed particles of gold sol. Add 1% so that the final concentration is 1%.
An aqueous solution of 0% bovine serum albumin (hereinafter, referred to as BSA) was added to block the surface of the colloidal gold particles bound to the antibody. Centrifuge this solution (5,6
(00 × G, 30 minutes) to collect the colloidal gold-labeled antibody in which the surface of the colloidal particles bound with the anti-MPB64 protein mouse monoclonal antibody was blocked with BSA. This gold colloid-labeled antibody was added to 10% saccharose
The suspension was resuspended in 50 mM Tris-HCl buffer (pH 7.4) containing 1% BSA and 0.5% Triton-X10 to obtain a purified gold colloid-labeled antibody solution.

(3) Preparation of blue latex-labeled antibody 0.25 mg of the anti-MPB64 protein mouse monoclonal antibody obtained in the above and 10% blue latex (particle size 0.2 μm)
m, a product of Nippon Paint Co., Ltd.) 1 ml of a 1% blue latex solution obtained by diluting the solution with purified water
After allowing to stand at room temperature for 30 minutes to bind the anti-MPB64 protein mouse monoclonal antibody to the blue latex, centrifugation (5,000 × G, 30 minutes) was performed to bind the anti-MPB64 protein mouse monoclonal to the blue latex. The crude blue latex-labeled antibody, which was a clonal antibody, was precipitated and collected. This crude blue latex-labeled antibody was redispersed in 1 ml of 10 mM phosphate buffer (pH 7.4) containing 1% BSA solution and 0.85% sodium chloride solution, and centrifuged (5,000 × G, 30 minutes). ) Was repeated three times. The thus treated blue latex-labeled antibody was treated with a 10 mM phosphate buffer (pH 7.4) containing 1% BSA and 0.85% sodium chloride solution.
0 ml to obtain a purified blue latex-labeled antibody solution having a solid content of about 0.05%.

4. Preparation of chromatographic test strip (1) A chromatographic test strip was prepared according to the description of Japanese Patent Publication No. Hei 7-186405. That is, width 5
An elongated strip-shaped nitrocellulose membrane (membrane filter) having a length of 36 mm and a length of 36 mm was used as a membrane carrier for chromatographic development of a chromatographic medium. The second anti-MPB64 protein was placed 7.5 mm from the end in the direction opposite to the starting point of the chromatographic exhibition. Antibody 3.
0.5 μl of an antibody solution containing 0 mg / ml was applied in the form of a spot, and dried at room temperature to obtain a supplemented site.
In the immunological reaction with the second anti-MPB64 protein antibody, the binding site for the antigen MPT64 protein or MPB64 protein is as described in 3. above. Refers to an antibody having a binding specificity different from that used in the preparation of a colloidal gold-labeled antibody or a blue latex-labeled antibody.

(2) A 5 mm × 15 mm strip of glass fiber non-woven fabric was impregnated with 37.5 μl of the above-mentioned gold colloid-labeled antibody solution, and dried at room temperature to obtain a gold colloid-labeled antibody-impregnated member. (3) Further, 37.5 μl of the above blue gold latex-labeled antibody solution was impregnated into a 5 mm × 15 mm strip-shaped glass fiber nonwoven fabric, and dried at room temperature to obtain a blue latex-labeled antibody-impregnated member.

(4) Next, a cotton cloth serving as a sample addition member, the above-described member impregnated with a gold colloid-labeled antibody or a blue latex-labeled antibody, a nitrocellulose membrane serving as a membrane carrier for chromatography, and a filter paper serving as an absorption member Were adhered to predetermined positions on the adhesive surface of the adhesive sheet to prepare a chromatographic test strip. That is, the chromatographic development membrane carrier is stuck on the adhesive surface of the pressure-sensitive adhesive sheet, and at the end opposite to the starting point of the chromatographic development of the chromatographic development membrane carrier,
The above-described colloidal gold-labeled antibody solution or blue latex-labeled antibody solution is impregnated and dried, and the labeled antibody-impregnated member is arranged such that the end opposite to the starting point of the chromatographic development is overlapped and arranged. The sample addition member was arranged so as to cover the entire portion from the portion or the overlapping portion of the blue latex-labeled antibody-impregnated portion. Further, on the other end of the nitrocellulose membrane, a part of the absorbing member was overlapped and arranged to form a chromatographic strip.

[0062] 5. Detection of Mycobacterium tuberculosis from a subject (1) Preparation of criteria Criteria of purified MPB64 protein is diluted with physiological saline, and MPB6
The concentrations of the four proteins were 0.25 ng / ml, 0.5 ng / ml,
1.0 ng / ml, 2.0 ng / ml, 4.0 ng / ml, 8.0 ng / ml
And a solution consisting of 16.0 ng / ml.
The B64 protein solution was used as a standard sample. Using these standard samples, an assay using the above-described chromatographic test strip and an assay using the RPHA method were performed, and the results obtained for both methods were compared for the detection sensitivity of both methods with respect to the MPB64 protein concentration.

The antibody-sensitized blood cells were analyzed by the method described in JP-A-7-110332.
Prepared according to the method described in Japanese Patent Application Publication No. That is, 1 volume part of a 0.5% 10 mM phosphate buffer suspension of glutaraldehyde-fixed sheep erythrocytes and 1 mg of tannin (US N
BC) was dissolved in 100 ml of physiological saline, mixed with 1 part by volume, left at room temperature for 15 minutes, tannin-treated erythrocytes were centrifuged, and tannin-treated erythrocytes were washed three times with physiological saline. The suspension was suspended in physiological saline so as to be 1 part by volume to obtain a 0.5% tannin erythrocyte physiological saline solution.

An antibody obtained by immunizing a goat according to a conventional method was purified by affinity column chromatography, and a purified anti-MPB64 protein polyclonal antibody solution (2 μg / m 2)
l) One part by volume and 1 part by volume of the above 0.5% tannin erythrocyte physiological saline solution were mixed, left at 37 ° C. for 30 minutes, and then this liquid was centrifuged (1,500 × G, 10 minutes). To separate antibody-sensitized red blood cells. This antibody-sensitized red blood cell 10
After washing three times with mM phosphate buffer, suspend in 0.5 mM phosphate buffer containing 0.1% BSA and add 0.5%
An antibody-sensitized red blood cell saline solution was prepared.

In a chromatographic test strip using a gold colloid-labeled antibody-impregnated member, 100 μl of a standard sample at each concentration was developed by chromatography, and after 15 minutes, the presence or absence of red-purple coloring at the supplemented site in the test strip was visually observed. Judgment, uncolored is defined as-, and coloring is represented by the density from ± (false positive) to +++ ((dark colored),
±, +, ++, +++ were classified into four stages and used as criteria. Table 1 shows the relationship between the visual criterion of the chromatographic test strip and the protein concentration of the standard sample.
Needless to say, the presence / absence of red-purple coloring and the shade of coloring at the capture site indicate the presence / absence of tuberculosis bacteria and the approximate number of tuberculosis bacteria in the subject.

When the same operation as above was performed using the blue latex-labeled antibody-impregnated portion, the same result as above was obtained.

On the other hand, in the RPHA method, 5 μl of a standard sample of each concentration was added to each well of a 96-well plastic microtiter plate (a product of Sumitomo Bakelite Co., Ltd.), and then the antibody-sensitized erythrocyte physiology described above was added to each well. 25 μl of a saline solution was added dropwise at room temperature.
The mixture was allowed to stand for 0 minutes to generate an immune reaction, and the degree of hemagglutination in each well after the reaction was visually determined.
It was divided into five stages of-, ±, +, ++, +++ from (no aggregation) to ++ (very good aggregation). This. R
The results of the macroscopic determination in the PHA method were compared with the determination criteria based on the purified MPB64 protein concentration of the standard sample on the above-described chromatographic test strip, and the results are shown in Table 1. As shown in Table 1, the method for detecting M. tuberculosis of the present invention is RPHA
It can be seen that the sensitivity is higher than that of the method and that it is semi-quantitative.

[0068]

[Table 1]

(2) Example 1 for Detection of Mycobacterium tuberculosis from a Subject Human sputum was used as a tuberculosis subject (eight cases), and the detection of M. tuberculosis from the subject was examined. That is, the subject 5 ml
And 5 ml of a 2% aqueous sodium hydroxide solution. The mixture is allowed to stand at room temperature for 30 minutes, and then centrifuged (1,
The precipitate formed by collecting the precipitate was collected by mixing the precipitate with physiological saline, and the mixture was centrifuged (1,500 × G, 30 minutes) to remove the precipitate again. Collect, mix this sediment with 200 μl of saline,
This was used as a test sample.

100 μl of this test sample was placed in a sterile test tube, and 5 ml of Dubos liquid medium (a product of Becton Dickinson Co., Ltd.) was added to the test tube, and cultured at 37 ° C. A tuberculosis detection method using the chromatographic test strip of the present invention and a tuberculosis bacterium was detected by the RPHA method. In addition, about the culture solution on the 7th day from the start of the culture, M. tuberculosis was detected only by the RPHA method.

As a control, 5 sputum samples of each of the 8 subjects described above were used.
and 5 ml of a 2% aqueous sodium hydroxide solution,
After allowing this mixture to stand at room temperature for 30 minutes, the solution 10
0 μl is uniformly inoculated on the surface of a test tube Kudo PD solid slant medium (a product of Kyowa Pharmaceutical Co., Ltd.), and 5 μl at 37 ° C.
A niacin production test was performed on the Mycobacterium tuberculosis grown and cultured for 6 days.

That is, 1 ml of boiling distilled water was added to the surface of the above-mentioned test tube Kudo PD solid slant medium, the surface was made substantially horizontal, and the mixture was allowed to stand for 5 minutes to extract niacin from Mycobacterium tuberculosis cells. 200 μl was collected from this extract and mixed with 100 μl of a 4% by volume aniline / ethanol solution and a 10% by volume bromocyan solution. Five minutes after mixing, if niacin derived from Mycobacterium tuberculosis is present in the mixture, the mixture develops a yellow color. If the mixture develops a yellow color as determined with the naked eye, the subject is considered positive (+ ). Table 2
Shows the test results.

[0073]

[Table 2]

From Table 2, it can be seen that in the niacin production test, culturing in the test tube Kudo PD solid slant medium requires a long period of 56 days, whereas, in the detection method of the present invention, culturing in the Dubos liquid medium requires 3 days. In a short period of time, the detection method of the present invention has a much higher sensitivity than the RPHA method.

(3) Example 2 for Detection of Mycobacterium tuberculosis from the Subject The sputum of each of eight patients with suspected tuberculosis by X-ray diagnosis was examined for M. tuberculosis.
That is, each of the above sputum was mixed with an equal amount of a 2% aqueous sodium hydroxide solution, and the mixture was allowed to stand at room temperature for 30 minutes to obtain a culture sample.

In a test tube, Kudo P
D: Inoculate uniformly on the surface of the solid slant medium, culture at 37 ° C., and on the 7th, 14th, 21st, 28th, and 56th days from the start of culture, test tube Kudo PD solid slant medium 500 μl of distilled water was added to the surface of each sample and shaken, and the liquid in the test tube was used as a cell extract sample. This cell extract sample 100
μl was charged to a chromatographic test strip and developed by chromatography, and the presence or absence of red-purple coloration at the capture site after 15 minutes and the density of the color were visually observed, and judged according to the above criteria. For comparison, a niacin production test was carried out on each of the above-mentioned samples in the same manner as described above for M. tuberculosis grown and cultured in the same manner as above except that the culture period was changed to 56 days. Table 3 shows the results.

[0077]

[Table 3]

In Table 3, (-) and (+) indicate that "no colonies" and "formed", respectively, on the surface of the above-mentioned test tube Kudo PD solid slant medium.

Table 3 shows that when the test tube Kudo PD solid slant medium was used for culturing the sample, it took 56 days for the niacin production test to reveal the test results, whereas the nyasin production test required a long time. The method shows that the presence of Mycobacterium tuberculosis in a subject can be determined at the 28th day of culture, which is half the number of days of the niacin production test, and that the method is extremely sensitive.

Next, typical examples of the chromatographic test strip suitably used in the method for detecting Mycobacterium tuberculosis of the present invention will be described more specifically with reference to the drawings. That is, FIG.
Shows a chromatographic test strip, a is a plan view, b
FIG. 2 is an end view of a vertical section of a chromatographic test strip indicated by a. FIG. 2 shows a case for accommodating the chromatographic test strip shown in FIG.
Is a plan view, and b is a longitudinal section end view of the case indicated by a. The drawings are only for illustrating the principle, and dimensions and the like are not accurately shown.

This chromatographic test strip is applied to the adhesive surface of the adhesive sheet 1 sequentially from the upstream side (hereinafter, the left side in the drawing is referred to as the upstream side, and the right side in the drawing is referred to as the downstream side). A member 5, a colored labeled antibody-impregnated member 2, a chromatographic developing membrane carrier 3, and an absorbing member 4 are adhered. Thus, the color-labeled antibody-impregnated member 2 has a color-labeled anti-MPB64 protein antibody solution all over its surface,
The second anti-MPB64 protein antibody solution is impregnated into the chromatographic membrane carrier 3 in the form of spots (the portions where the second anti-MPB64 protein is impregnated in the spot form on the chromatographic membrane carrier 3 are described below). This is referred to as a capturing unit 31). The sample addition member 5 is laminated on the color-labeled antibody-impregnated member 2. The chromatographic membrane carrier
The upstream end 3 is covered with the above-mentioned colored antibody-impregnated member 2, and is connected to the colored antibody-impregnated member 2.

The absorption member 4 has its upstream end laminated on the downstream end of the chromatographic development membrane carrier 3. These adhesive sheets 1, color-labeled antibody-impregnated members 2,
Each of the chromatographic development membrane carrier 3, the absorption member 4, and the sample addition member 5 is formed in an elongated rectangular band shape.
The chromatographic development membrane carrier 3 automatically moves the specimen and other various samples charged in the form of a spot on the sample addition member 5 to the downstream side in the chromatographic development direction by its own capillary action during the chromatographic development. What is necessary is just to have the material which can be obtained.

Thus, the fiber-labeled nonwoven fabric as the colored-labeled antibody-impregnated member 2, the nitrocellulose membrane as the chromatographic developing membrane carrier 3, the filter paper as the absorbing member 4, and the material as the sample-adding member 5 are not limited. Most preferred, however, are sheets or films of porous synthetic resins such as porous polyethylene and porous polypropylene, and cellulose paper or woven or non-woven fabrics such as filter paper and cotton cloth.

Chromatographic development membrane carrier 3 having a capture part 31
It is preferable to use a color combination of which the color of the colored combination supplemented by the capturing unit 31 is clearly visible. For example, when the color-marking substance is colloidal gold particles, the colloidal gold particles exhibit a purple-red color, and the color-conjugated substance also exhibits a purple-red color. Preferably, white is particularly preferred.

The above-mentioned chromatographic test strip is housed in a case 6. Case 6 is usually made of synthetic resin. The case 6 includes a container body 61 and a lid 62. A sample injection port 621 and a judgment hole 622 are formed on the upper surface of the lid 62 at positions corresponding to the sample addition member 5 and the capture section 31 of the chromatographic development membrane carrier 3 of the chromatographic test strip, respectively. Has been established.

Samples such as a subject, a culture of the subject, and various extracts from these are irrigated into the sample addition member 5 from a sample inlet 621 of the lid 62. The irrigated sample infiltrates the sample addition member 5 toward the downstream side, and at the same time, is transferred to the color-labeled antibody-impregnated member 2 below and developed toward the downstream side of the color-labeled antibody-impregnated member 2. Can be MPT produced by M. tuberculosis in various samples
64 protein or MPB64 protein, the MPT64 or MPB64 protein and the color-labeled anti-MPB64 protein immunoreaction during this time.
T64 protein or MPB64 protein and color-labeled anti-MPB6
A color conjugate with the 4-protein antibody is produced.

The sample is further developed with the chromatographic development membrane carrier 3 toward the downstream side. The color-coupled product is captured by the capture section 31 of the chromatographic development membrane carrier 3 during the development and is accumulated and colored. This color is determined by the judgment hole 622.
Is observed with the naked eye.

[0088]

According to the present invention, the presence or absence of M. tuberculosis can be determined with a simple method, in a short time, safely and with high accuracy, and the number of M. tuberculosis bacteria present in a sample can be determined. Can also be semi-quantified.

[Brief description of the drawings]

1 shows a chromatographic test strip, wherein a is a plan view and b is a longitudinal section end view of the chromatographic test strip indicated by a.

2 shows a case for accommodating the chromatographic test strip shown in FIG. 1, wherein a is a plan view and b is a longitudinal section end view of the case shown by a.

[Explanation of symbols]

 1 Adhesive sheet 2 Color-labeled antibody-impregnated member 3 Chromatographic membrane carrier 31 Capture unit 4 Absorption member 5 Sample addition member 6 Case 61 Container body 62 Lid 621 Sample inlet 622 Judgment hole

Claims (9)

[Claims] 1. A test sample is cultured on a solid medium and grown, and the MPT64 protein mixed with M. tuberculosis or the MPB64 protein mixed with M. bovis is combined with a color-labeled anti-MPB64 protein antibody by immunoreaction. A method for detecting Mycobacterium tuberculosis, comprising capturing the conjugate with a second anti-MPB64 protein antibody. 2. A culture obtained by culturing a subject and a color-labeled anti-MPB64 protein antibody are mixed, and the mixed solution is then brought into contact with a second anti-MPB64 protein antibody.
T64 protein or MTB64 protein and color-labeled anti-MPB6
The second anti-MPB against these conjugates with the 4 protein antibody
A method for detecting Mycobacterium tuberculosis, comprising determining the presence or absence of Mycobacterium tuberculosis or Mycobacterium bovis based on the presence or absence of coloration caused by capture with 64 antibody. 3. The method for detecting Mycobacterium tuberculosis according to claim 1, wherein in the color-labeled anti-MPB64 protein antibody, the color-labeling substance bound to the anti-MPB64 protein antibody is a colloidal metal. 4. The method for detecting Mycobacterium tuberculosis according to claim 1, wherein in the color-labeled anti-MPB64 protein antibody, the color-labeling substance bound to the anti-MPB64 protein antibody is a colored latex. 5. The method for detecting Mycobacterium tuberculosis according to claim 1, wherein the method is performed using a test strip for chromatography. 6. A tuberculosis comprising at least a chromatographic test strip or a chromatographic test strip lacking a color-marked antibody member housed in a case and a color-labeled anti-MPB64 protein antibody housed in a container. Bacteria detection kit. 7. At least a sample addition member, a color-labeled antibody-impregnated member, a chromatographic development membrane carrier and an absorption member, and the color-labeled antibody-impregnated member and the chromatographic development membrane carrier are provided with a color-labeled antibody. MPB64 protein antibody and second anti-M
Each of the PB64 protein antibodies is impregnated, the sample addition member is laminated on the color-marked antibody-impregnated member, and the chromatographic development membrane carrier has its upstream end coated with the color-marked antibody-impregnated member. And the absorption member is connected to the member for impregnating the color-labeled antibody, and the absorption member has its upstream end laminated to the membrane carrier for chromatography development and connected to the membrane carrier for chromatography development. The kit for detecting Mycobacterium tuberculosis according to claim 6, wherein the test strip is contained in a case. 8. The kit for detecting Mycobacterium tuberculosis according to claim 6, wherein in the color-labeled anti-MPB64 protein antibody, the color-labeling substance that is bound to the anti-MPB64 protein antibody is a colloidal metal. 9. The kit for detecting Mycobacterium tuberculosis according to claim 6, wherein in the color-labeled anti-MPB64 protein antibody, the color-labeling substance to be bound to the anti-MPB64 protein antibody is a colored latex.