JPH07110332A - Inspection reagent and detection method for tubercle bacillus - Google Patents

Abstract

PURPOSE:To make it possible to detect tubercle bacillus quickly by incubating a specimen in a liquid culture medium to detect MPB64 secreted into a culture liquid immunologically. CONSTITUTION:Colibacillus stock containing fused protein manifestation vector pMAL64c is incubated, the culture bacillus body is crushed by an ultrasonic wave and the top of the crushed liquid is taken centrifugally to prepare MPB64 purely from the supernatant fraction. Antiserum can be obtained easily by immunizing an animal able to produce antibodies, for example, guinea pigs, rabbits and sheep with the MPB64. To detect tubercle bacillus using this anti- MPB64 antiserum, the tubercle bacillus in a specimen is incubated in a liquid culture medium. For example, after one week, the MPB64 secreted into the culture liquid is detected immunologically. As for the detection method for the purpose, reversed passive blood cell agglutination reaction and reversed passive latex agglutination reaction are ideal.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a reagent for specifically detecting Mycobacterium tuberculosis by immunologically detecting MPT64. More specifically, a tubercle bacillus test reagent and a method for culturing M. tuberculosis in a sample in a liquid medium and immunologically detecting MPT64 produced in the culture medium by an anti-MPB64 antibody against MPB64 which is immunologically identical Regarding the method.

[0002]

2. Description of the Related Art Mycobacterium tuberculosis is mycobacterium tuberculosis.
It is well known that this is an infectious disease of the respiratory tract caused by the infection of uberculosis). Pulmonary tuberculosis, which makes up the majority of tuberculosis, is often difficult to distinguish clinically from lung cancer, pulmonary mycosis, paragonimiasis, etc., and the definitive diagnosis is solely based on the method of detecting Mycobacterium tuberculosis by culturing specimens. There is. However, the culture of Mycobacterium tuberculosis is usually performed using Ogawa's medium.
~ 8 weeks are required, and the identification of the detected bacteria is essential.

[0003]

As described above, it takes a long time to detect Mycobacterium tuberculosis by the conventional method.
Not only is it a major obstacle to the definite diagnosis and early treatment of tuberculosis, but it also poses a serious public health problem, such as infection of people around by the bacillus. Further, no method for immunologically detecting Mycobacterium tuberculosis has been known so far. The present invention provides a test reagent and a detection method that can detect Mycobacterium tuberculosis immunologically specifically and rapidly.

[0004]

In order to solve the above problems, the present inventors have found that MPB, a protein produced only by Mycobacterium tuberculosis,
Focusing on 64, by culturing a specimen such as sputum in a liquid medium and immunologically detecting MPB64 secreted in the culture medium, a reagent for detecting Mycobacterium tuberculosis rapidly and reliably was successfully developed, The present invention has been completed. Furthermore, this specific detection method can be applied to the identification of cultured unknown isolates and rapid drug susceptibility testing.

The present invention is a tubercle bacillus test reagent for immunologically detecting tubercle bacillus comprising an anti-MPB64 antibody, and a method for detecting tubercle bacillus by an immunoassay using the reagent. MPB64 was isolated and purified from the protein produced by BCG bacteria by M. Harboe et al. In 1986, and the amino acid sequence near its N-terminus has been reported (Infection and Immun
ity vol.52, 293-302, 1986). Mycobacterium tuberculosis secretes several hundred kinds of proteins into the culture medium, but MPT64 is a species-specific protein produced only in Mycobacterium tuberculosis and MPB64 is produced in BCG bacterium, and both are immunologically identical.

It takes a great deal of labor and time to purely separate MPB64 from several hundred kinds of proteins in the culture solution of Mycobacterium tuberculosis, and the yield thereof is very small. Although it was extremely difficult, the present inventors
MPB64 gene was cloned in Japanese Patent Laid-Open No. 1-247094, the entire base sequence was determined, and MPB was isolated in E. coli.
We have succeeded in expressing 64 directly. With further improvement, we have developed a technology that enables greater expression. That is, an Escherichia coli strain (FERMP-13762) containing the fusion protein expression vector pMAL64c was cultured, the cultured bacterial cells were disrupted by ultrasonic waves, and the disrupted liquid supernatant was collected by centrifugation.
MPB64 can be prepared purely from this supernatant fraction. It is also possible to prepare a large amount of MPB64 purely from the culture supernatant by using the above-mentioned MPB64 gene as a host and using a related strain of Mycobacterium tuberculosis such as Smegma as a host to directly secrete and express it. is there.

The antisera for immunologically detecting MPB64 can be easily obtained by immunizing animals having antibody-producing ability, such as guinea pig, rabbit, sheep and goat, with MPB64 by a conventional method. This anti-MPB64
In order to detect Mycobacterium tuberculosis using an antiserum, the Mycobacterium tuberculosis in a sample may be cultured in a liquid medium and, for example, one week later, MPB64 secreted in the culture medium may be immunologically detected. Reverse passive hemagglutination (RPH)
Many immunological assay methods can be applied including A), reverse passive latex agglutination reaction (RPLA), enzyme immunoassay method (EIA) and the like. MP with high sensitivity by these methods
B64 can be detected, but RPHA and RPLA are particularly preferable because it is desirable that they can be tested in a safety cabinet as a biohazard countermeasure against Mycobacterium tuberculosis. In order to perform the RPHA method, anti-MPB64 antibody was sensitized to tannic acid-treated fixed human O-type red blood cells or sheep red blood cells, and this was added to a tubercle bacillus culture solution or its diluted solution in a test tube or a microplate. It can be determined as a tube bottom aggregation image after 1 to 3 hours. RPLA using a latex such as polystyrene as a carrier instead of red blood cells is also a useful method. In any method, if tube bottom aggregation occurs, the presence of MPB64 can be confirmed, demonstrating that the tuberculosis bacterium has been cultured. This reaction is also particularly suitable for clinical examinations because it can be performed very easily and in a safety cabinet.

[0008]

INDUSTRIAL APPLICABILITY As described above, it is possible to use the conventional 4
It took ~ 8 weeks, but if immunologically detected using anti-MPB64 antibody, one week of culture is sufficient, and at the same time, it can be surely confirmed that it is Mycobacterium tuberculosis. It enables early definite diagnosis and early treatment.

[0009]

EXAMPLES Example 1. Production of MPB64 E. coli JM containing fusion protein expression vector pMAL64c
The 109 strain (FERMP-13762) was added to 4 liters of L-broth medium (containing 100 mg / l ampicillin) of 40 ml of a culture solution obtained by overnight culture. After culturing at 37 ° C. for 4 hours, isopropyl-1-thio-β-D-galactoside (IPTG) was added to a final concentration of 0.3 mM, and the cells were further cultured for 3 hours. This culture solution is ice-cooled and centrifuged (8,
After collecting the cells at 000 rpm for 10 minutes, 200 ml of column buffer (10 mM Tris-Cl (pH7.4), 200 mM NaCl, 1 mM)
EDTA, 1 mM sodium azide, 1 mM dithiothreitol)
And suspended at −20 ° C. overnight. After thawing, the cells were disrupted by an ultrasonic cell disruption device (Tosho Denki UCD-200T type). The mixture was centrifuged again and the supernatant of the disrupted solution was collected. The supernatant was passed through 20 ml of an amylose resin column (manufactured by NEB, USA, capable of adsorbing maltose binding protein) and washed with 8 times the column buffer. Then 1
The adsorbed fusion protein was eluted with a column buffer containing 0 mM maltose, and the protein-containing fraction (150 mg equivalent amount) was collected using the absorbance at 280 nm as an index.

Embodiment 2. Purification of MPB64 Protease Factor Xa was added to the protein-containing fraction obtained in Example 1.
Add 250 μg (manufactured by NEB, USA), and add 72 at 25 ° C
I had time. The cleavage solution was directly applied to 1 g of a hydroxyapatite column (manufactured by Bio-Rad, USA). 80
Maltose was removed by flowing ml of column buffer, and then 0.5M sodium phosphate solution (pH 7.0).
To elute the protein. The protein-containing fraction is directly applied to an amylose column to adsorb the cleaved maltose binding protein, and the target recombinant MPB6
4 proteins were passed through. Fractions containing MPB64 protein were collected, concentrated, and concentrated to 25 mM NaC containing 20 mM Tris-Cl (pH 8.0).
The solution was dialyzed against 1 solution. This is FPLC (Pharmacia, Sweden) (Conditions: Mono Q ion exchange column is used, and 20 mM Tris-Cl buffer (p
H8.0) in a concentration gradient solution consisting of NaCl) to obtain 3.0 mg of a purified standard product.

Embodiment 3. Preparation of anti-MPB64 goat serum MPB64 prepared in Example 2 was added to physiological saline at 1 mg / ml.
A solution was prepared by mixing 2 ml of this solution with 2 ml of Freund's complete adjuvant and then forming an emulsion, which was then injected subcutaneously into the dorsal skin of the goat and into the muscle of the thigh to immunize. MPB64, 1 mg / ml after 4 weeks, 6 weeks and 8 weeks
The final booster immunization was 7
After a day, whole blood was collected and the serum was separated to obtain 800 ml of anti-MPB64 goat serum.

Embodiment 4. Preparation of Purified Anti-MPB64 Antibody 1 volume of saturated ammonium sulfate aqueous solution was added to 2 volumes of anti-MPB64 goat serum (hereinafter referred to as antiserum) prepared in Example 3, treated at room temperature for 1 hour, and then centrifuged at 3,000 rpm for 30 minutes. The ammonium sulfate precipitation fraction was collected and dialyzed against physiological saline to obtain a 1/3 saturated fraction of the antibody. Then B
MPB64 was adsorbed on a column packed with rCN-Sepharose 4B (manufactured by Pharmacia, Sweden), washed, and then adsorbed through a 1/3 saturated fraction of the above antibody.
Acetic acid buffer (pH 2.5) was flown to dissociate the antibody, which was then collected and corrected to pH 7.2 to obtain a purified anti-MPB64 antibody.

Embodiment 5. Preparation of antibody-sensitized blood cells 0.5% PB of glutaraldehyde-fixed sheep red blood cells
In one volume of S suspension, 1: 100,000 tannin (US,
(Manufactured by NBC Co., Ltd.) and treated at room temperature for 15 minutes,
Wash 3 times with PBS and suspend in 1 volume of PBS to 0.5%
A tannin blood cell was obtained. Example 4 was added to 1 volume of this tannin blood cell.
1 volume of the purified anti-MPB64 antibody (4 μg / ml) obtained in
The cells were treated at 37 ° C. for 30 minutes, washed 3 times with PBS, and then suspended in 1 volume of a diluent (0.1% bovine serum albumin + PBS) to obtain antibody-sensitized blood cells. Using this antibody-sensitized blood cell, a reverse passive hemagglutination reaction was performed.
The above showed tube bottom aggregation.

Example of detection of Mycobacterium tuberculosis Sputum was used as a test sample for Mycobacterium tuberculosis, and cultured in a Dubos liquid medium by a conventional method, and after 1 week, the presence or absence of MPB64 was examined as follows. 25 μl of the diluting solution was added dropwise to the microplate, 5 μl of the liquid culture solution was added thereto, and the antibody-sensitized blood cells 25 prepared in Example 5 were further added thereto.
μl was dropped, and after 3 hours, the presence or absence of tube bottom aggregation was visually determined. As a control, it was cultured for 8 weeks by a conventional method using Ogawa medium. The results are shown in the table.

[0015]

[Table 1]

As can be seen from the table, the detection result of MPB64 in the liquid cultured for 1 week and the detection result of Mycobacterium tuberculosis by the culture for 8 weeks in Ogawa medium are in good agreement.
It was shown that the detection method could completely replace the Ogawa culture method. As described above, the MPB64 detection method was able to obtain rapid and reliable results as a detection method for Mycobacterium tuberculosis, and was shown to be an appropriate method as a clinical test method.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Kazuhiro Matsuo Kazuhiro Matsuo 1-1, Suzuki-cho, Kawasaki-ku, Kanagawa Prefecture Ajinomoto Co., Inc. Central Research Laboratory (72) Akihiro Yamazaki 1-Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa -1 Central Research Institute of Ajinomoto Co., Inc.

Claims (2)

[Claims] 1. A Mycobacterium tuberculosis test reagent for immunologically detecting Mycobacterium tuberculosis, which comprises an anti-MPB64 antibody. 2. A method for detecting Mycobacterium tuberculosis by an immunoassay using an anti-MPB64 antibody.