JP2001181298A - Anti-M. Tuberculosis 38 kDa protein monoclonal antibody and immunological analysis method and reagent for M. tuberculosis 38 kDa protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunological analysis of a 38 kDa protein of Mycobacterium tuberculosis, which can detect Mycobacterium tuberculosis quickly and easily and can distinguish Mycobacterium tuberculosis from atypical mycobacteria. Provided are a method and an analytical reagent, and an anti-M. Tuberculosis 38 kDa protein monoclonal antibody that can be used for the method and the analytical reagent. The monoclonal antibody is Mycobacterium tuberculosis 38.
It reacts specifically with kDa protein and does not react with atypical mycobacteria. In the immunological analysis method, the monoclonal antibody or a fragment thereof is brought into contact with a test sample, and a complex of the monoclonal antibody or a fragment thereof and the Mycobacterium tuberculosis 38 kDa protein is detected. The reagent for immunological analysis contains the monoclonal antibody or a fragment thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a 38 kD antituberculous bacterium.
The present invention relates to a protein monoclonal antibody or a fragment thereof, and an immunological analysis method and an analytical reagent for the 38 kDa protein of Mycobacterium tuberculosis.

[0002]

2. Description of the Related Art Mycobacteri.
um tuberculosis (hereinafter simply referred to as "M. tuberculosis") is an infectious agent that causes human tuberculosis disease. All the genes have already been elucidated,
411,529 bases [Na
cure 393, pp. 537-544 (199
8)]. Diagnosis of tuberculosis is based on specimens (eg, sputum, blood,
Pleural and ascites fluid, urine, stool, cerebrospinal fluid, biopsy tissue, or M. tuberculosis culture)
Smearing, PCR, or culture methods have been used as known methods for detecting M. tuberculosis. Recently, M. tuberculosis protein antigens (A60, T
Methods for immunological detection of blood antibody titers using BGL, Kp90, or 38 kDa have been studied.

[0003] In the above-mentioned culturing method, it takes about 15 hours for division of M. tuberculosis, and it takes 3 to 8 weeks to find M. tuberculosis in a specimen by culturing. When treating patients with suspected tuberculosis infection, development of a rapid method for identifying the causative organism is desirable because the treatment and epidemiological approaches are different between M. tuberculosis and atypical mycobacteria. It is.

At present, the Chile-Neelsen method is used as a rapid test method for M. tuberculosis in tuberculosis patient specimens, and the result of microscopic examination of a smear-stained specimen is indicated by the number of Gaffkey. However, this smear method has a drawback that it is impossible to distinguish between Mycobacterium tuberculosis and atypical mycobacteria. Identification of Mycobacterium tuberculosis is performed by culturing Mycobacterium tuberculosis present in a test sample and determining whether the niacin test is positive or negative. Because this niacin test requires a large amount of cells,
After isolation of M. tuberculosis, further enrichment is required and takes considerable time. Also, some atypical mycobacteria have been reported to be positive for the niacin test.

[0005] Recently, M. tuberculosis has been identified by extracting a gene from a patient sample, amplifying a part of the M. tuberculosis-specific gene by PCR, and hybridizing with a labeled DNA probe. . However, skill is required in the operation of extracting a gene from a test sample or in the technique of the PCR method, and further, it is necessary to prevent DNA contamination. Even in this case, it takes 5 to 6 hours to obtain a result. Genetic diagnostic methods are becoming popular as rapid diagnostic methods for tuberculosis, but interest in simpler diagnostic methods is still high.

[0006] Furthermore, the immunological detection method using a 38 kDa polyclonal antibody against Mycobacterium tuberculosis has the advantage that results can be obtained easily and in a short time, and distinguishes Mycobacterium tuberculosis from atypical mycobacteria. There are advantages that can be.
For example, a 38 kDa protein derived from anti-tuberculosis is known to be present only in TB groups that cause infection in humans (M. tuberculosis and M. bovis) [J. Infe
ct. Dis. , 166, pp. 874-884 (1
992)].

[0007] In connection with this, Infection a
nd Immunity, 57, pp. 2481 to 24
Page 88 (1989) discloses various anti-M. Tuberculosis 38 kDa protein monoclonal antibodies prepared using the M. tuberculosis 38 kDa protein as an immunogen, and their epitope regions. That is, in the literature, the epitope recognized by the monoclonal antibody HGT3 exists in the C-terminal region (region comprising the 285th to 374th amino acid sequence) of the Mycobacterium tuberculosis 38 kDa protein, while the monoclonal antibody HBT12 and the monoclonal antibody HGT3 It is disclosed that the epitope of antibody TB71 is present in the central region (region consisting of the 117th to 284th amino acid sequences) of the Mycobacterium tuberculosis 38 kDa protein.

The monoclonal antibody HGT3 having an epitope in the C-terminal region (region comprising the 285th to 374th amino acid sequence) of the Mycobacterium tuberculosis 38 kDa protein cross-reacts with M. tuberculosis and M. bovis. , Its subclass is known to be IgM [J. Immunol. 139, p.
451 (1987)].

[0009]

SUMMARY OF THE INVENTION The present inventor has solved the above-mentioned drawbacks of the prior art, and has established a tuberculosis bacterium capable of quickly and easily distinguishing between mycobacterium tuberculosis and atypical mycobacteria. For the purpose of providing a detection method, M. tuberculosis 38 kDa
A polypeptide containing the C-terminal region of the protein is genetically engineered, and this polypeptide is
When an anti-tuberculosis 38 kDa protein monoclonal antibody was prepared using an immunogen instead of the Da protein, it was found that it was possible to distinguish between Mycobacterium tuberculosis and atypical mycobacteria. The present invention is based on such findings.

[0010]

The object of the present invention is achieved by a monoclonal antibody or a fragment thereof according to the present invention, which specifically reacts with the 38 kDa protein of Mycobacterium tuberculosis and does not react with atypical mycobacteria. can do. The present invention also relates to a hybridoma that produces the monoclonal antibody. Also, the present invention provides a method for detecting a M. tuberculosis bacterium comprising contacting the monoclonal antibody or a fragment thereof with a test sample and detecting a complex of the monoclonal antibody or a fragment thereof and a M. tuberculosis 38 kDa protein.
The present invention relates to a method for immunological analysis of kDa protein. Furthermore,
The present invention relates to a reagent for immunological analysis of the Mycobacterium tuberculosis 38 kDa protein, which comprises the monoclonal antibody or a fragment thereof.

[0011] In the present specification, "M. tuberculosis" refers to Mycobacterium tuberculosis.
ulosis). In this specification,
“Atypical mycobacteria” refers to the genus Mycobacterium (Mycob).
acteria), among the microorganisms belonging to the TB group, the aforementioned M. tuberculosis and M. bovis belonging to the TB group.
Means microorganisms excluding. Examples of the atypical mycobacteria include Mycobacterium avium and Mycobacterium intracellulare.
ulure), Mycobacterium kansasii (M.
kansasii), M. chelonei, M. fortuitum, M. gastri, M. gardonae, M. M. marinum, Mycobacterium nonchromogenum (M. nonchromogen)
genicum), M. scroflaceum, M. simiae, M. triviale, M. terrae, or M. teraee. Um Zenopi (M. xenopi).

As used herein, the term "fragment" of an antibody refers to a part of an antibody capable of binding to an antigen, for example, Fab fragment, Fab 'fragment, F
(Ab ') ₂ fragment, Fv fragment and the like.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The monoclonal antibody of the present invention or a fragment thereof specifically reacts with the 38 kDa protein of Mycobacterium tuberculosis and does not react with atypical mycobacteria.

The monoclonal antibody of the present invention can be prepared using a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing as an immunogen. Said polypeptide is itself a novel compound. The amino acid sequence represented by SEQ ID NO: 1 in the sequence listing comprises 91
And the second to 91st amino acid sequences are the 285th to 374th amino acid sequences of the 38 kDa protein of M. tuberculosis (the number of constituent amino acid residues = 374).
Is identical to the amino acid sequence at position 1. The amino acid sequence at positions 285 to 374 in the Mycobacterium tuberculosis 38 kDa protein is an epitope of the monoclonal antibody HGT3, which is a known monoclonal antibody that specifically reacts with the Mycobacterium tuberculosis 38 kDa protein (ie, an anti-M. Tuberculosis 38 kDa protein monoclonal antibody). Is known to be an area [Infection and Imm
unity, 57, pp. 2481 to 2488 (19
89)]. In addition, the 285-th to 374-th amino acid sequence in the 38 kDa Mycobacterium tuberculosis protein does not include a cysteine (SH group-containing amino acid) residue, and furthermore, does not include an amino acid sequence to which a sugar chain is bound. .

The polypeptide used as an immunogen when preparing the monoclonal antibody of the present invention is not particularly limited as long as it contains the amino acid sequence represented by SEQ ID NO: 1 in the Sequence Listing. At the N-terminal and / or C-terminal of the amino acid sequence represented by the sequence of SEQ ID NO: 1
It can be a polypeptide consisting of an amino acid sequence to which an arbitrary amino acid sequence is added. Examples of the amino acid sequence that can be added include, for example, all or a part of the amino acid sequence of glutathione S-transferase (GST).

The polypeptide used as an immunogen when preparing the monoclonal antibody of the present invention includes, for example, a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing (the number of constituent amino acid residues = 91, molecular weight = 9 kDa; hereinafter, sometimes referred to as 91AA polypeptide) or a fusion protein of 91AA polypeptide and other polypeptides. As the immunogen for preparing a monoclonal antibody, it is desirable that the molecular weight be 20 kDa or more, so that a fusion protein of 91AA polypeptide and other polypeptides (for example, all of GST or a fragment thereof and 91AA polypeptide) Or a fusion protein of LacZα or a fragment thereof with the 91AA polypeptide) is preferably used as the immunogen.

The hybridoma producing the monoclonal antibody of the present invention is prepared by a method known per se except that a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing is used as an immunogen. can do. For example, a mouse is immunized with the polypeptide, and spleen cells obtained from the mouse and mouse myeloma cells are isolated from Nature, 256, 495 (19).
75) or the cell fusion method described in J. 75). Immun
ol. Method, 100, 181-189 (198
By performing cell fusion using the electric cell fusion method described in 7), a hybridoma producing the monoclonal antibody of the present invention can be obtained. Examples of a screening antigen for screening a hybridoma producing the monoclonal antibody of the present invention include, for example, the aforementioned 91AA polypeptide, ie, SEQ ID NO: 1 in the sequence listing.
(A number of constituent amino acid residues = 91, molecular weight = 9 kDa) having an amino acid sequence represented by the following sequence:

The monoclonal antibody of the present invention can be prepared by culturing the thus obtained hybridoma (preferably mouse hybridoma) in a medium or intraperitoneally of a mammal (particularly mouse). . When a hybridoma is cultured in a medium, for example, L-glutamine, L-pyruvic acid, and antibiotics (penicillin G and streptomycin) are added to Dulbecco's modified Eagle's minimum essential medium containing 5 to 10% fetal bovine serum. Medium in a culture vessel such as a flask.
When 5-5 × 10 ⁵ hybridoma cell lines are planted,
It grows 10-20 times in 2-4 days. The antibody can be collected from the supernatant.

On the other hand, when hybridomas are cultured in the abdominal cavity of a mammal, for example, a mineral oil (for example, 2,6,10,14-tetramethylpentadecane or the like) is inoculated in the abdominal cavity of a syngeneic mouse one week before. Inoculate. Thereafter, 10 ⁵ to 10 ⁷ hybridoma cell lines cultured in a culture vessel were
When inoculated intraperitoneally into syngeneic mice, and 7 to 20 days later, when the hybridoma cell line proliferated and the tumor became large,
Ascites may be collected and the antibody may be collected from the supernatant.

The monoclonal antibody of the present invention can be separated and purified from the thus-collected culture solution or mouse ascites by a known method generally used for protein isolation and purification. Examples of such a method include salting out with ammonium sulfate, ion exchange column chromatography using ion exchange cellulose, protein A binding affinity chromatography, dialysis, and lyophilization.

The antibody fragment of the present invention can be obtained, for example, by digesting the monoclonal antibody of the present invention with a proteolytic enzyme by a conventional method, and then separating and purifying the protein according to a conventional method.

When the monoclonal antibody of the present invention or a fragment thereof is used, the 38 kDa protein of Mycobacterium tuberculosis present in a test sample derived from a patient infected with Mycobacterium tuberculosis is immunologically analyzed ("detection to determine the presence or absence of the antibody" And "measurement" for quantitatively or semi-quantitatively determining the amount of the antibody). As an analysis method using the monoclonal antibody or a fragment thereof of the present invention, a conventionally known immunological analysis method, for example, ELISA or immunochromatography can be used. Less than,
The immunological analysis method of the present invention will be described for the case where the monoclonal antibody of the present invention is used, but these descriptions also apply to the case where the fragment is used instead of the monoclonal antibody.

In the immunological analysis method according to the present invention, the monoclonal antibody of the present invention is brought into contact with a test sample, and incubated under conditions capable of forming an antigen-antibody complex by an antigen-antibody reaction. Mycobacterium tuberculosis 38
An antigen-antibody complex formed by binding the kDa protein and the monoclonal antibody is detected.

The test sample is not particularly limited as long as the sample may contain the 38 kDa protein of Mycobacterium tuberculosis. For example, sputum, blood, pleural ascites, urine, stool, cerebrospinal fluid, biopsy tissue, or tuberculosis Bacterial cultures can be mentioned.

[0025] As in the conventional method for detecting Mycobacterium tuberculosis, in the immunological analysis method of the present invention, the destruction of leukocytes and the killing of various bacteria contained in the test sample are analyzed before analyzing the test sample. The intended pretreatment can be performed. Especially in sputum, due to uneven distribution of bacteria in sputum,
First, it is preferable to homogenize the test sample. Examples of the pretreatment method include (1) a method using N-acetyl-L-cysteine, (2) a method using dithiothreitol, (3) a method using weak alkaline protease (Spouterzyme), and (4) A method of treating with a strong alkali or a weak alkali, or (5) a surfactant (for example, Tween
n20 or NP40).

For example, a test sample is treated with the 4% sodium hydroxide method or the N-acetyl-L-cysteine / sodium hydroxide method (NALC-NaOH method), and neutralized with a phosphate buffer. Centrifugation (3,000 rpm ×
20 minutes), smear the sediment on a glass slide,
The immunological analysis method of the present invention can be performed. When a biopsy tissue specimen is used as a test sample, it is deparaffinized, and a 10 mmol / l aqueous sodium citrate solution (p
H6.0) in a pressure jar (or 1
After autoclaving at 15 ° C. for 15 minutes), the immunological analysis method of the present invention can be performed. Further, as generally performed by an immunostaining method, the slide glass is subjected to APES (3-Aminopropyltrie).
thoxysilane (Sigma)).

[0027] In the immunological analysis method of the present invention, the EL
When ISA, fluorescence immunoassay, luminescence immunoassay, or radioimmunoassay is used, for example, a test sample is immobilized on a suitable insoluble carrier (for example, an ELISA plate), and the monoclonal antibody of the present invention is used. In addition to the insoluble carrier, the mixture is incubated under conditions allowing an antigen-antibody reaction (for example, at about 37 ° C. for about 30 minutes to 4 hours), the monoclonal antibody is washed, and a labeled second antibody (for example,
Labeled anti-mouse IgG antibody), and the same antigen-
After incubation under conditions that allow an antibody reaction, the labeled secondary antibody is washed, and a signal derived from the label of the labeled secondary antibody is measured.

When the ELISA, the fluorescence immunoassay, the luminescence immunoassay or the radioimmunoassay is used, examples of the label include various labels known in immunoassays, for example, enzymes (eg, peroxidase or alkaline phosphatase). A fluorescent dye [eg, fluorescein isothiocyanate (FITC)], a luminescent substance,
Alternatively, a radioactive substance can be used. Instead of the labeled second antibody, a combination of a biotinylated second antibody and labeled avidin (for example, peroxidase-labeled avidin or alkaline phosphatase-labeled avidin) can be used, or the monoclonal antibody of the present invention can be used in combination. Instead of the combination with the labeled second antibody, a labeled monoclonal antibody of the present invention alone or a combination of the monoclonal antibody of the present invention biotinylated and labeled avidin can be used. It should be noted that labeled streptavidin can be used in place of the labeled avidin. Further, in order to block non-specific adsorption of proteins, it is preferable to block with, for example, 1% bovine serum albumin (BSA) / phosphate buffered saline (PBS).

When an immunochromatography method is used in the immunological analysis method of the present invention, it can be carried out, for example, as follows. That is,
The monoclonal antibody (anti-M. Tuberculosis 38 kDa protein monoclonal antibody) of the present invention is labeled with colored particles [eg, metal colloid (eg, gold colloid), non-metal colloid (eg, polystyrene colloid), or liposome], and these labeled antibodies are used. (Hereinafter referred to as a labeled first antibody) with a water-absorbing pad [for example, Loprosorb (Po
le))] and dried, and this water-absorbing pad is coated with a flaky chromatography substrate (for example, polyamide,
Nitrocellulose, cellulose acetate, or glass fiber filter).

On the other hand, on the flaky chromatography substrate, an end opposite to the end to which the water-absorbing pad is attached is provided with a rabbit anti-tuberculosis polyclonal antibody (for example, manufactured by Novocastra; hereinafter, a second antibody). ) And a rabbit anti-mouse Ig antibody (for example, Dako) are immobilized in the length direction of the flaky chromatography base material (hereinafter, referred to as a solid support). Here, the second antibody is used as an antibody for detecting Mycobacterium tuberculosis, and the rabbit anti-mouse Ig antibody is used as a positive control antibody.

Subsequently, colored particles (for example, gold colloid)
When a test sample aqueous solution (for example, a treatment solution of a test sample or a Mycobacterium tuberculosis suspension) is brought into contact with the water-absorbing pad coated with a labeled monoclonal antibody (labeled first antibody), the test sample aqueous solution becomes Absorbed and developed on the solid support, rabbit anti-M. Tuberculosis polyclonal antibody and rabbit anti-mouse I
g antibody penetrates toward the immobilized tip. When the tuberculosis 38 kDa protein is present in the test sample aqueous solution, a complex of the labeled first antibody and the tuberculosis 38 kDa protein (labeled first antibody-tuberculosis 38 kDa protein complex) is formed. It moves on the solid support. When the labeled first antibody-M. Tuberculosis 38 kDa protein complex reaches the region where the second antibody is immobilized, the labeled first antibody-M. Tuberculosis 38 kDa protein-second
An antibody complex is formed and trapped at that position. As a result, the labeled first antibody and the Mycobacterium tuberculosis 38 kDa protein stop in this region as a labeled first antibody-M. Tuberculosis 38 kDa protein-second antibody complex and do not move any further. The substance contained in the test sample aqueous solution is further developed on the solid support, and the unreacted labeled primary antibody is a positive control antibody (rabbit anti-mouse Ig antibody)
In the area coated with, the labeled primary antibody-rabbit anti-mouse Ig
Form an antibody complex. In the immunochromatography method, the accumulation of the colored particles on the solid support is visually observed, and a positive control is used as a control to determine whether the test sample is positive or negative.

The role of the solid phase carrier in the immunochromatography method depends on the compound to be analyzed (M. tuberculosis 38 kDa).
Protein) and a labeled first antibody (eg, colloidal gold-labeled anti-tuberculosis bacterium 38 kDa protein monoclonal antibody), and reacted with the immobilized antibody (second antibody)
Another object is to separate unreacted substances.

The binding of the second antibody to the solid support in the immunochromatography method is, for example, about 3 mg / m 2.
1 ml of the antibody solution is applied to the solid phase carrier in an amount of 0.5 ml and left at room temperature for 1 hour. Also, to block non-specific adsorption of proteins,
For example, 0.5% casein (for example, Hammersten; manufactured by Merck) /0.1 mol / l maleate buffer (pH
It is preferable to block by immersing in 7.5) for 30 minutes. Further, it is preferable to provide a region for immobilizing a rabbit anti-mouse immunoglobulin antibody against the monoclonal antibody of the present invention (for example, mouse IgG) in the upper region where the second antibody is immobilized. By providing a region for immobilizing an anti-immunoglobulin antibody, it is possible to more accurately determine whether the test sample is positive or negative.

The reagent for immunological analysis according to the present invention is constituted using the same components as the conventionally known reagents for immunological analysis of the same kind except that it contains the monoclonal antibody of the present invention or a fragment thereof. be able to.

[0035]

EXAMPLES The present invention will be described below in more detail with reference to examples, but these examples do not limit the scope of the present invention.

Example 1 << Preparation of Hybridoma Producing Anti-M. Tuberculosis 38 kDa Protein Monoclonal Antibody >> Glutathione S-transferase (GST) / 91AA polypeptide fusion protein (molecular weight = 37 kDa) prepared in Preparation Examples 1 and 2 described below. 5 mg (dissolved in 0.5 ml of physiological saline) was sufficiently dispersed in 0.5 ml of Freund's complete adjuvant, and Balb / c mice were immunized with 100 μl of the dispersion. The immunization was performed four times every two weeks. After sacrificing the immunized mice, spleens were removed, to obtain a 10 ⁸ splenocytes. The obtained spleen cells and mouse myeloma cells P3 × 63Ag8.653 were subjected to an electrofusion device SSH.
-2 (Shimadzu Corporation) for cell fusion and culture.

The supernatant of the grown cells was collected, and the 91AA polypeptide (molecular weight 9 kD) prepared in Preparation Example 2 described later was used.
The presence or absence of anti-M. tuberculosis 38 kDa protein antibody production was examined by ELISA using a) as an antigen. Said E
Cells whose production of the antibody was confirmed by the LISA method were
After cloning by the limiting dilution method, ELISA
As confirmed by the method A, three types of hybridomas producing the anti-M. Tuberculosis 38 kDa protein monoclonal antibody were obtained. Each of the obtained three types of hybridomas was designated as hybridoma No. 5, hybridoma no. 1
8, and hybridoma no. 35. Less than,
In the present specification, each monoclonal antibody produced by each hybridoma is also named based on its hybridoma number. For example, hybridoma No. 5 produced monoclonal antibody No. 5 5
Called.

[0037]

Example 2 << Reaction specificity of anti-M. Tuberculosis 38 kDa protein monoclonal antibody >> (1) Reaction specificity to 91AA polypeptide 96-well ELISA plate (Dynatech)
First, the 91AA polypeptide prepared in Preparation Example 2 (1.0
(μg / ml) was dispensed at 100 μl each, and left at 4 ° C. overnight. Next, each well of the plate was filled with 1% bovine serum albumin (BSA) / phosphate buffered saline (P
BS) for 1 hour. After removing the supernatant, 50 μl of the culture supernatant of each mouse hybridoma obtained in Example 1 (that is, hybridoma No. 5, hybridoma No. 18, and hybridoma No. 35) was added. The culture supernatant was diluted 5-fold with PBS before adding to the wells. After standing at room temperature for 2 hours, 0.2% BSA / 0.2% Tween2
Washed 5 times with 0 / PBS.

Subsequently, alkaline phosphatase (AL
P) 100 μl of labeled anti-mouse IgG antibody (goat) (10
0 ng / ml) and allowed to stand at room temperature for 2 hours, then again with 0.2% BSA / 0.2% Tween 20 / PBS.
And washed 5 times. In each well of this plate, a diethanolamine buffer solution containing p-nitrophenyl phosphate (p
H9.6) 50 μl was added, and the mixture was allowed to stand at room temperature for 30 minutes to develop color. Then, the absorbance at 405 nm was measured. Table 1 shows the results.

<< Table 1 >> Culture Solution Absorbance 405 nm (x5) C-terminal polypeptide No. of 38 kDa protein 5 0.939 No. 18 0.301 No. 35 0.751

(2) Reaction specificity against Mycobacterium tuberculosis 100 μl of 91AA polypeptide (1.0 μg / ml)
Was replaced with 100 μl of heat-denatured Mycobacterium tuberculosis (1 μg / ml) at 115 ° C. for 15 minutes by using an autoclave, and the above step (1) of this example was repeated.
Table 2 shows the results.

<< Table 2 >> Culture solution Absorbance 405 nm (x5) 5 0.435 No. 18 0.145 No. 35 0.326

[0042]

Example 3 << Confirmation of Subtype of Monoclonal Antibody Produced by Each Hybridoma >> Each mouse hybridoma obtained in Example 1 (that is, hybridoma N
o. 5, hybridoma no. 18, and hybridoma No. 18 The subclass of each monoclonal antibody produced by 35) was determined using a clone typing system (South
rn Biotechnology Associate
eInc. ). Table 3 shows the results.

<< Table 3 >> Culture Solution Subclass No. 5 IgG1 λ No. 18 IgM No. 35 IgG1 κ

[0044]

Example 4 << Purification of Monoclonal Antibody >> (1) Monoclonal antibody No. Purification of Hybridoma No. 35 In this example, hybridoma No. 35 obtained in Example 1 was used. No. 35 was cultured, and monoclonal antibody No. 35 against Mycobacterium tuberculosis 38 kDa protein was used. 35 samples were collected.
First, in a Dulbecco's modified Eagle's minimum essential medium containing 10% fetal bovine serum, a medium containing L-glutamine, L-pyruvic acid, and antibiotics (penicillin G and streptomycin) was used. 35 (1 × 1
0 ⁵ / ml) were cultured. Cell concentration of 2 × 10 ⁶ cells / m
l, 200 ml of the culture is centrifuged,
The crude antibody fraction was separated from the obtained supernatant by 50% saturated ammonium sulfate fractionation. After dialysis, it was adsorbed on a protein A Sepharose 4B column (1 × 4 cm, pH 8.6),
By eluting with citrate buffer (pH 5.5),
A monoclonal antibody against the Mycobacterium tuberculosis 38 kDa protein (monoclonal antibody No. 35) was obtained. FIG. 1 shows the purification results of the monoclonal antibody. As shown in FIG. 1, after the protein was eluted, the IgG antibody bound to protein A was replaced with a citrate buffer (pH 5.5).
Eluted by 5).

(2) Monoclonal antibody no. 5 and monoclonal antibody no. Purification of hybridoma No. 35 obtained in Example 1 5 and hybridoma no. 35 are cultured in large quantities, respectively.
The mice were injected intraperitoneally. Two weeks after the injection, ascites was collected every two days. This ascites was treated with protein A Sepharose 4 in the same manner as in step (1) of this example.
B column (pH 8.6), and
8 kDa protein monoclonal antibody (ie, monoclonal antibody No. 5 and monoclonal antibody N
o. 35) was obtained.

[0046]

Example 5 << Confirmation of Mycobacterium tuberculosis by microscopy after staining with monoclonal antibody >> In this example, the cultured Mycobacterium tuberculosis was suspended in PBS, heat-denatured by autoclaving at 115 ° C. for 15 minutes, 50 μl each on a glass slide
Each sample was dispensed and flame-fixed, and used as a test sample. In addition, as a reagent for immunostaining,
Of the anti-M. Tuberculosis 38 kDa protein monoclonal antibody No. 1 purified in step (1) of 35 and an immunohistochemical staining kit (manufactured by ScyTek; sold by Diatron).

Four drops of a super block solution (vial # 1) were dropped on the test sample, and blocking was performed at room temperature for 5 minutes. Buffer [composition: 50 mmol / l-Tris-HC
1 (pH 7.6)] and diluted with PBS and diluted with PBS.
35 (100 μl) was added dropwise and reacted at room temperature for 20 minutes. Next, after washing four times with the above buffer solution, 4 drops of a biotin-labeled anti-mouse IgG antibody solution were added dropwise, and reacted at room temperature for 10 minutes. After washing four times with the buffer solution, streptavidin / HRP (peroxidase) -labeled antibody was further added dropwise, and reacted at room temperature for 10 minutes. After washing four times with the buffer solution, 4,3 drops of 3,3'-diaminobenzidine (DAB) color developing solution (4 drops of DAB chromogen was added to the DAB substrate solution and mixed to adjust the color developing solution) was added thereto, followed by staining. . The slide glass specimen was observed under a microscope to determine whether or not the cells of M. tuberculosis stained. As a result, the tuberculosis bacterium component was stained brown.

[0048]

Example 6 << Detection of Mycobacterium tuberculosis by Fluorescence Microscopy Using a Fluorescently Labeled Monoclonal Antibody >> Also in this example, a test sample and an anti-tuberculosis 38 kDa protein monoclonal antibody were used in Example 5 above. The same was used.

(1) Detection of Mycobacterium tuberculosis by Direct Fluorescent Antibody Method First, anti-M. Tuberculosis 38 kDa protein monoclonal antibody 35 was labeled with fluorescein isothiocyanate (FITC) [Goldman, M .; : Flu
oresence Antibody Metho
d, 101, Academic Press, New
York (1968)], FITC-labeled anti-tuberculosis bacterium 38k
A Da protein monoclonal antibody was prepared.

[0050] 1% BSA / PBS 200μ on the test sample
1 was added dropwise and blocking was performed for 30 minutes. On top of this, the previously prepared FITC-labeled anti-tuberculosis bacterium 38 kDa
Protein monoclonal antibody (0.5 μg / ml) 2
00 μl was added dropwise and reacted at room temperature for 30 minutes. Buffer solution (0.2% BSA / 0.2% Tween 20 / PBS)
After washing three times with, the slide glass specimen was observed with a fluorescence microscope to confirm the fluorescence of the tubercle bacillus constituents.

(2) Detection of Mycobacterium tuberculosis by the indirect fluorescent antibody method 200 μl of 1% BSA / PBS was dropped on the test sample,
Blocked for 30 minutes. On this, as a primary antibody, anti-M. Tuberculosis 38 kDa protein monoclonal antibody N
o. 200 μl of 35 (0.5 μg / ml) was added dropwise and reacted at room temperature for 30 minutes. Buffer (0.2% BSA /
After washing three times with 0.2% Tween 20 / PBS),
A commercially available FI as a secondary antibody was added to this slide glass specimen.
TC-labeled rabbit anti-mouse IgG antibody (0.5 μg / m
l) 200 μl was added dropwise and reacted at room temperature for 30 minutes.
Buffer (0.2% BSA / 0.2% Tween20 / P
After washing three times with BS), the mixture was observed with a fluorescence microscope to confirm the fluorescence of tubercle bacillus constituents.

[0052]

Preparation Example 1 Purification of Glutathione-S-Transferase (GST) / 91AA Polypeptide Fusion Protein The DNA encoding the 91AA polypeptide containing the epitope sequence of the 38 kDa protein of Mycobacterium tuberculosis is disclosed in Japanese Patent Application No. 11-160176. Are the same as those described above. As a Mycobacterium tuberculosis 38 kDa protein antigen, a glutathione-S-transferase (GST) / 91AA polypeptide fusion protein was used as a mouse immunizing antigen,
The 91AA polypeptide (molecular weight 9 kDa) was used as an antigen for screening. Preparation of this peptide protein was performed as follows.

1. Culture Glutathione-S-transferase (GST) / 9
The recombinant plasmid containing the 1AA polypeptide fusion protein gene was transformed into E. coli DH5. This transformant was inoculated into 100 ml of L-broth containing ampicillin (50 μg / ml) and cultured at 37 ° C. overnight. 100 ml of this culture solution was added to ampicillin (50 μg
/ Ml) was transferred to a spinner flask (volume = 3 liters) containing 3 liters of L-broth and cultured with stirring at 37 ° C. for 4 hours. Next, IPTG (700 mg) was added, and the cells were further cultured for 4 hours. The culture was returned to room temperature and stirring was continued overnight.

2. Preparation of GST / 91AA polypeptide fusion protein crude fraction The culture was centrifuged at 4 ° C (6,000 rpm; 1
5 minutes) [Do this twice using six 500 ml centrifuge tubes], and precipitate the cells at 20 mmol / l-Tri.
s-HCl (pH 8.0) / 1% Triton X-1
Suspended in about 40 ml, and centrifuged again at 4 ° C. (1
(2,000 rpm × 10 minutes). The cells obtained here were lysed in Lysis Buffer [50 mmol / l
-Tris-HCl (pH 8.0) / 1 mmol / l-
[EDTA / 100 mmol / l-NaCl], and suspended in 9 ml, 50 μl of 5 mmol / l-phenylmethanesulfonyl fluoride (PMSF) and lysozyme (10 mg).
/ Ml) and lysed in ice for about 20 minutes. When the viscosity appears, DNase I (3 mg / m
l) The DNA was degraded by adding 20 μl. PBS / 1% Triton X-100 was added to make the total volume about 40 ml, and freezing and thawing were repeated twice.
This was centrifuged (12,000; 10 minutes) to obtain a supernatant.

3. Separation and Purification by Glutathione-Sepharose 4B Column The fraction obtained in the step 2 was applied to a glutathione-Sepharose 4B column (2.5 × 4 cm), and PB
S / 1% Triton X-100 (10ml) and P
After washing with about 20 ml of BS, 5 mmol / l glutathione / 50 mmol / l-Tris-HCl (pH 8.
0) Elution was carried out with 50 ml and 20 ml of 3 mol / l-NaCl. The eluted fraction of the GST / 91AA polypeptide fusion protein was confirmed by performing Coomassie staining after electrophoresis on a 12.5% polyacrylamide gel (in the presence of 0.4% SDS).

4. Separation and purification using a butyl-Sepharose 4B column About 80 ml of the fraction obtained in the step 3 was subjected to 2 mol / l
When dialysis was performed overnight in a low-temperature room with-(NH ₄ ) ₂ SO ₄ (500 ml), the amount of the solution was reduced to about 50 ml.
This was applied to a butyl-Sepharose 4B column (2.5 × 3c
m) and washed with about 20 ml of 2 mol / l ammonium sulfate aqueous solution, then 30 ml of 1 mol / l ammonium sulfate aqueous solution, 60 ml of 0.4 mol / l ammonium sulfate aqueous solution, and 50 mmol / l phosphate buffer (PB) (pH 6.5) Elution was at 20 ml. GST /
The elution fraction of the 91AA polypeptide fusion protein was 1
After electrophoresis on a 2.5% polyacrylamide gel (in the presence of 0.4% SDS), it was confirmed by performing Coomassie staining.

5. Separation and purification by Sephaacryl S-200 column 10 ml of the peak fraction of the GST / 91AA polypeptide fusion protein obtained in the above step 4 was separated by Sephaacryl S.
Apply to a -200 column (2.5 x 95 cm),
0.2 mol / l-Tris-HCl (pH 9.5) /
It was eluted with 0.2 mol / l-NaCl. GST / 91
The elution fraction of the AA polypeptide fusion protein was 12.
5% polyacrylamide gel (in the presence of 0.4% SDS)
After electrophoresis, the reaction was confirmed by performing Coomassie staining.

[0058]

Preparation Example 2 << Separation and Purification of 91AA Polypeptide >> After dissolving 0.5 mg of the GST / 91AA polypeptide fusion protein purified in Preparation Example 1 above in PBS, 50 μg of thrombin (Boehringer Mannheim; derived from human plasma) was added. At 37 ° C. for 2 hours. This reaction solution was applied to a glutathione-Sepharose 4B column (2.5 × 4 cm), and about 20 ml of PBS was applied.
And the fractions collected were passed through. 16. eluted fractions
Tricine-SDS using 5% polyacrylamide gel
After performing electrophoresis, Coomassie staining was performed to confirm that the polypeptide was a 91AA polypeptide.

[0059]

As described above, the use of the monoclonal antibody of the present invention makes it possible to detect Mycobacterium tuberculosis quickly and easily, and to distinguish between Mycobacterium tuberculosis and atypical mycobacteria.

[0060]

[Sequence List Free Text] The 2nd to 91st amino acid sequences in the amino acid sequence described in SEQ ID NO: 1 in the sequence listing are the same as those of Mycobacterium tuberculosis.
Is an amino acid sequence derived from

[0061]

[Sequence List] <110> Iatron Laboratories, Inc <120> Anti-Mycobaterium tuberculosis 38 kDa Protein Antibody and Method and Reagent for Immunologically Assaying Mycobaterium tuberculosis 38 kDa Protein <130> IAT99010P <160> 1 <210> 1 <211> 91 <212> PRT <213> Artificial Sequence <220> <222> (2) .. (91) <223> This is an amino acid sequence derived from Mycobacterium tuberculosis. <400> 1 Met Ala Ala Gly Phe Ala Ser Lys Thr Pro Ala Asn Gln Ala ILe 1 5 10 15 Ser Met ILe Asp Gly Pro Ala Pro Asp Gly Tyr Pro ILe ILe Asn 20 25 30 Tyr Glu Tyr Ala ILe Val Asn Asn Arg Gln Lys Asp Ala Ala Thr 35 40 45 Ala Gln Thr Leu Gln Ala Phe Leu His Trp Ala ILe Thr Asp Gly 50 55 60 Asn Lys Ala Ser Phe Leu Asp Gln Val His Phe Gln Pro Leu Pro 65 70 75 Pro Ala Val Val Lys Leu Ser Asp Ala Leu ILe Ala Thr Ile Ser 80 85 90 Ser

[Brief description of the drawings]

FIG. 1 is a graph showing the purification results of a monoclonal antibody.

FIG. 2: D encoding the designed polypeptide of the present invention
FIG. 2 is an explanatory diagram showing a base sequence of NA.

FIG. 3 is an explanatory diagram showing a step of preparing a plasmid pGEX-2T · Pep1.

FIG. 4 is an explanatory diagram showing the structure of a fusion protein.

Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat II (reference) // C07K 14/35 C12R 1:91) (C12P 21/08 C12N 15/00 C C12R 1:91) ZNAA (72) Inventor Hidenori Nogi 1-11-4 Higashikanda, Chiyoda-ku, Tokyo F-term in Yatron Co., Ltd. 4B024 AA13 BA31 BA50 CA04 DA02 DA06 EA04 GA03 GA11 HA15 4B064 AG26 AG27 BA14 CA10 CA20 CC24 CE06 CE12 DA15 4H045 AA11 AA30 BA21 BA41 CA11 DA76 DA86 FA72 FA74 GA10 GA15 GA22 GA25 GA26 HA05

Claims (3)

[Claims] 1. A monoclonal antibody or a fragment thereof, which specifically reacts with the Mycobacterium tuberculosis 38 kDa protein and does not react with atypical mycobacteria. 2. The monoclonal antibody or a fragment thereof according to claim 1 is brought into contact with a test sample, and the monoclonal antibody or a fragment thereof is brought into contact with M. tuberculosis 38k.
An immunological analysis method for the Mycobacterium tuberculosis 38 kDa protein, which comprises detecting a complex with a Da protein. 3. A reagent for immunological analysis of the Mycobacterium tuberculosis 38 kDa protein, comprising the monoclonal antibody or a fragment thereof according to claim 1.