JP2002372531A - Diagnostic method of respiratory tract infection utilizing digestion fragment of causative fungus in sputum - Google Patents

Abstract

PROBLEM TO BE SOLVED: To establish an immunological detection method capable of detecting, quickly and simply, causative fungus of a respiratory tract infection including tuberculosis, which is desired on a field of medical treatment. SOLUTION: As the result of earnest and repeated research, it is found out that the causative fungus of the respiratory tract infection can be detected simply, quickly and surely, which has been impossible by a conventional method, by identifying digestion fragments included in large quantities in sputum of a respiratory tract infection patient by paying attention to existence of the digestion fragments of the causative fungus which has not been noted by the conventional method.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunological detection method for detecting pathogens causing respiratory infections that is simple, rapid, and reliable for diagnosing all respiratory infections including tuberculosis and the like. It relates to an immunological detection kit.

[0002]

BACKGROUND OF THE INVENTION In recent years, tuberculosis, which has become an epidemic of re-emerging infectious diseases, is the deadliest of all respiratory infections. According to recent WHO statistics, 3 million Nearly as many patients have died. Nearly 30,000 patients die each year in Japan, and the Ministry of Health and Welfare declared a tuberculosis emergency on July 26, 1999. In this tuberculosis, almost all tissues and organs of the whole body become affected sites of Mycobacterium tuberculosis, especially in the lungs. Patients with pulmonary tuberculosis constantly excrete the bacteria themselves into the air due to coughing or the like, and a delay in diagnosis will contribute to the spread of infection.

In order to treat these respiratory infections rationally and effectively, it is necessary to quickly and surely identify the causative bacteria causing respiratory infections and to select an appropriate treatment method. is necessary.

[0004] Here, since the pathogenic bacteria that have invaded the living body are phagocytosed and digested by macrophages, very few remain as complete cells without being destroyed when excreted from the living body. Therefore, most of the cells excreted together with sputum are not complete cells but are digested cell fragments.
However, current methods for detecting the causative bacteria of respiratory tract infections are based on staining and identification of undigested intact cells and cultivation of live bacteria.Recently, PCR amplification targeting bacterial-specific nucleic acid genes has been performed. At present, no attention has been paid to digested bacterial fragments.

For example, taking the diagnosis of tuberculosis as an example, M. tuberculosis contained in a sputum sample of a subject is identified as Ziehl-Neel.
sen staining (Chiel-Neelsen staining), M. tuberculosis detection by culture (culture method), P.
It has been performed by a method of detecting Mycobacterium tuberculosis by the CR method or the like.

[0006] The Thiel-Neelsen staining method is a method of staining with fuchsin. Diagnosis is made by the fact that fuchsin taken into acid-fast bacterium is stained red even when decolorized with alcoholic hydrochloric acid. However, only intact cells covered with hard lipid cell walls can be stained and miss digested bacterial fragments contained in sputum. For this reason, the detection rate is as low as 35% or less, which results in overlooking many patients with evacuation and there is a problem of lack of certainty.

[0007] Culture of Mycobacterium tuberculosis by a culture method is usually carried out.
The specimen is cultured for 4 to 8 weeks using the Ogawa medium, and the bacteria to be detected are identified to determine the M. tuberculosis. However, it takes a long time to judge the results and lacks agility, which is a major obstacle to the definitive diagnosis of tuberculosis and early treatment. Infection is a major problem.

[0008] Detection by the PCR method is a test method targeting the nucleic acid gene of M. tuberculosis, which detects M. tuberculosis using a nucleic acid primer capable of amplifying a specific region of the M. tuberculosis gene. However, when judging from sputum samples that do not incorporate culture, accuracy remains a problem, and there are many cases where false negatives are indicated. Furthermore, due to the nature of the amplification method, there is a problem in the reliability of the determination because false positives may occur due to the contamination of a small amount of sample.

As is apparent from the above-mentioned examples of tuberculosis diagnosis, there is no report on the use of digested bacterial fragments for the diagnosis of respiratory tract infections, and each of the conventional methods solves the problem. There is a problem to be solved. There is still no diagnostic method that can detect tuberculosis and other respiratory tract infections quickly, simply, and reliably, and establishment of such a diagnostic method is eagerly sought from medical sites.

[0010]

SUMMARY OF THE INVENTION Accordingly, the present invention provides an immunological detection that can quickly and simply and reliably detect the causative bacteria of respiratory infections such as tuberculosis, which has been desired in medical practice. The aim is to establish a method.

The present inventors have conducted intensive studies to solve the above problems, and as a result, have noticed the presence of digested fragments of pathogenic bacteria, which the conventional method has not paid attention to. By identifying digestive fragments abundantly contained in the present invention, it was found that pathogens causing respiratory tract infections that could not be achieved by conventional methods could be simply, quickly and reliably detected, and completed the present invention. .

[0012]

That is, according to the present invention,
The sputum collected from the subject is brought into contact with an antibody that recognizes a pathogenic bacterium causing respiratory tract infection to form an immune complex, and the pathogen causing the respiratory tract infection contained in the sputum Provided is a method for detecting a causative microorganism of a respiratory tract infection, which comprises detecting not only intact whole intact cells but also digested cell fragments.

As the antibody used in the present invention, a polyclonal antibody recognizing a pathogen causing respiratory infection or a monoclonal antibody recognizing as an epitope a different component of the pathogen causing respiratory infection. Multiple combinations of cocktail antibodies are preferred. The large number preferably means 50 or more types, more preferably 100 to 300 types.

The respiratory tract infection which can be used in the present invention is not particularly limited as long as it is caused by pathogenic bacteria and causes respiratory inflammation, but it can be suitably used particularly for tuberculosis.

The method for detecting the causative organism of respiratory tract infection according to the present invention preferably utilizes colloidal gold / silver sensitization.

Further, the present invention provides a cocktail comprising a combination of polyclonal antibodies recognizing pathogens causing respiratory infections or monoclonal antibodies recognizing epitopes of different components of pathogens causing respiratory infections as epitopes. It is intended to provide a diagnostic kit for respiratory tract infections for detecting not only undigested intact whole cells of the pathogen contained in sputum but also digested cell fragments containing antibodies. Is an antibody modified with colloidal gold, and more preferably has silver sensitizing means.

Most of the cells excreted together with sputum are not complete cells but digested fragments thereof, and the present invention focuses on such digested fragments to provide respiratory infections such as Mycobacterium tuberculosis. It enables rapid and reliable diagnosis of the disease, has very high clinical value, and is expected to be widely used clinically.

The term "digested fragment" as used in the present invention refers to a fragment of a bacterial cell in which cells are digested by digestion with macrophages and are immunogenic lipids, proteins, glycoproteins, and the like.
It shall include the components of all pathogenic bacteria, including glycoproteins.

[0019]

BEST MODE FOR CARRYING OUT THE INVENTION The antibody used in the present invention and an immunological detection method for detecting pathogenic bacteria causing respiratory infections will be described in detail below.

The antibody used in the present invention can be appropriately selected from antibodies that can be obtained by using, as an immunogen, a pathogenic bacterium that causes respiratory tract infection and recognize the pathogenic bacterium. In particular,
It is preferable to use polyclonal antibodies that recognize pathogens that cause respiratory infections, and use cocktail antibodies that combine multiple monoclonal antibodies that recognize as epitopes different components of pathogens that cause respiratory infections It is also possible. It is also possible to use a mixture of a polyclonal antibody and a monoclonal antibody. The epitope recognized by the monoclonal antibody that can be used in the present invention may be present on any antigenic substance constituting a pathogenic bacterium.

However, it is impossible to use only one kind of a monoclonal antibody that recognizes a part of a specific component as an epitope among the constituents of a bacterial cell alone, or to use only a few kinds in combination. Digested fragments contained in sputum excreted from the body contain various components, such as proteins, lipids, glycoproteins, glycolipids, etc., which are the components of the pathogenic bacterium, in different compositions, respectively. It is inappropriate to use a monoclonal antibody having a specific component as an epitope alone or to use only a combination of only a few types among them, since many digested fragments containing no specific component are missed. That is, it is significant that the epitope recognized by the antibody is not limited to any component in the bacterial cell.

As these antibodies, commercially available antibodies may be used, or antibodies prepared according to a conventional method may be used. Methods for producing these antibodies are well known to those skilled in the art, and can obtain a pathogenic bacterium causing respiratory tract infection as an immunizing antigen. The pathogenic bacteria used as an immunizing antigen may be not only complete cells but also any antigenic substance (cell fragment) constituting the pathogenic bacteria,
Cell fragments obtained by disrupting cells by means such as homogenization may be used, but from the viewpoint that antibodies need to recognize many digested fragments, those obtained by isolating and purifying a single component are suitable. Absent. As the cells, both dead cells and live cells can be used.

Immunization of a warm-blooded animal is carried out, for example, by administering the above-mentioned immunizing antigen itself or a carrier or diluent to a site of the warm-blooded animal where an antibody can be produced. Upon administration, complete Freund's adjuvant or incomplete Freund's adjuvant may be administered in order to enhance the antibody-producing ability. The administration can usually be performed once every 2 to 6 weeks, for a total of about 3 to 10 times. The dose is generally 1 to 10 per kg of animal body weight.
It can be performed in the range of 0 mg.

The polyclonal antibody can be prepared by collecting blood from a warm-blooded animal immunized with an immunizing antigen and separating and purifying an IgG fraction from an antiserum obtained by collecting serum. For separation and purification, for example, an IgG fraction can be collected from serum to obtain a polyclonal IgG according to affinity chromatography using a protein G or protein A column, gel filtration or electrophoresis, DEAE ion exchange chromatography, or the like. .

The monoclonal antibody is fused with an antibody-producing cell collected from a warm-blooded animal immunized with an immunizing antigen and an immortalized cell such as a myeloma cell of the warm-blooded animal.
It can be obtained by preparing a fused cell (hybridoma), selecting a desired hybridoma that produces an antibody recognizing a desired immunizing antigen, and culturing the hybridoma. The production of hybridomas is described, for example, in Kohler and Milstein, Natu.
re, Vol. 256. p495 (1975), and examples of the fusion promoter include PEG and Sendai virus. The resulting hybridoma is selected by culturing it in a conventional selection medium, for example, a HAT medium. Separation and purification of a monoclonal antibody can be performed in the same manner as separation and purification of a polyclonal antibody.

In a group of bacteria belonging to the same genus, the cells of a specific strain and the cells of other species often have similar antigens, respectively. When a polyclonal antibody against the body is prepared, it shows cross-reactivity with cells of other strains having a similar antigen. Therefore, a polyclonal antibody that can remove non-specific components by absorbing an antibody against cells of a specific bacterial species with cells of another bacterial species and specifically reacts with cells of a specific bacterial species Can be obtained. Such an antibody can also be suitably used in the present invention, and by using such an antibody, a digestion fragment specific to a specific bacterial species can be detected. It becomes possible. In addition, absorption treatment methods for removing non-specific components are known to those skilled in the art. It is possible to use any of such known methods, and it is also possible to perform the absorption treatment using a cell column described in 2001-72541. Specifically, a cell column supporting cells having an antigen similar to the target cells for antibody production was prepared, and the cells were obtained from the serum immunoglobulin fraction of an animal immunized with the target cells for antibody production. By passing the obtained polyclonal antibody through the column, an antibody that reacts with a cell having an antigen similar to the target cell of the antibody is absorbed and removed, thereby producing an antibody that specifically reacts with the target cell. can do.

The term "microorganism column" as used herein means a method for adsorbing an antibody substance contained in a sample by utilizing an interaction (affinity) between substances such as an antigen and an antibody. The method of the present invention uses an antigen-antibody reaction, that is, a method of producing an animal immunized with a target cell for producing an antibody by utilizing the affinity between a cell as an antigen and an antibody reactive with the cell. It absorbs and removes, from the polyclonal antibody obtained from the serum immunoglobulin fraction, an antibody having a cross-reactivity to a bacterial species having a shape similar to that of the target cell for producing the antibody.

The cell column is specifically composed of a cellulose-based carrier, an agarose-based carrier, a polyacrylamide-based carrier,
Bacteria and their fragments are immobilized on an insoluble carrier such as a dextran-based carrier, a polystyrene-based carrier, a polyvinyl alcohol-based carrier, a polyamino acid-based carrier, or a porous silica-based carrier by a conventional method (by physical adsorption and crosslinking). Polymerized, sealed in a matrix, or immobilized by non-covalent bonding, etc.), and filled with the insoluble carrier in a column made of glass, plastic, stainless steel, or the like.

As the insoluble carrier used in the method of the present invention, any insoluble carrier which can immobilize cells or cell fragments can be used. For example, Amersham, a commercially available product, may be used. Pharmacia Biotech's Sepharose 2B, Sepharose 4B, Sepharose 6B, CN
Br-activated Sepharose 4B, AH-Sepharose 4B, CH-Sep
harose 4B, Activated CH-Sepharose 4B, Epoxy-activa
ted Sepharose 6B, Activated thiol-Sepharose 4B, Se
phadex, CM-Sephadex, ECH-Sepharose 4B, EAH-Sepharo
se 4B, NHS-activated Sepharose or Thiopropyl S
Bio-Gel manufactured by Bio-Rad, such as epharose 6B
A, Cellex, Cellex AE, Cellex-CM, Cellex PAB, Bio-G
el P, Hydrozide Bio-Gel P, Aminoethyl Bio-Gel P, B
io-Gel CM, Affi-Gel 10, Affi-Gel 15, Affi-Prep 1
0, Affi-Gel Hz, Affi-Prep Hz, Affi-Gel 102, CM Bio
-Gel A, Affi-Gel heparin, Affi-Gel 501 or Affi
-Chromagel A, Chromagel P, Enzafix P-HZ, Enzafix PS manufactured by Wako Pure Chemical Industries, such as -Gel601
AE-Cellulose, CM-Cellulose or PAB Cellulose manufactured by Selva (Serva), such as H or Enzafix P-AB
Etc., among these, CNBr-activ
ated Sepharose 4B is preferred.

For example, in the case of Mycobacterium tuberculosis, an antibody that can be used is Mycobacterium tuberculosis Aoyama B (Mycobacterium) described in Japanese Patent Application No. 2001-72541.
Bacterium tuberculosis Aoy
ama B, hereinafter abbreviated as MTAB. ), An anti-M. Tuberculosis polyclonal antibody (MTB01R1), which is a rabbit IgG purified polyclonal antibody obtained from a rabbit immunized with
L / S, Japan Biological Science Center, Inc.), Mycobacterium kansasii (Mycobacterium k.)
ansasii, hereinafter abbreviated as MK. )), A mycobacterial (MK) polyclonal antibody (MKA), which is a rabbit IgG purified polyclonal antibody obtained from a rabbit immunized with
01R1L / S, Japan Biological Science Center) Mycobacterium avium complex (Mycobac)
terium avium complex, M
Abbreviated as AC. ) Is a rabbit IgG purified polyclonal antibody obtained from a rabbit immunized with
C) Polyclonal antibody (MAC01R1L / S,
It is possible to suitably use a commercially available antibody such as (Japan Bioscience Center Co., Ltd.), or the antibody may be produced according to the antibody production method described above.

The anti-M. Tuberculosis polyclonal antibody described above shows a cross-reactivity with MK and MAC.
As described in Japanese Patent Application No. 2001-72541,
An MK cell column supporting the cross-reactive MK cells and a MAC cell column supporting the MAC cells were prepared, and an anti-tuberculosis polyclonal antibody was passed through each of the cell columns. And an antibody having specific reactivity to MTAB (hereinafter abbreviated as MTAB-specific antibody) by absorbing and removing an antibody having cross-reactivity with MAC cells.
Can be prepared. The antibody produced in this manner can be used in the present invention, whereby the diagnosis of the type of bacteria can be performed more quickly and reliably.

Similarly, an antibody having specific reactivity with MK (hereinafter, abbreviated as MK-specific antibody) is obtained by absorbing an antibody that reacts with MAC and MTAB from an acid-fast bacillus (MK) polyclonal antibody. ) Is an acid-fast bacterium (MA)
C) An antibody having specific reactivity with MAC (hereinafter abbreviated as MAC-specific antibody) can be prepared by absorbing an antibody that reacts with MK and MTAB from the polyclonal antibody.

The method for detecting an immune complex formed by the reaction between an antigen and an antibody is not particularly limited as long as the antigen can be specifically detected without the presence of contaminants. In particular, a gold colloid / silver sensitization method can be suitably used. In order to detect a digested fragment distributed as a fine fragment using gold colloid, it is necessary to perform silver sensitization in order to facilitate visual judgment. Therefore, any metal colloid can be used as long as it can sensitize silver.
In addition, the fluorescent staining method has a poor construct because sputum contains a component that emits fluorescence, and specifically identifies bacterial fragments that have formed an immune complex with these contaminants and antibodies modified with colloidal gold. It is not suitable because it is difficult to recognize them separately. Similarly, the enzyme immunoassay cannot be used in the present invention because it is not suitable for visually judging small fragments.

The particle size of the gold colloid is about 5 to 10 nm from the viewpoint of storage stability and ease of adjustment. If the particle size is too small, the degree of coloring per particle is small, so that the degree of coloring is poor and visual confirmation becomes poor. If the particles are too large, a non-specific reaction may occur.

A method for preparing an antibody modified with colloidal gold is known, and commercially available colloidal gold particles or a conventional method, for example, the method of France et al., Nature Phys. Sc
i. , Vol. 241 p20-22 (1973), etc., and colloidal gold particles prepared according to the method of the present invention and the antibody of interest, for example, Geoghegan and A
Kerman, J .; Histochem. Cyt
ochem. vol. 25 p1187-1200
(1977), an antibody modified with colloidal gold can be prepared.

For silver sensitization, a commercially available kit can be used. For example, Silver Enhancer Ki can be used.
t (Sigma) or the like is used.

The present invention also provides a diagnostic kit for respiratory infection suitable for the method for immunologically detecting the causative bacteria of respiratory infection. The kit is a polyclonal antibody having specific reactivity to a pathogen causing respiratory infection, or a cocktail antibody comprising a large number of monoclonal antibodies recognizing as epitopes different components of the pathogen causing respiratory infection. Is an essential component. The antibody may be in the form of a solution dissolved in an appropriate buffer or a lyophilized product, and may further contain a stabilizer, a preservative, and the like.

The antibody may be modified as necessary with, for example, colloidal gold or the like, and may further include a means for silver sensitization. However, these are not essential components because commercially available products can be suitably used.

[0039]

EXAMPLES The present invention will be described in detail with reference to examples. However, the present invention is significant in that a digested fragment of a bacterial cell, which has not been focused on in the past, was used as a target for diagnosis, and is limited to the following examples. It is not something to be done. Hereinafter, an example in which a diagnosis of M. tuberculosis was performed by using sputum as a sample and reacting it with an anti-tuberculosis polyclonal antibody modified with colloidal gold and sensitizing silver, will be described in detail.

Example 1 Preparation of Colloidal Gold-Modified Antituberculous Polyclonal Antibody Dissolved in 50 μl of 0.02 M phosphate buffer (pH 7.3)
1 mg of the anti-M. Tuberculosis polyclonal antibody (MTB0
1R1L / S, Japan Biological Science Center)
0 μl of gold colloid solution (GOLD COLLOID)
5NM, SIGMA) and mix at 4 ° C.
Labeling was allowed to stand for 16 hours. After labeling, ultrafiltration membrane
0.0 for (ULTRAFREE, MILLIPORE)
2% NaN _ThreeAfter replacing the buffer with PBS (-),
It was concentrated to 100 μl.

Example 2 Detection of Mycobacterium tuberculosis

As a sample, sputum collected from 13 subjects suspected of having tuberculosis was used. The sputum collected from the subject was wiped off with a flame-sterilized platinum loop, smeared on a slide glass, and then flame-fixed.

First, tuberculosis diagnosis was performed by the Thiel-Neelsen staining method. Fuchsin carbonate solution was dropped on the slide glass, heated at 60 ° C. for 5 minutes, washed with running water, decolorized with 3% hydrochloric acid alcohol, washed with running water, and reacted with methylene blue for 10 seconds. And washed with running water. After the slide glass was air-dried, the presence or absence of staining was observed with an optical microscope, and the slide glass was judged using the Gaffkey number.

Next, antibody staining and silver sensitization were performed. Next, the slide glass diagnosed by the Thiel-Neelsen staining method was used in a 0.05 M TRIS-HCl buffer solution (pH 7.6) containing 0.01% Tween 20.
After immersion for 5 minutes, the rabbit normal immunoglobulin fraction (Immunoglobulin Fraction,
Normal rabbit, Dako)
Blocking was performed by allowing the reaction to proceed for 5 minutes.
This slide glass was washed three times for 5 minutes with a 0.05 M TRIS-HCl buffer solution (pH 7.6) containing 0.01% Tween 20 and then diluted 200-fold with the same buffer solution. Colloidal gold-modified anti-tuberculosis rabbit polyclonal antibody was dropped on the same sample,
After reacting for minutes, wash with water and use a commercially available silver sensitization kit Sil.
Sensitization was performed using the ver Enhancer Kit (Sigma) according to the manufacturer's instructions. For silver sensitization, specifically, immediately before use, solutions A and B are mixed, and a mixed solution obtained by mixing is dropped on a slide glass, sensitized to an appropriate concentration while observing, washed with water, and fixed. Liquid
After immersion for 2 minutes, washing was performed. The slide glass was sealed with GelMount, and silver particles obtained by silver sensitization were counted at 400 times magnification with an optical microscope. In addition, the number of observed particles was 20 to 0.13 mm ² .
If the number is less than or equal to 50, it is determined as negative (-), if more than 50, it is determined as positive (+), and if it is 21 to 49, it is determined as false positive (+-). All of the above were performed at room temperature.

As a comparative example, the above sputum was subjected to a tuberculosis diagnosis by the PCR method. The PCR method was performed using a commercially available Amplicor TB (Roche).

The results are summarized in Table 1, and FIGS. 1 to 3 show photographs of microscope images of Sample Nos. 3, 6 and 11 by the colloidal gold / silver sensitization method. Specimen 3 is an example in which M. tuberculosis was not detected by the Thiel-Neelsen staining method and PCR, and was determined to be negative also by the method of the present invention. In sample 6, no M. tuberculosis was detected by the Thiel-Neelsen staining method.
CR is an example in which M. tuberculosis has been detected, and was determined to be positive also by the method of the present invention. Specimen 11 is an example in which M. tuberculosis was detected by the Thiel-Neelsen staining method, and was determined to be tuberculosis without performing PCR, and was also determined to be positive by the method of the present invention. It is.

[0047]

[Table 1]

From the above results, it is understood that the sensitivity is as high as that of the PCR method. It is also understood that a sample diagnosed by the Thiel-Neelsen staining method can be used, and that M. tuberculosis can be detected with higher sensitivity than the diagnosis by the Thiel-Neelsen staining method.

[Example 3] Next, a specific example of the production of a bacterial species antibody which can be suitably used in the present invention will be described. These antibodies can also be used in the method of the present invention for detecting the causative bacteria of respiratory infections and the diagnostic kit for respiratory infections.

Preparation of MTAB Specific Antibody First, the MK cells used to remove antibodies having cross-reactivity with MK and MK cells carrying cell fragments
A cell column was prepared. (1) MK (ATCC No. 12) having a wet weight of 400 mg
478) in a coupling buffer (0.1
M bicarbonate buffer (pH 8.3) + 0.5 M Na
Cl) was suspended in 4 mL, and ultrasonically treated in an ultrasonic washer for 15 to 60 minutes to homogenize to obtain a cell treatment solution. (2) 1 g of CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech) was swelled in 1 mM HCl for 15 minutes. (3) CNBr-activated Sepharose swollen in (2)
4B was packed in a PD-10 column (Amersham Pharmacia Biotech), and washed with 50 mL of 1 mM HCl and 50 mL of the coupling buffer in this order. (4) The bacterial cell treatment solution prepared in (1) was added to the column of (3), and reacted at room temperature for 16 hours with a rotator. (5) The column of (4) is coupled with a coupling buffer of 20 m.
L, after washing in this order with 10 mL of a blocking buffer (0.2 M glycine buffer (pH 8.3)), filling the column with a blocking buffer,
The reaction was performed at room temperature for 2 hours. (6) Coupling buffer 30 mL, washing buffer (0.1 M acetate buffer ((pH 4.0) +0.5
M NaCl), 30 mL, elution buffer (0.1 M
Washing was performed in this order with 30 mL of a glycine buffer (pH 2.5) and 30 mL of a binding buffer (PBS (-)). (7) Fill the column with the binding buffer, and use 4
Stored at ° C.

2. Preparation of MAC Cell Column A MAC cell column that absorbs the MAC antibody was replaced with MAC (ATCC No. 19078) instead of MK-killed cells.
It was prepared in the same manner as described above, except that heat-killed bacteria were used.

3. Next, absorption treatment was performed on the anti-M. Tuberculosis polyclonal antibody to prepare an MTAB-specific antibody, that is, an antibody that does not show a cross-reactivity with MK and MAC but specifically reacts with MTAB by the following procedure. (1) Anti-tuberculosis polyclonal antibody (MTB01R1L
/ S, Japan Biological Science Center) 10 mg in PBS
Replaced with (-). (2) The MK cell column (3.5 mL) prepared by the method described in 1 above is equilibrated with PBS (−),
(1) Anti-tuberculosis polyclonal antibody / PBS solution
The solution was passed through the MK cell column at a flow rate of 0.6 mL / min. (3) The protein fraction passed through the MK cell column was recovered. (4) The MAC cell column (3.5 mL) prepared by the method described in 2 above is equilibrated with PBS (−),
The protein fraction collected in (3) was passed again through the MAC cell column. (5) The protein fraction that passed through the MAC cell column was collected. (6) The protein fraction collected in (5) was concentrated using an ultrafiltration membrane (fraction molecular weight: 50,000) to obtain an MTAB-specific antibody.

In the above example, the MAC cell column was used after the MK cell column was used for the absorption treatment.
After using the C cell column, the MK cell column may be used.

4. By examining the cross-reactivity between the MK and MAC of the anti-M. Tuberculosis polyclonal antibody absorption-treated antibody and the non-absorption-treated antibody, the performance of the anti-M. Tuberculosis polyclonal antibody was compared with the absorption-untreated antibody.

The unabsorbed anti-tuberculosis polyclonal antibody had an antibody titer at the time of blood collection of 25600 (Ab)
s. 0.659). The anti-tuberculosis polyclonal antibody-absorbed antibody is the MTAB-specific antibody obtained in 3 above.

The examination was performed by the indirect ELISA method. (1) 100 mg / mL MTAB suspended in PBS
(ATCC No. 31726), heat-killed bacteria of MK and MAC were coated with a coating buffer at 100 μg / mL.
(PH 9.6), each dead cell was dispensed to each well of each microplate in a volume of 60 μL, and the temperature was adjusted to 37 ° C.
For 1.5 hours and adsorbed to the wells.
These plates were discarded immediately before use, washed twice with a plate washer, and then 160 μl of a blocking solution was dispensed into each well and incubated at 37 ° C. for 1 hour. (3) The blocking solution in each well was discarded and washed three times with a plate washer. (4) As the primary antibody, an unabsorbed anti-tuberculosis polyclonal antibody and an absorption-treated anti-tuberculosis polyclonal antibody were used. These antibodies are serially diluted two-fold from 100 μg / mL to 2 × 10 −7 with a sample diluent, and the respective serial dilutions of the unabsorbed anti-tuberculosis polyclonal antibody and the absorption-treated antibody of the anti-tuberculosis polyclonal antibody Dispense 80 μL of each serially diluted solution into each well of a microplate at 37 ° C.
For 1 hour. (5) After incubation, the reaction solution was discarded and washed three times with a plate washer. (6) Biotin-labeled anti-rabbit Ig as a secondary antibody
80 μL of G antibody solution to each well of each microplate
Dispense and incubate at 37 ° C for 1 hour. (7) After washing the plate after the reaction of the secondary antibody with a plate washer, 80 μL of the enzyme substrate is dispensed into each well of each microplate, and 30 minutes after the start of the reaction, the absorbance at a wavelength of 405 nm is read by a plate reader. Measured. The performance of the unabsorbed antibody and the absorbed antibody was compared at the highest dilution ratio at which the absorbance difference between the target sample and the control cleared 0.1 or more. Table 2 shows the composition of each reagent.

[0057]

TABLE 2 20x coating buffer _{Na 2 CO 3 1.272 g NaHCO 3} 2.344 g NaN 3 0.160 g milli-Q water at constant volume 25xPBS in 40 ml (-) KCl 5g KH 2 PO 4 5g Na 2 HPO 4 72.5g NaCl 200g millimeter 1000mL to volume cleaning solution 25XPBS with Q water (-) 400 ml Tween 20 10 ml NaN 3 10 g milli-Q water constant volume blocking 10L solution Geratin 10 g 25xPBS (-) to 1000mL with 40 ml NaN3 1 g milli-Q water Dissolve Geratin by heating in constant volume warm water. Sample diluent Dilute the blocking solution 10-fold with the washing solution. Enzyme substrate (for AP): p-Nitrophenylphosphate disodium salt 100 mg 0.5 mM MgCl2 solution 100 ml Diethanolamine 100 μl

The unabsorbed antibody to the anti-M. Tuberculosis polyclonal antibody was determined by comparing the titer of each dead cell.
For the reactivity with the dead TAB cells, the MK dead cells and 2
5% (2560/10240), 5% of the dead cells of the MAC
It had a cross-reactivity of 0% (5120/10240). On the other hand, comparing the titer of each killed cell with the antibody treated with the anti-M. Tuberculosis polyclonal antibody, the MK-killed cell showed 0.4% (20/20)
5120), and 0.2% (10/51)
20). Thereby, the above 3.
It was confirmed that the MTAB-specific antibody prepared in Example 1 did not have cross-reactivity with MK and MAC, and was an antibody that specifically reacted with MTAB.

Example 4 Preparation of MK-Specific Antibody Preparation of MATB Cell Column A MATB cell column that absorbs MATB antibody that reacts with MATB is replaced with MATB (A
TCC No. 31726) It was prepared in the same manner as described in Example 3, except that heat-killed bacteria were used.

2. Absorption treatment of acid-fast bacillus (MK) polyclonal antibody Acid-fast bacterium (MK) polyclonal antibody (MKA01R1)
L / S, Japan Bioscience Center Co., Ltd.) does not show cross-reactivity with MTAB and MAC by absorbing the antibody that reacts with MTAB and the antibody that reacts with MAC,
An antibody that specifically reacts with MK, that is, an MK-specific antibody was obtained. The absorption treatment of the acid-fast bacillus (MK) polyclonal antibody is performed in the above-mentioned method of absorption treatment of the anti-tuberculosis bacterium polyclonal antibody, instead of combining the MK cell column and the MAC cell column in the above 1. The procedure was performed in accordance with the above-described method, except that the MTAB cell column prepared in the above and the MAC cell column prepared in Example 3 were combined.

3. MTAB and MAC of Absorbed and Unabsorbed Antibodies to Mycobacterium (MK) Polyclonal Antibodies
By examining the cross-reactivity with the antibody, the performance of the absorption-treated antibody and the unabsorbed antibody of the acid-fast bacillus (MK) polyclonal antibody was compared.

The mycobacterial (MK) polyclonal antibody is
The antibody titer at the time of blood collection was 25600 (Abs.
119). The absorption-treated antibody of the acid-fast bacterium (MK) polyclonal antibody is the MK-specific antibody obtained in the above item 2.

The absorbance of the mycobacterial (MK) polyclonal antibody after absorption and the absorbance of the unabsorbed antibody were measured by ELISA.
This was performed using Method A, and the antibody titer was determined. The experimental procedure was such that the conditions for adsorption of each dead cell to the wells were 1 ° C at 4 ° C.
Let stand for 6 hours and use the above-mentioned acid-fast bacterium (MK) as the primary antibody
The procedure was performed according to the method described in Example 3 except that the unabsorbed polyclonal antibody and the MK-specific antibody were used.

The unabsorbed antibody to the acid-fast mycobacterial (MK) polyclonal antibody was compared with the dead MTAB cells by comparing the titer of each dead cell with the MTAB dead cells. 12.5% (5120/40)
960). On the other hand, the antibody treated with the acid-fast bacterium (MK) polyclonal antibody showed that the MTAB killed cells were 0.8%
(80/10240), 0.1% of the dead cells of the MAC
(10/10240). Thus, the MK-specific antibody prepared in 3 above
Almost no cross-reactivity with TAB and MAC was observed, confirming that the antibody specifically binds to MK.

Example 5 Preparation of MAC-Specific Antibody Absorption treatment of acid-fast bacillus (MAC) polyclonal antibody Acid-fast bacillus (MAC) polyclonal antibody (MAC01R)
MTB from 1L / S, Japan Biological Science Center)
Absorbing the antibody that reacts with MAB and the antibody that reacts with MK does not show cross-reactivity with MTAB and MK,
Abbreviated as an antibody that specifically reacts with AC, that is, a MAC-specific antibody. ). In the method for absorbing the acid-fast bacillus (MAC) polyclonal antibody, in the method described in Example 3, instead of combining the MK cell column and the MAC cell column, the MTAB cell column and the MK cell column are combined. Except that, it carried out according to the said method.

2. MTAB and MK of Absorbed and Unabsorbed Antibodies to Mycobacterium (MAC) Polyclonal Antibodies
By examining the cross-reactivity with mycobacteria (MA)
C) Performance comparison between the absorption-treated antibody and the unabsorbed-treated antibody of the polyclonal antibody was performed.

The unabsorbed antibody to the acid-fast bacillus (MAC) polyclonal antibody has an antibody titer at the time of blood collection of 256 cells.
00 (Abs. 0.776). The anti-acid-fast bacterium (MAC) polyclonal antibody-absorbed antibody is the anti-MAC-specific antibody obtained in 1 above.

The experimental procedure was such that the dead bacteria were adsorbed to the wells at 4 ° C. for 16 hours, and the anti-M was used as a primary antibody.
The procedure was performed in accordance with the method described in Example 3, except that the unabsorbed AC rabbit IgG-treated antibody and the anti-MAC rabbit IgG-absorbed antibody were used.

The absorbance (0.
In comparison with the titer reaching 118), the unabsorbed treated antibody of the anti-acid-fast bacterium (MAC) polyclonal antibody showed 12.5% (2560/2048) of the dead MTAB cells.
0), 50% (10240/2048)
0). As a result, in the unabsorbed antibody of the acid-fast bacillus (MAC) polyclonal antibody, cross-activity with the dead MTAB cells and the dead MK cells was observed. On the other hand, the absorption-treated antibody of the acid-fast mycobacterial (MAC) polyclonal antibody shows that the reactivity of the dead cells with the M is higher than that of the M cells by comparing the titers of the dead cells.
0.8% (20/2560) dead TAB cells, MK
The dead cells were only 0.4% (10/2560).
Thereby, 1. The MAC-specific antibody prepared in
Little cross-reactivity to AB and MK was observed,
It was confirmed that the antibody specifically reacted with MAC.

[0070]

The present invention provides a method for detecting the causative bacteria of respiratory tract infections and a kit for diagnosing respiratory tract infections, which enables rapid, simple and highly sensitive diagnosis of respiratory tract infections. Therefore, it is expected to be widely applied in the diagnosis of respiratory tract infections, and greatly contributes to diagnosis of respiratory tract infections and early treatment of respiratory tract infections. Particularly in the case of M. tuberculosis diagnosis, in the diagnosis of tuberculosis, the sample stained by the most widely used Tiel-Neilsen staining method can be used as it is, so that it is not necessary to prepare the sample again, which is economically excellent. ing.

[Brief description of the drawings]

FIG. 1 is a diagram showing a microscope image of a specimen 3 diagnosed by the method for detecting a causative microorganism of a respiratory tract infection of the present invention.

FIG. 2 is a view showing a microscope image of a specimen 6 diagnosed by the method for detecting a causative microorganism of a respiratory tract infection of the present invention.

FIG. 3 is a diagram showing a microscope image of a specimen 11 diagnosed by the method of detecting a causative microorganism of a respiratory infection according to the present invention.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Riharu Nakajima 461-1 Isshiki, Kazu-cho, Kazu-cho, Kaizu-gun, Gifu Biotechnology Division, Japan Biological Science Center Inc. (72) Inventor Tomohiro Samori Kumiyama-cho, Kuse-gun, Kyoto Ohashibe small letter 16-18 Ohashibe F-term (in reference) 2G045 AA28 BA11 BB50 CB08 DA77 FA18 FB03 FB11 4B063 QA01 QA18 QA19 QQ03 QQ06 QR48 QS03 QS11 QS33 QS36 QX02

Claims (7)

[Claims] 1. A method for detecting a causative bacterium of a respiratory tract infection,
The sputum collected from the subject is brought into contact with an antibody that recognizes a pathogenic bacterium causing respiratory tract infection to form an immune complex, and the pathogen causing the respiratory tract infection contained in the sputum A method for detecting a causative microorganism of a respiratory tract infection, comprising detecting not only intact complete cells but also digested cell fragments. 2. The antibody according to claim 1, wherein said antibody is a combination of a polyclonal antibody recognizing a pathogen causing respiratory tract infection, or a plurality of monoclonal antibodies recognizing different components of the pathogen causing respiratory infection as epitopes. The method for detecting a causative microorganism of respiratory infection according to claim 1, which is a cocktail antibody. 3. The respiratory tract infection is tuberculosis.
3. The method for detecting a causative bacterium of a respiratory tract infection according to any one of claims 2 to 2. 4. The respiratory system according to claim 1, wherein the antibody is an antibody modified with colloidal gold, and the formed immune complex is subjected to a silver sensitization treatment. Method of detecting the causative bacteria of infectious disease. 5. A polyclonal antibody recognizing a pathogen causing respiratory tract infection or a cocktail antibody comprising a large number of monoclonal antibodies recognizing as epitopes different components of the pathogen causing respiratory infection. A respiratory infection diagnostic kit for detecting not only undigested intact cells of pathogenic bacteria causing respiratory infection contained in sputum but also digested bacterial fragments. 6. The diagnostic kit for respiratory tract infection according to claim 5, wherein the antibody is an antibody modified with colloidal gold. 7. The diagnostic kit for respiratory tract infection according to claim 6, further comprising silver sensitizing means.