JP2019158627A - Method and device for detecting peculiar antigen of mycoplasma pneumoniae - Google Patents

Abstract

To provide a method and a device for measuring mycoplasma pneumoniae easily and rapidly by finding an antigen protein peculiar to mycoplasma pneumoniae of which antibody can detect mycoplasma pneumoniae sensitively and by preparing and using the antigen.SOLUTION: There are provided a method for detecting mycoplasma pneumoniae which detects one or at least two of proteins coded by at least one of mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574, and a method for detecting mycoplasma pneumoniae using the antibody of the detected protein by an immunological technique. The immunological technique may include using ELISA or immunochromatographic assay. There is also provided a vaccine which uses the protein as an antigen.SELECTED DRAWING: None

Description

  The present invention relates to a detection method and a detection device for Mycoplasma pneumoniae using a newly found Mycoplasma pneumoniae-specific antigen.

  In recent years, the separation rate of Mycoplasma pneumoniae (M. pneumoniae) from patients with atypical pneumonia has increased. Mycoplasma pneumoniae is one of the causative bacteria that cause pneumonia and bronchitis, especially in children, and is the smallest microorganism that can self-grow on the medium. Since this pathogen does not have a cell wall, β-lactam antibacterial agents, which are cell wall synthesis inhibitors, do not work. Oral β-lactam antibacterial drugs are frequently used for upper respiratory tract infections in children, but as described above, they are ineffective for Mycoplasma pneumoniae and may be severely refractory. Therefore, it is very important to diagnose upper respiratory tract infections caused by Mycoplasma pneumoniae.

  Diagnosis of mycoplasma pneumonia includes complement binding reaction (CF), passive agglutination reaction (PA), fluorescent antibody method, genetic test, and culture method. However, since it takes several weeks for the antibody to rise, the result of CF or PA is only a definitive diagnosis after the end of treatment. Furthermore, in recent years, diagnostic methods using gene detection techniques have been implemented. However, since special equipment and skilled operation techniques are necessary, the number of medical facilities that can be implemented is limited. . In addition, detection of the pathogen itself is the most reliable diagnostic method, but it takes several weeks for its growth, so the culture method is not suitable as a diagnostic method for therapeutic purposes.

At present, in order to solve the above problems, a method for detecting Mycoplasma pneumoniae in a short time by detecting an antigen specific to Mycoplasma pneumoniae using an antigen-antibody reaction has been studied or put into practical use.
For example, a monoclonal specific to P1 protein synthesized from MPN141 (P1) of MPN688 to Mycoplasma pneumoniae gene MPN1 described in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/NC_000912.1) A detection method using an antibody is described in Patent Document 1.
In Patent Document 2, 50s ribosomal protein synthesized from MPN539 (rplL) of MPN688 from Mycoplasma pneumoniae gene MPN1 described in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/NC_000912.1) A detection method using an antibody specific for L7 / L12 is described.

JP 2013-72663 A JP 2009-131174 A

  An object of the present invention is to discover a Mycoplasma pneumoniae-specific antigen protein that can detect Mycoplasma pneumoniae with high sensitivity, and to prepare and use the antibody, thereby easily and quickly measuring the Mycoplasma pneumoniae. And to provide a measuring device.

  In order to achieve the above object, the present inventor has arrived at the present invention by finding a mycoplasma pneumoniae-specific antigen protein using genomic information of mycoplasma pneumoniae. That is, the present invention is as follows.

1. A method for detecting Mycoplasma pneumoniae, comprising detecting one or more of the following proteins (i) or (ii):
(I) The protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574 (ii) In the amino acid sequence of the protein of (i), one or more amino acids are deleted, substituted or added A mycoplasma pneumoniae-specific and antigenic protein 2. Use one or more of the following antibodies (i) or (ii) to detect mycoplasma pneumoniae by an immunological technique Method.
(I) Antibody against a protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574 (ii) Amino acids of the protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574 An antibody against an antigenic protein that is specific for Mycoplasma pneumoniae and has one or more amino acids deleted, substituted or added in the sequence 3. The immunological technique is Enzyme-Linked ImmunoSorbent Assay (ELISA) or immunochromatography 2. The method for detecting Mycoplasma pneumoniae according to 2 above, which is a graphic assay.
4. The method for detecting Mycoplasma pneumoniae according to 2 or 3 above, wherein the immunological technique uses a sandwich method.
5. A Mycoplasma pneumoniae detection device that detects Mycoplasma pneumoniae by the detection method according to any one of 1 to 4 above.
6. In the amino acid sequence of the protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574, or the protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574, Alternatively, a vaccine in which a plurality of amino acids are deleted, substituted or added, and is Mycoplasma pneumoniae-specific protein having antigenicity, or a partial peptide thereof.

  By using the detection method and detection device for Mycoplasma pneumoniae of the present invention, it becomes possible to detect Mycoplasma pneumoniae in a short time using a different antigen protein or an antibody against it.

The results of measuring the mycoplasma pneumoniae specificity of each antigen by measuring various microbial cells by ELISA using the antibodies against the newly found four antigens are shown. The example of a structure of the test strip which is a detection device at the time of measuring by an immunochromatographic assay is shown.

The present invention is described in detail below.
The protein of the present invention is a newly discovered Mycoplasma pneumoniae-specific antigen protein, Mycoplasma pneumoniae M129 described in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/NC_000912.1). Proteins encoded by the strain MPN295, MPN314, MPN479, and MPN574 genes, respectively. By detecting these proteins, the presence or absence of Mycoplasma pneumoniae can be examined. These proteins are preferably detected by immunological techniques using an antigen-antibody reaction for convenience, rapidness, and detection, but they can also be detected by other protein analysis methods such as mass spectrometry. The detection of a protein includes a case where the presence of a protein is clarified by detecting a plurality of peptide fragments or characteristic peptide sequences. The detected protein does not need to match the sequence table exactly, and even in the same Mycoplasma pneumoniae, the sequence may be slightly different from strain to strain, and it can be determined that the protein is the same within the range of differences between strains. For example, detecting them is also included. Further, if an existing protein used for detection of Mycoplasma pneumoniae, for example, P1 protein or L7 / L12 ribosomal protein is also measured and detected, specificity and sensitivity may be improved.

  As the antibody against the protein, any antibody may be used as long as it is an antibody of each protein. A polyclonal antibody or a monoclonal antibody may be used. From the viewpoint of quality uniformity, a monoclonal antibody is used. It is more preferable. One type or two or more types of antibodies may be used. Of course, if the type is an antibody against a different protein, it can be said that it is a different type of antibody, and even if it is against the same protein, it can be said that it is a different type of antibody if it is different. Polyclonal antibodies are considered different types of antibodies each time they are immunized.

  Examples of methods for detecting Mycoplasma pneumoniae by immunological techniques using antibodies include ELISA (Enzyme-Linked ImmunoSorbent Assay) and immunochromatographic assays.

  For the ELISA or immunochromatographic assay, a sandwich method in which an antigen is sandwiched between two antibodies may be used. In this case, the combination of the two antibodies to be sandwiched is not particularly limited as long as it is specific for Mycoplasma pneumoniae, but when a monoclonal antibody is used, the combination may be such that the epitopes do not overlap or are not close together. preferable. Furthermore, it may be used in combination with an existing antibody against Mycoplasma pneumoniae-specific protein. For example, an antibody against a protein encoded by MPN295 and an anti-P1 antibody may be used in combination.

  The detection device is, for example, an ELISA device in which a sample is placed in a well of a plate that has been coated with a solid-phased antibody in advance and a primary reaction is performed, then an enzyme-labeled second antibody is added after washing, and an enzyme reaction is performed, An antibody pre-labeled with a metal colloid etc. in the form of a membrane and a capture antibody is laid on the membrane destination, and a sample droplet is simply dropped onto the membrane-like metal colloid-labeled antibody. And a detection device that uses an immunochromatographic assay.

  The immunochromatographic assay using an antibody against a Mycoplasma pneumoniae-derived specific antigen in the present invention is a method in which high specificity and sensitivity of an antigen-antibody reaction are combined with an excellent chromatographic separation technique. The device is inexpensive and easy to operate, and no special equipment or reagents are required. In addition, since the diagnosis result can be obtained in a short time, it can be examined in the outpatient clinic, and appropriate antibacterial treatment can be performed using the result.

  The specific antigen found in the present invention can also be used as a vaccine for preventing Mycoplasma pneumoniae. One or two or more specific antigens found by the present invention are used, and after inactivating this antigen, it can be used for immunization in combination with an adjuvant. It is preferable to select and use those having higher immunogenicity depending on the types and combinations of specific antigens.

  Next, a method for detecting the presence of Mycoplasma pneumoniae by detecting Mycoplasma pneumoniae-specific antigen, preparing an antibody against the antigen, and detecting the antigen using this antibody will be described.

  As the Mycoplasma pneumoniae-specific antigen, a protein having a low homology with other bacterial species and a high expression level in Mycoplasma pneumoniae is selected using genomic information. Manufacture or obtain selected proteins, inoculate mice and make polyclonal antibodies. Compare ELISA with other bacterial species and reactivity with Mycoplasma pneumoniae using Mycoplasma. -It is possible to narrow down proteins highly specific to pneumoniae and find the best candidates.

By constructing an expression system for this optimal candidate antigen protein, a genetically engineered antigen protein can be obtained, an antibody against this can be prepared, and its specificity and sensitivity can be examined.
Immunization at the time of antibody production can be performed by a general method. For example, the above-mentioned recombinant antigen protein is dissolved in a solvent such as Phosphate buffered saline (PBS), and the dissolved antigen and adjuvant are emulsified and administered subcutaneously or peritoneally to the immunized subject. Mammals and birds are suitable as immunization targets, and mice and rats are preferred. Further, examples of the adjuvant include sodium hydroxide, aluminum hydroxide, calcium phosphate, aluminum phosphate, and liquid paraffin. Preferably, Freund's complete adjuvant or Freund's incomplete adjuvant is used. Although it is desirable to select the dose appropriately for each animal to be immunized, it is preferably about 10 to 100 μg / ml.
In addition, the antigen used for immunization is not necessarily limited to the antigen protein, and a partial peptide thereof can also be used.

  The spleen or lymph node of the immunized animal is taken out, these cells and myeloma cells are fused, and further screened and cloned by a predetermined method, whereby a hybridoma producing the target antibody can be obtained.

  The cloned hybridoma may be grown by the mouse ascites method or by cell culture. The grown antibody can be purified and purified by affinity chromatography.

In order to produce an immunochromatographic assay device using a purified and purified antibody, a labeled antibody, a membrane corresponding to a chromatography carrier, a capture antibody, and the like are required.
There are no particular restrictions on the antibody carrier used for labeling when producing labeled antibodies, but colored latex particles, silica particles, magnetic particles, colloidal gold particles, colloidal platinum particles and the like are preferred, and colloidal gold particles are particularly preferred. When gold colloidal particles are used, the particle size is preferably about 20 to 80 nm, more preferably 30 to 60 nm, and particularly preferably 40 nm.
Examples of the membrane include microporous materials such as glass fiber, nylon, nitrocellulose, polyvinylidene difluoride (PVDF), and cellulose acetate, and microporous materials made of nitrocellulose are particularly preferable.

  In the present invention, for treatment of the sample suspension, which is a sample, in order to isolate Mycoplasma pneumoniae cells in a sample collected from a patient and expose and suspend the antigen recognition site, It is desirable to contain an agent, a buffer solution, and inorganic salts.

  Nonionic surfactants include Tween20 (polyoxyethylene sorbitan monolaurate), TritonX-100 (polyethylene glycol mono-p-isooctylphenyl ether), Brij35 (polyoxyethylene (23) lauryl ether), Brij58 (Polyoxyethylene (20) cetyl ether), NP-40 (Nonidet P40) and the like.

  Examples of the buffer solution include, but are not limited to, a Tris-HCl buffer solution, a Tricine buffer solution, and a Bicine buffer solution. The concentration is preferably about 10 mM to 200 mM.

  The inorganic salts to be contained are not particularly limited, and examples thereof include sodium salts, potassium salts, magnesium salts, and calcium salts. Further, among them, sodium chloride is more preferable.

This sample treated with a surfactant or the like can be subjected to an immunochromatographic assay device to examine the presence or absence of Mycoplasma pneumoniae.

  Hereinafter, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

Example 1
1. Narrowing down Mycoplasma pneumoniae-specific antigenic proteins
Concerning the mycoplasma pneumoniae genes MPN1 to MPN688 described in GenBank (https://www.ncbi.nlm.nih.gov/nuccore/NC_000912.1), the homology of the sequence with other bacterial species was confirmed by BLAST search. A protein with low expression and a high expression level in Mycoplasma pneumoniae was selected with reference to Reference Material 1. The selected proteins were produced and inoculated into mice to produce antibodies. Using these antibodies, cross-reactivity with other bacterial species and reactivity with Mycoplasma pneumoniae were compared by ELISA. As a result, proteins synthesized from MPN295, MPN314, MPN479 and MPN574, which had extremely high Mycoplasma pneumoniae specificity, were found as antigens.
Reference 1: Tobias Maier, Alexander Schmidt et al., Molecular Systems Biology 511, (2011). Quantification of mRNA and protein and integration with protein turnover in a bacterium

2. Production of antigenic protein
DNA was extracted from M. pneumoniae, and the gene of interest encoding the protein antigen was amplified by PCR. The amplified product was incorporated into pETBlue2 plasmid vector and introduced into E. coli NovaBlue to construct an expression plasmid. This Escherichia coli was cultured, the constructed plasmid was extracted and introduced into Escherichia coli Tuner (DE3) pLacI to construct a protein expression system in Eschelichia coli. Furthermore, this E. coli was cultured to produce an antigen protein, and the protein was purified with an affinity column to obtain a recombinant antigen protein.

3. Immunization to mice
Equal amounts of the recombinant antigen protein prepared at 0.2 to 1.0 mg / mL and Freund's Complete Adjuvant (FCA) were mixed and vortexed, and then an emulsion was prepared with an ultrasonic crusher (sonics vibracell VC-505). 100-500 μl of the immunogen was subcutaneously administered to the mice under BALB / cAJcl (Claire Japan) twice for 2 weeks, and then 2 times later, once for a total of 3 times. One week later, it was confirmed that the antibody titer was elevated by ELISA using blood containing antibody of this mouse, and spleen or lymph nodes were collected.

4). Culture of myeloma cells Roswell Park Memorial Institute 1640 (RPMI1640) medium supplemented with penicillin, streptomycin, amphotericin B, Fetal Bovine Serum (FBS) added at a rate of 10% of mouse myeloma cells (P3X63-Ag8.653) Culture was performed.

5). Preparation of Myeloma Cells and Mouse-Derived Cells Spleens or lymph nodes collected from mice were shredded with scissors and passed through a 75 μm mesh with a rubber policeman while washing with serum-free medium. After washing at 1,000 rpm for 5 minutes, the cells were counted. Thereafter, spleen cells or lymph node cells: myeloma cells were prepared at about 5: 1, centrifuged at 1,000 rpm for 5 minutes, and the cell pellet was incubated at 37 ° C. in warm water for 2 minutes.

6). Cell fusion
1 mL of polyethylene glycol 1500 (PEG1500; Roche) kept at 37 ° C. was added to the prepared cell pellet with slow stirring over 1 minute, and then stirring was continued for 1 minute. Furthermore, 4.5 mL of RPMI1640 medium supplemented with penicillin, streptomycin and amphotericin B kept at 37 ° C. was added with gentle stirring for 3 minutes, and further 4.5 mL of the same medium was added with gentle stirring for 2 minutes. Centrifugation was performed at 1,000 rpm for 5 minutes, penicillin, streptomycin, amphotericin B and 20 mL of GIT medium supplemented with 10% FBS were added to the cell pellet and suspended, followed by centrifugation at 1,000 rpm for 5 minutes. Then, cells were suspended in a GIT medium containing 10% FBS supplemented with hypoxanthine-aminopterin-thymidine (HAT) supplement (SIGMA) in a medium containing BM condimedH1 (Roche), dispensed into a 96-well plate, 37 ° C, Cultivation was performed for 7 to 10 days under 5% CO ₂ .

7). Hybridoma screening (ELISA)
M. pneumoniae (ATCC29342) bacterial cell disruption solution diluted to 5 μg / ml with carbonate-bicarbonate buffer (pH 9.3) was dispensed into a 96-well plate for ELISA at 50 μl / well and allowed to react overnight by refrigeration. After washing twice with carbonate-bicarbonate buffer (pH 9.3), 100 μl / well of 0.5% Bovine Serum Albumin (BSA) -added carbonate-bicarbonate buffer (pH 9.3) was added, and the mixture was added at room temperature. The reaction was allowed to proceed for blocking. Subsequently, the culture supernatant after culturing the hybridoma for about 7 days to 10 days was diluted 2-fold with a 0.1% Triton X-100 added buffer, dispensed at 50 μl / well, and reacted at room temperature for 2 hours. Next, the plate was washed 4 times with 0.1% Triton X-100 added buffer, and enzyme-labeled anti-mouse Ig antibody diluted 5,000 times with the same buffer was reacted at room temperature for 1.5 hours. The plate was washed again with 0.1% Triton X-100 addition buffer, and the TMB substrate solution was dispensed at 50 μl / well. After 15 minutes of reaction, 1 M phosphoric acid was added at 50 μl / well to stop the reaction. Subsequently, the absorbance at 450 nm was measured with a microplate reader.

8). Hybridoma cloning
Hybridomas for which antibody production was confirmed by ELISA were cloned by the limiting dilution method. The medium used was a GIT medium supplemented with 10% FBS and hypoxanthine-thymidine (HT) supplement (SIGMA).

9. Mouse ascites
0.5 ml of ascites inducing reagent pristane was inoculated intraperitoneally into mice 10 days and 3 days before hybridoma inoculation, and 1 × 10 ⁶ to 10 ⁷ hybridoma cells were inoculated. And ascites was collected after 1-2 weeks.

10. Antibody purification Ab-Rapid SPiN EX (Protenova) was used for antibody purification. PBS was used for binding to the column and washing, 0.1 M sodium citrate buffer pH 4.0 was used for antibody elution, and 1 MTris was used for neutralization of the eluted IgG antibody. The purified IgG antibody was dialyzed with PBS and stored at -20 ° C.

11. Detection of Mycoplasma pneumoniae by ELISA method Cell disruption antigen is 5 μg / ml (Here, the protein concentration in the cell disruption solution is the concentration of the cell disruption antigen. The same shall apply hereinafter.) Recombinant protein antigen is 1 μg / ml. Diluted with carbonate-bicarbonate buffer (pH 9.3), dispensed 50 μl per well, left at 4 ° C. overnight, and then shaken at room temperature for 4 hours, carbonate-bicarbonate buffer (pH 9.3). ) Was dispensed 100 μl per well and shaken for 1 minute at room temperature twice. Dispense 100 μl of 0.5% BSA-added carbonate-bicarbonate buffer (pH 9.3) per well, shake for 1 hour at room temperature, and add Mycoplasma pneumoniae-specific antibody to 0.1% TritonX-100 + 1% BSA-added Tris buffer. The diluted solution was diluted 50 μl per well and allowed to react for 2 hours. After the reaction, 100 μl of 0.1% TritonX-100-added Tris buffer was dispensed per well, and washing was repeated 4 times at room temperature for 1 minute. The HRP-labeled anti-mouse antibody was diluted 5,000-fold with 0.1% TritonX-100 + 1% BSA-added Tris buffer, dispensed at 50 μl per well, and allowed to react at room temperature for 1.5 hours. Washing was performed by dispensing 100 μl of 0.1% TritonX-100-added Tris buffer per well and repeating shaking four times for 1 minute at room temperature. Dispense 50 μl of TMB reagent solution per well, and after 10-15 minutes at room temperature, dispense 50 μl of 1M phosphoric acid per well to stop the reaction. Absorbance at 450 nm using a microplate reader (BIO-TEK) Was measured. These results are shown in FIG.

  From the results shown in FIG. 1, the Mycoplasma pneumoniae-specific antibody in MPN295, 314, 479, and 574 showed a positive reaction to Mycoplasma pneumoniae cell antigen and recombinant protein. None of the mycoplasma other than Mycoplasma pneumoniae, general bacteria, fungal cell antigens, etc. showed any cross reaction. That is, the protein antigen found this time was found to be extremely high in Mycoplasma pneumoniae specificity.

12 Preparation of antibody-immobilized membrane A nitrocellulose membrane (MILLIPORE; HF180) was used as the membrane. A 10 mM sodium phosphate buffer solution (pH 7.0) containing 5% 2-propanol and a monoclonal antibody prepared to a final concentration of 1 mg / ml was applied linearly on the membrane and dried at room temperature for 1 hour. . After drying, immerse the membrane in 200 ml of 20 mM sodium phosphate buffer (pH 7.8) containing 0.5% sodium caseinate, shake gently for 20 minutes, discard the solution, and then wash (purify gently with purified water for 5 minutes) ) Was performed twice. Then, it was immersed in a 3% sucrose solution, shaken slowly for 10 minutes, the solution was dropped firmly and dried overnight at room temperature.

13. Production of test strip as detection device (Figure 2)
A backing sheet as a base material was bonded to the side of the nitrocellulose membrane carrier opposite to the side on which the antibody was applied. Next, an absorbent pad was attached so as to overlap 5 mm on the upstream side (control line side) of the nitrocellulose membrane, and the joint between the nitrocellulose membrane and the absorbent pad was fixed with cellophane tape. Finally, it was cut to a width of 5 mm along the longitudinal direction to obtain a test strip for immunochromatographic assay.

14 Preparation of colloidal gold-labeled antibody A purified monoclonal antibody was prepared at 60 μg / mL with 20 mM MOPS (pH 7.0). 100 μl of 0.2M MOPS (pH 7.0) was added to and mixed with 900 μl of a gold colloid solution (Tanaka Kikinzoku Kogyo: average particle size 40 nm), and then 100 μl of the purified monoclonal antibody was added and mixed, and then allowed to stand at room temperature for 20 minutes. Next, 250 μl of 20 mM MOPS (pH 7.0) to which 5% sodium caseinate was added was added and left at room temperature for 20 minutes, followed by centrifugation at 12,000 × g for 5 minutes. After removing the supernatant, 1 ml of 10 mM Tris-HCl buffer (pH 8.2) supplemented with 0.1% casein Na was added and centrifuged at 12,000 × g for 5 minutes. After repeating this operation twice, 200 μl of 0.1% casein Na-added 10 mM Tris-HCl buffer (pH 8.2) was added and dispersed well to obtain a colloidal gold labeled antibody solution.

15. Detection of Mycoplasma pneumoniae antigen by immunochromatographic assay [antigen preparation]
The stock bacterial solution was added to PPLO medium and cultured for about 10 days under an aerobic condition at 37 ° C. The culture solution was centrifuged at 10,000 × g for 20 minutes, the supernatant was removed, resuspended in PBS, and centrifuged at 25,000 × g for 20 minutes. After centrifugation, the suspension was resuspended in a small amount of PBS, transferred to a microtube, and centrifuged at 20,000 × g for 20 minutes. After centrifugation, the cells were resuspended in PBS about 9 times the wet weight of the cells, and the cells were crushed with an ultrasonic crusher (sonics vibracell VC-505) to obtain an antigen. The cells were prepared and used at the time of the experiment so that the mycoplasma pneumoniae concentration was about the same as that of infected patients (10 ⁵ to 10 ⁸ CFU / mL).

[Detection of antigen]
The antigen was prepared according to the above preparation method. The test strip prepared by the method described above was used. The colloidal gold labeled antibody was prepared by the method described above. The antigen was prepared to 50 μg / mL with 50 mM Tris-HCl (pH 8.0) buffer containing 0.1% Triton-X100 and 0.1 M NaCl. Add 50 μl / well of the prepared antigen solution to a 96-well microplate, add 10 μl / well of gold colloid-labeled antibody, mix well, and immerse one end of the test strip on the liquid-immobilized membrane side (left side of FIG. 2) for 15 minutes to react. It was. After the reaction, it was determined that “+” indicates that the red line was visually confirmed in the antibody-immobilized line part (FIG. 2 test line) on the membrane, and “−” indicates that it was not confirmed.
The results are shown in Table 1.

From the results shown in Table 1, antibodies against Mycoplasma pneumoniae-specific antigen encoded by MP314 and MPN574 showed a positive reaction against any Mycoplasma pneumoniae. No other cross-reactions were observed with other mycoplasmas except for Mycoplasma pneumoniae and general bacteria and fungi.
That is, it was found that Mycoplasma pneumoniae can be detected very specifically in a short time by measuring the protein antigen found this time using an immunochromatographic assay.

  In addition, Table 2 shows the results of detecting the antigen by immunochromatography using an anti-MPN574 protein antibody and detecting the antigen by varying the cell disruption antigen concentration and examining the sensitivity.

From the results shown in Table 2, mycoplasma pneumoniae-infected patients (10 ⁵ to 10 ⁸ CFU / mL) can be detected at a cell disruption antigen concentration of 0.0024 μg / mL (equivalent to approximately 4 × 10 ⁴ CFU / mL). It was found that the detection was possible at a concentration sufficiently lower than the concentration in the sample, and the sensitivity was sufficiently high.

According to the present invention, Mycoplasma pneumoniae can be detected simply and quickly,
Examination can be performed at the outpatient clinic, and appropriate antibacterial therapy can be performed using the results.

Claims (6)

A method for detecting Mycoplasma pneumoniae, wherein one or more of the following proteins (i) or (ii) are detected.
(I) The protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574 (ii) In the amino acid sequence of the protein of (i), one or more amino acids are deleted, substituted or added And Mycoplasma pneumoniae specific and antigenic protein A method for detecting Mycoplasma pneumoniae by an immunological technique using one or more of the following antibodies (i) or (ii).
(I) Antibody against a protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574 (ii) Amino acids of the protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, and MPN574 An antibody against a protein having antigenicity, wherein one or a plurality of amino acids are deleted, substituted or added in the sequence, and are mycoplasma pneumoniae-specific   The method for detecting Mycoplasma pneumoniae according to claim 2, wherein the immunological technique is Enzyme-Linked ImmunoSorbent Assay (ELISA) or immunochromatographic assay.   The method for detecting Mycoplasma pneumoniae according to claim 2 or 3, wherein the immunological technique uses a sandwich method. A mycoplasma pneumoniae detection device for detecting mycoplasma pneumoniae by the detection method according to claim 1. One or more amino acid sequences of the protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, MPN574, or the protein encoded by any one of the Mycoplasma pneumoniae genes MPN295, MPN314, MPN479, MPN574 A vaccine in which a single amino acid is deleted, substituted or added, and is Mycoplasma pneumoniae-specific, antigenic protein, or a partial peptide thereof.