METHOD FOR DETECTING MICROORGANISMS BELONGING TO MYCOPLASMA PNEUMONIAE AND/OR MYCOPLASMA GENITALIUM

The present invention relates to a method and a reagent kit for detecting microorganisms belonging to Mycoplasma pneumoniae which is, in general, a pathogenic microorganism for pneumonia, and/or Mycoplasma genitalium, using a molecule specific to the detection of the microorganisms as an indicator.

(1) Patient Ratio and Symptoms of Mycoplasma pneumoniae Pneumonia

Mycoplasma pneumoniae infections are classified as a community-acquired atypical pneumonia, and it is said that the proportion of Mycoplasma pneumoniae infections in community-acquired pneumonia amounts to 30 to 40% in adults and to even 60 to 70% when the adults are limited to young adults aged 15 to 25. The infection route of Mycoplasma pneumoniae is a respiratory tract infection, and it is not rare that such infections spread in facilities such as schools, and in families. In addition, in the Mycoplasma pneumoniae infections, pneumonia occurs in about 3 to 5% of the infections, and the remainder is bronchitis, upper respiratory tract inflammation, or inapparent infection. Characteristic symptoms include an obstinate cough that is not accompanied by expectoration from an early period of infection, and may be sometimes accompanied by symptoms such as fever, headache, pharyngeal pain, chills, or general malaise.

(2) Current Status of Screening for Mycoplasma pneumoniae Infections

A screening test of the culture from a pharyngeal swab sample in patients and an antibody screening test using a patient's serum are common as screening tests for Mycoplasma pneumoniae infections. Since Mycoplasma pneumoniae per se grows only in a special culture medium, the culturing is difficult to execute, and it is necessary to perform a PCR test for the final identification of Mycoplasma pneumoniae, the culture screening can be carried out only in limited facilities, and this is a current status of the screening test of the culture. In addition, a screening test to quickly obtain the results has been demanded, because several weeks are needed for the culture.

On the other hand, because the antibody screening test is generally easy in the procedure and provides results more quickly compared to the screening test of the culture, such an antibody screening test is a test that has been well used. But there are problems such that it is difficult to determine whether the infection is a previous one or a current one because IgM antibody titers of Mycoplasma are long-lasting, and it takes a long time to increase the antibody titers. In order to solve the above problems, the judgment based on rise in the antibody titers between the acute phase of the infection and the convalescent phase of the infection over time is recommended, but since it takes a very long time to perform an antibody testing until the convalescent phase, therapy is delayed, so that its delay may cause prolongation and worsening of symptoms, as well as may cause the adverse effect of infection expansion due to secondary infection.

In addition, in order to solve the above problems, antibodies and detection methods for specifically detecting a microorganism belonging to Mycoplasma pneumoniae, which are useful for diagnosing Mycoplasma pneumoniae infections, have been disclosed.

For example, patent literature 1 describes an immunodetection method using a monoclonal antibody against a membrane protein antigen of Mycoplasma pneumoniae of about 43 kilodaltons (kDa). Also, patent literature 2 describes that detection of Mycoplasma pneumoniae can be performed with high accuracy by using an antibody against ribosomal protein L7/L12. In addition, patent literature 3 describes that a quick and specific diagnosis of Mycoplasma pneumoniae infection is possible by using a monoclonal antibody against protein P1 of Mycoplasma pneumoniae, the monoclonal antibody having a cross-reactivity of only 1% or less to other species of the genus Mycoplasma or other pathogenic species of coexisting flora.

However, in order to detect a microorganism belonging to Mycoplasma pneumoniae in clinical specimens, the antibody described above and the detection method using the antibody may require a complicated pretreatment of the specimens containing the microorganism, and have a problem such that they are insufficient for a specific diagnosis of Mycoplasma pneumoniae because of still low specificity and sensitivity.

(3) Mycoplasma genitalium and Diseases

Chlamydia trachomatis is known as the major causative bacteria of nongonococcal urethritis. However, Chlamydia trachomatis is detected in about 30 to 40% of patients with nongonococcal urethritis, and, in most cases, it is not clear where their symptoms originate. In addition to Chlamydia trachomatis, microorganisms of the genera Mycoplasma and Ureaplasma have attracted attention, and Mycoplasma genitalium in particular is shown as one of the causative bacteria of nongonococcal urethritis and sexually transmitted disease.

(4) Current Status of Screening for Mycoplasma genitalium Infections

Reports of Mycoplasma genitalium infections by the culture method or the PCR method have been published in papers, but since a quick diagnosis cannot be performed by these methods, a method for detecting quickly and specifically a microorganism belonging to Mycoplasma genitalium in clinical specimens has been demanded.

• [Patent literature 1] Japanese Unexamined Patent Publication (Kokai) No. 63-298
• [Patent literature 2] WO2001/057199
• [Patent literature 3] Japanese Unexamined Patent Publication (Kokai) No. 5-304990

According to conventional methods, microorganisms belonging to Mycoplasma pneumoniae and/or Mycoplasma genitalium could not be quickly and specifically detected. Therefore, since it is not possible to quickly diagnose the infections from Mycoplasma pneumoniae and/or Mycoplasma genitalium, therapy is delayed, and its delay may cause prolongation and worsening of symptoms, as well as the adverse effect of infection expansion due to secondary infection. This is the current situation. If infections with these Mycoplasmas can be quickly detected/diagnosed, it becomes possible to administer a macrolide antibiotic effective for the Mycoplasmas and start the correct treatment in the early infection stage.

Further, Mycoplasma pneumoniae and Mycoplasma genitalium are known to be serologically very close to each other, but since the infection site (such as tissues and organs) of Mycoplasma pneumoniae is different from that of Mycoplasma genitalium as mentioned above, if it is possible to identify a molecule capable of specifically detecting the two microorganisms, it was considered that diagnosis of both infectious diseases becomes possible using the molecule as an indicator.

The present invention has been made in view of the problems. The object of the present invention is to specify a molecule to rapidly and specifically diagnose the Mycoplasma pneumoniae and/or Mycoplasma genitalium infections, and to provide a detection method and a detection kit using the molecule as an indicator.

Under these circumstances, the present inventors have conducted intensive studies, and have found that DnaK of microorganisms belonging to Mycoplasma pneumoniae and/or Mycoplasma genitalium can be used as an indicator to rapidly and specifically detect Mycoplasma pneumoniae and/or Mycoplasma genitalium infections. The DnaK protein, which is also called Heat Shock Protein 70 (Hsp70), was found as a group of proteins the expression of which was increased when cells were exposed to stress conditions such as heat to protect the cells, and is currently known to participate in the intracellular transport or refolding (molecular chaperone function) of proteins translated. The advantages in using the DnaK protein as an index in immunological analysis methods reside in the facts that:

(1) the DnaK protein is always expressed, because it participates in the transport or refolding of proteins,
(2) DnaK accounts for approximately 1% of the total proteins, and
(3) DnaK is present, not as a monomer, but as a trimer, a hexamer, or further multimers.

The present invention has been achieved based on these findings.

The present invention provides as follows:

[1] A method for detecting Mycoplasma pneumoniae or Mycoplasma genitalium, characterized by using DnaK of Mycoplasma pneumoniae or Mycoplasma genitalium as an indicator.
[2] The method of, wherein a DnaK protein is immunologically analyzed.
[3] An anti-DnaK antibody specific to Mycoplasma pneumoniae or Mycoplasma genitalium.
[4] A kit for detecting Mycoplasma pneumoniae or Mycoplasma genitalium, comprising the anti-DnaK antibody of.
[5] The method of, using a DnaK gene as an indicator.
[6] A primer or probe specific to Mycoplasma pneumoniae or Mycoplasma genitalium.
[7] A kit for detecting Mycoplasma pneumoniae or Mycoplasma genitalium, comprising the primer or probe of.

The term “the microorganisms” as used herein means Mycoplasma pneumoniae or Mycoplasma genitalium, in particular, microorganisms which have pathogenicity and are significant to be diagnosed as microorganisms causative of the diseases described below.

The term “antibody which specifically reacts with the microorganisms” as used herein means an antibody which specifically reacts with the species or the genus of the microorganisms. An antibody which specifically reacts with the species of the microorganisms is particularly useful in the diagnosis of microorganism infections.

According to the method for detecting microorganisms belonging to Mycoplasma pneumoniae and/or Mycoplasma genitalium using the specific molecule in the present invention as an indicator, Mycoplasma pneumoniae and/or Mycoplasma genitalium infections caused by the microorganisms can be rapidly and specifically diagnosed.

The present invention now will be further illustrated in detail by, but is by no means limited to, the following embodiments of the present invention as typical examples.

The method of the present invention for detecting Mycoplasma pneumoniae or Mycoplasma genitalium is characterized in that DnaK of the microorganisms is used as an indicator to detect Mycoplasma pneumoniae and/or Mycoplasma genitalium (i.e., either of Mycoplasma pneumoniae or Mycoplasma genitalium, or both Mycoplasma pneumoniae and Mycoplasma genitalium, most preferably, both Mycoplasma pneumoniae and Mycoplasma genitalium). Mycoplasma pneumoniae and/or Mycoplasma genitalium infections can be diagnosed by detecting these microorganisms.

The Mycoplasma pneumoniae infection which may be diagnosed by the present invention is mycoplasmal pneumonia. The Mycoplasma genitalium infection is non-gonococcal non-chlamydial urethritis or cervicitis.

In the diagnosis of the Mycoplasma pneumoniae infection, a sample in which Mycoplasma pneumoniae may exist may be used. Examples of the sample include pharyngeal swab, nasopharyngeal swab, nasal aspirate, nasal mucus, sputum, and bronchoalveolar lavage fluid. When the Mycoplasma genitalium infection is to be diagnosed, a sample in which Mycoplasma genitalium may exist may be used. Example of the sample include urine, urethral swab specimens, and cervical swab specimens. The identification of the two infections can be decided by the collection site of the sample as a target for measurement.

DnaK which is used as an indicator in the present invention is a DnaK protein (NCBI number: NP_—110122) or a DnaK gene (NCBI number: NC_—000912 REGION: 521837.523624) derived from Mycoplasma pneumoniae, or a DnaK protein (NCBI number: AAC71527) or a DnaK gene (NCBI number: L43967 REGION: 374919.376706) derived from Mycoplasma genitalium. The above-mentioned proteins and genes are examples of a strain belonging to the microorganisms, and the DnaK sequences of the microorganisms within the scope of the present invention include sequences corresponding to the DnaK proteins and genes described above.

As shown in Example 8 described below, the DnaK genes derived from different Mycoplasma pneumoniae strains absolutely (100%) accorded with each other, even among strains in which the types of the P1 gene of Mycoplasma pneumoniae were different, and no variations were detected among strains collected from various places and over the past 50 years. From this, it is considered that the sequences of the DnaK gene and the DnaK protein of Mycoplasma pneumoniae are stable. Therefore, it is preferable to refer to the nucleotide sequences or the amino acid sequences published by NCBI as described above.

The first embodiment of the method for detecting Mycoplasma pneumoniae or Mycoplasma genitalium of the present invention is characterized by using an anti-DnaK antibody specific to the microorganisms. When the specific antibody is selected, the specificity to the microorganisms is at least 10⁵CFU/mL or higher, preferably 10⁴CFU/mL or higher, and more preferably 10³CFU/mL, and the specificity to the other microorganisms is at least 10⁷CFU/mL or lower, and preferably 10⁸CFU/mL or lower.

The antibody which may be used in the present invention may be a polyclonal antibody or a monoclonal antibody. These antibodies may be obtained by the following methods or other similar methods, but the method is not limited to the same.

As the first embodiment of the method for preparing the antibody, the complete length of the DnaK protein or its partial peptide may be used to prepare the antibody. With respect to microorganisms of which the nucleotide sequence and the amino acid sequence of the DnaK protein are known, a peptide fragment may be synthesized based on a region which shows less similarity to the amino acid sequences of DnaK proteins of other microorganisms. The length of the peptide for preparing the antibody is not limited, but in the case of the antibody against the DnaK protein, a peptide having a length capable of characterizing the protein, preferably 5 amino acids or more, and most preferably 8 amino acids or more, may be used. This peptide or the complete length of the protein alone, or a conjugate thereof crosslinked with a carrier protein such as KLH (keyhole-limpet hemocyanin) or BSA (bovine serum albumin), may be inoculated into an animal, optionally along with an adjuvant, and a serum is collected from the animal to obtain an antiserum containing an antibody (polyclonal antibody) which recognizes the DnaK protein. An antibody which is purified from the antiserum may be used. Examples of the animal which may be inoculated include a sheep, a horse, a goat, a rabbit, a mouse, and a rat, and a rabbit or a goat is preferable for preparing a polyclonal antibody. A monoclonal antibody may be obtained in accordance with a known method for preparing hybridoma cells, and a mouse is preferable in this case.

A fusion protein of the complete length or an amino acid sequence consisting of 5 residues or more (preferably 8 residues or more) of the protein with glutathione S-transferase or the like may be used as an antigen, after purification of the fusion protein, or without purification. The antibody may be also prepared by a genetically recombinant antibody expressed in culture cells using an immunoglobulin gene isolated by a gene cloning method and various methods described in the publication: Antibodies; A laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory Press.

From the antibodies prepared as described above, an antibody having a high specificity may be prepared by selecting an antibody which specifically reacts with Mycoplasma pneumoniae and/or Mycoplasma genitalium (i.e., either of Mycoplasma pneumoniae or Mycoplasma genitalium, or both Mycoplasma pneumoniae and Mycoplasma genitalium, most preferably, both Mycoplasma pneumoniae and Mycoplasma genitalium), and does not react with other pathogenic microorganisms, in accordance with a known method.

The antibody against the DnaK which may be used as the marker antigen of the present invention may be obtained by the following methods or other similar methods, but the method is not limited to the same.

a) With respect to microorganisms of which the nucleotide sequence and the amino acid sequence of the DnaK protein are known, a peptide fragment may be synthesized based on a region which shows less similarity to the amino acid sequences of DnaK proteins of other microorganisms, and a polyclonal or monoclonal antibody may be prepared using the peptide fragment as an antigen to obtain the antibody of interest.

The complete length of the nucleotide sequence of the gene may be obtained using common genetic engineering techniques, such as gene amplification by a PCR method using DNA sequences at both the termini of the known gene as primers, or hybridization using a homologous sequence as a template probe.

Next, a protein antigen of interest may be obtained by constructing a fusion gene with other protein genes, introducing the fusion gene into a host such as E. coli by a known gene introduction method, overexpressing the fusion protein, and purifying the expressed protein by an affinity column chromatography method using an antibody against the protein used for preparing the fusion protein. In this case, since the complete length of the DnaK protein becomes antigens, if an antibody against an amino acid region which is conserved between microorganisms outside the scope is obtained, such an antibody cannot be used in the present invention. Therefore, with respect to an antigen obtained by this method, the antibody of interest may be obtained by obtaining hybridomas producing monoclonal antibodies and selecting a clone producing an antibody which specifically reacts with the microorganisms.

b) With respect to microorganisms of which the amino acid sequence of the DnaK protein is unknown, since the amino acid sequences of the DnaK protein have a homology of 80-1000, preferably 90-100%, between different species, the protein gene of interest may be easily obtained using common genetic engineering techniques, such as gene amplification of a specific sequence region by a PCR method based on a sequence homologous to the amino acid sequence, or hybridization using a homologous sequence as a template probe.

The protein antigen of interest may be obtained by constructing a fusion gene of the protein gene with other protein genes, introducing the fusion gene into a host such as E. coli by a known gene introduction method, overexpressing the fusion protein, and purifying the expressed protein by an affinity column method using an antibody against the protein used for preparing the fusion protein. In this case, since the complete length of the DnaK protein becomes antigens, if an antibody against an amino acid region which is conserved between microorganisms outside the scope is obtained, such an antibody cannot be used in the present invention. Therefore, with respect to an antigen obtained by this method, the antibody of interest may be obtained by obtaining hybridomas producing monoclonal antibodies and selecting a clone producing an antibody which specifically reacts with the microorganisms.

c) As another method in the case that the amino acid sequence of the DnaK protein is unknown, a synthetic peptide consisting of 5-30 amino acids corresponding to a common sequence region which are conserved between microorganisms in known amino acid sequences of the DnaK protein is prepared, and a polyclonal or monoclonal antibody is prepared using the peptide sequence in accordance with a known method. A highly purified DnaK protein may be obtained by purifying a cell homogenate of a microorganism of interest by affinity column chromatography using the antibody. When the purity of the protein is not sufficient, the purity may be improved by a known purification method, such as ion-exchange chromatography, hydrophobic chromatography, or gel filtration. The antibody of interest may be obtained by obtaining hybridomas using the obtained purified DnaK protein antibody, and selecting a hybridoma producing an antibody which specifically reacts with the microorganisms.

As the second embodiment of the method for preparing the antibody, Mycoplasma pneumoniae may be used as an antigen to prepare an antibody which reacts with the DnaK protein and is specific to Mycoplasma pneumoniae and/or Mycoplasma genitalium, as shown in Example 1.

Similarly, Mycoplasma genitalium may be used as an antigen to prepare the antibody. In the case that the microorganism is used as an antigen, the antigen may be prepared in accordance with a known method. Examples of the method include sonication, heat treatment, surfactant treatment, formalin treatment, freezing and thawing treatment, and treatment with hydrochloric acid.

The antibody of the present invention which is obtained by the methods described above and is specific to the microorganisms may be used in various immunological assays, and various detection reagents and kits specific to the microorganisms of interest may be provided.

The antibody may be used in any of the known immunological assays, for example, an agglutination method using polystyrene latex particles onto which the antibody is bound, an ELISA method carried out in a microtiter plate, immunochromatography, or a sandwich method using the antibody labeled with colored particles, particles capable of developing a color, magnetic particles, an enzyme, or a fluorescent substance, alone or as a combination.

In the detection method using DnaK as an indicator of the present invention, Mycoplasma pneumoniae and/or Mycoplasma genitalium may be specifically detected without intentionally disrupting the cells, or a known method for treating microorganisms may be used to carry out detection with high sensitivity. More particularly, a treatment method using an extraction reagent comprising various surfactants such as Triton X-100, Tween-20, or SDS, an enzyme treatment method using an appropriate enzyme such as a protease, or a known method for disrupting a cell structure, such as a disruption of microorganism cells by a physical method, may be used. It is preferable that optimum conditions for extraction are selected for each microorganism by examining the combination of reagents such as surfactants.

The reagent kit for detecting the microorganism using the antibody of the present invention corresponds to the reagent kit for detection using the detection method.

The kit is not limited, so long as it contains at least one antibody of the present invention. The number, type, and combination of the antibodies used may be appropriately changed in accordance with the immunological assay to be used. The kit may contain a liquid for pretreatment in the extraction method described above, as a pretreatment of a sample.

As a method for extracting DNA, a known method may be used. Examples of the method include a solubilization of a sample with a surfactant, or deproteinization using a deproteinization agent, to obtain DNA. Preferably, so long as the DnaK gene as described below may be analyzed, for example, when the gene extracted is next amplified by a PCR method, the DNA preferably contains no inhibitors of PCR reaction.

As a method for pretreating a sample, a similar approach as described in the method for detecting the microorganisms using an antibody may be used.

The amount of DNA extracted is not limited so long as an amount capable of analyzing the DnaK gene is extracted. When the DNA is subjected to a PCR method, the amount is, for example, 5 to 50 fg or more per reaction.

The DNA extracted is used to analyze the DnaK gene. The analysis of the DnaK gene may be carried out in accordance with a known method. Examples of the method include a method for detecting the amplification of the DnaK gene by a PCR method, and a method for specifying the DnaK gene by a probe method. For example, any method for amplifying the DnaK gene by a PCR method may be used, so long as the nucleotide sequence of interest may be amplified. Any method for specifying the DnaK gene by a probe method may be used, so long as the nucleotide sequence of interest may be specified.

To amplify or specify the desired nucleotide sequence of the DnaK gene, a sequence having an 80-100% homology with respect to Mycoplasma pneumoniae and/or Mycoplasma genitalium and having a homology of preferably 60% or less with respect to other pathogenic microorganisms may be appropriately selected. The primer or probe may contain one or more variations, deletions, or additions in its nucleotide sequence, so long as the DNA fragment of interest may be amplified.

For example, when the DnaK gene of Mycoplasma pneumoniae is to be amplified, PCR amplification primers may be designed on the basis of the DnaK gene sequence of Mycoplasma pneumoniae (NCBI number: NC_—000912 REGION: 521837.523624) published in NCBI, as described in the Examples below. More particularly, sense primer MpDnaK_S and antisense primer MpDnaK_A may be used to amplify the complete length of the DnaK gene.

When the DnaK gene of Mycoplasma genitalium is to be amplified, PCR amplification primers may be designed on the basis of the DnaK gene sequence of Mycoplasma genitalium (NCBI number: L43967 REGION: 374919.376706) published in NCBI.

As shown in Example 8, the DnaK genes derived from different Mycoplasma pneumoniae strains absolutely (100%) accorded with each other, even among strains in which the types of the P1 gene of Mycoplasma pneumoniae were different, and no variations were detected among strains collected from various places and over the past 50 years. From this, it is not necessary to take into consideration the difference between Mycoplasma pneumoniae strains in order to specifically detect the DnaK gene of Mycoplasma pneumoniae, and the primers or probe may be designed by taking into consideration the differences among the strains other than Mycoplasma pneumoniae.

Further, because it is considered that the sequence of the DnaK protein of Mycoplasma pneumoniae is also conservative, antibodies prepared using the DnaK protein are considered to show no difference in reactivity with respect to the genotype, the place for collection, and the time of collection, and thus, may be used over a wide area and time.

The reagent kit for detecting the microorganisms using the gene of the present invention corresponds to the reagent kit for detection using the detection method. This is a kit which is used for the method for specifically detecting Mycoplasma pneumoniae and/or Mycoplasma genitalium and which is characterized by comprising at least two types of primers for amplifying a nucleotide sequence specific to the DnaK gene of interest.

As another embodiment, the kit is characterized by comprising at least one type of probe for specifying a nucleotide sequence specific to the DnaK gene of interest.

These kits may further contain a liquid for pretreatment in the extraction method described above, as a pretreatment of a sample.

The present invention now will be further illustrated by, but is by no means limited to, the following Examples.

(1) Preparation of Monoclonal Antibodies Specific to Mycoplasma pneumoniae and Mycoplasma genitalium
(1-1) Cultivation of strains for immunization and preparation of immunogens

PPLO glucose broths (containing horse serum, fresh yeast extract, and thallium acetate) were each separately inoculated with one of 6 strains of Mycoplasma pneumoniae (FH, Bru, Mac, M52, PI1428, and M129-B7 strains: purchased from ATCC), and cultivation was carried out at 37° C. for 7 days under aerobic conditions. Each strain collected by centrifugation was washed and suspended in PBS. These suspensions were frozen and thawed to prepare immunogens.

Six-week-old female Balb/c mice (CREA Japan, Inc.) were used for immunization. Each immunogen solution derived from a strain was emulsified with Freund's complete adjuvant (SIGMA). Each emulsion (100 μg of antigen) was subcutaneously injected into a mouse. Until an increase in antibody titer against immunogen was observed in each mouse, 50 μg of each antigen emulsified with Freund's incomplete adjuvant (SIGMA) was subcutaneously injected into the mouse every two weeks. Further, 25 μg of each antigen diluted with PBS was intraperitoneally injected into the mouse three days before cell fusion.

The following procedures were carried out in accordance with a conventional method. Spleen cells aseptically collected from immunized mice were fused with myeloma cells (P3U1) using polyethylene glycol 1500 (Roche), and inoculated into wells of 96-well plates. Hybridoma cells were selectively cultivated using a HAT medium, and their culture supernatants were screened under the following ELISA conditions. Immobilization for ELISA was carried out using Mycoplasma pneumoniae antigen (1 μg/mL) derived from each of the 6 strains used as immunogens. After a blocking treatment for wells, each culture supernatant was added to the wells and incubated at 4° C. overnight. The wells were washed with a washing liquid three times, and a 2000-fold diluted HRP-labeled rabbit anti-mouse Ig antibody (Dako) was added to the wells and incubated at room temperature for 1 hour. The wells were washed with a washing liquid three times, and a substrate (TMBZ) solution was added to the wells and incubated at room temperature for 10 minutes. After the reaction was stopped, an absorbance at 450 nm was measured. The selected hybridomas were further screened by a limiting dilution method to establish clone strains. With respect to monoclonal antibodies produced from 16 strains in the established clone strains, the following experiments were carried out. The monoclonal antibodies produced from the 16 clone strains reacted with all the immunogens derived from the 6 strains.

The molecular weight of each protein recognized by the 16 monoclonal antibodies was determined by Western blotting. First, 10 μg of Mycoplasma pneumoniae antigen (FH strain) was electrophoresed by SDS-PAGE and blotted onto nitrocellulose membranes. Each culture supernatant of the 16 clones was added to the membranes and incubated at room temperature for 1 hour. The membranes were washed with a washing liquid three times, and a 1000-fold diluted HRP-labeled rabbit anti-mouse Ig antibody was added to the membranes and incubated at room temperature for 1 hour. The membranes were washed with a washing liquid three times, and a substrate (4-chloro-1-naphthol) solution was added to the membranes and incubated at room temperature for 10 minutes. After the development, the membranes were washed with a distilled water to stop the reaction.

As a result, it was found that 10 monoclonal antibodies recognized a molecule having a molecular weight of 62-69 kDa and 6 monoclonal antibodies recognized a molecule having a molecular weight of 40-45 kDa. From this result, we attempted to identify the antigen with respect to the molecule of 62-69 kDa which was considered to have a high immunogenicity because many clones were obtained.

Iso Strip (Roche) was used to determine the subclass of 10 monoclonal antibodies which recognized the molecule of 62-69 kDa. It was found that 6 antibodies were H chain G1/L chain κ, 1 antibody was H chain G1/L chain A, λ antibody was H chain 2b/L chain κ, 1 antibody was H chain 2b/L chain λ, and 1 antibody was H chain 2a/L chain λ.

(2) Identification of Antigen Specific to Mycoplasma pneumoniae and Mycoplasma genitalium

A Mycoplasma pneumoniae M129-B7 strain, of which the entire gene sequence had been already determined, was used to purify an antigen. Mycoplasma pneumoniae (M129-B7 strain) was inoculated into a PPLO glucose broth (containing horse serum, fresh yeast extract, and thallium acetate), and cultivated at 37° C. for 7 days under aerobic conditions. The strain collected by centrifugation was washed and suspended in PBS. The suspension was frozen.

The monoclonal antibody MCM12 obtained in (1) was bound to CNBr-activated Sepharose 4B (GE healthcare) as a column carrier to prepare an affinity column for antigen purification. The binding to the column carrier was carried out by reacting IgG 5 mg/mL gel in 0.1 mol/L NaHCO₃—NaOH and 0.5 mol/L NaCl (pH 8.3) at 4° C. overnight. Unreacted groups were blocked using a 0.2 mol/L glycine buffer (pH 8).

Proteins extracted from the Mycoplasma pneumoniae strain were applied to the column. After a non-adsorbed fraction was eluted, a column-adsorbed fraction was eluted using 3 mol/L sodium thiocyanate and collected. This fraction was dialyzed against 50 mmol/L PBS (pH 7) to obtain a purified product.

The purified antigen was analyzed by SDS-PAGE and Western blotting. The purified antigen (0.1 μg) was electrophoresed by SDS-PAGE and blotted onto nitrocellulose membranes. Monoclonal antibody MCM12 or monoclonal antibody MCM19 (10 μg/mL IgG solution) was separately added to the membranes and incubated at room temperature for 1 hour. The membranes were washed with a washing liquid three times, and a 1000-fold diluted HRP-labeled rabbit anti-mouse Ig antibody was added to the membranes and incubated at room temperature for 1 hour. The membranes were washed with a washing liquid three times, and a substrate (4-chloro-1-naphthol) solution was added to the membranes and incubated at room temperature. After the development, the membranes were washed with a distilled water to stop the reaction.

It was confirmed that both antibodies recognized the purified antigen.

The N-terminal 10 amino acid residues of the purified antigen protein were analyzed in accordance with a conventional method. The purified antigen was electrophoresed by SDS-PAGE. A PVDF membrane on which the sample was blotted was washed with 50% methanol/0.1% trifluoroacetic acid and methanol and dried, and 10 cycles of amino acid sequencing was carried out from the N-terminus. A protein sequencer PPSQ-23A (Shimadzu) and a PTH analyzer SPD-10A (Shimadzu) were used as analyzers.

As a result, the following sequence was obtained:

A search was carried out using the database Swiss-Prot in accordance with a conventional method, and the obtained sequence completely accorded with the sequence consisting of the 2nd to 11th amino acid residues of chaperone protein DnaK of Mycoplasma pneumoniae. The molecular weight of the DnaK deduced from its amino acid sequence was 65 kDa, which nearly accorded with the molecular weight of the antibody-recognized antigen determined by Western blotting.

As described above, it was confirmed that the antibodies obtained above were anti-DnaK antibodies specific to Mycoplasma pneumoniae and Mycoplasma genitalium.

In the monoclonal antibodies obtained in Example 1, monoclonal antibody MCM12 and monoclonal antibody MCM19 were used to examine the sensitivity and cross-reactivity of the antibodies.

PPLO glucose broths (containing horse serum, fresh yeast extract, and thallium acetate) were each separately inoculated with one of the 8 strains of Mycoplasma pneumoniae shown in Table 1, and cultivation was carried out at 37° C. for 4 days under aerobic conditions. Strains in which the broth reached pH 6.8 were used as test strains. To determine the number of each strain in the broth, 10-step dilution series were prepared with sterilized PBS, and 10 μL of each dilution was inoculated onto PPLO (containing horse serum, fresh yeast extract, and thallium acetate) agar media and incubated at 37° C. for 10 days. Growth colonies on the agar media were counted under an optical microscope having a magnification of 40 to calculate the colony forming unit of each strain.

Strains belonging to the genus Mycoplasma other than Mycoplasma pneumoniae shown in (1-1), the genus Ureaplasma, and the genus Acholeplasma were cultivated in accordance with the broths and the culture conditions shown in Table 2. The cultivation was carried out at 37° C. The terms “aerobic” and “anaerobic” in Table 2 mean aerobic cultivation and anaerobic cultivation, respectively. To determine the number of each strain in the broth, 10-step dilution series were prepared with sterilized PBS, and 10 μL of each dilution was inoculated onto PPLO (containing horse serum, fresh yeast extract, and thallium acetate) agar and incubated at 37° C. for 10 days. Growth colonies on the agar were counted under an optical microscope having a magnification of 40 to calculate the colony forming unit of each strain. The test was carried out at a number of 10⁶to 10⁷cfu/mL.

Table 3 to Table 6 show microorganisms which were used in a cross-reactivity test of bacteria and fungi other than the genus Mycoplasma, the genus Ureaplasma, and the genus Acholeplasma used in (1-1) and (1-2), and the culture conditions. Heart infusion agar (Difco), trypticase soy agarII with 5% sheep blood (Becton, Dickinson and Company), chocolate agar (NISSUI), modified GAM agar (NISSUI), skirrows medium (Becton, Dickinson and Company), and Sabouraud-dextrose agar (Difco) were used as media.

These strains were cultivated on agar, and suspended in sterilized PBS at a concentration of 10⁷to 10⁸cfu/mL to prepare test strains. To determine the number of each strain, each test suspension in which each strain was suspended in sterilized PBS was stepwisely (10-step) diluted with the same PBS, and 50 μL of each dilution was inoculated onto agar media. Growth colonies on the media were counted by the naked eye.

The blank spaces in the “Strain No.” column of the tables mean strains which were isolated and identified from clinical specimens.

Ascites fluid containing monoclonal antibody MCM19 was applied to ammonium sulfate fractionation, IgG was purified using rProteinA Sepharose FF (GE healthcare), and a quantitative analysis of protein was carried out by a BCA method. The purified IgG antibody (10 μg/mL) was immobilized on a 96-well microplate.

(2-1-2) Method for Preparation of Antibody for Labeling with Alkaline Phosphatase and Method for Preparation of Labeled Antibody

Ascites fluid containing monoclonal antibody MCM12 was applied to ammonium sulfate fractionation, and IgG was purified using MEP Hypercel (Pall Corporation). The IgG was digested with pepsin to prepare F(ab′)₂, and F(ab′)₂was crosslinked with alkaline phosphatase to prepare an alkaline-phosphatase-labeled antibody.

The immobilized 96-well microplate was washed, and blocked with 0.1 mmol/L TBS (pH 7.5) containing 1% BSA at room temperature for 1 hour. Each strain suspension to be tested (100 μL) was added to the microplate, and incubated at room temperature for 1 hour. The microplate was washed, and the alkaline-phosphatase-labeled antibody (10 μg/mL) was added and incubated at room temperature for 1 hour. The microplate was washed, and development was carried out using a substrate (pNPP) solution for 30 minutes. The reaction was stopped, and an absorbance at 405 nm was measured.

Test strains (1-1) were applied to the ELISA described above, and a test dilution which showed an absorbance of 0.05 or higher and a maximum dilution magnification was used to calculate the number of each strain. The results are shown in Table 7.

It was found from the results shown in Table 7 that the sensitivity against Mycoplasma pneumoniae was 10³to 10⁴cfu/mL by the ELISA using the monoclonal antibodies.

Test strains (1-2)[the genus Mycoplasma other than Mycoplasma pneumoniae, the genus Ureaplasma, and the genus Acholeplasma shown in Table 2] and test strains (1-3) [other bacteria and fungi shown in Table 3 to Table 6] were applied to the ELISA described above.

All the microorganisms other than Mycoplasma genitalium showed an absorbance of less than 0.010. With respect to Mycoplasma genitalium, the number thereof calculated from a test dilution which showed an absorbance of 0.05 or higher and a maximum dilution magnification was 6.9×10⁴cfu/mL.

As shown in these results, it was found that the ELISA using the monoclonal antibodies showed a cross-reactivity to Mycoplasma genitalium, but did not show a cross-reactivity to other microorganisms.

As described above, it was confirmed that the ELISA did not show a cross-reactivity to many bacteria and fungi which might disturb the diagnosis of a Mycoplasma pneumoniae or Mycoplasma genitalium infection.

To a colloidal gold solution, of which pH was previously adjusted by adding 2 mL of a 50 mmol/L phosphate buffer (pH 11) to 18 mL of a colloidal gold solution (Tanaka Kikinzoku) having a diameter of 40 nm, 2.5 mL of 100 μg/mL monoclonal antibody MCM12 solution was added and stirred. After the mixture was stirred for 1 hour, 1 mL of 1 mass % polyethylene glycol (Mw. 20000, Wako Pure Chemical Industries) aqueous solution was added and stirred, and 2 mL of 10 mass % BSA aqueous solution (SIGMA) was added and stirred. This solution was centrifuged at 4° C. and 8000G for 15 minutes, and almost all the supernatant was removed so that approximately 1 mL of the supernatant was left. Colloidal gold was re-dispersed using an ultrasonic generator. The dispersed colloidal gold was dispersed into 20 mL of a phosphate buffer containing BSA, and centrifuged at 4° C. and 8000 G for 15 minutes. Almost all the supernatant was removed so that approximately 1 mL of the supernatant was left, and colloidal gold was re-dispersed using an ultrasonic generator to prepare an antibody-conjugated colloidal gold solution.

The antibody-conjugated colloidal gold solution prepared in (1-1) was diluted with the phosphate buffer containing BSA, and impregnated into a glass fiber pad (Millipore) which was previously cut to a size of 20 mm×300 mm. The pad was dried at room temperature overnight to prepare a pad carrying the colloidal gold antibody.

Onto a nitrocellulose membrane (Millipore) which was cut to a size of 30 mm×300 mm, an antibody was immobilized in accordance with the following method to prepare an antibody-immobilized membrane. A solution of monoclonal antibody MCM19 for immobilization (5 mg/mL) was applied in a line with a width of approximately 1 mm, using a coater (BioDot), at a position 16 mm from one of the long sides of the membrane as the bottom, and dried to prepare the antibody-immobilized membrane.

The antibody-immobilized membrane, the pad carrying colloidal gold, and an absorbent pad (Pall corporation) were attached to an adhesive back sheet so that adjacent pieces overlapped with each other. The resulting overlapped structure was cut along the long side with a width of 6 mm, using a cutter, to prepare test strips for immunochromatography. Each test strip was put into a housing case to prepare test kits for immunochromatography.

Cultivated strains, PBS-washed strains, culture supernatants, and pellets of cultivated strains were dissolved with a phosphate buffer containing Triton X-100 to prepare Mycoplasma pneumoniae antigen (or strain) solutions for test at predetermined concentrations. To each immunochromatographic kit for test, 100 μL of Mycoplasma pneumoniae antigen (or strain) solution for test was added dropwise. After 15 minutes from the addition, cases where a development was detected by the naked eye at the position on which the anti-Mycoplasma-pneumoniae antibody was coated of each antibody-immobilized membrane were judged as “positive”, and cases where no development was detected were judged as “negative”.

Test strains (1-1) in Example 2 were applied to the immunochromatography described above, and a test dilution which showed a development generated on the test line and a maximum dilution magnification was used to calculate the number of each strain. The results are shown in Table 8.

It was found from the results shown in Table 8 that the sensitivity against Mycoplasma pneumoniae was 10³to 10⁴cfu/mL by immunochromatography using the monoclonal antibodies.

Test strains (1-2)[the genus Mycoplasma other than Mycoplasma pneumoniae, the genus Ureaplasma, and the genus Acholeplasma shown in Table 2] and test strains (1-3) [other bacteria and fungi shown in Table 3 to Table 6] of Example 2 were applied to the immunochromatography described above.

All the microorganisms other than Mycoplasma genitalium were negative, i.e., did not show any developments. By contrast, a development was detected in Mycoplasma genitalium, and the number thereof calculated from a test dilution showing a maximum dilution magnification was 6.9×10⁴cfu/mL.

As shown in these results, it was found that the immunochromatography using the monoclonal antibodies showed cross-reactivity to Mycoplasma genitalium, but did not show a cross-reactivity to other microorganisms.

As described above, it was confirmed that the immunochromatography did not show cross-reactivity to many bacteria and fungi which might disturb the diagnosis for a Mycoplasma pneumoniae or Mycoplasma genitalium infection.

Pharyngeal swabs were collected from 3 patients suspected of suffering with a mycoplasma infection and 33 healthy persons, and a detection of Mycoplasma pneumoniae was carried out in accordance with the immunochromatography of Example 3. As a result, a positive reaction was observed in the 3 patients suspected of suffering with a mycoplasma infection, and the 33 healthy persons were negative, as shown in Table 9.

DNAs were extracted from the same samples in accordance with a conventional method, and a gene detection of Mycoplasma pneumoniae was carried out using a modified method derived from the qualitative PCR method of Jensen et al. (APMIS. 1989; 97(11): 1046-8.), in which part of a Mycoplasma pneumoniae P1 gene (M. pneumoniae M129-B7 NCBI number: NC_—000912) was amplified, and both were compared to each other. Both positive and negative results accorded with each other, as shown in Table 9.

Next, DNAs derived from the samples which showed positive by both the immunochromatography and the qualitative PCR method was used, and a gene detection of Mycoplasma genitalium was carried out using a modified method derived from the qualitative PCR method of Yoshida et al. (J Clin Microbiol. 2002; 40(4): 1451-5.) for Mycoplasma genitalium, in which part of a Mycoplasma genitalium 16s rRNA region (M. genitalium G7 NCBI number: L43967) was amplified. As a result, the gene derived from Mycoplasma genitalium was not detected in any of the samples, as shown in Table 10.

In this manner, it was confirmed that the gene derived from Mycoplasma genitalium could be amplified by this method.

As described above, it was shown that the antibody of the present invention was used to specifically detect Mycoplasma pneumoniae, and a mycoplasma infection can be diagnosed.

As samples to be measured, 8 strains of Mycoplasma pneumoniae purchased from ATCC (M. pneumoniae FH: ATCC No. 15531, M. pneumoniae Bru: ATCC No. 15377, M. pneumoniae Mac:

ATCC No. 15492, M. pneumoniae Mutant 22: ATCC No. 39505, M. pneumoniae M52: ATCC No. 15293, M. pneumoniae PI1428: ATCC No. 29085, M. pneumoniae M129-B7: ATCC No. 29342, and M. pneumoniae UTMB-10P: ATCC No. 49894) were used. These 8 strains of Mycoplasma pneumoniae were cultivated in a PPLO medium, and DNAs were extracted.

The DNA extraction was carried out using a Sumitest EX-R&D kit (Medical & Biological Laboratories), and each DNA was suspended in 10 mmol/L Tris-HCl, 1 mmol/L EDTA Buffer pH 8.0 (Nippon Gene)(hereinafter referred to as TE Buffer) and cryopreserved at −40° C.

With respect to the extracted DNAs, the number of gene copies was determined by a mycoplasma common quantitative PCR for 16s rRNA region. Each DNA was diluted with TE buffer to prepare 10-fold diluted preparations from 2×10⁶to 2×10⁰copies/μL. These were used in detecting the DnaK gene.

The mycoplasma common quantitative PCR for 16s rRNA region was carried out as follows.

Primers which were common to the genus Mycoplasma for amplifying the 16s rRNA region were designed, and the number of gene copies in each extracted M. pneumoniae DNA was calculated by a real-time PCR method using a standard. The real-time PCR was carried out using LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science).

The following primer sequences were used. M. pneumoniae M129-B7 complete genome: GenBank Accession No. NC_—000912

With regard to the PCR conditions, a reaction at 95° C. for 10 minutes was carried out and a cycle composed of reactions at 94° C. for 10 seconds for denaturing, at 60° C. for 2 seconds for annealing, and at 72° C. for 12 seconds was repeated 50 times.

As the standard, a diluted series (10⁷, 10⁵, 10³, 10², and 10¹copies/test) of pT7Blue T-Vector (Takara Bio) in which part of 16s rRNA (771 bp: 302-1072 for 16s rRNA) of M. pneumoniae (M129 strain) was recombined was used. The number of copies in the standard was calculated on the basis of the following equations:

DNAconcentration(µg/mL)=ABS(260nm)×501pmolofkbpDNA=0.66µgcopy(copies/mL)=10.66×{1000bpLp+Lr×(A260×50)×(6.02×1023)}×10-12Lp:LengthofplasmidLr:LengthofrecombinantDNA[Math.1]

Next, the DnaK gene of Mycoplasma pneumoniae was amplified by PCR as follows. With regard to a PCR reaction liquid, 25 μL of Premix EX Taq Hot Start Version (TaKaRa), 1 μL of 10 pmol/μL sense primer MpDnaK_S, and 1 μL of 10 μmol/μL antisense primer MpDnaK_A were added to 18 μL of Otsuka distilled water (Otsuka Pharmaceutical) to prepare 45 μL of a master mixture, and 5 μL of each extracted DNA was added to the master mixture to adjust the total volume to 50 μL. TE buffer was used as a PCR negative control. To amplify the DnaK gene having a complete length of 1,788 bp, the sense primer was designed at 81 bp 5′-upstream from the starting codon of the DnaK gene, and the antisense primer was designed at 53 bp 3′-downstream from the stop codon. More particularly, sense primer MpDnaK_S corresponded to the 521,756-521,782 nucleotide sequence of M. peumoniae M129 (GenBank Acc No. NC_—000912), and antisense primer MpDnaK_A corresponded to the 523,655-523,677 nucleotide sequence.

In the PCR reaction, using a Mastercycler (Eppendorf), a cycle composed of reactions at 94° C. for 30 seconds for denaturing, at 50° C. for 30 seconds for annealing, and at 72° C. for 2 minutes was repeated 50 times, and finally a reaction at 72° C. for 5 minutes was carried out. After the PCR reaction, 5 μL of each PCR product was subjected to 2% agarose electrophoresis, and the agarose gel was stained with ethidium bromide and irradiated with ultraviolet light to confirm an amplified band of approximately 1,900 bp.

The 8 Mycoplasma pneumoniae stains prepared above were examined to confirm that all the 8 strains could be amplified up to 10²copies/test.

As samples to be measured, 17 mycoplasma strains purchased from ATCC (M. genitalium: ATCC No. 33530, M. hominis: ATCC No. 23114, Ureaplasma parvum: ATCC No. 700970, U. urealyticum: ATCC No. 27618, M. fermentans: ATCC No. 19989, Acholeplasma laidlawii: ATCC No. 23206, A. oculi: ATCC No. 51735, M. penetrans: ATCC No. 55252, M. pirum: ATCC No. 25960, M. orale: ATCC No. 23714, M. salivarium: ATCC No. 23064, M. arthritidis: ATCC No. 19611, M. buccale: ATCC No. 23636, M. faucium: ATCC No. 25293, M. lipophilum: ATCC No. 27104, M. primatum: ATCC No. 25948, and M. spermatophilum: ATCC No. 49695) were used. These 17 mycoplasma strains were cultivated in a PPLO medium. Similar to Example 5, DNAs were extracted, the number of gene copies was determined by the quantitative PCR for 16s rRNA region, and each DNA was diluted to 2×10⁵copies/μL.

The procedures described in Example 5 were repeated, except that the 17 Mycoplasma strains were used as the samples to be measured, to carry out the PCR for the DnaK gene of Mycoplasma pneumoniae, and no amplified band was detected in any of the 17 strains. Because no cross-reactivity was detected when the concentration of the DNA sample was 10,000 times that of DNA capable of amplifying the DnaK gene of M. pneumoniae, it was found that the PCR for the DnaK gene of Mycoplasma pneumoniae had an extremely high specificity.

As samples to be measured, extracted DNAs from 46 cases of positive clinical specimens (40 cases of pharyngeal swabs, 2 cases of nasal mucus, 1 case of nasopharyngeal aspirates, and 3 cases of nasopharyngeal swabs) and 30 cases of negative specimens (10 cases of pharyngeal swabs from healthy persons, 10 cases of pharyngeal swab from clinical specimens, 4 cases of nasal mucus, 3 cases of nasopharyngeal aspirates, and 3 cases of nasopharyngeal swabs) were tested by a nested PCR for the Mycoplasma pneumoniae P1 gene region, described in the “Mycoplasma pneumonia” section of National Institute of Infectious Diseases, “Pathogen Detection Manual” p. 1309-1344.

The PCR for the DnaK gene of Mycoplasma pneumoniae was carried out to confirm that the DnaK gene was amplified in all 46 cases of P1 gene PCR positive. By contrast, the DnaK gene was not amplified in any of the 30 cases of P1 gene PCR negative.

The nucleotide sequences of PCR products from the 8 ATCC strains of Example 5 and the 8 clinical specimens (7 cases of pharyngeal swabs and 1 case of nasopharyngeal swabs) of Example 7, in which the amplification was detected by the PCR for DnaK gene, were determined using a BigDye Terminator v3.1 (Applied Biosystems) and a 3130×1 Genetic Analyzer (Applied Biosystems).

As a result, with respect to the DnaK gene (1,788 bp, SEQ ID NO: 6), the PCR products from the 8 ATCC strains and the 8 clinical specimens absolutely (100%) accorded with each other, and also absolutely (100%) accorded with the M129 strain (Acc No. NC_—000912) and FH strain (Acc No. CP002077) registered in GenBank. The alignment between the M129 stain and the FH strain is shown in FIGS. 1 to 3.

With respect to the P1 gene, differential typing was carried out by a PCR-RLFP method in accordance with the reference: JOURNAL OF CLINICAL MICROBIOLOGY, 1996, p. 447-449 Vol. 34, No. 2, and the 8 ATTC strains of Example 5 were classified into two groups. More particularly, 4 strains including M129-B7, M52, PI1428, and Mutant 22 were classified into Type I, and 4 strains including FH, Bru, Mac, and UTMB-10P were classified into Type II. The alignment between the M129 strain (SEQ ID NO: 7) and the FH strain (SEQ ID NO: 8) as typical strains is shown in FIGS. 4 to 10.

It was considered from these results that the obtained antibodies show no difference in reactivity with respect to the genotype, the place for collection, and the time of collection, because the DnaK genes absolutely (100%) accorded with each other, even among strains in which the types of the P1 gene were different, and no variations in the nucleotide sequence were detected among strains collected from various places and over the past 50 years.

According to the present invention, Mycoplasma pneumoniae and/or Mycoplasma genitalium can be specifically detected with high sensitivity in specimens such as oral swab specimens, nasal cavity swab specimens, urine, tissue samples, or body fluids, or samples derived from culture. In particular, the present invention is important for the diagnosis of atypical pneumonia caused by Mycoplasma pneumoniae or the diagnosis of nongonococcal urethritis and sexually transmitted disease caused by Mycoplasma genitalium, and is industrially applicable to the manufacture of pharmaceuticals.

Although the present invention has been described with reference to specific embodiments, various changes and modifications obvious to those skilled in the art are possible without departing from the scope of the appended claims.