CA2398469C

CA2398469C - Antibody for detecting mycoplasma pneumoniae

Info

Publication number: CA2398469C
Application number: CA002398469A
Authority: CA
Inventors: Monzur Rahman; Takashi Etoh
Original assignee: Asahi Kasei Corp
Current assignee: Asahi Kasei Corp
Priority date: 2000-01-31
Filing date: 2001-01-31
Publication date: 2009-04-21
Anticipated expiration: 2021-01-31
Also published as: KR20020073192A; CN100516210C; CA2398469A1; CN1406279A; JP5331285B2; WO2001057199A1; KR100734995B1; AU2001230532A1; TWI282368B; JP2012006968A

Abstract

A method of specifically, quickly and highly sensitively detecting a microorganism belonging to Mycoplasma pneumoniae; an antibody to be used in the detection; a detection reagent kit; and a process for producing the antibody to be used in the detection. Namely, an antibody against the ribosomal protein of a microorganism belonging to Mycoplasma pneumoniae which reacts specifically with this microorganism; a method of detecting this microorganism in a specimen by using this antibody; and a detection reaction kit containing this antibody. The ribosomal protein is exemplified by Ribosomal Protein L7L 12 employed in detecting the infection with a microorganism causative of pneumonia.</SDO AB>

Description

CA 02398469 2002-07-23 i DESCRIPTION
ANTIBODY FOR DETECTING MYCOPLASMA PNEUMONIAE
Field of the Invention The present invention relates to antibody useful to a detection of microorganism that belongs to Mycoplasma pneumoniae, that is -the cause microorganism of common pneumonia, a detection method of the microorganism, a reagent kit for the detection of the microorganism, and a method for preparing the antibody useful to a detection of the microorganism.

The invention is important to pharmaceutical industry specifically to the diagnosis of atypical pneumonia caused by Mycoplasma pneumoniae, and has industrial applicability in the art.

The invention can be useful for detecting the species Mycoplasma pneumoniae in test samples, such as from throat swabs, tissue samples, body fluids, experimental solutions and cultures.

Background Art Diagnosis of microbial infections can be confirmed either by detection of the causative pathogen from the infection site or by detection of antibodies to the disease contributing microorganisms in serum and body fluids. The diagnosis, i.e. the detection of the causative pathogen, is particularly important in the sense that it makes possible quick treatment available to the patient.

Detection of the causative pathogen of infections can be generally classified as cultivation and identification methods, that the causative pathogen is selectively. cultivated and then identified based on its physiological, biochemical or structural properties; genetic diagnosis, that the causative pathogen is amplified by PCR or target specific nucleic acid hybridization etc., thus the causative pathogen is detected; or immunological methods, that the causative pathogen is detected using a specific reaction of antibody with antigen marker of the pathogen.

2 However, it takes time to obtain results by cultivation and identification methods or genetic diagnosis methods. Therefore, diagnosis by immunological methods is commonly used because the pathogen can be detected within a short time with high sensitivity thus the patient can be quickly and appropriately treated.

Depending on the species of microorganism, a combination of a variety of marker antigens and antibody or antibodies can be used for detecting the causative pathogen of infections by conventional immunological methods.

Mycoplasma pneumoniae is the common causative pathogen of pneumonia, tracheobronchitis and pharygitis. It is small Gram-negative bacteria that invade selectively human host and cause diseases. It causes approximately 15-20% of all community-acquired pneumonias in general populations and up to 50% of pneumonias in certain confmed groups (Ragnar Norrby 1999).

The microorganism is very small in size, fastidious in culture and forms very small colony on enriched media. Growth of Mycoplasma pneumoniae on enriched agar plates are very slow, and might take at least 21 days or more for the identification (Granato, Poe et al.

1980). Several Mycoplasma species of human origin can produce similar biochemical reactions. On the other hand, lacks of cell wall add further difficulties to recognize them under light microscope (Knudson and MacLeod 1979). Therefore, Gram staining and culture methods are not practiced for detecting this pathogen quickly.

DNA hybridization assay probes directed to genomic sequences for detecting Mycoplasma pneumoniae are mentioned by Hyman et al., Buck et al., and Bernet et al.
(Hyman, Yogev et al. 1987; Bernet, Garret et al. 1993; Buck, O'Hara et al.
1993). Probes directed to ribosomal RNA (rRNA) sequences of Mycoplasma pneumoniae are mentioned by Tilton et al. (Tilton, Dias et al. 1980), Yogev et al. (Yogev, Halachmi et al. 1988), Gobel et al. (Gobel, Geiser et al. 1987), Zivin and Monahan (EPO 305145, and Gobel and Stanbridge (EPO 250662). Kai et al. (Kai, Kamiya et al. 1987) and Jensen et al. (Jensen, Sondergard-Andersen et al. 1993) describe primers directed to 16S rRNA
sequences of M_ycoplasma pneumoniae. All mention probes are designed to DNA of Mycoplasma pneumoniae or DNA of Mycoplasma pneumoniae and Mycoplasma genitalium. As these

3 probes are relatively insensitive, the more recently available amplified techniques e.g.
PCR have largely captured them. PCR is highly sensitive and can be done within shorter time than hybridization. However, problems of false positive often rise among asymptomatic carriers who are negative to cultured Mycoplasma pneumoniae, or persons who indicate positive after their disease.
Japanese Patent Application Laid-open No. 63(1988)-298 discusses an immuno-detection method based on the western blotting method, that uses a monoclonal antibody to an approximately 43 kilo Dalton membrane protein antigen of Mycoplasma pneumonia.
However, there are problems with the said antibody and detection methods based on that antibody in that species specificity to Mycoplasma pneumoniae is insufficient for proper diagnosis due to low specificity. Moreover, the diagnostic method takes at least five hours to complete (Madsen et al. 1988).
In Chest. 1999 Apr;115(4):1188-94, Chan ED et al. concluded that in adults, Mycoplasma-associated bronchiolitis without pneumonia is rarely reported, but in hospitalized patients, it may be more common than expected and may be associated with severe physiologic disturbances. Also, in Pediatr Rev. 1998 Oct;19(10):327-31, Cimolai N.
discussed about Mycoplasma pneumoniae respiratory infections.

Disclosure of the Invention The present invention has been achieved to solve the above problems.
Specifically, an object of the present invention is to provide a method for specifically, high- sensitively and rapidly detecting a microorganism that belongs to Mycoplasma pneumoniae, a detection antibody using for the detection and a reagent kit for the detection. Furthermore, another object of the present invention is to provide a method for manufacturing the detection antibody using for the detection.
The inventors have identified a useful protein antigen that is conserved same function in all bacteria. Generally, the structural change of said protein is expected to be very low. Surprisingly, it has been found that the antibody to the protein is specific to bacterial species or genus, has a protean property enable to use for discrimination specific to bacterial species or genus, and object microorganisms may be detected all serotypes thereof. In more detail, said proteins are useful proteins exhibiting species-specific property that the structures, through the change of the amino acid sequence, are completely same in same species, but structural changes are accompanied in the case 3a species are different.

The inventors focused on intracellular molecules that are present as molecules

4 having same function in all microorganism cells and somewhat differ between microorganisms in terms of it's amino acid sequence, particularly Ribosomal Protein L7/L12 that is one kind of ribosomal protein. Ribosomal Protein L7/L12 is a protein with a molecular weight of approximately 13 kilo Daltons and is known to exist as an essential ribosomal protein in protein synthesis. Progress has been made in understanding the complete amino acid sequence of Ribosomal Protein L7/L12 in several microorganisms including Mycoplasma pneumoniae.
The inventors focused on the fact that even though there are similarities between different microorganisms in terms of said molecule, this molecule also has a structural segment that is unique to each microorganism and discovered that, it is possible to detect various microorganisms with species specificity and to detect all serotypes within the same species by using antibody to said protein.
The inventors completed the present invention upon discovering that antibody specific to the protein of Mycoplasma pneumoniae can be obtained and species-specific 15 detection of Mycoplasma pneumoniae is possible using said antibody.
In accordance with the present invention a monoclonal antibody specific to Ribosomal Protein L7/L12 of Mycoplasma pneumoniae has been discovered and developed. The antibody is novel and different than those previously described and has property of reacting specifically to the said protein.
The present invention relates to an antibody which is an antibody to ribosomal protein L7/L12 comprising SEQ ID NO:2 of microorganism belonging to Mycoplasma pneumoniae and which reacts specifically with ribosomal protein L7/L12 comprising SEQ
ID NO:2 of said microorganism.
Sequences No. I and No. 2 in the Sequence List are the DNA sequence of the Ribosomal Protein L7/L12 gene of Mycoplasma pneumoniae and the corresponding amino acid sequence (NCBI database accession# NC_000912). The left terminal and right terminal of the amino acid sequences entered in the Sequence List are amino group (referred to below as the N terminal) and carboxyl group terminals (referred to below as the C terminal), respectively, and the left terminal and right terminal of the base sequence is the 5' terminal and the 3' terminal, respectively. Amino acid in the sequence of closest match test is expressed by one letter notation. The notation "+" in closest match test indicates that it is different amino acid but amino acid with similar properties such as 4a hydrophobic. The notation (blank) indicates that it is entirely different amino acid including properties j thereof. Moreover, the series of bio-molecular experiments of gene preparation mentioned in this text can be performed by methods entered in standard experimental manuals.
"Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press, Sambrook, J. et al. (1989), is given as an example of the well-known standard experimental manual.

Table I

Closest Match Test:

Mp: 1 MAKLDKNQLIESLKEMTIMEIDEIIKAVEEAFGVSATPVVAAGAVGGTQEAASEVTVKVT 60 1 M KLDK QLIESLKEMTI+EIDEIIKAVEEAFGV+ATP+VAAGA G TQEAASEV+VKVT
Mg: 1 MGKLDKKQLIESLKEMTIVEIDEIIKAVEEAFGVTATPIVAAGAAGATQEAASEVSVKVT 60 Mp: 61 GYTDNAKLAVLKLYREIAGVGLMEAKTAVEKLPCVVKQDIKPEEAEELKKRFVEVGATVE 120 GY DNAKLAVLKLYREI GVGLMEAKTAVEKLPCVVKQDIKPEEAEELKKRFVEVGATVE

Mg: 61 GYADNAKLAVLKLYREITGVGLMEAKTAVEKLPCVVKQDIKPEEAEELKKRFVEVGATVE 120 Mp: 121 IK 122 +K
Mg: 121 VK 122 In the present invention, the term "microorganism", means Mycoplasma pneumoniae, specifically, indicates microorganism having a pathogenic property in respiratory organ and high significance in diagnosis as a causative pathogen of Mycoplasma infections. In the present invention, the term "antibody specifically reacting with microorganism" indicates an antibody that can specifically react with species or genus of microorganism, and antibody specifically reacting with species is especially useful in diagnosis of bacterial infections.

The term "antibody" in the present invention means a polyclonal antibody or monoclonal antibody that can be made using the entire length or only a partial peptide of said Ribosomal Protein L7/L12. Although there are no special restrictions to the peptide length for making the antibody, in the case the antibody corresponds to Ribosomal Protein L7/L12, the segment may be of the length characterizing the Ribosomal Protein L7/L12, and a peptide of 5 amino acids or longer, particularly 8 amino acids or longer, is preferred.
Antiserum containing antibody (polyclonal antibody) that identifies Ribosomal Protein L7/L12 can be obtained by inoculating laboratory animals with adjuvant and a peptide or the full length protein, as is or, when necessary, after being cross-linked with a carrier protein such as KLH (keyhole-limpet hemocyanin) and BSA (bovine serum albumin) and recovering the serum. Moreover, the antibody can be used after it has been purified from the antiserum. The laboratory animals that are inoculated include sheep, horses, goats, rabbits, mice, rats, etc., and sheep, rabbits, etc., are particularly preferred for preparation of monoclonal antibody. Moreover, monoclonal antibody can also be obtained by conventional methods of making hybridoma cells, but mice are preferred in this case.

The entire length of said protein, or its amino acid sequence of 5 or more, preferably 8 or more, residues that has been fused with glutathione S-transferase (GST), etc., can be purified and used as antigen, or it can be used as antigen without being purified. The antibody can also be produced from the genetic recombination antibody expressed in cultured cell using immunoglobulin genes that have been separated by a variety of methods in published documents ("Antibodies: A Laboratory manual," E. Harlow et al., Cold Spring Harbor Laboratory), cloning methods, etc.

Antibody to Ribosomal Protein L7/L12 that can be employed as the marker antigen of the present invention can be obtained by the following methods, and other similar methods as well, though not to be limited within these methods:

a) The desired antibody can be acquired by synthesizing a peptide fragment, in the case microorganism has a known Ribosomal Protein L7/L12 genetic sequence and amino acid sequence, using the region least similar to the amino acid sequence of said protein of another microorcanism and making polyclonal antibody, or monoclonal antibody, using this peptide fragment as the immune source.

Moreover, it is possible to acquire the entire sequence of said gene by using a conventional genetic procedure, such as gene amplification by PCR using the DNA
sequence at both terminals of said laiown genetic sequence as the probe, or hybridization using the sequence of a homologous segment as the template probe.

Then, a fused gene with another protein gene is constructed and said fused gene is inserted into the host by conventional gene insertion methods using Escherichia coli, etc., as the host and expressed in large quantities. The desired protein antigen can then be acquired by purifying the expressed protein by affmity column methods with antibody to the protein that was used as the fusion protein. In such a case, even if antibody to the amino acid segment retained within the microorganisms is acquired, it does not coincide with the purpose of the present invention because the full length of Ribosomal Protein becomes the antigen. Consequently, hybridoma that produces monoclonal antibody to the antigen that has been obtained by this method is acquired by conventional methods and the desired antibody can be obtained by selecting a clone, which produces antibody that will react only with the desired microorganism.

b) For microorganisms that the amino acid sequence of the Ribosomal Protein L71L12 is unknown, as the amino acid sequence of the Ribosomal Protein L7/L12 has 50 -60 % of homology between microorganisms, the protein gene can easily obtained by using a conventional genetic procedure, such as gene amplification of specific sequence moiety by PCR based on the sequence of a homologous segment of the amino acid sequence, or hybridization using the sequence of a homologous segment as the template probe. Then, a fused ;ene with another protein gene is constructed and said fused gene is inserted into the host such as Escherichia coli, and the like by conventional gene insertion methods, and expressed in large quantities. The desired protein antigen can then be acquired by purifying the expressed protein by affmity column methods with antibody to the protein that was used as the fusion protein. In such a case, even if antibody to the amino acid segment retained within the microorcyanisms is acquired, it does not coincide with the purpose of the present invention because the full lenQth of Ribosomal Protein L7/L12 becomes the antigen.

Consequently, hybridoma that produces monoclonal antibody to the antigen that has been obtained by this method is acquired by conventional methods and the desired antibody can be obtained by selecting a clone, which produces antibody that will react only with the desired microorganism.

c) Altematively, as another method that is suitable for the case where the amino acid sequence of the Ribosomal Protein L7/L12 is unknown, a peptide of 5 to 30 amino acids corresponding to the common sequence segment retained in the microorganisms is synthesized from the known amino acid sequence of the Ribosomal Protein L7/L12, and polyclonal antibody or monoclonal antibody to this peptide sequence is made by conventional methods. Then Ribosomal Protein L7/L12 highly purified can be obtained by purifying the disrupted liquid of bacterial cells through affinity column chromatography using said antibody. If purity of the protein is insufficient, it can be purified by conventional methods, such as ion exchange chromatography, hydrophobic chromatography, gel filtration, etc., after which the eluted fraction of Ribosomal Protein L7/L12 is identified by method such as westem blotting using antibody that was made, and purified protein can be obtained. The desired antibody can be obtained by acquiring hybridoma by conventional methods using the purified Ribosomal Protein L7/L12 antigen that has been obtained, and selecting hybridoma reactable specifically with the desired microorganism.

The antibody of the present invention specific to various microorganisms that has been obtained by the methods in a), b) and c) can be used in a variety of immunoassay methods to provide various diagnostic reagents and kits specific to the desired microorganism. For example, this antibody can be used in aggregation reactions, that is one of known measuring method and where antibody is adsorbed on polystyrene latex particles, ELISA, which is a conventional technology performed in a microtiter plate, conventional immunochromatography methods, and sandwich assay, whereby said antibody labeled with colored particles or particles having coloring capability, or with enzyme or fluorescence substances, and magnetic micro-particles coated with capture antibody, etc., are used, etc.

The term "microorganism diagnosis methods using antibody" means diagnostics methods using any known conventional immunoassay, such as aggregation whereby said antibody is adsorbed on polystyrene latex particles, ELISA, which is a conventional method performed in a microtiter plate, conventional immunochromatography methods, or sandwich assay, whereby said antibody labeled with colored particles or particles having coloring capability, or with enzyme or fluorescence substances, and magnetic micro-particles coated with capture antibody, etc., are used.
Moreover, the optical immunoassay (OIA) technology described in Japanese (via Intemational) Patent Application Laid-open No. 07-509565, in which microorganisms are detected by the principle of an optical interference induced by an antibody reaction on the optical thin film which is formed by silicone, silicon nitride or the like, is a useful as a high sensible diagnostic method, especially as a diagnostic method of microorganisms using an antibody.
Moreover, as a method for extracting intracellular marker antigen from the desired microorganism in aforementioned diagnostic method, reagent treatment using an extraction reagent(s) comprising various surfactants, such as typically Triton X-100TM and Tween-20TM, enzyme treatment using an appropriate enzyme, such as protease, etc., and physical treatment using known cell structure crushing methods, typically cell-crushing of microorganism, can be used. It is preferred that the most suitable conditions for extracting with reagent are set to each kind of microorganism using a proper combination of surfactants, etc.
Moreover, in the present invention the term "reagent kit for diagnosis of microorganisms using antibody" means a diagnostic kit that uses the above-mentioned diagnostic method.
The amino acid and DNA sequence of Ribosomal Protein L7/L12 of Mycoplasma pneumoniae are shown in Sequence List. Consequently, in the case of this microorganism, it is possible to compare the amino acid sequence of Ribosomal Protein L7/L12 with the same protein of closely related microorganisms. Which is shown in Sequence List under the heading "Closest Match". Synthesizing a peptide with the segment of low homology and making polyclonal or monoclonal antibody to that could short cut the selection of those having specificity to the microorganism.

Especially in the case of a polyclonal antibody, it is preferred that IgG
fraction be obtained by purification of the antiserum of immunized laboratory animals with a protein A
column, etc., and affinity purification be performed with the synthetic peptide used in immunization of the laboratory animals.

5 Moreover, PCR primers are formed based on the sequences of N-terminal and C-terminal from the DNA sequence of Ribosomal Protein L7/L12 of the microorganism.
Utilizing homology of the PCR primers, DNA fragments are amplified by the PCR

method using genomic DNA and extracted, the fragments of Mvcoplasma pneumoniae can be thus acquired according to a conventional method. The entire length of the gene for 10 Ribosomal Protein L7/L12 of Mycoplasma pneumoniae can be acquired through the analysis of the DNA sequence information of these fragments.

The Ribosomal Protein L7/L12 gene of Mycoplasma pneumoniae thus acquired forms a fusion protein gene with, for example, GST, etc., and an expression vector is built using an appropriate expression plasmid, Escherichia coli is transformed and a large quan-tity of said protein can be expressed. A suitable amount of the transformed Escherichia coli is cultivated and disrupted bacterial fluid is subjected to purification by an affinity column using GST to obtain the GST fusion Ribosomal Protein L7/L12 of Mycoplasma pneumoniae.

It is also possible to acquire the target specific monoclonal antibody by establishing a multiple clone of hybridomas using said protein as is or GST moiety deleted protein as an antigen protein, and selecting the antibody which exhibits a specific response to Mycoplasma pneumoniae bacteria, a homogenized fluid of the bacteria, or Ribosomal Protein L7/L12 of Mycoplasma pneumoniae.

Antibody made based on the present invention can be used in all known types of immunoassay, such as known aggregation reaction whereby said antibody is adsorbed on polystyrene latex particles, ELISA, which is a conventional technology performed in a microtiter plate, conventional immunochromatography, and sandwich assay, whereby said antibody labeled with colored particles or particles that have coloring capability, or enzymes or fluorescence substances, and magnetic particles coated with capture antibody are used, etc.

Moreover, antibody that is made based on the present invention can simultaneously function as a so-called capture antibody that captures said antigen protein in solid or liquid phase and as a detecting antibody that is a so-called enzyme-labeled antibody by modificating an enzyme, such as peroxidase and alkali phosphatase, etc., by conventional methods in any of these immunoassay procedure.

Preferred Embodiment of the Invention The following examples are given to explain specifically the present invention; the present invention the principle of is not being restricted to these examples.

Example 1 Cloning of Ribosomal Protein L7/L12 genes from Mycoplasma pneumoniae After inoculating an appropriate amount of Mycoplasma pneumoniae.(ATCC15531, distributed and purchased from ATCC) on PPLO agar (DIFCO: 0412-17-3) supplemented with Mycoplasma Supplement (DIFCO: 0836-68-9), the niicroorganism was cultivated for 5 hours in a CO2 incubator under conditions of 37 C and 5% CO2. The grown colonies were suspended in a TE buffer (Wako Pure Chemical Industries, Ltd.) to a fmal concentration of approximately 5 x 109 CFU/ml. Approximately 1.5 ml of this suspension was transferred to a microcentrifuge tube and centrifuged for 2 minutes at 10,000 rpm. The supematant was discarded. The sediment was resuspended in 567 l of TE buffer. Then 30 l of 10% of SDS and 3 l of 20 mg/mi proteinase K solution were added and thoroughly mixed, followed by incubating for 1 hour at 37 C. The suspension was incubated for further 1 hour at 56 C.
After 80 1 of 10% acetyl trimethyl ammonium bromide/0.7 M NaCI solution were added and mixed, it was incubated for 10 minutes at 65 C. Equal volume of chloroform-isoamyl alcohol mixed solution at a volume ratio of 24:1 was added and stirred well.

The solution was centrifuged for 5 minutes at 4 C and 12,000 rpm with a microcentrifuge and the aqueous fraction was transferred to a new microcentrifuge tube.
Isopropanol was added to the fraction at 0.6-times its volume and the tube was vigorously shaken to form sediment of the DNA. The white DNA sediment was scooped with a glass rod and transferred to a different microcentrifugation tube containing I ml of 70% ethanol (cooled to -20 ). The tube was centrifuged for 5 minutes at 10,000 rpm and the supernatant was gently removed. Then another 1 ml of 70% ethanol was added and the mixture was centrifuged for 5 more minutes.
Once the supernatant had been removed, the sediment was dissolved in 100 1 of TE buffer to obtain the DNA solution. The concentration of the genomic DNA
solution was determined quantitatively in accordance with E5: Spectrophotometric determination of the Amount of DNA or RNA, "Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press, Sambrook, J. et al. (1989).
PCR (polymerase chain reaction) was performed using lOng of this genomic DNA. Taq polymerase (Takara Co., Ltd., code ROOIA) was used for PCR Five 1 of buffer attached to the enzyme, 4 l of a dNTP mixture attached to the enzyme, and 200 pmol of each oligonucleotide (shown in Sequence No. 3 and 4 of the Sequence List) were added to the enzyme. Purified water was added to bring the final volume to 50 l.
The mixture was performed for 5 cycles using TaKaRa PCR Thermal Cycler 480, where one cycle was consisted of treatments for 1 minute at 95 C, 2 minutes at 50 C, and 3 minutes at 72 C. Then, 25 cycles of treatments for 1 minute at 95 C, 2 minutes at 60 C, and 3 minutes at 72 C per one cycle were carried out. Electrophoresis was performed in 1.5% agarose gel using a part of the PCR product. This product was then stained with ethidium bromide (Nippon Gene Co., Ltd.) and observed under ultraviolet rays to confirm amplification of approximately 400 by cDNA. After fragmentation treatment with restriction endonucleases BamHI and Xhol, electrophoresis was performed in 1.5%
agarose gel and staining with ethidium bromide was carried out. An approximately 400 by band was cut out from the gel. This band was purified with Suprecol (Takara Co., Ltd.) and then inserted into pGEX-6P-lTM (Pharmacia), which is a common vector. The vector can function as an expression vector for the desired molecule, which can express fused protein with GS.T protein, by insertion of the desired gene fragment into the appropriate restriction endonuclease site.

Actually, vector pGEX-6P-1 TM and the previous DNA were mixed together at a molar ratio of 1:3 and DNA was inserted into the vector with DNA lipase (Invitrogen Co.).
Vector pGEX-6P-1TM into which DNA had been inserted was genetically introduced to Escherichia coli one-shot competent cells and then inoculated in a plate of LBL-broth agar (Takara Co., ltd.) that was a semi-sold culture plate containing 50 g/ml ampicillin (Sigma). The plate was then incubated at 37 C for 12 hours and the grown colonies were selected at random and inoculated into L-Broth liquid culture medium containing the same concentration of ampicillin. Shake cultivation was performed at 37 C for 8 hours and the bacteria were recovered and the plasmid was separated using Wizard MiniprepTM in accordance with the attached literature. The plasmid was cleaved with restriction endonuclease BamHUXhoI.
Insertion of said PCR product was confirmed by cutting out approximately 370 by DNA.
The DNA base sequence of the DNA that had been inserted was determined using said clone.

Determination of the base sequence of the inserted DNA fragment was performed using the Fluorescence Sequencer of Applied Biosystems.

The sequence sample was prepared using PRISMTM, Ready Reaction Dye Terminator Cycle Sequencing Kit (Applied Biosystems). First, 9.5 ) l of reaction stock solution, 4.0 l of 0.8 pmol/pl T7 promoter primer (Gibco BRL) and 6.5 I of 0.16 i_tg/ l template DNA were added to a microtube of 0.5 ml and mixed. After covering the mixture with a double layer of 100 gl of mineral oil, PCR amplification was performed for cycles, where one cycle was consisted of treatments for 30 seconds at 96 C, 15 seconds at 55 C, and 4 minutes at 60 C. The resulting product was then kept at 4 C for 5 minutes. After the reaction was completed, 80 gl of sterilized pure water was added and stirred. The product was centrifuged and the aqueous layer was extracted 3 times with 25 phenol-chloroform mixed solution. Ten ill of 3M-sodium acetate with pH 5.2 and 300 1 of ethanol were added to 100 1 aqueous layer and stirred. The product was then centrifuged for 15 minutes at room temperature and 14,000 rpm and the sediment was recovered. Once the sediment was washed with 75% ethanol, it was dried under a vacuum for 2 minutes to obtain the sequencing sample. The sequencing sample was dissolved in formamide containing 4 gI of 10 mM EDTA and denatured for 2 minutes at 90 C.
This was then cooled in ice and submitted to sequencing.

Two out of the 5 randomly selected clones had homology of the sequence with the probe used for PCR. In addition, a DNA sequence was evidently identical to the gene sequence of Ribosomal Protein L7/L12. The entire base sequence and the corresponding amino acid sequence of the structural gene moiety are as shown in Sequence No.
1 and No. 2 of the Sequence List. This gene fragment clearly codes for the gene of Mycoplasma pneumoniae Ribosomal Protein L7/L12.

Example 2 Mass expression in Escherichia coil and purification of Ribosomal Protein L7/L12 from Mycoplasma pneumoniae Escherichia coli into which expression vector had been inserted was cultivated overnight in 50 ml of LB medium at 37 C. Then 500 ml of 2-times concentrated YT medium was heated at 37 C for 1 hour. Fifty milliliters of the Escherichia coli solution that had been cultivated overnight were introduced to 500 ml of the aforementioned medium.
One hour later, 550 l of 100 mM isopropyl-(3-D(-)-thiogalactopyranoside (IPTG) were introduced and cultivated for 4 hours. The product was then recovered and introduced to 250 ml centrifugation tubes and centrifuged for 10 minutes at 7,000 rpm. The supernatant was discarded and dissolved in 25 ml each of Lysis buffer containing 25% sucrose in 50 mM Tris buffer, pH7.4. The product was then added with 1.25m1 of 10% NP-40 and 125 1 of 1M

MgCI2, and transferred to a plastic tube. The product.was submitted to 5 times of ultrasonic treatment for 1 minute. After that, the product was centrifuged for 15 minutes at 12.000 rpm, and the supernatant was recovered.

Next, the aforementioned supernatant was adsorbed on a glutathione agarose column conditioned with PBS. Then the column was washed using 2 bed volume of a washing solution containing 4.2 mM MgCI2 and 1 mM dithiothreitol (DTT) in 20 mM Tris buffer, pH7.4. Elution was performed with 50 mM Tris buffer, pH 9.6, containing 5 mM
glutathione. The protein content in the eluted fraction was determined by the pijment bonding method (Bradford method; BioRad Co.) and the main fraction was acquired.

Purity of the purified GST fusion Ribosomal Protein L7/L12 that was obtained was confirmed by electrophoresis to be approximately 75%, showing that purity satisfactory for an immunogen.

Example 3 Preparation of monoclonal antibody to Ribosomal Protein L7/L12 of Mycoplasma 5 pneumoniae First, regarding to immunization of mice, 100 g of the GST fusion Ribosomal Protein L7/L12 antigen of Mycoplasma pneumoniae were dissolved in 200 l of PBS and then 200 l of Freund's complete adjuvant were added and mixed and emulsification was performed. Two hundred microliters of the emulsion were injected intraperitoneally to 10 immunize mice. Then the same emulsion antigen was intraperitoneally injected after 2 weeks, after 4 weeks, and after 6 weeks. Two-fold the concentration of antigen emulsion was injected intraperitoneally after 10 weeks and after 14 weeks. The spleen was excised on 3 days after the final immunization and submitted to cell fusion.

After thoroughly mixing 2 x 107 myeloma cells per 108 spleen cells from mice, which 15 had been taken out aseptically, in a glass tube, the mixture was centrifuged for 5 minutes at 1,500 rpm and the supernatant was discarded. The cells were thoroughly mixed.

The myeloma cells used for cell fusion were obtained by cultivation of cell strain NS-1 in an RPMI 1640 culture medium containing 10% bovine fetal serum, cultivating this product beginning 2 weeks before cell fusion using an RPMI 1640 medium containing 0.13 mM azaguanine, 0.5 g/ml MC-210, and 10% bovine fetal serum for 1 weeks, and then further cultivating the cell strain for 1 week in an RPMI 1640 medium containing 10%
bovine fetal serum.

Fifty ml of RPMI 1640 culture medium that had been kept at 37 C were added to the mixed cell sample and centrifuged at 1,500 rpm. After removing the supernatant, 1 ml of 50% polyethylene glycol that had been kept at 37 C was added and stirred for 1 minute. Ten ml of RPMI 1640 medium kept at 37 were added and the solution was vigorously mixed for approximately 5 minutes while it was suctioned and evacuated from a sterile pipette.

After centrifugation for 5 minutes at 1,000 rpm and removal of the supernatant, 30 ml of HAT culture medium were added to bring the cell concentration to 5 x 10 cells/ml.

This mixture was stirred till uniform and then poured, 0.1 ml per each well, into a 96-well culture plate and cultivated at 37 C in and under condition of 7% carbon dioxide gas. HAT
culture was added, 0.1 ml at a time, on the first day and at after first week and after second week. Then ELISA screened the cells that had produced the desired antibody.

GST fusion Ribosomal Protein L7/L12 and GST of were dissolved in PBS
containing 0.05% sodium azide and diluted to 10 g/ml, and the diluted solution was poured, 100 l each, into separate 96-well plates and adsorbed overnight at 4 C.

After removing the supernatant, 200 .l 1% of bovine serum albumin solution in PBS
were added and the mixture was reacted and blocked for 1 hour at room temperature. After removing the supernatant, the product was washed with washing solution containing 0.02%

Tween 20, in PBS. One hundred milliliters of culture solution of fused cells were added to this and the mixture was reacted for 2 hours at room temperature. The supernatant was removed and sediment was washed with washing solution. Next, 100 l of 50 ng/ml peroxidase labeled goat anti-mouse IgG antibody solution were added and the solution was reacted for 1 hour at room temperature. The supernatant was removed and the product was again washed with washing solution. Then TMB solution (KPL Inc.) was added, 100 l at a time, and the mixture was reacted for 20 minutes at room temperature. After coloration, 100 l of 1 N sulfuric acid were added to stop the reaction and absorbance at 450 nm was determined.

As a result, positive wells that only reacted with GST Ribosomal Protein L7/L12, but did not react with GST were detected, and it was concluded that antibody to Ribosomal ~
Protein L7/L12 is produced.

Therefore, the cells in the positive wells were recovered and cultivated with HAT
medium in a 24-well plastic plate.

The fused medium that had been cultivated was diluted with HT medium to a cell count of approximately 20 cells/ml and then 50 1 of the diluted product was mixed with 106 thymus cells of six-week-old mouse suspended in HT culture medium, in a 96-well culture plate. The mixture was then cultivated for 2 weeks at 37 C under condition of 7% carbon dioxide gas.

Antibody activity in the culture supernatant was similarly determined by the aforementioned ELISA method and the cells that showed positive reaction with Ribosomal Protein L7/L12 were recovered. Furthen.nore, the same dilution and cloning procedure was repeated to obtain a total of 5 clones of hybridoma MPRB-1 - 5.

Example 4 Selection of monoclonal antibody that detect Ribosomal Protein L7/L12 of Mycoplasma pneumoniae.
Monoclonal antibody was produced and recovered in accordance with standard methods using the positive hybridoma cells obtained as previously described.
Specifically, 5 x 106 cells in PBS that had been subcultured using RPMI 1640 culture medium containing 10% FCS were intraperitoneally injected into Balb/C
mice that had been intraperitoneally injected with 0.5 ml of PristaneTM before 2 weeks previously.
After 3 weeks, ascites was recovered and the centrifugation supernatant was obtained.
The solution containing antibody that was obtained was adsorbed in a Protein A
column (5 ml bed, Pharmacia) and rinsed with PBS at 3-times of the bed volume.
Then elution with citrate buffer, pH 3 was performed. The antibody fraction was recovered and the monoclonal antibody that produced by each hybridoma was obtained. The monoclonal antibody derived from these 5 strains of hybridoma was evaluated using ELISA
method.
The sandwich assay method was used to assess the monoclonal antibody. The monoclonal antibody that was prepared was used as antibody for detection by being chemically bound to peroxidase.
That is, enzyme labeling was performed using horseradish peroxidase (Sigma Grade VI) in accordance with the method described in "Analytical Biochemistry (1983), 68-73" with the reagent S-acetylthioacetic acid N-hydroxysuccinimide for binding. In the ELISA reaction, a solution of commercial anti-Mycoplasma pneumoniae polyclonal antibody (Biodesign, rabbit) diluted to a concentration of 10 pg/ml was poured, 100 l each, into a separate 96-well plate and adsorbed overnight at 4 C.
After removing the supematant, 200 l of 1% bovine serum albumin solution in PBS were added and the mixture was reacted and blocked for 1 hour at room temperature.
The supematant was removed and the product was washed with washing solution containing 0.02% Tween 20, in PBS. One hundred microliters of antigen solution, which had been obtained by adding Triton X=100 to culture solutions of each species of microorganism in an amount to a concentration of 0.3% and then extracting the solution for 5 minutes at room temperature, were added to this and the mixture was reacted for 2 hours at room temperature.
The supematant was removed and the product was further washed with washing solution.
Then 100 l of 5 g/ml peroxidase-labeled anti-Ribosomal Protein L7/L12 antibody solution were added and the mixture was reacted for 1 hour at room temperature. The supernatant was removed and the product was washed again with washing solution. TMB

solution (KPL Inc.) was added, 100 l each, and the mixture was reacted for 20 minutes at room temperature. After coloration, 100 l of 1 N sulfuric acid were added to stop the reaction. Absorbance at 450 nm was determined.

It is clearly evident that when monoclonal antibody derived from hybridoma MPRB-1 was used as the enzyme-labeled antibody, all strains of Mycoplasma pneumoniae tested were detected at a sensitivity of 106 cells/mi, while reactivity of other microorganisms e.g., Haemophilus influenzae, Klebsiella pneumoniae, Chlamydia pneumoniae and Neisseria meningitides could not exhibit reactivity, even at high concentrations of 10g cells/ml and therefore, antibody with specific reactivity to Mycoplasma pneumoniae can be obtained by using monoclonal antibody to Ribosomal Protein L7/L12. The antibody was named as AMMP-1. Table 2 shows only those results with AMMP-1. Results with other antibodies, which cross-reacted with other microorganisms are not mentioned here.

Table 2 Result of Detection (10 cells/mi) M. pneumoniae +
Results of Detection (10 cells/ml) N. meningitides -N. lactamica -N. mucosa -N. sicca -H. influenzae -B. catarrharis -N. gonorrhoeae -E. coli -K. pneumoniae -(+: Positive, -: Negative) Example 5 Acquisition of a polyclonal antibody, which specifically reacts with Ribosomal Protein L7/L12 of Mycoplasma pneumoniae usinj a Ribosomal Protein L7/L12-immobilized affmity column The Ribosomal Protein L7/Ll2 of Mycoplasma pneumoniae, which was acquired by the method described in Examples 1, or the supernatant of Mycoplasma pneumoniae bacteria treated with Triton X-100 was used as an antioen. About 1.2 ml of a physiological saline solution containina 100 g of antigen was emulsified with the addition of 1.5 ml of Freund's adjuvant. The emulsion was subcutaneously injected into SPF Japanese White Rabbit to immunize the animal. The rabbit was immunized 5 to 6 times, once every two weeks, and the antibody titer was confirmed.

The antibody titer was confirmed by the ELISA method. Solutions of Ribosomal Protein L7/L12 of Mycoplasma pneumoniae dissolved in PBS containing 0.05%
sodium azide diluted to 10 910,1/ml were poured, 100 l each, into 96-well plates and adsorbed overnight at 4 C. After removing the supematant, 200 l of 1% bovine serum albumin solution in PBS were added and the mixture was reacted and blocked for 1 hour at room temperature. The supernatant was removed and the product was washed with a washino, solution containinc, 0.02% Tween 20, in PBS. One hundred l of a solution obtained by diluting normal rabbit serum and immunized rabbit antiserum was added and the mixture was reacted for two hours at room temperature. The supernatant was removed and the product was further washed with a washin~ solution. Then, 100 l of 50 ng/ml peroxidase-labeled goat anti-rabbit IgG antibody solution was added and the mixture was 5 reacted for one hour at room temperature. The supernatant was removed and the product was washed again with a washing solution. OPD solution (Sigma Co.) was added, 100 l each, and the mixture was reacted for 20 minutes at room temperature. After coloration, 100 l of 1 N sulfuric acid was added to stop the reaction. Absorbance at 492 nm was determined.

10 After confirming that the antibody titer had increased, a large quantity of blood was collected. Blood was collected in a glass centrifuge tube from the ear artery, allowed to stand for one hour at 37 C, and then overnight at 4 C. The mixture was centrifuged at 3000 rpm for 5 minutes and the supematant was recovered. The resulting anti-serum was preserved at 4 C.

15 An affmity column immobilized Ribosomal Protein L7/L12 of Mycoplasma pneumoniae was prepared. HiTrap NHS-activated column (1 ml, manufactured by Pharmacia) was used. Immediately after replacing the column with 1 mM HCI, a PBS
solution of Ribosomal Protein L7/L12 (1 mg/ml) was charged. The column was allowed to stand for 30 minutes and a blocking reagent was charged, followed by equilibration with 20 PBS.

Using the affmity column immobilized Ribosomal Protein L7/L12 of Mycoplasma pneumoniae, the polyclonal antibody in the resulting anti-serum obtained as an antigen from the supematant of Triton X- 100 treated bacteria of Mycoplasma pneumoniae as an antigen was purified. This antiserum was diluted with PBS to a volume of 5 times, passed through a 0.45 m filter, then adsorbed in the column immobilized with Ribosomal Protein L7/L12 of Mycoplas aa pneumoniae at a flow rate of 0.5 ml/min. After elution from the colum.n with 0.1 M glycine buffer, pH 2.1 and immediately neutralizing with 1 M Tris buffer, pH 9.0, eluted fractions of the target antibody were recovered by the ELISA method, the same as the antibody titer measuring method.

The polyclonal antibody obtained in this manner was evaluated by the same OIA
method as described in Japanese Patent Application Laid Open No.07-509565.

The purified antibody was used as a capture antibody for the OIA method.
Moreover, AMMP-1 monoclonal antibody described in Example 4 was labeled with peroxidase and used as the detect antibody. The enzyme labeling was performed using horseradish peroxidase (Sigma Grade VI) and the reagent S-acetylthioacetic acid N-hydroxysuccinimide for binding in accordance with the method in "Analytical Bio-chemistry 132 (1983), 68-73".

In the OIA reaction, the purified polyclonal antibody in PBS containing 0.05%
sodium azide was diluted with 0.1 M HEPES buffer, pH 8.0, to a concentration of 10 g/ml and the diluted solution was added onto a silicon wafer, 50 l at a time, to react for 30 minutes at room temperature, followed by washing with distilled water and coated with a coating solution including sucrose and alkali-treated casein.

Fifteen l of antigen solution, which had been obtained by adding Triton X-100 to culture solutions of various species of microorganism to a concentration of 0.5% and then extracting for 5 minutes at room temperature, was added onto the above-described silicon wafer and reacted for 10 minutes at room temperature. Then, 15 l of 20 g/ml peroxidase-labeled monoclonal antibody was added and the mixture was reacted for 10 minutes. After washing with distilled water, TMB solution (KPL Inc.) was added, 15 l at a time, and the niixture was reacted for 5 minutes at room temperature. The product was washed with distilled water and observed the blue colored as a result of enzyme reaction.
As shown in Table 3 as a result, it is clear that when the purified polyclonal antibody of APMP-1 is used as the capture antibody, Mycoplasma pneumoniae can be detected at a sensitivity of 108 cells/ml, while reactivity of other microorganisms cannot be detected.
Thus, it was confirmed that an affmity column immobilized with the Ribosomal Protein L7/L12 of Mycoplasma pneumoniae obtained the polyclonal antibody with specific reactivity to Mycoplasma pneumoniae.

Table 3 Results of Detection (10 cells/ml) M. pneumoniae +

H. influenzae ATCC10211 _ E. coli ATCC25922 E. faecalis ATCC19433 _ K. pneumoniae ATCC13883 -N. gonorrhoeae IID821 _ N. lactamica ATCC23970 -N. meningitidis ATCC13090 -P. aeruginosa ATCC27853 -Group B Streptococcus ATCC12386 -S. aureus ATCC25923 -S. pneumoniae ATCC27336 -S. pyogenes ATCC19615 -(+: Positive, -: Negative) Industrial Applicability According to the present invention, not only microorganisms can be detected species specifically, but also microorganisms of all serotypes in the same species can be detected at a high precision, by using antibodies to the evolutionary and functionally conserved intracellular molecules.

By using antibodies to Ribosomal Proteins L7/L12 of microorganisms as such antibodies, Mycoplasma pneumoniae can be detected precisely.

Moreover, detection of microorganisms can be performed with higher precision and wider applicability by using the reagent kit for detecting microorganisms comprising such an antibody.

References Cited:
Patent Documents United States Patent 5,552,279 Weisburg, et al. September 3, 1996. Nucleic acid probes for the detection of Mycoplasma pneumoniae and Mvcoplasma genitalium.

EPO 305145, Application No. 88307793.5 Zivin and Monahan. March 1,1989.

Methods and probes for detecting nucleic acid.

EPO 250662, Application No. 86304919.3. Gobel and Stanbridge. January 7, 1988.
Detection of Mycoplasma by DNA hybridization.

Japanese Patent Application Laid Open No. 63(1988)-298 Other Documents Bernet, C., M. Garret, et al. (1993). "Detection of Mycoplasma pneumoniae by using the polymerase chain reaction." J Clin Microbiol 31(4): 1013-5.

Buck, G. E., L. C. O'Hara, et al. (1993). "Rapid, sensitive detection of Mycoplasma pneumoniae in simulated clinical specimens by DNA amplification." J Clin Microbiol 31(4): 1013-5.

Gobel, U. B., A. Geiser, et al. (1987). "Oligonucleotide probes complementary to variable regions of ribosomal RNA discriminate between Mycoplasma species".

Granato, P. A., L. Poe, et al.(1980). "Use of modified New York City medium for growth of Mycoplasma pneumoniae. A preliminary report." Zh Mikrobiol Epidemiol Immunobiol(8): 50-3.

Hyman, H. C., D. Yogev, et al.(1987). "DNA probes for detection and identification of Mycoplasma pneumoniae and Mycoplasma genitalium" .

Jensen, J. S., J. Sondergard-Andersen, et al. (1993). "Detection of Mycoplasma pneumoniae in simulated clinical samples by polymerase chain reaction. Brief report." J
Med Microbio138(3): 166-70.

Kai, M., S. Kamiya, et al. (1987). "Rapid detection of Mycoplasma pneumoniae in clinical samples by the polymerase chain reaction." J Gen Microbiol 133(Pt 7):
1969-74.
Knudson, D. L. and R. MacLeod (1979). "Mycoplasma pneumoniae and Mycoplasma salivarium: electron microscopy of colony growth in agar." Sci Rep Res Inst Tohoku Univ [Med] 26(3-4): 71-91.

Madsen RD, Weiner LB, McMillan JA, Saeed FA, North JA, Coates SR. (1988).
Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody immunoblot assay. Am J Clin Pathol 89(1):95-9 Ragnar Norrby, S. (1999). "Atypical pneumonia in the Nordic countries:
aetiology and clinical results of a trial comparing fleroxacin and doxycycline. Nordic Atypical Pneumonia Study Group." J Med Microbiol 48(12): 1115-22.

Tilton, R. C., F. Dias, et al. (1980). "DNA probe versus culture for detection of Mycoplasma pneumoniae in clinical specimens." J Clin Microbiol 12(6): 748-52.

Yogev, D., D. Halachmi, et al. (1988). "Distinction of species and strains of mycoplasmas (mollicutes) by genomic DNA fmgerprints- with an rRNA gene probe."
J Clin Microbiol 26(11): 2266-9.

Chan ED, Kalayanamit T, Lynch DA, Tuder R, Arndt P, Winn R, Schwarz MI.
"Mycoplasma pneumoniae-associated bronchiolitis causing severe restrictive lung disease in adults: report of three cases and literature review". Chest. 1999;115(4): 1188-94.

NCBI database accession #NC#000912., Himmelreich,R., Hilbert, H., Plagens, H., Pirkl, E., Li, B. C. and Herrmann, R.

Cimolai N. Mycoplasma pneumoniae respiratory infection. Pediatr Rev. 1998;
19(10): 327-31.

Harlow, E., and D. Lane (1988). "Antibodies: A laboratory manual." New York.
Cold Spring Harbor Laboratory Press.

Shambrook, J., E. F. Fritsch, and T. Maniatis. (1989). "Molecular Cloning: A
laboratory Manual (2 d ed.)." Cold Spring Harbor Laboratory Press.

SEQUENCE LISTING
<110> ASAHI KASEI KABUSHIKI KAISHA

<120> ANTIBOY FOR DETECTING MYCOPLASMA PNEUMONIAE
<130> ASAHI-8 <150> JP 2000 062729 <151> 2000-01-31 <160> 4 <210> 1 <211> 369 <212> DNA

<213> Mycoplasma pneumoniae <400> 1 atg gca aaa cta gat aaa aac caa tta att gaa tcg ttg aag gaa atg 48 Met Ala Lys Leu Asp Lys Asn Gln Leu Ile Glu Ser Leu Lys Glu Met acc atc atg gaa atc gat gaa atc att aag gct gta gaa gaa gct ttt 96 Thr Ile Met Glu Ile Asp Glu Ile Ile Lys Ala Val Glu Glu Ala Phe gga gta tcg gca aca cct gta gta gct gct ggt gct gtt ggt ggt aca 144 Gly Val Ser Ala Thr Pro Val Val Ala Ala Gly Ala Val Gly Gly Thr caa gaa gct gct agc gaa gtg act gtg aaa gtt act ggt tac act gac 192 Gln Glu Ala Ala Ser Glu Val Thr Val Lys Val Thr Gly Tyr Thr Asp aac gct aaa tta gct gtg tta aag ctt tac cgc gaa att gct ggt gtt 240 Asn Ala Lys Leu Ala Val Leu Lys Leu Tyr Arg Glu Ile Ala Gly Val ggt tta atg gaa gct aaa act gct gtg gaa aaa ctt cct tgt gtt gtt 288 Gly Leu Met Glu Ala Lys Thr Ala Val Glu Lys Leu Pro Cys Val Val aag caa gac atc aaa cct gaa gaa gct gaa gaa ctt aaa aag cgt ttc 336 Lys Gln Asp Ile Lys Pro Glu Glu Ala Glu Glu Leu Lys Lys Arg Phe gtt gaa gtt gga gca act gtt gaa atc aaa taa 369 Val Glu Val Gly Ala Thr Val Glu Ile Lys <210> 2 <211> 122 <212> PRT

<213> Mycoplasma pneumoniae <400> 2 Met Ala Lys Leu Asp Lys Asn Gln Leu Ile Glu Ser Leu Lys Glu Met = 3/4 Thr Ile Met Glu Ile Asp Glu Ile Ile Lys Ala Val Glu Glu Ala Phe Gly Val Ser Ala Thr Pro Val Val Ala Ala Gly Ala Val Gly Gly Thr Gln Glu Ala Ala Ser Glu Val Thr Val Lys Val Thr Gly Tyr Thr Asp Asn Ala Lys Leu Ala Val Leu Lys Leu Tyr Arg Glu Ile Ala Gly Val Gly Leu Met Glu Ala Lys Thr Ala Val Glu Lys Leu Pro Cys Val Val Lys Gln Asp Ile Lys Pro Glu Glu Ala Glu Glu Leu Lys Lys Arg Phe Val Glu Val Gly Ala Thr Val Glu Ile Lys <210> 3 <211> 29 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer DNA used for obtaining Ribosomal Protein L7/L12 gene from Hycoplasma pneumoniae <400> 3 aatggatcca tggcaaaact agataaaaa 29 <210> 4 <211> 30 <212> DNA

<213> Artificial Sequence <220>

<223> PCR primer DNA used for obtaining Ribosomal Protein L7/L12 gene from Mycoplasma pneumoniae <400> 4 tgactcgagt tatttgattt caacagttgc 30

Claims

1. An antibody which is an antibody to ribosomal protein L7/L12 comprising SEQ ID NO:2 of a microorganism belonging to Mycoplasma pneumoniae and which reacts specifically with ribosomal protein L7/L12 comprising SEQ ID NO:2 of said microorganism.

2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

3. The antibody according to claim 1 or 2, wherein the antibody is conjugated with an enzyme.

4. A method of detecting a microorganism belonging to Mycoplasma pneumoniae in a test sample, comprising:
a) contacting a test sample with a lysing solution, and extracting ribosomal protein from the microorganism b) contacting the extracted test sample with a capture antibody, wherein the capture antibody is the antibody defined in any one of claims 1 to 3 fixed on a solid surface, and forming an antigen-antibody complex from the ribosomal protein and the capture antibody, and c) detecting the antigen-antibody complex using a detection antibody, wherein the detection antibody is the antibody defined in any one of claims 1 to 3.

5. The method according to claim 4, wherein the detection antibody is an antibody conjugated with an enzyme and the antigen-antibody complex is detected with a substrate specific to the enzyme.

6. A reagent kit for detecting a microorganism belonging to Mycoplasma pneumoniae, comprising the antibody defined in any one of claims 1 to 3 together with instructions for detecting a microorganism belonging to Mycoplasma pneumoniae in a test sample.

7. A method of preparing the antibody defined in any one of claims 1 to 3, comprising using as an immunogen, (A) Ribosomal Protein L7/L12 of microorganism belonging to Mycoplasma pneumoniae and obtained by a gene manipulation procedure or by isolation and purification from the microorganism, (B) a peptide moiety thereof, (C) or a synthesized peptide corresponding to the peptide moiety to elicit an immunological response and generate the antibody defined in any one of claims 1 to 3.