CA2398467C

CA2398467C - Antibody for detecting chlamydia pneumoniae

Info

Publication number: CA2398467C
Application number: CA2398467A
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: 2010-06-29
Anticipated expiration: 2021-01-31
Also published as: KR100734994B1; JP5331284B2; CA2398467A1; AU2001230531A1; CN1962694A; JP2012017334A; KR20020073191A; WO2001057089A1; CN1406248A

Abstract

A method for specifically, highly sensitively and quickly detecting a microorganism belonging to Chlamydia penumoniae; 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 C. penumoniae which reacts specifically with this microorganism; a method of detecting the microorganism in a specimen by using this antibody; and a detection reagent kit containing this antibody. The ribosomal protein is exemplified by Ribosomal Protein L7/L12 and this method is usable in detecting the infection with a microorganism causative of pneumonia.

Description

DESCRIPTION
ANTIBODY FOR DETECTING CHLAMYDIA PNEUMONIAE
Field of the Invention The present invention relates to antibody useful to a detection of microorganism that belongs to Chlamydia 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 importanf to medication, specifically to the diagnosis of atypical pneumonia caused by Chlamydia pneumoniae.
The invention can be useful for detecting the species Chlamydia 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.
However, it takes time to obtain results by cultivation and identification methods or

2 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.
Chlamydia pneumoniae is the common causative pathogen of pneumonia through out the world. It is small non-motile Gram-negative bacteria that invade selectively human host and cause diseases without any known animal reservoir. The sero-prevalence is 40 to SO% in the 30-to 40-year old age group(Hyman, Roblin et al. 1995). It causes pharyngitis, bronchitis and mild pneumonia.
The microorganism is very small, obligate parasite and grow within the cytoplasm of host cells. Growth of Chlamydia pneumoniae in tissue culture medium is slow (Godzik, O'Brien et al. I995), and might take at least 3-5 days or more for the identification of the bacterium in the medium (Essig, Zucs et al. 1997). Therefore, Gram staining method and culture method are not pertinent for the diagnostic method rapidly detecting the causative pathogen. As a rapid diagnosis for Chlamydia, the immunological method using antibody is often used.
It is known that lipopolysaccharide (LPS), which is a genus-specific antigen of Chlamydia, is present as an antigen determinant (Verkooyen, Van Lent ~et al.
1998), and antibodies to LPS are used as the reagent antibody in various diagnostic kits, particularly for detection of Chlamydia trachomatis.
Moreover, Peterson et al.(Peterson, Cheng et al. 1993; Peterson, de la Maza et al.
1998), and Batteiger et al. (Batteiger, Newhall et al. 1986) have both reported on monoclonal antibody to major outer membrane protein (MOMP) of the genus Chlamydia.
These antibodies were found to be useful to distinguish Chlamydia pneumoniae and Chlamydia trachomatis. Later, these antibodies played an important role in unveiling antigenic differences within the species of Chlamydia pneumoniae. Antigens like these might have advantage for serotyping within the species of Chlamydia pneumoniae, but not

3 for routine diagnosis where all strains of the species are needed to be detect. A common antigen with common function, that most of the structure is evolutionarily protected in the species of the microorganism, but the antigen that could be used for selectively detecting the differences, was not known hitherto.
S The present invention relates to useful protein commonly existing in all microorganisms as molecules having same function, and as protein antigen for obtaining antibody. Generally, these molecules do structurally change in a small scale.
If these same functional common molecules structurally change in a large scale, it may bring important bad influence to the survival of the microorganisms.
Though there is a few numbers of commercially available monoclonal antibodies for the detection of the pathogen of Chlam~=dia, those are not enough. Till not long ago it was believed that only TWAR strain causes pneumonia (Thorn and Grayston 1991;
United States Patent No. 5,008,186). Recently several sero-variety of the pathogen of Chlamydia have been reported. It is found that LPS or MOMP differ from strain to strain and antibodies to only one serotype do not cover all.
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 Chlamydia 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 microorganisms. 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 species or genus of microorganism, has a protean property enable to use for discrimination specific to species or genus of microorganism, and object microorganisms may be detected all serotypes thereof.

The inventors focused on intracellular molecules that are present as molecules 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/LI2 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 Chlamydia 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 Chlamydia pneumoniae can be obtained and species-specific detection of Chlamydia pneumoniae is possible using said antibody.
In accordance with the present invention a monoclonal antibody specific to Ribosomal Protein L7/L12 of Chlamydia pneumoniae has been discovered and developed.
The antibody is novel and different from any antibody previously known and has property of reacting specifically to the said protein.
Sequences No. l and No. 2 in the Sequence List are the DNA sequence (NCBI
database accession #NC#000922) of the Ribosomal Protein L7/L12 gene of Chlamydia pneumoniae and the corresponding amino acid sequence (NCBI database accession #AE001593.1, NCBI data base). 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 of amino acid. The notation "+" in closest match test indicates that it is different S
amino acid but amino acid with similar properties such as hydrophobic. The notation " "
(blank) indicates that it is entirely different amino acid including properties 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 S 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 1 Closest Match Test:
Ct: I MTTESLETLVEQLSGLTVLELSQLKKLLEEKWDVTAAAPWAVAGAAAAGDAPASAEPTE 60 +TTESLETLVE+LS LTVLELSQLKKLLEEKWDVTA+APWAVA A G+AP +AEPTE
Cp: 1 VTTESLETLVEKLSNLTVLELSQLKKLLEEKWDVTASAPWAVA-AGGGGEAPVAAEPTE 59 IS Ct: 61 FAVILEDVPSDKKIGVLKVVREVTGLALKEAKEMTEGLPKTVKEKTSKSDAEDTVKKLQE 120 FAV LEDVP+DKKIGVLKWREVTGLALKEAKEMTEGLPKTVKEKTSKSDAEDTVKKLQ+
Cp: 60 FAVTLEDVPADKKIGVLKVVREVTGLALKEAKEMTEGLPKTVKEKTSKSDAEDTVKKLQD 119 Ct: 121 AGAKAVAKGL 130 Cp: 120 AGAKASFKGL 129 Ct= Chlamydia trachomatis Cp= Chlamydia pneumoniae In the present invention, the term "microorganism" means Chlamydia pneumoniae, specifically, indicates microorganism having a pathogenic property in respiratory organ and high significance in diagnosis as a causative pathogen of Chlamydia 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 to Ribosomal Protein L7/L12, the segment may be of the length characterizing the Ribosomal Protein L7IL12, 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 L7lLl2 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 bacteria 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 known 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 affinity 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 microorganism is acquired, it does not coincide with the purpose of the present invention because the full length 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.
b) For microorganism that the amino acid sequence of the Ribosomal Protein is unknown, as the amino acid sequence of the Ribosomal Protein L7/L12 has 50 -60 °70 of homology between microorganism, 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 gene 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 affinity column methods with antibody to the protein g that was used as the fusion protein. In such a case, even if antibody to the amino acid segment retained within the microorganism is acquired, it does not coincide with the purpose of the present invention because the full length 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) Alternatively, 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 microorganism 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 western 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 various diagnostic reagents and kits specific to microorganisms, and can be used in a variety of immunoassay methods. 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 substance, 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 (01A) technology described in Japanese (via International) Patent Application Laid-open No. 07(1995)-509565, in which microorganisms are detected by the principle of an optical interference induced by an antibody reaction on the optical thin film which in 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 reagents) comprising various surfactants, such as typically Triton X-100 and Tween-20, 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 Chlamydia pneumoniae are shown as Sequence Numbers l and 2 in Sequence List, respectively.
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 5 microorganisms.
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.
10 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 Chlamydia pneumoniae can be thus acquired according to a conventional method. The entire length of the gene for Ribosomal Protein L7/L12 of Chlamydia pneumoniae can be acquired through the analysis of the DNA
sequence information of these fragments.
The Ribosomal Protein L7/L12 gene of Chlamydia 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 quantity 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 Chlamydia 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 Chlamydia pneumoniae bacteria, a homogenized fluid of the bacteria, or Ribosomal Protein L7/L12 of Chlamydia 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 Embodiments 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.
1 ~ Example 1 Cloning of Ribosomal Protein L7/L12 genes from Chlamydia pneumoniae Chlamydia pneumoniae (ATCC VR-1310; distributed and purchased from ATCC) was cultured on a monolayer of HL cell line. Detailed procedure for culturing Chlamydia pneumoniae is described by Kuo et al. and the like (Cles and Stamm 1990; Kuo and Grayston 1990; Yoshizawa, Dairiki et al. -1992). The microorganism was cultivated for 5 days in a COZ incubator under conditions of 37°C and 5% C02. Infected cells were collected by centrifugation and suspended in a TE buffer (Wako Pure Chemical Industries, Ltd.) to a final concentration of approximately 5 x 107 cells/ml. Approximately 1.5 ml of this suspension was transferred to a microcentrifuge tube and centrifuged for 2 minutes at 10,000 rpm. The supernatant was discarded. The sediment was resuspended in 567 ~.tl of TE buffer.
Then 30 N,I of 10% SDS and 3 ~tl of 20 mg/ml Proteinase K solution were added and thoroughly mixed, and the suspension was incubated for one hour at 37°C. The suspension was incubated for another one hour at ~6°C. After adding 80 ~tl of 10%
acetyl trimethyl ammonium bromide/0.7 M NaCI solution, it was incubated for 10 minutes at 6~°C. 700 ~.tl of chloroform-isoamyl alcohol 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 using a microcentrifugation device 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 1 ml of 70% ethanol (cooled to -20°C). 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 p1 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 in "Molecular cloning: A laboratory manual", Cold Spring Harbor Laboratory Press, Sambrook, J. et al. (1989).
PCR (polymerise chain reaction) was performed using long of this genomic DNA.
Taq polymerise (Takara Co., ltd., code R001A) was used for PCR Five N,I of buffer attached the enzyme, 4 E.d of a dNTP mixture attached the enzyme, and 200 pmol of each synthetic 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 p1.
This mixture was cycled 5 times with a TaKaRa PCR Thermal Cycler 480 for 1 minute at 95°C, 2 minutes at 50°C, and 3 minutes at 72°C
and was then cycled 25 times for 1 minute at 95°C, 2 minutes at 60°C, and 3 minutes at 72°C.
Electrophoresis was performed in 1.5% agarose gel using a part of this PCR product. This product was then stained with ethidium bromide (Nippon Gene Co., Ltd.) and observed under ultraviolet ray to confirm amplification of approximately 400 by DNA. 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-1 (Pharmacia), which is a common vector. This vector can function as an expression vector for the desired molecule, which can express fused protein with GST
protein, by insertion of the desired gene fragment into the appropriate restriction endonuclease site.
Specifically, vector pGEX-6P-1 and the previous DNA were mixed together at a molar ratio of 1:3 and DNA was inserted into the vector with T4 DNA ligase (Invitrogen Co:). Vector pGEX-6P-1 into which DNA had been inserted was genetically introduced to Escherichia coli one-shot competent cells and then inoculated in a plate of LB
L-broth agar (Takara Co., ltd.) which was semi-sold culture plate containing 50 p,g/ml ampicillin (S manna).
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 ampicilIin. Shake cultivation was performed at 37°C
for 8 hours and the bacteria were recovered and the plasmid was separated using Wizard Miniprep in accordance with the attached description. The plasmid was cleaved with restriction endonuclease BamHI/XhoI. Insertion of said PCR product was confirmed by cutting out approximately 370 by DNA. The 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 PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (Applied Biosystems). First, 9.5 E1.1 of reaction stock solution, 4.0 ~.tl of 0.8 pmol/~,l T7 promoter primer (Gibco BRL) and 6.5 ~tl of 0.16 p,g/~tl template DNA
were added to a microtube with a capacity of 0.5 ml and mixed. After covering the mixture with a double layer of 100p.1 mineral oil, PCR amplification was performed for 25 cycles, where one cycle consisted of 30 seconds at 96°C, 15 seconds at 55°C, and 4 minutes at 60°C.
The product was then kept at 4°C for 5 minutes. After the reaction was completed, 80 Etl sterilized pure water was added and stirred. The product was centrifuged and the aqueous layer was extracted 3 times with phenol-chloroform mixed solution. Ten microliters 3M-sodium acetate (pH 5.2) and 300~t.1 ethanol were added to 100111 aqueous layers 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 containling 4 E,tl 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. 4 and No. 5 of the Sequence List. This gene fragment clearly codes for Chlamydia pneumoniae Ribosomal Protein L7/L12.
Example 2 Mass expression in Escherichia coil and purification of Ribosomal Protein L7/L12 from Chlamydia pneumoniae Escherichia coli into which expression vector had been inserted was cultivated overnight in 50 ml of LB medium at 37°C. Then 500 mI 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 ~d 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, pH 7.4. Furthermore, 1.2~ ml of 10% NP-40 and 125 ~t.l of 1M MgCl2 were added and the mixture was transferred to a plastic tube. Ultrasonication was performed 1 minute x 5 times while ice cold. 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 with twice bed volume of washing solution containing 4.2 mM MgCl2 and 1 mM dithiothreitol (DTT) in 20 mM Tris buffer, pH
7.4. Elution was performed in 50 mM Tris buffer, pH 9.6, containing 5 mM
glutathione.
The protein content in the elution fraction was determined by the pigment bonding method (Bradford method; BioRad Co.) and the main fraction was acquired.
Purity of the purified GST fusion Ribosomal Protein L7/LI2 that was obtained was confirmed by electrophoresis to be approximately 75%, showing the purity satisfactory for an immunogen.
5 Example 3 Preparation of monoclonal antibody to Ribosomal Protein L7/LI2 of Chlamydia pneumoniae First, regarding to the immunization of mice, 100 p,g of the GST fusion of Ribosomal Protein L7/L12 antigen of Chlamydia pneumoniae were dissolved in 200 p.1 of PBS and then 10 200 E.d of Freund's complete adjuvant were added and mixed and emulsification was performed. Two hundred microliters . of the emulsion were injected intraperitoneally to immunize mice. Then the same emulsion antigen was iniraperitoneally injected after 2 weeks, after 4 weeks, and after 6 weeks. Two-fold the concentration of antigen emulsion was injected intraperitoneally after IO weeks and after I4 weeks. The spleen was excised 15 out 3 days after the final immunization and submitted to cell fusion.
After thoroughly mixing 2 x 10~ myeloma cells per 10g spleen cells, which had been recovered aseptically from mice, in a glass tube, the mixture was centrifuged for 5 minutes at 1,500 rpm and the supernatant was discarded. The cells were then thoroughly mixed.
The myeloma cells used for cell fusion were obtained by cultivation of cell strain NS-1 with an RPMI 1640 culture medium containing 10% bovine fetal serum, cultivating this product using an RPMI 1640 medium containing 0.13 mM azaguanine, 0.5 p.g/ml MC-210, and 10% bovine fetal serum for 1 weeks from 2 weeks before the cell fusion, and then further cultivating the cell strain for I week with 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 mixed solution was vigorously mixed for approximately 5 minutes by sucking and discharging the mixed solution with 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 106cells/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 and under condition of 7% carbon dioxide gas. HAT
culture was added, 0.1 ml at a time, on the first day, at after 1 week and after 2 week, respectively. Then the cells that had produced the desired antibody were screened by ELISA.
GST fusion Ribosomal Protein L7/L12 and GST protein were dissolved in PBS
containing 0.05% sodium azide and diluted to 10 ~.g/ml. The diluted solutions were separately poured, 100 u1 per each well, into 96-well plates and adsorbed overnight at 4°C.
After removing the supernatant, 200 E,tl of 1 % 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 (0.02%
Tween 20, PBS). One hundred microliters of culture solution of fused cells were added to this and reacted for 2 hours at room temperature. The supernatant was removed and the sediment was washed with washing solution. Next, 100 N.l of 50 ng/ml peroxidase-labeled goat anti-mouse IDG 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. Then TMB solution (KPL Co., Ltd.) was added, 100 Etl each, and the.mix~ture was reacted for 20 minutes at room temperature. After coloration, 100 p1 of 1N
sulfuric acid were added to stop the.reaction and absorbance at 450 nm was determined.
As a result, positive cells that reacted only with GST fusion Ribosomal Protein L7/L12 but did not react with GST protein were detected, thus it could be 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 number of approximately 20 cells/ml and then 50 ~tl of the diluted medium was mixed with 106 six-week-old mouse thymus cells suspended in HT culture medium in a 96-well culture plate. The culture was then cultivated for 2 weeks at 37°C and under conditions 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. Furthermore, the same dilution detection and cloning procedure was repeated to obtain 5 clones in total as hybridoma CPRB-1 - 5.
Example 4 Selection of monoclonal antibody that detects Ribosomal Protein L7/L12 of Chlamydia pneumoniae.
Monoclonal antibody was produced and recovered in accordance with conventional 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 Pristane before 2 weeks in advance.
Ascites was recovered 3 weeks later and the centrifugation supernatant was obtained.
The obtained solution containing antibody was adsorbed in a Protein A column (5 ml, Pharmacia) and rinsed with PBS at 3-times 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 by ELISA method.
The sandwich assay method was used to evaluate 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 132 (1983), 68-73" using the reagent S-acetylthioacetic acid N-hydroxysuccinimide for binding. In the ELISA reaction, a solution of a commercially available anti-Chlamydia pneumoniae polyclonal antibody (rabbit} diluted to a concentration of 10 ~.g/ml was separately poured, 100 E,tl per each well, into a 96-well plate and adsorbed overnight at

4°C.
After removing the supernatant, 200 N.1 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 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 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 supernatant was removed and the product was washed again with washing solution. Then IO 100 1.L1 of 5 p,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 with washing solution. TMB (KPL) solution was added, 100 Etl each, and the mixture was reacted for 20 minutes at room temperature. After coloration, 100 ~t.l of 1N sulfuric acid were added to stop the reaction.
Absorbance at 450 nm was determined.
It is evident that when monoclonal antibody derived from hybridoma CPRB-1 was used as the enzyme-labeled antibody, all strains of Chlamydia pneumoniae tested were detected at a sensitivity of 106 cells/ml, while reactivity of other microorganisms, such as Haemophilus influen~ae, Klebsiella pneumoniae, Mycoplasma pneumoniae and Ner.'sseria meningitides could not be detected, even at high concentrations of 10$
cells/ml and therefore, antibody with specific reactivity to Chlamydia pneumoniae can be obtained by using monoclonal antibody to Ribosomal Protein.L7/L12. The antibody was named as AMCP-1.
Table 2 shows only those results with AMCP-1. Results with other antibodies that cross-reacted with other-microorganisms are not mentioned here.

Table 2 Result of Detection (106 cells/ml) C. pneumoniae +

Results of Detection (10$ cells/ml) N. meningitides -N. lact:amica -N. mucosa -N. si cca -M. pneumoniae -H. influenzae -B. cat:arrharis -N. gonorrhoeae -E. coli -K. pneumoniae -(+ : Positive, - : Negative) Example 5 Acquisition of a polyclonal antibody, which specifically reacts with Ribosomal Protein L7/L12 of Chlamydia pneumoniae using a Ribosomal Protein L7/L12-immobilized affinity column Ribosomal Protein L7/L12 of Chlamydia pneumoniae , which was acquired by the method described in Examples l, or the supernatant of Chlamydia pneumoniae bacteria treated with Triton X-100 was used as an antigen. About 1.2 ml of a physiological saline solution containing 100 pg 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 rabbit. 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. Ribosomal Protein L7/L12 of Chlamydia pneumoniae was dissolved in PBS containing 0.0~% sodium azide and diluted to concentration of 10 ~tg/ml. The diluted solution was poured, 100 p1 per each well, into 96-well plates and adsorbed overnight at 4°C. After removing the supernatant, 200 E.d 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 washing solution containing 0.02% Tween 20, in PBS. One hundred ~tl of a solution obtained by diluting normal rabbit serum and immunized rabbit antiserum was added and the

5 mixture was reacted for two hours at room temperature. The supernatant was removed and the product was washed again with a washing solution. Then, 100 ~t.l of 50 ng/ml peroxidase-labeled goat anti-rabbit IgG antibody solution was added and the mixture was reacted for one hour at room temperature. The supernatant was removed and the product was washed with a washing solution. OPD solution (Sigma Co.) was added, 100 ~.tl each, 10 and each mixture was reacted for 20 minutes at room temperature. After coloration, 100 p.1 of 1N sulfuric acid was added to stop the reaction. Absorbance at 492 nm was determined.
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 blood was centrifuged at 3,000 15 rpm for 5 minutes and the supernatant was recovered. The resulting anti-serum was stored at 4°C.
An affinity column immobilized Ribosomal Protein L7/L12 of Chlamydia pneumoniae was prepared. HiTrap NHS-activated column ( 1 ml, manufactured by Pharmacia was used. Immediately after replacing the column with 1 mM HCI, a solution of 20 Ribosomal Protein L7/L12 in PBS (1 mg/ml) was charged. The column was allowed to stand for 30 minutes and a blocking reagent was charged, followed by equilibration with PBS.
Using the affinity column immobilized Ribosomal Protein L7/L12 of Chlamydia pneumoniae, the polyclonal antibody in the resulting anti-serum obtained as an antigen from the supernatant of Triton X-100 treated bacteria of Chlamydia pneumoniae was purified.
This antiserum was diluted with PBS to 5 times of its volume, passed through a 0.45 u.m filter, and then adsorbed in the column immobilized Ribosomal Protein L7/L12 of Chlamydia pneumoniae at a flow rate of 0.5 ml/min. After elution from the column with 0.1 M glycine, pH 2.1, the eluted fraction was immediately neutralized with 1 M
Tris buffer, pH

9.0, the target antibody in eluted fraction was then recovered by the ELISA
method similar to the antibody-titer measuring method.
The polyclonal antibody obtained in this manner was evaluated by the OIA
method as described in Japanese (via International} Patent Application Laid-open No.
07(1995)-509565.
The purified antibody was used as a capture antibody for the OIA method.
Moreover, peroxidase-labeled AMCP-1 monoclonal antibody described in Example-4 was used as the detect antibody. That is, enzyme labeling was performed in accordance with the method described in "Analytical Biochemistry 132 (1983}, 68-73" using horseradish peroxidase (Sigma Grade VI) and the reagent S-acetylthioacetic acid N-hydroxysuccinimide for binding.
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 p.g/ml and the diluted was added onto a silicon wafer, 50 E.d at a time, to react for 30 minutes at room temperature, followed by washing with distilled water and coating with a coating solution including sucrose and alkali treated casein.
Fifteen N.l of antigen solution, which had been obtained by adding Triton X-100 to culture solutions of each bacterium to a concentration of 0.5% and then extracting the solution for 5 minutes at room temperature, was added onto the above silicon wafer and reacted for 10 minutes at room temperature. Then, 15 ~tl of 20 p.g/ml peroxidase-labeled monoclonal antibody was added and reacted for 10 minutes. After washing with distilled water, TMB solution (KPL} was added, 15 uI at a time, and reacted for 5 minutes at room temperature. The product was washed with distilled water and observed the blue generated from the enzyme reaction.
As a result, as shown in Table 3 it is clear that when the purified polyclonal antibody APCP-1 is used as the capture antibody, Chlamydia pneumoniae can be detected in a sensitivity of 10g cells/ml, while reactivity of other microorganisms cannot be detected.
Thus, an affinity column immobilized with the Ribosomal Protein L7/L12 of Chlamydia pneumoniae confirmed the capturing an antibody having specific reactivity to Chlamydia pneumonaae.
Table 3 Results of Detection _ (108 cells/ml) C. 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 ATCC12386 -B
Streptococcus 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, Chlamydia 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 No. x,008,186, Grayston, et al.. 1991 Detection of unique Chlamydia strain associated with acute respiratory disease United States Patent No. 5,281,518 Campbell, et al. 1994 Detection of a unique Chlamydia strain associated with acute respiratory disease United States Patent No. 5,350,673 Campbell, et al. 1994 Detection of a unique Chlamydia strain associated with acute respiratory disease Other Documents:
Batteiger, B. E., W. J. t. Newhall, et al. (1986). "Antigenic analysis of the major outer membrane protein of Chlamydia trachomatis with murine monoclonal antibodies."
Infect Immun 53(3): 646-50.
Cles, L. D. and W. E. Stamen (1990). "Use of HL cells for improved isolation and passage of Chlamydia pneumoniae." J Clin Microbiol 28(5): 938-40.
Essig, A., P. Zucs, et al. (1997). "Diagnosis of omithosis by, cell culture and polymerase chain reaction in a patient with chronic pneumonia." Clin Diagn Lab Immunol 4(2j: 213-6.
Godzik, K. L., E. R. O'Brien, et al. (1995). "In vitro susceptibility of human vascular wall cells to infection with Chlamydia pneumoniae." J Clin Microbiol 33(9):

Hyman, C. L., P. M. Roblin, et al. (1995). "Prevalence of asymptomatic nasopharyngeal carnage of Chlamydia pneumoniae in subjectively healthy adults:
assessment by polymerise chain reaction-enzyme immunoassay and culture." Clin Infect Dis 20(5): 1174-8.
Kuo, C. C. and J. T. Grayston (1990}. "A sensitive cell line, HL cells, for isolation and propagation of Chlamydia pneumoniae strain TWAR." J Infect Dis 162(3}: 755-8.
Peterson, E. M., X. Cheng, et al. { 1993). "Functional and structural mapping of Chlamydia trachomatis species- specific major outer membrane protein epitopes by use of neutralizing monoclonal antibodies." J Gen Microbiol 139(Pt 11): 2621-6.
Peterson, E. M., L. M. de la Maza, et al. (1998}. "Characterization of a neutralizing monoclonal antibody directed at the lipopolysaccharide of Chlamydia pneumoniae." Scand J
Infect Dis 30(4): 381-6.
Thom, D. H. and J. T. Grayston ( 1991 ). "Infections with Chlamydia pneumoniae strain TWAR." Clin Chest Med 12(2): 245-56.
Verkooyen, R P., N. A. Van Lent, et al. (1998). "Diagnosis of Chlamydia pneumoniae infection in patients with chronic obstructive pulmonary disease by micro-immunofluorescence and ELISA." Am Heart J 135(1): 15-20.
Yoshizawa, H., K. Dairiki, et al. (I992). "Comparison of sensitivity of Hep-2 cells with that of HL cells against Chlamydia pneumoniae." Kansenshogaku Zasshi 66(8):
1037-41.
NCBI database accession #NC_000922. Kalman, S., Mitchell, W., Marathe, R., Lammel, C., Fan, J., Olinger, L., Grimwood, J., Davis, R. W. and Stephens, R.
S.
NCBI database accession #AE001593.1. Kalman, S., Mitchell, W., Marathe, R, Lammel, C., Fan, J., Olinger, L., Grimwood> J., Davis, R. W. and Stephens, R.
S.
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 RAISHA
<120> ANTIBOY FOR DETECTING CHLAMYDIA PNELGifONIAE
<130> ASAHI-9 <150> JP 2000 062684 <151> 2000-O1-31 <160> 4 <210> 1 <211> 39 <212> DNA
<213> Chlamydia pnevmoniae <400> 1 gtg aca aca gaa agt ttg gaa act tta gta gag aag tta agt aat tta 48 Val Thr Thr Glu Ser Leu Glu Thr Leu Val Glu Lys Leu Ser Asn Leu act gta cta gaa ctc tct caa ttg aaa aaa tta tta gaa gag aag tgg 96 Thr Val Leu Glu Leu Ser Gln Leu Lys Lys Leu Leu Glu Glu Lys Trp gat gtt act get tct get ccc gta gtt get gtt get get ggt ggt ggc 144 Asp Val Thr Ala Ser Ala Pro Val Val Ala Val Ala Ala Gly Gly Gly gga gaa get cct gtt get gcc gaa cct aca gaa ttt gca gta acc ctc 192 Gly Glu Ala Pro Val Ala Ala Glu Pro Thr Glu Phe Ala Val Thr Leu gaa gat gtt cct gca gat aaa aaa atc ggc gtc tta aaa gtc gtt agg 240 Glu Asp Val Pro Ala Asp Lys Lys Ile Gly Val Leu Lys Yal Val Arg 65 70 75 g0 gaa gta act gga tta get tta aaa gaa get aaa gaa atg aca gaa ggt 288 Glu Val Thr Gly Leu Ala Leu Lys Glu Ala Lys Glu Met Thr Glu Gly tta cct aaa act gtt aaa gaa aaa act tct aaa agt gat get gaa gat 336 Leu Pro Lys Thr Val Lys Glu Lys Thr Ser Lys Ser Asp Ala Glu Asp act gtt aag aag tta caa gat get ggc gca aaa gcc tca ttt aag gga 384 Thr Val Lys Lys Leu Gln Asp Ala Gly Ala Lys Ala Ser Phe Lys Gly ctg taa 390 Leu <210> 2 <211> 122 <212> PRT
<213> Chlamydia pneumoniae <400> 2 Val Thr Thr Glu Ser Leu Glu Thr Leu Val Glu Lys Leu_Ser Asn Leu Thr Val Leu Glu Leu Ser Gln Leu Lys Lys Leu Leu Glu Glu Lys Trp Asp Val Thr Ala Ser Ala Pro Val Val Ala Va1 Ala Ala GIy Gly,Gly Gly Glu Ala Pro Val Ala AIa Glu Pro Thr Glu Phe Ala Val Thr Leu Glu Asp Val Pro Ala Asp Lys Lys Ile Gly Val Leu Lys Val Val Arg Glu Val Thr Gly Leu Ala Leu Lys Glu Ala Lys Glu Met Thr Glu Gly Leu Pro Lys Thr Val Lys GIu Lys Thr Ser Lys Ser Asp Ala Glu Asp Thr Val Lys Lys Leu Gln Asp Ala Gly Ala Lys Ala Ser Phe Lys Gly Leu <210> 3 <211> 33 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer DNA used for obtaining Ribosomal Protein L7/L12 gene from Chlamydia pneumoniae <400> 3 gtgggatccg tgacaacaga aagtttggaa act 33 <2i0> 4 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> PCR primer DNA used for obtaining Ribosomal Protein L7/L12 gene from Chlamydia pneumonsae <400> 4 ttactcgagt tacagtccct taaatgaggc ttttg 35

Claims

1. An antibody which is antibody to ribosomal protein of microorganism belonging to Chlamydia pneumoniae and which reacts specifically with ribosomal protein of said microorganism.

2. The antibody according to claim 1, wherein the ribosomal protein of the microorganism belonging to Chlamydia pneumoniae is Ribosomal Protein L7/L12.

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

4. The antibody according to any one of claims 1 to 3, wherein the antibody is conjugated with an enzyme.

5. A method of detecting the microorganism belonging to Chlamydia pneumoniae, characterized by using the antibody defined in any one of claims 1 to 4.

6. A method of detecting the microorganism belonging to Chlamydia pneumoniae, 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 4 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 4.

7. The method according to claim 6, 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.

8. A reagent kit for detecting the microorganism belonging to Chlamydia pneumoniae, characterized by using the antibody defined in any one of claims 1 to 4.

9. A method of preparing the antibody defined in any one of claims 1 to 3, characterized by using as an immunogen, Ribosomal Protein L7/L12 of microorganism belonging to Chlamydia pneumoniae and obtained by a gene manipulation procedure or by isolation and purification from the microorganism, a peptide moiety thereof or a synthesized peptide corresponding to the peptide moiety.