WO2001057199A1 - Antibody for detecting mycoplasma pneumoniae - Google Patents

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 L7/L12 employed in detecting the infection with a microorganism causative of pneumonia.

Description

 Specification

 Mycoplasma '' antibody for pneumonia detection

 The present invention provides an antibody useful for detecting a microorganism belonging to Mycoplasma pneumoniae, which is a common cause of pneumonia, a method for detecting the microorganism, a reagent kit for detecting the microorganism, and an antibody for detecting the microorganism. And a method for producing the same.

 INDUSTRIAL APPLICABILITY The present invention is important for the diagnosis of atypical pneumonia caused by Mycoplasma pneumoniae, particularly in the pharmaceutical industry, and is useful in the pharmaceutical industry.

 The present invention is useful for detecting microorganisms, Mycoplasma pneumoniae, contained in specimens such as throat swabs, tissue samples: body fluids, laboratory solutions, and specimens collected from cultures. Background art

 Diagnosis of microbial infection is usually confirmed by detecting the causative organism at the site of infection, or by detecting antibodies to the causative organism in serum or body fluids. In particular, this diagnosis is important in that the detection of the causative organism enables rapid treatment of the patient.

 In general, infectious disease-causing bacteria are detected by separating and culturing the causative bacteria and then identifying them based on their physiological, biochemical or structural characteristics. (PCR) or specific nucleic acid hybridization to amplify and detect this by genetic diagnosis and immunological method to detect the causative organism using the specific reaction between the antibody and the antigen marker of the causative organism Classified by method.

 However, when using culture identification or genetic diagnostics, it takes a long time to obtain results. Therefore, diagnosis by immunological methods that can quickly detect causative bacteria with high sensitivity and that leads to prompt and appropriate treatment of patients is widely used.

 Conventionally, various marker-antigen and antibody combinations have been used for detection of infectious disease-causing bacteria by immunological methods.

¾coplasra £ neumoniae is a common cause of pneumonia, bronchitis and pharyngitis. small It is a fungus-like gram-negative bacterium that selectively enters the human body and causes disease. Approximately 15-20% of population-induced pneumonia in the general public and up to 50% of pneumonia in a restricted population are said to be caused by Mycoplasma pneumoniae (Ragnar Norrby 1999). This bacterium is a very small microorganism that is difficult to culture and forms small colonies in a concentrated medium. The growth of Mycoplasma pneumoniae in nutrient-enriched agar is very slow, requiring at least 21 days to identify cells in the medium (Granato, Poe, et al. 1980). Microorganisms of some Mycoplasma species originating in the human body also undergo similar biochemical reactions. On the other hand, the lack of a cell wall makes observation under a bright microscope more difficult (Knudson and MacLeod 1979). Therefore, the Gram stain method and the culture method cannot be applied as a diagnostic method for rapid detection as a pathogen.

 The DNA hybridization analysis probe used for the gene sequence for Mycoplasma pneumoniae detection is described in 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 used for Mycoplasma pneumoniae ribosomal manore RNA (rRNA) detection include those described by Tilton et al. (Tilton, Dias et al. 1980), Yogev ζ> (Yogev, Halachmi et al. 1988), Gobel et al. (Gobel, Geiser et al. 1987), Zivin and Monahan (EPO 305145) and Gobeland and Stanbridge (EP0250662). Kai et al. (Kai, Kamiya et al. 1987) and Jensen et al. (Jensen, Sondergard-Andersen et al. 1993) describe primers for the 16S rRNA-located IJ of Mycoplasma pneumoniae. All of the probes described herein are designed for Mycoplasma pneumoniae DNA or Mycoplasma pneumoniae Mycoplasma ggnitalium DNA. Because these probes are relatively sensitive, they are captured using techniques that have become available recently, such as the PCR method. The PCR method is very sensitive and can be performed in a shorter time than hybridization. However, there are often false-positive problems that are positive in asymptomatic asymptomatic Mycoplasma pneumoniae negative and in carriers after the disease has been cured.

Japanese Patent Application Laid-Open No. 63-298 describes an immunodetection method based on Western blotting using a monoclonal antibody against a membrane antigen protein of Mycoplasma pneumoniae of about 43 kilodaltons. However, the above antibody and the detection method using the antibody are specific It is difficult to determine the specificity of Mycoplasma pneumoniae for species-specific diagnosis. Another problem is that this diagnostic method requires at least 5 hours to produce a result (Madsen et al. 1988).

 The present invention has been made to solve the above problems. That is, an object of the present invention is to provide a method for quickly and specifically detecting a microorganism belonging to Mycoplasma pneumoniae with high sensitivity, a detection antibody used for the detection, and a detection reagent kit. Still another object of the present invention is to provide a method for producing a detection antibody used for the detection.

 The present inventors have found a protein in which the same function is conserved in all microorganisms as a useful antigen protein. Usually, it is expected that the structural change of such a protein is extremely small. Surprisingly, however, antibodies against the protein are specific to the species or genus of the microorganism, and antibodies against the protein have a variety that can be used to identify the species or genus of the microorganism. The target microorganism was found to be capable of detecting all its serotypes. More specifically, the protein is a useful protein that has the same structure between identical species through amino acid sequence changes, and exhibits species specificity accompanied by structural changes when the species is different.

 The present inventors have found that all microbial cells exist as molecules having the same function, and that the amino acid structure of the cells has some differences between microorganisms, especially Ribosomal Protein L7 / L12 protein, which is a kind of ribosomal protein. We paid attention to. Ribosoraal Protein L7 / L12 protein is a protein with a molecular weight of about 13 kDa and is known to exist as a ribosomal protein essential for protein synthesis. In particular, the entire amino acid sequence of Ribosomal Protein L7 / L12 protein has been analyzed for some microorganisms including Mycoplasma pneumoniae.

 The present inventors have noted that although this molecule is similar between microorganisms, a part of the molecule has a structural portion unique to each microorganism, and an antibody against this Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein was identified. It has been found that the use of this method makes it possible to detect species specific to various microorganisms and bacteria and to detect serotypes in all the same species.

The present inventors have ascertained that an antibody specific to the protein of Mycoplasma eneunroniae can be obtained. The present inventors have also found that Mycoplasma pneumoniae can be specifically detected by using the antibody, and completed the present invention.

 According to the present invention, a specific monoclonal antibody against the Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae has been found and developed. This antibody is novel and differs from any conventionally known antibodies in that it has the property of specifically reacting with the protein.

Rooster S歹¹ Ja5 your Rere飞酉himself歹¹ J No. 1及Hi 2 is a DNA sequence and corresponding amino acid sequence of Ribosomal Protein L7 / L12 gene of MYco lasma pneumoniae (NCBI database accession # NC 000912) 0 Note The left and right ends of the amino acid sequence described in the sequence listing are the amino terminal (hereinafter referred to as N-terminal) and the carboxyl terminal (hereinafter referred to as C-terminal), respectively. And: T-terminal. Amino acids in the amino acid sequence of the cross match test are represented by one letter abbreviation. The notation “+” in the crossmatch test indicates that the amino acid is a different amino acid but has similar properties such as hydrophobicity, and the blank “” indicates that the amino acid is completely different including the property. In addition, a series of molecular biological experiments of the genetic manipulation described in the present invention can be performed by a method described in an ordinary experimental manual. Examples of the above-mentioned ordinary experimental book include, for example, Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Shambrook, J. et al. (1989).

table 1

Cross Match Test:

 Mp: 1 AKLDKNQL IESLKEMT IME I DE 11 KAVEEAFGVSATPWAAGAVGGTQEAASEVTVKVT 60

 1 M KLDK QLIESLKEMTI + EIDEI IKAVEEAFGV-l-ATP + VAAGA G TQEAASEV + VKVT

Mg: 1 MGKLDKKQLIESL EMTIVEIDEI I AVEEAFGVTATPIVAAGAAGATQEAASEVSVKVT 60

Mp: 61 GYTDNAKLAVLKLYREIAGVGLMEAKTAVEKLPCWKQDIKPEEAEEL KRFVEVGATVE 120

 GY DNAKLAVLKLYREI GVGLMEAKTAVEKLPCW QDIKPEEAEELKKRFVEVGATVE

Mg: 61 GYADNAKLAVLKLYREITGVGLMEAKTAVE LPCWKQDIKPEEAEELK RFVEVGATVE 120

Mp: 121 IK 122 + K

Mg: 121 VK 122

In the present invention, the term "microorganism" means Mycoplasma pneumoniae, and particularly refers to a microorganism having pathogenicity in the respiratory tract and having a high diagnostic significance as a causative microorganism of mycoplasma infection. In the present invention, “an antibody that specifically reacts with a microorganism” refers to an antibody that specifically reacts with a species or genus of a microorganism, but an antibody that specifically reacts with a species of a microorganism in diagnosing a microbial infectious disease. Is particularly useful.

 In the present invention, the antibody refers to a polyclonal antibody or a monoclonal antibody, and can be prepared using the full-length Ribosomal Protein L7 / L12 protein or a partial peptide thereof. The length of the peptide for preparing the antibody is not particularly limited, but in the case of an antibody against the Ribosomal Protein L7 / L12 protein, it is sufficient if the length is characterized by this protein, preferably 5 amino acids or more, and particularly preferably A peptide having at least 8 amino acids may be used. This peptide or full-length protein can be used as is, or after cross-linking with a carrier protein such as KLH (keyhole-l-impet hemocyanin) or BSA (bovine serum albumin), and inoculated into an animal with an adjuvant if necessary, and the serum can be collected. An antiserum containing an antibody (polyclonal antibody) that recognizes Ribosomal Protein L7 / L12 protein can be obtained. Further, the antibody can be purified from the antiserum and used. The animals to be inoculated include sheep, poma, goat, peas, mice, rats and the like, and in particular, sheep, peas, etc. are preferred for producing polyclonal antibodies. It is also possible to obtain a monoclonal antibody by a known method for producing hybridoma cells. In this case, a mouse is preferable.

 In addition, a fusion protein of daltathione S-transferase (GST) or the like with the amino acid sequence of full length or 5 residues or more, preferably 8 residues or more of the protein is purified, or unpurified. As it is, it can be used as an antigen.

In various methods shown in Antibodies (Antibodies; A laboratory manual, E. Harlow et al., Old spring Harbor Laboratory Press), cells cultured using the immunoglobulin gene isolated by the gene closing method, etc. Expressed recombinant antibody Can also be produced.

 An antibody against Ribosomal Protein L7 / L12 that can be used as the marker antigen of the present invention can be obtained by the following method or other similar methods, but is not limited to these methods.

 a) For microorganisms of which the gene sequence and amino acid sequence of Ribosomal Protein L7 / L12 protein are known, peptide fragments are synthesized in regions of other microorganisms with low similarity to the amino acid sequence of the protein and used as immunogens. The desired antibody can be obtained by preparing a polyclonal antibody or a monoclonal antibody.

 In addition, the gene can be amplified by a conventional gene manipulation technique such as gene amplification by PCR using a DNA sequence at both ends of the known gene as a probe, and hybridization using a homologous partial sequence as a プ ロ ー ブ -type probe. A full-length sequence can be obtained.

 After that, a fusion gene or the like with another protein gene is constructed, and the fusion gene is inserted into a host by a known gene transfer technique using E. coli or the like as a host, expressed in a large amount, and then used as a fusion protein. The target protein antigen can be obtained by purifying the expressed protein by an antibody affinity column method or the like. In this case, since the full-length protein of Ribosomal Protein L7 / L12 protein serves as an antigen, obtaining an antibody against an amino acid portion conserved between microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, a hybridoma that produces a monoclonal antibody is obtained by a known method, and the desired antibody is obtained by selecting a clone that produces an antibody that reacts only with the relevant microorganism. be able to.

b) For microorganisms for which the amino acid sequence of Ribosomal Protein L7 / L12 protein is unknown, one of the reasons is that the amino acid sequence of Ribosomal Protein L7 / L12 protein is 50-60% homologous between strains. Based on the sequence of the homologous part of the sequence, the gene of the protein can be easily obtained by using a general gene manipulation technique such as the gene amplification of the specific sequence part by PCR and the hybridization method using the homologous part sequence as a type II probe. Can be obtained.

After that, a fusion gene with another protein gene is constructed, and the relevant fusion gene is inserted into the host by a known gene transfer method using Escherichia coli or the like as a host, expressed in a large amount, and then used for the protein used as the fusion protein. Antibody affinity column method, etc. The target protein antigen can be obtained by further purifying the expressed protein. In this case, since the full-length protein of Ribosomal Protein L7 / L12 protein serves as an antigen, obtaining an antibody against an amino acid portion conserved between microorganisms does not meet the purpose of the present invention. Therefore, for the antigen obtained by this method, a hybridoma that produces a monoclonal antibody is obtained by a known method, and a clone that produces an antibody that reacts only with the relevant microorganism is selected. Can be obtained.

 c) Alternatively, if the amino acid sequence of the Ribosoraal Protein L7 / L12 protein is unknown, another method is to use the common amino acid sequence of the known Ribosomal Protein 7 / L12 protein that is conserved between microorganisms. A corresponding synthetic peptide of 5 to 30 amino acids is prepared, and a polyclonal antibody or a monoclonal antibody is prepared for the peptide sequence by a known method. By purifying the target microorganism cell lysate by affinity column chromatography using the antibody, highly purified Ribosomal Protein L7 / L12 protein can be obtained. If the degree of protein purification is insufficient, Ribosomal is purified by known methods such as ion-exchange chromatography, hydrophobic chromatography, and genole filtration, followed by Western blotting using an antibody prepared. The purified protein can be obtained by identifying the elution fraction of Protein L7 / L12 protein. Obtaining a hybridoma by a known method based on the obtained purified Ribosomal Protein L7 / L12 protein antigen and obtaining a desired antibody by selecting a hybridoma that specifically reacts with a desired microorganism. Can be.

 The antibodies of the present invention specific to various microorganisms obtained by the above-mentioned methods a), b) and c) can be used in various immunological analysis methods to produce various microorganisms specific to the target microorganism. Diagnostic reagents and kits can be provided. For example, this antibody can be obtained by a known measurement method such as an agglutination reaction in which the antibody is adsorbed on polystyrene latex particles, an ELISA method which is a known technique performed in a microtiter plate, an existing immunochromatography method, or a colored particle. Can be used in all known immunoassay techniques, such as sandwich assays using particles having a chromogenic ability, or magnetic microparticles coated with a capture antibody together with the antibody labeled with an enzyme or a fluorescent substance. "

Microbial diagnostic methods using antibodies include an agglutination reaction in which the antibody is adsorbed on polystyrene latex particles, an ELISA method that is a known technique performed in a microtiter plate, and an existing immunological method. Diagnosis using all known immunoassay techniques such as chromatography, colored or colored particles, or sandwiches using magnetic particles coated with a capture antibody together with the antibody labeled with an enzyme or a fluorescent substance. Means the way.

 In addition, a particularly useful microbial diagnostic method using an antibody is described in Japanese Patent Application Laid-Open Publication No. Hei 7-509655, which is a method for performing an antibody reaction on an optical thin film formed of silicon, silicon nitride, or the like. A so-called optical immunoassay (0IA, Optical Immunoassay) or the like, which is detected by the principle of interference or the like, is useful as a highly sensitive diagnostic method.

 In addition, as a method for extracting a cell / marker-antigen from a microorganism required in the detection method, a treatment method using an extraction reagent using various surfactants such as Triton X-100, Tween-20, and an appropriate method Known cell structure disruption techniques, including enzymatic treatment using enzymes such as proteases and disruption of microbial cells by physical methods, are used. It is desirable to set the optimal extraction conditions with the reagent for each microorganism by combining a surfactant and the like.

 Further, the reagent kit for detecting a microorganism using an antibody in the present invention corresponds to a reagent kit for detection using the detection method.

 The amino acid sequence of Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae and the sequence of the rooster are shown in the Sequence Listing. Therefore, in the case of this microorganism, it is possible to compare the amino acid sequence of the Ribosomal Protein L7 / L12 protein with the homologous protein of a similar microorganism described in the sequence listing as “cross-match”. By synthesizing a peptide having a segment with low homology and preparing a polyclonal or monoclonal antibody against the peptide, it becomes possible to omit the selection of those having specificity for microorganisms.

 In particular, in the case of polyclonal antibodies, the antiserum of the immunized animal is purified using a Protein A column, etc. to obtain the IgG fraction, and then affinity purification is performed using the synthetic peptide used for animal immunization. It is desirable.

 Furthermore, PCR primers were prepared based on the N-terminal and C-terminal sequences from the DNA sequence of the Ribosomal Protein L7 / L12 protein of the microorganism.

Utilizing the homology of this PCR primer, a DNA fragment is propagated by PCR using genomic DNA, extracted, and a fragment of the Mycoplasma pneumoniae Ribosomal Protein L7 / L12 gene is obtained by a conventional method. S can. Mycoplasma E. neumoniae (D Ribosomal Protein The full length of the L7 / L12 gene can be determined by analyzing the DNA sequence information of these fragments.

 The obtained Ribosomal Protein L7 / L12 gene of Mycoplasma pneumoniae constitutes a fusion protein gene with, for example, GST, constructs an expression vector using an appropriate expression plasmid, and then transforms E. coli, etc. Can be expressed in large quantities. ^ Transformed E. coli is cultured in an appropriate amount, and the cell lysate is purified by affinity column using GST to obtain Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein. Fusion protein of white matter and GST .

 This protein is used as is, or after cutting the GST portion, a number of hybridoma clones are established by a known method as an antigen protein, and Mycoplasma and pneui ^ onia cells are recovered from cell lysate or Mycoplasma pneumoniae. By selecting an antibody that shows a specific reaction to the Ribosomal Protein L7 / L12 protein, a specific monoclonal antibody of interest can be obtained. .

 Antibodies prepared according to the present invention can be obtained by a known measurement technique such as agglutination reaction in which the antibody is adsorbed on polystyrene latex particles, a known technique performed in a microtiter plate, an ELISA method, an existing immunochromatography method, or a colored particle. Alternatively, it can be used in all known immunoassay techniques such as sandwiches using particles having a coloring ability, or magnetic particles coated with a capture antibody together with the antibody labeled with an enzyme or a fluorescent substance.

 In addition, an antibody prepared according to the present invention can function as a so-called capture antibody that captures the antigen protein in a solid phase or a liquid phase in all immunoassay techniques, and at the same time, an enzyme such as peroxidase or alkaline phosphatase is known. By modifying the antibody by the method described above to obtain a so-called enzyme-labeled antibody, it can function as a detection antibody. BEST MODE FOR CARRYING OUT THE INVENTION

 The following examples are for the purpose of specifically describing the present invention and do not limit the scope of the present invention in any way.

Example 1

Mycoplasma. Dneumoniae force et al.'S Ribosomal Protein L7 / L12¾fe Mycoplasma pneumoniae (ATCC15531)

Supplement (DIFCO, 0836-68-9) PPLO agar medium supplemented with (DIFC0, 0412-17-3) After inoculating an appropriate amount on, at 37 ° C, 5% C0 ₂ condition in C0 ₂ incubator one The cells were cultured for 5 hours. The grown colonies were suspended in TE buffer (manufactured by Wako Pure Chemical Industries) so as to have a final concentration of about 5 × 10 10 Fll / ml. About 1.5 ml of this suspension was transferred to a microcentrifuge tube, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was discarded. The precipitate was resuspended in 567 TE buffer. Further, 30 μl of 10% SDS and 31 of 20 mg / ml Proteinase K solution were added, mixed well, and incubated at 37 for 1 hour. The suspension was incubated for an additional hour at 56t :. Next, 80 1 of 10% cetyltrimethylammonium bromide / 0.7 M NaCl solution was added, mixed well, and incubated at 65 for 10 minutes. The same volume of a 24: 1 chloroform-isoamyl alcohol mixture was added to 700 / xl and stirred well.

 This solution was centrifuged at 12,000 rpiB for 5 minutes at 4 in a microcentrifuge, and the aqueous fraction was transferred to a new microcentrifuge tube. A 0.6-fold amount of isopropanol was added thereto, and the tube was shaken well to form a DNA precipitate. The white DNA precipitate was scooped with a glass rod and transferred to another microfuge tube containing 1 ml of 70% ethanol (chilled to -20 ^). The tubes were then centrifuged at 10,000 rpni for 5 minutes and the supernatant was gently removed. An additional 1 ml of 70% ethanol was added and the mixture was centrifuged for another 5 minutes. After removing the supernatant again, the precipitate was dissolved in ΙΟΟμΙ of TE buffer to obtain a DNA solution. The concentration of this genomic solution was determined by Molecular Cloning, A laboratory manual, 1989, Eds.Shambrook, J., Fritsch, EF, and Maniatis, T., Cold Spring harbor Laboratory Press E5, Spectrophotometric Determination of the Amount of DNA or Quantification was performed according to RNA. PCR (polymerase chain reaction) was performed using lOng of this genomic DNA. PCR used Taq polymerase (Takara Shuzo, code R001A). 5 μl of the buffer attached to the enzyme, 41 of the dNTP mixture attached to the enzyme, and 200 pniol of each oligonucleotide (shown in SEQ ID NOs: 3 and 4) were added to the enzyme. Purified water was added so that the total volume was 50 μl.

This mixture was subjected to 5 cycles of 1 minute at 95, 50 ^ 2 minutes, and 3 minutes at 72 ° C using a TaKaRa PCR Thermal Cycler 480, and then 95 = 01 minutes, 2 minutes at 60, and 72: 3 minutes for 25 minutes. Cycled. Using a part of this PCR product, electrophoresis was performed in a 1.5% agarose gel. Etc. After staining with (Nippon Gene Co., Ltd.), it was observed under ultraviolet light, and it was confirmed that cDNA of about 400 bp was amplified. After digestion with restriction enzymes BamHI and Xhol, electrophoresis was performed in a 1.5% agarose gel, and staining with ethidium drug was performed. An approximately 400 bp band was cut out of the gel. This band was purified with SuprecOl (Takara Shuzo Co., Ltd.) and inserted into a general vector, PGEX-6P-1 (Pharmacia). The vector can function as an expression vector for a target molecule capable of expressing a fusion protein with a GST protein by incorporating a target gene fragment into an appropriate restriction enzyme site.

 Specifically, the vector pGEX-6P-1 and the above DNA were mixed at a molar ratio of 1: 3, and the DNA was incorporated into the vector using T4 DNA ligase (Invitrogen). The vector containing the DNA, pGEX-6P-1, was introduced into Escherichia coli one-shot competent cells by a genetic method, and then LB, a semi-solid culture plate containing 50 ig / ml ampicillin (Sigma). L-broth agar (Takara Shuzo) was inoculated. Plates were incubated at 37 ° for 12 hours and grown colonies were randomly selected and inoculated into broth broth containing the same concentration of ampicillin. After shaking culture at 37 ^ for 8 hours · After collecting the bacteria, the plasmid was separated using Wizard Miniprep according to the attached instructions. Plasmid was digested with restriction enzymes BamHI / XhoI. The insertion of the PCR product was confirmed by cutting about 370 bp of DNA. The nucleotide sequence of the inserted DNA was determined using the above clones.

 The nucleotide sequence of the inserted DNA fragment was determined using a fluorescent probe manufactured by Applied Biosystems.

The preparation of the sequence sample was performed using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems). First, add 9.51 reaction solution, 4.0 μl of 0.8 pmol / 1 T7 promoter primer (Gibco BRL), and 6.5 μl of 0.16 μg / μΐ template DNA. The mixture was added to a 5 ml microtube and mixed. After the mixture was covered with two layers of 100 1 mineral oil, 25 cycles of PCR amplification were performed. Here, one cycle consists of a treatment at 96 for 30 seconds, a treatment at 55 ° for 15 seconds, and a treatment at 60 for 4 minutes. The product was held at 4 for 5 minutes. After the reaction was completed, 80 sterile purified water was added and stirred. The product was centrifuged, and the aqueous layer was extracted three times with a phenol-chloroform mixture. .10 y1 sodium acetate ρ5.2 and 3001 ethanol were added to the 100 / ^ 1 aqueous layer and stirred. Then 14,000 rpm, The precipitate was collected by centrifugation at room temperature for 15 minutes. After washing the precipitate with 75% ethanol, it was dried by leaving it to stand under vacuum for 2 minutes to obtain a sample for sequence. Sequence samples were dissolved in formamide containing 4 μl lOmM EDTA and denatured at 90 for 2 minutes. This was cooled in water and subjected to a sequence.

 Two of the five randomly selected clones had sequence homology with the probe used for PCR. Also, a DNA sequence consistent with the gene sequence of Ribosomal Protein L7 / L12 protein was apparent. The entire nucleotide sequence and the corresponding amino acid sequence of the structural gene portion were as shown in SEQ ID NOs: 1 and 2 in the Sequence Listing. This gene fragment clearly encodes the gene of Ribosomal Protein L7 / L12 protein of Micoplasma pneumoniae.

Example 2

 Large-scale expression and purification of Ribosomal Protein L7 / L12 gene from Mycoplasma pneumoniae in Escherichia coli

Escherichia coli incorporating the expression vector was cultured in an LB medium at 50 ml, 37 for 24 hours. 500 ml of 2x YT medium was heated at 37 for 1 hour. 1 ml of the cultured E. coli solution was placed in 500 ml of the aforementioned medium. One hour later, 550 μl of lOOmM isopropyl i3-D (-)-thiogalactopyranoside (IPTG) was introduced, and the cells were cultured for 4 hours. The product was collected, transferred to a 250ml centrifuge tube and centrifuged 10 min at 7000R _P m. The supernatant was discarded, and the cells were dissolved in 25 ml of 50 mM Tris buffer pH 7.4, 25 ml of Lysis buffer containing 25% sucrose. Further, 10% NP-40 1.25ral and 1M MgCl ₂ 125 / zl were added and transferred to a plastic tube. Ultrasonic treatment was performed 5 times for 1 minute under ice. Thereafter, the mixture was centrifuged at 12,000 rpm for 15 minutes, and the supernatant was collected.

Next, the supernatant was adsorbed onto a daltathione agarose column conditioned with PBS. Next, the column was washed with a washing solution containing 20 mM Tris buffer pH 7.4, 4.2 mM MgCl ₂ lm dithiothreitol (DTT) for 2 bed volumes. Elution was performed in 50 mM Tris buffer pH 9.6 containing 5 mM glutathione. The protein content of the eluted fraction was determined by the 'pigment binding method (Bradford method; BioRad Co.)', and the main fraction was obtained.

 The purity of the obtained purified GST protein Ribosomal Protein L7 / L12 protein was confirmed by electrophoresis to be about 75%, and sufficient purity as an immunogen could be secured.

Example 3 Preparation of monoclonal antibody against Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein

 First, with regard to immunization of mice, 100 μg of GST fusion Ribosomal Protein L7 / L12 protein antigen of Mycoplasma pneumoniae was dissolved in PBS of 200; z1 and 200 μl of Freund's complete adjuvant was added and mixed. After emulsification, 200 // 1 was injected intraperitoneally. The same emulsion antigen was injected intraperitoneally at 2, 4 and 6 weeks. Two-fold concentrations of the emulsified antigen were injected intraperitoneally after 10 and 14 weeks. Three days after the end of the final immunization, the spleen was removed and cell fusion was performed.

2 × 10 ⁷ myeloma cells were placed in a glass tube and mixed well with 10 ⁸ spleen cells of the mouse aseptically removed, centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were mixed well. The myeloma cells used for cell fusion were cultured in RPMI 1640 medium containing 10% fetal bovine serum using an NS-1 cell line.From 2 weeks before cell fusion, 0.13raM azaguanine was added. 5; ig / nil MC-210, RPMI1640 medium containing 10% fetal bovine serum for 1 week, and further cultured for 1 week in RPMI1640 medium containing 10% fetal bovine serum. .

 50 ml of RPMI 1640 culture maintained at 37 was added to the mixed cell sample and centrifuged at 1,500 rpm. After removing the supernatant, 1 ml of 50% polyethylene dalicol maintained at 37 was added and stirred for 1 minute. 10 ml of RPMI1640 culture solution maintained at 37X: was added, and the mixture was vigorously stirred by sucking and discharging the mixture with a sterilized pipette for about 5 minutes.

 After centrifugation at 1,000 rpm for 5 minutes and removing the supernatant, 30 ml HAT culture solution was added so that the cell concentration was 5 × 10 V ml. The mixture was stirred until uniform, 0.1 ml was poured into a 96-well culture plate, and cultured at 37 under a 7% carbon dioxide atmosphere. HAT medium was added in an amount of 0.1 ml each on the first day, the first week, and the second week, and cells producing the desired antibody were screened by ELISA.

 GST fusion Ribosomal Protein L7 / L12 protein and GST protein dissolved in PBS containing 0.05% sodium azide at a concentration of 10 g / ml, respectively, in 100 / zl 96-well plates separately And adsorbed 1 晚 with 4.

After the supernatant was removed and pro Kkingu react 1 hour 1% bovine serum albumin solution (in PBS) 200 _mu 1 added at room temperature. After removing the supernatant, the product was washed with a washing solution (0.02% Tween 20, PBS). this 100 ml of a culture solution of the fused cells was added thereto, and reacted at room temperature for 2 hours. The supernatant was removed, and the precipitate was washed with a washing solution. Then, 100 μl of a goat anti-mouse IgG antibody solution labeled with peroxidase at a concentration of 50 ng / ml was added, and reacted at room temperature for 1 hour. The supernatant was removed, and the product was washed again with a washing solution. 1 The solution (! (Manufactured by Nishisha Co., Ltd.) was added for 10 (^ 1), and the mixture was allowed to react at room temperature for 20 minutes. The absorbance was measured.

 As a result, a positive peptide that reacted only with GST fusion Ribosomal Protein L7 / L12 protein and did not react with GST protein was found, and it was found that antibodies to Ribosomal Protein L7 / L12 protein were included.

 Therefore, the cells in the positive well were collected and cultured in a HAT medium in a 24-well plastic plate.

The 50 mu 1 was diluted with HT medium to the cultured fusion medium is about 20 1 number of cells, 10 ⁶ murine thymocytes 6 week old suspended in HT medium and a 96-well culture flop L ^ one And mixed. After mixing, 7% C0 ₂ under the conditions, and cultured for 2 weeks at 37.

 The antibody activity in the culture supernatant was similarly assayed by the ELISA method described above, and cells positive for reaction with the Ribosomal Protein L7 / L12 protein were collected. Further, the same dilution test and the same closing operation were repeated to obtain a total of 5 clones of hybridomas MPRB-1 to MPRB-5.

Example 4

 Selection of monoclonal antibody to detect Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein

 Monoclonal antibodies were produced and recovered using the positive hybridoma cells obtained as described above according to a standard method.

Specifically, cells subcultured using RPMI 1640 medium (containing 10% FCS) were injected intraperitoneally with Blab / C mice, in which 0.5 ml of pristane was injected intraperitoneally 2 weeks ago. ^Six (in PBS) were injected. Three weeks later, ascites was collected, and the centrifuged supernatant was obtained.

The obtained solution containing the antibody was absorbed by a Protein A column (5ral, manufactured by Pharmacia) and washed with 3 times the volume of PBS. Subsequently, elution was carried out with citrate buffer pH3. The antibody fraction was collected to obtain a monoclonal antibody produced by each hybridoma. These 5 strains of Hypri-Dorma Evaluation was performed by ELISA using the derived monoclonal antibody.

 For evaluation of the monoclonal antibody, a sandwich analysis method was used. The prepared monoclonal antibody was used as a detection antibody by binding to peroxidase. Enzyme labeling uses horseradish peroxidase (Sigma grade VI) and coupling uses reagent S acetylthiothioacetate N-hydroxysuccinimide and is described in Analytical Bio-chemistry 132 (1983), 68-73. Performed according to method. In the ELISA reaction, a solution obtained by diluting a commercially available anti-Mycoplasma pneumoniae polyclonal antibody (Biodesign, egret) at a concentration of 10 μg / ml was separately dispensed into 96-well plates in ΙΟΟμΙ and 4 1 晚 adsorbed.

 After removing the supernatant, 200 µl of a 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour for blocking. After removing the supernatant, the product was washed with a washing solution (0.02% Tween 20, PBS). To this, add an amount of 0.3% Triton X-100 to the culture of each microorganism, extract at room temperature for 5 minutes, add antigen solution (μμΙΟΟ), and allow to react at room temperature for 2 hours. Was. The supernatant was removed and the product was washed again with washing solution. Next, 100 μl of a peroxidase-labeled anti-Ribosomal Protein L7 / L12 protein antibody solution having a concentration of 5 g / ml was added, and reacted at room temperature for 1 hour. The supernatant was removed and the product was washed again with washing solution. (4) 100 μl of the solution (manufactured by KPL) was added thereto, and the mixture was reacted at room temperature for 20 minutes. After coloring, the reaction was stopped by adding 100 µl of 1N sulfuric acid. The absorbance at 450 nm was measured.

When a monoclonal antibody derived from Hypri-doma MPRB-1 is used as the enzyme-labeled antibody, all strains of Mycoplasma pneumoniae tested can be detected with a sensitivity of 10 ⁶ cells / ml and other Haemophi JLUs influenzae, Klebsiella Dneumoniae, Chlamydia pneumoniae Oyohi Neisseria microorganism has acquired the antibody with the 10 ⁸ cells / ml of highly concentrated Mycoplasma pneumoniae in the use of monoclonal antibodies against Ribosomal protein L7 / L12 protein showed no reactivity with specific reactivity for such meningitides This was clearly confirmed. This antibody was named AMMP-1. Table 2 shows only the results using AMP-1. The results using other antibodies that cross-react with other microorganisms are not mentioned here. Table 2

 Detection result (10 ° cells / ml)

M. pneumoniae Detection result (10 ⁸ cells / ml)

N. meningx tides

 N. lactamica

 N. mucosa

 . sicca

 H. Influenzae

 B. catarrharis

 N. gonorrhoeae

 E. col 丄

 K. pneumoniae

 (+; Positive,-; negative) Example 5

 Use of an affinity column immobilized with Ribosomal Protein L7 / L12 protein to obtain a polyclonal antibody that specifically reacts with Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein

 Example 1 Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein or Triton X-100-treated cell supernatant obtained as described above was used as an antigen. About 1.2 ml of physiological saline containing 100 / ig of antigen was emulsified with 1.5 ml of Freund's adjuvant. Emulsion was injected subcutaneously into SPF Japanese White Egret to immunize Egret. Immunization was performed 5 to 6 times every two weeks, and the antibody titer was confirmed.

Confirmation of the antibody titer was performed by the ELISA method. Dissolve 10 μg / ml of Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein dissolved in PBS containing 0.05% sodium azide at a concentration of 10 μg / ml into a 96-well plate in ΙΟΟμΙ, and absorb with 1 4 with 4 I let it. After removing the supernatant, 200% 1% bovine serum albumin solution (in PBS) was added, and the mixture was reacted at room temperature for 1 hour for blocking. After removing the supernatant, the product was washed with a washing solution (0.02% Tween 20, PBS). A solution (ΙΟΟμΙ) obtained by diluting normal serum of rabbits and antiserum of immunized rabbits was added and reacted at room temperature for 2 hours. The supernatant was removed and the product was washed again with the washing solution. Next, 100 ペ ル of a peroxidase-labeled anti- ゥ egan IgG antibody solution at a concentration of 50 ng / ml was added and reacted at room temperature for 1 hour. The supernatant was removed and the product was washed again with washing solution. 0PD solution (manufactured by Sigma) was added in {μ} portions, and the mixture was reacted at room temperature for 20 minutes. The reaction was stopped by applying 100 μl of 1N sulfuric acid at the coloration. The absorbance at 492 nm was measured. After confirming the increase in antibody titer, a large amount of blood was collected. Blood was collected from the ear artery into a glass centrifuge tube, allowed to stand at 37 for 1 hour, and allowed to stand at 4 for 10 minutes. Thereafter, the mixture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was recovered. The obtained antiserum was stored at 4¾.

 An affinity column on which the Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae was immobilized was prepared. A HiTrap NHS activation column (1 ml, manufactured by Pharmacia) was used. Immediately after the column was replaced with ImHC1, a PBS solution (lmg / ml) of Ribosomal Protein L7 / L12 protein was added. After allowing the force ram to stand for 30 minutes, the blocking reagent was added and equilibrated with PBS.

 Using this Mycoplasma pneumoniae Ribosomal Protein L7 / L12 protein-fixed affinity column, polyclonal antibodies in antiserum obtained using the supernatant of Mycoplasma pneumoniae Triton X-100 treated cells as antigen Purification was performed. This antiserum was diluted 5-fold with PBS, passed through a 0.45 ix m finole letter, and adsorbed onto a Mycoplasma pneumoniae <D Ribosomal Protein L7 / L12 protein immobilized column at a flow rate of 0.5 ral / min. The column was eluted with 0.1M daricin buffer (pH 2.1) and immediately neutralized with 1M Tris buffer (pH 9.0). Collected.

 The polyclonal antibody thus obtained was evaluated by the 0IA method described in Japanese Patent Application Laid-Open Publication No. Hei 7-5099565.

 The purified antibody was used as a capture antibody in the 0IA method. As the detection antibody, the ACT-1 monoclonal antibody described in Example 4 was used, which was enzymatically labeled with peroxidase. Enzyme labeling was performed using horseradish peroxidase (Sigma grade VI) and binding was performed using the reagent S-acetylthioacetic acid N-hydroxysuccinimide. Analytical Biochemistry 132 (1983), 68-73 Performed according to the method described.

 In the 0IA reaction, purified polyclonal antibody in PBS containing 0.05% sodium azide was diluted to a concentration of 10 μg / ml with 0.1 M HEPES buffer, pH 8.0. After reacting at room temperature for 30 minutes, it was washed with distilled water, coated with a coating solution containing saccharose and alkali-treated casein, and then used.

The antigen solution 15 / X1 obtained by adding Triton X-100 in a concentration of 0.5% to the culture solution of each microorganism obtained in the above operation and extracting at room temperature for 5 minutes is placed on the silicon wafer. The mixture was added and reacted at room temperature for 10 minutes. Then, 20 μg / ml peroxidase-labeled monoclonal antibody Was added and allowed to react for 10 minutes. After washing with distilled water, a TMB solution (produced by KPL) was added in 151 portions, and the mixture was reacted at room temperature for 5 minutes. The product was washed with distilled water, and the blue color produced by the enzyme reaction was visually observed.

As a result, as shown in Table 3, by using a purified polyclonal antibody APMP- 1 as the capture antibody, can be detected with a sensitivity of Mycoplasma pneumoniae 10 ⁸ cells / ml, and reactivity of other microorganisms can not be detected Is evident. In this way Mycoplasma

It was confirmed that a polyclonal antibody specifically reacting with Mycoplasma pneumoniae was obtained using an affinity column immobilized with Ribosomal Protein L7 / L12 protein of pneumoniae. Table 3

Detection result (10 ⁸ cells / ml)

 M. pneumoniae

H. Infl uenzae ATCG10211

 E. Col 丄 ATCC25922

 E. faecali s ATCC19433

 K. pneumoniae ATCC13883

 N. gonorrhoeae I ID821

 N. la ctami ca ATCC23970

 N. meningi tidis ATCC13090

 P. aeruginosa ATCC27853

 GroupB Streptococcus ATCC12386

 S. Aureus ATCC25923

 S. Pneumoniae ATCC27336

 S. pyogenes ATCC19615

 (+; Positive;-; negative) Industrial applicability

According to the present invention, it is not only possible to specifically detect a microorganism of a specific species using an antibody against an intracellular molecule that is functionally retained during the evolution of the microorganism, but also to detect all sera in the same species. Microorganisms of the type can be accurately detected.

 As such an antibody, an antibody against a ribosomal protein of a microorganism, Ribosomal Protein L7 / L12 protein can be used to detect Mycoplasma pneumoniae with high accuracy.

 Further, by using a reagent kit for detecting microorganisms comprising such an antibody as a component, detection of microorganisms can be performed more generally and accurately. References:

Patent literature

 * U.S. Patent No. 5, 552 279 Weisburg, et al., September 3, 1996 ["Mycoplasma pneumoniae and I Nucleic acid probes for detection of Mycoplasma genitalium I

 * EP0 305145, Application No. 88307793.5 Zivin and Monahan.March 1, 1989.Methods and probes for detecting nucleic acid.

 * EP0 250662, Application No. 86304919, 3 Gobel and Stanbridge. January 7 1988, Detection of Mycoplasma by DNA hybridization.

* JP 63 (1988) -298

Other patent documents

 * Bernet, C. M. Garret, et al. (1993) "Detection of Mycoplasma pneumoniae using polymerase chain reaction. I J Cl in Microbiol 31 (4), 1013-5.

 * Buck, G.E., L.C. 0 'Hara, et al. (1993) "A rapid and sensitive method for detecting Mycoplasma pneumoniae in simulated clinical samples by DNA amplification. I J Clin Microbiol 31 (4), 1013-5

* Gobel, U.B., A. Geiser, et al., (1987) "Oligonucleotide probes complementary to the variable region that differentiates ribosomal RNA between Mycoplasma species /

 * Granato, P.A., L. Poe, et al., (1980) "Improvement in Mycqplasma.rtneumoniae growth report on the use of New York City medium j jh Mikrobiol Epidemiol I unobiol (8), 50-3

 * Hyman, H.C.D.Yogev, et al., (1987) `` ^ ycoplasm neumoniae ¾ J; 1 Detection of Mycoplasma genitalium

* Jensen, JSJ Sondergard Andersen, et al. (1993) "Polymerase chain reaction Detection of Mycoplasma pneumoniae in pseudoclinical samples used ”J Med Microbiol 38 (3), 166-70

 * Kai, M., S. Kamiya, et al., (1987) "Rapid detection of Mycoplasma pneumoniae using polymerase chain reaction" J Gen Microbiol 133 (Pt7), 1969-74

 * Knudson, DL and R. MacLeod (1979) "^ ycoplasma Dneumoniae and Myco lasma salivarium-electron micrographs of colony growth in agar" Sci Rep Res Inst Tohoku Univ [Med] 26 (3-4), 71-91

 * Madsen RD, Weiner LB, McMillan JA, Saeed JA, North JA, Coates SR. (1988) .Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody i unoblot assay Am J Cl in Pathol 89 (1): 95 -9

 * Ragnar Norrby, S. (1999) "Representative pneumonia in Nordic countries, its etiology and comparative clinical results of fleroxacin and doxycycline, Nordic Atypical Pneumoniae Study Group" J Med Microbiol 48 (12), 1115-22

 * Tilton, R.G., F. Dias, et al., (1980) `` Culture with DNA probes for detecting Mycoplasma pneumoniae in clinical samples. '' J Cl in Microbiol 12 (6 748-52)

* Yogev, D., D. Halachmi, et al. (1988) _"r RNA gene probe and detection of Mycoplasma species due to genomic DNA fingerprints and Mycoplasma strains (mol l icutes)" J Cl in Microbiol 26 (11) , 2266- 9

 * Chan ED, Kalaya it T Lynch DA, Tuder R Arndt P, WinnR. Schwarz MI. "A literature review of bronchiolitis associated with Mycoplasma pnenmoniae causing severe lung disease in adults" Chest., 1999; 115 (4), 1188-94

 * NCBI database # NC # 000912., Hinunelreich, R., Hibelt, H., Plagens, H., Pirkl, E., Li, B.C., and Herrmann, R.

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Claims

The scope of the claims 1. An antibody against a ribosomal protein of a microorganism belonging to Mycoplasma pneumoniae, which specifically reacts with the microorganism.  2. The antibody according to claim 1, wherein the ribosomal protein of a microorganism belonging to Mycoplasma pneumoniae is Ribosomal Protein L7 / L12.  3. The antibody according to claim 1, wherein the antibody is monoclonal or polyclonal.  4. The antibody according to any one of claims 1 to 3, wherein the antibody is an antibody bound to an enzyme. 5. A method for detecting a microorganism belonging to Mycoplasma pneumoniae, comprising using the antibody according to any one of claims 1-4.  6.a) contacting the sample with a lysis solution to extract ribosomal proteins from the microorganism; b) transferring the extracted sample to a capture antibody, that is, an antibody according to any one of claims 14 to 14 on a solid surface. The antibody immobilized on the liposomal protein to form an antigen-antibody complex between the capture antibody and the antibody, and c) a detection antibody, that is, any one of claims 1-4. A method for detecting a microorganism belonging to Mycoplasma pneumoniae in a sample, comprising detecting an antigen-antibody complex using the antibody and the antigen. 7. The method according to claim 6, wherein the detection antibody is an antibody bound to an enzyme, and the antigen-antibody complex is detected by a specific substrate of the enzyme.  8. A reagent kit for detecting a microorganism associated with Mycoplasma pneumoniae, which comprises using the antibody according to any one of claims 1-4.  9. Ribosomal Protein L7 / L12 protein of a microorganism belonging to Mycoplasma pneumoniae obtained by genetic manipulation or isolation and purification from a microorganism, or its synthesis corresponding to its partial peptide 14. The method for producing an antibody according to claim 13, wherein the peptide is used as an immunogen.