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WO2001057199A1 - Anticorps servant a detecter le mycoplasma pneumoniae - Google Patents

Anticorps servant a detecter le mycoplasma pneumoniae Download PDF

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Publication number
WO2001057199A1
WO2001057199A1 PCT/JP2001/000626 JP0100626W WO0157199A1 WO 2001057199 A1 WO2001057199 A1 WO 2001057199A1 JP 0100626 W JP0100626 W JP 0100626W WO 0157199 A1 WO0157199 A1 WO 0157199A1
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WO
WIPO (PCT)
Prior art keywords
antibody
protein
mycoplasma pneumoniae
microorganism
ribosomal protein
Prior art date
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Ceased
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PCT/JP2001/000626
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English (en)
Japanese (ja)
Inventor
Monzur Rahman
Takashi Etoh
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Asahi Kasei Corp
Asahi Chemical Industry Co Ltd
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Asahi Kasei Corp
Asahi Chemical Industry Co Ltd
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Priority to CA002398469A priority Critical patent/CA2398469C/fr
Priority to AU2001230532A priority patent/AU2001230532A1/en
Priority to JP2001558014A priority patent/JP5331285B2/ja
Publication of WO2001057199A1 publication Critical patent/WO2001057199A1/fr
Anticipated expiration legal-status Critical
Priority to US10/386,050 priority patent/US20040014943A1/en
Priority to US12/424,370 priority patent/US20090269789A1/en
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56927—Chlamydia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1217—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1242—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
    • C07K16/1253—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C07K16/1275—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria from Streptococcus (G)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56933—Mycoplasma

Definitions

  • 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.
  • 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.
  • 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.
  • PCR PCR
  • 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.
  • 3 ⁇ 4coplasra £ 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).
  • Probes used for Mycoplasma pneumoniae ribosomal manore RNA (rRNA) detection include those described by Tilton et al. (Tilton, Dias et al. 1980), Yogev ⁇ > (Yogev, Halachmi et al. 1988), Gobel et al. (Gobel, Geiser et al.
  • 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.
  • 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 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.
  • Ribosomal Protein L7 / L12 protein which is a kind of ribosomal protein.
  • 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.
  • 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.
  • 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 ⁇ 1 Ja5 your Rere ⁇ himself ⁇ 1 J No. 1 ⁇ 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • a carrier protein such as KLH (keyhole-l-impet hemocyanin) or BSA (bovine serum albumin)
  • 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
  • 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.
  • GST daltathione S-transferase
  • Antibodies 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.
  • 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.
  • 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 ⁇ ⁇ ⁇ ⁇ -type probe. A full-length sequence can be obtained.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • highly purified Ribosomal Protein L7 / L12 protein can be obtained.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Ribosomal Protein L7 / L12 protein of Mycoplasma pneumoniae 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an enzyme such as peroxidase or alkaline phosphatase is known.
  • 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.
  • TE buffer manufactured by Wako Pure Chemical Industries
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ l of 0.8 pmol / 1 T7 promoter primer (Gibco BRL), and 6.5 ⁇ l of 0.16 ⁇ g / ⁇ 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.
  • 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 ⁇ l 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.
  • IPTG isopropyl i3-D (-)-thiogalactopyranoside
  • 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.
  • the supernatant was adsorbed onto a daltathione agarose column conditioned with PBS.
  • 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.
  • mice 100 ⁇ g of GST fusion Ribosomal Protein L7 / L12 protein antigen of Mycoplasma pneumoniae was dissolved in PBS of 200; z1 and 200 ⁇ l 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.
  • 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. .
  • RPMI 1640 culture maintained at 37 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.
  • 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.
  • 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.
  • Monoclonal antibodies were produced and recovered using the positive hybridoma cells obtained as described above according to a standard method.
  • cells subcultured using RPMI 1640 medium 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.
  • 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.
  • 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.
  • 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 43 ⁇ 4.
  • 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.
  • 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.
  • 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.
  • purified polyclonal antibody in PBS containing 0.05% sodium azide was diluted to a concentration of 10 ⁇ g / 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 ⁇ g / 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.
  • microorganisms of the type can be accurately detected.
  • an antibody against a ribosomal protein of a microorganism Ribosomal Protein L7 / L12 protein can be used to detect Mycoplasma pneumoniae with high accuracy.

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Abstract

L'invention porte sur un procédé spécifique, rapide et très sensible de détection d'un micro-organisme du type Mycoplasma pneumoniae sur un anticorps servant à cette détection; sur une trousse de réactifs de détection; et sur le procédé d'obtention dudit anticorps, et en particulier sur un anticorps agissant contre la protéine ribosomale d'un micro-organisme du type Mycoplasma pneumoniae, et réagissant spécifiquement avec ledit micro-organisme; sur un procédé de détection du micro-organisme dans un échantillon à l'aide dudit anticorps, et sur une trousse de réactifs de détection contenant ledit anticorps. La protéine ribosomale est par exemple la PR L7/L12 qui sert à détecter les infections dues à un micro-organisme cause de pneumonies.
PCT/JP2001/000626 1998-07-31 2001-01-31 Anticorps servant a detecter le mycoplasma pneumoniae Ceased WO2001057199A1 (fr)

Priority Applications (5)

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CA002398469A CA2398469C (fr) 2000-01-31 2001-01-31 Anticorps servant a detecter le mycoplasma pneumoniae
AU2001230532A AU2001230532A1 (en) 2000-01-31 2001-01-31 Antibody for detecting mycoplasma pneumoniae
JP2001558014A JP5331285B2 (ja) 2000-01-31 2001-01-31 マイコプラズマ・ニューモニア検出用抗体
US10/386,050 US20040014943A1 (en) 1998-07-31 2003-03-12 Antibodies for detecting microorganisms
US12/424,370 US20090269789A1 (en) 1998-07-31 2009-04-15 Antibodies for detecting microorganisms

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JP2000-062729 2000-01-31
JP2000062729 2000-01-31

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PCT/JP2001/000625 Continuation-In-Part WO2001057089A1 (fr) 1998-07-31 2001-01-31 Anticorps de détection de chlamydia pneumoniae
US10/386,050 Continuation-In-Part US20040014943A1 (en) 1998-07-31 2003-03-12 Antibodies for detecting microorganisms

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Cited By (6)

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JP2004201605A (ja) * 2002-12-26 2004-07-22 Asahi Kasei Corp レジオネラ属菌リボソームｌ７／ｌ１２タンパク質をコードするｄｎａ
JP2009180580A (ja) * 2008-01-30 2009-08-13 Asahi Kasei Corp 抗体固定化担体
WO2011068189A1 (fr) 2009-12-04 2011-06-09 三菱化学メディエンス株式会社 Procédé de détection de micro-organismes appartenant à mycoplasma pneumoniae et/ou mycoplasma genitalium
JP2014167439A (ja) * 2013-02-28 2014-09-11 Asahi Kasei Corp マイコプラズマ・ニューモニアの検出方法
JP2021069355A (ja) * 2019-11-01 2021-05-06 旭化成株式会社 検体中の肺炎マイコプラズマを検出するための抗体、並びに斯かる抗体を用いて肺炎マイコプラズマを検出するための方法、試薬、及びキット
JP2021177184A (ja) * 2013-08-23 2021-11-11 株式会社タウンズ マイコプラズマ・ニューモニエの免疫学的検出法およびキット

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KR100905066B1 (ko) * 2007-12-04 2009-06-30 주식회사 바이오랜드 마이코플라즈마 뉴모니아성 폐렴 진단을 위한 재조합단백질 및 이를 이용한 진단 키트
JP2016031353A (ja) 2014-07-30 2016-03-07 株式会社 富山研究所 肺炎マイコプラズマ検出試薬及びその用途

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Cited By (11)

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Publication number Priority date Publication date Assignee Title
JP2004201605A (ja) * 2002-12-26 2004-07-22 Asahi Kasei Corp レジオネラ属菌リボソームｌ７／ｌ１２タンパク質をコードするｄｎａ
JP2009180580A (ja) * 2008-01-30 2009-08-13 Asahi Kasei Corp 抗体固定化担体
WO2011068189A1 (fr) 2009-12-04 2011-06-09 三菱化学メディエンス株式会社 Procédé de détection de micro-organismes appartenant à mycoplasma pneumoniae et/ou mycoplasma genitalium
KR20120107983A (ko) 2009-12-04 2012-10-04 미쓰비시 가가쿠 메디엔스 가부시키가이샤 마이코플라즈마·뉴모니에 및/또는 마이코플라즈마·제니탈륨에 속하는 미생물을 검출하는 방법
US8940496B2 (en) 2009-12-04 2015-01-27 Lsi Medience Corporation Method for detecting microorganisms belonging to Mycoplasma pneumoniae and/or Mycoplasma genitalium
JP5712140B2 (ja) * 2009-12-04 2015-05-07 株式会社Ｌｓｉメディエンス マイコプラズマ・ニューモニエ及び／又はマイコプラズマ・ジェニタリウムに属する微生物を検出する方法
JP2014167439A (ja) * 2013-02-28 2014-09-11 Asahi Kasei Corp マイコプラズマ・ニューモニアの検出方法
JP2021177184A (ja) * 2013-08-23 2021-11-11 株式会社タウンズ マイコプラズマ・ニューモニエの免疫学的検出法およびキット
JP7260192B2 (ja) 2013-08-23 2023-04-18 株式会社タウンズ マイコプラズマ・ニューモニエの免疫学的検出法およびキット
JP2021069355A (ja) * 2019-11-01 2021-05-06 旭化成株式会社 検体中の肺炎マイコプラズマを検出するための抗体、並びに斯かる抗体を用いて肺炎マイコプラズマを検出するための方法、試薬、及びキット
JP7402659B2 (ja) 2019-11-01 2023-12-21 旭化成株式会社 検体中の肺炎マイコプラズマを検出するための抗体、並びに斯かる抗体を用いて肺炎マイコプラズマを検出するための方法、試薬、及びキット

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JP2012006968A (ja) 2012-01-12
CN1406279A (zh) 2003-03-26
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AU2001230532A1 (en) 2001-08-14
JP5331285B2 (ja) 2013-10-30
CN100516210C (zh) 2009-07-22

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