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WO2016194986A1 - Analyseur immunochromatographique pour la détection de mycoplasma pneumoniae - Google Patents

Analyseur immunochromatographique pour la détection de mycoplasma pneumoniae Download PDF

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Publication number
WO2016194986A1
WO2016194986A1 PCT/JP2016/066310 JP2016066310W WO2016194986A1 WO 2016194986 A1 WO2016194986 A1 WO 2016194986A1 JP 2016066310 W JP2016066310 W JP 2016066310W WO 2016194986 A1 WO2016194986 A1 WO 2016194986A1
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WIPO (PCT)
Prior art keywords
antibody
mycoplasma pneumoniae
sample
labeling substance
protein
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Application number
PCT/JP2016/066310
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English (en)
Japanese (ja)
Inventor
鈴木　啓太
久彦 岩本
Original Assignee
田中貴金属工業株式会社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2015171163A external-priority patent/JP6143818B2/ja
Application filed by 田中貴金属工業株式会社 filed Critical 田中貴金属工業株式会社
Priority to CN201680031731.XA priority Critical patent/CN107709995A/zh
Priority to EP16803420.5A priority patent/EP3306320B1/fr
Priority to US15/578,493 priority patent/US10852300B2/en
Priority to KR1020177034687A priority patent/KR20180002752A/ko
Publication of WO2016194986A1 publication Critical patent/WO2016194986A1/fr

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    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/30—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]

Definitions

  • the present invention relates to an immunochromatography analyzer for detecting Mycoplasma pneumoniae, an immunochromatography kit, and a detection method thereof.
  • Mycoplasma pneumonia is a respiratory infection mainly caused by Mycoplasma pneumoniae.
  • Mycoplasma pneumoniae is a typical causative agent of atypical pneumonia.
  • the age of mycoplasma pneumonia patients is mainly in early childhood, schoolchildhood, and adolescence (5 to 35 years).
  • the initial symptoms are cold syndrome-like symptoms, so-called cold-like symptoms, but cough over time. May continue and may continue for about a month after the fever.
  • mycoplasma pneumonia there is a high need in the medical field to determine the presence or absence of Mycoplasma pneumoniae infection at the early stage of infection for the selection of antibiotics, but by applying it to an immunochromatographic analysis method that is easy to operate, mycoplasma can be quickly and easily performed. ⁇ Pneumoniae can be detected and mycoplasma pneumonia can be diagnosed.
  • the simplest structure of an immunochromatographic analyzer used for immunochromatographic analysis is a structure in which a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part, and an absorption part are connected to each other.
  • Patent Document 1 discloses an immunochromatographic analyzer using a monoclonal antibody specific for the P1 protein of Mycoplasma pneumoniae.
  • Patent Document 2 discloses an immunochromatographic analyzer using an antibody specific for Mycoplasma pneumoniae ribosomal protein Ribosomal Protein L7 / L12.
  • the present inventors have focused on the P30 protein of Mycoplasma pneumoniae as a target protein that can be detected with higher sensitivity than the P1 protein of Mycoplasma pneumoniae, and in particular, recognized a specific site of the P30 protein. It was found that the antibody to detect Mycoplasma pneumoniae with high sensitivity.
  • mycoplasma pneumoniae infection can be detected quickly and easily with high sensitivity according to an immunochromatography analyzer using an antibody that binds to a specific site of the P30 protein of Mycoplasma pneumoniae, and the present invention has been achieved.
  • An immunochromatography analyzer for detecting Mycoplasma pneumoniae comprising a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part and an absorption part, The immunochromatographic analyzer, wherein the labeling substance holding part and the detection part contain an antibody that strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae consisting of the amino acid sequence of SEQ ID NO: 2.
  • An immunochromatography analysis kit comprising the immunochromatography analyzer according to any one of 1 to 3 above and a sample diluent for diluting and developing the sample. 5.
  • a method for detecting Mycoplasma pneumoniae in a specimen using an immunochromatographic analyzer including a sample addition unit, a labeled substance holding unit, a chromatographic medium unit having a detection unit, and an absorption unit, the following step (1)
  • An immunochromatographic analysis method comprising (4).
  • a step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to a sample addition portion (2) P30 protein of Mycoplasma pneumoniae having the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding portion
  • a step of recognizing Mycoplasma pneumoniae by an antibody that strongly recognizes domain III (hereinafter referred to as antibody P30 (A))
  • a step of developing the specimen and antibody P30 (A) as a mobile phase in a chromatographic medium part (4)
  • a step of detecting Mycoplasma pneumoniae in the developed mobile phase with the antibody P30 (A) contained in the detection unit
  • Mycoplasma pneumoniae can be detected quickly and easily with high sensitivity by using an immunochromatographic analyzer using an antibody that binds to a specific site of the P30 protein of Mycoplasma pneumoniae. That is, the diagnosis of pneumonia mycoplasma can be performed more reliably and quickly.
  • FIG. 1 is a cross-sectional view for explaining the structure of an immunochromatographic analyzer according to an embodiment of the present invention.
  • FIG. 2 is a graph showing the results of detection of Mycoplasma pneumoniae using the immunochromatography analyzer of the present invention and measuring the color intensity.
  • FIG. 3 is a graph showing the results of detection of Mycoplasma pneumoniae using the immunochromatography analyzer of the present invention and measuring the color intensity.
  • FIG. 4 is a graph showing the results of the detection of Mycoplasma pneumoniae P30 protein using the immunochromatography analyzer of the present invention and the measurement of its color intensity.
  • FIG. 1 is a cross-sectional view for explaining the structure of an immunochromatographic analyzer according to an embodiment of the present invention.
  • FIG. 2 is a graph showing the results of detection of Mycoplasma pneumoniae using the immunochromatography analyzer of the present invention and measuring the color intensity.
  • FIG. 3 is a graph showing the results of detection of Mycoplasma pneumoniae using the immunochromatography analyzer of the present invention
  • FIG. 5 is a graph showing the results of performing a competitive inhibition ELISA test using peptide 1 or peptide 2 as an antigen and measuring the color intensity in Test Example 4 using antibody P30 (a).
  • FIG. 6 is a graph showing the results of a competition inhibition ELISA test using peptide 1 or peptide 2 as an antigen and the color intensity measured in test example 4 using antibody P30 (b).
  • the immunochromatography analyzer for detecting Mycoplasma pneumoniae of the present invention is a chromatograph having a sample addition part for adding a specimen, a labeling substance holding part for holding a labeling substance, and a detection part for detecting Mycoplasma pneumoniae. A medium part and an absorption part that absorbs the liquid that has passed through the detection part, wherein the labeling substance holding part and the detection part contain an antibody that strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae .
  • the antibody used in the immunochromatography analyzer of the present invention is an antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae (hereinafter also referred to as antibody P30 (A)).
  • Mycoplasma pneumoniae P30 protein is an adhesion protein consisting of 274 amino acids shown in SEQ ID NO: 1, rich in proline (20.7%), and having a molecular weight of 29,743.
  • domain III of the P30 protein shown in SEQ ID NO: 2 is a region corresponding to the 177th to 274th amino acid sequence of the P30 protein (SEQ ID NO: 1), and is a domain rich in a specific amino acid repetitive sequence.
  • There are three types of amino acid repetitive sequences in domain III 7 amino acid sequences shown in SEQ ID NO: 3 (PGMAPR), 3 amino acid sequences shown in SEQ ID NO: 4 (PGMPPH), and shown in SEQ ID NO: 5 (PGGFPPQ). There are three amino acid sequences.
  • the antibody P30 (A) used in the present invention recognizes domain III rich in this amino acid repetitive sequence. Therefore, by performing analysis using the immunochromatography analyzer of the present invention, the sandwich structure as shown below is obtained. It is estimated that the detection sensitivity is remarkably improved and the effect is remarkably exhibited.
  • antibody P30 (A) when antibody P30 (A) is contained in the labeling substance holding part and the detection part of the immunochromatography analyzer of the present invention, first, antibody P30 (A) held in the labeling substance holding part is Mycoplasma pneumoniae. It binds to part of the repeat sequence of domain III in the P30 protein.
  • the antibody P30 (A) immobilized on the detection unit binds to the same repetitive sequence present at a location different from the location where the antibody P30 (A) held in the labeling substance holding unit is bound, A sandwich structure is formed by sandwiching the P30 protein with the antibody P30 (A), and Mycoplasma pneumoniae is detected.
  • antibody P30 (A) recognizes domain III having the above-mentioned repetitive sequence, it can be estimated that it can recognize a plurality of locations of P30 protein and can increase the detection sensitivity of Mycoplasma pneumoniae. Is done.
  • the antibody P30 (A) used in the present invention preferably recognizes at least the amino acid sequence of SEQ ID NO: 3 (PGMAPR) among the repeated sequences of amino acids in the domain III. As shown in the results of Examples below, the antibody recognizing the above sequence more strongly recognizes the domain III of the P30 protein of Mycoplasma pneumoniae, so that Mycoplasma pneumoniae can be detected with high sensitivity.
  • PMAPR amino acid sequence of SEQ ID NO: 3
  • the antibody P30 (A) in the present invention is an antibody that "recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae" as an antibody having an absorbance at 450 nm of 0.2 Abs or more when performing an ELISA test. Can be defined.
  • ELISA test is performed according to the following protocol.
  • Mycoplasma pneumonia P30 ((amino acids 96-274) was expressed and purified in a conventional manner using Escherichia coli in a 96-well plate for Nunc Immuno modules (Thermo Fisher Scientific, code 469949)
  • ELISA 4Pg / n4 Add 100 ⁇ L of mL in 50 mM carbonate buffer pH 9.5 and incubate at 4 ° C. for 16 hours. After 16 hours, the P30 solution is removed, and the wells are washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.
  • PBST 5% BSA in PBST
  • BSA manufactured by Oriental Yeast
  • anti-Mycoplasma pneumoniae P30 antibody As a primary antibody, anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (A)) 5 ⁇ g / mL in 50% blocking solution 100 ⁇ L is added to the well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution is removed, and the wells are washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • An antibody having a value obtained by subtracting the absorbance of a blank (secondary antibody without adding a primary antibody, well in which color development reaction was performed) by the above method to be 0.2 Abs or more was designated as “Mycoplasma pneumoniae P30 protein domain III. Antibody ".
  • the antibody P30 (A) in the present invention is an antibody that "recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae", specifically, the full-length Mycoplasma pneumonia P30 protein (amino acids 1-274: SEQ ID NO: 1) or aminoplasm 96-274 fragment of Mycoplasma pneumonia P30 protein (these proteins are referred to as protein A) and one or more of three types of repeated sequences of amino acids in domain III or domain III (SEQ ID NOs: 3 to 5)
  • a competitive inhibition test competitive inhibition ELISA test
  • the competitive inhibition ELISA test can be defined by either a direct competitive method or an indirect competitive method.
  • ELISA 96-well plate Mycoplasma pneumonia P30 (amino acid 1-274: SEQ ID NO: 1) 4 ngm 50 mL Add 100 ⁇ L of carbonate buffer pH 9.5 and incubate at 4 ° C. for 16 hours. After 16 hours, the P30 solution is removed, and the wells are washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS). Remove the liquid remaining in the well by tapping on a paper towel.
  • PBST 5% BSA in PBST
  • BSA manufactured by Oriental Yeast
  • the BSA solution is removed, and the wells are washed 3 times with 300 ⁇ L PBST (0.05% Tween20 in PBS).
  • the liquid remaining in the well is removed by tapping on a paper towel to create a full-length protein-fixed well.
  • anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (A)) 5 ⁇ g / mL in 50% blocking solution is added to the well as a primary antibody in the full-length protein-fixed well, and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution is removed, and the wells are washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • the primary antibody anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (A)) 5 ⁇ g / mL, and the primary antibody amount 40 times or 80 times the domain III or 100 ⁇ L of a 50% blocking solution containing a domain III fragment containing one or more of the three repeating sequences of amino acids in domain III is added to the wells and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution is removed, and the wells are washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • PBST 0.05% Tween 20 in PBS
  • the absorbance is reduced by 30% or more when the domain III or a fragment of domain III containing one or more of three types of repeating sequences of amino acids in domain III is used 40 times the amount of the primary antibody, or 80 times that of the primary antibody. It can be confirmed that the domain III of the P30 protein of Mycoplasma pneumoniae is more strongly recognized if the absorbance when used in an amount decreases by 50% or more.
  • Examples of the antibody P30 (A) used in the present invention include polyclonal antibodies and monoclonal antibodies.
  • a monoclonal antibody is preferable from the viewpoint of sensitivity.
  • the antibody P30 (A) can be obtained by purchasing, for example, trade name: Mp-P30-3-AB from Fucal Research.
  • fixed means that the antibody is arranged on a carrier such as a membrane so that it does not move
  • hold means that the inside or the surface of the carrier such as a membrane can move. It means to be placed.
  • a sample addition part also called a sample pad
  • a labeling substance holding part also called a conjugate pad
  • a chromatograph It is comprised from the graph medium part (3), the detection part (4), the absorption part (5), and the backing sheet (6).
  • the sample addition part (1) is a part to which a sample containing a specimen is added in the immunochromatography analyzer.
  • the sample is rapidly absorbed, but it can be constituted by a porous sheet having such a property that the sample moves quickly.
  • the porous sheet include cellulose filter paper, glass fiber, polyurethane, polyacetate, cellulose acetate, nylon, and cotton cloth.
  • the labeling substance holding part (2) contains a labeling substance (marker substance) described later, and the labeling substance is held as a labeled antibody (hereinafter also simply referred to as a labeled antibody) bound to the antibody P30 (A). Held in the part (2).
  • a labeled antibody hereinafter also simply referred to as a labeled antibody bound to the antibody P30 (A).
  • a film of glass fiber, cellulose or the like is usually used for the labeling substance holding part (2).
  • the content of the labeled antibody in the labeling substance holding part is usually 0.06 to 0.25 ⁇ g / device, preferably 0.1 to 0.2 ⁇ g / device, more preferably 0.1 to 0.15 ⁇ g. / Device.
  • the content of the labeled antibody per unit area of the labeling substance holding part is usually 0.1 to 0.42 ⁇ g / cm 2 , preferably 0.17 to 0.33 ⁇ g / cm 2 , more preferably 0.17 to 0.25 ⁇ g / cm 2 .
  • An enzyme or the like is generally used for labeling a detection reagent in immunochromatographic analysis, but it is preferable to use an insoluble carrier as the labeling substance because it is suitable for visually determining the presence of the substance to be detected.
  • a labeled detection reagent can be prepared by sensitizing the antibody P30 (A) to an insoluble carrier.
  • the means for sensitizing the antibody P30 (A) to the insoluble carrier may be a known method.
  • the insoluble carrier as a labeling substance, metal particles such as gold, silver or platinum, metal oxide particles such as iron oxide, non-metallic particles such as sulfur and latex particles made of synthetic polymer, or other insoluble carriers are used. be able to.
  • the insoluble carrier is a labeling substance suitable for visually determining the presence of the substance to be detected, and is preferably colored to facilitate visual determination.
  • the metal particles and the metal oxide particles themselves exhibit a specific natural color corresponding to the particle diameter, and the color can be used as a label.
  • the average particle diameter of the gold particles is, for example, 10 nm to 250 nm, preferably 35 nm to 120 nm.
  • the average particle diameter was measured with a transmission electron microscope (TEM: manufactured by JEOL Ltd., JEM-2010), and the projected area circle equivalent diameter of 100 particles was measured randomly using a photograph taken. It can be calculated from the average value.
  • the gold particles contained in the labeling substance holding part are usually 0.25 to 0.7 ⁇ g / cm 2 , preferably 0.3 to 0.65 ⁇ g / cm 2 per unit area of the labeling substance holding part, Preferably, it is 0.4 to 0.6 ⁇ g / cm 2 . This is because, by setting the range, the labeled particles are developed in a dispersed state, and the recognition site of the antibody is not hindered and the sensitivity can be increased.
  • the chromatographic medium part (3) is a developed part of the chromatograph.
  • the chromatographic medium part (3) is an inert film made of a microporous material that exhibits capillary action.
  • a membrane made of nitrocellulose hereinafter, Nitrocellulose membrane
  • cellulose acetate membrane hereinafter also referred to as cellulose acetate membrane
  • Cellulose membranes, nylon membranes and porous plastic cloth can also be used.
  • nitrocellulose membrane it is only necessary to mainly contain nitrocellulose, and a membrane mainly composed of nitrocellulose such as a pure product or a nitrocellulose mixed product can be used.
  • the nitrocellulose membrane may further contain a substance that promotes capillary action.
  • a substance that lowers the surface tension of the film surface and brings about hydrophilicity is preferable.
  • a substance having an amphipathic action such as saccharides, amino acid derivatives, fatty acid esters, various synthetic surfactants or alcohols, which does not affect the movement of the detected substance and does not affect the coloring of the labeling substance. No material is preferred.
  • Nitrocellulose membrane is porous and exhibits capillary action.
  • the index of the capillary phenomenon can be confirmed by measuring the water absorption rate (water absorption time: capillary flow time).
  • the water absorption rate affects detection sensitivity and inspection time.
  • the form and size of the chromatographic medium part (3) represented by the above nitrocellulose membrane and cellulose acetate membrane are not particularly limited, and are appropriate in terms of actual operation and reaction results. If it is.
  • the support made of plastic or the like on the back surface of the chromatographic medium part (3).
  • the properties of the support are not particularly limited, but when the measurement result is observed by visual judgment, the support preferably has a color that is not similar to the color caused by the labeling substance. Usually, it is preferably colorless or white.
  • the detection part (4) is formed on the chromatographic medium part (3). That is, the antibody P30 (A) that binds to the substance to be detected Mycoplasma pneumoniae is immobilized at an arbitrary position on the chromatographic medium part (3). Immobilization of the antibody P30 (A) can be performed according to a conventional method.
  • the content of antibody P30 (A) in the detection section (4) is usually 0.1 to 2.5 ⁇ g / device, preferably 0.3 to 2.0 ⁇ g / device, more preferably 0.3 to 1.0 ⁇ g / device. Further, the content of the antibody P30 (A) per unit area of the detection unit (4) is usually 0.04 to 1.0 g / cm 2 , preferably 0.125 to 0.8 ⁇ g / cm 2 . More preferably, it is 0.125 to 0.42 ⁇ g / cm 2 .
  • the chromatographic medium part (3) is blocked by a known method as necessary. Processing can be performed. Generally, proteins such as bovine serum albumin, skim milk, casein or gelatin are preferably used for the blocking treatment. After such blocking treatment, if necessary, for example, one or a combination of two or more surfactants such as Tween 20, Triton X-100, or SDS may be washed.
  • a surfactant such as Tween 20, Triton X-100, or SDS may be washed.
  • the absorption part (5) is installed at the end of the chromatographic medium part (3) in order to absorb a liquid such as a specimen or a developing solution that has passed through the detection part (4).
  • the absorption part (5) contains, for example, glass fiber, pulp, cellulose fiber, or the like, or a non-woven fabric containing a polymer such as an acrylic acid polymer, a hydrophilic drug having an ethylene oxide group or the like. What was made to use is used, Especially preferably, it is a glass fiber.
  • the absorption part (5) is made of glass fiber, the return of the sample liquid can be greatly reduced.
  • the backing sheet (6) is a base material. By applying an adhesive on one side or sticking an adhesive tape, one side has adhesiveness, and the sample addition part (1), labeling substance holding part (2), chromatographic medium part ( 3) A part or all of the detection unit (4) and the absorption unit (5) are provided in close contact with each other.
  • the backing sheet (6) is not particularly limited as long as the backing sheet (6) becomes impermeable and impermeable to the sample solution by the adhesive.
  • the immunochromatographic analyzer produced as described above is usually subjected to a drying process before commercialization.
  • the drying temperature is, for example, 20 to 50 ° C., and the drying time is 0.5 to 1 hour.
  • the immunochromatography analysis kit of the present invention includes the above immunochromatography analyzer and a sample diluent for diluting and developing the sample.
  • the sample diluent can also be used as a developing solution, but usually water is used as a solvent, to which a buffer solution, salt, and nonionic surfactant are further added.
  • a buffer solution for example, one or more of proteins, polymer compounds (PVP, etc.), ionic surfactants or polyanions, or antibacterial agents, chelating agents, etc. for promoting antigen-antibody reactions or suppressing nonspecific reactions May be added.
  • P30 was isolated from the protein complex (P1, P90, P40, P30) present in the cell adhesion organ of Mycoplasma pneumonia, and a nonionic surfactant was diluted with the sample to expose the antigen recognition site of the anti-P30 antibody. It is preferable to make it contain in a liquid.
  • Nonionic surfactants include, for example, Triton X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether), Tween 20 (trade name, polyoxyethylene sorbitan monolaurate), NP-40 (product) Name Nonit 40) and Brij 35 may be mentioned, and one or more may be added.
  • the nonionic surfactant contained in the sample diluent is a nonionic surfactant having an HLB value of 13 to 18. More preferably, 60% or more of the nonionic surfactant contained in the sample diluent is a nonionic surfactant having an HLB value of 13 to 17, and particularly preferably the nonionic surfactant contained in the sample diluent. 100% of the ionic surfactant is a nonionic surfactant having an HLB value of 13-17. Specifically, it is preferable to contain two types of Triton X-100 (HLB value: 13.7) and Tween 20 (HLB value: 16.7) at a mass ratio of 1: 1.
  • the sample and developing solution mixed in advance can be supplied and dropped onto the sample addition unit, or the sample can be supplied to the sample addition unit first. -After dripping, you may make it expand
  • the immunochromatographic analysis method of the present invention includes the following steps (1) to (4), and detects Mycoplasma pneumoniae to be detected contained in a specimen using the above immunochromatographic analyzer.
  • a step of adding a sample-containing solution obtained by diluting a sample with a sample dilution solution to a sample addition portion (2) P30 protein of Mycoplasma pneumoniae having the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding portion
  • a step of recognizing Mycoplasma pneumoniae by an antibody that strongly recognizes domain III hereinafter referred to as antibody P30 (A)
  • (3) a step of developing the specimen and antibody P30 (A) as a mobile phase in a chromatographic medium part (4) Process of detecting Mycoplasma pneumoniae in the developed mobile phase with antibody P30 (A) contained in the detection unit Each process will be described below.
  • Step (1) Step of adding a specimen-containing solution obtained by diluting a specimen with a specimen diluent to the sample addition section
  • the specimen is smoothly passed through the immunochromatographic medium without degrading measurement accuracy. It is preferable to adjust or dilute with a sample diluent to a concentration that allows the sample to be transferred to a concentration of 1).
  • the specimen dilution liquid described above can be used.
  • a predetermined amount (usually 0.1 to 2 mL) of the specimen-containing liquid is dropped onto the sample addition part (1). When the specimen-containing liquid is dropped, the specimen-containing liquid starts to move in the sample addition part (1).
  • the specimen used in the present invention is a specimen that may contain Mycoplasma pneumoniae to be detected.
  • step (2) An antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae consisting of the amino acid sequence of SEQ ID NO: 2 held in the labeling substance holding part (hereinafter referred to as antibody P30 (A)).
  • step (2) the sample-containing liquid added to the sample addition part in step (1) is moved to the labeling substance holding part (2), and the label held in the labeling substance holding part.
  • step (3) the sample-containing liquid added to the sample addition part in step (1) is moved to the labeling substance holding part (2), and the label held in the labeling substance holding part
  • This is a step of recognizing the domain III of the P30 protein in Mycoplasma pneumoniae, which is the substance to be detected in the specimen, by the antibody P30 (A) to which the substance is bound.
  • the above-mentioned labeling substances can be used.
  • the antibody P30 (A) to which the labeling substance is bound recognizes and binds to a part of the specific repeating sequence in the amino acid sequence of domain III of the P30 protein.
  • step (3) Step of developing the sample and antibody P30 (A) as a mobile phase in a chromatographic medium part
  • Mycoplasma pneumoniae which is a substance to be detected in step (2), is labeled in the labeling substance holding part. Is recognized by the antibody P30 (A) bound to the sample, and then the specimen and the antibody P30 (A) are passed over the chromatographic medium as a mobile phase.
  • Step (4) is a step of detecting the mycoplasma in the sample that has passed over the chromatographic medium as the mobile phase.
  • Pneumonier is sandwiched between antibody P30 (A) immobilized on the detection part and antibody P30 (A) to which the labeling substance is bound in the above step (2) by a specific antigen-antibody binding reaction. This is a step of specifically reacting and binding to color the detection part.
  • the antibody P30 (A) immobilized on the detection part is sandwiched between the same repetitive sequences present at a different place from the place where the antibody P30 (A) held in the labeling substance holding part is bound. Combined so as to be sandwiched in a shape.
  • the labeling reagent dissolved in the sample water does not cause a specific binding reaction even if it passes through the detection part on the chromatographic medium part. Do not color.
  • the above peptide 1 is a peptide having the amino acid sequence shown in SEQ ID NO: 4 (PGMPPH) and the amino acid sequence shown in SEQ ID NO: 3 (PGMAPR).
  • Peptide 2 is a peptide having the amino acid sequence shown in SEQ ID NO: 5 (PGGFPPQ) and the amino acid sequence shown in SEQ ID NO: 3 (PGMAPR).
  • sample addition part The nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.
  • the labeling substance solution was prepared by the above procedure. After adding 300 ⁇ L of a 10% by mass trehalose aqueous solution and 1.8 mL of distilled water to 300 ⁇ L of the prepared labeling substance solution, uniformly added to a 12 mm ⁇ 300 mm glass fiber pad (Millipore), vacuum is added.
  • the labeling substance holding part was produced by drying with a dryer.
  • chromatographic medium part and detection part As a membrane, a sheet made of nitrocellulose (manufactured by Millipore, trade name: HF120, 300 mm ⁇ 25 mm) was used. Next, 150 ⁇ L of a solution obtained by diluting the antibody P30 (a) with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol so as to have a concentration of 1.0 mg / mL was dried with a membrane. An upper chromatographic dispenser “XYZ3050” (manufactured by BIODOT) was applied to the upper detection site (detection line) at a width of 1 mm in an amount of 1 ⁇ L / mm (25 ⁇ L per sheet).
  • a substrate consisting of a backing sheet, a sample addition part, a labeling substance holding part, a chromatographic medium part having a detection part, absorption for absorbing the developed sample and labeling substance Glass fiber non-woven fabrics were bonded together as a part. And it cut
  • the length of the labeling substance holding part in the sample developing direction was 12 mm.
  • Specimen diluent 1% by mass of nonionic surfactant NP-40 (trade name: Nonidet P-40, HLB value 17.7) manufactured by Nacalai Tesque Co., Ltd., trade name Nonidet MN-811 (manufactured by NOF Corporation)
  • NP-40 trade name: Nonidet P-40, HLB value 17.7
  • Nonidet MN-811 manufactured by NOF Corporation
  • a 50 mM HEPES buffer solution (pH 7.5) containing a 1: 1 mixture of HLB values 9.3) was prepared, and used as a sample diluent for subjecting the sample to dilution treatment.
  • Mycoplasma pneumonia P30 ((amino acid 96-274) was expressed and purified by a conventional method using Escherichia coli P30Ag) was used. This P30Ag was adjusted to the target concentration of 10 pg / mL with the above extract, and peptide 1 or peptide 2 was prepared so that 400 ng of peptide 1 or peptide 2 was developed, and used as a sample sample. 150 ⁇ L of the sample sample was placed on the sample addition part of the immunochromatography analyzer and developed, and the degree of coloring (coloring intensity) of the detection part was measured with a densitometer (the unit in the table is mAbs).
  • the number of binding sites is small or nonexistent, and a large amount of peptide flows to the absorption unit without being captured by the detection unit. As a result, the amount of labeling substance that is captured and deposited by the detection unit decreases, thereby reducing the color intensity. .
  • the antibody held in the labeling substance holding part binds to Peptide 1 and Peptide 2 prior to P30Ag, so that the amount of the antibody that binds to P30Ag developed later is present in Peptide 1 or Peptide 2. Therefore, the amount of P30Ag captured by the detection unit is relatively higher when the antibody is not bound, and as a result, the color intensity caused by P30Ag detected by the detection unit is low. It will be a hindrance.
  • the antibody P30 (a) used in Test Example 1 is an antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae, according to the protocol described in paragraphs [0025] to [0030]
  • the test by the ELISA method was performed. Specifically, whether or not the antibody P30 (a) was an antibody having an absorbance at 450 nm of 0.2 Abs or more with a blank drawn was examined as follows.
  • Mycoplasma pneumonia P30 ((amino acids 96-274) was expressed and purified in an ordinary manner using E. coli 4 g / n (4 Pg / mL). 100 ⁇ L of in 50 mM carbonate buffer pH 9.5 was added and incubated at 4 ° C. for 16 hours. After 16 hours, the P30 solution was removed, the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS), and the liquid remaining in the wells was removed by tapping on a paper towel.
  • PBST 0.05% Tween 20 in PBS
  • PBST 5% BSA in PBST
  • BSA manufactured by Oriental Yeast
  • a primary antibody 100 ⁇ L of the antibody P30 (a) 5 ⁇ g / mL in 50% blocking solution (primary antibody solution) was added to the well, incubated at 37 ° C. for 1 hour, the primary antibody solution was removed, and the well was removed with 300 ⁇ L PBST (0.05 % Tween20 in PBS).
  • PBST 0.05 % Tween20 in PBS.
  • a secondary antibody 1 mg / mL of Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemical Industries, Ltd., code 04-117611) was added to 100 ⁇ L of the well and incubated at 37 ° C. for 1.5 hours.
  • the absorbance at 450 nm obtained by subtracting the absorbance of the blank (secondary antibody without adding the primary antibody, well in which the color reaction was performed) was 0.403 Abs, which was 0.2 Abs or more. I was able to confirm. That is, it was confirmed that the antibody P30 (a) “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.
  • this antibody P30 (a) is an antibody that "recognizes strongly domain III of the P30 protein of Mycoplasma pneumoniae"
  • competition inhibition ELISA test indirect competition method was performed. Specifically, as a result of competitive inhibition ELISA test using antibody P30 (a), whether or not the absorbance decreased by 30% or more was tested as follows.
  • Nunc Immuno modules manufactured by Thermo Fisher Scientific, code 469949
  • ELISA 96-well plate full length protein Mycoplasma pneumonia P30 (amino acid 1-274: SEQ ID NO: 1) 4 ng / mL in 50 mM carbonate buffer L9.
  • P30 solution was removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween20 in PBS). The liquid remaining in the well was removed by tapping on a paper towel.
  • BSA 5% BSA in PBST
  • BSA manufactured by Oriental Yeast
  • the BSA solution was then removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • the liquid remaining in the well was removed by tapping on a paper towel to create a full-length protein-fixed well.
  • an anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (a)) 5 ⁇ g / mL in 50% blocking solution was added to the well as a primary antibody in the full-length protein-fixed well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • the primary antibody anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (a)) 5 ⁇ g / mL and 40 times the amount of the primary antibody P30 (a) 100 ⁇ L of a 50% blocking solution containing the peptide 1 (SEQ ID NO: 6: PGMAPRPGMPPHPGMAPR) or peptide 2 (SEQ ID NO: 7: PGMAPRPGFPPQPGMAPR) used in 1 was added to the well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • a 50% blocking solution containing the peptide 1 SEQ ID NO: 6: PGMAPRPGMPPHPGMAPR
  • peptide 2 SEQ ID NO: 7: PGMAPRPGFPPQPGMAPR
  • Example 3 ⁇ Preparation of immunochromatography analyzer of the present invention> [Example 1] (1) Preparation of sample addition part The nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.
  • the nonwoven fabric (Millipore company_made: 300 mm x 30 mm) which consists of glass fiber was used as a sample addition part.
  • a phosphate buffer solution pH 7.4 containing 1% by mass of bovine serum albumin (BSA) was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then, after sufficiently stirring, centrifugation was performed at 8000 ⁇ g for 15 minutes, and the supernatant was removed. Then, 0.1 mL of a phosphate buffer (pH 7.4) containing 1% by mass of BSA was added.
  • the labeling substance solution was prepared by the above procedure.
  • the labeling substance holding part was produced by drying with a dryer.
  • chromatographic medium part and detection part As a membrane, a sheet made of nitrocellulose (manufactured by Millipore, trade name: HF120, 300 mm ⁇ 25 mm) was used. Next, 150 ⁇ L of a solution obtained by diluting the antibody P30 (a) with a phosphate buffer solution (pH 7.4) containing 5% by mass of isopropyl alcohol so as to have a concentration of 1.0 mg / mL was dried with a membrane. An upper chromatographic dispenser “XYZ3050” (manufactured by BIODOT) was applied to the upper detection site (detection line) at a width of 1 mm in an amount of 1 ⁇ L / mm (25 ⁇ L per sheet).
  • a goat-derived antiserum having a wide affinity for the gold particle labeling substance dilute a goat-derived antiserum having a wide affinity for the gold particle labeling substance with a phosphate buffer (pH 7.4) downstream of the detection site.
  • the solution was applied to the control site (control line). Then, it was dried at 50 ° C. for 30 minutes and dried overnight at room temperature to prepare a chromatographic medium part and a detection part.
  • Specimen diluent 1% by mass of nonionic surfactant NP-40 (trade name: Nonidet P-40, HLB value 17.7) manufactured by Nacalai Tesque Co., Ltd., trade name Nonidet MN-811 (manufactured by NOF Corporation)
  • NP-40 trade name: Nonidet P-40, HLB value 17.7
  • Nonidet MN-811 manufactured by NOF Corporation
  • a 50 mM HEPES buffer solution (pH 7.5) containing a 1: 1 mixture of HLB values 9.3) was prepared, and used as a sample diluent for subjecting the sample to dilution treatment.
  • a specimen sample containing Mycoplasma pneumoniae the color intensity at the detection part was measured when the prepared immunochromatography analyzer was used.
  • a specimen sample containing Mycoplasma pneumoniae a commercially available inactivated Mycoplasma pneumoniae (manufactured by Meridian, trade name Mycoplasma pneumoniae Antigen (FH)), or a pharyngeal wipe from a pneumonia mycoplasma infected person was used.
  • the pharyngeal swab was collected from two subjects with pneumonia mycoplasma infection by wiping the pharynx with a commercially available cotton swab.
  • a commercially available inactivated mycoplasma pneumoniae was adjusted to the target concentration with the above extract and used as a specimen sample (described as a commercially available specimen in Table 3). Further, the collected pharyngeal wiping solution was diluted 20-fold with the above-described sample diluent, and used as sample samples (described as subject 1 and subject 2 in Table 3).
  • the antibody P30 (b) is an antibody that strongly recognizes domain III of the P30 protein of Mycoplasma pneumoniae
  • an ELISA test was performed according to the protocol described in paragraphs [0025] to [0030]. It was. Specifically, whether or not the antibody P30 (b) was an antibody having an absorbance at 450 nm with a blank drawn of 0.2 Abs or more was examined as follows.
  • PBST 5% BSA in PBST
  • BSA manufactured by Oriental Yeast
  • the antibody P30 (b) 5 ⁇ g / mL in 50% blocking solution 100 ⁇ L was added to the well as a primary antibody and incubated at 37 ° C. for 1 hour. Then, the primary antibody solution antibody P30 (b) was removed, and the well was removed with 300 ⁇ L PBST (0.05 % Tween20 in PBS). As a secondary antibody, 1 mg / mL of Anti Mouse IgG (H + L), Rabbit, IgG Whole, Peroxidase Cojugated (manufactured by Wako Pure Chemical Industries, Ltd., code 04-117611) was added to 100 ⁇ L of the well and incubated at 37 ° C. for 1.5 hours.
  • the absorbance at 450 nm obtained by subtracting the absorbance of the blank was 0.061 Abs and less than 0.2 Abs. I was able to confirm. That is, it was confirmed that the antibody P30 (b) was not an antibody that “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.
  • this antibody P30 (b) is an antibody “recognizes domain III of P30 protein of Mycoplasma pneumoniae”
  • the protocol described in paragraphs [0032] to [0039] is followed.
  • a competitive inhibition ELISA test (indirect competitive method) was performed, and whether or not the absorbance decreased to 30% or less was examined as follows.
  • Nunc Immuno modules manufactured by Thermo Fisher Scientific, code 469949
  • ELISA 96-well plate full length protein Mycoplasma pneumonia P30 (amino acid 1-274: SEQ ID NO: 1) 10 ng / mL in 50 mM carbonate buffer L9.
  • P30 solution was removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween20 in PBS). The liquid remaining in the well was removed by tapping on a paper towel.
  • BSA 5% BSA in PBST
  • BSA manufactured by Oriental Yeast
  • the BSA solution was then removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • the liquid remaining in the well was removed by tapping on a paper towel to create a full-length protein-fixed well.
  • an anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (a)) 5 ⁇ g / mL in 50% blocking solution was added to the well as a primary antibody in the full-length protein-fixed well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • the primary antibody anti-Mycoplasma pneumoniae P30 antibody (antibody P30 (b)) 5 ⁇ g / mL and the amount that is 40 times the amount of the primary antibody P30 (b)
  • test 100 ⁇ L of a 50% blocking solution containing Peptide 1 (SEQ ID NO: 6: PGMAPRPGMPPHPGMAPR) or Peptide 2 (SEQ ID NO: 7: PGMAPRPGFPPQPGMAPR) used in Example 1 was added to the well and incubated at 37 ° C. for 1 hour. Thereafter, the primary antibody solution was removed, and the wells were washed 3 times with 300 ⁇ L PBST (0.05% Tween 20 in PBS).
  • the absorbance of the well added with the primary antibody and the peptide 1 or peptide 2 was not decreased in the peptide 1 compared with the absorbance of the well added only with the primary antibody, and only about 22% in the peptide 2 It was confirmed that the decrease rate was less than 30%. That is, it was confirmed that the antibody P30 (b) was not an antibody that “recognizes strongly the domain III of the P30 protein of Mycoplasma pneumoniae”.
  • Example 2 Except that both the antibody P30 (a) in the labeling substance solution added to the labeling substance holding part and the antibody P30 (a) in the antibody diluent applied to the detection part were changed to the antibody P30 (b), Example 1 was repeated. The results are shown in Table 3 and FIG.
  • Example 2 The composition of the sample diluent was Triton X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether, HLB value: 13.7, manufactured by Wako Pure Chemical Industries, Ltd.), Tween 20 (trade name, manufactured by Wako Pure Chemical Industries, Ltd.).
  • Example 1 was repeated except that polyoxyethylene sorbitan monolaurate, HLB value: 16.7) was changed. The results are shown in Table 4 and FIG. [Comparative Example 6]
  • Example 2 was repeated except that the antibody P30 (a) in the antibody diluent applied to the detection unit was changed to the antibody P30 (b). The results are shown in Table 4 and FIG.
  • Example 3 The detection sample was changed to P30 protein ((amino acid 96-274) was expressed and purified by a conventional method using E. coli P30Ag), and the composition of the sample dilution was changed to Triton.
  • X-100 (trade name, polyethylene glycol mono-p-isooctylphenyl ether, HLB value: 13.7), manufactured by Wako Pure Chemical Industries, Ltd., Tween 20 (trade name, polyoxyethylene sorbitan monolaurate, manufactured by Wako Pure Chemical Industries, Ltd.) , HLB value: 16.7)
  • Example 1 was repeated except that it was changed to a 1: 1 mixture. The results are shown in Table 5 and FIG.
  • Example 3 was repeated except that the antibody P30 (a) in the antibody diluent applied to the detection unit was changed to the antibody P30 (b). The results are shown in Table 5 and FIG.
  • Example 3 was repeated except that the antibody P30 (a) in the antibody dilution solution applied to the detection unit was changed to the antibody P1 (b). The results are shown in Table 5 and FIG.
  • Example 11 Example 3 was repeated except that the antibody P30 (a) in the labeling substance solution added to the labeling substance holding part was changed to the antibody P1 (a). The results are shown in Table 5 and FIG.
  • Mycoplasma pneumoniae can be detected quickly and easily with high sensitivity by using an immunochromatography analyzer using an antibody that binds to a specific site of the P30 protein of Mycoplasma pneumoniae. It can be used for early diagnosis.
  • Sample pad 2 Labeling substance holding part 3 Chromatographic medium part 4 Detection part 5 Absorption part 6 Backing sheet

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Abstract

La présente invention vise à fournir un analyseur immunochromatographique, permettant la détection rapide et facile de Mycoplasma pneumoniae avec une sensibilité élevée, de sorte que la pneumonie à mycoplasme puisse être diagnostiquée plus sûrement et plus rapidement . La présente invention concerne un analyseur immunochromatographique pour la détection de Mycoplasma pneumoniae, ledit analyseur immunochromatographique comportant une partie d'addition d'échantillon, une partie de maintien de substance marqueur, une partie de milieu de chromatographie comprenant une section de détection, et une partie d'absorption, la partie de maintien de substance marqueur et la section de détection contenant un anticorps qui réalise une identification précise du domaine III de la protéine P30 de Mycoplasma pneumoniae comprenant la séquence d'acides aminés représentée par SEQ ID NO: 2.
PCT/JP2016/066310 2015-06-01 2016-06-01 Analyseur immunochromatographique pour la détection de mycoplasma pneumoniae WO2016194986A1 (fr)

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CN201680031731.XA CN107709995A (zh) 2015-06-01 2016-06-01 肺炎支原体检测用免疫层析分析装置
EP16803420.5A EP3306320B1 (fr) 2015-06-01 2016-06-01 Analyseur immunochromatographique pour la détection demycoplasma pneumoniae
US15/578,493 US10852300B2 (en) 2015-06-01 2016-06-01 Immunochromatographic analyzer for Mycoplasma pneumoniae detection
KR1020177034687A KR20180002752A (ko) 2015-06-01 2016-06-01 마이코플라스마ㆍ뉴모니에 검출용 면역 크로마토그래피 분석 장치

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JPS61274687A (ja) * 1985-03-27 1986-12-04 バイオジェン　インコーポレイテッド 豚ミコプラスマの組換表面抗原およびワクチン並びにそれらに基づく診断法
JP2010019796A (ja) * 2008-07-14 2010-01-28 Clarion Co Ltd ナビゲーション装置、ナビゲーション装置の表示制御方法及び制御プログラム
CN104198703A (zh) * 2014-08-18 2014-12-10 湖北工业大学 人肺炎支原体金标银染免疫层析检测试剂盒及其制备方法和应用
WO2015025968A1 (fr) * 2013-08-23 2015-02-26 株式会社ビーエル Méthode et kit de détection immunologique de mycoplasma pneumoniae
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JPS61274687A (ja) * 1985-03-27 1986-12-04 バイオジェン　インコーポレイテッド 豚ミコプラスマの組換表面抗原およびワクチン並びにそれらに基づく診断法
JP2010019796A (ja) * 2008-07-14 2010-01-28 Clarion Co Ltd ナビゲーション装置、ナビゲーション装置の表示制御方法及び制御プログラム
WO2015025968A1 (fr) * 2013-08-23 2015-02-26 株式会社ビーエル Méthode et kit de détection immunologique de mycoplasma pneumoniae
WO2016017598A1 (fr) * 2014-07-30 2016-02-04 田中貴金属工業株式会社 Agent de détection de mycoplasma pneumoniae, et application de celui-ci
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CHANG HY ET AL.: "DOMAIN ANALYSIS OF PROTEIN P30 IN MYCOPLASMA PNEUMONIAE CYTADHERENCE AND GLIDING MOTILITY", J BACTERIOL, vol. 193, no. 7, 2011, pages 1726 - 1733, XP055334317 *

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