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WO2019064463A1 - Procédé et dispositif pour la concentration de virus ou de bactérie par bioaffinité - Google Patents

Procédé et dispositif pour la concentration de virus ou de bactérie par bioaffinité Download PDF

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Publication number
WO2019064463A1
WO2019064463A1 PCT/JP2017/035358 JP2017035358W WO2019064463A1 WO 2019064463 A1 WO2019064463 A1 WO 2019064463A1 JP 2017035358 W JP2017035358 W JP 2017035358W WO 2019064463 A1 WO2019064463 A1 WO 2019064463A1
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Prior art keywords
carrier
virus
target
binding molecule
ligand binding
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Ceased
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PCT/JP2017/035358
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English (en)
Japanese (ja)
Inventor
和秀 上村
敏男 河原
小林　正彦
賢洋 成瀬
智彦 藤井
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nidek Co Ltd
Chubu University
Original Assignee
Nidek Co Ltd
Chubu University
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Priority to PCT/JP2017/035358 priority Critical patent/WO2019064463A1/fr
Publication of WO2019064463A1 publication Critical patent/WO2019064463A1/fr
Anticipated expiration legal-status Critical
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
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/02—Separating microorganisms from their culture media
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C12N7/02—Recovery or purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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

Definitions

  • the present invention relates to virus or bacterial concentration techniques.
  • the present invention relates to a method and apparatus for concentrating target viruses or bacteria utilizing bioaffinity (biological affinity).
  • MBP Mannan-binding protein
  • adsorption agent which consists of a calcium-phosphate type compound (for example, patent document 1), use of a lectin (for example, refer patent documents 2 and 3), etc. are proposed.
  • concentration of the detection target in the sample prior to detection is important for achieving high sensitivity detection. In clinical application or practical application, it is required to concentrate easily and quickly. On the other hand, assuming use in clinical examinations etc., it is desirable to concentrate the virus or bacteria to be detected while maintaining (that is, without destroying) its structure (form) as much as possible. Therefore, it is an object of the present invention to provide means for concentrating viruses and bacteria while maintaining the structure simply and quickly.
  • the present inventors have conceived a strategy of concentrating viruses or bacteria by capturing and recovering in a metal ion-dependent manner in the course of investigations in view of the above-mentioned problems, and mannan binding as a metal ion-dependent binding molecule
  • MBP protein
  • antibodies have high binding specificity and are extremely useful as a means for capturing a target molecule. If it is an antibody which shows metal ion dependence, it can be said that it can be used for the said strategy like MBP. With antibodies, more selective enrichment of targets can be expected. In addition, the high specificity of the antibody also contributes to the improvement of the concentration efficiency.
  • the following invention is based on the above results and considerations. [1] A target virus or bacterium is captured on a carrier via a metal ion-dependent ligand binding molecule, and then the target is released from the ligand binding molecule by treatment with a chelating agent, and then recovered. , Methods of concentrating the target virus or bacteria.
  • [2] The method according to [1], comprising the following steps (1) and (2): (1) By contacting a target virus or bacteria-containing sample with a carrier having the metal ion-dependent ligand binding molecule immobilized on the surface in the presence of metal ions, the target virus or bacteria can be used as a carrier Capturing step; (2) treating the carrier that has captured the target virus or bacteria with a chelating agent. [3] The method according to [2], wherein the step of washing the carrier is performed between step (1) and step (2). [4] The method according to any one of [1] to [3], wherein the ligand binding molecule is a mannan binding protein.
  • [5] The method according to any one of [1] to [3], wherein the ligand binding molecule is an antibody.
  • [6] The method according to [4] or [5], wherein the metal ion is a calcium ion.
  • the target is an influenza virus.
  • [8] The method according to any one of [1] to [7], wherein the sample is a mucosal washing solution or a body fluid.
  • the carrier is a particle comprising an inorganic substance or an organic substance having a hydroxyl group on the surface.
  • a particulate carrier on which a metal ion-dependent ligand binding molecule is immobilized on a surface or a carrier of an integral porous material A columnar container including an inlet at one end and an outlet at the other end, the carrier being filled; A device for concentrating target viruses or bacteria.
  • FIG. Sectional drawing of the virus concentration device 1.
  • FIG. Calibration curve showing the relationship between the virus concentration and the amount of chemiluminescence. The virus concentration was measured by virus binding assay and a standard curve was generated. Virus elution curve.
  • the first aspect of the present invention relates to a method of concentrating target virus or bacteria (hereinafter also referred to as "the method of the present invention”). According to the method of the present invention, it is possible to obtain a liquid (referred to as a "target concentrate") in which the target virus or bacteria is concentrated (ie, the abundance is increased).
  • the target concentrate is used, for example, as an examination / diagnosis of an infectious disease or as a sample or sample for various studies.
  • the "target” is one to be concentrated by the method of the present invention (concentrated object).
  • Virus or bacteria are targeted.
  • the virus and bacteria are not particularly limited. Examples of viruses that can be targeted include influenza virus, respiratory syncytial virus, mumps virus, hepatitis C virus, hepatitis B virus, human immunodeficiency virus, and dengue fever virus virus).
  • viruses that can be targets include chlamydia (Chlamydia), Neisseria gonorrhoeae, Neisseria meningitidis, Shigella (Shigella), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Salmonella typhi (S.
  • MBP metal ion-dependent ligand binding molecule
  • a virus influenza virus having a sugar chain (usually a sugar chain having a mannose, fucose or N-acetylglucosamine at the end) to which MBP exhibits binding properties
  • Mumps virus Mumps virus etc.
  • Enrichment is to increase the concentration (presence rate) of the target virus or bacteria, and in the concentrated sample, the target virus or bacteria is present at a higher concentration than before concentration. Obviously, the target virus or bacteria is present at a higher concentration than before concentration. Obviously, the target virus or bacteria is present at a higher concentration than before concentration. Obviously, the target virus or bacteria is present at a higher concentration than before concentration. Obviously, the target virus or bacteria is present at a higher concentration than before concentration. Become.
  • the method of the present invention utilizes bioaffinity to concentrate a target virus or bacteria, and the target virus or bacteria is captured on a carrier via a metal ion dependent ligand binding molecule and then treated with a chelating agent. It is characterized by the operation of removing the target from the ligand binding molecule and recovering it.
  • the method of the present invention performs the following two steps. (1) By contacting a target virus or bacteria-containing sample with a carrier having the metal ion-dependent ligand binding molecule immobilized on the surface in the presence of metal ions, the target virus or bacteria can be used as a carrier Step of capturing (2) treating the carrier which captured the target virus or bacteria with a chelating agent
  • Step (1) a sample to be subjected to the method of the present invention is prepared.
  • the sample is a solution (target-containing solution) containing target virus or bacteria.
  • a solution target-containing solution
  • the resulting fluid referred to herein as "mucosal lavage fluid" or bodily fluid (saliva, whole blood, plasma, serum, urine, sweat, tears, breast milk, etc.) is used as a sample.
  • samples of human origin are used, but samples of non-human animals such as monkeys, pigs, cattle, horses, goats, sheep, dogs, cats, mice, rats, guinea pigs, hamsters, etc. can also be used.
  • water preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water
  • a mucous membrane lavage fluid or body fluid subjected to pretreatment such as removal of insoluble components by filtration, centrifugation or the like, dilution, etc. may be used as a sample.
  • the sample prepared as described above is brought into contact with a carrier having the metal ion-dependent ligand binding molecule immobilized on the surface in the presence of a metal ion, and the target virus or bacteria is captured on the carrier.
  • a carrier having the metal ion-dependent ligand binding molecule immobilized on the surface in the presence of a metal ion
  • the target virus or bacteria is captured on the carrier.
  • the column method is preferably used for this treatment from the viewpoint of convenience and concentration efficiency.
  • a column in which the carrier is packed is prepared in a columnar container having an inlet at one end and an outlet at the other end, and the sample and the carrier are brought into contact in the column.
  • the carrier is a support made of an insoluble material, and can be composed of an inorganic substance or an organic substance having a hydroxyl group on the surface.
  • the material are glass, silica, various resins (polystyrene resins, polyacrylic resins, etc.), hydroxyl group-containing inorganic materials (ultraviolet-treated titanium oxide, alumina, etc.).
  • the shape of the carrier is preferably particulate (beads).
  • the particle size of the particulate carrier defines the size of the voids between the particles.
  • the average particle size of the particulate carrier is preferably 30 ⁇ m to 110 ⁇ m, more preferably 30 ⁇ m to 60 ⁇ m, such that a void is formed to allow efficient contact between the target and the carrier surface. If the particle size of the carrier is too large, the contact efficiency with the target is reduced, which in turn affects the concentration efficiency. Even if the particle size is too small, sufficient concentration efficiency can not be obtained.
  • the average particle diameter can be determined by volume averaging using a laser diffraction / scattering method (microtrack) to measure the particle diameter.
  • a carrier having such a property that it does not deform even when a pressure of 127 / ⁇ 2 (N / cm 2 ) (where ⁇ is the inner diameter (cm) of the columnar container filled with the carrier).
  • N / cm 2 the inner diameter of the columnar container filled with the carrier.
  • ⁇ the inner diameter of the columnar container filled with the carrier.
  • the columnar container filled with the carrier for example, one having an inner diameter of 0.4 cm to 1 cm can be used as the columnar container filled with the carrier.
  • a carrier consisting of an integral porous material can also be used.
  • the integral porous material is a porous material having both through holes and pores or only through holes, which is formed by pressure bonding of particles, and may be referred to as a monolith.
  • a pore-free silica carrier is used, which is suitable for efficient capture of the target (virus or bacteria).
  • the penetration pore size of the integral porous material suitable for the present invention is, for example, 500 nm to 10,000 nm, preferably 500 nm to 2,000 nm for influenza virus.
  • Metal ion dependent ligand binding molecules are immobilized on the surface of the carrier.
  • a metal ion dependent ligand binding molecule is a molecule that exhibits binding to a ligand specifically in the presence of a specific metal ion. In other words, the metal ion used and the metal ion dependent ligand binding molecule are in a corresponding relationship.
  • MBP and antibodies can be used as metal ion dependent ligand binding molecules.
  • MBP is one of animal lectins and specifically binds to a sugar chain having mannose, fucose and N-acetylglucosamine at the end. In living organisms, MBP is mainly present in serum and liver. Recombinant MBP has also been developed (see, eg, Vorup-Jensen T et al., International Immunopharmacology 1, 677-687. (2001)). MBP can be prepared from serum of animals such as rabbits and humans according to a conventional method (for preparation, see, for example, the literature Uemura et al., J. Biol. Chem. 1996, 271: 4581-4584. become).
  • human-derived MBP can be prepared as a recombinant form according to a conventional method.
  • preparation method reference is made, for example, to the document Ma et al., Proc. Natl. Acad. Sci. USA, 1999, Vol. 96, 371-375.
  • Influenza virus is an RNA virus having an envelope consisting of the same lipid bilayer membrane as general cells, and has hemagglutinin and sialidase as membrane proteins on its surface. Among them, hemagglutinin is known to be subjected to high mannose type sugar chain modification, and MBP is known to strongly bind to influenza virus through the high mannose type sugar chain of hemagglutinin.
  • an antibody When an antibody is employed as a metal ion-dependent ligand binding molecule, one having specific binding to a target (specific virus or bacteria) and exhibiting metal ion dependence is used. Such an antibody can be prepared using an immunological technique, a phage display method, a ribosome display method or the like using a target or a part thereof (which may be a recombinant) as an antigen.
  • the antibody As a metal ion dependent ligand binding molecule, the antibody may be an antibody fragment such as Fab, Fab ′, F (ab ′) 2 , scFv or dsFv antibody.
  • the method of immobilizing the metal ion-dependent ligand binding molecule on the carrier is not particularly limited, and may be immobilized by a conventional method.
  • a solid phase formation method using a silane coupling method and an amine coupling method first, an amino group is imparted using aminopropyltriethoxysilane (for example, Bioconjugate Technique page 539 or the like is a reference), and then, Immobilization of the succinylimide group using N, N'-disuccinimidyl carbonate (for example, referring to Bioconjugate Technique page 542 or the like) or the like can be performed.
  • a biotin-avidin bond may be used to immobilize the metal ion-dependent ligand binding molecule on a carrier.
  • biotin or a biotin analog iminobiotin, desthiobiotin, biotin sulfoxide
  • a biotin binding protein is bound to the surface of the carrier.
  • a commercially available biotinylation reagent for example, Sulfo-NHS-LC-Biotin of Thermo Scientific or Biotin Labeling Kit-NH 2 of Dojin Science Laboratories
  • Sulfo-NHS-LC-Biotin of Thermo Scientific or Biotin Labeling Kit-NH 2 of Dojin Science Laboratories can be used for biotinylation of the metal ion-dependent ligand binding molecule.
  • biotin-binding protein is avidin and streptavidin, but neutravidin, bradavidin, lizavidin and the like can also be used.
  • the biotinylated metal ion-dependent ligand-binding molecule prepared as described above is brought into contact with a carrier to which a biotin-binding protein is bound under appropriate conditions, and the metal ion-dependent ligand-binding molecule is a carrier via a biotin-avidin bond. Immobilize on
  • metal ions or their solutions (metal salt solutions) that produce the desired metal ions are subjected to contact operation after being added to the sample, (ii) metals
  • the contact operation is performed after adding the sample to the salt solution, or (iii) the contact operation is performed after diluting the sample with the metal salt solution.
  • water preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water
  • the temperature conditions for the contact operation are, for example, 4 ° C. to 50 ° C., preferably 10 ° C. to 40 ° C., and more preferably 15 ° C. to 30 ° C.
  • Examples of the "metal ion” in the present invention are calcium ion, magnesium ion, zinc ion, selenium ion, nickel ion, copper ion, iron ion, manganese ion and molybdenum ion, but the metal ion-dependent ligand binding molecule to be used A corresponding one is adopted.
  • calcium ion (Ca 2+ ) is suitable for MBP as a metal ion dependent ligand binding molecule.
  • the concentration of the metal ion is not particularly limited as long as the metal ion-dependent ligand binding molecule exerts its function (ie, the ability to bind to a target that is a ligand).
  • the contact as described in (i) to (iii) above may be carried out in the presence of 2 mM to 10 mM of metal ions.
  • Step (2) By step (1), the target in the sample is captured on the support surface via the metal ion dependent ligand binding molecule.
  • the carrier that has captured the target is treated with a chelating agent. By this treatment, the binding ability of the metal ion dependent ligand binding molecule is reduced or lost, and the target is detached. The detached target is recovered to obtain a solution containing the target at high concentration.
  • the carrier after step (1) may be washed prior to chelator treatment to remove unwanted components and to increase the concentration of target recovered.
  • this washing operation in order to maintain the capture state, it is preferable to use the solution containing the metal ion used in step (1). Therefore, in a preferred embodiment, the metal ions used in step (1) are washed with a solution (washing solution) dissolved in a solvent such as water (preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water).
  • a solvent such as water (preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water).
  • the washing operation may be performed multiple times.
  • the chelating agent used in step (2) is not particularly limited as long as it can release the binding between the metal ion-dependent ligand binding molecule and the target.
  • chelating agents for aminocarboxylic acid-based chelating agents specifically examples are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and hydroxyethylethylenediaminetriacetic acid (HEDTA) can be used.
  • the solution to which the chelating agent is added (chelating agent solution) is brought into contact with the carrier, and the concentration of the chelating agent in the solution is, for example, 50 mM to 200 mM, preferably 100 mM to 120. It is mM.
  • the chelating agent solution will be injected into the column and eluted.
  • the elution pattern is not uniform depending on the type of target, the material and form of the carrier, and the type and concentration of the chelating agent, the elution pattern can be confirmed by, for example, preliminary experiments.
  • a second aspect of the present invention provides a device which can be used in the method of the present invention, ie a device for concentrating a target virus or bacteria (a device of the present invention).
  • a device for concentrating a target virus or bacteria a device of the present invention.
  • the method of the present invention can be carried out more simply.
  • the same items as those in the first aspect of the present invention for example, a metal ion-dependent ligand binding molecule, a carrier, a target, etc. are the same, and thus the description thereof is omitted.
  • the apparatus of the present invention is roughly divided into two elements, namely, a carrier and a column (column). On the surface of the carrier, a metal ion dependent ligand binding molecule is immobilized.
  • the carrier is preferably particulate.
  • the carrier may be composed of an integral porous material. The material of the carrier, the particle size (in the case of a particulate carrier) and the like are as described in the first aspect.
  • the material of the columnar container is not particularly limited. Examples of the material include plastic (eg, polypropylene, polystyrene, polycarbonate, methyl methacrylate), glass, metal (eg, stainless steel, titanium).
  • the size of the columnar container is also not particularly limited. For example, a columnar container having an inner diameter of 0.4 cm to 1 cm and a total length of 2 cm to 5 cm can be used.
  • the columnar container is provided with an inlet and an outlet. Specifically, a columnar container provided with an inlet at one end and an outlet at the other end is used. Although a frit (filter) is provided for the purpose of holding the carrier, etc., a connector, a pre-filter, a cock or the like may be provided in addition thereto.
  • the columnar container is filled with the above carrier.
  • the carrier filler
  • the carrier is filled so that its volume is 0.05 cm 3 to 1 cm 3 (bed height is, for example, 0.4 cm to 5 cm).
  • the method of packing the carrier is preferably a wet packing method.
  • Calcium chloride is added to the eluate to 20 mM, and the solution is again applied to a mannan sepharose column, and washing and elution are similarly performed, calcium chloride is added to the eluate again to 20 mM, and the solution is applied again to the mannan sepharose column.
  • the MBP was purified by eluting with dissociation buffer II (20 mM imidazole, 100 mL mannose, 1.25 M sodium chloride, pH 7.8).
  • glass bead carrier having succinyl imide group A mixed solution of hydrogen peroxide water and concentrated sulfuric acid mixed in a ratio of 3: 7 and glass beads are mixed by inverting for 10 minutes, transferred to a vacuum filter unit (ADVANTEC, VH 050 P), It was washed with pure water while suctioning. The washed glass beads were placed in an oven at 80 ° C. for 1 hour to dry, and then invert-mixed with phosphate buffer containing 4% 3-aminopropyltriethoxysilane for 15 minutes. The glass beads after reaction were transferred to a vacuum filter unit, washed with pure water and anhydrous acetone, and dried at 80 ° C.
  • FIG. 1 is a front view showing the appearance of the virus concentration device 1
  • FIG. 2 is a cross-sectional view of the virus concentration device 1.
  • the luer adapter 3 is connected to the column body 2.
  • a frit (filter) 5 is attached to the column body 2 and a carrier (glass beads to which MBP is bound) 4 is packed.
  • Influenza virus strain A / Aichi / 75/2008 was diluted to about 1,500 pfu / mL with virus buffer (20 mM hydroxyethyl piperazine ethane sulfonic acid, 5 mM calcium chloride, 150 mM sodium chloride) did. This virus dilution was injected at a rate of approximately 0.1 mL per second into a virus concentration device previously equilibrated with virus dilution buffer.
  • a virus solution (20 mM hydroxyethyl piperazine ethane sulfonic acid, 100 mM EDTA, 150 mM sodium chloride) was injected, and the solution discharged from the device was recovered as a virus concentrate.
  • the virus concentration factor is calculated by dividing the number of viruses (pfu) contained in the eluate by the number of viruses (pfu) contained in the injected virus solution, and the number of viruses (pfu) is the virus concentration measured by the method of the next paragraph ( It calculated by multiplying the volume (mL) of the virus liquid by pfu / mL).
  • the plate was washed with a phosphate buffer containing 0.1% Tween 20, 100 ⁇ L of a chemiluminescent substrate (Thermo Scientific SuperSignal ELISA Femto Substrate) was added and left for 2 minutes, and then the luminescence amount was measured with a luminescence plate reader.
  • a virus standard solution an influenza virus strain A / Aichi / 75/2008 (H3N2) solution whose concentration was previously measured using a plaque assay was used.
  • the calibration curve actually obtained is shown in FIG. A calibration curve was prepared for each experiment, and it was used to calculate the virus concentration from the amount of chemiluminescence of each virus solution.
  • target viruses and bacteria can be concentrated easily and quickly. Moreover, by processing under mild conditions, it is possible to concentrate while maintaining the structure of the target.
  • targets can be detected even from samples (for example, gargle) in which targets are diluted, and various aspects such as prevention and early treatment of infectious diseases, surveillance of epidemic, and spread prevention, etc. Contribution is expected.
  • Influenza viruses cause seasonal epidemics and cause many infected patients every year.
  • An immunochromatographic method using a nasal swab is used for the diagnosis.
  • Nasal swabs can be used to obtain specimens containing high concentrations of virus, but their invasiveness is often a problem, often accompanied by pain and bleeding.
  • Another method for collecting the virus is to use a pharyngeal gargle, which is not invasive, but because the concentration of the virus is only about 1/100 of that of the nasal swab, the current immunochromatographic method is Not used in However, gargle generally has a volume of about 20 mL, and if it can be conveniently concentrated to about 100 times, non-invasive influenza virus can be obtained by combining it with the currently used immunochromatographic method. Can be diagnosed.
  • virus particles such as influenza virus can be efficiently concentrated.
  • virus can be detected from a sample diluted by virus particles collected by coughing or mouthwash. That is, by applying the present invention, while maintaining the concentration of virus to be detected, a milder, clinically desirable sample collection method is realized, and the noninvasive diagnosis as described above becomes possible.

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Abstract

La présente invention a pour objet un moyen pour la concentration d'un virus ou d'une bactérie de manière simple et rapide tout en conservant la structure de celui-ci ou de celle-ci. À cet effet l'invention concerne un procédé pour la concentration d'un virus ou d'une bactérie cible, le procédé étant caractérisé en ce que le virus ou la bactérie cible est capturé(e) par un support par l'intermédiaire d'une molécule de liaison de type ligand dépendante d'un ion métallique, est isolé(e) de la molécule de liaison de type ligand par traitement avec un agent chélatant et est ensuite récupéré(e).
PCT/JP2017/035358 2017-09-28 2017-09-28 Procédé et dispositif pour la concentration de virus ou de bactérie par bioaffinité Ceased WO2019064463A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7651105B2 (ja) 2020-09-28 2025-03-26 学校法人中部大学 ウイルス濃縮材、濃縮装置及びウイルス濃縮材の製造方法
WO2025132598A1 (fr) 2023-12-19 2025-06-26 The Antibody Lab Gmbh Test à base de lectine pour détection de contamination microbienne

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JP7651105B2 (ja) 2020-09-28 2025-03-26 学校法人中部大学 ウイルス濃縮材、濃縮装置及びウイルス濃縮材の製造方法
WO2025132598A1 (fr) 2023-12-19 2025-06-26 The Antibody Lab Gmbh Test à base de lectine pour détection de contamination microbienne

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