WO2019064463A1 - Method and device for concentrating virus or bacterium using bioaffinity - Google Patents
Method and device for concentrating virus or bacterium using bioaffinity Download PDFInfo
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- 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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- carrier
- virus
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- binding molecule
- ligand binding
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- 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
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- 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
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- 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
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- 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
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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
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.
Abstract
The present invention addresses the problem of providing a means for concentrating a virus or a bacterium simply and rapidly while maintaining the structure thereof. Provided is a method for concentrating a target virus or bacterium, the method characterized in that the target virus or bacterium is captured by a carrier via a metal ion-dependent ligand binding molecule, is isolated from the ligand binding molecule by treatment with a chelating agent, and is then recovered.
Description
本発明はウイルス又は細菌の濃縮技術に関する。詳細には、バイオアフィニティー（生物学的親和性）を利用して標的のウイルス又は細菌を濃縮する方法及び装置に関する。
The present invention relates to virus or bacterial concentration techniques. In particular, the present invention relates to a method and apparatus for concentrating target viruses or bacteria utilizing bioaffinity (biological affinity).
生体に備わっている微生物感染防御機構には自己と非自己を識別するための生体分子が存在する。獲得免疫では抗体やT細胞受容体が抗原と特異的に結合する役割を担い、自然免疫ではC型レクチンやToll様受容体などがその役割を担う。マンナン結合タンパク質（MBP）は哺乳動物の血清に存在するC型レクチンの一種でカルシウムイオン依存的にマンノース、フコース、およびN-アセチルグルコサミンと結合する。MBPはこれらの糖鎖を末端に有する種々の微生物と結合し補体系活性化を介して殺菌作用を示すことにより、これらに対する感染防御を担っている。
There are biological molecules for distinguishing between self and non-self in the microbial infection defense mechanism provided in the living body. In adaptive immunity, antibodies and T cell receptors play a role to specifically bind to an antigen, and in innate immunity, C-type lectins and Toll-like receptors play a role. Mannan-binding protein (MBP) is a type of C-type lectin present in mammalian serum, and binds calcium, mannose, fucose, and N-acetylglucosamine in a calcium ion-dependent manner. MBP plays a protective role against infection by binding to various microorganisms having these sugar chains at the end and exerting bactericidal action through activation of the complement system.
ウイルスや細菌による感染の早期治療や予防のためには、より高感度でウイルス／細菌を検出することが重要である。検出感度の向上のためには、当然ながら検出方法の改良も有効であるが、検出方法に供する試料の調製が重要な鍵となる。試料中の検出対象（ウイルス／細菌）の濃度を高めることができれば、実質的に検出感度の大幅な上昇を望める。尚、ウイルスを濃縮するための手段として、リン酸カルシウム系化合物からなる吸着剤の使用（例えば特許文献１）、レクチンの使用（例えば特許文献２、３を参照）などが提案されている。
For early treatment and prevention of viral and bacterial infections, it is important to detect viruses / bacteria with higher sensitivity. Of course, improvement of the detection method is also effective for improving detection sensitivity, but preparation of a sample to be used for the detection method is an important key. If the concentration of the detection target (virus / bacteria) in the sample can be increased, a substantial increase in detection sensitivity can be expected substantially. In addition, as means for concentrating a virus, use of the 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.
上記の通り、検出に先立って試料中の検出対象を濃縮しておくことは、高感度の検出を実現するために重要となる。臨床応用ないし実用化する上では簡便且つ迅速に濃縮することが要求される。一方、臨床検査等での利用を想定した場合、検出対象のウイルスや細菌を可能な限りその構造（形態）を維持しつつ（即ち破壊することなく）濃縮することが望まれる。そこで本発明は、簡便且つ迅速に、しかもその構造の維持を図りつつ、ウイルスや細菌を濃縮する手段を提供することを課題とする。
As described above, 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.
上記課題に鑑み検討を進める中で本発明者らは、金属イオン依存的に捕捉し、回収することによってウイルス又は細菌を濃縮するという戦略を着想するとともに、金属イオン依存性結合分子としてのマンナン結合タンパク質（MBP）に着眼し、その実効性を検証した。その結果、MBPを利用することで簡便且つ迅速にインフルエンザウイルスを濃縮することに成功した。また、上記戦略の実用化に有益且つ重要な各種知見も得られた。
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 We focused on protein (MBP) and verified its effectiveness. As a result, it succeeded in concentrating influenza virus simply and rapidly by using MBP. In addition, various useful and important findings were obtained for the practical application of the above strategy.
ところで、抗体は結合特異性が高く、標的分子を捕捉するための手段として極めて有用である。金属イオン依存性を示す抗体であれば、MBPと同様、上記戦略に利用可能といえる。抗体によれば、より選択的な標的の濃縮を期待できる。また、抗体の特異性の高さは濃縮効率の向上にも寄与する。
以下の発明は上記の成果及び考察に基づく。
［１］金属イオン依存性リガンド結合分子を介して標的のウイルス又は細菌を担体に捕捉した後、キレート剤で処理することによって前記リガンド結合分子から前記標的を脱離し、回収することを特徴とする、標的のウイルス又は細菌を濃縮する方法。
［２］以下のステップ（１）及び（２）を含む、［１］に記載の方法：
（１）標的のウイルス又は細菌を含有する試料を、金属イオンの存在下、該金属イオン依存性リガンド結合分子を表面に固相化した担体に接触させることにより、標的のウイルス又は細菌を担体に捕捉するステップ；
（２）標的のウイルス又は細菌を捕捉した前記担体をキレート剤で処理するステップ。
［３］ステップ（１）とステップ（２）の間に、前記担体を洗浄するステップを行う、［２］に記載の方法。
［４］前記リガンド結合分子がマンナン結合タンパク質である、［１］～［３］のいずれか一項に記載の方法。
［５］前記リガンド結合分子が抗体である、［１］～［３］のいずれか一項に記載の方法。
［６］前記金属イオンがカルシウムイオンである、［４］又は［５］に記載の方法。
［７］前記標的がインフルエンザウイルスである、［１］～［６］のいずれか一項に記載の方法。
［８］前記試料が粘膜洗浄液又は体液である、［１］～［７］のいずれか一項に記載の方法。
［９］前記担体が、表面に水酸基を有する無機物質又は有機物質からなる粒子である、［１］～［８］のいずれか一項に記載の方法。
［１０］前記粒子の粒径が30μm～110μmである、［９］に記載の方法。
［１１］前記担体が、一端に注入口を他端に排出口を備えた柱状容器に充填されている、［１］～［１０］のいずれか一項に記載の方法。
［１２］前記担体は127/φ2（N/cm2）（但し、φは担体を充填する柱状容器の内径（cm））の圧力をかけても変形しない性質を有する、［１１］に記載の方法。
［１３］前記キレート剤がアミノカルボン酸系キレート剤である、［１］～［１２］のいずれか一項に記載の方法。
［１４］金属イオン依存性リガンド結合分子を表面に固相化した粒子状の担体又は一体型多孔質材料の担体と、
一端に注入口を他端に排出口を備え、前記担体が充填された柱状容器と、
を備えた、標的のウイルス又は細菌を濃縮するための装置。 By the way, 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.
[7] The method according to any one of [1] to [6], wherein 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.
[9] The method according to any one of [1] to [8], wherein the carrier is a particle comprising an inorganic substance or an organic substance having a hydroxyl group on the surface.
[10] The method according to [9], wherein the particle size of the particles is 30 μm to 110 μm.
[11] The method according to any one of [1] to [10], wherein the carrier is packed in a columnar container provided with an inlet at one end and an outlet at the other end.
[12] The carrier according to [11], wherein the carrier has the property of not deforming under pressure of 127 / φ 2 (N / cm 2 ) (where φ is the inner diameter (cm) of the columnar container filled with carrier) the method of.
[13] The method according to any one of [1] to [12], wherein the chelating agent is an aminocarboxylic acid chelating agent.
[14] 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.
以下の発明は上記の成果及び考察に基づく。
［１］金属イオン依存性リガンド結合分子を介して標的のウイルス又は細菌を担体に捕捉した後、キレート剤で処理することによって前記リガンド結合分子から前記標的を脱離し、回収することを特徴とする、標的のウイルス又は細菌を濃縮する方法。
［２］以下のステップ（１）及び（２）を含む、［１］に記載の方法：
（１）標的のウイルス又は細菌を含有する試料を、金属イオンの存在下、該金属イオン依存性リガンド結合分子を表面に固相化した担体に接触させることにより、標的のウイルス又は細菌を担体に捕捉するステップ；
（２）標的のウイルス又は細菌を捕捉した前記担体をキレート剤で処理するステップ。
［３］ステップ（１）とステップ（２）の間に、前記担体を洗浄するステップを行う、［２］に記載の方法。
［４］前記リガンド結合分子がマンナン結合タンパク質である、［１］～［３］のいずれか一項に記載の方法。
［５］前記リガンド結合分子が抗体である、［１］～［３］のいずれか一項に記載の方法。
［６］前記金属イオンがカルシウムイオンである、［４］又は［５］に記載の方法。
［７］前記標的がインフルエンザウイルスである、［１］～［６］のいずれか一項に記載の方法。
［８］前記試料が粘膜洗浄液又は体液である、［１］～［７］のいずれか一項に記載の方法。
［９］前記担体が、表面に水酸基を有する無機物質又は有機物質からなる粒子である、［１］～［８］のいずれか一項に記載の方法。
［１０］前記粒子の粒径が30μm～110μmである、［９］に記載の方法。
［１１］前記担体が、一端に注入口を他端に排出口を備えた柱状容器に充填されている、［１］～［１０］のいずれか一項に記載の方法。
［１２］前記担体は127/φ2（N/cm2）（但し、φは担体を充填する柱状容器の内径（cm））の圧力をかけても変形しない性質を有する、［１１］に記載の方法。
［１３］前記キレート剤がアミノカルボン酸系キレート剤である、［１］～［１２］のいずれか一項に記載の方法。
［１４］金属イオン依存性リガンド結合分子を表面に固相化した粒子状の担体又は一体型多孔質材料の担体と、
一端に注入口を他端に排出口を備え、前記担体が充填された柱状容器と、
を備えた、標的のウイルス又は細菌を濃縮するための装置。 By the way, 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.
[7] The method according to any one of [1] to [6], wherein 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.
[9] The method according to any one of [1] to [8], wherein the carrier is a particle comprising an inorganic substance or an organic substance having a hydroxyl group on the surface.
[10] The method according to [9], wherein the particle size of the particles is 30 μm to 110 μm.
[11] The method according to any one of [1] to [10], wherein the carrier is packed in a columnar container provided with an inlet at one end and an outlet at the other end.
[12] The carrier according to [11], wherein the carrier has the property of not deforming under pressure of 127 / φ 2 (N / cm 2 ) (where φ is the inner diameter (cm) of the columnar container filled with carrier) the method of.
[13] The method according to any one of [1] to [12], wherein the chelating agent is an aminocarboxylic acid chelating agent.
[14] 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.
１．標的のウイルス又は細菌を濃縮する方法
本発明の第１の局面は標的のウイルス又は細菌を濃縮する方法（以下、「本発明の方法」とも呼ぶ）に関する。本発明の方法によれば標的のウイルス又は細菌が濃縮された（即ち、存在率が上昇した）液（「標的濃縮液」と呼ぶ）を得ることができる。標的濃縮液は例えば、感染症の検査／診断、或いは各種研究用の検体ないし試料として利用される。 1. Method of concentrating target virus or bacteria 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.
本発明の第１の局面は標的のウイルス又は細菌を濃縮する方法（以下、「本発明の方法」とも呼ぶ）に関する。本発明の方法によれば標的のウイルス又は細菌が濃縮された（即ち、存在率が上昇した）液（「標的濃縮液」と呼ぶ）を得ることができる。標的濃縮液は例えば、感染症の検査／診断、或いは各種研究用の検体ないし試料として利用される。 1. Method of concentrating target virus or bacteria 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.
「標的」とは、本発明の方法によって濃縮されるもの（濃縮対象物）である。ウイルス又は細菌が標的となる。ウイルス及び細菌は特に限定されない。標的となり得るウイルスを例示すると、インフルエンザウイルス、ＲＳウイルス（Respiratory syncytial virus）、ムンプスウイルス（Mumps virus）、Ｃ型肝炎ウイルス（Hepatitis C Virus）、ヒト免疫不全ウイルス（Human Immunodeficiency virus）、デング熱ウイルス（Dengue virus）である。標的となり得る細菌を例示すると、クラミジア（Chlamydia)、淋菌（Neisseria gonorrhoeae）、髄膜炎菌（Neisseria meningitidis）、赤痢菌（Shigella）、大腸菌（Escherichia coli）、サルモネラ菌（Salmonella）、チフス菌（S. typhi）、インフルエンザ菌（Haemophilus influenzae）、肺炎かん菌（Klebsiella pneumoniae)、百日咳菌(Bordetella pertussis)、コレラ菌(Vibrio cholerae）、カンピロバクター（Campylobacter）、緑膿菌（Pseudomonas aeruginosa）、レジオネラ（Legionella pneumophila）である。尚、金属イオン依存性リガンド結合分子としてMBPを用いる場合には、MBPが結合性を示す糖鎖（通常、マンノース、フコース又はN-アセチルグルコサミンを末端に有する糖鎖）を有するウイルス（インフルエンザウイルス、ムンプスウイルス（Mumps virus）等）が好適な標的となる。
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). Examples of bacteria that can be targets include chlamydia (Chlamydia), Neisseria gonorrhoeae, Neisseria meningitidis, Shigella (Shigella), Escherichia coli (Escherichia coli), Salmonella (Salmonella), Salmonella typhi (S. typhi), Haemophilus influenzae, Klebsiella pneumoniae, Bordetella pertussis, Vibrio cholerae, Campylobacter, Pseudomonas aeruginosa, Legionella pneumella It is. When MBP is used as the 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.) is a suitable target.
「濃縮」とは、標的のウイルス又は細菌の濃度（存在率）を高めることであり、濃縮された試料中には標的のウイルス又は細菌が、濃縮前に比較して高濃度で存在することになる。
"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. Become.
本発明の方法は、バイオアフィニティーを利用して標的のウイルス又は細菌を濃縮するものであり、金属イオン依存性リガンド結合分子を介して標的のウイルス又は細菌を担体に捕捉した後、キレート剤で処理し、前記リガンド結合分子から前記標的を脱離し、回収する、という操作によって特徴付けられる。典型的には、本発明の方法では以下の２つのステップを行う。
（１）標的のウイルス又は細菌を含有する試料を、金属イオンの存在下、該金属イオン依存性リガンド結合分子を表面に固相化した担体に接触させることにより、標的のウイルス又は細菌を担体に捕捉するステップ
（２）標的のウイルス又は細菌を捕捉した前記担体をキレート剤で処理するステップ 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. Typically, 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
（１）標的のウイルス又は細菌を含有する試料を、金属イオンの存在下、該金属イオン依存性リガンド結合分子を表面に固相化した担体に接触させることにより、標的のウイルス又は細菌を担体に捕捉するステップ
（２）標的のウイルス又は細菌を捕捉した前記担体をキレート剤で処理するステップ 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. Typically, 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
ステップ（１）
このステップではまず、本発明の方法に供する試料を用意する。試料は、標的のウイルス又は細菌を含有する溶液（標的含有液）である。例えば、うがい液（口腔粘膜洗浄液、鼻粘膜洗浄液）、洗眼液（眼表面の粘膜上皮）、膣洗浄液等、ウイルス又は細菌の感染経路ないし感染部位として重要な、外界に露出する粘膜を洗浄して得られた液（本明細書中では「粘膜洗浄液」と呼ぶ）、或いは体液（唾液、全血、血漿、血清、尿、汗、涙、母乳等）を試料として用いる。典型的にはヒト由来の試料が用いられるが、サル、ブタ、ウシ、ウマ、ヤギ、ヒツジ、イヌ、ネコ、マウス、ラット、モルモット、ハムスター等、非ヒト動物由来の試料を用いることも可能である。粘膜洗浄液を得るための洗浄に用いる液体には例えば、水（好ましくはイオン交換水、精製水、蒸留水、純粋又は超純水）等を用いることができる。また、検体採取後に金属イオンを含む中性緩衝液の10倍濃縮液を9対１の割合で混合するとよい。ろ過や遠心分離等による不溶成分の除去、希釈等の前処理を経た粘膜洗浄液又は体液を試料として用いることにしてもよい。 Step (1)
In this step, first, 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. For example, cleanse the mucous membrane exposed to the outside, which is important as a route of infection or infection of virus or bacteria, such as gargle (oral mucous membrane lavage, nasal mucous membrane lavage), eye wash (mucous epithelium of ocular surface), vaginal lavage, etc. 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. Typically, 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. is there. For example, water (preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water) or the like can be used as a liquid used for washing to obtain a mucosal washing solution. In addition, it is preferable to mix a 10-fold concentrate of a neutral buffer solution containing metal ions at a ratio of 9 to 1 after sample collection. 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.
このステップではまず、本発明の方法に供する試料を用意する。試料は、標的のウイルス又は細菌を含有する溶液（標的含有液）である。例えば、うがい液（口腔粘膜洗浄液、鼻粘膜洗浄液）、洗眼液（眼表面の粘膜上皮）、膣洗浄液等、ウイルス又は細菌の感染経路ないし感染部位として重要な、外界に露出する粘膜を洗浄して得られた液（本明細書中では「粘膜洗浄液」と呼ぶ）、或いは体液（唾液、全血、血漿、血清、尿、汗、涙、母乳等）を試料として用いる。典型的にはヒト由来の試料が用いられるが、サル、ブタ、ウシ、ウマ、ヤギ、ヒツジ、イヌ、ネコ、マウス、ラット、モルモット、ハムスター等、非ヒト動物由来の試料を用いることも可能である。粘膜洗浄液を得るための洗浄に用いる液体には例えば、水（好ましくはイオン交換水、精製水、蒸留水、純粋又は超純水）等を用いることができる。また、検体採取後に金属イオンを含む中性緩衝液の10倍濃縮液を9対１の割合で混合するとよい。ろ過や遠心分離等による不溶成分の除去、希釈等の前処理を経た粘膜洗浄液又は体液を試料として用いることにしてもよい。 Step (1)
In this step, first, 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. For example, cleanse the mucous membrane exposed to the outside, which is important as a route of infection or infection of virus or bacteria, such as gargle (oral mucous membrane lavage, nasal mucous membrane lavage), eye wash (mucous epithelium of ocular surface), vaginal lavage, etc. 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. Typically, 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. is there. For example, water (preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water) or the like can be used as a liquid used for washing to obtain a mucosal washing solution. In addition, it is preferable to mix a 10-fold concentrate of a neutral buffer solution containing metal ions at a ratio of 9 to 1 after sample collection. 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.
以上のようにして用意した試料を、金属イオンの存在下、当該金属イオン依存性リガンド結合分子を表面に固相化した担体に接触させ、標的のウイルス又は細菌を担体に捕捉する。バッチ法の利用を排除するものではないが、簡便性や濃縮効率の点などから、好ましくは、この処理にはカラム法を利用する。カラム法では一端に注入口を他端に排出口を備えた柱状容器に担体を充填したカラムを用意し、カラム内で試料と担体の接触が行われる。
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. Although the use of the batch method is not excluded, the column method is preferably used for this treatment from the viewpoint of convenience and concentration efficiency. In the column method, 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.
担体は不溶性材料からなる支持体であり、表面に水酸基を有する無機物質又は有機物質によって構成することができる。その材質の例は、ガラス、シリカ、各種樹脂（ポリスチレン系樹脂、ポリアクリル系樹脂など）、水酸基含有無機材料（紫外線処理した酸化チタン、アルミナなど）である。担体の形状は好ましくは粒子状（ビーズ）である。粒子状担体の粒径は粒子間の空隙のサイズを規定する。標的と担体表面の効率的な接触を可能にする空隙が形成されるように、粒子状担体の平均粒径は、好ましくは30μm～110μmであり、更に好ましくは30μm～60μmである。担体の粒径が大きすぎれば、標的との接触効率が低下し、ひいては濃縮効率に影響する。粒径が小さすぎる場合も十分な濃縮効率が得られなくなる。尚、粒径の測定にはレーザー回折・散乱法（マイクロトラック）を用い、体積平均によって平均粒径が求められる。
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. Examples of 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 μm to 110 μm, more preferably 30 μm to 60 μm, 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.
簡便にウイルスを濃縮するために担体が備えるべき性質の一つとして、耐圧性能が重要である。担体を柱状容器（カラム）に充填して使用する場合を想定すると、簡便に濃縮するために求められる耐圧性能は、親指と人差し指／中指でものを挟んだ時に発生する力が印加されても担体（粒子）が破壊されないような強度を有すること、と考えることができる。指で発生させることのできる力を＜約100 Nとし、担体を充填する柱状容器の内径がφ（cm）（断面積S（cm2）＝0.785φ2）と仮定すると、求められる耐圧は約127/φ2（N/cm2）となる。そこで、127/φ2（N/cm2）（但し、φは担体を充填する柱状容器の内径（cm））の圧力をかけても変形しない性質を有する担体を用いることが好ましい。例えば、内径が0.4cmの柱状容器を使用した場合、793 N/cm2の圧力に耐えられる担体を採用することが望まれる。尚、担体を充填する柱状容器として、例えば、その内径が0.4cm～1cmのものを用いることができる。
The pressure resistance is important as one of the properties that the carrier should have in order to easily concentrate the virus. Assuming that the carrier is packed and used in a columnar container (column), the pressure resistance required for easy concentration is the carrier even when the force generated when sandwiching the thumb and the index finger / middle finger is applied. It can be considered to have such a strength that the (particles) are not broken. Assuming that the force that can be generated with a finger is <about 100 N and the inner diameter of the columnar container filled with the carrier is φ (cm) (cross-sectional area S (cm 2 ) = 0.785 φ 2 ), the required withstand pressure is about It is 127 / φ 2 (N / cm 2 ). Therefore, it is preferable to use a carrier having such a property that it does not deform even when a pressure of 127 / φ 2 (N / cm 2 ) (where φ is the inner diameter (cm) of the columnar container filled with the carrier). For example, when using a columnar container with an inner diameter of 0.4 cm, it is desirable to adopt a support that can withstand a pressure of 793 N / cm 2 . In addition, as the columnar container filled with the carrier, for example, one having an inner diameter of 0.4 cm to 1 cm can be used.
一体型多孔質材料からなる担体を用いることもできる。一体型多孔質材料とは粒子を圧着することなどにより形成した、貫通孔と細孔の両方または貫通孔のみを有する多孔質材料であり、モノリスと呼ばれることもある。好ましくは、標的（ウイルス又は細菌）の効率的な捕捉に適するように、細孔を有しないシリカ担体を用いる。本発明に適した一体型多孔質材料の貫通孔径は例えばインフルエンザウイルスの場合、500 nm～10,000 nm、好ましくは500 nm～2,000 nmである。
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. Preferably, 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.
担体の表面には金属イオン依存性リガンド結合分子が固相化されている。金属イオン依存性リガンド結合分子とは、特定の金属イオン存在下において特異的に、リガンドに対する結合性を発揮する分子である。換言すれば、使用する金属イオンと金属イオン依存性リガンド結合分子は対応関係にある。
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.
例えば、MBPや抗体を金属イオン依存性リガンド結合分子として用いることができる。MBPは動物レクチンの一つであり、マンノース、フコース、N-アセチルグルコサミンを末端に有する糖鎖と特異的に結合する。生体においてMBPは主に血清及び肝臓に存在する。組換え型MBPも開発されている（例えば、Vorup-Jensen T et al., International Immunopharmacology 1, 677-687. (2001)を参照）。MBPは、ウサギ、ヒト等の動物の血清から常法に従い調製することができる（調製法については、例えば、文献Uemura et al., J. Biol. Chem. 1996, 271:4581-4584.が参考になる）。また、ヒト由来のMBPは常法に従い、遺伝子組み換え型として調製することができる。（調製法については、例えば、文献Ma et al., Proc. Natl. Acad. Sci. USA, 1999, Vol.96, 371-375が参考になる。）
For example, 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). Moreover, human-derived MBP can be prepared as a recombinant form according to a conventional method. (For the preparation method, reference is made, for example, to the document Ma et al., Proc. Natl. Acad. Sci. USA, 1999, Vol. 96, 371-375.)
インフルエンザウイルスは一般の細胞と同じ脂質二重膜からなるエンベロープを有するRNAウイルスであり、その表面にはヘマグルチニンとシアリダダーゼを膜タンパク質として有する。このうちヘマグルチニンは高マンノース型糖鎖修飾を受けていることが知られており、MBPはこのヘマグルチニンの高マンノース型糖鎖を介してインフルエンザウイルスと強く結合することが知られている。
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.
金属イオン依存性リガンド結合分子として抗体を採用する場合には、標的（特定のウイルス又は細菌）に対して特異的結合性を有し、且つ金属イオン依存性を示すものを用いる。このような抗体は、標的又はその一部（組換え体であってもよい）を抗原として用いた免疫学的手法、ファージディスプレイ法、リボソームディスプレイ法などを利用して調製することができる。金属イオン依存性リガンド結合分子として抗体が、Fab、Fab'、F(ab')2、scFv、dsFv抗体などの抗体断片であってもよい。
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. 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.
金属イオン依存性リガンド結合分子の担体への固相化の方法は特に限定されず、常法によって固相化すればよい。例えば、シランカップリング法とアミンカップリング法を利用した固相化方法、最初にアミノプロピルトリエトキシシランを用いたアミノ基の付与を行い（例えばBioconjugate Technique 539頁等が参考になる）、次いで、炭酸 N,N'-ジスクシンイミジルを用いたスクシニルイミド基の付与を行う（例えばBioconjugate Technique 542頁等が参考になる）、等によって固定化することができる。
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. For example, 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.
金属イオン依存性リガンド結合分子の担体への固相化にビオチン・アビジン結合を利用することにしてもよい。この場合、金属イオン依存性リガンド結合分子にビオチン又はビオチン類縁体（イミノビオチン、デスチオビオチン、ビオチンスルホキシド）を結合しておき、担体の表面にはビオチン結合性タンパク質を結合させておく。金属イオン依存性リガンド結合分子のビオチン化には市販のビオチン化試薬（例えば、Thermo Scientific社のSulfo-NHS-LC-Biotinや同仁科学研究所社のBiotin Labeling Kit - NH2）を用いることができる。また、ビオチン結合性タンパク質の代表はアビジン及びストレプトアビジンであるが、ニュートラアビジン、ブラダビジン、リザビジン等を用いることもできる。上記のようにして用意したビオチン化金属イオン依存性リガンド結合分子と、ビオチン結合タンパク質が結合した担体を適当な条件下で接触させ、ビオチン・アビジン結合を介して金属イオン依存性リガンド結合分子を担体に固相化する。
A biotin-avidin bond may be used to immobilize the metal ion-dependent ligand binding molecule on a carrier. In this case, biotin or a biotin analog (iminobiotin, desthiobiotin, biotin sulfoxide) is bound to the metal ion-dependent ligand binding molecule, and 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) can be used for biotinylation of the metal ion-dependent ligand binding molecule. . In addition, a representative of the 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
金属イオンの存在下で試料と担体を接触させるため、例えば、(i)所望の金属イオンを生じる金属塩又はその溶液（金属塩溶液）を試料に添加した後に接触操作を行う、(ii)金属塩溶液に試料を添加した後に接触操作を行う、或いは、(iii)金属塩溶液で試料を希釈した後に接触操作を行う。尚、MBP及びウイルスの活性保持の点から、金属塩溶液の調製に用いる溶媒には水（好ましくはイオン交換水、精製水、蒸留水、純粋又は超純水）を用いるとよい。接触操作を行う際の温度条件は例えば4℃～50℃、好ましくは10℃～40℃、更に好ましくは15℃～30℃である。
In order to contact the sample and the carrier in the presence of metal ions, for example, (i) 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. From the viewpoint of retention of MBP and virus activity, water (preferably ion-exchanged water, purified water, distilled water, pure or ultrapure water) is preferably used as a solvent for preparing the metal salt solution. 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.
本発明における「金属イオン」の例はカルシウムイオン、マグネシウムイオン、亜鉛イオン、セレンイオン、ニッケルイオン、銅イオン、鉄イオン、マンガンイオン、モリブデンイオンであるが、使用する金属イオン依存性リガンド結合分子に対応するものが採用される。例えば、金属イオン依存性リガンド結合分子としてのMBPにはカルシウムイオン（Ca2+）が適切である。
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. For example, calcium ion (Ca 2+ ) is suitable for MBP as a metal ion dependent ligand binding molecule.
金属イオンの濃度は、金属イオン依存性リガンド結合分子がその機能（即ち、リガンドである標的への結合能）を発揮する限りにおいて特に限定されない。例えば、2 mM～10 mMの金属イオンが存在する環境で上記(i)～(iii)のごとき接触を実施するとよい。
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). For example, 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.
ステップ（２）
ステップ（１）によって、試料中の標的が金属イオン依存性リガンド結合分子を介して担体表面に捕捉される。ステップ（２）では、標的を捕捉した担体をキレート剤で処理する。この処理によって、金属イオン依存性リガンド結合分子の結合能が低下又は喪失し、標的が脱離する。脱離した標的を回収し、高濃度で標的を含有する溶液を得る。 Step (2)
By step (1), the target in the sample is captured on the support surface via the metal ion dependent ligand binding molecule. In step (2), 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.
ステップ（１）によって、試料中の標的が金属イオン依存性リガンド結合分子を介して担体表面に捕捉される。ステップ（２）では、標的を捕捉した担体をキレート剤で処理する。この処理によって、金属イオン依存性リガンド結合分子の結合能が低下又は喪失し、標的が脱離する。脱離した標的を回収し、高濃度で標的を含有する溶液を得る。 Step (2)
By step (1), the target in the sample is captured on the support surface via the metal ion dependent ligand binding molecule. In step (2), 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.
不要成分を除去するため、及び回収される標的の濃度を高めるために、キレート剤処理の前に、ステップ（１）後の担体（表面に標的が捕捉されている）を洗浄するとよい。この洗浄操作には、捕捉状態を維持するため、ステップ（１）に使用した金属イオンを含有する溶液を用いることが好ましい。従って、好ましい態様では、ステップ（１）に使用した金属イオンを水（好ましくはイオン交換水、精製水、蒸留水、純粋又は超純水）等の溶媒に溶解した溶液（洗浄液）で洗浄する。洗浄操作を複数回行うことにしてもよい。
The carrier after step (1) (the target is trapped on the surface) may be washed prior to chelator treatment to remove unwanted components and to increase the concentration of target recovered. In 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). The washing operation may be performed multiple times.
ステップ（２）に使用するキレート剤は、金属イオン依存性リガンド結合分子と標的との間の結合を解除できるものであれば特に限定されない。例えばアミノカルボン酸系キレート剤のキレート剤（具体例はエチレンジアミン四酢酸（EDTA）、ジエチレントリアミン五酢酸（DTPA）、ヒドロキシエチルエチレンジアミン三酢酸（HEDTA））を用いることができる。
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. For example, chelating agents for aminocarboxylic acid-based chelating agents (specific examples are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), and hydroxyethylethylenediaminetriacetic acid (HEDTA)) can be used.
ステップ（２）の処理では、キレート剤を添加した溶液（キレート剤溶液）を担体に接触させることになるが、当該溶液におけるキレート剤の濃度は例えば50 mM～200 mM、好ましくは100 mM～120 mMである。
In the treatment of step (2), 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.
担体がカラムに充填されている場合には、キレート剤溶液をカラムに注入して溶出することになる。濃縮効率を高めるためには、高濃度で標的が溶出する画分を分取するとよい。尚、溶出パターンは、標的の種類、担体の材質・形態、キレート剤の種類・濃度等に依存し一律ではないが、例えば予備実験によって溶出パターンを確認することができる。
If the carrier is packed in a column, the chelating agent solution will be injected into the column and eluted. In order to increase the concentration efficiency, it is preferable to fractionate fractions in which the target is eluted at high concentration. Although 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.
２．濃縮装置
本発明の第２の局面は、本発明の方法に利用可能な装置、即ち、標的のウイルス又は細菌を濃縮する装置（本発明の装置）を提供する。本発明の装置を利用することで、本発明の方法をより簡便に実施することができる。尚、本発明の第１の局面と重複する事項（例えば、金属イオン依存性リガンド結合分子、担体、標的など）については同様であるため、その説明を省略する。 2. Concentrating device 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). By utilizing the apparatus 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.
本発明の第２の局面は、本発明の方法に利用可能な装置、即ち、標的のウイルス又は細菌を濃縮する装置（本発明の装置）を提供する。本発明の装置を利用することで、本発明の方法をより簡便に実施することができる。尚、本発明の第１の局面と重複する事項（例えば、金属イオン依存性リガンド結合分子、担体、標的など）については同様であるため、その説明を省略する。 2. Concentrating device 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). By utilizing the apparatus 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.
本発明の装置は大別して二つの要素、即ち、担体及び柱状容器（カラム）を備える。担体の表面には、金属イオン依存性リガンド結合分子が固相化されている。担体は好ましくは粒子状である。一体型多孔質材料で担体を構成してもよい。担体の材質や粒径（粒子状の担体の場合）等は第１の局面で説明した通りである。
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.
柱状容器の材質は特に限定されない。材質の例として、プラスチック（例えばポリプロピレン、ポリスチレン、ポリカーボネート、メタクリル酸メチル）、ガラス、金属（例えばステンレス、チタン）を挙げることができる。柱状容器のサイズも特に限定されないが、例えば、内径が0.4 cm～1 cm、全長が 2 cm～5 cmの柱状容器を用いることができる。
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.
柱状容器には注入口と排出口が備えられる。具体的には一端に注入口、他端に排出口を備えた柱状容器が用いられることになる。担体の保持などを目的としてフリット（フィルター）が備えられるが、それ以外にコネクタ、プレフィルター、コック等を備えることにしてもよい。
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.
柱状容器には上記担体が充填される。例えばその体積が0.05 cm3～1 cm3（ベッド高としては、例えば、0.4 cm～5 cm）となるように担体（充填剤）を充填する。担体の充填方法は湿式充填法が望ましい。
The columnar container is filled with the above carrier. For example, the carrier (filler) 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.
簡便にウイルス（インフルエンザウイルスなど）を濃縮できる装置の開発を目指し、以下の検討を行った。
１．ウサギMBPの精製
ウサギ血清500mLに対して等量の高塩濃度中性緩衝液（40mMイミダゾール、40mM塩化カルシウム、2.5M塩化ナトリウム、pH7.8）を加えて混合し、あらかじめ結合用緩衝液（20mMイミダゾール、20mM塩化カルシウム、1.25M塩化ナトリウム、pH7.8）で平衡化したマンナンセファロースカラムにかけた。カラムを結合用緩衝液で洗浄した後、結合物を解離用緩衝液I（20mMイミダゾール、2mL EDTA、1.25M塩化ナトリウム、pH7.8）で溶出した。溶出物に20mMになるように塩化カルシウムを加えて、再びマンナンセファロースカラムにかけ、同様に洗浄と溶出を行い、その溶出物に再び20mMになるように塩化カルシウムを加えて、さらにもう一度マンナンセファロースカラムにかけ、解離用緩衝液II（20mMイミダゾール、100mL マンノース、1.25M塩化ナトリウム、pH7.8）で溶出することにより、MBPを精製した。 In order to develop a device that can easily concentrate viruses (such as influenza virus), the following study was conducted.
1. Purification of rabbit MBP An equal volume of high-salt neutral buffer (40 mM imidazole, 40 mM calcium chloride, 2.5 M sodium chloride, pH 7.8) is added to 500 mL of rabbit serum and mixed, and the buffer for binding (20 mM in advance) The mixture was applied to a mannan sepharose column equilibrated with imidazole, 20 mM calcium chloride, 1.25 M sodium chloride, pH 7.8. After washing the column with binding buffer, the conjugate was eluted with dissociation buffer I (20 mM imidazole, 2 mL EDTA, 1.25 M sodium chloride, pH 7.8). 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).
１．ウサギMBPの精製
ウサギ血清500mLに対して等量の高塩濃度中性緩衝液（40mMイミダゾール、40mM塩化カルシウム、2.5M塩化ナトリウム、pH7.8）を加えて混合し、あらかじめ結合用緩衝液（20mMイミダゾール、20mM塩化カルシウム、1.25M塩化ナトリウム、pH7.8）で平衡化したマンナンセファロースカラムにかけた。カラムを結合用緩衝液で洗浄した後、結合物を解離用緩衝液I（20mMイミダゾール、2mL EDTA、1.25M塩化ナトリウム、pH7.8）で溶出した。溶出物に20mMになるように塩化カルシウムを加えて、再びマンナンセファロースカラムにかけ、同様に洗浄と溶出を行い、その溶出物に再び20mMになるように塩化カルシウムを加えて、さらにもう一度マンナンセファロースカラムにかけ、解離用緩衝液II（20mMイミダゾール、100mL マンノース、1.25M塩化ナトリウム、pH7.8）で溶出することにより、MBPを精製した。 In order to develop a device that can easily concentrate viruses (such as influenza virus), the following study was conducted.
1. Purification of rabbit MBP An equal volume of high-salt neutral buffer (40 mM imidazole, 40 mM calcium chloride, 2.5 M sodium chloride, pH 7.8) is added to 500 mL of rabbit serum and mixed, and the buffer for binding (20 mM in advance) The mixture was applied to a mannan sepharose column equilibrated with imidazole, 20 mM calcium chloride, 1.25 M sodium chloride, pH 7.8. After washing the column with binding buffer, the conjugate was eluted with dissociation buffer I (20 mM imidazole, 2 mL EDTA, 1.25 M sodium chloride, pH 7.8). 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).
２．スクシニルイミド基を有するガラスビーズ担体の作成
過酸化水素水と濃硫酸を3:7の割合で混合した混合液とガラスビーズを10分間転倒混和し、減圧フィルターユニット（アドバンテック、VH050P）に移して、吸引しながら純水で洗浄した。洗浄したガラスビーズを80℃のオーブンに1時間いれて乾燥させた後、4％の3-アミノプロピルトリエトキシシランを含むリン酸緩衝液とともに15分間転倒混和した。反応後のガラスビーズを減圧フィルターユニットに移して純水および無水アセトンで洗浄し、80℃で乾燥させた。次に、ジイソプロピルエチルアミンとアセトンを1:32で混合した混合液に溶解した1% 炭酸ジ(N-スクシンイミジル)溶液とシランコートしたガラスビーズを2時間転倒混和した。反応後のガラスビーズを減圧フィルターユニットに移して、アセトンで洗浄したのち、80℃で乾燥させた。 2. Preparation of 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. Next, a 1% solution of di (N-succinimidyl carbonate) in a mixed solution of diisopropylethylamine and acetone mixed at 1:32 and silane-coated glass beads were mixed by inversion for 2 hours. The glass beads after reaction were transferred to a vacuum filter unit, washed with acetone, and dried at 80 ° C.
過酸化水素水と濃硫酸を3:7の割合で混合した混合液とガラスビーズを10分間転倒混和し、減圧フィルターユニット（アドバンテック、VH050P）に移して、吸引しながら純水で洗浄した。洗浄したガラスビーズを80℃のオーブンに1時間いれて乾燥させた後、4％の3-アミノプロピルトリエトキシシランを含むリン酸緩衝液とともに15分間転倒混和した。反応後のガラスビーズを減圧フィルターユニットに移して純水および無水アセトンで洗浄し、80℃で乾燥させた。次に、ジイソプロピルエチルアミンとアセトンを1:32で混合した混合液に溶解した1% 炭酸ジ(N-スクシンイミジル)溶液とシランコートしたガラスビーズを2時間転倒混和した。反応後のガラスビーズを減圧フィルターユニットに移して、アセトンで洗浄したのち、80℃で乾燥させた。 2. Preparation of 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. Next, a 1% solution of di (N-succinimidyl carbonate) in a mixed solution of diisopropylethylamine and acetone mixed at 1:32 and silane-coated glass beads were mixed by inversion for 2 hours. The glass beads after reaction were transferred to a vacuum filter unit, washed with acetone, and dried at 80 ° C.
３．MBPをカップリングさせたガラスビーズ担体の作成
精製したウサギMBPをカップリング用緩衝液（20mM ヒドロキシエチルピペラジンエタンスルホン酸, 20mM 塩化カルシウム, 1.25M 塩化ナトリウム）に対して透析し、カップリング反応を阻害するイミダゾールを除去した後、限外濾過膜（アミコンウルトラ50）を用いて、10mg/mLになるように濃縮した。10mg/mL MBP溶液にスクシニルイミド化ガラスビーズを加え、1時間転倒混和した。遠心してMBP溶液を除去して、ブロッキング液（20mMトリスヒドロキシメチルアミノメタン、20mM 塩化カルシウム、1.25M 塩化ナトリウム）で洗浄した後、ブロッキング液を加えて一晩放置した。最後に、固相抽出用エンプティーカートリッジType mini（巴製作所）にMBPを結合させたガラスビーズを100μL充填して、ウイルス濃縮デバイス１（図１、２）とした。カートリッジのキャップ部分はメスルアープラグとなっており、ウイルス希釈液注入時及び溶出時にはシリンジを接続して使用することができる。尚、図１はウイルス濃縮デバイス１の外観を示す正面図であり、図２はウイルス濃縮デバイス１の断面図である。ウイルス濃縮デバイス１ではカラム本体２にルアーアダプター３が接続される。カラム本体２にはフリット（フィルター）５が装着されており、担体（MBPを結合させたガラスビーズ）４が充填される。 3. Preparation of MBP coupled glass bead carrier Purified rabbit MBP is dialyzed against coupling buffer (20 mM hydroxyethyl piperazine ethane sulfonic acid, 20 mM calcium chloride, 1.25 M sodium chloride) to inhibit the coupling reaction After removing the imidazole, the solution was concentrated to 10 mg / mL using an ultrafiltration membrane (Amicon Ultra 50). The succinyl imidated glass beads were added to 10 mg / mL MBP solution and mixed by inversion for 1 hour. After centrifugation to remove the MBP solution and washing with a blocking solution (20 mM trishydroxymethylaminomethane, 20 mM calcium chloride, 1.25 M sodium chloride), the blocking solution was added and left overnight. Finally, 100 μL of MBP-bound glass beads were packed into a solid phase extraction empty cartridge Type mini (manufactured by Sakai Mfg. Co., Ltd.) to obtain virus concentration device 1 (FIGS. 1 and 2). The cap portion of the cartridge is a female luer plug, which can be used by connecting a syringe at the time of virus dilution solution injection and elution. FIG. 1 is a front view showing the appearance of thevirus concentration device 1, and FIG. 2 is a cross-sectional view of the virus concentration device 1. In 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.
精製したウサギMBPをカップリング用緩衝液（20mM ヒドロキシエチルピペラジンエタンスルホン酸, 20mM 塩化カルシウム, 1.25M 塩化ナトリウム）に対して透析し、カップリング反応を阻害するイミダゾールを除去した後、限外濾過膜（アミコンウルトラ50）を用いて、10mg/mLになるように濃縮した。10mg/mL MBP溶液にスクシニルイミド化ガラスビーズを加え、1時間転倒混和した。遠心してMBP溶液を除去して、ブロッキング液（20mMトリスヒドロキシメチルアミノメタン、20mM 塩化カルシウム、1.25M 塩化ナトリウム）で洗浄した後、ブロッキング液を加えて一晩放置した。最後に、固相抽出用エンプティーカートリッジType mini（巴製作所）にMBPを結合させたガラスビーズを100μL充填して、ウイルス濃縮デバイス１（図１、２）とした。カートリッジのキャップ部分はメスルアープラグとなっており、ウイルス希釈液注入時及び溶出時にはシリンジを接続して使用することができる。尚、図１はウイルス濃縮デバイス１の外観を示す正面図であり、図２はウイルス濃縮デバイス１の断面図である。ウイルス濃縮デバイス１ではカラム本体２にルアーアダプター３が接続される。カラム本体２にはフリット（フィルター）５が装着されており、担体（MBPを結合させたガラスビーズ）４が充填される。 3. Preparation of MBP coupled glass bead carrier Purified rabbit MBP is dialyzed against coupling buffer (20 mM hydroxyethyl piperazine ethane sulfonic acid, 20 mM calcium chloride, 1.25 M sodium chloride) to inhibit the coupling reaction After removing the imidazole, the solution was concentrated to 10 mg / mL using an ultrafiltration membrane (Amicon Ultra 50). The succinyl imidated glass beads were added to 10 mg / mL MBP solution and mixed by inversion for 1 hour. After centrifugation to remove the MBP solution and washing with a blocking solution (20 mM trishydroxymethylaminomethane, 20 mM calcium chloride, 1.25 M sodium chloride), the blocking solution was added and left overnight. Finally, 100 μL of MBP-bound glass beads were packed into a solid phase extraction empty cartridge Type mini (manufactured by Sakai Mfg. Co., Ltd.) to obtain virus concentration device 1 (FIGS. 1 and 2). The cap portion of the cartridge is a female luer plug, which can be used by connecting a syringe at the time of virus dilution solution injection and elution. FIG. 1 is a front view showing the appearance of the
４．ウイルス濃縮実験
インフルエンザウイルス株 A/Aichi/75/2008(H3N2) をウイルス希釈用緩衝液（20mM ヒドロキシエチルピペラジンエタンスルホン酸, 5mM 塩化カルシウム, 150mM 塩化ナトリウム）で約1,500 pfu/mLになるように希釈した。このウイルス希釈液を、あらかじめウイルス希釈用緩衝液で平衡化したウイルス濃縮デバイスにおよそ毎秒0.1mLの速度で20mL注入した。次に、ウイルス脱離液（20mM ヒドロキシエチルピペラジンエタンスルホン酸, 100mM EDTA, 150mM 塩化ナトリウム）を注入し、デバイスから排出される溶液をウイルス濃縮液として回収した。ウイルス濃縮倍率は、溶出液に含まれるウイルス数（pfu）を注入したウイルス液に含まれるウイルス数（pfu）で除して算出し、ウイルス数（pfu）は次項の方法で測定したウイルス濃度（pfu/mL）にウイルス液の体積（mL）を乗じて算出した。 4. Virus concentration experiment Influenza virus strain A / Aichi / 75/2008 (H3N2) 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. Next, 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).
インフルエンザウイルス株 A/Aichi/75/2008(H3N2) をウイルス希釈用緩衝液（20mM ヒドロキシエチルピペラジンエタンスルホン酸, 5mM 塩化カルシウム, 150mM 塩化ナトリウム）で約1,500 pfu/mLになるように希釈した。このウイルス希釈液を、あらかじめウイルス希釈用緩衝液で平衡化したウイルス濃縮デバイスにおよそ毎秒0.1mLの速度で20mL注入した。次に、ウイルス脱離液（20mM ヒドロキシエチルピペラジンエタンスルホン酸, 100mM EDTA, 150mM 塩化ナトリウム）を注入し、デバイスから排出される溶液をウイルス濃縮液として回収した。ウイルス濃縮倍率は、溶出液に含まれるウイルス数（pfu）を注入したウイルス液に含まれるウイルス数（pfu）で除して算出し、ウイルス数（pfu）は次項の方法で測定したウイルス濃度（pfu/mL）にウイルス液の体積（mL）を乗じて算出した。 4. Virus concentration experiment Influenza virus strain A / Aichi / 75/2008 (H3N2) 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. Next, 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).
５．インフルエンザウイルス濃度の測定
96穴白色プレート（Thermofisher社MaxiSoap）の各ウェルに1μg/mLのフェツインを50μL加えて2時間放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、50μLのウイルス液を加えて、4℃で一晩放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、100μLのメタノールを加えて、1分放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、100μLの10%中性ホルマリン溶液を加えて、30分放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、0.1% Tween20を含むリン酸緩衝液で4000倍に希釈した抗インフルエンザ抗体液（タカラバイオ社M149）を加えて、1時間放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、0.1% Tween20を含むリン酸緩衝液で5000倍に希釈したペルオキダーゼ標識２次抗体（ニチレイ社ヒストファインMAX-PO）を加えて、30分放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、化学発光基質（Thermo Scientific SuperSignal ELISA Femto Substrate）を100μL加えて2分放置した後、発光プレートリーダーにて発光量を測定した。ウイルス標準液としてはプラークアッセイを用いてあらかじめ濃度を測定したインフルエンザウイルス株A/Aichi/75/2008(H3N2)液を使用した。実際に得られた検量線を図３に示す。検量線は実験の都度作成し、それを用いて各々のウイルス液の化学発光量からウイルス濃度を算出した。 5. Measurement of influenza virus concentration 50 μl of 1 μg / ml fetuin was added to each well of a 96-well white plate (MaxiSoap from Thermofisher) and left for 2 hours. After washing the plate with phosphate buffer containing 0.1% Tween 20, 50 μL of virus solution was added and left overnight at 4 ° C. After washing the plate with phosphate buffer containing 0.1% Tween 20, 100 μL of methanol was added and left for 1 minute. After washing the plate with phosphate buffer containing 0.1% Tween 20, 100 μL of 10% neutral formalin solution was added and left for 30 minutes. After washing the plate with phosphate buffer containing 0.1% Tween 20, anti-influenza antibody solution (Takara Bio M149) diluted 4,000 times with phosphate buffer containing 0.1% Tween 20 was added and left for 1 hour. After washing the plate with phosphate buffer containing 0.1% Tween 20, add peroxidase-labeled secondary antibody (Nichirei Histofine MAX-PO) diluted 5000-fold with phosphate buffer containing 0.1% Tween 20, and add 30 I left for a minute. The plate was washed with a phosphate buffer containing 0.1% Tween 20, 100 μL 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. As 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.
96穴白色プレート（Thermofisher社MaxiSoap）の各ウェルに1μg/mLのフェツインを50μL加えて2時間放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、50μLのウイルス液を加えて、4℃で一晩放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、100μLのメタノールを加えて、1分放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、100μLの10%中性ホルマリン溶液を加えて、30分放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、0.1% Tween20を含むリン酸緩衝液で4000倍に希釈した抗インフルエンザ抗体液（タカラバイオ社M149）を加えて、1時間放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、0.1% Tween20を含むリン酸緩衝液で5000倍に希釈したペルオキダーゼ標識２次抗体（ニチレイ社ヒストファインMAX-PO）を加えて、30分放置した。プレートを0.1% Tween20を含むリン酸緩衝液で洗浄した後、化学発光基質（Thermo Scientific SuperSignal ELISA Femto Substrate）を100μL加えて2分放置した後、発光プレートリーダーにて発光量を測定した。ウイルス標準液としてはプラークアッセイを用いてあらかじめ濃度を測定したインフルエンザウイルス株A/Aichi/75/2008(H3N2)液を使用した。実際に得られた検量線を図３に示す。検量線は実験の都度作成し、それを用いて各々のウイルス液の化学発光量からウイルス濃度を算出した。 5. Measurement of influenza virus concentration 50 μl of 1 μg / ml fetuin was added to each well of a 96-well white plate (MaxiSoap from Thermofisher) and left for 2 hours. After washing the plate with phosphate buffer containing 0.1
＜結果＞
１．ガラスビーズの直径と濃縮倍率の関係
市販の大きさの異なる各種ガラスビーズを用いて、ウイルス濃縮倍率を測定したところ、表1に示すように、直径が53～38μmのガラスビーズ（研磨用320番手ガラスビーズ）を用いた場合に、最も高い濃縮倍率が得られることが分かった。
ガラスビーズの直径とウイルス濃度倍率の関係
<Result>
1. Relationship between diameter of glass beads and concentration ratio When concentration ratios of viruses were measured using various commercially available glass beads having different sizes, as shown in Table 1, glass beads having a diameter of 53 to 38 μm (number 320 for polishing) It was found that the highest concentration ratio was obtained when glass beads were used.
Relationship between glass bead diameter and virus concentration magnification
１．ガラスビーズの直径と濃縮倍率の関係
市販の大きさの異なる各種ガラスビーズを用いて、ウイルス濃縮倍率を測定したところ、表1に示すように、直径が53～38μmのガラスビーズ（研磨用320番手ガラスビーズ）を用いた場合に、最も高い濃縮倍率が得られることが分かった。
1. Relationship between diameter of glass beads and concentration ratio When concentration ratios of viruses were measured using various commercially available glass beads having different sizes, as shown in Table 1, glass beads having a diameter of 53 to 38 μm (number 320 for polishing) It was found that the highest concentration ratio was obtained when glass beads were used.
２．ウイルス溶出パターンの検証
脱離液を注入して濃縮デバイスからウイルスを溶出する際に、1滴（50μL）ずつ分取して、その1滴ごとのウイルス濃度を測定したところ、図４のような溶出曲線が得られ、脱離液注入後の2滴目（50-100μLに相当）にピークがあり、その濃度はウイルス希釈液の50倍に達した。すなわち、デバイスのサイズや形状に合わせた適切な溶出体積を決定することにより、注入時の50倍の濃度のウイルス液を得ることが可能であることが示された。また、このことは、デバイス内部全体に広がるガラスビーズ上のMBPに結合したウイルスは脱離液の作用により直ちに解離して、脱離液のほぼ先頭に集積したことを示唆している。 2. Verification of virus elution pattern When injecting a desorbent and eluting virus from the concentration device, it was taken one drop (50 μL) at a time, and the virus concentration was measured for each drop, as shown in FIG. An elution curve was obtained, and there was a peak in the second drop (corresponding to 50-100 μL) after injection of the desorbent, and the concentration reached 50 times that of the virus dilution. That is, it was shown that it is possible to obtain a 50-fold concentration of virus solution at the time of injection by determining an appropriate elution volume according to the size and shape of the device. This also suggests that the virus bound to the MBP on the glass beads spread throughout the inside of the device was immediately dissociated by the action of the desorbing solution and accumulated almost at the top of the desorbing solution.
脱離液を注入して濃縮デバイスからウイルスを溶出する際に、1滴（50μL）ずつ分取して、その1滴ごとのウイルス濃度を測定したところ、図４のような溶出曲線が得られ、脱離液注入後の2滴目（50-100μLに相当）にピークがあり、その濃度はウイルス希釈液の50倍に達した。すなわち、デバイスのサイズや形状に合わせた適切な溶出体積を決定することにより、注入時の50倍の濃度のウイルス液を得ることが可能であることが示された。また、このことは、デバイス内部全体に広がるガラスビーズ上のMBPに結合したウイルスは脱離液の作用により直ちに解離して、脱離液のほぼ先頭に集積したことを示唆している。 2. Verification of virus elution pattern When injecting a desorbent and eluting virus from the concentration device, it was taken one drop (50 μL) at a time, and the virus concentration was measured for each drop, as shown in FIG. An elution curve was obtained, and there was a peak in the second drop (corresponding to 50-100 μL) after injection of the desorbent, and the concentration reached 50 times that of the virus dilution. That is, it was shown that it is possible to obtain a 50-fold concentration of virus solution at the time of injection by determining an appropriate elution volume according to the size and shape of the device. This also suggests that the virus bound to the MBP on the glass beads spread throughout the inside of the device was immediately dissociated by the action of the desorbing solution and accumulated almost at the top of the desorbing solution.
本発明によれば、簡便且つ迅速に標的のウイルスや細菌を濃縮することができる。また、温和な条件で処理することで標的の構造を維持しつつ濃縮することができる。本発明を利用・活用することにより、標的が希薄に存在する試料（例えばうがい液）からでも標的の検出が可能となり、感染症の予防や早期治療、流行の監視や拡大防止など、様々な面での貢献が期待される。
According to the present invention, 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. By using and utilizing the present invention, 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.
インフルエンザウイルスは季節性流行を引き起こし毎年多くの感染患者を出している。その診断には鼻腔ぬぐい液を用いた免疫クロマトグラフィー法が用いられている。鼻腔ぬぐい液からは高濃度のウイルスを含む検体が得られるが、しばしば痛みや出血を伴うなどその侵襲性が問題となっている。ウイルスを採取する方法としてはその他に咽頭うがい液を用いる方法があり、この方法は侵襲性がないものの、ウイルスの濃度が鼻腔ぬぐい液の1/100程度しかないことから、現在の免疫クロマトグラフィー法では用いられていない。しかしながら、うがい液は一般に20mL程度の体積を有しており、これを簡便に100倍程度に濃縮することができれば、現在用いられている免疫クロマトグラフィー法と組み合わせることにより、非侵襲的なインフルエンザウイルスの診断が可能となる。本発明を利用すればインフルエンザウイルス等のウイルス粒子を効率的に濃縮でき、例えば、咳嗽あるいは洗口により採取した、ウイルス粒子が希薄な検体からもウイルスの検出が可能となる。即ち、本発明を適用することにより、検出に供するウイルス濃度を維持しつつ、より温和で臨床上望ましい検体採取法が実現し、上記の如き非侵襲的な診断が可能となる。
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. If the present invention is used, virus particles such as influenza virus can be efficiently concentrated. For example, 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.
この発明は、上記発明の実施の形態及び実施例の説明に何ら限定されるものではない。特許請求の範囲の記載を逸脱せず、当業者が容易に想到できる範囲で種々の変形態様もこの発明に含まれる。本明細書の中で明示した論文、公開特許公報、及び特許公報などの内容は、その全ての内容を援用によって引用することとする。
The present invention is not limited to the description of the embodiments and examples of the above-mentioned invention. Various modifications are also included in the present invention as long as those skilled in the art can easily conceive of the claims without departing from the scope of the claims. The contents of articles, published patent publications, patent publications, etc. specified in the present specification are incorporated by reference in their entirety.
１ ウイルス濃縮デバイス
２ カラム本体
３ ルアーアダプター
４ 担体
５ フリット（フィルター） 1virus concentration device 2 column main body 3 luer adapter 4 carrier 5 frit (filter)
２ カラム本体
３ ルアーアダプター
４ 担体
５ フリット（フィルター） 1
Claims (14)
- 金属イオン依存性リガンド結合分子を介して標的のウイルス又は細菌を担体に捕捉した後、キレート剤で処理することによって前記リガンド結合分子から前記標的を脱離し、回収することを特徴とする、標的のウイルス又は細菌を濃縮する方法。 A target virus or bacterium is captured on a carrier via a metal ion-dependent ligand binding molecule, and then the target is detached from the ligand binding molecule by treatment with a chelating agent, and the target is recovered and recovered. Method of concentrating virus or bacteria.
- 以下のステップ（１）及び（２）を含む、請求項１に記載の方法：
（１）標的のウイルス又は細菌を含有する試料を、金属イオンの存在下、該金属イオン依存性リガンド結合分子を表面に固相化した担体に接触させることにより、標的のウイルス又は細菌を担体に捕捉するステップ；
（２）標的のウイルス又は細菌を捕捉した前記担体をキレート剤で処理するステップ。 The method according to claim 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. - ステップ（１）とステップ（２）の間に、前記担体を洗浄するステップを行う、請求項２に記載の方法。 The method according to claim 2, wherein the step of washing the carrier is performed between step (1) and step (2).
- 前記リガンド結合分子がマンナン結合タンパク質である、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein the ligand binding molecule is a mannan binding protein.
- 前記リガンド結合分子が抗体である、請求項１～３のいずれか一項に記載の方法。 The method according to any one of claims 1 to 3, wherein said ligand binding molecule is an antibody.
- 前記金属イオンがカルシウムイオンである、請求項４又は５に記載の方法。 The method according to claim 4 or 5, wherein the metal ion is a calcium ion.
- 前記標的がインフルエンザウイルスである、請求項１～６のいずれか一項に記載の方法。 The method according to any one of claims 1 to 6, wherein the target is an influenza virus.
- 前記試料が粘膜洗浄液又は体液である、請求項１～７のいずれか一項に記載の方法。 The method according to any one of claims 1 to 7, wherein the sample is a mucosal washing solution or a body fluid.
- 前記担体が、表面に水酸基を有する無機物質又は有機物質からなる粒子である、請求項１～８のいずれか一項に記載の方法。 The method according to any one of claims 1 to 8, wherein the carrier is a particle comprising an inorganic substance or an organic substance having a hydroxyl group on the surface.
- 前記粒子の粒径が30μm～110μmである、請求項９に記載の方法。 The method according to claim 9, wherein the particle size of the particles is 30 μm to 110 μm.
- 前記担体が、一端に注入口を他端に排出口を備えた柱状容器に充填されている、請求項１～１０のいずれか一項に記載の方法。 The method according to any one of the preceding claims, wherein the carrier is packed in a pillared container provided at one end with an inlet and at the other end with an outlet.
- 前記担体は127/φ2（N/cm2）（但し、φは担体を充填する柱状容器の内径（cm））の圧力をかけても変形しない性質を有する、請求項１１に記載の方法。 The method according to claim 11, wherein the carrier has a property such that it does not deform under pressure of 127 / φ 2 (N / cm 2 ) (where φ is the inner diameter (cm) of the columnar container filled with the carrier).
- 前記キレート剤がアミノカルボン酸系キレート剤である、請求項１～１２のいずれか一項に記載の方法。 The method according to any one of claims 1 to 12, wherein the chelating agent is an aminocarboxylic acid chelating agent.
- 金属イオン依存性リガンド結合分子を表面に固相化した粒子状の担体又は一体型多孔質材料の担体と、
一端に注入口を他端に排出口を備え、前記担体が充填された柱状容器と、
を備えた、標的のウイルス又は細菌を濃縮するための装置。 A particulate carrier or a carrier of an integral porous material in which a metal ion-dependent ligand binding molecule is immobilized on the surface;
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.
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| WO2013046722A1 (en) * | 2011-09-30 | 2013-04-04 | 中外製薬株式会社 | Ion concentration-dependent binding molecule library |
| JP2013517327A (en) * | 2010-01-19 | 2013-05-16 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Modified opsonin for pathogen detection and treatment |
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| JP2013517327A (en) * | 2010-01-19 | 2013-05-16 | プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ | Modified opsonin for pathogen detection and treatment |
| WO2013046722A1 (en) * | 2011-09-30 | 2013-04-04 | 中外製薬株式会社 | Ion concentration-dependent binding molecule library |
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