Virus concentration material, concentration device, and method for producing virus concentration material

Abstract

To provide a virus concentrate that is applicable to a virus deficient in high-mannose glycans of hemagglutinin.SOLUTION: The inventive virus concentrate 1 includes a chimera molecule 4 supported on a carrier 5, wherein the chimera molecule includes a C lectin 2 having calcium ion-dependent glycosylation activity and a glycoconjugate 3 including sialo-glycans 3.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a virus concentrate, a concentrating device and a method for producing a virus concentrate. [Background technology]

[0002] Influenza viruses cause seasonal epidemics every year, resulting in significant socio-economic losses. There is also concern that the emergence of mutated influenza viruses may lead to a global pandemic in the near future.

[0003] Currently, the diagnosis of influenza virus infection (so-called influenza) is performed by immunochromatography using nasal swabs as specimens. The nasal swabs collected from subjects suffering from influenza contain high concentrations of influenza viruses (e.g., 10 4 ~10 5 Since the virus is present in the blood at high concentrations (approximately pfu / mL) and is easy to detect, nasal swabs are used to diagnose influenza.

[0004] However, in order to collect this nasal swab, a swab must be inserted deep into the subject's nasal cavity, rubbed against that area several times with the swab, and then removed from the cavity, which can often cause discomfort or severe pain to the subject, posing a problem.

[0005] Therefore, the use of gargling fluid has been attracting attention as an alternative specimen to nasal swabs, which does not cause pain to subjects when collected. However, the concentration of influenza virus in gargling fluid is up to 10 2 ~10 3pfu / mL, which is very low compared to nasal swabs. Specifically, the influenza virus concentration in gargling fluid is about 1 / 100 of that in nasal swabs. Therefore, attempts have been made to increase the virus concentration in gargling fluid by using a virus concentrator rather than using gargling fluid as a specimen as is (see, for example, Patent Document 1).

[0006] A conventional virus concentration material is, for example, a material in which serum mannan-binding protein (MBP) is supported on a carrier made of glass beads. It is known that the hemagglutinin present on the surface of influenza viruses is modified with high-mannose glycans. Conventional virus concentration materials capture influenza viruses by specifically binding serum mannan-binding protein to the high-mannose glycans. The binding site of serum mannan-binding protein is calcium ion-dependent, and by appropriately adjusting the calcium ion concentration, it is possible to break the bond between the serum mannan-binding protein of the virus concentration material and influenza viruses (high-mannose glycans), thereby liberating influenza from the virus concentration material. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] International Publication No. 2019 / 064463 Summary of the Invention [Problem to be solved by the invention]

[0008] If a virus that lacks the high-mannose glycan of hemagglutinin (e.g., a new mutated influenza virus) occurs in the future, conventional virus concentration materials will not be able to be used against that new influenza virus.

[0009] An object of the present invention is to provide a virus concentration material etc. that can be applied to viruses lacking the high-mannose type sugar chain of hemagglutinin. [Means for solving the problem]

[0010] <1> A chimeric molecule having a C-type lectin having calcium ion-dependent sugar chain binding activity and a sugar compound containing a sialo-sugar chain is supported on a carrier. the law of nature, The chimeric molecule has a structure derived from a linker having two succinylimide groups in the molecule, one of the succinyl imide groups reacts with the C-type lectin and the other succinyl imide group reacts with the sugar compound, thereby binding the C-type lectin to the sugar compound via the linker; a sugar ligand layer formed on the surface of the carrier and containing a sugar ligand that specifically binds to the binding site of the C-type lectin; The chimeric molecule is supported on the carrier by the binding site of the C-type lectin binding to the sugar ligand. Virus concentrate.

[0013] < 2 The method is applicable to a virus that specifically binds to the sialo-glycan as a receptor, and the test liquid in which the concentration of the virus is 10,000 pfu / mL or less is concentrated. > The viral concentrate described herein.

[0014] <3> The above <1> or < 2> a cylindrical container having an inlet at one open end, an outlet at the other open end, and a storage section disposed between the inlet and the outlet for storing the virus concentration material in a state in which a test liquid injected from the inlet side can pass through to the outlet side.

[0015] < 4A method for producing a virus concentration material in which a chimeric molecule having a C-type lectin having calcium ion-dependent glycan binding activity and a glycan compound containing a sialo-glycan is supported on a carrier, the method comprising the steps of: forming a glycan ligand layer on a surface of the carrier, the glycan ligand layer containing a glycan ligand that specifically binds to a binding site of the C-type lectin; contacting a C-type lectin-containing buffer solution obtained by dissolving the C-type lectin in a buffer solution containing calcium ions with the carrier on which the glycan ligand layer has been formed, thereby binding the C-type lectin to the glycan ligand, thereby forming a C-type lectin layer containing the C-type lectin on the glycan ligand layer; a linker layer forming step of contacting the carrier on which the C-type lectin layer has been formed with a linker-containing buffer solution prepared by dissolving a linker having two succinylimide groups in a buffer solution containing the linker, thereby binding the linker to the C-type lectin, thereby forming a linker layer containing the linker on the C-type lectin layer; and a sugar compound layer forming step of contacting the carrier on which the linker layer has been formed with a sugar compound-containing buffer solution prepared by dissolving the sugar compound containing the sialo-glycan in a buffer solution containing calcium ions, thereby binding the sugar compound to the linker, thereby forming a sugar compound layer containing the sugar compound on the linker layer. Effect of the Invention

[0016] According to the present invention, it is possible to provide a virus concentration material and the like that is applicable to viruses lacking the high-mannose type sugar chain of hemagglutinin. [Brief description of the drawings]

[0017] [Figure 1] Schematic cross-sectional view of the virus concentration material [Diagram 2] A schematic enlarged view of a portion of the virus concentrate [Diagram 3] An explanatory diagram showing a specific example of a linker [Figure 4] Flow diagram showing an example of a method for producing a virus concentrate [Diagram 5] FIG. 1 is a schematic diagram illustrating the cross-sectional configuration of a concentration device. [Figure 6] A schematic diagram of influenza viruses concentrated using a virus concentration material. [Figure 7] An explanatory diagram showing the linker used in the comparative example DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] [Virus concentrate] FIG. 1 is a cross-sectional view that shows a schematic configuration of a virus concentration material 1, and FIG. 2 is an enlarged view that shows a schematic view of a part of the virus concentration material 1. As shown in FIG.

[0019] The virus concentration material 1 is composed of a chimeric molecule 4 having a C-type lectin 2 with calcium ion-dependent glycan binding activity and a glycan compound 3 containing a sialo-glycan 3a, supported on a carrier 5. It is known that influenza viruses use sialo-glycans expressed on the cell surface as receptors and are adsorbed to the cell surface via the binding between hemagglutinin on the virus surface and the sialo-glycan.

[0020] In this specification, the term "concentration" refers to increasing the concentration of a target virus, and the target virus is present in a concentrated sample at a higher concentration than before concentration.

[0021] The carrier 5 is a support made of an insoluble material, and is preferably made of an inorganic or organic material having a hydroxyl group on the surface from the viewpoint of easiness of forming a sugar ligand layer described later. Specific materials constituting the carrier 5 are not particularly limited as long as the object of the present invention is not impaired, and examples thereof include glass, silica, various resins (styrene-based resins, acrylic-based resins, etc.), and hydroxyl-containing inorganic materials (ultraviolet-treated titanium oxide, alumina, other ceramics, etc.). The shape of the carrier 5 is not particularly limited as long as the object of the present invention is not impaired, and is preferably particulate (bead-like). The particle size of the particulate carrier determines the total surface area to which influenza viruses bind and the water permeability. That is, the smaller the particle size, the larger the total surface area, which is advantageous for influenza virus binding, but the smaller the particle size, the lower the water permeability, making the concentration operation difficult due to an increase in back pressure. The desired particle size is as small as possible while having sufficient water permeability, and is preferably, for example, 30 μm to 110 μm, and more preferably 30 μm to 60 μm. The average particle size is measured by a laser diffraction / scattering method (Microtrac) and the average particle size is determined by volume averaging. Beads (carriers) of as uniform a diameter as possible may be used so that the average particle size of the carrier 5 falls within the above-mentioned particle size range, or beads (carriers) of different particle sizes may be mixed so that the particle size distribution is intentionally bimodal.

[0022] An integral porous material formed by pressing a large number of particles together may be used as the carrier 5. This type of integral porous material is a porous material having both through holes and pores or only through holes, and is sometimes called a "monolith" or the like.

[0023] The chimeric molecule 4 is a compound having a C-type lectin (hereinafter simply referred to as "C-type lectin") 2 having calcium ion-dependent glycan binding activity and a sugar compound 3 containing a sialo-glycan 3a, and is supported on the surface of a carrier 5. In addition to the C-type lectin 2 and the sugar compound 3, the chimeric molecule 4 has a structure derived from a linker (bivalent functional linker) 6 having two succinylimide groups in the molecule. The chimeric molecule 4 of this embodiment is supported on the carrier 5 in a dissociable (separable) state via a sugar ligand 7 that binds to the carrier 5.

[0024] C-type lectin 2 is a protein having calcium ion-dependent glycan-binding activity and has one or more binding sites (sugar recognition domains) 2a. The binding site 2a of C-type lectin 2 has calcium ion-dependent glycan-binding activity and specifically binds to a saccharide ligand 7 described below. As such a C-type lectin 2, for example, mannan-binding protein (MBP) can be used.

[0025] MBP is a type of animal lectin that specifically binds to sugar chains having mannose, fucose, and N-acetylglucosamine at the terminus. In the living body, MBP is mainly present in serum and liver. Recombinant MBP can also be used as C-type lectin 2 (see, for example, Vorup-Jensen T et al., International Immunopharmacology 1, 677-687. (2001)).

[0026] MBP can be prepared from serum of animals such as rabbits and humans according to standard methods. Human-derived MBP can also be prepared as a recombinant form according to standard methods. For the sake of convenience, only one binding site of C-type lectin 2 is shown in Fig. 2.

[0027] The sugar compound 3 is a compound containing a sialo-glycan 3a. The sialo-glycan 3a is a sugar chain containing sialic acid 3a1, and is known as a substance to which hemagglutinin present on the surface of influenza viruses specifically binds as a receptor. The sugar compound 3 may be a chemically synthesized synthetic sialo-glycan or a substance derived from a natural product (for example, fetuin, a glycoprotein containing a sialo-glycan).

[0028] As a synthetic sialo-oligosaccharide, for example, Neu5Acα(2-6)Galβ(1-4)GlcNAc-β-ethylamine (Tokyo Chemical Industry Co., Ltd.) can be used.

[0029] The structure of the sialo-glycan 3a may be appropriately set depending on the type of target virus (influenza virus). For example, when the target is an avian influenza virus, a sialo-glycan in which sialic acid and galactose are bonded by α2-3 bond may be used.

[0030] Moreover, the sugar compound 3 has a functional group 3b capable of binding to the linker 6 described below. For example, in the case of a synthetic sialo-glycan, the aglycon portion has a functional group (such as an amino group) capable of reacting with the functional group of the linker 6 (succinylimide group).

[0031] The linker 6 is used to bind the C-type lectin 2 to the sugar compound 3 containing the sialo-glycan 3a when preparing the chimeric molecule 4. The linker 6 is made of a compound having two succinyl imide groups in the molecule. One succinyl imide group of the linker 6 reacts with a functional group such as an amino group of the C-type lectin 2, and the other succinyl imide group reacts with a functional group such as an amino group of the sugar compound 3, thereby binding the C-type lectin 2 to the sugar compound 3 via the linker 6. Therefore, a structure derived from the linker 6 is present in the chimeric molecule 4.

[0032] Examples of the linker 6 include BS3 (bis(sulfosuccinimidyl)suberate) and Sulfo-EGS (3,3'-[ethylenebis(oxycarbonyl)]bis[2,5-dioxo-3-(sodiosulfo)pyrrolidin-1-yl propionate]) shown in FIG.

[0033] The chimeric molecule 4 is supported on the carrier 5 by the binding site of the C-type lectin 2 binding to a sugar ligand 7 formed on the surface of the carrier 5. The sugar ligand 7 is a substance containing a sugar (mannose, etc.) to which the binding site of the C-type lectin 2 specifically binds, and for example, mannan (a polysaccharide whose main constituent unit is mannose) is used.

[0034] The method for solid-phasing (immobilizing) the sugar ligand 7 on the support 5 is not particularly limited as long as it does not impair the objectives of the present invention, and examples thereof include a solid-phasing method using a silane coupling method and an amine coupling method, and a method in which an amino group is first added using aminopropyltriethoxysilane, and then a succinylimide group is added using N,N'-disuccinimidyl carbonate (see, for example, Bioconjugate Technique, p. 542, etc.).

[0035] The binding of C-type lectin 2 to carbohydrate ligand 7 is mediated by calcium ions (Ca 2+ The binding between the C-type lectin 2 and the sugar ligand 7 in the virus concentration material is maintained in an environment in which, for example, 2 mM to 10 mM calcium ions are present. The calcium ions (Ca 2+ ) can dissociate the binding between C-type lectin 2 and carbohydrate ligand 7.

[0036] As shown in FIG. 1, when viewed macroscopically, the virus concentration material 1 comprises a saccharide ligand layer 70 consisting of saccharide ligands 7 formed on the surface of the carrier 5, a C-type lectin layer 20 consisting of C-type lectin 2 formed on the saccharide ligand layer 70, a linker layer 60 consisting of linker 6 formed on the C-type lectin layer 20, and a saccharide compound layer 30 consisting of saccharide compound 3 formed on the linker layer 60.

[0037] FIG. 2 shows a microscopic view of a portion of the virus concentration material 1, and illustrates the binding of hemagglutinin HA of influenza virus V to sialoglycan 3a (sialic acid 3a1).

[0038] [Method of manufacturing virus concentrate] Next, an example of a method for producing a virus concentrated material will be described. Fig. 4 is a flow diagram showing an example of a method for producing a virus concentrated material. As shown in Fig. 4, the method for producing a virus concentrated material includes a sugar ligand layer forming step S1, a C-type lectin layer forming step S2, a linker layer forming step S3, and a sugar compound layer forming step S4.

[0039] The sugar ligand layer formation step S1 is a step of forming a sugar ligand layer 70 containing a sugar ligand 7 that specifically binds to the binding site 2a of C-type lectin 2 on the surface of a carrier 5. In the sugar ligand layer formation step S1, a sugar ligand layer 70 made of mannan, which is the sugar ligand 7, is formed so as to cover the surface of the carrier 5 by a solid-phase method using, for example, a silane coupling method and an amine coupling method.

[0040] The C-type lectin layer forming step S2 is a step of contacting a C-type lectin-containing buffer solution obtained by dissolving C-type lectin 2 in a buffer solution containing calcium ions with the carrier 5 on which the sugar ligand layer 70 has been formed, and binding the C-type lectin 2 to the sugar ligand 7, thereby forming a C-type lectin layer 20 containing C-type lectin 2 on the sugar ligand layer 70. In the C-type lectin layer forming step S2, the binding site of C-type lectin 2 is bound to the sugar ligand 7 formed on the surface of the carrier 5, thereby forming the C-type lectin layer 20 on the sugar ligand layer 70.

[0041] The linker layer formation step S3 is a step of contacting a linker-containing buffer solution, obtained by dissolving a linker 6 having two succinylimide groups in the molecule in a buffer solution containing calcium ions, with the support 5 on which the C-type lectin layer 20 has been formed, to bind the linker 6 to the C-type lectin 2, thereby forming a linker layer 60 containing the linker 6 on the C-type lectin layer 20.

[0042] The sugar compound layer formation step S4 is a step of forming a sugar compound layer 30 containing the sugar compound 3 on the linker layer 60 by contacting a sugar compound-containing buffer solution, which is prepared by dissolving a sugar compound 3 containing a sialo-glycan 3a in a buffer solution containing calcium ions, with the support 5 on which the linker layer 60 is formed, and binding the sugar compound 3 to the linker 6.

[0043] The virus concentrated material 1 can be manufactured through the steps described above.

[0044] The steps from the C-type lectin layer forming step S2 onwards may be carried out sequentially in a state in which the carrier 5 is sealed in a predetermined container (for example). The container may be a cylindrical container having an inlet, an outlet, and a container section between the inlet and the outlet for containing a buffer solution such as a C-type lectin-containing buffer solution in a permeable (flowable) state. By carrying out the steps from the C-type lectin layer forming step S2 onwards in such a cylindrical container, the solid-phase reaction in each step can be carried out sequentially. As the cylindrical container, for example, one used in the concentrator 100 described later may be used.

[0045] [Concentrator] The virus concentration material 1 is used, for example, by filling it into a specific cylindrical container 200. In this specification, the cylindrical container 200 filled with the virus concentration material 1 is referred to as a concentrating device 100. Fig. 5 is a cross-sectional view that shows a schematic cross-sectional configuration of the concentrating device 100. As shown in Fig. 5, the concentrating device 100 includes the virus concentration material 1 and a cylindrical container 200.

[0046] The cylindrical container 200 has a shape in which two cylindrical parts are connected in the vertical direction. The cylindrical container 200 has a first cylindrical body (female luer cock connector) 210 arranged at the top and a second cylindrical body (male luer cock connector) 220 arranged at the bottom, and has a total length of about 27.5 mm.

[0047] The inlet 200a of the cylindrical container 200 is formed from the open end of the first cylindrical body 210, and the outlet 200b of the cylindrical container 200 is formed from the open end of the second cylindrical body 220. The cylindrical container 200 is provided with a storage section 200c that is disposed between the inlet 200a and the outlet 200b and that stores the virus concentration material 1 in a state in which the test liquid injected from the inlet 200a side can permeate to the outlet 200b side. The storage section 200c is formed inside the second cylindrical body 220. A disk-shaped frit (e.g., a polyethylene filter) 221 is disposed on the bottom side (the outlet 200b side) of the storage section 200c, and the portion surrounded by the frit 221 and the inner peripheral surface of the second cylindrical body 220 constitutes the storage section 200c. The virus concentration material 1 filled in the storage section 200c is pressed down by the cylindrical lower end of the first cylindrical body 210. The virus concentration material 1 is in the form of beads, and an aggregate of these beads is filled in the storage section 200c.

[0048] The inner diameter of the storage section 200c is set to, for example, about 4 mm to 8 mm, and the inner diameter of the outlet 200b is set to about 1 mm to 3 mm. In the case of this cylindrical container, about 0.1 mL to 0.5 mL of the virus concentration material 1 is enclosed in the storage section 200c.

[0049] [Method of concentrating test liquid using virus concentration material] Next, a method for concentrating a test liquid using the virus concentration material 1 will be described. Here, a method for concentrating viruses in a test liquid using a concentrating device 100 shown in Fig. 5 will be described. The virus concentration material 1 is applicable to influenza viruses and the like that specifically bind to sialo-glycan 3a as a receptor. The virus concentration material 1 can concentrate a test liquid having a virus concentration of, for example, 10,000 pfu / mL or less.

[0050] For example, the test liquid is a gargle liquid collected from a subject, and the test liquid is injected into the cylindrical container 200 through the injection port 200a using a syringe or the like, and the virus is captured by the virus concentration material 1. The test liquid injected through the injection port 200a passes through the virus concentration material 1 filled in the container 200c, and then through the frit 221, and is discharged to the outside through the outlet 200b. When the test liquid passes through the virus concentration material 1, the virus in the test liquid is captured by the sugar compound 3 having the sialo-glycan 3a. After the injection of the test liquid is completed, a buffer solution containing EDTA or the like is supplied into the cylindrical container 200 through the injection port 200a, which dissociates the bond between the hemagglutinin of the influenza virus and the sialo-glycan 3a of the sugar compound 3, and the influenza virus is eluted from the outlet 200b of the concentrator 100.

[0051] 6 is an explanatory diagram showing a schematic representation of influenza viruses V concentrated by the virus concentration material 1. A part of the virus concentration material 1 remains bound to the influenza viruses V captured by the virus concentration material 1 and dissociated from the carrier 5. Specifically, even after the bond between the C-type lectin 2 and the sugar ligand 7 of the virus concentration material 1 is dissociated, the chimeric molecule 4 remains bound to the influenza viruses V.

[0052] The virus concentration material 1 utilizes the mechanism by which influenza virus hemagglutinin binds to the sialoglycan 3a as a receptor, and can be used without any problems against a new influenza virus that lacks the high-mannose glycan of hemagglutinin.

[0053] Furthermore, when the chimeric molecule 4 is bound to influenza virus V, it is possible to gather influenza viruses V into clusters by appropriately adjusting the calcium ion concentration, so that mannose 9 present in the hemagglutinin HA of influenza virus V can be bound to the binding site 2a of the C-type lectin 2 of the chimeric molecule 4.

[0054] In addition, by appropriately adjusting the calcium ion concentration, it is possible to bind the C-type lectin 2 of the chimeric molecule 4 to a ligand (mannan) or the like immobilized on a substrate, thereby purifying influenza virus V.

[0055] In addition, since the calcium ion concentration is set low when influenza viruses V are recovered from the concentrating device 100, it is presumed that the influenza viruses V do not aggregate with each other in the recovered liquid (concentrated liquid) but remain individually suspended.

[0056] As described above, the virus concentration material 1 has high virus concentration performance and can concentrate influenza viruses and the like to high concentrations.

[0057] Furthermore, the virus concentration material 1 can be applied without any problems to new influenza viruses that lack the high-mannose type glycan of hemagglutinin.

[0058] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples in any way.

[0059] [Purification of rabbit MBP] An equal volume of buffer (imidazole: 40 mM, calcium chloride: 40 mM, sodium chloride: 2.5 M, pH = 7.8) was added to 500 mL of rabbit serum and mixed, and the mixture was applied to a mannan-Sepharose column equilibrated in binding buffer (imidazole: 20 mM, calcium chloride: 20 mM, sodium chloride: 1.25 M, pH = 7.8). After washing the column with binding buffer, the bound product (MBP) was eluted with dissociation buffer I (imidazole: 20 mM, EDTA: 20 mM, sodium chloride: 1.25 M, pH = 7.8). Among the eluted fractions, fractions with high protein concentrations were collected, calcium chloride was added to them, and the mixture was again applied to a mannan-Sepharose column. Washing and elution were performed in the same manner, calcium chloride was added to the eluate again, and the mixture was applied to a mannan-Sepharose column once more. The final purified product, MBP, was obtained by elution with dissociation buffer II (imidazole: 20 mM, mannose: 10 mM, calcium chloride: 20 mM, sodium chloride: 1.25 M, pH = 7.8).

[0060] [Preparation of mannan-glass beads] Washed glass beads were prepared, reacted with 3-aminopropylethoxysilane (APTES), and then washed with acetone. After washing with acetone, DSC (disuccinyl carbonate) was reacted, washed with acetone, and then dried. After drying, yeast mannan dissolved at a concentration of 100 μg / mL in Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4) was added and reacted. After the reaction, the glass beads were washed with Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4), and then the glass beads were treated with Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, Tris: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4) to block unreacted succinyl imide groups. The glass beads were then washed again with Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4), and furthermore, sulfo-NHS-Acetate (sulfosuccinimidyl acetate) dissolved in Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM) was allowed to act on the glass beads to block the amino groups contained in yeast mannan. In this way, glass beads to which yeast mannan was bound (mannan-glass beads) were obtained.

[0061] The amino groups of yeast mannan were blocked to prevent the bifunctional linker used later from reacting with sialo-glycans, which would bind directly to yeast mannan without MBP. If sialo-glycans were to bind directly to yeast mannan, influenza viruses bound to glass beads via sialo-glycans would not be eluted with EDTA, resulting in a significant decrease in concentration performance.

[0062] [Preparation of chimeric molecules] The purified rabbit serum MBP was dialyzed against Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4) to remove imidazole. Note that imidazole is a secondary amine and must be removed because it inhibits the next coupling reaction between the succinylimide group and the amino group. The rabbit serum MBP after dialysis was diluted with Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4) and the concentration was adjusted to 100 μg / mL to obtain a buffer (buffer containing C-type lectin). The buffer was contacted with mannan-glass beads, and the MBP (C-type lectin) in the buffer was bound to the mannan of the mannan-glass beads. Thereafter, the glass beads were washed with Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH=7.4) to remove unreacted MBP. Next, various bifunctional linkers (BS3 (see FIG. 3), Sulfo-EGS (see FIG. 3), Sulfo-SMCC (see FIG. 7)) were reacted with the glass beads to which MBP was bound. Each linker was reacted in a state dissolved in a buffer (Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH=7.4)) (linker-containing buffer). Furthermore, a sugar compound containing sialo-glycan dissolved in Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 20 mM, pH = 7.4) (i.e., sugar compound-containing buffer) was reacted. As the sugar compounds, sugar compound I (Neu5Acα(2-6)Galβ(1-4)GlcNAc-β-ethylamie) and sugar compound II (bovine serum fetuin) were prepared, and sugar compound I and sugar compound II were reacted with various bivalent functional linkers, respectively. In this way, six types of virus concentration materials in which chimeric molecules were bound to glass beads via yeast mannan (sugar ligand) were obtained.

[0063] [Evaluation 1: Evaluation of virus concentration performance] The virus concentration performance of the six obtained virus concentration materials was evaluated by the concentration experiment shown below. Influenza virus A / Memphis / 1 / 7 was grown in MDCK cells (growth liquid) and then aliquoted into 1 mL portions. The virus concentration was then determined by plaque assay, and was found to be 5.0 x 10 6 pfu / mL. In the concentration experiment, 20 mL of the virus liquid was used, which was diluted 500 times with Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, calcium chloride: 5 mM, pH = 7.4). Next, the virus concentration material was filled in a predetermined cartridge with a volume of 0.1 mL to prepare a virus concentration device (concentration apparatus). 20 mL of the virus liquid was injected from the inlet of this virus concentration device to bind the virus to the virus concentration material. Then, Hepes buffer (hydroxyethylpiperazineethanesulfonic acid: 20 mM, sodium chloride: 0.15 M, EDTA: 100 mM, pH = 7.4) was injected to cleave the binding portion between MBP and yeast mannan in the virus concentration material, and the virus captured in the virus concentration material was eluted. The concentration ratio was calculated from the virus concentration of the appropriate elution fraction (200 μL) and the virus concentration before concentration. The virus concentrations before and after concentration were measured by a known virus binding assay using a microtiter plate coated with fetuin, a glycoprotein having sialo-glycan, and an influenza virus-specific antibody. The results are shown in Table 1.

[0064] [Table 1]

[0065] As shown in Table 1, the virus concentration materials of Examples 1 to 4 use homobifunctional linkers BS3 and Sulfo-EGS as linkers. Such virus concentration materials of Examples 1 to 4 were shown to exhibit high virus concentration performance.

[0066] In contrast, the virus concentration materials of Comparative Examples 1 and 2 used a heterobifunctional linker, Sulfo-SMCC, as the linker. In Comparative Examples 1 and 2, chimeric molecules were not successfully produced (i.e., the binding between the sugar compound containing sialo-glycan and MBP was not successful), and the virus concentration performance of the obtained virus concentration materials was low.

[0067] [Evaluation 2: Evaluation of the volume of virus concentrate] To evaluate the difference in the volume of the virus concentration material, a virus concentration material similar to that of Example 3 described above was prepared. The obtained virus concentration material was filled into a predetermined cartridge in the volume (mL) shown in Table 2 to prepare the virus concentration devices (concentration apparatus) of Examples 5 to 7. Then, for Examples 5 to 7, the virus concentration performance was evaluated in the same manner as for Example 1 described above. The results are shown in Table 2.

[0068] [Table 2]

[0069] As shown in Table 2, the highest virus concentration performance was achieved when the volume of the virus concentrator was 0.5 mL. [Explanation of symbols]

[0070] 1...virus concentration material, 2...C-type lectin, 2a...binding site, 3...sugar compound, 3a1...sialic acid, 3a...sialoglycan, 3b...functional group, 4...chimeric molecule, 5...carrier, 6...linker, 7...sugar ligand, 20...C-type lectin layer, 30...sugar compound layer, 60...linker layer, 70...sugar ligand layer, V...influenza virus, HA...hemagglutinin

Claims

1. A chimeric molecule having a C-type lectin having calcium ion-dependent sugar chain binding activity and a sugar compound having a sialo-sugar chain is supported on a carrier, The chimeric molecule has a structure derived from a linker having two succinylimide groups in the molecule, one of the succinyl imide groups reacts with the C-type lectin and the other succinyl imide group reacts with the sugar compound, thereby binding the C-type lectin to the sugar compound via the linker; a sugar ligand layer formed on the surface of the carrier and containing a sugar ligand that specifically binds to a binding site of the C-type lectin; The chimeric molecule is a virus concentration material that is supported on the carrier by the binding site of the C-type lectin binding to the sugar ligand.

2. 2. The virus concentration material according to claim 1, which is applicable to a virus that specifically binds to the sialo-glycan as a receptor, and which concentrates a test liquid having a virus concentration of 10,000 pfu / mL or less.

3. A virus concentration material according to claim 1 or 2, A concentration device comprising a cylindrical container having an inlet at one open end, an outlet at the other open end, and a storage section disposed between the inlet and the outlet for storing the virus concentration material in a state in which the test liquid injected from the inlet side can pass through to the outlet side.

4. A method for producing a virus concentration material comprising a carrier carrying a chimeric molecule having a C-type lectin having calcium ion-dependent sugar chain binding activity and a sugar compound having a sialo-sugar chain, the method comprising the steps of: a sugar ligand layer forming step of forming a sugar ligand layer on the surface of the carrier, the sugar ligand layer including a sugar ligand that specifically binds to the binding site of the C-type lectin; a C-type lectin layer forming step of contacting a C-type lectin-containing buffer solution obtained by dissolving the C-type lectin in a buffer solution containing calcium ions with the carrier on which the sugar ligand layer has been formed, thereby binding the C-type lectin to the sugar ligand, thereby forming a C-type lectin layer containing the C-type lectin on the sugar ligand layer; a linker layer forming step of contacting a linker-containing buffer solution obtained by dissolving a linker having two succinylimide groups in a buffer solution containing calcium ions with the carrier on which the C-type lectin layer has been formed, thereby binding the linker to the C-type lectin, thereby forming a linker layer containing the linker on the C-type lectin layer; and a sugar compound layer formation step of contacting the carrier on which the linker layer is formed with a sugar compound-containing buffer solution obtained by dissolving the sugar compound containing the sialo-glycan in a buffer solution containing calcium ions, thereby binding the sugar compound to the linker, thereby forming a sugar compound layer containing the sugar compound on the linker layer.

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