JP2013159567A - Method of capturing virus by sulfated sugar chain immobilized gold nanoparticle and dextran sulfate immobilized gold nanoparticle, fractionating method, immunizing method, and method of producing antiviral antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel method of fractionating an antigen protein of a virus particle surface, which specifically binds to a sugar chain, by sugar chain immobilized gold nanoparticles, and directly using the antigen protein for production of an antiviral antibody.SOLUTION: Using gold nanoparticles, wherein a sulfated sugar chain having high virus binding properties is immobilized by using a uniquely developed linker compound, an antigen protein of a virus particle surface is fractionated using binding between the sugar chain and a virus antigen. In this way, an antibody is obtained by simply and efficiently using the antigen protein as an immunogen.

Description

  The present invention is a technique relating to a method for capturing an antigen protein on the surface of a virus particle by using gold nanoparticles having a sugar chain immobilized thereon and a method for producing an antibody against the captured antigen.

  Infections with pathogenic viruses cause many diseases. Examples of countermeasures against viral infection include administration of antiviral vaccines, antiviral synthetic compounds, and antiviral antibodies. Antiviral vaccines include live attenuated vaccines and inactivated vaccines. In the former, attenuated viruses themselves are administered into the body, and in the latter, inactivated viruses or virus fragment mixtures (split vaccines) are administered. Administration of these vaccines promotes antibody production in the body and prevents viral infection. Many of the antiviral synthetic compounds are not drugs that directly destroy viruses, but mainly function to suppress virus growth. Diseases are prevented by preventing the spread of the virus into the host cell, such as Tamiflu, which suppresses the release of virus particles from within the host cell, and Amantadine, which suppresses the cleavage of the virus particle within the host cell. On the other hand, antiviral antibodies are intended to suppress viral activity and activate host immunity by antibodies, and one currently on the market is palivizumab (synergis) against respiratory infectious RS virus. Although many antiviral antibodies have not yet been clinically applied at present, they are expected to occupy an important position in future countermeasures against viral infections (Non-patent Document 1).

  Advantages of antiviral antibodies include high specificity, high commonality of production / purification methods, and use as a diagnostic agent. Conventional methods for producing antiviral antibodies are: 1. Create an antibody using the virus particle itself as an antigen. Based on the steps of purifying a specific antigen from a virus using the antibody obtained by the above method and preparing an antibody against the specific antigen by a conventional animal immunization method or a phage display method. Therefore, as a disadvantage, the antigen recognized by the antibody becomes a constituent molecule of all virus particles, so the work of screening and purification of the antibody becomes enormous, making it possible to produce an antibody that can be used as an antiviral drug with neutralizing activity. Takes more time, effort, and cost. Simplification of procedures required for antibody production, reduction of time, and cost reduction are problems that should be addressed promptly in developing antiviral antibodies.

  Nanoparticles are materials applied in various fields such as life science, medical diagnosis, and biotechnology. Nanoparticles on which proteins such as antibodies are immobilized are useful for immunostaining by antigen-antibody reaction and for detection, separation and purification of antigens. In addition, drug delivery systems (DDS) using nanoparticles with immobilized drugs and genes, analytical markers using labeled nanoparticles, and analytical reagents such as tracers are also widely used. The nanoparticles used in the present invention are gold nanoparticles modified with a sugar chain, but the gold nanoparticles can be surface-treated with other metals, chemical compounds, peptides, lipids, enzymes, etc. by chemical modification. Is possible. High-functional materials using gold nanoparticles have been widely used in the field of biotechnology, and have been used for immunosensors and tumor therapy methods that detect nucleic acids, proteins, and sugar chains (Non-patent Documents) 2, 3). There is already known a method of immobilizing a sugar chain having high binding property to a virus on the surface of a gold nanoparticle and using it for capturing and concentrating the virus (Patent Document 1). Efficient protein fractionation and direct use in the production of antiviral antibodies has not yet been performed.

JP 2009-148246 A

Froude JW, MAbs, 3 (6), Epubahead of print, 2011 Daniel MC and Astruc D, Chemrev, 104, 294-346, 2004 Mitsunaga M, Nat Med, 17, 1685-1691, 2111

  The present invention was devised to solve the problems of the procedure, time, and cost required for the production of an antibody against a specific antigen when the virus particle itself was used as an antigen during the development of the above-described antiviral antibody. Is. Fractionation and fractionation of viral antigen protein that shows specific binding to sugar chains by gold nanoparticles with uniform particle size and high dispersibility, which can easily immobilize sugar chains on the particle surface An object of the present invention is to provide a novel method in which a complex of an antigen and nanoparticles is directly used for the production of an antiviral antibody.

  The sugar chain-immobilized gold nanoparticle according to the present invention is produced by adding a water-soluble sugar chain ligand complex treated with a reducing agent to the gold nanoparticle surface, and exhibits high dispersibility in an aqueous solution. The surface of the sugar chain-immobilized gold nanoparticles is preferably modified with a sugar chain ligand complex, and the average particle size is preferably 1 to 10 nm. Further, the sugar chain-immobilized gold nanoparticles according to the present invention bind to a sugar chain having a reducing end at the amino group present at one end of the above-mentioned sugar chain ligand complex, that is, a linker compound having a hydrocarbon-derived chain. In addition, the sugar chain-immobilized gold nanoparticles are bonded to the gold particles with a hydrocarbon structure containing sulfur atoms present at multiple ends of the linker compound.

  The sugar chain of the sugar chain-immobilized gold nanoparticles according to the present invention is a sulfated polysaccharide. In particular, dextran sulfate having a molecular weight of about 2500 is preferred. That is, dextran sulfate (DS25) whose molecular weight is adjusted to about 2500 out of dextran sulfate mainly substituted with sulfate groups at the 2nd, 3rd and 4th positions of dextran, which is a polysaccharide in which glucose is condensed by α1-6 bonds. Indicates. DS25 is highly negatively charged due to a large number of sulfate groups and exhibits strong binding to the surface of virus particles such as influenza and herpes. Thereby, the capture | acquisition of the virus through a sugar chain by a gold nanoparticle is enabled.

  Virus-captured DS25-immobilized gold nanoparticles (DS25-GNP) are particularly bound to proteins that can serve as antigens on the surface of the virus particles. Even if the virus is physically destroyed, DS25-GNP is bound to the viral antigen protein. This conjugate of DS25-GNP and antigen protein is fractionated. For fractionation, density gradient centrifugation using ultracentrifugation is used to separate the envelope protein / nanoparticle complex from other proteins by using the property that nanoparticles form a layer according to their weight. To do.

  By directly immunizing an animal with a conjugate of the virus antigen protein and DS25-GNP fractionated by density gradient centrifugation, production of an antibody specific to the virus antigen protein can be induced. This makes it possible to produce antiviral antibodies that use proteins on the surface layer of target virus particles as antigens, and antibodies that can also be used as drugs as neutralizing antibodies that block adsorption (binding) to cells during virus infection Can be manufactured. At this time, the target virus may be any type of virus as long as it is capable of binding to DS25-GNP. For example, various viruses such as influenza virus, dengue virus, herpes virus, and AIDS virus (HIV). Can be given. In addition, if a sugar chain that specifically binds to the virus is used, it can be applied to any sugar chain-binding virus in the same manner, such as Norovirus, adult T cell leukemia virus (HTLV-1), bovine diarrhea virus (BVDV). ).

  As described above, the sugar chain-immobilized gold nanoparticles used in the present invention can be prepared by simply mixing the reducing agent-treated sugar chain ligand complex and the gold nanoparticles. Thereby, it is possible to immobilize a sugar chain having a sulfate group, which has a high binding property to a virus, and it is possible to immobilize a large number of sugar chains, thereby providing an effect of providing high stability. In addition, fractionation of virus particle surface antigen protein using the sulfated sugar chain-immobilized gold nanoparticles can be performed in a short time and simply by performing physical disruption of the virus and density gradient centrifugation. The purification step can be simplified. This also makes it easy to produce an antibody that specifically recognizes the antigen protein on the surface of the virus particle.

An antibody is a glycoprotein produced by B cells in the body of vertebrates, including humans, and generally recognizes and binds to one epitope in a specific antigen. The action of antibodies that bind to pathogenic viruses includes opsonization that activates phagocytes, complement activation that activates complement, and neutralization that reduces infectivity and toxicity. It exhibits a protective function against diseases caused by viruses. In addition, antibodies are used not only as therapeutic agents but also for simple diagnosis of pathogenic viruses. By realizing a simple fraction of virus antigen protein according to the present invention, it is possible to reduce the time, labor, and cost required for antibody production and vaccine development.

Ligand complex containing dextran sulfate 2500 (DS25) and linker compound Transmission electron micrograph and average particle size of dextran sulfate-immobilized gold nanoparticles Dynamic light scattering measurements and average particle size of dextran sulfate immobilized gold nanoparticles Density gradient centrifugation of virus-sugar chain-immobilized gold nanoparticle complex Polyacrylamide electrophoresis diagram of each fraction separated by density gradient centrifugation Mass spectrometry of protein contained in fraction 2 Antibody titer in mouse serum when mice were immunized with virus-sugar chain-immobilized gold nanoparticle complex Antibody titer in culture supernatant of hybridoma in each screening Class and subclass of antibodies obtained from hybridoma culture supernatant Concentration-dependent antibody titer of purified antibody Confirmation of binding between virus antigen protein and purified antibody by immunoprecipitation Mass spectrometry of viral antigen proteins bound by antibodies

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this. Hereinafter, a method for producing sugar chain-immobilized gold nanoparticles, a method for fractionating virus antigens, a method for identifying virus antigens, and a method for identifying antibodies will be described in detail.

[1. Method for producing sugar chain-immobilized gold nanoparticles]
The present invention uses a method for producing gold nanoparticles having a sugar chain immobilized thereon. Gold nanoparticles produced by this method have the characteristic of being dispersed in an aqueous solution. That is, this production method is also a method for preparing a colloidal solution of sugar chain-immobilized gold nanoparticles.

  In the present specification, “sugar chain-immobilized gold nanoparticles” are those in which a sugar chain ligand complex, which will be described in detail below, and gold nanoparticles are bound. In the present specification, “nanoparticles” are intended to be dispersed in an aqueous solution to form a colloidal solution. Therefore, the average particle diameter of the nanoparticles is preferably in the range of 0.5 to 400 nm, and more preferably in the range of 1 to 10 nm. The average particle size may be less than 0.5 nm, but the production of such particles is expensive and impractical, and if it exceeds 400 nm, the dispersion stability of the particles tends to change over time, which is not preferable. . Moreover, since the capture efficiency is remarkably increased when nanoparticles having a size smaller than the target virus particle are used, nanoparticles having an average particle diameter of 1 to 10 nm are preferable as described later.

  The sugar chain ligand complex, which is a component of the sugar chain-immobilized gold nanoparticle according to the present invention, specifically includes a linker compound that can bind to any gold nanoparticle and a virus protein to be captured. It consists of sugar chains that can interact. Therefore, the sugar chain ligand complex is required not to form a non-specific interaction based on hydrophobicity with a substance such as a protein.

  The linker compound has a hydrocarbon chain or a hydrocarbon-derived chain in the main chain, and has an amino group at one end of the main chain. Due to the presence of this terminal amino group, a sugar chain molecule can be easily introduced. The amino group may be a modified amino group (for example, an amino group modified with an acetyl group, a methyl group, or a formyl group), an aromatic amino group, or an unmodified amino group. May be.

  The linker compound has a hydrocarbon structure containing an S—S bond or an SH group at the other end of the main chain in that a gold-sulfur (Au—S) bond can be easily formed. Yes. As a result, the linker compound can be assembled by arranging sugar chain molecules on the gold nanoparticles. Sulfur in the disulfide bond or SH group forms a metal-sulfur bond with Au in the gold nanoparticle, and can strengthen the bond with the metal.

  In the sugar chain ligand complex, which is a complex of the linker compound and sugar chain, a sugar chain having a reducing end is introduced in the amino group of the linker compound. That is, the sugar chain ligand complex has a structure in which the linker compound and a sugar chain having a reducing end are bonded via an amino group. This sugar chain can be introduced by a reductive amination reaction between the amino group of the linker compound and the sugar chain. That is, the aldehyde group or ketone group in the sugar chain generated by equilibrium reacts with the amino group of the linker compound. By subsequently reducing the Schiff base formed by this reaction, it is possible to easily introduce a sugar chain into the amino group.

  The “sugar chain having a reducing end” is a saccharide that can be formed by a reducing ketone or aldehyde at the molecular end, and particularly refers to dextran sulfate (DS25) having a molecular weight of about 2500. Dextran sulfate is a polyanionic dextran derivative mainly composed of a glucose skeleton linked by α1-6 bonds, and is synthesized from various high-purity dextran fractions. Among these dextran sulfates, DS25 adjusts the molecular weight to about 2500 to retain a large number of sulfate groups necessary for binding to viruses and to control the particle size of the product nanoparticles to around 10 nm. It is possible. The DS25 may be a commercially available or natural polysaccharide chain prepared by degradation.

  The ligand complex in which the linker compound and DS25 are bonded can be firmly and easily bonded to the gold nanoparticles by the sulfur of the SH group. For this reason, DS25 can be easily immobilized on the gold nanoparticle surface. Furthermore, the high hydration property of DS25 bonded on the gold nanoparticles in this way makes it possible to stabilize the gold nanoparticles in a colloidal state.

  The said linker compound is manufactured by the manufacturing method shown below, for example. That is, the linker compound is produced by performing a condensation reaction between thioctic acid and an aromatic amino group end.

By the condensation reaction between the thioctic acid and the aromatic amino group, the carboxyl group of the thioctic acid and the aromatic amino group are condensed to form an amide bond, whereby the linker compound can be obtained. That is, as the linker compound that can be used in the present invention, the general formula (1)
However, it is not limited to this. The above-mentioned ligand complex is obtained by introducing a sugar chain having a reducing end into the linker compound thus prepared.

  The sugar chain-immobilized gold nanoparticles according to the present invention can be obtained by mixing a solution containing gold nanoparticles with the above-described ligand complex treated with a reducing agent, and each S atom of the S—S bond of the ligand complex. Are bound by gold and gold-sulfur bonds on the nanoparticles. Specifically, for example, by adding a solution containing the DS25 ligand complex treated with a reducing agent to a solution of gold nanoparticles, the SS bond of the DS25 ligand complex is changed to a metal-sulfur bond on the nanoparticle. DS25-immobilized gold nanoparticles can be obtained.

  The reducing agent used for the reducing agent treatment is not particularly limited, and examples thereof include salts such as sodium borohydride and sodium cyanoborohydride and salts having different cation components.

  The solvent used in the solution containing the gold nanoparticles and the ligand complex is not particularly limited, and examples thereof include ultrapure water, methanol, ethanol, propanol, and mixed solvents thereof. In addition, sugar chain-immobilized gold nanoparticles that are stable in a solution state can be obtained by centrifugally filtering the sugar chain-immobilized gold nanoparticles obtained by the above mixing and removing components such as low-molecular salts.

  The mixing ratio of gold used for adjusting the sugar chain-immobilized gold nanoparticles, the ligand complex, and the reducing agent is not particularly limited, but the gold concentration in the solution is 0.1-1 mM in the final concentration. It is preferable that The concentration of the ligand complex is preferably 0.1-1 mM in the final concentration in the solution. Moreover, it is preferable that the density | concentration of the reducing agent used is 1-10 mM by the final concentration in a solution.

[2. Isolation method of virus antigen using sugar chain-immobilized gold nanoparticles]
In the virus isolation method using sugar chain-immobilized gold nanoparticles according to the present invention, the solution containing the sugar chain-immobilized gold nanoparticles interacts with the solution containing the virus particles, and then the conjugates are different in specific gravity. Fractionation is performed by density gradient centrifugation using a solution consisting of several layers.

  The above-mentioned “solution containing sugar chain-immobilized gold nanoparticles” is intended to be a colloidal solution in which sugar chain-immobilized gold nanoparticles according to the present invention are dispersed in a liquid. As long as the sugar chain-immobilized gold nanoparticles are included, other salts may be included. As the liquid, for example, ultrapure water or a buffer solution can be used.

  In addition, the above-mentioned “solution containing virus particles” is intended to be a colloidal solution in which viruses used for recovering virus antigens in the present invention are dispersed in a liquid. The virus is intended to be a virus that exhibits binding properties to the sugar chain ligand complex of the sugar chain-immobilized gold nanoparticles according to the present invention. The particle size of the virus particles is preferably 10-100 nm. As the liquid of the solution in which the virus particles are dispersed, ultrapure water, a buffer solution, or the like can be used as long as it does not contain a substance that may inhibit the binding between the sugar chain ligand complex and the virus particles. In addition, the concentration of virus particles in the solution is preferably 100 to 10,000 HAU (erythrocyte aggregation unit) in the case of a virus that induces hemagglutination.

  In addition, the above-mentioned “solution consisting of several layers having different specific gravities” is intended to be a solution formed of a multilayer solution in which several layers of sucrose solutions having different concentrations are stacked, but is not limited to this. is not. Moreover, said density gradient centrifugation is based on the centrifugation method using ultracentrifugation. The gravity by centrifugation is preferably around 30000 g. In addition, the above-mentioned fraction is intended to be separated in a solution after density gradient centrifugation of a conjugate of sugar chain-immobilized gold nanoparticles and virus particles. Moreover, said isolation intends to collect | recover the layer in which the sugar chain fixed gold nanoparticle is contained among the solution layers isolate | separated by density gradient centrifugation. Moreover, said refinement | purification intends carrying out centrifugal filtration of the solution layer collect | recovered after density gradient centrifugation, and removing low molecular components, such as sucrose contained in a solution layer.

  The fraction fractionated in the above step is defined as a nanoparticle-virus antigen conjugate.

[3. Method for producing antiviral antibody using nanoparticle-virus antigen conjugate]
The method for producing an antiviral antibody using the nanoparticle-virus antigen conjugate according to the present invention comprises immunizing an animal with the nanoparticle-virus antigen complex produced by the above-described process, whereby a specific virus antigen is produced in the animal body. And a step of recovering an antibody specifically recognizing a viral antigen using the immune reaction.

  Examples of animals used for inducing the immune response include mice, but are not limited thereto. In addition, the dose at the time of administering the nanoparticle-virus antigen complex to the animal is not particularly limited, but for intraperitoneal administration, 10-50 μg (* nano When the molecular weight of the particles is about 200,000, it is preferably 0.1-0.5 nmol per dose.

  During the period of administration of the above-mentioned nanoparticle-virus antigen complex, serum is collected from the administered animal 3 days after each administration day. For example, if the administered animal is a mouse, serum is collected by collecting blood from the tail. The collected serum is used for determination of the binding to the virus antigen contained in the nanoparticle-virus antigen conjugate used for administration, that is, for the measurement of the antibody titer. To collect. For confirmation of the antibody titer, measurement using Enzyme-Linked-Immunosorbent-Assay (ELISA) or the like is performed, but it is not limited thereto.

  Administration of the above-mentioned nanoparticle-virus antigen complex is carried out continuously every other week for 1 month to 2 months, and within 3 days from the final administration, the administered animal is euthanized and immune tissues such as the spleen Is preferably recovered. Antibody-producing cells such as plasma cells are collected from the tissue obtained from the administered animal and cell fusion with B lymphocyte osteosarcoma cells is performed. Although not particularly limited, the osteoblastoma cell used for cell fusion is preferably a cell line lacking its ability to produce antibodies. Cells formed by cell fusion are called hybridomas and are continuously cultured until antibody production is confirmed.

  During the period of preparing and culturing the hybridoma in the above steps, screening for selecting cells exhibiting a high antibody titer is performed using the cell culture supernatant. In this screening, evaluation by the aforementioned ELISA or the like is performed, but the present invention is not limited to this. Further, the cells showing a high antibody titer as a result of the evaluation are sorted to form single cells using a limiting dilution method or the like. Using the above-described evaluation method or the like, the cells that have become single after sorting are newly screened 2-3 times to select the cells.

  After performing the above cell selection, the resulting high antibody titer cell is defined as an antibody-producing cell and a cell for producing an antibody recognizing a viral antigen according to the present invention.

  The antibody recognizing the viral antigen according to the present invention is recovered using the culture supernatant of the antibody-producing cells. For fractionation at the time of recovery, magnetic beads or columns or the like to which Protein A protein or the like that binds to the antibody constant region is immobilized are used, but the invention is not limited to this. The fractionated antibody is a monoclonal antibody consisting of a single cell, and is an antibody that specifically recognizes the viral antigen according to the present invention.

  The present invention will be described more specifically based on examples, but the present invention is not limited to this. Preparation of glycan-immobilized gold nanoparticles suitable for virus capture, capture of influenza virus proteins by glycan-immobilized gold nanoparticles, immune induction by glycan-immobilized gold nanoparticles and virus antigen protein conjugate, glycan immobilization This will be described in four steps: production of antibodies against viral antigen proteins captured with gold nanoparticles.

[Preparation of sugar-immobilized gold nanoparticles suitable for virus capture]
26 ml of 20 mM sodium chloroaurate (manufactured by Nacalai Tesque) was mixed with 900 μl of 5 mM solution of sugar chain ligand complex (FIG. 1) prepared from dextran sulfate having an average molecular weight of 2500 and 3 ml of 50 mM sodium borohydride aqueous solution. . This mixed solution was stirred under light shielding to prepare a crude colloidal solution of sugar chain-immobilized gold nanoparticles. Place the solution in a dialysis membrane with a molecular weight of 3500 cut (Spectrumlab) and perform dialysis with phosphate buffer containing ultrapure water and 0.05% polyoxyethylene sorbitan monolaurate (Tween 20, Nacalai Tesque). The solution after dialysis was lyophilized. After drying, the powder containing gold nanoparticles was adjusted to 10 mg / ml, and the solution was ultracentrifuged at 100000 g, 30 min, 4 ° C. The solution after ultracentrifugation is divided into a precipitated fraction (a fraction containing large gold nanoparticles) and a non-precipitated fraction (a fraction containing small gold nanoparticles). DS25-GNP-large was used. The solution of the fraction that did not precipitate was further precipitated by ultracentrifugation under conditions of 300000 g, 50 min, 4 ° C., and this fraction was designated as DS25-GNP-small. The above DS25-GNP-large and DS25-GNP-small were suspended in ultrapure water to give 5 mg / ml. FIG. 2 shows transmission electron microscope images of DS25-GNP-large and DS25-GNP-small and average particle diameters. FIG. 3 shows the average particle diameter when these nanoparticles are measured by the dynamic light scattering method.

[Capture influenza virus protein by sugar chain-immobilized gold nanoparticles]
Viral particles were captured using a 5 mg / ml solution of the sugar chain-immobilized gold nanoparticles DS25-GNP-large and DS25-GNP-small. As a virus, a solution (15.4 HAU / μl) in which a New-Caledonia strain of influenza A virus (A / New-Caledonia / 20/1999 / H1N1) was suspended in a phosphate buffer was used. 25 μl of the sugar chain-immobilized gold nanoparticle solution and 25 μl of the influenza virus solution were mixed, and the mixture was stirred overnight at 4 ° C. with light shielding and allowed to interact. The same volume of 6% Triton X-100 (manufactured by Nacalai Tesque) was added to the interacted solution, and the mixture was crushed by ultrasonic waves.

  A solution containing disrupted influenza virus and sugar chain-immobilized gold nanoparticles is placed on a sucrose solution adjusted to 20/35/40/50%, and sucrose density gradient by ultracentrifugation at 30000 g, 90 min, 15 ° C. Centrifugation was performed. As shown in FIG. 4, as a result of density gradient centrifugation, a converged nanoparticle layer was formed in the virus solution containing DS25-GNP-small.

After density gradient centrifugation, the solution containing influenza virus is divided into four fractions, each of which is subjected to removal of low molecules and concentration of the solution using a 10 kD (kilo Dalton) ultrafiltration centrifugal filter (Millipore). It was. The solution concentrated to 50 μl was developed on an acrylamide gel by sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE). This developed gel was stained with Coomassie Brilliant Blue staining solution (Nacalai Tesque) or silver staining solution (Wako Pure Chemicals), and the electrophoresis pattern of each fraction was detected (FIG. 5).

  Among the stained parts obtained by the above electrophoresis pattern, a gel containing a protein in the vicinity of 60 kD (arrowhead in the figure) of fraction 2 is collected, and the protein in the gel is digested using the proteolytic enzyme trypsin (Promega). It was. The peptide obtained by digesting the protein in the gel was desalted and purified with a hydrophobic column (Zip-tip C16, manufactured by Millipore). The obtained peptide was subjected to mass spectrometry using an ion trap matrix-assisted laser ionization-time-of-flight mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Corporation). Data obtained by mass spectrometry was searched with a protein sequence identification database MASCOT to identify proteins. The mass peak obtained by mass spectrometry and the results of protein identification are shown in FIG. As a result of data analysis, the influenza-derived protein captured by DS25-GNP-small was mainly hemagglutinin, a virus particle surface protein.

  From the above, the sugar chain-immobilized gold nanoparticles DS25-GNP-small captures the virus via hemagglutinin, which is an antigenic protein on the surface of influenza virus, and efficiently separates hemagglutinin using DS25-GNP-small. I understood that I can draw.

[Immune induction by glycan-immobilized gold nanoparticles and virus antigen protein conjugate]
A fraction (HA-GNP fraction) containing a conjugate of the above sugar chain-immobilized gold nanoparticles DS25-GNP-small and influenza virus-derived hemagglutinin was used as an immunizing antigen. For the immunization experiment, mouse Balb / cNsea (6 weeks old, male) was used, no adjuvant was used, and antigen administration was performed by intraperitoneal administration. The antigen was converted to DS25-GNP-small amount and administered at 30 μg per dose. On the other hand, administration using only phosphate buffer or DS25-GNP-small as an antigen as a target experiment was performed on another mouse. The number of animals in each treatment group was 4 animals. Antigen was administered every other week, for a total of 4 administrations. Three days after the 2nd to 4th administration except the first time, blood was collected from the tip of the tail of the mouse, and serum was collected.

  The antibody titer was measured by ELISA using each collected serum to determine the presence or absence of antibodies. At this time, as an antigen of ELISA, a protein solution obtained by crushing influenza virus by Triton-X100 and ultrasonic treatment was used, and prepared to be 1 HAU per well of a 96-well plate for measurement. In addition, an ELISA using only DS25-GNP-small as an antigen was performed as a target experiment, and prepared to be 5 μg per well of a 96-well plate for measurement. The results of measuring the antibody titer of each serum during the administration period by ELISA are shown in FIG. In the measurement using the influenza virus crushing protein as the antigen of ELISA, the antibody titer of the group administered with the HA-GNP fraction increased after the fourth administration (FIG. 7a). On the other hand, in the measurement using only DS25-GNP-small as an antigen for ELISA, no increase in antibody titer was observed in any group (FIG. 7b).

  From the above, it was found that HA-GNP induced immunity against influenza virus in mice. On the other hand, it was found that DS25-GNP-small alone does not induce an immune response.

[Production of antibody against viral antigen protein bound with sugar chain-immobilized gold nanoparticles]
In Example 3, spleen tissue was collected from the mice of the group administered with the HA-GNP fraction. This group is a group in which the antibody titer against influenza virus disruption protein increased after the fourth antigen administration. Spleen cells were isolated from the collected spleen tissue, and cell fusion with B lymphocyte osteomyeloma cells (myeloma cells) was performed. As myeloma cells, the strain SP2 / 0-AG14 having no antibody production ability was used. The fused cells were cultured in a 96-well culture plate and sequentially screened by ELISA (FIG. 8).

  First, when cells in each well formed colonies, an ELISA was performed using influenza virus disrupted protein as an antigen (screening 1). The result is shown in FIG. 8a. Next, cells with antibody titers exceeding an arbitrary threshold in screening 1 were collected, and cells were divided by limiting dilution. In this method, the cell density in the solution is reduced by dilution, and only one cell is added per well. When each cell formed a colony again, ELISA with the same antigen as described above was performed (screening 2). The result is shown in FIG. Ten additional cell colonies were selected from the cells whose antibody titers exceeded an arbitrary threshold value in screening 2, and used as antibody-producing hybridomas for antibody recovery.

  The antibody was collected using the culture supernatant of the antibody-producing hybridoma. First, immunochromatography was performed using the culture supernatant to determine the isotype of the monoclonal antibody. At this time, a mouse monoclonal antibody isotype determination kit (Sigma) was used. As a result, the obtained antibody was found to belong to the immunoglobulin gamma 2b subclass and the kappa class (IgG2b-kappa) (FIG. 9). Next, affinity chromatography was performed using the culture supernatant to purify the monoclonal antibody. For purification, a Protein A column (Thermochemical) was used. The purified antibody was subjected to ultrafiltration (10 kD) to remove low molecules and exchange buffers. When the purified monoclonal antibody was used to carry out ELISA using the influenza virus crushing protein as an antigen, a concentration-dependent increase in antibody titer was observed (FIG. 10).

  The purified monoclonal antibody was subjected to an immunoprecipitation experiment in order to confirm the binding property to the antigen. After binding the antibody to Protein A-bound magnetic beads (Invitrogen), the solution was mixed in a solution containing a disrupted protein of influenza virus and allowed to act, and then the antigen-antibody complex was recovered by magnetic separation and separated by SDS-PAGE. did. As a result of examining the protein by silver staining, an antigenic protein was found at a position of about 60 kD (FIG. 11). The gel of the antigen protein portion was collected, and the protein in the gel was digested using the protease protease trypsin (Promega). The peptide obtained by digesting the protein in the gel was desalted and purified with a hydrophobic column (Zip-tip C18, manufactured by Millipore). The obtained peptide was subjected to mass spectrometry using an ion trap type matrix-assisted laser ionization-time-of-flight mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Corporation). Data obtained by mass spectrometry was searched with a protein sequence identification database Mascot to identify proteins. The mass peak obtained by mass spectrometry and the results of protein identification are shown in FIG. As a result of data analysis, the antigen protein of influenza virus to which the monoclonal antibody was bound was hemagglutinin. From the above, it was revealed that the antibody obtained by immunizing the sugar chain-immobilized gold nanoparticle-influenza virus antigen protein complex is an antibody that binds to hemagglutinin, which is a protein on the surface layer of influenza virus particles. .

Claims (7)

Carbohydrate immobilization in which the amino group present at one end of a linker compound having a hydrocarbon-derived chain is bonded to a sulfated sugar having a reducing end and the sulfur atom present at the other end of the linker compound is bonded to gold. Metallized nanoparticles. 2. The sulfated saccharide is dextran sulfate mainly substituted with sulfate groups at the 2nd, 3rd and 4th positions of dextran, which is a polysaccharide in which glucose is condensed with an α1-6 bond. Sugar chain-immobilized gold nanoparticles. The sugar chain-immobilized gold nanoparticles according to claim 2, wherein the dextran sulfate has a molecular weight of about 2500. A virus capturing method comprising a step of capturing a virus by binding the sugar chain-immobilized gold nanoparticles according to any one of claims 1 to 3 to a protein on the surface of the virus particle. The fractionation method which has the process of crushing the virus capture | acquired by the virus capture method of Claim 4, and fractionating the protein of the virus particle surface couple | bonded with the sugar chain fixed gold nanoparticle. An immunization method comprising a step of using a conjugate of a virus protein and a sugar chain-immobilized gold nanoparticle fractionated by the fractionation method according to claim 5 as an animal immunizing antigen. An antiviral antibody production method comprising a step of producing an antibody that recognizes an antigen protein on the surface of a virus particle from an animal immunized by the immunization method of claim 6.