WO2012144554A1 - Method and kit for assessing virus concentration - Google Patents

Abstract

The purpose of the present invention is to provide a method for capturing a virus from a solution containing the virus in a simple manner without causing clogging in a process of capturing or concentrating the virus from the solution containing the virus, and assessing the quantity of the captured virus accurately and in a simple manner. As a means for achieving the purposes, provided is a method for assessing the concentration of a virus in a solution containing the virus by bringing the solution containing the virus into contact with a surface-treated hollow yarn or allowing the solution to pass through the hollow yarn to capture the virus and then calculating the quantity of the captured virus.

Description

Method and kit for evaluating virus concentration

The present invention relates to a method for evaluating the concentration of virus contained in a liquid by capturing the virus through a liquid containing a virus through or in contact with a surface-treated hollow fiber, and calculating the amount of the captured virus, and The present invention relates to a virus concentration evaluation kit having a function of evaluating the virus concentration by a method for evaluating the virus concentration.

When sewage, river water, tap water, etc. are contaminated by various viruses, the risk of harming the health of neighboring residents increases. In particular, viral contamination of living water in developing regions is a major threat to health problems for residents. Moreover, in public facilities such as a pool used by an unspecified large number of people, when the water is contaminated with an orally infectious virus, the virus infection may spread at a stretch. In view of these, it is very important to constantly monitor the virus contamination in the water environment in order to maintain the safety and security of people's lives. To that end, simple and accurate viral load assessment technology is essential. is there.

To date, as a method for evaluating the amount of virus, for example, concentration of environmental water containing virus is carried out by negative charge membrane method (mixed membrane of nitrocellulose and cellulose acetate, flat membrane cartridge). There is known a method for detecting the above (Non-patent Document 1). In that case, secondary concentration may be further performed using an ultrafiltration membrane as necessary. Although this method has a high virus recovery rate, depending on the origin of the environmental water, the membrane may become clogged during concentration, which is problematic.

Also, Patent Document 1 describes an apparatus for detecting an analyte in a biological sample or an analog thereof, including a solid support provided with several adjacent zones.

Patent Document 2 describes an apparatus, a method, and a kit for detecting an analyte in a biological sample. An analyte that requires extraction from a biological sample before detection can be performed in one stage. It is described that it can provide a means to be extracted and detected.

Patent Document 3 describes a method of recovering a surface sample (including microorganisms and other analytes) by wiping within a certain range of the surface by a predetermined method using a recovery device means made of an absorbent or an adsorbent. Yes.

In addition, Patent Document 4 describes a virus detection method in which a test sample that can contain a virus is brought into contact with a virus-separating cell, and then in situ hybridization is performed on the obtained cell. As test samples, nasal discharge, nasal wipe, ocular conjunctival wipe, pharyngeal wipe, sputum, stool, blood (serum, plasma), spinal fluid, saliva, urine, sweat, semen or tissue are described.

Special table 2009-518636 JP-T 9-501494 JP-T-2002-514916 JP 2003-024077 A

The conventional technology has a problem of causing problems such as clogging in the process of capturing or concentrating the virus from the liquid containing the virus.
Therefore, an object of the present invention is to easily capture a virus from a solution containing virus without causing clogging in the step of capturing or concentrating the virus from the solution containing virus, and to accurately and easily determine the amount of virus captured. It is to provide a way to evaluate.

In order to solve the above problems, the present inventors have studied a method for capturing a virus and a method for evaluating the supplemented virus, and have completed the present invention.

That is, the present invention captures a virus by bringing a liquid containing a virus into contact with or passes through a surface-treated hollow fiber, and calculates the amount of the captured virus, thereby calculating the concentration of the virus contained in the liquid. On how to evaluate

ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to evaluate accurately and simply the method of capture | acquiring a virus simply from the liquid containing a virus, and the amount of captured viruses.

That is, the present invention
1. In the liquid containing the virus by capturing the virus through the liquid containing the virus through or in contact with the hollow fiber surface-treated with a compound having a charge or a compound having no charge, and calculating the amount of the captured virus A method for evaluating the concentration of viruses contained in
2.1. In the method for evaluating the virus concentration described in
1) First step of collecting a certain amount of liquid (liquid B) from the liquid containing liquid (liquid A) 2) The liquid B is passed through or brought into contact with the hollow fiber to capture the virus in the hollow fiber. Two steps 3) a method for evaluating the concentration of virus, comprising each step of the third step of calculating the amount of virus in the B liquid trapped in the hollow fiber,
3. 1. The hollow fiber is composed of polyolefin, polyethersulfone, or cellulose mixed ester. Or 2. A method for evaluating the concentration of the virus described in
4). The hollow fiber is a porous hollow fiber; ~ 3. A method for evaluating the concentration of the virus according to any one of
5. 2. The polyolefin is polyethylene, polypropylene or poly-4-methylpentene Or 4. A method for evaluating the concentration of the virus described in
6). The compound having a charge is a (meth) acrylate, a sulfonate, a compound having an ammonium group, a compound having a sulfonium group, a compound having a carboxylate group, or a compound having a phosphate group. ~ 5. A method for evaluating the concentration of the virus according to any one of
7. The compound having no charge is (meth) acrylic acid, sulfonic acid, a compound having an amino group, a compound having a sulfo group, a compound having a carboxy group, or a compound having a phospho group. ~ 5. A method for evaluating the concentration of the virus according to any one of
8). 5. A sulfonium group or a compound having a sulfo group is a sulfated sugar. Or 7. A method for evaluating the concentration of the virus described in
9. Sulfated sugar is heparin, a heparin derivative in which the primary or secondary hydroxyl group of heparin is sulfated, a heparin derivative in which an N-acetyl group-elimination product of heparin is N-sulfate-esterified, dextran sulfate, or fucoidan 8. A method for evaluating the concentration of the virus described in
10. The method for calculating the amount of virus is by polymerase chain reaction, antigen-antibody reaction, or culture method. ~ 9. A method for evaluating the concentration of the virus according to any one of
11. 1. The virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, adenovirus, enterovirus, norovirus, influenza virus, human immunodeficiency virus, rotavirus, or poliovirus. ~ 10. A method for evaluating the concentration of the virus according to any one of
12 3) In the third step of calculating the amount of virus in the B liquid trapped in the hollow fiber,
1. a step of recovering the virus from the hollow fiber capturing the virus with a phenol aqueous solution, an alkaline aqueous solution, or an inorganic salt aqueous solution; ~ 11. A method for evaluating the concentration of the virus described in
13.1. ~ 12. A virus concentration evaluation kit having a function of evaluating the virus concentration by the method according to any one of
14.1. ~ 12. A hollow fiber for capturing a virus, to which a compound having an amino group or a compound having an ammonium group is used, which is used in the method for evaluating the virus concentration according to any one of
15. 13. The compound having an amino group or the compound having an ammonium group is a compound selected from polyethyleneimine, polyallylamine, amino acids, and salts thereof. The hollow fiber for capturing a virus according to claim 1,
16.14. Or 15. In the hollow fiber for capturing a virus to which an amino group-containing compound or an ammonium group-containing compound is immobilized,
A compound having an amino group or a compound having an ammonium group is immobilized on a hollow fiber surface-treated with a polymer material having a hydroxyl group via a compound having a group capable of reacting with a hydroxyl group and an amino group or an ammonium group. A hollow fiber for capturing a virus,
17. A hollow fiber surface-treated with a polymer material having a hydroxyl group is an ethylene-vinyl alcohol copolymer, an ethylene-vinyl alcohol-vinyl acetate copolymer, a partially saponified product of an ethylene-vinyl acetate copolymer, or a vinyl alcohol-acetic acid. 15. A hollow fiber surface-treated with a vinyl copolymer, a hydroxymethacrylate copolymer, a cellulose acetate partial saponified product, or a glycerin derivative. ~ 16. A hollow fiber for capturing a virus according to any one of
18. 15. The compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group is epichlorohydrin or a diepoxy compound. Or 17. The hollow fiber for capturing a virus according to claim 1,
19.14. Or 15. In the hollow fiber for capturing a virus to which an amino group-containing compound or an ammonium group-containing compound is immobilized,
A virus-capturing hollow fiber in which a compound having an amino group or a compound having an ammonium group is immobilized on a hollow fiber by a graft polymerization reaction,
20.14. Or 15. In the hollow fiber for capturing a virus to which an amino group-containing compound or an ammonium group-containing compound is immobilized,
A virus-capturing hollow fiber in which a compound having an amino group or a compound having an ammonium group is immobilized by surface-treating the hollow fiber with a compound having an amino group or a compound having an ammonium group,
21. 15. The hollow fiber is a porous hollow fiber. ~ 20. A hollow fiber for capturing a virus according to any one of
22. The hollow fiber is based on polyethylene, polypropylene or poly-4-methylpentene.14. 21. A hollow fiber for capturing a virus according to any one of
23. 13. Immobilization amount of a compound having an amino group or a compound having an ammonium group is in the range of 1 to 100 μg / cm ² . ~ 22. A hollow fiber for capturing a virus according to any one of
24. 13. The virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, adenovirus, enterovirus, norovirus, influenza virus, human immunodeficiency virus, rotavirus, or poliovirus. 23. Or a virus-capturing hollow fiber according to any one of the above.

Hereinafter, the surface treatment of the hollow fiber performed in the present invention will be described.
-Hollow fiber The hollow fiber used in the present invention can be used without limitation as long as it captures the virus targeted in the present invention.
As a base material constituting the hollow fiber, from the viewpoint of capturing viruses, olefin resin, styrene resin, sulfone resin, acrylic resin, urethane resin, ester resin, ether resin, cellulose mixed ester, etc. More specifically, examples include polyethylene terephthalate, ethylene vinyl alcohol copolymer, polymethyl methacrylate, polysulfone, polyether sulfone, polyacrylonitrile, polyethylene, polypropylene, and poly-4-methylpentene.
The hollow fiber that can be used in the present invention is not particularly limited, but a porous hollow fiber is preferable in that it can efficiently trap viruses. In view of reducing clogging, a non-filtration type hollow fiber having a skin layer on the outer wall surface while the hollow fiber inner wall surface is porous may be used. The said porous hollow fiber can be manufactured by well-known and usual manufacturing methods, such as an extending | stretching method.

・ Compound used for surface treatment The compound used for the surface treatment of the hollow fiber is not particularly limited as long as it has the ability to trap viruses, and it may be either a charged compound or a non-charged compound. May be. Examples of such compounds include the following. Examples of those having a charge include (meth) acrylates, sulfonates, compounds having an ammonium group, compounds having a sulfonium group, compounds having a carboxylate group, and compounds having a phosphate group. Examples of those having no charge include (meth) acrylic acid, sulfonic acid, compounds having an amino group, compounds having a sulfo group, compounds having a carboxy group, and compounds having a phospho group. Examples of the compound having an amino group or the compound having an ammonium group include compounds selected from polyethyleneimine, polyallylamine, amino acids, and salts thereof. Regarding amino acids, those in which a salt is formed in the molecule are also included in the present invention.

The sulfonium group or the compound having a sulfo group may be a sulfated sugar. More specifically, heparin, a heparin derivative in which a primary or secondary hydroxyl group of heparin is sulfated, and deacetylation of the N-acetyl group of heparin. It may be a heparin derivative obtained by N-sulfate esterification, dextran sulfate, or fucoidan.

-Surface treatment method As a method of performing the surface treatment of the hollow fiber with the above-mentioned compound, a publicly known and commonly used method can be used. Examples of such a method include graft polymerization with a radical polymerizable compound and sulfonation with concentrated sulfuric acid, and the like. Examples thereof include a method of reacting a compound capable of reacting with the graft chain after graft polymerization.

The hollow fiber can also be surface-treated with a polymer material having a hydroxyl group. In that case, a compound having an amino group or a compound having an ammonium group may also be immobilized via a compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group by utilizing a hydroxyl group. Is possible.

Hereinafter, each case will be described in detail.
.. Treatment of a compound having a radical polymerizable group by graft polymerization A treatment method by a graft polymerization reaction with a compound having a radical polymerizable group by irradiating with ionizing radiation can be carried out as follows.
For example, an ethylenically unsaturated group of a compound having a radical polymerizable group such as (meth) acrylic acid is graft-polymerized to the hollow fiber by radicals generated by irradiating the hollow fiber with ionizing radiation. Examples of the ionizing radiation source used in the graft polymerization include known and commonly used α-rays, β-rays, γ-rays, accelerated electron beams, X-rays and the like, and practically preferred are γ-rays and accelerated electron beams.

The graft polymerization method includes a simultaneous irradiation graft polymerization method in which the hollow fiber is brought into contact with a compound having a radical polymerizable group and irradiated with ionizing radiation, and the hollow fiber is preliminarily irradiated with a compound having a radical polymerizable group. Any pre-irradiation graft polymerization method can be used and can be selected according to the purpose.

In the graft polymerization method using ionizing radiation used in the present invention, the optimum irradiation dose and accelerating voltage differ depending on the hollow fiber, so the range cannot be determined unconditionally, and it is adjusted as appropriate considering the material and thickness of the hollow fiber. It is necessary to. For example, when the irradiation amount is large, dielectric breakdown due to charging occurs, and when the irradiation amount is small, the polymerization reaction does not proceed. For this reason, in consideration of the material and form of the hollow fiber, the amount of irradiation that does not cause dielectric breakdown due to charging and the polymerization reaction proceeds sufficiently may be adjusted as appropriate. Further, the acceleration voltage is related to the permeability and varies depending on the thickness of the hollow fiber. When the thickness is small, the acceleration voltage is generally small, and when it is thick, it needs to be large.

For example, when the hollow fiber is made of poly-4-methylpentene and has a thickness of 10 μm to 100 μm, it may be 10 kGy or more, 300 kGy or less, more preferably 90 kGy or less, and the acceleration voltage may be appropriately selected. it can.
In addition, even when the hollow fiber is made of polyethylene or polypropylene, graft polymerization can be similarly performed.

The radicals in the hollow fiber base material after irradiation with the ionizing radiation are quickly inactivated by temperature rise and contact with oxygen. Therefore, after irradiation, it is preferable to store at a low temperature in a state where oxygen is sufficiently removed, and to carry out the graft polymerization reaction promptly. For the above reasons, it is desirable to carry out the reaction under deoxygenation or in an inert gas. The hollow fiber after the graft polymerization reaction may be removed from the compound having an unreacted radical polymerizable group by various methods such as washing with a solvent.

The introduction of the compound having a radical polymerizable group into the hollow fiber can be performed by selecting either the inner surface or the outer surface of the hollow fiber, or both, depending on the embodiment. For example, when the virus is captured by passing through the inner surface of the hollow fiber, the compound may be introduced into the inner surface of the hollow fiber. Conversely, when used by passing through the outer surface, the compound is introduced into the outer surface of the hollow fiber. Just keep it. As the hollow fiber base material, a polymer compound having a methylene group in the main chain is particularly preferred because radicals are easily generated by irradiation with ionizing radiation.
The compound introduced into the hollow fiber by the graft polymerization reaction can be converted into a salt by a known and usual method. Such salts are not particularly limited as long as they can capture viruses, but salts formed with metal ions such as sodium, potassium, calcium and magnesium, trialkylamines, aromatic amines And salts formed as preferred salts.

.. Introduction of sulfonic acid or sulfonate by sulfonation The sulfonation of the hollow fiber of the present invention can be carried out by a generally known method using a sulfonating agent such as concentrated sulfuric acid or fuming sulfuric acid. The obtained sulfonic acid group can be converted to a sulfonate by a known and commonly used method. Such salts are not particularly limited as long as they can capture viruses, but salts formed with metal ions such as sodium, potassium, calcium, and magnesium salts of sulfonic acids, trialkyls Examples of preferred salts include amines and salts formed with aromatic amines.

.. Introduction of compound having amino group or ammonium group In the present invention, the introduction method of the amino group is not limited, but a preferable introduction method is a radical polymerizable group capable of graft polymerization and a group capable of reacting with the amino group. Examples thereof include a method of introducing a compound (I) having an amino group into a hollow fiber and then reacting with a compound (II) having an amino group or ammonia. The compound (II) having an amino group may have a functional group such as a hydroxyl group in addition to the amino group.

Preferred compounds as the compound (I) having a radically polymerizable group capable of graft polymerization and a group capable of reacting with an amino group include epoxy groups such as (meth) acrylic acid and glycidyl (meth) acrylate (meta And (meth) acrylate having an isocyanate group such as acrylate and (meth) acryloyloxyalkyl isocyanate.
Preferred examples of the compound (II) having an amino group include a hydroxyalkylamine derivative in which a hydroxyl group or an amino group may have a protecting group, or at least two in a molecule in which an amino group may have a protecting group. 2-aminoethanol, ethylenediamine, butylenediamine, hexamethylenediamine, 1,2-bis (2-aminoethoxy), which is an amine derivative having an amino group of which hydroxyl group or amino group may have a protecting group Examples include ethane, polyethyleneimine, polyallylamine, and the like.
Moreover, ammonia can also be mentioned as a compound that reacts with the compound (I) having a radical polymerizable group and a group capable of reacting with an amino group.
Examples of the protecting group used include, but are not limited to, a group protecting with a carbamate bond, a group protecting with an ester bond, a group protecting with an amide bond, or a group protecting with an ether bond.

Examples of the group protected by a carbamate bond include t-butyl carbamate (Boc group) and benzyl carbamate. Examples of the group protected by an ester bond include an acetyl group and a benzoyl group. Examples of the protecting group include an acetylamide group and a benzoylamide group, and examples of the group to be protected by an ether bond include a methoxymethyl ether group and a benzyl ether group. These groups can be appropriately selected depending on the type and conditions of the reaction performed with the compound having a radical polymerizable group.

When (meth) acrylic acid is used as the compound (I) having a radical polymerizable group capable of graft polymerization and a group capable of reacting with an amino group, the reaction with the compound (II) having an amino group is amidation. Just do the law. Amidation reaction methods include, for example, amidation with an active ester, amidation with a condensing agent, a combination thereof, a mixed acid anhydride method, an azide method, a redox method, a DPPA method, a Woodward method, peptide synthesis, etc. What is necessary is just to perform the well-known and usual amidation reaction used suitably.

Examples of amidation with an active ester include NHS (N-hydroxysuccinimide), nitrophenol, pentafluorophenol, DMAP (4-dimethylaminopyridine), HOBT (1-hydroxybenzotriazole), and HOAT (hydroxyazabenzotriazole). And the like to form an active ester obtained by once condensing a group having a high leaving ability with a carboxy group, and reacting this with an amino group. Amidation with a condensing agent may be used alone or in combination with the active ester. As the condensing agent, EDC (1- (3-dimethylaminopropyl-3-ethyl-carbodiimide hydrochloride), HONB (endo-N-hydroxy-5-norbornene-2,3-dicarboxamide), DCC (dicyclohexylcarbodiimide) , BOP (benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate), HBTU (O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium hexafluorophosphate) TBTU (O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyluronium tetrafluoroborate), HOBt (1-hydroxybenzotriazole), HOOBt (3,4-dihydro-3- Hydroxy-4-oxo- , 2,3-benzotriazine), di-p-trioylcarbodiimide, DIC (diisopropylcarbodiimide), BDP (1-benzotriazole diethyl phosphate-1-cyclohexyl-3- (2-morpholinylethyl) carbodiimide), fluorine Cyanuric chloride, cyanuric chloride, TFFH (tetramethylfluoroformamidinium hexafluorophosphophosphate), DPPA (diphenylphosphoradidate), TSTU (O- (N-succinimidyl) -N, N, N ′, N′-tetramethyl Uronium tetrafluoroborate), HATU (N-[(dimethylamino) -1-H-1,2,3-triazolo [4,5,6] -pyridin-1-ylmethylene] -N-methylmethanaminium Hexafluorophosphate / N-oxide) BOP-Cl (bis (2-oxo-3-oxazolidinyl) phosphine chloride), PyBOP ((1-H-1,2,3-benzotriazol-1-yloxy) -tris (pyrrolidino) phosphonium tetrafluorophosphate), BrOP (bromotris (dimethylamino) phosphonium hexafluorophosphate), DEPBT (3- (diethoxyphosphoryloxy) -1,2,3-benzotriazin-4 (3H) -one), PyBrOP (bromotris (pyrrolidino) phosphonium Hexafluorophosphate) and the like.

Among these, a method in which the carboxy group of (meth) acrylic acid is once NHS and then reacted with the amino group of the compound (II) having an amino group for amidation is preferred.

As a solvent that can be used in these amidation methods, water and an organic solvent used for peptide synthesis can be used. For example, dimethylformamide (DMF), dimethyl sulfoxide (DMSO), hexaphosphoroamide, dioxane, tetrahydrofuran ( THF), ethyl acetate, and the like, and mixed solvents and aqueous solutions containing these.

The proportion of the activated ester residue introduced into the carboxy group depends on the type of activator used and the amount of reagent used. In general, compared to the reaction in solution, the polymer obtained from the polymerizable compound is bonded, and the reactivity is lowered. Therefore, in order to increase the introduction amount, it is necessary to use a considerably excessive amount of the reaction reagent. It is thought that there is. Therefore, the ratio of the activator to the condensing agent reaction reagent relative to the carboxy group of (meth) acrylic acid polymerized on the hollow fiber cannot be defined unconditionally, but the active ester group is introduced into the carboxyl group in an equal amount. Is preferably about 1 to 10 in molar ratio. Furthermore, by adjusting the excess amount of the reaction reagent, it is possible to arbitrarily adjust the introduction ratio of the active ester group in the range of 0.01 to 100%.

When glycidyl (meth) acrylate is used as the compound (I) having a radical polymerizable group and a group capable of reacting with an amino group, the hydroxyl group or amino group of the compound (II) having an amino group, and a glycidyl group Can be bound by reacting with.
Examples of the reaction conditions include a solvent used in the reaction of a normal hydroxyl group or amino group and a glycidyl group, a reaction temperature, and a reaction time. As an example of preferable conditions, when the compound having a hydroxyl group or an amino group is a solution, it may be used directly. When a solvent is used, a solvent that does not dissolve or swell the hollow fiber, more preferably a hollow A solvent capable of dissolving a polymer of a compound having a radical polymerizable group without dissolving and swelling the yarn can be used, and the concentration of the compound having a hydroxyl group or an amino group can be arbitrarily selected. Can be used by adjusting to a concentration of 1 to 50%.

The reaction temperature can be selected from the temperature at which the compound (II) having an amino group and the solvent do not vaporize, and is preferably 0 to 70 ° C., more preferably 20 to 60 ° C. Although the reaction time varies depending on the type, concentration, and reaction temperature of the compound (II) having an amino group, the content of the compound (II) having an amino group in the reaction solution can be determined by gas chromatography, liquid chromatography, titration, etc. Monitoring is preferably performed until the compound (II) having an amino group is not consumed. Generally, the reaction can be completed in 30 minutes to 12 hours, but is not limited thereto, and reaction conditions can be appropriately selected and set.

When (meth) acryloyloxyalkyl isocyanate is used as the compound (I) having a radical polymerizable group and a group capable of reacting with an amino group, the hydroxyl group or amino group of the compound (II) having an amino group; It can couple | bond by making it react with the isocyanate group which the compound which has a radically polymerizable group has.
Examples of the reaction conditions include a solvent used in a reaction between a normal hydroxyl group or amino group and an isocyanate group, a reaction temperature, and a reaction time. As an example of preferable conditions, the solvent can be appropriately selected from solvents such as ethyl acetate and acetonitrile that do not dissolve and swell the hollow fiber and do not react with isocyanate. The reaction concentration can be arbitrarily selected. Specifically, the reaction concentration can be selected from 0.5 to 15% by weight. The reaction temperature is preferably 0 to 50 ° C. because the reaction with isocyanate is an exothermic reaction. The reaction time can be 2 to 24 hours, but is not limited thereto, and reaction conditions can be set by appropriately selecting.

The amino group introduced by the above method can be converted to an ammonium salt by a known and conventional method.
A preferable ammonium salt is not particularly limited as long as it has a function of capturing a virus, but inorganic salts such as hydrochloride, sulfate, phosphate, formate, acetate, oxalate, succinate, benzoic acid Organic salts such as salts can be mentioned.

After surface treatment of the hollow fiber with a polymer material having a hydroxyl group, a compound having an amino group or a compound having an ammonium group is immobilized via a compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group. The method includes the following.

Examples of the polymer material having a hydroxyl group that can be used in the present invention include an ethylene-vinyl alcohol copolymer, an ethylene-vinyl alcohol-vinyl acetate copolymer, a partially saponified product of ethylene-vinyl acetate copolymer, vinyl, and the like. Examples include those containing a vinyl alcohol copolymer such as an alcohol-vinyl acetate copolymer, those containing a hydroxymethacrylate copolymer, partially saponified cellulose acetate, or glycerin derivatives. However, in addition to those exemplified, there is no particular limitation as long as the resin has a hydroxyl group.

The surface treatment with the polymer material having a hydroxyl group can be carried out by a known and usual method. For example, the porous polyolefin is dipped in a solution in which the polymer material having a hydroxyl group is dissolved, pulled up and then dried. Can be mentioned as a preferred method. Among these, an ethylene-vinyl alcohol copolymer is preferable in that the polyolefin porous hollow fiber can be easily hydrophilized as disclosed in JP-A-61-271003. Alternatively, it is also possible to use a method of making a porous mixture of polyolefin or the like and a polymer material having a hydroxyl group in advance.

The compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group used in the present invention has a group capable of reacting with a hydroxyl group present on the surface of the surface-treated hollow fiber, and the amino group after immobilization. Alternatively, there is no particular limitation as long as it has a functional group that easily reacts with an ammonium group. For example, compounds such as epichlorohydrin, carboxylic anhydride, dicarboxylic acid, dicarboxylic acid chloride, diisocyanate, diepoxy compound and the like can be mentioned. Examples of the compound that can be graft-polymerized on the substrate include (meth) acrylic acid, glycidyl (meth) acrylate, (meth) acryloyloxyalkyl isocyanate, and maleic anhydride.

When the hydroxyl group present on the surface of the hollow fiber is reacted with a hydroxyl group and a compound having a group capable of reacting with an amino group or an ammonium group and immobilized, for example, a reaction between a known and commonly used hydroxyl group and an epoxy group, The reaction can be carried out under conditions such as a reaction with a carboxy group, a reaction between a hydroxyl group and a carboxylic acid anhydride, a reaction between a hydroxyl group and an isocyanate.
When a compound having an ethylenically unsaturated group is graft polymerized, (meth) acrylic acid, glycidyl (meth) acrylate, (meta ) It is possible to immobilize acryloyloxyalkyl isocyanate or maleic anhydride.
Examples of the method for generating radicals include a method using a reagent such as diammonium cerium nitrate which is widely used as a radical polymerization initiator for a polymer material having a hydroxyl group such as cellulose or polyvinyl alcohol.

In addition, when fixing the compound which has a group which can react with a hydroxyl group and an amino group or an ammonium group, it can fix to either the inner surface of a thread | yarn, an outer surface, or both according to embodiment. For example, when blood is perfused on the inner surface of the hollow fiber, a compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group may be contacted and immobilized on the inner surface of the hollow fiber. Sometimes, a compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group may be brought into contact with the outer surface of the hollow fiber. When it is desired to immobilize on the inner and outer surfaces including inside the pores of the yarn, the yarn may be bundled and immersed in the reaction solution, or the reaction solution may be circulated after assembling as a module.

The compound having an amino group or an ammonium group used in the present invention is not particularly limited as long as it can react with a compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group. Examples of such compounds include polyallylamine, ammonia, 2-aminoethanol, ethylenediamine, butylenediamine, hexamethylenediamine, 1,2-bis (2-aminoethoxy) ethane, 3,3′-diaminodipropylamine. And amines such as diethylenetriamine, phenylenediamine or polyethyleneimine, or ammonium salts thereof.

The reaction of a compound having an amino group or an ammonium group with a hydroxyl group and a compound having a group capable of reacting with an amino group or an ammonium group is a reaction of a known and commonly used amino group or ammonium group with an epoxy group, an amino group or an ammonium group. It can be suitably carried out under the conditions used for the reaction between the carboxy group and the carboxyl group, the reaction between the amino group or ammonium group and the carboxylic acid anhydride, and the reaction between the amino group or ammonium group and the isocyanate.

.. Introduction of Sulfated Sugar Sulfated sugar can be used as the compound having a sulfonium group or the compound having a sulfo group of the present invention. As the sulfated saccharide used in the present invention, heparin, a heparin derivative in which primary or secondary hydroxyl groups of heparin are sulfated, and an acetyl group-eliminated product of N-acetyl group of heparin are converted to N-sulfate. Examples include heparin derivatives, dextran sulfate, sulfonium groups such as fucoidan, and sulfated sugars having a sulfo group.
As the heparin used in the present invention, generally known heparins can be used without limitation. Heparin is a kind of heparan sulfate that is widely present in the body such as small intestine, muscles, lungs, spleen and mast cells, and is chemically a glycosaminoglycan, β-D-glucuronic acid or α-L-iduron. It is a polymer in which an acid and D-glucosamine are polymerized by 1,4-bonds, and has a feature that the degree of sulfation is particularly high compared to heparan sulfate. The average molecular weight of heparin is not particularly limited, but when the average molecular weight is large, the reactivity with a compound having a hydroxyl group or an amino group is lowered, so that the efficiency of immobilizing heparin is considered to be poor. Accordingly, the molecular weight of heparin is preferably about 500 to 500,000 daltons, more preferably 1,200 to 50,000 daltons, and even more preferably 5,000 to 20,000 daltons.

Examples of sulfated sugars that can be used in addition to heparin include heparin derivatives in which the primary or secondary hydroxyl group of heparin is sulfated or heparin in which an acetyl group-elimination product of the N-acetyl group of the heparin is N-sulfated. Derivatives are exemplified. The sulfuric esterification can be usually performed by a known method.

When synthesizing a heparin derivative in which a primary or secondary hydroxyl group of heparin is sulfated, for example, heparin amine salt is obtained by treating an alkali salt of heparin with an ion exchange resin (H ⁺ ) or the like and treating with an amine. Adjust. Thereafter, it can be treated with a sulfating agent to obtain the desired heparin derivative. As the sulfating agent, known and commonly used SO ₃ • pyridine is preferable.

In the case of synthesizing a heparin derivative obtained by N-sulfate esterification of an acetyl group-eliminated heparin N-acetyl group, for example, after deacetylating the heparin N-acetyl group with hydrazine or the like, To obtain the desired heparin derivative. As the sulfating agent, known and commonly used SO ₃ .NMe _{3 and the} like are preferable.
Moreover, a well-known and usual thing can be used for dextran sulfate or fucoidan.

Here, the compound (I) having a radical polymerizable group and a group capable of reacting with an amino group is subjected to a graft polymerization reaction, and then reacted with a compound (II) having an amino group or ammonia to fix sulfated sugar. Alternatively, the compound (I) having a group capable of reacting with a radical polymerizable group and an amino group and the compound (II) having an amino group or ammonia are reacted, and then a graft polymerization reaction is carried out, followed by sulfation. Sugar may be immobilized. The coupling | bonding of the sulfated sugar of this invention and the compound (II) which has an amino group can be performed by the said amidation reaction, for example.

The average pore size of the hollow fiber used in the present invention is not particularly limited as long as it has a pore size capable of efficiently removing viruses. Considering the efficiency of capturing the virus, it varies depending on the type of virus to be captured and can be appropriately selected and used. For example, the average flow pore size is preferably in the range of 50 to 500 nm.
The inner diameter of the porous hollow fiber used is not particularly limited as long as it has an inner diameter capable of efficiently removing viruses. When the inner diameter is large, the number of yarns that can be put into the module is reduced, so that the contact area may be reduced, and the test substance (environmental water or the like) may be retained due to inferior linear velocity. On the other hand, if the inner diameter becomes too small, the blood cell component is likely to be clogged. Considering these points, the inner diameter is preferably 150 to 500 μm, more preferably 160 to 400 μm, and further preferably 170 to 350 μm.

The thickness of the porous hollow fiber used is not particularly limited as long as it has a thickness that can efficiently remove the virus. Preferable film thickness is 30 to 100 μm, more preferably 35 to 80 μm, and further preferably 40 to 60 μm.
The amount of the compound used for the surface treatment of the hollow fiber of the present invention is not particularly limited as long as it is an amount capable of efficiently removing viruses. Considering the efficiency of capturing the virus, it varies depending on the type of virus to be captured and can be appropriately selected and used. For example, in the case of a hollow fiber based on polyolefin, 1 × The case of 10 ⁻⁶ to 1 × 10 ⁻² mol / g can be mentioned.

The water vapor adsorption amount of the compound used for the surface treatment of the hollow fiber of the present invention is not particularly limited as long as it is an amount capable of efficiently removing viruses. Considering the efficiency of capturing the virus, it varies depending on the type of virus to be captured and can be appropriately selected and used, but a preferable water vapor adsorption amount is 1 to 100 mL / g. Here, the water vapor adsorption amount can be measured by a publicly known and commonly used method.

An example is shown below.
50 mg of the pulverized hollow fiber sample was precisely weighed and placed in a test tube and dried under reduced pressure at room temperature for 5 hours. Then, using a gas adsorption amount measuring device (BELSORP-max, manufactured by Nippon Bell Co., Ltd.), the adsorption isotherm using a water vapor molecule as a probe is measured, and the water vapor adsorption amount at a relative pressure of 0.5 is compared. The hydrophilicity of the sample was evaluated.

-Recovery of trapped virus The present invention is characterized in that the virus trapped in the hollow fiber is recovered, the amount is calculated, and the amount of virus contained in the liquid containing the original virus is evaluated. Have.

The method for recovering the virus is not particularly limited as long as it can recover the virus efficiently, but the hollow fiber is washed with a phenol aqueous solution, an alkaline aqueous solution, or an inorganic salt aqueous solution (inorganic salt aqueous solution). Is particularly preferred.

The aqueous phenol solution is a mixed solution of phenol and water, and the content of phenol contained in the solution can be appropriately adjusted and used, but phenol / water = 4/1 to 2/3 (mass conversion) Can be mentioned as a preferred mass ratio.

In addition, the alkaline aqueous solution can be used without limitation as long as it is an alkaline aqueous solution. However, since the virus recovery rate is good, an alkali metal hydroxide such as sodium hydroxide or potassium hydroxide is used. And carbonates such as sodium carbonate and sodium hydrogen carbonate. Examples of the inorganic salt aqueous solution include a sodium chloride aqueous solution.

The method for recovering the virus can be carried out by washing the hollow fiber in which the virus is trapped, either alone or in combination with an aqueous organic polymer solution such as phenol aqueous solution, alkaline aqueous solution, beef extract, or inorganic salt aqueous solution. . The washing method is not particularly limited as long as the virus can be eluted from the hollow fiber. Preferably, the washing method can be carried out by immersion of the hollow fiber or by passing the solution in a hollow fiber module.

Hereinafter, the kit and the evaluation method of the present invention will be described.
-Kit form The form of the evaluation kit comprising the hollow fiber of the present invention is not particularly limited as long as it is a shape applicable to the above-mentioned use, and examples thereof include a hollow fiber module. In the hollow fiber module, the shape and material of the container are not particularly limited, and may be any convenient size, shape, and material according to the amount and properties of the liquid containing the virus to be used.

-Virus to be evaluated The virus targeted by the present invention is a virus that can be captured by the hollow fiber of the present invention. Examples of such viruses include hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, adenovirus, enterovirus, norovirus, influenza virus, human immunodeficiency virus, rotavirus, and poliovirus. Can be mentioned.

-Evaluation target liquid Examples of the liquid containing virus targeted in the present invention include environmental water that can contain virus, body fluid that is a human body fluid component, culture liquid containing virus, and water for daily use. More specific examples of body fluids include blood, saliva, sweat, urine, runny nose, semen, plasma, lymph, tissue fluid and the like. Specific examples of environmental water include river water, lake water, groundwater, seawater, and the like. Examples of water for daily use include tap water, well water, hot springs, and pool water.

Evaluation method The concentration of the virus of the present invention is evaluated by each of the following: “collecting a solution containing virus” → “capturing virus” → (“freeing virus” → “removing unwanted substances” →) “calculating the amount of virus” It goes through the process.
The virus can be captured by passing a solution containing the virus through the surface-treated hollow fiber or by bringing it into contact. In this step, clogging can be reduced by using a non-filtration-type hollow fiber as the hollow fiber, or by suppressing the pressure at the time of liquid passing or contacting.
After capturing the virus, a step of releasing the virus from the hollow fiber may be added prior to calculating the amount of virus. Examples of the releasing step include a method of treating with a salt, acid, alkali, surfactant or the like. Further, a step of removing the substance added in the release step may be added thereafter.

The method for calculating the amount of virus is not particularly limited as long as it is a method capable of detecting the above virus. Preferred methods include polymerase chain reaction (PCR) method, antigen-antibody reaction method (envelope protein detection method, core protein detection). Method), culture methods and the like. Among these, the PCR method is a principle for amplifying a nucleic acid or a technique using the same, and has the following excellent features.
1) It is possible to selectively amplify only a specific nucleic acid fragment (several tens to thousands of base pairs) desired from a very long nucleic acid molecule and achieve the object with a very small amount of nucleic acid solution. .
2) The time required for amplification is as short as about 2 hours.
3) The process is simple and can be amplified by a fully automatic desktop device.

More specifically, a certain amount of liquid (liquid B) is collected from the virus-containing liquid (liquid A), and the virus in the liquid B is captured by the method shown in the examples below, and the capture rate (X %). Thereafter, the captured virus is collected by the method shown in the following examples, and the recovery rate (Y%) is calculated.
Furthermore, in order to calculate the concentration of virus in the solution A (for example, virus copy number / L), for example, calculation may be performed using the following equation.
Here, the dilution rate indicates the dilution rate when the B solution is prepared from the A solution.

Virus concentration in solution A (number / L) = calculated number of virus copies in solution B (number / L) / {recovery rate (%) × capture rate (%)} × dilution rate

Here, the virus copy number represents the amount of virus in the specimen measured and calculated by the PCR method.

The following examples illustrate the invention in more detail.

(Preparation Example 1) <Production of heparin surface-treated hollow fiber module>
A test tube containing 20 mL of acetone, 16 mL of epichlorohydrin, and 4 mL of 40% NaOH aqueous solution, and a filtration-type hollow fiber (about 13 cm, hydrophilized with ethylene / vinyl alcohol copolymer (hereinafter referred to as EVOH) therein. About 150 pieces (351 mg) were immersed. The reaction was carried out at 30 to 40 ° C. for 5 hours while applying ultrasonic waves. After completion of the reaction, the reaction product was washed with acetone and water and vacuum dried to obtain a hollow fiber having an epoxy group introduced (354 mg).

A hollow fiber having an epoxy group introduced into 28% ammonia water was immersed and reacted at 40 ° C. for 2 hours. After completion of the reaction, it was washed with water to obtain a hollow fiber having a primary amino group introduced.

Heparin 150 mg and sodium cyanoborohydride 20 mg were put into a test tube, dissolved in 30 mL of PBS, the hollow fiber was immersed and reacted at 40 ° C. for 3 days. After completion of the reaction, it was washed with water. 26 mL of 0.2 M AcONa aqueous solution was added to the hollow fiber, and 13 mL of acetic anhydride was slowly added dropwise while cooling with ice. Then, it was made to react with an ultrasonic wave for 30 minutes under ice cooling. Furthermore, it was made to react for 30 minutes, returning to room temperature. After completion of the reaction, the mixture was washed with 20% NaCl, 0.1 M NaHCO ₃ aqueous solution, water, and PBS to obtain a heparin surface-treated hollow fiber.

Using this heparin surface-treated hollow fiber (13 cm × 3), a small sample module (inner diameter 3 mm, outer diameter 5 mm, length 7.4 mm) was produced.

(Example 1) <Capture of hepatitis B virus-like particles>
For a small sample prepared in Preparation Example 2 with 80 mL of 0.002% BSA-PBS containing hepatitis B virus-like particles (hepatitis B virus surface antigen HBsAg-XT, manufactured by Vehicle Co., Ltd.) at a concentration of 0.1 ng / mL The solution was passed through the module, and a filtrate fraction of 42 mL and a flow-through fraction of 38 mL were collected. The hepatitis B virus-like particle concentration of each fraction was measured using a microwell ELISA HBsAg for hepatitis B surface antigen (manufactured by HOPE Laboratory). As a result, it was confirmed that 57% of hepatitis B virus-like particles were trapped in the hollow fiber in the module.

(Preparation Example 2) <Production of polyacrylic acid surface-treated hollow fiber module>
1 g of acrylic acid monomer was dissolved in 100 g of water to make a 1% by mass aqueous solution, and the dissolved oxygen in the aqueous solution was removed by reducing the pressure while stirring.

On the other hand, an unfiltered hollow fiber bundle (hollow fiber surface area: 200 cm ² , manufactured by DIC Corporation) made of poly-4-methylpentene (hereinafter referred to as PMP) is placed in a glass test tube and sealed with a rubber stopper. The inside of the test tube was replaced with nitrogen. Thereafter, an electron beam of 90 kGy was irradiated at an acceleration energy of 4.8 MeV with an electron beam irradiation apparatus “DYNAMITRON 5 MeV-150 kW” manufactured by RDI.

Next, the inside of the test tube containing the hollow fiber bundle irradiated with the electron beam was evacuated, and the deoxygenated acrylic acid monomer aqueous solution was added under 23 ° C. to start graft polymerization. After standing for 4 hours, the hollow fiber bundle was taken out, washed repeatedly with water until unreacted monomers and the like were below the detection limit (1 μg / mL) by GPC measurement, and then dried in vacuo. In the obtained polyacrylic acid surface-treated hollow fiber, the amount of polyacrylic acid bonded is 25 mg, and the bond density (in terms of monomer) is 1.7 × 10 ⁻⁶ mol / cm ² (1.0 × 10 ⁻³ mol / g). Met.

(Preparation Example 3) <Preparation of polyethyleneimine surface-treated hollow fiber module>
Hollow fiber bundle made of PMP (hollow fiber surface area: 200 cm ^2, DIC (Ltd.)) was placed in a glass test tube, sealed with a rubber stopper, the interior tube after nitrogen substitution, RDI Co. Electronic The electron beam of 90 kGy was irradiated with the acceleration energy of 4.8 MeV with the line irradiation apparatus "Dynamitron 5MeV-150kW".

Next, a deoxygenated 50 mg / mL glycidyl methacrylate (hereinafter referred to as GMA) methanol solution was poured into the test tube, and allowed to react at 23 ° C. for a predetermined time. The hollow fiber bundle was taken out, washed with methanol, and vacuum dried to obtain a polyGMA surface-treated hollow fiber. The amount of polyGMA introduced calculated from the weight increase of the hollow fiber was 0.190 mg per 1 cm ² of the hollow fiber.
The poly-GMA surface-treated hollow fiber is immersed in a 30% by mass polyethyleneimine (molecular weight 600, manufactured by Wako Pure Chemical Industries) acetonitrile solution, heated at 60 ° C. for 3 hours, washed with methanol, and vacuum-dried, and the surface-treated hollow fiber Got. The amount of polyethyleneimine introduced was 8.58 μg (8.5 × 10 ⁻⁶ mol / g) per 1 cm ^{2 of} hollow fiber.

(Preparation Example 4) <Production of polyethyleneimine (molecular weight 600) surface-treated hollow fiber>
On the other hand, a hollow fiber bundle made of PMP (hollow fiber surface area: 200 cm ² , manufactured by DIC Corporation) was placed in a glass test tube, sealed with a rubber stopper, and the inside of the test tube was purged with nitrogen. Thereafter, an electron beam of 90 kGy was irradiated with an acceleration energy of 4.8 MeV while cooling with dry ice.

Next, the inside of the hollow fiber bundle test tube irradiated with the electron beam was evacuated, and deoxygenated 50 mg / mL GMA / methanol solution was added to the test tube at 23 ° C. to start graft polymerization. After standing for 3 hours, the hollow fiber bundle was taken out, washed with ethyl acetate and methanol, and vacuum dried to obtain a polyGMA surface-treated hollow fiber. The amount of polyGMA introduced from the increase in the weight of the hollow fiber was about 4.0 mg (1.7 × 10 ⁻² mol / g) per 1 cm ² of the hollow fiber.

The polyGMA surface-treated hollow fiber was immersed in a 30% by mass polyethyleneimine (molecular weight 600, manufactured by Wako Pure Chemical Industries) acetonitrile solution, heated at 60 ° C. for 3 hours, washed with methanol and acetonitrile, and vacuum-dried. An imine surface-treated hollow fiber was obtained. The amount of polyethyleneimine introduced was about 1.0 mg (1.0 × 10 ⁻³ mol / g) per 1 cm ^{2 of} hollow fiber.

(Example 2) <Polyethyleneimine (molecular weight 600) HEV capture of surface-treated hollow fiber>
Porcine liver-derived hepatitis E virus (hereinafter referred to as HEV) was mixed with DMEM (manufactured by Wako Pure Chemical Industries, Ltd.) and 199 medium (manufactured by Life Technologies) in 1: 1, and the medium was supplemented with 2% fetal calf serum. / PRF / 5 (human hepatoma-derived cells) HEV culture solution infected with cells and proliferated was prepared. As a result of measuring real-time reverse transcription PCR (hereinafter referred to as real-time RT-PCR), HEV in the culture solution was 2 × 10 ⁵ copies / μL.

The hollow fiber 5 cm ² produced in Preparation Example 4 was cut out, immersed in 1 mL of PBS (−) for 16 hours, further washed twice with PBS (−), and used for the test. This was transferred to a 1.5 mL capacity microtube, 1 mL of the diluted virus solution was added, and the mixture was shaken back and forth at room temperature for 1 hour. Thereafter, the supernatant was recovered, and HEV-RNA in the supernatant was extracted with ISOGEN (manufactured by Nippon Gene Co., Ltd., 40% phenol aqueous solution) and isolated with chloroform and isopropanol. Isolated HEV-RNA was measured by real-time RT-PCR. As a result, it was confirmed that 74% HEV was trapped in the hollow fiber.

Further, the hollow fiber was recovered and washed three times with PBS (−), and then HEV-RNA was isolated directly from the hollow fiber with ISOGEN, chloroform and isopropanol. Isolated HEV-RNA was measured by real-time RT-PCR. As a result, it was confirmed that 42% of the captured HEV was recovered.

(Example 3) <Production of polyethyleneimine (molecular weight 1,800) surface-treated hollow fiber>
In the same manner as in Preparation Example 4, a polyGMA surface-treated hollow fiber was obtained. Thereafter, a polyethyleneimine surface-treated hollow fiber was obtained in the same manner as in Preparation Example 4 using polyethyleneimine (molecular weight 1,800, manufactured by Wako Pure Chemical Industries, Ltd.). Introduction amount of polyethyleneimine was hollow fiber 1 cm ² per about ^{0.9mg (2.9 × 10 -4 mol /} g). The hollow fiber was subjected to the HEV capture test in the same manner as in Example 2. As a result, it was confirmed that 76% HEV was trapped in the hollow fiber. The trapped virus was recovered and isolated in the same manner as in Example 2. (Recovery rate: 22%)

(Example 4) <HEV capture of acrylic acid surface-treated hollow fiber>
The acrylic acid surface hollow fiber produced in Preparation Example 2 was subjected to the HEV capture test in the same manner as in Example 2. As a result, it was confirmed that 30% HEV was trapped in the hollow fiber.

(Preparation Example 5) <Production of polyethyleneimine (molecular weight 1,800) immobilized hollow fiber>
According to JP-A-2-0259260, 26 mL of acetone, 21 mL of epichlorohydrin, and 5 mL of 40% NaOH aqueous solution are put into a test tube, and into this, an EVOH hydrophilized polyethylene porous hollow fiber having an inner diameter of 287 μm, a film thickness of 52 μm, and an average pore diameter of 102 nm Soaked. The reaction was carried out at 30 to 40 ° C. for 5 hours while applying ultrasonic waves. After completion of the reaction, the reaction product was washed with acetone and water to obtain a hollow fiber having an epoxy group introduced therein. The amount of epoxy groups introduced was about 0.01 μmol / cm ² (4.8 × 10 ⁻⁶ mol / g) as a result of titration with NaOH using a Na ₂ S ₂ O ₃ solution. Was confirmed.

Next, the hollow fiber which introduce | transduced the epoxy group was immersed in the 3.2 mass% polyethyleneimine (molecular weight 1,800 Wako Purechemical make) aqueous solution, and it was made to react at 40 degreeC for 3 days. After completion of the reaction, it was washed with water to obtain a hollow fiber introduced with polyethyleneimine. When the amount immobilized was measured, it was confirmed that about 27 μg / cm ² (7.1 × 10 ⁻⁶ mol / g) was introduced.

(Example 5) <HEV capture and recovery test of polyethyleneimine (molecular weight 1,800)>
The hollow fiber produced above was subjected to the HEV capture test in the same manner as in Example 1. As a result, it was confirmed that 95% HEV was trapped in the hollow fiber.

Next, the hollow fiber is recovered, washed three times with PBS (−), immersed in a 10 mM NaOH aqueous solution to recover the supernatant, neutralized by adding 1/10 volume of 100 mM HCl aqueous solution, and HEV-RNA is recovered. It was measured by isolation and real-time RT-PCR. As a result, it was found that HEV could be recovered 100% from the captured hollow fiber.
Next, the concentration of virus contained in the liquid used in this embodiment, which corresponds to a certain amount of liquid (B liquid), 6.84 × 10 ⁷ copies number /mL×100/95=7.2×10 Calculated as ⁷ copies / mL. Furthermore, since the dilution rate at the time of collecting B liquid from the virus liquid (A liquid) was 2.8 times, the virus concentration contained in the virus liquid (A liquid) used in this example was 7.2 × 10. It was calculated that ⁷ copies / mL × 2.8 = 2.0 × 10 ⁸ copies / mL.

(Example 6)
<HEV capture / recovery test of polyethyleneimine (molecular weight 10,000) immobilized hollow fiber>
Except for using polyethyleneimine (molecular weight 10,000, manufactured by Wako Pure Chemical Industries, Ltd.), the same operation as in Example 4 was performed to obtain a hollow fiber into which polyethyleneimine (molecular weight 10,000) was introduced. When the immobilized amount was measured, it was found that about 67 μg / cm ² (3.2 × 10 ⁻⁶ mol / g) was introduced.

The hollow fiber produced above was subjected to the HEV capture test in the same manner as in Preparation Example 3. As a result, it was confirmed that 96% HEV was trapped in the hollow fiber.

Next, the hollow fiber is recovered, washed three times with PBS (−), immersed in a 10 mM NaOH aqueous solution to recover the supernatant, neutralized by adding 1/10 volume of 100 mM HCl aqueous solution, and using ISOGEN. HEV-RNA was isolated and measured by real-time RT-PCR. As a result, it was found that 12% of HEV could be recovered from the captured hollow fiber.

Next, a certain amount of liquid concentrations of virus contained in the liquid used in this embodiment corresponding to (B solution), 8.23 × 10 ⁷ copies number /ML/(0.96×0.12) = 7.2 × 10 ⁷ copies / mL. Furthermore, since the dilution rate at the time of collecting B liquid from the virus liquid (A liquid) was 2.8 times, the virus concentration contained in the virus liquid (A liquid) used in this example was 7.2 × 10. It was calculated that ⁷ copies / mL × 2.8 = 2.0 × 10 ⁸ copies / mL.

Further, the hollow fiber was washed three times with PBS (−), and HEV-RNA was isolated directly from the hollow fiber with ISOGEN, chloroform and isopropanol. Isolated HEV-RNA was measured by real-time RT-PCR. As a result, it was confirmed that 88% of the captured HEV was recovered.
Next, the concentration of the virus contained in the liquid used in this example corresponding to a certain amount of liquid (B liquid) was 6.08 × 10 ⁷ copies / mL / (0.96 × 0.88). = 7.2 × 10 ⁷ copies / mL. Furthermore, since the dilution rate at the time of collecting B liquid from the virus liquid (A liquid) was 2.8 times, the virus concentration contained in the virus liquid (A liquid) used in this example was 7.2 × 10. ⁷ was calculated to be copy number /ML×2.8=2.0×10 ⁸ copy number / mL.

(Example 7)
<HEV capture, elution, and recovery test of phenylalanine-immobilized hollow fiber>
A hollow fiber into which phenylalanine was introduced was obtained in the same manner as in Preparation Example 5 except that 3.0 mass% phenylalanine / 0.1 M sodium carbonate aqueous solution was used instead of polyethyleneimine. When the amount of immobilization was measured, it was found that about 7 μg / cm ² (1.1 × 10 ⁻⁵ mol / g) was introduced. The hollow fiber was subjected to the HEV capture test in the same manner as in Example 2. As a result, it was confirmed that 41% of HEV was captured by the hollow fiber. The trapped virus was recovered and isolated in the same manner as in Example 2. (Recovery rate: 13%)

(Example 8)
<HEV capture / elution / recovery test of cetylamine-immobilized hollow fiber>
A hollow fiber into which phenylalanine was introduced was obtained in the same manner as in Preparation Example 3, except that a 10% by mass cetylamine (C ₁₆ —NH ₂ manufactured by Tokyo Chemical Industry) / ethanol solution was used instead of polyethyleneimine. Measurement of the amount of immobilization, it was confirmed to be introduced about ^{16μg / cm 2 (3.3 × 10} -5 mol / g). The hollow fiber was subjected to the HEV capture test in the same manner as in Example 2. As a result, it was confirmed that 58% of HEV was trapped in the hollow fiber.

Next, the hollow fiber was recovered, washed 3 times with PBS (−), and HEV-RNA was isolated directly from the hollow fiber with ISOGEN, chloroform, and isopropanol. Isolated HEV-RNA was measured by real-time RT-PCR. As a result, it was confirmed that 80% of the captured HEV was recovered.

(Example 9) <HAV capture / recovery test of polyethyleneimine (molecular weight 1,800)>
Hepatitis A virus (hereinafter referred to as HAV) was added to Eagle's MEM (manufactured by Wako Pure Chemical Industries, Ltd.) with sodium bicarbonate added to a final concentration of 0.15% in a medium containing fetal bovine serum 2% (final concentration). Then, GL37 (African green monkey kidney-derived cultured cells) cells were infected and propagated to prepare a virus culture solution. Further, the cells were disrupted and HAV solution purified by sucrose density gradient centrifugation was prepared. As a result of RT-PCR measurement, the amount of HAV in the culture broth was 2.0 × 10 ⁶ copies / μL.

The hollow fiber 5 cm ² produced in Preparation Example 5 was cut out, immersed in 1 mL of PBS (−) for 16 hours, further washed twice with PBS (−) and used for the test. This was transferred to a 1.5 mL capacity microtube, 1 mL of the diluted virus solution was added, and the mixture was shaken back and forth at room temperature for 1 hour. Thereafter, the supernatant was recovered, and HAV-RNA in the supernatant was extracted with ISOGEN and isolated with chloroform and isopropanol. Isolated HAV-RNA was measured by real-time RT-PCR. As a result, it was confirmed that 88% of HEV was trapped in the hollow fiber.

Next, the hollow fiber is recovered, washed three times with PBS (−), immersed in a 10 mM NaOH aqueous solution to recover the supernatant, neutralized by adding 1/10 volume of 100 mM HCl aqueous solution, and using ISOGEN. HAV-RNA was isolated and measured by real-time RT-PCR. As a result, it was found that 66% of HEV could be recovered from the captured hollow fiber.

The concentration of the virus contained in the liquid used in this example corresponding to a certain amount of liquid (liquid B) was 1.14 × 10 ⁸ copies / mL / (0.88 × 0.66) = 1. It was calculated to be 9 × 10 ⁸ copy number / mL. Furthermore, since the dilution ratio at the time of collecting B solution from virus solution (A solution) was 10 times, the virus concentration contained in virus solution (A solution) used in this Example was 1.9 × 10 ⁸ copies. Calculated to be number / mL × ¹⁰ = 2.0 × 10 ⁹ copies / mL.

Further, the hollow fiber was washed three times with PBS (−), and HAV-RNA was isolated directly from the hollow fiber with ISOGEN, chloroform and isopropanol. Isolated HAV-RNA was measured by real-time RT-PCR. As a result, it was confirmed that 34% of the captured HAV was recovered.

The concentration of the virus contained in the liquid used in this example corresponding to a certain amount of liquid (liquid B) was 5.9 × 10 ⁷ copies / mL / (0.88 × 0.34) = 1. Calculated as 9 × 10 ⁸ copies / mL. Furthermore, since the dilution rate at the time of collecting B liquid from virus liquid (A liquid) was 10 times, the virus concentration contained in the virus liquid (A liquid) used in this example was 1.9 × 10 ⁷ copies. It was calculated to be the number /ML×10=1.9×10 ⁹ copy number / mL.

(Comparative Example 1) <HEV capture / elution / recovery test of poly-4-methylpentene hollow fiber>
The poly-4-methylpentene hollow fiber into which poly GMA had been introduced was subjected to the HEV capture test in the same manner as in Example 1. As a result, HEV was not captured at all in the hollow fiber.

(Comparative Example 2) <HEV capture and recovery test of polyethylene hollow fiber>
The epoxy group-introduced EVOH hydrophilized polyethylene porous hollow fiber was subjected to the HEV capture test in the same manner as in Example 1. As a result, HEV was not captured at all in the hollow fiber.

Table 1 shows various substrates and ligands, virus capture ability, and recovery performance.
In Example 6, a recovery result using a sodium hydroxide aqueous solution (NaOH aqueous solution recovery) and a recovery result using a phenol aqueous solution (phenol aqueous solution recovery) are shown.

The evaluation method and kit of the present invention can be used for evaluation of virus contamination in environmental water, body fluid, domestic water and the like.

Claims (24)

1. In the liquid containing the virus by capturing the virus through the liquid containing the virus through or in contact with the hollow fiber surface-treated with a compound having a charge or a compound having no charge, and calculating the amount of the captured virus Of assessing the concentration of viruses contained in the.
2. A method for evaluating the virus concentration according to claim 1,
   1) First step of collecting a certain amount of liquid (liquid B) from the liquid containing liquid (liquid A) 2) The liquid B is passed through or brought into contact with the hollow fiber to capture the virus in the hollow fiber. Two steps 3) A method for evaluating the virus concentration, comprising each step of the third step of calculating the amount of virus in the B liquid trapped in the hollow fiber.
3. The method for evaluating the virus concentration according to claim 1 or 2, wherein the hollow fiber is composed of polyolefin, polyethersulfone, or cellulose mixed ester.
4. The method for evaluating the virus concentration according to any one of claims 1 to 3, wherein the hollow fiber is a porous hollow fiber.
5. The method for evaluating the virus concentration according to claim 3 or 4, wherein the polyolefin is polyethylene, polypropylene or poly-4-methylpentene.
6. The compound having a charge is a (meth) acrylate, a sulfonate, a compound having an ammonium group, a compound having a sulfonium group, a compound having a carboxylate group, or a compound having a phosphate group. The method to evaluate the density | concentration of the virus in any one.
7. 6. The compound having no charge is (meth) acrylic acid, sulfonic acid, a compound having an amino group, a compound having a sulfo group, a compound having a carboxy group, or a compound having a phospho group. A method for evaluating the concentration of the virus described in 1.
8. The method for evaluating the virus concentration according to claim 6 or 7, wherein the sulfonium group or the compound having a sulfo group is a sulfated sugar.
9. Sulfated sugar is heparin, a heparin derivative in which the primary or secondary hydroxyl group of heparin is sulfate esterified, a heparin derivative in which an N-acetyl group-elimination product of heparin is N-sulfate ester, dextran sulfate, or fucoidan The method for evaluating the virus concentration according to claim 8.
10. The method for evaluating the virus concentration according to any one of claims 1 to 9, wherein the method for calculating the amount of virus is by polymerase chain reaction, antigen-antibody reaction, or culture method.
11. The virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, adenovirus, enterovirus, norovirus, influenza virus, human immunodeficiency virus, rotavirus, or poliovirus. A method for evaluating the concentration of the virus according to any one of 10.
12. 3) In the third step of calculating the amount of virus in the B liquid trapped in the hollow fiber,
    The method for evaluating the virus concentration according to any one of claims 2 to 11, further comprising a step of recovering the virus from a hollow fiber in which the virus is trapped, with an aqueous phenol solution, an alkaline aqueous solution, or an inorganic salt aqueous solution.
13. A virus concentration evaluation kit having a function of evaluating the virus concentration by the method according to any one of claims 1 to 12.
14. A virus-capturing hollow fiber to which a compound having an amino group or a compound having an ammonium group is used, which is used in the method for evaluating the virus concentration according to any one of claims 1 to 12.
15. The virus-capturing hollow fiber according to claim 14, wherein the compound having an amino group or the compound having an ammonium group is a compound selected from polyethyleneimine, polyallylamine, amino acids, and salts thereof.
16. In the hollow fiber for capturing a virus, wherein the compound having an amino group according to claim 14 or 15 or the compound having an ammonium group is immobilized,
    A compound having an amino group or a compound having an ammonium group is immobilized on a hollow fiber surface-treated with a polymer material having a hydroxyl group via a compound having a group capable of reacting with a hydroxyl group and an amino group or an ammonium group. A hollow fiber for capturing a virus, characterized by
17. A hollow fiber surface-treated with a polymer material having a hydroxyl group is an ethylene-vinyl alcohol copolymer, an ethylene-vinyl alcohol-vinyl acetate copolymer, a partially saponified product of an ethylene-vinyl acetate copolymer, or a vinyl alcohol-acetic acid. The virus-capturing hollow fiber according to any one of claims 14 to 16, which is a hollow fiber surface-treated with a vinyl copolymer, a hydroxymethacrylate copolymer, a partially saponified cellulose acetate, or a glycerin derivative.
18. The virus-capturing hollow fiber according to claim 16 or 17, wherein the compound having a hydroxyl group and a group capable of reacting with an amino group or an ammonium group is epichlorohydrin or a diepoxy compound.
19. In the hollow fiber for capturing a virus, wherein the compound having an amino group according to claim 14 or 15 or the compound having an ammonium group is immobilized,
    A virus-capturing hollow fiber in which a compound having an amino group or a compound having an ammonium group is immobilized on a hollow fiber by a graft polymerization reaction.
20. In the hollow fiber for capturing a virus, wherein the compound having an amino group according to claim 14 or 15 or the compound having an ammonium group is immobilized,
    A virus-capturing hollow fiber in which a compound having an amino group or a compound having an ammonium group is immobilized by surface-treating the hollow fiber with a compound having an amino group or a compound having an ammonium group.
21. The virus-capturing hollow fiber according to any one of claims 14 to 20, wherein the hollow fiber is a porous hollow fiber.
22. The virus-capturing hollow fiber according to any one of claims 14 to 21, wherein the hollow fiber comprises polyethylene, polypropylene, or poly-4-methylpentene as a substrate.
23. Compounds having an amino group, or a virus retention hollow fiber according to any one of claims 14-22 immobilization amount of a compound having an ammonium group is in the range of 1 ~ 100μg / cm ^2.
24. The virus is hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, adenovirus, enterovirus, norovirus, influenza virus, human immunodeficiency virus, rotavirus, or poliovirus. The hollow fiber for capturing a virus according to any one of 23.