JP2002116120A - Target substance detecting method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method capable of detecting a target substance included in a swimming pool made of plural samples even in an extremely low concentration. SOLUTION: This target substance detecting method is characterized in that after bringing an adsorbent capable of adsorbing the target substance into contact with the samples having the possibility of including the target substance mixed with plural specimens, a water insoluble solid is separated from the inside of the samples, and then, the target substance bonding to the water insoluble solid is detected.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for detecting a target substance contained in a plurality of samples at a low concentration.

[0002]

2. Description of the Related Art Viruses are one of the causes of various diseases of humans, animals and plants, and the identification of the causative virus is very important for the inspection and diagnosis thereof. As a conventional virus inspection / diagnosis method, immunological measurement of a virus antigen or an antiviral antibody is generally performed. However, several weeks to several months after virus infection, there is a period during which the amount of virus or the amount of anti-virus antibody is small and thus cannot be detected by these immunological assays. This undetectable period is called a window period (blank period). When such a patient makes a blood donation, the blood donation is often the source of infection, and an unspecified number of transfused patients May be dangerous to patients using blood products. Therefore, to keep the window period as short as possible,
There is an urgent need to develop a technology that can detect viruses below the immunological measurement limit with high sensitivity.

[0003] In recent years, the possibility of detecting even a trace amount of virus has been opened up by a nucleic acid amplification technique represented by the polymerase chain reaction method (hereinafter, PCR method). However, detection of a trace amount of virus by PCR or the like also requires special equipment and advanced technology, and it is extremely difficult to simply carry it out in a general facility. In addition, the amount of sample that can be used is also limited by the instrument, and since the amount is small, the virus may not be detected if the virus concentration is low even by the PCR method. Therefore,
In order to solve these problems, a technique of concentrating a virus in a sample has been used.

[0004] In order to efficiently test a test substance in a large number of specimens having a low detection rate of a target substance, a small pool, for example, a few milliliters, is collected from a plurality of specimens, and a pool is created. Techniques for detecting viruses in them are being studied. However, the current method has a very low virus concentration in the pool,
Detection may be difficult.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems of the virus detection method, and to solve the problem even if the target substance contained in a pool prepared from a plurality of samples has an extremely low concentration. It is to provide a method that can be detected.

[0006]

Means for Solving the Problems The present invention solves the above-mentioned problems by adsorbing a target substance to a sample which may contain a target substance, in which a plurality of specimens are mixed at a fixed amount. And then contacting the adsorbent with the adsorbent, separating the adsorbent from the sample, and then detecting a target substance bound to the adsorbent. .

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. [Adsorbent] The adsorbent (hereinafter, simply referred to as “target substance adsorbent”) that can be used in the present invention does not indicate the material itself, but various materials such as organic polymers,
It is made of an inorganic polymer, an inorganic compound, a natural polymer compound, or the like, or a combination thereof, and may have various shapes as described later. Since the adsorbent can easily separate the target substance, it must be water-insoluble. Aqueous material serving as a sample, such as blood,
It consists of substances that are insoluble in urine, aqueous protein solution and the like.

Examples of the organic polymer constituting the target substance adsorbent include (co) polymers of radically polymerizable monomers such as aromatic vinyl compounds, acrylates, methacrylates, and unsaturated carboxylic acids, polyesters,
Polyolefin, polyether, polycarbonate, polyimide, polyamide, cation exchange resin, anion exchange resin, polyethylene imine, polypropylene imine,
Polylysine, glucosamine, polyallylamine and the like can be mentioned.

Examples of the inorganic polymer include hydrolytic condensates such as alkoxysilane and organoalkoxysilane. Examples of the inorganic compound include silica, alumina, calcium phosphate and the like. Examples of natural polymers include dextran,
Examples include cellulose, agarose, and glucomannan.

When the material is, for example, an organic polymer, there is a water-soluble material. However, since the target substance adsorbent must be water-insoluble, it is cross-linked or held on a water-insoluble substrate. Is required.

In the present invention, the surface of the target substance adsorbent is preferably ionic in order to increase the adsorbing ability of the target substance. Here, that the surface is ionic indicates that the surface of the target substance adsorbent has at least one of an anionic charge and a cationic charge, or can be charged under a specific condition.

Examples of the target substance adsorbent having an anionic surface include those in which the target substance adsorbent is composed of a polymer obtained by copolymerizing a monomer having an anionic group, those in which the polymer is anionized, A polymer obtained by seed-bonding or graft-polymerizing a sulfonic acid-containing monomer to a synthetic polymer, one comprising a cation exchange resin,
Examples thereof include those in which a sulfated natural polymer is supported on a water-insoluble polymer carrier, those having a polymer having an anionic group or a sulfated natural polymer on the surface of an inorganic polymer or inorganic particles, and the like. Here, examples of the anionic group include a sulfone group and a carboxyl group.

The following can be mentioned as examples of the above. Examples of polymers obtained by copolymerizing monomers having an anionic group: (co) polymers of carboxylic acid-containing monomers. Here, the carboxylic acid-containing monomer (hereinafter referred to as “carboxylic acid monomer”) is a polymerizable monomer having an addition-polymerizable unsaturated bond and a carboxyl group in the molecule. Specific examples include acrylic acid, methacrylic acid,
Crotonic acid, itaconic acid, fumaric acid, maleic acid and the like.

Examples of the polymer obtained by anionizing (sulfonating): a sulfonable functional group at least on its surface, for example, an unsaturated double bond, an aromatic group, a primary or It consists of a (co) polymer having a secondary amino group, a primary halogenated alkyl group, an aliphatic aldehyde, an aliphatic ketone, an aliphatic carboxylic acid, an aliphatic carboxylic anhydride, a hydroxyl group, etc. Some are sulfonated. Surface sulfonable (co)
Examples of the polymer include (co) polymers of sulphonable monomers such as styrene, α-methylstyrene, vinylnaphthalene, divinylbenzene, butadiene, isoprene, and vinyl alcohol, and polymerization of these monomers and other polymers. Polymerization compounds such as (co) polymers with hydrophilic monomers; polycarbonates, polyesters, polyester ethers,
Polyarylether, polyalkylene oxide, polysulfone, polyethersulfone, polyamide, polyimide, polyetherimide, polyetherketone, polyurethane, acetaldehyde condensate of aromatic compound, condensation polymerization type high molecular compound such as polyether and the like. .

Examples of the cation exchange resin include divinylbenzene cross-linked sulfonated polystyrene, and the particle diameter may be such that the central particle diameter is about 0.1 mm to 1 mm. Examples of a sulfated natural polymer supported on a water-insoluble polymer carrier: sulfated polysaccharides include heparin, dextran sulfate, cellulose sulfate, guardlan sulfate, chondroitin sulfate, heparan sulfate, dextran sulfate,
Chitin sulfate, chitosan sulfate, dermatan sulfate, amylopectin sulfate, ketalan sulfate, xylan sulfate, karonin sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenin sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, Galactan sulfate, levan sulfate, mepesulfate,
Examples include polysaccharides having antiviral properties such as fucoidan and sulfated glycyrrhizin.

Sulfated polysaccharides include epoxy groups, amino groups,
It can be supported on a polymer having a functional group such as an aldehyde group, a carboxyl group, a hydroxyl group or an acid chloride group directly or via a coupling agent or a spacer.

Examples of the target substance adsorbent having a cationic surface include those in which the target substance adsorbent is composed of a polymer obtained by copolymerizing a monomer having a cationic group, and a polymerization initiator having a cationic group. From a polymer obtained by radical polymerization using a compound having a cationic group bonded to a polymer carrier, from an anion exchange resin, or from a water-soluble polymer containing a cationic group. Supported on a polymer carrier,
Examples thereof include a polymer having a cationic group or a polymer having a cationic group-containing water-soluble polymer on the surface of an inorganic polymer or an inorganic particle.

In the present specification, a cationic group refers to a group that forms a cation by itself and a cation that does not form a cation by itself but is easily converted to a cation by the binding of a proton near neutral (about pH 6 to 8). Amine and imine groups to be formed are meant. For example, a primary amino group, 2
And a quaternary amino group, a tertiary amino group, a quaternary ammonium group, and an imino group (-NH- group and = NH group).
The form in which these cationic groups are present is various and is not limited at all. For example, the cationic groups may be present in the compound in the form of an amidino group, an imidino group, a hydrazino group, a pyridyl group, or the like.

The following can be cited as examples of the above. Example of polymerization in which a monomer having a cationic group is copolymerized: Examples of the monomer having a cationic group include 2-dimethylaminoethyl (meth) acrylate, 2-diethylaminoethyl (meth) acrylate, and 2-dimethylaminopropyl (meth). Aminoalkyl group-containing (meth) acrylates such as acrylate and 3-dimethylaminopropyl (meth) acrylate, and quaternary salts thereof with methylene chloride, dimethyl sulfate, diethyl sulfate, and the like; 2
-(Dimethylaminoethoxy) ethyl (meth) acrylate, 2- (diethylaminoethoxy) ethyl (meth)
Acrylates, (meth) acrylates containing aminoalkoxyalkyl groups such as 3- (dimethylaminoethoxy) propyl (meth) acrylate and the like, and quaternary salts thereof with methylene chloride, dimethyl sulfate, diethyl sulfate and the like; N- (2- Dimethylaminoethyl) (meth) acrylamide, N- (2-diethylaminoethyl) (meth)
Acrylamide, N- (2-dimethylaminopropyl)
N-aminoalkyl group-containing (meth) acrylamides such as (meth) acrylamide and N- (3-dimethylaminopropyl) (meth) acrylamide, and quaternary salts thereof with methylene chloride, dimethyl sulfate, diethyl sulfate and the like. Can be Among them, 2-dimethylaminoethyl (meth) acrylate, N- (2-dimethylaminoethyl) (meth) acrylamide, and quaternary salts thereof with methylene chloride are preferable. These may be copolymerized with other polymerizable monomers.

Examples of the monomer copolymerizable with the cationic monomer include the following crosslinkable monomers and non-crosslinkable and nonionic monomers.

Examples of the crosslinking monomer include divinylbenzene, divinylbiphenyl, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, and propylene glycol. Di (meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di (meth) acrylate, tetrapropylene glycol di (meth) acrylate, 1,4-butanediol di (meth) acrylate,
1,6-hexanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, 2,
2'-bis [4- (meth) acryloyloxypropoxyoxyphenyl] propane, 2,2'-bis [4- (meth) acryloyloxydiethoxydiphenyl] propane, glycerin tri (meth) acrylate, trimethylolpropane tri ( Examples thereof include divinyl monomers such as meth) acrylate and pentaerythritol tetra (meth) acrylate, trivinyl monomers and tetravinyl monomers. Among them, divinylbenzene, ethylene glycol dimethacrylate and trimethylolpropane trimethacrylate are preferred.

The non-crosslinkable and nonionic monomer that can be copolymerized is a noncrosslinkable and nonionic monomer that can be copolymerized with either a cationic monomer or a crosslinkable monomer.

As such a monomer, styrene, α
Aromatic vinyl monomers such as methyl styrene, p-methyl styrene, halogenated styrene; unsaturated nitriles such as acrylonitrile; methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate;
Acrylates such as butyl acrylate, butyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, lauryl acrylate, lauryl methacrylate, glycidyl acrylate, glycidyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and methacrylic acid Esters; diolefins such as butadiene and isoprene; carboxylic acid vinyl esters such as vinyl acetate; and vinylidene halides such as vinyl chloride and vinylidene chloride. Among them, styrene, α-methylstyrene, acrylonitrile, methyl methacrylate, butyl methacrylate, 2-hydroxyethyl acrylate, and cyclohexyl methacrylate are preferred.

Examples of a polymer obtained by polymerizing a monomer using a polymerization initiator having a cationic group: A radical polymerization initiator having a cationic group is a polymer obtained by radical polymerization using the same. It has a cationic group derived from the radical polymerization initiator at its terminal. Preferred examples of the radical polymerization initiator having a cationic group include azobis-type initiators having an amidino group, an imidino group, or a pyridium group. Those having a 10-hour half-life temperature in the range of 40 to 95 ° C. are preferable because the polymerization can be carried out under mild conditions.

A carboxyl group, a hydroxyl group, an epoxy group, an amino group, an amide group and the like are introduced into the surface of a polymer particle obtained by binding a compound having a cationic group to a polymer carrier by copolymerization or seed polymerization. By reacting these groups as a bonding portion, a compound having a cationic group can be introduced to the polymer surface. Examples of the compound having a cationic group include polyethyleneamine, polypropyleneimine, polyallylamine, polyvinylamine, polyamine compounds such as polypropyleneamine, polylysine, polyarginine, free amino acids such as polyhistidine, and monomers having the above-described cationic group. Copolymers and the like can be mentioned.

Anion exchange resin A divinylbenzene cross-linked polystyrene has a quaternary ammonium group -CH ₂ N ⁺ (CH ₃ ) ₃ (type I) or -CH ₂ N ⁺ (CH ₃ ) ₃ (C ₂ H ₄
OH) (Type II) introduced strong basic anion exchange resin, -N
Weakly basic anions into which H (CH ₃ ) ₂ , —NH (CH ₂ CH ₂ NH) _n H, and —CONH (CH ₂ ) ₃ N (CH ₃ ) ₂ are introduced.

A polymer having a cationic group or a cationic group-containing water-soluble polymer on the surface of an inorganic polymer or inorganic compound. In addition, donor atoms (O, N,
Resins having S) can also be mentioned. Examples of the chelating resin include iminodiacetic acid type, iminopropionic acid type, ethylenediamine triacetic acid type, quinoline type, aminocarboxylic acid type, amine phosphoric acid type, amidoxime type, cryptant type, polyamine type, pyridine type, picolylamine type, and thiol type , Phosphoric acid type, polyphenol type, dithiocarbamic acid type, thiourea type, isothiuronium type and dithizone type.

In the present invention, the target substance adsorbent may contain a magnetic substance such as a ferromagnetic substance and a superparamagnetic substance, which are effective for automating the inspection and the separating operation. The shape of the target substance adsorbent is not particularly limited and may be various. For example, it may be in the form of particles, filters, fibers, sheets, tubes, plates, or the like. When the shape of the target substance adsorbent is particulate, the particle size is about 0.08 to 300 μm when dispersed and used in a sample, and about 0.1 to 1 mm when used by filling a column. It preferably has a particle size.

The filter is preferably a filter having an average pore diameter in the range of 0.1 to 50 μm, and examples thereof include a flat membrane, a hollow fiber membrane, a nonwoven fabric, and a woven fabric.
When the target is plasma, culture solution, or the like, the average pore size is 0.
1 μm to 10 μm is preferred. If the average pore size is 0.1 μm or less, a sufficient filtration amount cannot be obtained because the pore size becomes small. The average pore diameter is a value obtained by using a palm porometer (manufactured by Porous Materials) described in ASTM-F316, and is a value reflecting an actual transmission pore.
The amount of water passing through the filter was 10 mL / mi as a value measured at 25 ° C. ± 2 ° C. under a pressure of 0.7 kg / cm ^2.
n / m ² / mmHg or more, preferably 100 mL / min
/ M ² / mmHg or more, because the filtration pressure can be reduced. In the case of a nonwoven base material, the filaments forming the filter material may be monofilaments or multifilaments, but the average diameter (average of the major and minor diameters of the filaments observed with a scanning electron microscope) Value) is 100 μm or less, preferably 50 μm.
It is preferable that it is not more than m since the surface area of the film becomes large and the number of adsorption sites increases. Further, it may be a modified filament or a porous filament. Further, the ratio of the porosity of the filter (porosity) is 20%.
Or more, preferably 50% or more.

Examples of the target substance adsorbent having a sheet shape, a tube shape, or a plate shape include a blood collection tube, a vacuum blood collection tube, a test tube, a well plate, a microplate, a microtest tube, and an Eppendorf tube (trade name). Can be mentioned.

In order to obtain a target substance adsorbent having these shapes, a thermoplastic resin such as polystyrene, polypropylene, polyethylene, polyethylene terephthalate, norbornene-based resin among the organic polymers exemplified above is directly formed into a desired shape. be able to.

The target substance adsorbent suitable for the present invention can be obtained by ionizing the surface of a general-purpose blood collection tube made of glass, polystyrene, polymethyl methacrylate, or the like.

[Sample] The detection method of the present invention is applied to:
This is a sample in which a plurality of analytes are mixed and may contain a target substance. Samples include, for example, various body fluids such as blood, plasma, serum, urine, and saliva, cell lysates, and cultured cell lysates.

A sample obtained by collecting a predetermined amount from each of the plurality of samples and mixing the sample is called a "pool", and the first pool creation is called a primary pool. The number of samples collected in the creation of the primary pool is usually 5 to 500, preferably 10 to 50, and the amount of the sample collected from one sample is 0.01 to 1 ml, preferably 0.1 to 1 ml.
0.5 ml.

If necessary, samples may be collected from a plurality of primary pools to form a secondary pool. Similarly, there may be third or higher pools. The number of pools collected in the preparation of the secondary pool is usually 2 to 100, preferably 5 to 20, and the amount of a sample collected from one specimen is 0.01 to 1 ml, preferably 0.1 to 0 ml. .
5 ml.

In this manner, a primary or secondary pool is prepared. Here, by appropriately adjusting the amount of samples collected from each sample or pool and the number of collected samples or pools, a plurality of subjects can be prepared. The sample can be prepared in an amount suitable for the target substance detection means described later, in which a certain amount of the sample is contained in some cases.

[Contact / Binding] In the present invention, the target substance adsorbent is brought into contact with the sample prepared as described above.
There are various contact methods and there is no particular limitation. An appropriate method may be used depending on the shape, size, etc. of the insoluble carrier.

For example, the target substance adsorbent has a particle size of 200 μm.
In the case of particles having a particle size of less than 1, the target substance adsorbent is dispersed in the sample, and the sample is brought into contact with the insoluble carrier. This dispersing and contacting is optionally carried out by shaking. When the target substance adsorbent is a particle having a particle diameter of more than 200 μm, the column is packed and a sample is passed through the column. When the target substance adsorbent is a large one such as plastic beads having a diameter of 5 to 6 mm, one to several pieces are put into a test tube and brought into contact with the sample. When the target substance adsorbent is in the form of a filter or a fiber, the sample is filtered through the filter or the fiber so that the surface of the filter or the fiber sufficiently contacts the sample. When the target substance adsorbent is a test device such as a test tube or a well plate, a sample is placed in the test device and shaken.

When the sample is brought into contact with the target substance adsorbent, a polyvalent metal ion or the like can be further added as necessary. When the sample is brought into contact with the target substance adsorbent, the temperature of the sample is usually 5 to 37 ° C., and if the contact time is too long, precipitation of fibrin or the like occurs.

[Separation] After the target substance adsorbent is brought into contact with the sample, the target substance adsorbent is separated from the sample. At this time, when the target substance adsorbent is a particle having a particle size of less than 200 μm,
For example, it is separated by centrifugation. In addition, when the magnetic substance is contained in the target substance adsorbent, it can be easily separated from the sample by magnetism.

The target substance on the target substance adsorbent separated from the sample is concentrated. The target substance thus concentrated is then subjected to detection. However, if necessary, the target substance adsorbent may be washed with a buffer before being subjected to detection, and / or
Alternatively, detection may be started after the target substance is separated from the target substance adsorbent. For example, when the target substance is a virus or a bacterium, it can be separated, and when it is a nucleic acid, it can be extracted. Alternatively, detection can be performed while the target substance remains bound on the target substance adsorbent. An appropriate method may be selected according to the type of the target substance, the material of the target substance adsorbent, and the like.

As a method for separating the target substance from the target substance adsorbent, there is a method in which the target substance is dissociated from the target substance adsorbent by the action of a salt solution and a surfactant or a mixture of both. The salt solution has a high concentration, for example,
8 M potassium bromide, sodium bromide, sodium iodide, guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride, guanidine sulfate, sodium thiocyanate, aqueous solution of potassium thiocyanate and its salts, 1.5-6 M aqueous sodium chloride solution, 0.5-5 mM An aqueous solution of phosphotungstic acid or the like can be used.

Examples of the surfactant include SDS, Triton X-100, Tween 20, 80 and NP-40.

Further, nucleic acid extraction can be performed while the target substance remains bound to the target substance adsorbent. In order to effectively amplify and detect a target nucleic acid, the nucleic acid typically must be isolated from cells or other analyte residues.
Various separation methods are known, including a freezing method, a treatment method using digestive enzymes such as protease (eg, proteinase K), a boiling method, and a method using various detergents (for example, 1988
Higuchi U.S. Patent Application No. 178,202 filed on April 6, and European Patent Application Publication No. 078281 issued on May 22, 1991
No. 97), a solvent precipitation method and a heating protocol. It is also possible to directly extract the nucleic acid of the target substance by adding a small amount of buffer solution to the separated target substance adsorbent and heating it, or directly add a commercially available nucleic acid extraction reagent to remove the target substance. It is also possible to extract nucleic acids.

[Detection of Target Substance] The target substance which has been concentrated on the target substance adsorbent as described above and, if necessary, separated and extracted in a further concentrated state is detected. As the detection method, a known method can be adopted according to the type of the target substance, and examples thereof include a nucleic acid amplification test and an immunochemical assay.

Virus nucleic acid amplification test (NAT; nuclei
The method of c acid amplification test is not particularly limited. For example, PCR (Polymerization chain) of Roche
reaction) method, TMA (Transcript
ion Mediated amplification-hybridization protectio
n assay) method, Abbott's LCR (Ligase chain reacti
on) method, NASBA (Nucleic Acid Sequence-based Amp)
lification) (targeting RNA, characterized by constant temperature),
CPT (Cyclic Probe Tech) (Target DNA, D
Using NA / RNA chimera probe, biotin-labeled probe
Amplification), Qβ Replicase Amplification (target RNA, isolate RNA with streptavidin particles), In
A vader Assay or the like can be used.

Examples of the immunochemical measurement method include an enzyme immunoreaction (EIA) method, a chemiluminescence method (CLTA), and a fluorescence method (FIA). Target substances that can be detected by the target substance detection method of the present invention include viruses, nucleic acids, antigens, antibodies, proteins, and the like.

Examples of the virus include hepadnavirus (hepatitis B virus, etc.), adenovirus, flavivirus (Japanese encephalitis virus, etc.), herpes virus (herpes simplex virus, varicella-zoster virus, cytomegalovirus, EB virus) Etc.), poxvirus,
Parvovirus (Adeno-associated virus, etc.), Orthomyxovirus (Influenza virus, etc.), Rhabdovirus (Rabies virus, etc.), Retrovirus (Acquired immunodeficiency syndrome virus, etc.), Hepatitis C virus, Human T
Virus such as cell leukemia virus (HTLV-1), human immunodeficiency virus (HIV-1, HIV-2), and human parvovirus B-19.

The nucleic acids include DNA, RNA, mRN
A, tRNA and the like. Antigens and antibodies include ferritin, ASO, T ₃ , T ₄ , α-fetoprotein, HBs
Antigen, CEA (carcinoembryonic antigen), HCV surface antigen, HI
V-surface antigens, anti-Treponemal pallidum, anti-carbiolipin antigens, anti-HBC antibodies, anti-HIV antibodies, etc., all of which are measured by immunological reactions. Examples of the protein include α-lipoprotein and β-lipoprotein.

[0050]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.
In the following description, “parts” means “parts by weight” unless otherwise specified.

Reference Example 1 (1) Acetone was added to oily magnetic fluid ferricolloid HC50 [trade name, manufactured by Taiho Kogyo Co., Ltd.] to precipitate particles, which were then dried to obtain a lipophilic treatment. Ferrite-based superparamagnetic material (particle size:
0.01 μm). Then, the superparamagnetic material 4 obtained above
To 0 parts, 90 parts of cyclohexyl methacrylate, 10 parts of chloride of trimethylaminoethyl methacrylate and 3 parts of benzoyl peroxide (polymerization initiator) are added, and the system is mixed and stirred to uniformly disperse the superparamagnetic material, thereby obtaining a monomer composition. Was prepared.

On the other hand, an aqueous monomer composition was prepared by dissolving 10 parts of polyvinyl alcohol, 0.05 part of sodium lauryl sulfate and 0.1 part of polyethylene oxide nonylphenyl ether in 1000 parts of water. The above monomer composition was added to the aqueous monomer composition thus prepared, and the mixture was preliminarily stirred with a homogenizer, and then subjected to a dispersion treatment with an ultrasonic disperser, whereby oil droplets (oil phase) having an average particle diameter of 1 μm were formed in the aqueous medium. (Oil droplet dispersion) was prepared.

Next, the obtained suspension was charged into a two-liter three-necked flask equipped with a stirrer. The temperature of the system was raised to 75 ° C., and the system was stirred for 5 hours under a nitrogen atmosphere. By polymerizing the monomer (suspension polymerization), magnetic polymer particles were produced. The obtained particles were photographed with an optical microscope, the diameter of 200 particles was measured, and the average was determined. The result was 1.2 μm.

(2) The magnetic polymer particles obtained in (1) were dispersed in a 5 mM aqueous sodium hydroxide solution,
For 12 hours to obtain carboxy-modified magnetic polymer particles. (3) 1 g of the obtained carboxy-modified magnetic polymer particles were suspended in 20 mL of 10 mM MES buffer solution (pH 6.0), and a 30% aqueous solution of polyethyleneimine (Wako Pure Chemical Industries, Ltd., number average molecular weight 70,000), 33 μL, Water-soluble carbodiimide reagent EDC · hydrochloride (1-ethyl-3
0.05 g of (3-dimethylaminopropyl) carbodiimide hydrochloride) was added and reacted at room temperature for 2 hours to obtain an adsorbent.

Example 1 (1) The aqueous suspension of the target substance adsorbent obtained in Reference Example 1 was purified and then adjusted to a solid concentration of 5% with physiological saline. (2) 100 μL of 100 copies / mL of HBV-positive blood and 100 μL of each of 49 samples of normal human plasma (HBV-negative) were mixed to prepare a sample A having a total volume of 5 mL. (3) 100 μL of the target substance adsorbent aqueous suspension (5% by weight) prepared in the above (1) was added to 4.9 mL of this sample A,
The two were brought into contact with each other by rotating and stirring at room temperature for 10 minutes, and the target substance adsorbent was subjected to virus adsorption treatment.

After the virus adsorption treatment, the sample was set on a magnetic separation stand, the supernatant of the target substance adsorbent was separated from the supernatant, the supernatant was discarded, and Tris-HCl buffer (pH 7.4) was added. An adsorbent particle dispersion was obtained. From this particle dispersion, DN
Using A Extractor Kit (manufactured by Wako Pure Chemical Industries, Ltd.),
The nucleic acid was extracted according to the attached procedure manual. The extract (nucleic acid) obtained by the extraction is referred to as sample B. Extract the obtained extract into PC
By the R method, DNA was amplified by the amplification process of 35 times of PCR. PCR primer sequences are available from Hiroaki Okamoto, Igaku
no ayumi, (1992), 162 (9), 544-549. The obtained 1st-PCR and 2nd-PCR amplification products were subjected to electrophoresis using 3% agarose, DNA was visualized by ethidium bromide staining, and photographed on a polaroid photograph. From the fluorescence intensity of the band, the amount of the obtained target DNA was determined based on-, +, +
Judgment was made in four stages of + and +++.

Sample A 100 μL, original HBV positive blood 10
Nucleic acids were extracted from 0 μL (sample C) and 100 μL of normal human plasma (sample D) using the above DNA Extractor Kit.
A PCR reaction was performed in the same manner, and evaluation was similarly performed. Table 1 shows the above results.

[0058]

[Table 1]

Example 2 HCV (hepatitis C virus) was 2 × 10 ⁴ copies / mL
1 human plasma sample and 9 normal human plasma samples, 49
Samples and 99 samples were mixed at a fixed ratio to prepare samples of 10 sample pool, 50 sample pool, and 100 sample pool, respectively.

0.9 mL, 50
After taking 4.9 mL from the sample pool sample and 9.9 mL from the 100 sample pool sample, 100 μL of the adsorbent suspension (5% by weight) produced in Reference Example 1 was added thereto, and the mixture was rotated and stirred at room temperature for 10 minutes. After the adsorbent was magnetically aggregated and the supernatant was removed leaving 100 μL each, the magnetism was released and the adsorbent was redispersed in 100 mL of the supernatant to obtain a concentrated solution.

The obtained concentrate is enzymatically decomposed with a nucleic acid extraction reagent (trade name: Smythrite EX R & D, manufactured by Genome Science Laboratories), and the adsorbent is aggregated by magnetism. Was removed from the adsorbent.
Next, the extracted HCV nucleic acid was separated and subjected to a real-time detection PCR method (Proc. Nail. Acad. Sci. USA, Vol. 8).
8, pp7276-7280, 1991) by the TaqMan PCR method.
PRISM 7700 Sequence Detection System (PE Biosystem
HCV nucleic acid).

[0062] For the quantitative determination of DNA, the number of cycles (Th cycle) in which the fluorescence intensity exceeded a certain reference value (Threshhold: Th) during the 40 amplification steps was simultaneously measured using a dilution series of a sample with a known virus concentration. Created. DNA amount
Extrapolated to the Th cycle calibration curve. The results are shown in the column of “concentration 100 μL” in Table 2.

[0063]

[Table 2]

(2) The same 10-sample pool, 50-sample pool, and 100-sample pool from 100
Add the nucleic acid extraction reagent (product name: Suma Illustration EX R &
D, manufactured by Genome Science Laboratories)
The results of quantifying the amount of DNA in the same manner as in Table 2 are shown in Table 2 “Unconcentrated 1
00 μL ”. As a result, HCV showed a copy number of the viral nucleic acid which was about one digit higher than that of the pool specimen itself, indicating that concentration with the present particles was effective.

Example 3 Sample F (5 mL) obtained by mixing 100 μL of each sample with Sample A using HBs antigen-positive blood (sample E) and 49 normal human plasmas
Was prepared. 100 μL was taken from Sample F, 100 μL of the particles of Reference Example was added to the remaining 4.9 mL, and the mixture was stirred for 10 minutes to perform virus adsorption treatment. After this, the particles were made 0.1% BSA, Tween
After washing four times with n80 (including 0.01) solution, sodium styrenesulfonate (0.1%) was added to dissociate the virus. Thereafter, the particles were separated by magnetism to obtain 100 μL of a virus concentrate (sample G). These samples were measured with Fujirebi Orcipulse HBs antigen.

[0066]

[Table 3]

[0067]

According to the method for detecting a target substance of the present invention, the target substance contained in the original specimen or sample is extremely small because the target substance in the specimen subjected to the nucleic acid amplification test has already been concentrated. Even in this case, the target substance can be efficiently detected.

Continued on the front page (51) Int.Cl. ⁷ Identification FI FI Theme Court II (Reference) // G01N 27/447 G01N 27/26 315Z 325B (72) Inventor Tatsushi Tanaka 3-15-14- Yoga, Setagaya-ku, Tokyo 107 (72) Inventor Mitsuhiro Murata 2--11-24 Tsukiji, Chuo-ku, Tokyo Inside JSR Corporation (72) Inventor Mikio Hikami 2-11-11 Tsukiji 2-chome, Chuo-ku, Tokyo FSR term inside (Reference) 2G052 AA29 AD06 AD54 BA02 BA21 ED03 JA13 JA16

Claims (7)

[Claims] An adsorbent capable of adsorbing a target substance is brought into contact with a sample containing a plurality of analytes and possibly containing a target substance, and then the adsorbent is separated from the sample. And then detecting a target substance bound to the adsorbent. 2. The method for detecting a target substance according to claim 1, wherein the sample is a body fluid. 3. The method for detecting a target substance according to claim 1, wherein the target substance is a virus, a nucleic acid, an antigen, an antibody or a protein. 4. The adsorbent capable of adsorbing a target substance is in the form of a particle, a filter, a fiber, a sheet, a tube, or a plate. Target substance detection method. 5. The target according to claim 1, wherein the adsorbent capable of adsorbing the target substance comprises an organic polymer, an inorganic polymer, an inorganic compound or a natural polymer. Substance detection method. 6. The target substance detection method according to claim 1, wherein the target substance is detected by a nucleic acid amplification method or an immunochemical measurement method. 7. An adsorbent used for the detection method according to claim 1.