JP2002165591A - Magnetic particle and method for using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a material capable of specifically capturing a virus and utilizable for separation, concentration and detection of the virus and to provide a method for using the material. SOLUTION: This magnetic particle has a lectin bindable to the surface layer of the virus on the surface and 0.05-10 μm particle diameter.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a material which specifically captures a virus and can be used for the isolation, concentration and detection of the virus, and a method for using the same.

[0002]

2. Description of the Related Art A typical example of a conventional virus concentration method is an ultracentrifugation method. However, it requires expensive equipment, a long separation time, skilled workers, and it is difficult to process a large number of samples at the same time. It is hard to say that it is a simple method. In addition, since a hepatitis B virus surface antigen (HBs antigen) binds to heparin, a chromatography method using a heparin sepharose carrier has been reported, but it is also difficult to treat a large number of samples at the same time. In addition, ammonium sulfate, polyethylene glycol, polyanion and 2
Although there is a method of precipitating a virus by a combination of valent ions and the like, there is a drawback that sample purification after precipitation and separation of the virus is necessary due to problems such as PCR inhibition of the reagents to be mixed. On the other hand, as a method for eliminating the risk of virus infection, a method has been proposed in which a virus-containing sample is used by removing the virus with a filter or the like when using the sample. Materials used for such purposes include filters and microparticles that bind lectins that have an affinity for viruses and selectively eliminate viruses.
(JP-A-10-45601). Lectin-immobilized affinity chromatography columns are also commercially available, but these filters and column systems generally require a lower concentration of recovered virus than the batch method, and require improvement in applications for virus detection. In addition, the processing time of the column method is longer than that of the batch method, and the operation is at another stage, so that there are many limiting factors in realizing automation.

[0003]

SUMMARY OF THE INVENTION An object of the present invention is to solve the above-mentioned problems, to selectively adsorb a virus, and to facilitate the subsequent processing, and to use the magnetic particles. It provides a method.

[0004]

As a result of research, the present inventors have developed the following magnetic particles and a concentration method as means for solving the above-mentioned problems. That is, the present invention provides a magnetic particle having a mannose-binding lectin on its surface
"Magnetic particles"). The present invention also includes, secondly, an operation of bringing the virus-containing sample into contact with the above-described magnetic particles to bind the virus to the particles, and then separating and collecting the virus-bound particles from the sample. And methods for concentrating and detecting viruses.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The magnetic particles of the present invention contain a magnetic substance inside polymer particles. Such magnetic materials include various ferrites such as triiron tetroxide (Fe ₃ O ₄ ) and γ-iron sesquioxide (γ-Fe ₂ O ₃ ), iron, manganese, and the like.
Metals such as cobalt and chromium, and alloys of these metals can be used. The size of the magnetic material used for the magnetic particles of the present invention is preferably 20 nm or less. 20nm
Above, the influence of residual magnetization appears, and even after the magnetic field is removed, mutual coupling between the magnetic particles remains, which affects the particle dispersibility and the virus extraction efficiency, which is not preferable. It is preferable that this magnetic material is contained only in the inside of the particle and is not exposed on the particle surface.

[0006] The content of the magnetic material is 1 to the whole magnetic particles.
It is preferably at least 0% by weight, particularly preferably 20 to 90% by weight. Here, if the content of the magnetic substance is too small, good magnetic separability cannot be obtained for the magnetic particles, and as a result, in the virus separation / concentration method described below, the magnetic particles are separated from a sample such as blood or body fluid. For this reason, a considerably long time is required, so that high temporal efficiency may not be obtained, which is not preferable.

Although the particle size of the magnetic particles of the present invention is not particularly limited, it is generally 0.08 m to 300 μm, preferably 0.
It is 1 μm to 100 μm. When the particle size is less than 0.08 μm,
It is not preferable because the time required for magnetic separation becomes long. On the other hand, if the particle size exceeds 300 μm, the surface area of the particle per unit mass decreases, and the amount of immobilized lectin decreases in proportion. The shape of the magnetic particles of the present invention need not be spherical, and may be irregularly shaped particles. The particle diameter of the non-spherical particles is an average of the longest diameter and the shortest diameter of each particle.

[0008] The magnetic particles of the present invention can be prepared by the following method. (A) Polymerization is carried out by mixing a magnetic substance with a polymerization component containing a monomer constituting a polymer. As the polymerization method, an ordinary method for obtaining an aqueous polymer dispersion can be used, and examples thereof include emulsion polymerization, suspension polymerization, and dispersion polymerization. (B) A polymerization component containing a monomer constituting a polymer is polymerized by a method for obtaining a normal aqueous polymer dispersion to obtain an aqueous polymer dispersion. A magnetic layer is formed on the surface of the particles. (C) The polymer is dissolved in a solvent substantially insoluble in water and having a boiling point lower than that of water, and the magnetic substance is dispersed in this solution. This is dispersed in water using a suitable emulsifier or dispersant, and the solvent is removed by distillation. (D) Using the polymer aqueous dispersion obtained in (a), (b) or (c) above as a seed, adding a monomer and polymerizing to form a polymer layer on the particle surface. The magnetic particles of the present invention are preferably non-porous.
In the case of porous particles, in lectin fixation and virus extraction, the reaction rate in the pores and the reaction rate on the particle surface are extremely different, so that good reproducible results may not be obtained.

In the present invention, as the lectin capable of binding to the virus surface, it is preferable to use a mannose-binding lectin which is often present on the virus surface. The mannose-binding lectin used in the present invention is a lectin that mainly recognizes an α-mannosyl residue which is a constituent saccharide of a mother nucleus of an asparagine-linked sugar chain,
navalia ensiformis (ConA), Lens culinaris (LCA), Bow
ringia midbraedii (BMA), Dolichos lablab (DLA), Galant
hus nivalis (GNA), Gerardia savaglia (GSL), Machaer
ium biovulatum (MBA), Machaeriumu lunatus (MLA), Na
rcissus pseudonarcissus (NPA), Epipactis heleborine
(EHA) and Listera ovata (LOA). From the viewpoint of function and economy, ConA is particularly preferable among mannose-binding lectins. ConA that can be used is usually purified from jack bean. In order to bind the mannose-binding lectin compound to the surface of the magnetic particles, it is preferable to introduce a functional group such as an epoxy group, an amino group, an aldehyde group, a carboxyl group, a hydroxyl group, or an acid chloride group onto the surface of the magnetic particles. Examples of the method include a method of polymerizing a functional group-containing copolymerizable monomer as a copolymerizable monomer during the production of the magnetic particles, and a method of converting the monomer into a necessary functional group by a chemical reaction. Methods for binding a mannose-binding lectin to the active functional group include a cyanogen bromide activation method, a condensation reagent method using a carbodiimide reagent or a Woodward reagent, a diazo method via a diazonium compound, an acid azide derivative method, Any method such as a halogenated acetyl derivative method, a triazinyl derivative method, a halogenated methacrylic (acrylic) acid derivative method, and a cross-linking method using a polyfunctional cross-linking agent such as glutaraldehyde or a double-ended epoxy compound is possible. They can be bonded directly or via a coupling agent or a spacer.

The mannose-bound lectin-immobilized magnetic particles thus obtained may contain an emulsifier, a dispersant, an unreacted monomer, a water-soluble polymer, a decomposition product of a polymerization initiator, and the like in the dispersion medium. These substances, for example, when used in medical diagnostics, are highly likely to become reaction inhibitors in the nucleic acid amplification test stage, for example, Adv.Co.
It is preferable to remove the mannose-bound lectin-immobilized magnetic particles from the dispersion medium by a method such as that described in Lloid Interface Sci., 81, 77-165 (1999).

A dispersion of mannose-bound lectin-immobilized magnetic particles is prepared by mixing a certain amount of the dispersion with a sample, for example, at room temperature for 10 minutes.
The virus in the sample can be bound by incubating up and down for about a minute. Viruses to be concentrated in the product of the present invention include AIDS, hepatitis B, hepatitis C, and adult T-cell leukemia virus. In addition, filovirus that causes Ebola hemorrhagic fever, hantavirus that causes renal symptomatic hemorrhagic fever, and the like are also included. An appropriate buffer, chelating agent, metal ion or the like may be added to these individual virus concentrations depending on the situation.

[0012]

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples. In Examples, “parts” and “%” are expressed in terms of weight. Reference Example 1 Acetone was added to an oil-based magnetic fluid "Fericolloid HC50" (manufactured by Taiho Kogyo Co., Ltd.) to precipitate and precipitate, and then dried to obtain a ferritic material having a lipophilic surface. Superparamagnetic material (particle size: 0.01
μm). Next, 95 parts of cyclohexyl methacrylate, 5 parts of methacrylic acid, and 3 parts of benzoyl peroxide (polymerization initiator) were added to 40 parts of the superparamagnetic material, and the superparamagnetic material was uniformly dispersed by mixing and stirring the system to obtain a monomer. The composition was prepared. On the other hand, 10 parts of polyvinyl alcohol, 0.05 parts of sodium lauryl sulfate and 0.1 part of polyethylene oxide nonylphenyl ether were used.
One part was dissolved in 1000 parts of water to prepare an aqueous dispersion. The above-mentioned monomer composition was added to the obtained aqueous medium (aqueous phase), and the mixture was preliminarily stirred with a homogenizer, and then subjected to dispersion treatment with an ultrasonic disperser, whereby oil droplets (oil phase) having an average particle diameter of 1 μm were formed. A suspension (oil droplet dispersion) dispersed in an aqueous medium was prepared. Next, the obtained suspension was charged into a two-liter three-necked flask equipped with a stirrer, and
The temperature was raised to 0 ° C., and the monomers in the oil droplets were polymerized (suspension polymerization) for 5 hours while stirring under a nitrogen atmosphere to produce magnetic particles. 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. 1 g (dry weight) of the magnetic polymer particles was added to 10 ml of 10 mM MES.
EDC hydrochloride (1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride) which is dispersed in a buffer solution (pH 6) and is a water-soluble carbodiimide reagent
0.05 g was added and reacted at 20 ° C. for 1 hour. Then, 10m of 0.5% Concanavalin A (ConA, manufactured by SIGMA)
4 ml of M MES buffer solution was added and reacted at 20 ° C. for 2 hours. The magnetic particles were precipitated by centrifugation (5000 × g, 15 minutes), and the unreacted ConA in the supernatant was removed. Co in the supernatant
The amount of nA was quantified from a calibration curve prepared using ConA using BCA Protein Assay Reagent A manufactured by PIERCE, and the amount of ConA reacted with the particles was determined. As a result, the amount was 15 mg / g of the particles. The particles were redispersed in physiological saline to a concentration of 5% by weight.

Example 1 Evaluation of HIV-1 enrichment ability HIV-1 positive plasma (virus concentration 5 × 10 ⁶ copies / ml) was used for HI
Serial dilution with V-1 negative plasma, virus concentration 5 × 10 ⁴ c
opies / ml (sample A), 5 × 10 ³ copies / ml (sample B),
5 × 10 ² copies / ml (sample C), 5 × 10 ¹ copies / ml
Each sample of (Sample D) was prepared. For 1 ml of each sample,
100 μL of a physiological saline dispersion (5% by weight) of the magnetic particles obtained in Reference Example 1 was added, and mixed at room temperature for 10 minutes to adsorb the virus to the particles. The virus-adsorbed magnetic particles are separated from the supernatant by magnetic separation.
Was removed to obtain 100 μL of a magnetic particle dispersion (samples a, b, c, and d, respectively). From this dispersion, viral nucleic acids were extracted using a commercially available reagent (Smitest EX R & D, Genome Science Laboratories). However, the magnetic particles were removed by magnetic separation after the virus lysis reaction step. As a control, viral nucleic acids were extracted from 100 μL of Samples A to D by Sumitest EX R & D. Detection and quantification of the extracted viral nucleic acids were performed using the LightCycler System (Roche Diagnostics, Inc.) using RT.
The amplification reaction by -PCR was carried out by a method of quantitatively detecting the amplification reaction with a fluorescent reagent trade name SYBR green I. The specificity of the amplification was verified by measuring the melting temperature of the amplified product by measuring the melting curve. The base sequence of the primer pair in RT-PCR is 5 ′
-CCC ACA AGA TTT AAA CAC CA-3 ', 5'-TGA AGG CTA GTA
GTT CC-3 'was used. Quantification of viral nucleic acid amount is based on the number of cycles (Th cycle) in which the fluorescence intensity, which increases in correlation with the amount of amplification product, exceeds a certain reference value (Threshold: Th) in the amplification process of 45 times. It was extrapolated to a calibration curve of DNA amount-Th cycle, which was prepared by simultaneously measuring the dilution series of the product. As a result, the virus-concentrated magnetic particles are 5 ×
Virus-positive plasma in a wide concentration range of 10 ^{4 to} 5 × 10 ¹ copies / mL could be concentrated to 1/10 volume with a virus concentration efficiency of 80% or more (Table 1).

[0014]

[Table 1]

[0015]

The magnetic particles of the present invention contain a magnetic component,
In addition, since the lectin is bound to the surface, virus can be concentrated very easily by capturing the virus on the particles and magnetically separating the particles. Even if the amount of the contained virus is very small and it is difficult to detect it, the detection can be ensured by concentrating the virus using the present particles. In addition, since the concentration can be performed by magnetic separation, it can be easily applied to an automatic concentrator for processing multiple samples. The magnetic particles of the present invention specifically bind to a virus in a sample such as blood or body fluid, and are identified by a method such as an immunoreaction of the virus separated and concentrated by the particle, or nucleic acid extraction of the virus, and nucleic acid detection. , Can be quantified. The product of the present invention is particularly suitable for a nucleic acid detection method using a nucleic acid amplification method.

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Mikio Hijikata F-term (reference) 4J063 QA01 QA18 QA19 QQ03 QQ10 QR48 QR50 QR54 QS15 QX02 4B065 AA95X BA22 BA30 BD14 BD22 CA46 CA56 4C087 AA01 AA02 DA06 NA01 ZA51

Claims (6)

[Claims] 1. A magnetic particle having a lectin capable of binding to a virus surface layer and having a particle size of 0.05 μm to 10 μm on the surface. 2. The magnetic particle according to claim 1, wherein the lectin is a mannose-binding type. 3. The magnetic particle according to claim 2, wherein the mannose-binding lectin is concanavalin A. 4. A method for adsorbing a virus, comprising: bringing a magnetic particle according to claim 1 into contact with a sample which may contain a virus to bind the virus to the particle surface. 5. A method for removing a virus from a sample, comprising removing the virus bound to the magnetic particles by the method according to claim 4. 6. A method for concentrating a virus, comprising separating a virus bound to magnetic particles by the method according to claim 4 from a sample and collecting the virus.