JP2001158800A - Avidin particles - Google Patents

Abstract

(57) [Problem] To provide avidin particles on which avidin or streptavidin is immobilized and which is useful for an assay such as an immunoassay, an enzyme assay, and a nucleic acid assay. SOLUTION: The avidin particles of the present invention contain at least 30% by weight of an organic polymer substance, and a particle body having a carboxyl group exposed on the surface thereof, and avidin and strepto immobilized on the surface of the particle body. And at least one of avidin.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to avidin particles, and more particularly, to a carrier for diagnostic agents, a carrier for separating bacteria, a carrier for separating cells, a carrier for separating and purifying nucleic acids, a carrier for separating and purifying proteins,
The present invention relates to avidin particles useful as an immobilized enzyme carrier, drug delivery, and the like.

[0002]

BACKGROUND OF THE INVENTION It is known that avidin and biotin have a property of specifically binding to each other.
By utilizing such properties, affinity chromatography in which biotin is immobilized on cellulose and a stationary phase has been proposed, and assays (assays) such as immunoassays, enzyme assays, and nucleic acid assays have been proposed. Avidin is a protein having a molecular weight of about 260,000 and is difficult to synthesize artificially, whereas biotin has a molecular weight of 380
It can be easily and artificially synthesized, including its derivatives, and can easily label target substances such as specimens, enzymes, and nucleic acids. Therefore, if a method for capturing and separating / purifying a target substance labeled with biotin using avidin as a capture agent is established, it is extremely useful in the fields of biology, biochemistry, and medicine.

[0003] However, in the above-mentioned method, since biotin is used as a stationary phase, it is possible to capture and separate and purify a target substance labeled with avidin. It is not possible to separate and purify the target substance. Also,
Since the above-mentioned method uses a chromatography technique, it is necessary to use a relatively large amount of a target substance, and therefore, it is difficult to apply a small amount of the target substance.

On the other hand, as an assay method using avidin as a capturing agent, a means using a reaction vessel having avidin or streptavidin immobilized on the inner wall has been proposed (see Japanese Patent Application Laid-Open No. 24559/1990). However, in such means, the region for capturing the target substance is limited to the inner wall of the reaction vessel, and avidin or streptavidin is immobilized on the inner wall of the reaction vessel by physical adsorption means. For example, there is a problem that conditions such as a temperature, a type of a surfactant in a solution containing a target substance, and a salt concentration are limited.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made based on the above circumstances, and its object is to provide:
It is an object of the present invention to provide avidin particles obtained by immobilizing avidin or streptavidin and useful for an assay such as an immunoassay, an enzyme assay, or a nucleic acid assay. It is another object of the present invention to provide avidin particles that can be separated by magnetism.

[0006]

The avidin particles of the present invention contain at least 30% by weight of an organic polymer substance, and have a carboxyl group exposed on the surface of a particle body, and immobilized on the surface of the particle body. And at least one of avidin and streptavidin.

In the avidin particle of the present invention, it is preferable that the particle main body is non-porous. Also,
The organic polymer substance preferably contains at least 50% by weight of a structural unit derived from at least one monomer selected from acrylates and methacrylates. Further, it is preferable that the particle main body contains at least one of a paramagnetic substance and a strong paramagnetic substance. In the avidin particle of the present invention, a protein or oligonucleotide modified with biotin or a biotin derivative may be immobilized on the surface of the particle main body via avidin or streptavidin.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the avidin particles of the present invention will be described in detail. The avidin particle of the present invention comprises a particle body having a carboxyl group exposed on the surface, and at least one of avidin and streptavidin immobilized on the surface of the particle body.

The particle main body contains an organic polymer substance. As the organic polymer substance contained in the particle body, a water-insoluble substance is used, and as a monomer for obtaining such an organic polymer substance, an aromatic vinyl compound, an α, β-unsaturated Acid esters, α, β-
Unsaturated carboxylic acid amides, α, β-unsaturated nitrile compounds, vinyl halide compounds, conjugated diene compounds, lower fatty acid vinyl esters and the like can be used.

Specific examples of such a monomer include methyl acrylate, ethyl acrylate, n-butyl acrylate, 2-hydroxyethyl acrylate, polyoxyethylene acrylate, glycidyl acrylate, and ethylene glycol. Acrylic esters such as diacrylate, tribromophenyl acrylate, lauryl acrylate, tridecyl acrylate, stearyl acrylate, 2-ethylhexyl acrylate, cyclohexyl acrylate, benzyl acrylate, etc., methyl methacrylate, ethyl methacrylate N-butyl methacrylate, 2-hydroxyethyl methacrylate, polyoxyethylene methacrylate, glycidyl methacrylate, ethylene glycol dimethacrylate, tribromophenyl methacrylate, methacrylate Lauryl acrylate, tridecyl methacrylate, stearyl methacrylate, 2-ethylhexyl methacrylate, cyclohexyl methacrylate, methacrylate such as benzyl methacrylate, styrene, chlorostyrene, chloromethylstyrene, α-methylstyrene, divinylbenzene, styrene sulfone Sodium acrylate, acrylonitrile, methacrylonitrile, acrolein, methacrolein, acrylamide, methacrylamide, methylenebisacrylamide, methylenebismethacrylamide, butadiene, isoprene, vinyl acetate,
Vinylpyridine, N-vinylpyrrolidone, vinyl chloride,
And vinyl bromide. These can be used alone or in combination of two or more.

Among these, acrylates and methacrylates are preferred because of their low nonspecific adsorption of proteins, more preferably have 4 or more carbon atoms, more preferably 4 to 18 carbon atoms, particularly preferably 6 to 1 carbon atoms.
Acrylate and methacrylate having two hydrocarbon groups. Specifically, acrylic acid-n
-Butyl, lauryl acrylate, tridecyl acrylate, stearyl acrylate, 2-ethylhexyl acrylate, cyclohexyl acrylate, benzyl acrylate, n-butyl methacrylate, lauryl methacrylate, tridecyl methacrylate, stearyl methacrylate, methacryl 2-ethylhexyl acid, cyclohexyl methacrylate, benzyl methacrylate and the like, among which 2-ethylhexyl acrylate,
Particularly preferred are cyclohexyl acrylate, 2-ethylhexyl methacrylate, cyclohexyl methacrylate and the like.

In order to expose a carboxyl group on the surface of the particle body, a carboxyl group may be introduced into the organic polymer substance. The carboxyl group contributes to the dispersion stability of the particles and has a function of reacting with the amino group of avidin or streptavidin to immobilize them. Means for introducing a carboxyl group into an organic polymer substance include (a) means for copolymerizing a carboxyl group-containing unsaturated monomer with the above monomer, and (b) polymerizing the above monomer. Thereafter, means for modifying the obtained polymer with carboxylic acid can be used. In the case where a carboxyl group is introduced into the organic polymer substance by the means (a), specific examples of the carboxyl group-containing unsaturated monomer used include monocarboxylic acids such as acrylic acid, methacrylic acid, and crotonic acid; Examples thereof include dicarboxylic acids such as itaconic acid, maleic acid, and maleic anhydride and anhydrides thereof, and these can be used alone or in combination of two or more.

The amount of the carboxyl group exposed on the surface of the particle body is preferably in the range where the surface charge is 25 to 350 M equivalent / g, particularly 50 to 150 M equivalent / g. Surface charge by carboxyl group is 25M equivalent / g
If it is less than 1, it becomes difficult to immobilize a sufficient amount of avidin or streptavidin on the surface of the particle main body, and the obtained avidin particles may have low dispersion stability. On the other hand, when the surface charge by the carboxyl group exceeds 350 M equivalent / g, the polymerization method for obtaining the particles becomes complicated, and the reproducibility of the polymerization may decrease.

The content ratio of the organic high molecular substance in the particle body is 30% by weight or more, preferably 40% by weight or more. If the content is less than 30% by weight, the amount of components other than the organic polymer substance exposed on the surface of the particle body, for example, the amount of the inorganic component increases, so that when the particles are used, the inorganic component elutes. Or react with other substances used with the particles,
When used in assays, it may be difficult to perform the analysis with high accuracy.

The ratio of the area of the surface formed by the organic polymer substance to the area of the entire surface of the particle body is 9%.
0% or more, more preferably 95%
As described above, the content is particularly preferably 100%.

Although the particle body can be composed of only an organic polymer substance, it can be easily separated and recovered by a magnetic force. Therefore, the particle body is made of a magnetic material such as a paramagnetic material, a strong paramagnetic material, and a ferromagnetic material. (Hereinafter, these are collectively referred to simply as “magnetic material”). It is more preferable that both or one of the paramagnetic material and the strong paramagnetic material is contained. It is. In particular, it is preferable to use a strong paramagnetic material because there is no or little residual magnetization. Specific examples of such a magnetic material include triiron tetroxide (Fe ₃ O ₄ ), γ-iron trioxide (γ
—Fe ₂ O ₃ ), various ferrites, metals such as iron, manganese, cobalt, and chromium; and alloys such as cobalt, nickel, and manganese. Of these, triiron tetroxide is particularly preferable. It is preferable that such a magnetic substance has been subjected to lipophilic treatment.

The magnetic material used in the present invention is composed of particles having a small particle diameter, has excellent magnetic separability (the ability to separate in a short time by magnetism), and has a gentle vertical shaking operation. Is preferably redispersible. The particle diameter of the magnetic material is 40 to 300 °.
And more preferably 50 to 200.
Å, particularly preferably 60 to 150Å. When the particle size is less than 40 °, a treating agent (fatty acid, silane coupling agent,
A large amount of a titanium coupling agent) is required, and the proportion of the magnetic substance is reduced, which may result in poor magnetic responsiveness. On the other hand, when the particle diameter exceeds 300 °, the obtained particle bodies become magnetized, and the particle bodies are easily aggregated with each other and easily settle in the aqueous medium. The particle diameter of the non-spherical magnetic material is an average value of the longest diameter and the shortest diameter of each magnetic material. When a ferromagnetic material is used, it is particularly preferable that the magnetic domain is two to three times the size of a single magnetic domain.

The content of the magnetic substance in the particle body is set to 70% by weight or less, since the content of the organic polymer substance is 30% by weight or more, but is preferably 20 to 70% by weight.
%, More preferably 30 to 70% by weight. When this ratio is less than 20% by weight, sufficient magnetic responsiveness is not exhibited, and it may be difficult to separate particles in a short time by a required magnetic force. On the other hand, when this ratio exceeds 70% by weight, the amount of the magnetic material exposed on the surface of the particle main body increases, so that constituents of the magnetic material, for example, iron ions are eluted, and when used, other materials are used. This may have an adverse effect, and the particle main body may be brittle, so that practical strength may not be obtained.

When a magnetic substance is contained in the avidin particles of the present invention, the magnetic properties thereof are 15,000.
When the magnetic field of the Osted (Oe) is applied,
Coercive force of 50-2000 Oe, especially 100-1000 Oe
e, and the residual magnetic flux density is 1.0 to 70
emu / g, particularly preferably 2 to 20 emu / g. When the coercive force is less than 50 Oe or the residual magnetic flux density is less than 1 emu / g, a sufficiently large magnetic force acts between the particles even if the magnetic field is magnetically sensitized by a natural magnet (magnetic flux density is, for example, 1000 to 4000 Gauss). Therefore, the natural magnet separates particles with high efficiency.
It is difficult to recover, which is not practically preferable. On the other hand, when the coercive force exceeds 2,000 Oe or the residual magnetic flux density exceeds 70 emu / g, the magnetic force acting between the particles becomes excessively large.
It becomes difficult to redistribute.

The main body of the particle is preferably non-porous, so that a region capable of capturing a target substance such as a nucleic acid or a protein is limited to the surface, and as a result, the target can be obtained in a short time. Substances can be captured.
When the particle main body is porous, a considerably long time is required to capture the target substance because the process of entry of the target substance into the pores of the particle is governed by diffusion.

The number average particle diameter of the particle body is 0.1
It is preferably from 15 to 15 μm, particularly preferably from 0.5 to 10 μm. When the number average particle diameter is less than 0.1 μm, when the particle main body contains a magnetic substance, sufficient magnetic responsiveness is not exhibited, which is considerable for separating the particles. It takes a long time, and a considerably large magnetic force is required for separation, which is not preferable. On the other hand, when the number average particle size exceeds 15 μm,
Because the particles are likely to settle in the aqueous medium,
When capturing the target substance, an operation of stirring the medium is required, and the surface area of the particle body becomes small, so that it may be difficult to capture a sufficient amount of the target substance.

The particle body as described above can be produced by various methods. However, when producing a particle body containing a magnetic substance, the whole monomer for forming the organic polymer substance is used. A monomer composition is prepared by dispersing the lipophilic magnetic substance in a hydrophobic monomer of the body, and this monomer composition is used as an organic phase in an aqueous phase. It is preferable to adopt a method in which a suspension is prepared by emulsifying and dispersing the monomer in the suspension, and a monomer containing the hydrophobic monomer is polymerized in the suspension. Here, “hydrophobic monomer” refers to a monomer having a solubility in water at 20 ° C. of less than 0.1% by weight.

When a magnetic material that has not been subjected to lipophilic treatment is used in the preparation of the monomer composition, the magnetic material is unevenly distributed near the surface of the particle main body from which the magnetic material is obtained. Since the body is exposed, iron ions and the like may elute into the aqueous medium. The method for lipophilizing the magnetic material is not particularly limited, such as a method of treating with a surface treating agent such as a silane coupling agent or a titanium coupling agent, or a method of adsorbing a fatty acid salt or the like to the magnetic material. Further, a magnetic substance obtained by removing a dispersion medium from a commercially available magnetic fluid can also be used. Further, the amount of the magnetic substance dispersed in the hydrophobic monomer depends on the amount of the hydrophobic monomer.
It is usually 2 to 300 parts by weight based on parts by weight.

In the preparation of the suspension, when a monomer other than the hydrophobic monomer is used as the dispersion medium of the magnetic substance, the lipophilicized magnetic material is added to the lipophilic monomer. The particles cannot be uniformly dispersed, and coarse particles are precipitated by aggregation of the magnetic substance in the obtained monomer composition, which makes it difficult to emulsify and disperse the monomer composition in an aqueous phase.

As the disperser used for emulsifying and dispersing the monomer composition (organic phase) in the aqueous phase, conventionally known ultrasonic dispersers and dispersers having a high shear rate can be used. . The type of the ultrasonic dispersing machine is not particularly limited. For example, a dispersing machine having a horn-type oscillator, a dispersing machine having a plate-type oscillator, and a continuous dispersing machine for flowing a liquid through an oscillating unit. And the like. Examples of the high shear rate disperser include a homomixer, a colloid mill, a jet homogenizer, a high-pressure homogenizer, and the like.These dispersers may be appropriately selected according to the target dispersed particle diameter. Can be. Further, a membrane emulsification method in which an organic phase is emulsified and dispersed in an aqueous phase by extruding a monomer composition (organic phase) into an aqueous phase through a porous membrane or a porous filter can also be adopted.

The aqueous phase serving as a dispersion medium for the monomer composition contains an anionic surfactant, a nonionic surfactant,
A surfactant or an emulsifier such as a water-soluble polymer and an inorganic suspension protective agent may be added. Among these, partially saponified polyvinyl alcohol is preferred in that it can be applied to obtain particles having a wide range of particle diameters, and that particles having both positive and negative surface charges can be synthesized.

Then, after adding a monomer other than the hydrophobic monomer to the suspension thus prepared, the hydrophobic monomer and the other monomer are added to the suspension. Polymerize the monomer. The polymerization initiator for polymerizing the monomer is not particularly limited, and a conventionally known radical polymerization initiator, for example, an organic peroxide, an azo-based initiator, a persulfate-based initiator, or the like is used. can do. Among these, it is preferable to use an oil-soluble polymerization initiator. In particular, when preparing a monomer composition by dispersing a magnetic substance in a hydrophobic monomer, it is preferable to add an oil-soluble polymerization initiator, thereby producing a particle body containing no magnetic substance. Can be prevented.

The polymerization reaction of the monomer is preferably performed in an oxygen-free atmosphere. The reaction temperature varies depending on the decomposition temperature of the polymerization initiator to be used. For example, when benzoyl peroxide is used, the polymerization reaction can suitably proceed at 75 to 85 ° C.

In order to produce a particle body having no magnetic material exposed on the surface, (1) a monomer other than the hydrophobic monomer added to the suspension is divided into two parts. A method in which one is added to a suspension to polymerize, and then the other is added to further carry out polymerization. (2) After the polymerization reaction is completed, a strong acid is brought into contact with the obtained particle body to be present near the surface of the particle body. A method of eluting and removing a magnetic substance to be formed can be used.

The avidin particles of the present invention are those in which avidin and / or streptavidin are immobilized on the surface of the particle body as described above. As avidin and streptavidin, commercially available ones can be used.
It may be a product obtained by separating and purifying a product existing in a natural body, or a product produced artificially by a genetic engineering technique. As a method of immobilizing avidin or streptavidin on the surface of the particle body, for example, in the presence of a dehydration condensing agent such as a water-soluble carbodiimide, an amino group in the molecule of avidin or streptavidin,
There is a method in which avidin or streptavidin is immobilized on the surface of the particle main body by reacting with a carboxyl group exposed on the surface of the particle main body to form an amide bond. In such a method, a carboxyl group exposed on the surface of the particle main body may be previously reacted with a dehydration condensing agent, and then avidin or streptavidin may be added and reacted.

According to the above-mentioned avidin particles, since avidin or streptavidin is immobilized on the surface of the particle body, it is possible to reliably capture a target substance labeled with biotin, such as a nucleic acid or a protein. Can be. In addition, since avidin or streptavidin is immobilized by a chemical bond with a carboxyl group exposed on the surface of the particle main body, use conditions are rarely limited. Therefore, such avidin particles are useful as a carrier for diagnostic agents, a carrier for separating bacteria, a carrier for separating cells, a carrier for separating and purifying nucleic acids, a carrier for separating and purifying proteins, an immobilized enzyme carrier, drug delivery and the like.

The avidin particle of the present invention comprises a protein or an oligonucleotide modified with biotin or a biotin derivative (both are collectively referred to as “biotins”), and avidin or streptavidin. May be immobilized on the surface.

More specifically, nucleic acid-immobilized particles can be obtained by immobilizing oligonucleotides modified with biotin on the surface of avidin particles. Here, the length of the oligonucleotide to be immobilized is 10 to 100 bases, preferably 15 to 70 bases. The oligonucleotide to be immobilized is 1
It may be single-stranded DNA, double-stranded DNA, or RNA. Such a nucleic acid is
It can be prepared using a commercially available nucleic acid synthesizer. Such a nucleic acid-immobilized particle can be used for gene diagnosis and other genetic engineering in general. Specifically, messenger RNA can be directly recovered from a cell lysate using, for example, magnetic polymer particles to which oligo dT is bound. Also, by using a nucleic acid having a base sequence complementary to the AIDS virus nucleic acid as the nucleic acid to be immobilized, the AIDS virus is recovered from the biological sample and detected using a DNA probe method or a polymerase chain reaction method. can do.

Further, by immobilizing a bioactive protein modified with biotin on the surface of avidin particles, protein-immobilized particles carrying the bioactive protein can be prepared. The bioactive protein includes a protein having a general bioactivity or a complex thereof, and specific examples include immunoglobulins and various enzymes. Such antibody-bound particles and enzyme-bound particles can be used in the fields of diagnosis and research.

In the above, as the biotin derivative,
Biotin-ε-N-lindine, biocytin hydrazide,
2-iminobiotin, amino or sulfhydryl derivatives of biotinyl-ε-N-aminocaproic acid-N-hydroxysuccinimide ester, sulfosuccinimidiminodiotin, biotin bromoacetylhydrazide,
For example, p-diazobenzoylbiotin, 3- (N-maleimidopyrionyl) biotin, or the like can be used.

As a method for modifying a protein or an oligonucleotide with biotin, for example, an ester of biotin and N-hydroxyimide, for example, biotin-N-hydroxysuccinimide is reacted with an amino group of a protein molecule. A method of modifying a protein with biotins, binding phthalimidotriethylene glycol to the 5 'end of an oligonucleotide, and hydrolyzing it with ammonium hydroxide to form a primary amino group; For example, a method of modifying the 5 ′ end of an oligonucleotide with biotin by binding biotin-N-hydroxysuccinimide to the group may be mentioned, but not limited thereto. The way Can use, by an appropriate method, it is also possible to modify the 3 'end of the oligonucleotide by biotin.

As a method for immobilizing biotins modified to proteins or oligonucleotides on avidin or streptavidin immobilized on the surface of particles, a conventionally known method for immobilizing biotins on avidin is known. Can be applied. For example, in a phosphate buffer or a phosphate buffer containing 1 M sodium chloride, avidin particles and a protein or oligonucleotide modified with biotins are mixed for 10 minutes at room temperature.
After mixing for 1 hour, unreacted proteins or oligonucleotides are removed by a solid-liquid separation operation, whereby particles having the proteins or oligonucleotides immobilized thereon can be prepared.

[0038]

EXAMPLES The present invention will now be described by way of specific examples, which should not be construed as limiting the invention thereto.

Example 1 1 mL of a 1% dispersion (manufactured by JSR Co., Ltd.) of carboxyl group-introduced polystyrene particles (average particle size: 1.0 μm, carboxyl group surface charge: 70 M equivalent / g) was Centrifuge,
An operation of adding a 0.1 mM HCl solution (pH = 5.5) to a solid substance (polystyrene particles) and centrifuging the solid substance is described in 3 below.
By repeating the process twice, the polystyrene particles were washed. Then, 1-ethyl-3- was added to the polystyrene particles.
0.1 mM HCl solution in which 5 mg of dimethylaminopropylcarbodiimide hydrochloride (manufactured by Dojin Kagaku) is dissolved
After stirring at room temperature for 2 hours, 0.1 mg of streptavidin (manufactured by Sigma) 1 mg was dissolved.
Avidin particles were prepared by immobilizing streptavidin on the surface of polystyrene particles by adding 0.1 mL of an MHC1 solution and rotating and stirring at room temperature for 8 hours.

Next, the solution containing the avidin particles was centrifuged, and the solid substance (avidin particles) was added to a phosphate buffer solution (PBS, 0.1%) containing 0.1% bovine serum albumin.
% BSA / PBS, pH = 7.2) and centrifugation were repeated three times to remove unreacted streptavidin. And avidin particles,
A phosphate buffer (PBS, 0.1% BSA / PB) containing 0.1% bovine serum albumin so that the concentration becomes 1%.
S, pH = 7.2) to prepare an avidin particle dispersion.

Example 2 Instead of a 1% dispersion of polystyrene particles, magnetic polymer particles having carboxyl groups exposed on the surface [magnetic substance: γ-Fe ₂ O ₃ , organic polymer substance: polycyclohexyl methacrylate, magnetic Body: organic polymer substance = 50: 50 (weight ratio), average particle size 1.2
μm, ratio of organic polymer on particle surface:
An avidin particle dispersion was prepared in the same manner as in Example 1 except that a 10% dispersion of 99%, carboxyl group surface charge: 60 M equivalent / g) was used.

[Experimental Example 1] 100 μL of the avidin particle dispersion prepared in Examples 1 and 2 (avidin particle 1
0 μg) was placed in each of the centrifuge tubes, and each of these centrifuge tubes was filled with the fluorescent dye FAM for 5 ′.
200 pmol of a synthetic DNA oligomer (30mer) whose end is labeled and whose 3 ′ end is labeled with biotin
Was dissolved in 1 mL of a phosphate buffer (pH = 7.2), and the mixture was rotated and stirred at room temperature for 15 minutes. Thereafter, after separating the avidin particles from the phosphate buffer, the fluorescence intensity of the phosphate buffer is measured, and the fluorescence intensity of the stock solution of the phosphate buffer in which the synthetic DNA oligomer is dissolved is measured.
From these values, the capture amount of the synthetic DNA oligomer by the avidin particles (the capture amount per mg of particles) was calculated. As a comparative example, the amount of synthetic DNA oligomers captured was calculated in the same manner for the polystyrene particles (Comparative Example 1) and the magnetic polymer particles (Comparative Example 2) used in Example 1. The results are shown in Table 1.

[0043]

[Table 1]

Example 3 100 μL of the avidin particle dispersion prepared in Example 1 (corresponding to 1 mg of avidin particles) was placed in a centrifuge tube, and a synthetic DNA oligomer (base having a 3 ′ end modified with biotin) was added to the centrifuge tube. The sequence “5 ′ TTTTTTTTTTTTTTTTTTTTTT
TTTTTTTTTTTT 3 '", 30mer) 200
pmol dissolved phosphate buffer (pH = 7.2) 1
by adding 15 mL and rotating and stirring at room temperature for 15 minutes.
Avidin particles to which the synthetic DNA oligomer was immobilized were prepared. Next, the solution containing the avidin particles was centrifuged, and the solid substance (avidin particles) was added to a phosphate buffer solution (PBS, 0.1% B) containing 0.1% bovine serum albumin.
The operation of adding SA / PBS, pH = 7.2) and performing centrifugation was repeated three times to remove the unimmobilized synthetic DNA oligomer. Then, avidin particles were added to a phosphate buffer solution (PBS, 0.1% BS) containing 0.1% bovine serum albumin so that the concentration became 1%.
A / PBS, pH = 7.2) to prepare an avidin particle dispersion.

Experimental Example 2 100 μL of the avidin particle dispersion prepared in Example 3 (corresponding to 1 mg of avidin particles) was
Each of these centrifuge tubes was placed in a centrifuge tube, and a synthetic DNA oligomer (base sequence “5′AAAAAAAA”) labeled at the 5 ′ end with a fluorescent dye FAM was added to each of the centrifuge tubes.
AAAAAAAAAAAAAAAAAAAAAAAAA
3 '", 30mer) Buffer solution (10 mM Tris-HCl, 0.1 M sodium chloride, pH
= 7.6) 1 mL was added and the mixture was rotationally stirred at 37 ° C for 30 minutes. Thereafter, after separating the avidin particles from the buffer solution, the fluorescence intensity of the buffer solution was measured, and the fluorescence intensity of the stock solution of the buffer solution in which the synthetic DNA oligomer was dissolved was measured. The amount of oligomers captured (the amount captured per mg of particles) was calculated.
As a result, the capture amount of the synthetic DNA oligomer was 115 pm
ol / mg.

[0046]

According to the avidin particle of the present invention, since avidin or streptavidin is immobilized on the surface of the particle body, a target substance labeled with biotin, such as a nucleic acid or a protein, is reliably captured. be able to. In addition, since avidin or streptavidin is immobilized by a chemical bond with a carboxyl group exposed on the surface of the particle main body, use conditions are rarely limited. Such avidin particles are useful in assays such as immunoassays, enzyme assays, nucleic acid assays, and the like.

Further, by making the particle body non-porous, avidin particles that can reliably capture the target substance in a short time can be obtained. By adding a paramagnetic substance or a superparamagnetic substance to the particle body, avidin particles that can be easily separated by magnetism can be obtained. Further, by immobilizing a protein or oligonucleotide modified with biotin or a biotin derivative on the surface of the particle main body via avidin or streptavidin, it can be applied to a wide range, for example, as a diagnostic agent.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/545 G01N 33/545 Z F-term (Reference) 4B063 QA01 QA12 QA18 QQ42 QR32 QR48 QR54 QR56 QR83 QS13 QS34 QS36 QX02 4H045 AA10 BA62 EA61 FA81

Claims (5)

[Claims] 1. A particle body containing at least 30% by weight of an organic polymer substance and having a carboxyl group exposed on its surface;
Avidin particles comprising at least one of avidin and streptavidin immobilized on the surface of the particle body. 2. The avidin particle according to claim 1, wherein the particle body is non-porous. 3. The organic polymer substance comprises at least one selected from acrylates and methacrylates.
The avidin particles according to claim 1 or 2, comprising 50% by weight or more of a structural unit derived from a kind monomer. 4. The avidin particle according to claim 1, wherein the particle main body contains at least one of a paramagnetic substance and a ferroparamagnetic substance. 5. A protein or oligonucleotide modified with biotin or a biotin derivative,
The avidin particle according to any one of claims 1 to 4, wherein the avidin particle is immobilized on the surface of the particle main body via avidin or streptavidin.