JPH0587812A - Immunological diagnostic medicine carrier particle and its manufacture - Google Patents

Abstract

PURPOSE:To provide an immunological diagnostic medicine carrier particle excellent in sharpness of reaction and preservation stability and capable of fixing enzyme, protein, or immunity activating matter easily. CONSTITUTION:In a suspension of hydrophilic gel fine grain obtained by dispersing and polymerizing a monoethylene unsaturated amid of 20.0 to 94.8 % by weight, a cross linking thylene unsaturated monomer of 5.0 to 60.0% by weight, an ethylene unsaturated carboxylic acid of 0.1 to 30.0% by weight, (meta) acrylic acid ester of 0.1 to 50.0% by weight, and a monomer of 0 to 30.0% by weight capable of copolymerizing with these monomers, a fine grain hydrophobic monoethylene unsaturated monomer of 1 to 50% by weight is polymerized by water soluble radical polymerization start agent with a hydrophilic get fine grain as a soil grain.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to immunological diagnostic agent carrier particles and a method for producing the same, and more specifically, it has excellent reaction sensitivity and storage stability, and easily immobilizes enzymes, proteins or immunologically active substances. The present invention relates to an immunological diagnostic agent carrier particle and a method for producing the same.

[0002]

2. Description of the Related Art Among immunological test methods utilizing a specific antigen-antibody reaction, the particle agglutination method is significantly simpler than radioimmunoassay (RIA) method, enzyme-labeled immunosorbent assay (ELISA) method and the like. Therefore, there is an advantage that the time required for the inspection is short. In this particle agglutination method, red blood cells are used as carrier particles. However, red blood cells immobilized with an antigen such as glutaraldehyde are likely to cause nonspecific agglutination due to the antigen held by the red blood cells themselves. In addition, since red blood cells are biological substances, there are storage problems such as easy decomposition, and there are variations among individual particles and product lots, making it difficult to maintain the same quality when reagents are used. .. Gelatin has been proposed as a carrier for improving these drawbacks of red blood cells (Japanese Patent Laid-Open No. 58-11375).
6). However, since gelatin particles are also natural amphoteric polymers, their isoelectric points differ from lot to lot,
It is difficult to obtain products of the same quality. Further, gelatin particles are usually produced by dispersing an aqueous gelatin solution in an organic solvent that is immiscible with water to prepare aqueous gelatin droplets, and then reacting with a crosslinking agent such as glutaraldehyde to form particles. However, the obtained particles have a wide distribution of particle diameters and the particle diameters among product lots also vary. Therefore, there is a problem that the agglutination reaction using gelatin particles lacks sensitivity. Recently, synthetic latex particles have been developed as a material that overcomes the above-mentioned drawbacks of red blood cells and gelatin. However, since these particles have polystyrene as a main component, they are strongly hydrophobic, and when used in microplate particles mainly used for agglutination reaction, nonspecific agglutination is likely to occur, which is not preferable. Therefore, the synthetic latex particles are currently used mainly in the glass plate method, which requires less precision than the microtiter method. On the other hand, the immobilization of an antigen such as a protein on the surface of a particle can be relatively easily carried out by a so-called hydrophobic-hydrophobic bond utilizing the hydrophobic part of the protein in the case of highly hydrophobic polystyrene particles on the surface of the particle. .. On the other hand, in the case of red blood cells and gelatin particles, the surface of the particles is hydrophilic, so when immobilizing antigens such as proteins, covalent bonds must be formed by the carbodiimide method, glutaraldehyde method, cyanogen bromide method, etc. In addition, the process is complicated and quality reproducibility is poor.

[0003]

DISCLOSURE OF THE INVENTION As a result of intensive research conducted by the present inventors in order to develop a diagnostic drug particle carrier that does not have the above-mentioned drawbacks, the use of specific hydrophilic cross-linked particles results in a sharp reaction. It excels in convenience, storage stability, and lot-to-lot stability in quality,
It has been found that immobilization of proteins can be easily carried out, and the present invention has been completed based on this finding.

[0004]

Thus, according to the present invention, the hydrophilic gel microparticles are used as seed particles.
The hydrophobic monoethylenically unsaturated monomer is water-soluble in a suspension of the immunological diagnostic agent carrier particles, which are a polymer of 50% by weight of the hydrophobic monoethylenically unsaturated monomer, and hydrophilic gel particles. The method for producing carrier particles for an immunological diagnostic agent, which is polymerized with a polymerizable radical polymerization initiator, and the hydrophilic gel particles are monoethylenically unsaturated amide 20.0 to 94.8.
% By weight, crosslinkable ethylenically unsaturated monomer 5.0-60.
0% by weight, ethylenically unsaturated carboxylic acid 0.1-30.
0% by weight, (meth) acrylic acid ester 0.1 to 50.
0% by weight and 0 to 100% of monomers copolymerizable with these monomers
The carrier particles for immunological diagnostic agent, characterized in that they are fine particles having a particle diameter of 0.1 to 10 μm in a water-swelling state and a particle diameter dispersion of 1.2 or less, obtained by dispersion polymerization of 30.0% by weight. The manufacturing method is provided.

The present invention will be described in detail below. The immunological diagnostic agent carrier particles of the present invention are particles having a particle size in a water-swelling state of 0.1 to 10 μm and a particle size dispersion of 1.2 or less. Particle diameter in water swollen state is 0.1 μm
When it is less than, it is difficult to detect the aggregation of these particles and it cannot be used as a diagnostic agent. On the other hand, when the particle size exceeds 10 μm, the particles precipitate even though they do not undergo an agglutination reaction, and thus they cannot be used as a diagnostic agent. When the particle size dispersion exceeds 1.2, the agglutination reaction lacks sharpness.

[0006] The carrier particles for immunological diagnostic agent of the present invention are preferably a polymer of hydrophobic monoethylenically unsaturated monomer, which comprises 50% by weight or less of the hydrophilic gel particles as seed particles. .. When the amount of the hydrophobic monoethylenically unsaturated monomer exceeds 50% by weight, new polymer particles of the hydrophobic monoethylenically unsaturated monomer are generated during the polymerization, and aggregates are easily formed. The problem arises.

(Hydrophilic gel fine particles) The hydrophilic gel fine particles are produced by dispersion polymerization. Dispersion polymerization is a method of polymerizing a monomer in an organic solvent that dissolves the monomer but does not dissolve the polymer, using a polymerization initiator that is soluble in the organic solvent. The hydrophilic gel particles used in the present invention (hereinafter sometimes simply referred to as gel particles) include monoethylenically unsaturated amides, crosslinkable ethylenically unsaturated monomers,
It is obtained by dispersion polymerization of an ethylenically unsaturated carboxylic acid, a (meth) acrylic acid ester and a monomer copolymerizable with these monomers.

The monoethylenically unsaturated amide is a monomer that forms the skeleton of the gel particles used in the present invention, and is an essential component for imparting hydrophilicity to the gel particles.
This monomer is usually contained in an amount of 20.0 to 94.8% by weight, preferably 20.0% by weight of the total monomer mixture constituting the gel particles.
~ 88.0 wt% is used. If the amount used is less than 20.2% by weight, the hydrophilicity of the gel particles will be insufficient. On the other hand, if this amount exceeds 94.8% by weight, the gel particles become water-soluble, which is not preferable, and it becomes difficult to control the particle size dispersion. Examples of monoethylenically unsaturated amides include acrylamide, methacrylamide, diacetone acrylamide, and N-hydroxymethyl acrylamide.

The crosslinkable ethylenically unsaturated monomer is an essential component for making the gel particles insoluble and making them water-swellable. The amount of this monomer used is usually 5.0 to 60.0% by weight of the total monomer mixture, preferably 10.0 to 50.
It is 0% by weight. If it is less than 5% by weight, the water swelling property is insufficient, and if it exceeds 60% by weight, the monodispersibility of the particle size is impaired. Specific examples of the crosslinkable ethylenic monomer include crosslinkable ethylenically unsaturated amides such as methylenebisacrylamide; ethylene glycol di (meth) acrylate,
Examples thereof include polyfunctional (meth) acrylic acid esters such as triethylene glycol di (meth) acrylate, ethylene di (meth) acrylate, and trimethylolpropane tri (meth) acrylate. In the present specification, (meth) acrylic acid means acrylic acid and methacrylic acid, and (meth) acrylate means acrylate and methacrylate.

The ethylenically unsaturated carboxylic acid is an essential component for maintaining the monodispersity of the gel particles, and if this monomer is lacking, particles having a monodisperse particle size distribution cannot be obtained. The amount used is 0.1 to 30.0% by weight, preferably 2 to 20% by weight, based on the total monomer mixture. Outside this range, the distribution of particle diameters becomes wide. Specific examples of the ethylenically unsaturated carboxylic acid include monocarboxylic acids such as acrylic acid, methacrylic acid, crotonic acid, 3-butenoic acid, 4-pentenoic acid and cinnamic acid; itaconic acid, fumaric acid, maleic acid, butenetricarboxylic acid. It can represent a polycarboxylic acid such as an acid.

The (meth) acrylic acid ester is a component necessary for introducing a specific functional group into the hydrophilic gel particles and maintaining the monodispersity of the particle size. The amount of this monomer to be used needs to be adjusted depending on the purpose of use of the immunological diagnostic agent carrier particles, but it is usually 0.1 to 0.1% of the total monomer mixture.
It is 50.0% by weight, preferably 5.0 to 40.0% by weight. Examples of the (meth) acrylic acid ester include a hydroxyl group-containing (meth) acrylic acid ester such as ethylene glycol (meth) acrylate, triethylene glycol (meth) acrylate, hydroxymethyl (meth) acrylate, and hydroxypropyl (meth) acrylate. ; Epoxy group-containing (meth) such as glycidyl (meth) acrylate
Acrylic acid ester; methylaminoethyl (meth) acrylate, t-butylaminoethyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, dimethylaminopropyl (meth) acrylate, dimethylaminoethyl (meth) acrylate, dibutylaminoethyl ( In addition to amino group-containing (meth) acrylic acid esters such as (meth) acrylate, methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, and other non-substituted (meth) acrylic acid esters are exemplified. You can

In the synthesis of the gel fine particles of the present invention, a monomer copolymerizable with each of the above-mentioned monomers may be used for the purpose of adjusting the hydrophilicity and swelling degree of the gel fine particles, if necessary. it can. The amount of such a monomer used is 3% of the total monomer mixture.
It is 0% by weight or less. When the amount used exceeds 30% by weight, the particle size dispersion of the gel particles becomes large. Examples of these monomers include aromatic vinyl monomers such as styrene, α-methylstyrene, p-methylstyrene, chloromethylstyrene and halogenated styrene; butadiene,
Conjugated diolefins such as isoprene can be used, but they are not particularly limited as long as they are soluble in the polymerization reaction solvent.

The solvent used for dispersion polymerization is not particularly limited as long as it is an organic solvent that dissolves the monomers used for the synthesis of gel particles and does not dissolve the polymer from them. Specific examples thereof include lower alcohols such as methyl alcohol, ethyl alcohol, propyl alcohol and butyl alcohol, tetrahydrofuran, dimethylformamide, dioxane, methyl ethyl ketone, pyridine and dimethyl sulfoxide. These solvents can be used alone or as a mixture of two or more. Furthermore, if necessary, these solvents and water can be mixed and used. These solvents are preferably used in an amount such that the total monomer concentration during polymerization is 50% by weight or less, more preferably 1 to 20% by weight. When the monomer concentration exceeds 50% by weight, it is difficult to obtain primary particles, and the particles aggregate.

Polymerization of the gel fine particles used in the present invention is carried out using the above-mentioned solvent-soluble radical polymerization initiator. Examples thereof include azo compounds such as 2,2′-azobisisobutyronitrile and 2,2′-azobis (2-methyl) valeronitrile; acetyl peroxide, propionyl peroxide, isobutyryl peroxide,
Organic peroxides such as benzoyl peroxide can be mentioned. The amount of these initiators used varies depending on the type of the initiator, but is usually 0.005 per total monomer mixture.
Is in the range of up to 5% by weight.

The dispersion polymerization of the gel particles used in the present invention may be carried out according to a usual method. The temperature of dispersion polymerization varies depending on the type of solvent used and the type of polymerization initiator, but is usually 20 to 100 ° C.

When a product having a particle size smaller than the desired particle size is obtained or when the desired amount of the functional group cannot be introduced, the necessary monomer after the completion of the polymerization, if necessary together with the polymerization initiator or the solvent, is added. It is also possible to add and continue the polymerization reaction. Also in this case, the monomer concentration is preferably maintained at 50% by weight or less.

The fine gel particles obtained by dispersion polymerization are separated from the solvent by a technique such as filtration, decantation or evaporation, and dried. If necessary, the solvent and unreacted monomers can be removed by centrifugation once or more in a state of being dispersed in water to improve the degree of purification.

(Immunological Diagnostic Agent Carrier Particles) The immunological diagnostic agent carrier particles of the present invention are hydrophobic to a suspension of water-swollen gel particles obtained by redispersing the above-mentioned gel particles in water. It is obtained by adding a monomer and polymerizing it by using gel fine particles as seed particles.

The hydrophobic monomer means a monomer having a solubility in water at 20 ° C. of 1% by weight or less. Specific examples thereof include aromatic vinyl monomers such as styrene, α-methylstyrene, p-methylstyrene, chloromethylstyrene and halogenated styrene; conjugated diolefins such as butadiene and isoprene. The amount of the hydrophobic monomer used is 1 to 50% by weight, preferably 2 to 30% by weight, based on the gel particles. If this amount is less than 1% by weight, the hydrophobicity of the surface of the polymer particles is insufficient, and conversely, if it exceeds 50% by weight, the hydrophobicity becomes too strong and neither of them can achieve the object of the present invention. .. The method of adding the hydrophobic monomer is not particularly limited and may be temporary addition or sequential addition.

To polymerize the hydrophobic monomer, it is necessary to add a water-soluble polymerization initiator. Examples of the polymerization initiator include peroxides such as potassium persulfate, and azo compounds such as 2,2′-azobisamidinopropane hydrochloride. The amount of the polymerization initiator used is not particularly limited and varies depending on its type, but is usually 0.0
It is from 05 to 5% by weight. The polymerization temperature of the hydrophobic monomer varies depending on the type of solvent used and the type of polymerization initiator used, but is usually 20 to 100 ° C. At this time, if necessary, a polymerization stabilizer such as a surfactant or a buffer may be added.

(Immunological Diagnostic Agent) A diagnostic agent carrier can be obtained by immobilizing an immunoactive substance on the diagnostic agent carrier particles of the present invention.

Examples of immunoactive substances that can be used include denatured gamma globulin, rheumatoid factor, antinuclear factor, human albumin, antihuman albumin, and immunoglobulin G (Ig
G), immunoglobulin A (IgA), immunoglobulin M (IgM), streptolysin O, anti-streptolysin O antibody, C-reactive protein, anti-C-reactive protein antibody, alpha-fetoprotein (AFT), anti-AF
T antibody, carcinoembryonic antigen (CEA), anti-CEA antibody, human placental lactogen (HPL), anti-HPL antibody, human chorionic gonadotropin (HCG), anti-HCG antibody, anti-estrogen antibody, anti-insulin antibody, hepatitis B surface Antigen (H
BS), anti-HBS antibody, Treponema pallidum antigen, rubella antigen, complement component Clq, anti-complement component Clq antibody and the like. Immobilization of these enzymes, proteins or immunologically active substances on a diagnostic drug particle carrier is carried out by adding the protein to be immobilized to a suspension of the diagnostic drug particle carrier and then at about 2 ° C.
It can be performed by stirring for 4 hours. The protein-immobilized particles are then purified by centrifugation or the like, and if necessary, a blocking agent such as a surfactant or a water-soluble polymer may be added to obtain a diagnostic agent.

[0023]

EXAMPLES The present invention will be described in more detail with reference to the following examples. The parts and% in the examples are by weight unless otherwise specified.

In this example, each measuring method is as follows. (Polymerization conversion) By gravimetric method. (Particle size) Unless otherwise specified, Coulter Multisizer (manufactured by Coulter) was used for measurement. (Ξ potential) It was measured using a microscope type electrophoretic velocity measuring device (Mark II electrophoretic velocity measuring device manufactured by Rank Brothers). (Amount of Carboxyl Group in Particles) 2 g of polymer particles were added to 48 g of distilled water, 0.1N sodium hydroxide solution was added thereto until pH was 12, and the mixture was stirred for 6 minutes,
Conductivity titration (using CM-117 conductivity meter, manufactured by Kyoto Electronics Co., Ltd.) was performed by adding 0.5 ml of 0.1N hydrochloric acid solution every 30 seconds, and the hydrochloric acid consumption corresponding to the inflection point of the titration curve. Calculate from (Amount of epoxy group in particles) 2 g of polymer particles were added to distilled water 48
Then, 140 ml of 1.0 N hydrochloric acid was added thereto, and the mixture was stirred at 60 ° C. for 6 hours to react the epoxy groups in the particles with hydrochloric acid. Then, the residual hydrochloric acid was titrated with 1.0 N sodium hydroxide. , Calculated from the amount of hydrochloric acid consumed in the reaction with the epoxy group.

(Example 1) (gel fine particles) The inside of a 2 liter reactor equipped with a stirring blade, a cooling condenser, a nitrogen gas introducing tube and a thermometer was replaced with nitrogen, and ethyl alcohol 750 was placed in this reaction vessel.
g, acrylamide 67.1 g, glycidyl methacrylate 10 g, methylenebisacrylamide 16.2 g,
Methacrylic acid (10 g) and 2,2'-azobisisobutyronitrile (0.42 g) were added and stirred to homogenize. Then, after bubbling with nitrogen, the reactor was heated to 60 ° C. to start the reaction, kept for 24 hours, and then cooled. Ethyl alcohol was removed from the obtained gel particle suspension using an evaporator, and further dried in a vacuum dryer for 12 hours to completely remove ethyl alcohol to obtain a gel particle powder. The gel fine particle powder was added to distilled water to form a suspension, and 0.1N sodium hydroxide was added to adjust the pH of the solution to 9. The gel fine particles swollen in water had a weight average particle diameter of 2.05 μm, a number average particle diameter of 1.82 μm and a monodisperse particle diameter distribution of 1.13. The ξ potential was −32.5 mV. When the amount of carboxyl groups and the amount of epoxy groups in the obtained particles were measured, they were each 9.5.
The amounts corresponded to g of methacrylic acid and 9.8 g of glycidyl methacrylate. In addition, pyrolysis gas chromatography of the obtained gel fine particle powder (Hitachi, G
C-263, column PEG 20M x 2m, column temperature 1
50 ° C.), 15.3 g of acrylamide and 15.
2 g of methylenebisamide was detected. Infrared absorption spectrum of the resulting gel particles (IR-8, manufactured by JASCO Corporation)
The presence of an amide group, a carbonyl group and an epoxy group was confirmed by 10 uses, KBr tablet method). (Diagnostic Agent Carrier Particles) The hydrophilic gel particles (75 g) were added to distilled water (1425 g) to obtain a swollen particle suspension. This suspension was put into a 2 liter reactor equipped with a stirring blade, a cooling condenser, a nitrogen gas introducing tube and a thermometer, the inside of which was replaced with nitrogen in advance, and after bubbling with nitrogen while stirring, 60 ° C The reactor was warmed to. Then, 75 g of 3% potassium persulfate was added, 10 g of styrene was further added, and the polymerization reaction was carried out at 60 ° C. After 8 hours, the mixture was cooled and the polymerization conversion was measured and found to be 97%. The obtained polymer particles were suspended in water and centrifuged repeatedly to be purified. The obtained diagnostic agent carrier particles had a weight average particle diameter of 2.11 μm, a number average particle diameter of 1.98 μm, and a monodisperse particle diameter distribution of dispersion 1.07.

Example 2 The diagnostic agent carrier particles obtained in Example 1 were treated with 0.1N of pH 7 so that the particle concentration became 5%.
It was dispersed in a phosphate buffer (0.1N-PBS). On the other hand, Treponema pallidum, a syphilis pathogen suspended in 0.1N-PBS having a pH of 7.0, was destroyed by sonication to prepare an antigen solution. 20 ml of this antigen solution was added to 20 ml of the above particle suspension, and the mixture was stirred at 4 ° C. for 8 hours for immobilization treatment. The 0.1N-PBS dispersion of the antigen-sensitized particles thus obtained was washed twice in a centrifuge, and then the particle concentration was 5%.
And lyophilized to obtain a dried reagent. Distilled water was added to this lyophilized product to readjust the particle concentration to 5%, and the titer of the syphilis-positive serum was measured by the microplate method. The results are shown in Table 1. Further, when the diagnostic agent carrier particles before sensitization were stored at room temperature for 30 days, the same sensitization treatment as described above was performed again, and the titer was measured. The same result was obtained.

Comparative Example 1 The titer of the syphilis-positive serum was measured by the microplate method in the same manner as in Example 2 using a commercially available syphilis test reagent using sheep red blood cells. The results are shown in Table 1.

[0028]

[Table 1]

From the results shown in Table 1, it can be seen that the carrier particles of the immunological diagnostic agent of the present invention are more sensitive than conventional diagnostic agents.

Comparative Example 2 The amount of styrene used was 60
The same operation as in Example 1 was carried out except that the amount was changed to g, but the particles became agglomerated during the polymerization process of the diagnostic agent particles. When this aggregate was taken out and observed with an optical microscope, it was found that the gel particles were "grape-shaped" in series.

(Example 3) The amount of styrene used was 30
The same operation as in Example 1 was performed except that the value was changed to g. The obtained diagnostic agent carrier particles had a weight average particle diameter of 2.04 μm,
It had a number average particle size of 1.84 μm and a monodisperse particle size distribution of dispersion 1.11. The particles were subjected to the same antigen immobilization treatment as in Example 1, and the titer of the syphilis-positive serum was measured by the microplate method. Table 1 together with the results
Shown in.

(Comparative Example 3) The titer was measured in the same manner as in Example 2 except that commercially available high specific gravity polystyrene latex was used in place of the diagnostic agent carrier particles obtained in Example 1, and the titer was 80 times and 160 times. Agglutination occurred at doubling dilution and the detection limit was 2560 times.

Comparative Example 4 The titer was measured in the same manner as in Example 2 except that commercially available tannic acid-treated glutaraldehyde-fixed sheep red blood cells were used instead of the diagnostic agent carrier particles obtained in Example 1, and The detection limit was 5120 times. Further, sheep red blood cells before sensitization were stored at room temperature for 30 days, and then again subjected to the same sensitization treatment as above, and the titer was measured. As a result, aggregation was observed in syphilis-negative serum.

(Effects of the Invention) Thus, according to the present invention, compared to conventionally used carrier particles for immunological diagnostic agents, the sensitivity during the agglutination reaction is excellent, the storage stability of the particles, and the particles It is possible to obtain carrier particles for an immunological diagnostic agent which are excellent in reproducibility during production and which can easily immobilize proteins and the like.

Claims (4)

[Claims] 1. A carrier for an immunological diagnostic agent, which comprises a hydrophilic gel fine particle as a seed particle and is a polymer of 1 to 50% by weight of the fine particle of a hydrophobic monoethylenically unsaturated monomer. particle. 2. Hydrophilic gel particles comprising monoethylenically unsaturated amide 20.0-94.8% by weight, crosslinkable ethylenically unsaturated monomer 5.0-60.0% by weight, ethylenically unsaturated Disperse 0.1 to 30.0% by weight of carboxylic acid, 0.1 to 50.0% by weight of (meth) acrylic acid ester, and 0 to 30.0% by weight of a monomer copolymerizable with these monomers. Particle size of 0.1 to 10 in water swollen state obtained by polymerization
The immunological diagnostic agent carrier particles according to claim 1, wherein the particles are microparticles having a particle size dispersion of 1.2 or less. 3. The immunological diagnostic agent according to claim 1, wherein a hydrophobic monoethylenically unsaturated monomer is polymerized by a water-soluble radical polymerization initiator in a suspension of hydrophilic gel particles. A method for producing carrier particles. 4. Hydrophilic gel fine particles are monoethylenically unsaturated amide 20.0-94.8% by weight, crosslinkable ethylenically unsaturated monomer 5.0-60.0% by weight, ethylenically unsaturated Disperse 0.1 to 30.0% by weight of carboxylic acid, 0.1 to 50.0% by weight of (meth) acrylic acid ester, and 0 to 30.0% by weight of a monomer copolymerizable with these monomers. The method for producing carrier particles for immunological diagnostic agent according to claim 3, which is obtained by polymerization.