JPH0377464B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunoactive fine particles that are effective as reagents for immunological testing, and in particular to immunoactive particles in which an immunoactive substance is immobilized on a particulate carrier. The present invention relates to improvements in immunoactive microparticles that are effective as immunological test reagents for detecting or measuring components or identifying cells.

When using the reaction between an antigen and an antibody to immunologically detect or quantify one of them, the substance that binds to the substance to be measured is immobilized on a particulate carrier, and the particles become the substance to be measured. A highly sensitive measurement method that takes advantage of the phenomenon of agglutination in the presence of ions has become an important means for immunological clinical testing. Conversely, when the substance to be measured is immobilized on a particulate carrier, aggregation of the particles immobilized with the analyte due to the presence of antigens or antibodies that specifically react with the analyte can cause the analyte to be measured in the sample solution. Methods of detecting or quantifying a substance to be measured by inhibiting the presence of the substance are also widely used in immunological clinical tests. In addition, a method in which a substance that selectively binds to specific cells is immobilized on a particulate carrier and cells are identified based on whether or not the particles bind to the cells is often used as a means of immunological testing. It has been adopted.

Particulate carriers in immunological test reagents using such immunoactive particles have conventionally been red blood cells of mammals including humans and birds, inorganic particles such as kaolin and carbon, and organic polymers such as natural rubber latex and polystyrene. Latexes of molecular compounds are widely used for agglutination reactions. Among these, red blood cells can immobilize many types of antigens and antibodies and have the widest range of applications. However, there are problems such as differences in quality depending on the individual animal collected, poor stability and difficulty in storage, and non-specific agglutination by human serum. The most widely used particles of non-biological origin are polystyrene particles, which can be made of a synthetic polymer compound, have a constant quality, and are stable in themselves. Since polystyrene is hydrophobic and has the property of adsorbing various proteins, antigens or antibodies are usually immobilized on polystyrene by physical adsorption. However, when an antigen or antibody is immobilized by physical adsorption, an equilibrium exists between the immobilized antigen (or antibody) and the free antigen (or antibody), so that the corresponding target substance of measurement A competitive reaction occurs between the antigen (or antibody) immobilized on particles and the free antigen (or antibody), and the competitive reaction acts to suppress agglutination. As a result, a lack of sensitivity and stability has been noted in many cases. Also, as a matter of course, substances that are difficult to physically adsorb to polystyrene cannot be immobilized. Due to these problems, polystyrene particles have been put to practical use only to a limited extent compared to when red blood cells are used as a carrier. In order to solve these problems, we have recently developed a reagent (DT22649218) in which human chorionic gonadotropin is bound to a styrene-methacrylic acid copolymer latex using carbodiimide, carboxylated styrene-butadiene copolymer, carboxylated polystyrene, amino Various latex polymers such as carboxylated polystyrene acrylic acid polymers, acrylonitrile polymers, methacrylic acid polymers, anitrile-butadiene-styrene copolymers, polyvinyl acetate acrylate, polyvinyl pyridine, vinyl chloride-acrylate copolymers, etc. A reagent consisting of particles with a particle size of 0.01 to 0.9 microns condensed with chorionic gonadotropin, human serum albumin, or modified gamma globulin via an amide bond (Japanese Patent Publication No. 12966/1989) Methacrylic acid, 2
- A reagent in which a treponemal antigen is bound to a methyl methacrylate latex containing hydroxyl and carboxyl groups produced by copolymerizing hydroxyethyl methacrylate and methyl methacrylate using the cyanogen bromide or carbodiimide method (Clinical Pathology 27, Supplement, 522 (1978)), a reagent in which human chorionic gonadotropin or insulin is bound to a latex by reacting the free epoxy groups of a latex with polystyrene particles as a core and coated with a styrene-glycidyl methacrylate copolymer shell (particularly Published in 1984-110118)
Reagents in which an antigen or antibody is bound to a carrier through a covalent bond have been proposed. In these prior art methods, a method of binding an immunoactive substance to particles using carbodiimide is often used, but when carbodiimide is used, condensation reactions occur between and within molecules of the immunoactive substance. This is an undesirable side reaction that impairs the activity of the immunologically active substance. If cyanogen bromide is used, it is possible to avoid condensation reactions between and within molecules of the immunoactive substance, but in this case, it is difficult to obtain reproducibility of the reaction between a polymer having a hydroxyl group and cyanogen bromide. As a result, the immunoactivity of particles immobilized with immunoactive substances tends to fluctuate. Compared to these methods of immobilizing immunoactive substances, the method of reacting a free epoxy group introduced into a polymer with a protein or polypeptide causes less deactivation of immune activity and has good reproducibility. However, in the above-mentioned prior art techniques that utilize epoxy groups, since the polymer particle surface is a copolymer of a hydrophobic compound such as styrene, it tends to non-specifically adsorb proteins. In general, human or animal body fluids contain many types of proteins, and plasma or serum in particular contains these proteins at high concentrations. When proteins from a sample body fluid are adsorbed onto carrier particles, they may interfere with the target immunological reaction such as an antigen-antibody reaction, leading to a decrease in the selectivity and sensitivity of the agglutination reaction.

The inventors of the present invention have conducted intensive studies to develop a new carrier material for immobilizing an immunoactive substance that overcomes the drawbacks of the prior art, and as a result, have arrived at the present invention, which is highly effective.

That is, the present invention provides an immunoactive substance immobilized product in which the immunoactive substance is bound to the surface of a solid carrier, in which a surface reduction product of acrylonitrile or methacrylonitrile polymer fine particles is used as the solid carrier. Such an immunoactive substance immobilized product is produced by reducing the surface of acrylonitrile or methacrylonitrile polymer fine particles, and utilizing the reactivity of the functional group generated by the reduction, directly or by bonding other functional groups to this. After that, it is produced by binding an immunologically active substance.

In the method of the present invention, first, polymer fine particles containing acrylonitrile or methacrylonitrile as a main component are produced. Alternatively, the two may be mixed in any ratio and polymerized. In either case, a crosslinkable monomer having at least two carbon-carbon double bonds in the molecule may be added for the purpose of crosslinking the polymer. Compounds suitable as crosslinking agents for this purpose are, for example, divinylbenzene, divinyltoluene, diallyl ether, N,N'-methylenebisacrylamide, ethylene glycol dimethacrylate, ethylene glycol diacrylate, divinyl succinate, diallyl succinate,
These include vinyl methacrylate, triallylisocyanurate, and divinyl sulfone. The amount of crosslinking monomer added is usually 30 mol% or less based on the total monomers. Furthermore, addition of a crosslinking monomer is not an essential condition. Further, in the present invention, it is also possible to add and copolymerize a polymerizable monomer other than acrylonitrile or methacrylonitrile or a crosslinkable monomer having two or more carbon-carbon double bonds in the molecule. Preferred as such copolymerization components are hydrophilic unsaturated monomers or compounds that are introduced into the polymer through copolymerization and are simultaneously reduced and converted to hydrophilic properties when the nitrile is reduced. Examples of preferred compounds as hydrophilic monomers include 2-oxyethyl acrylate, 2-oxyethyl methacrylate, 2-oxypropyl acrylate, 2-
Oxypropyl methacrylate, degree of polymerization 2 or more
100 acrylic or methacrylic esters of polyethylene glycol monoalkyl ether, acrylamide, methacrylamide, N-
These include vinylpyrrolidone and glyceryl methacrylate. Examples of monomers useful for introducing hydrophilic groups into the polymer by post-polymerization reduction include lower alkyl esters of acrylic acid or methacrylic acid such as methyl, ethyl, n-propyl or isopropyl; acetic acid; or vinyl esters of lower carboxylic acids such as propionic acid, vinylene carbonate, maleic anhydride, etc. Two or more of these hydrophilic monomers or monomers that can be converted into hydrophilic properties may be used in combination. The molar ratio of the sum of hydrophilic monomers and monomers convertible to hydrophilicity to the sum of acrylonitrile and methacrylonitrile is 95:
It can be varied from 5 to 5:95.

Further, the ratio of acrylonitrile to methacrylonitrile used may be 100:0 to 0:100 (molar ratio), ie, any ratio.

The polymer fine particles of the present invention can be produced, for example, by the following method.

The polymerization reaction is usually preferably carried out by emulsion polymerization, precipitation polymerization or suspension polymerization. All of these methods are suitable for the purpose of the present invention because the polymer is precipitated in the form of particles at the same time as the polymerization. Precipitation polymerization is a method in which polymerization is carried out in a medium that dissolves monomers but does not dissolve the polymer produced by polymerization, and is produced by selecting the combination of monomer and polymerization medium. The average diameter of polymer particles is 0.1
It is relatively easy to adjust the particle size to fall within the range of 10 μm to 10 μm, and the particle size distribution is also relatively narrow. Another advantage of precipitation polymerization, unlike emulsion polymerization or suspension polymerization, is that it does not use emulsifiers or suspension stabilizers, so there is no need to remove these additives after the polymerization reaction.

Suitable media for precipitation polymerization include esters such as ethyl acetate, n-propyl acetate, isopropyl acetate, butyl acetate isomers and the above-mentioned corresponding esters of propionic acid, methyl ethyl ketone, methyl n-propyl ketone, and methyl isopropyl ketone. , ketones such as methyl butyl ketone isomers, benzene, toluene, o-xylene, m-xylene, p-xylene, carbon tetrachloride, and water.

The particle size of the polymer fine particles obtained by this method is excellent in uniformity, and the average diameter is within the range of 0.1 μm to 10 μm. The average diameter can be adjusted by monomer composition and choice of polymerization medium.

As the polymerization initiator, common radical polymerization initiators such as 2,2'-azobisisobutyronitrile, 2,2'-azobis(2,4-dimethylvaleronitrile), 2,2'-azobis(2,4 -dimethyl-
Azo compounds such as 4-methoxyvaleronitrile), peroxides such as benzoyl peroxide, dilauroyl peroxide, di-tert-butyl peroxide, potassium persulfate, and sodium persulfate can be used. The polymerization temperature may also be within the normal radical polymerization temperature range, with 20°C to 80°C being particularly preferred. The concentration of the polymerization initiator in the polymerization mixture is usually
It is about 0.001 to 0.03 mol/1. The concentration of the monomer in the polymerization mixture is usually preferably in the range of 5 to 50% by weight. When the monomer concentration exceeds 50% by weight, the resulting polymer particles tend to aggregate. Although the present invention can be carried out even if the monomer concentration is less than 5% by weight, productivity will be lower because fewer polymer particles will be obtained. In addition, it is preferable that the polymerization is carried out while the mixture is left standing while the atmosphere is replaced with an inert gas such as nitrogen or argon.

Emulsion polymerization and suspension polymerization are suitable polymerization methods for polymerizing monomers that normally have low solubility in water in aqueous media, but since acronitrile is approximately 7% soluble in water at room temperature, these polymerization Among the legal methods, some ingenuity is required when applying the suspension polymerization method. In other words, when carrying out suspension polymerization of acrylonitrile, a neutral salt such as sodium chloride or sodium sulfate is dissolved in an aqueous medium to lower the solubility of acrylonitrile through a salting-out effect and disperse it as oil droplets. is used as a polymerization initiator and polymerized with sufficient stirring. In this case, it is preferable to add and dissolve a water-soluble polymer compound such as polyvinyl alcohol, polyacrylic acid, or polyN-vinylpyrrolidone as a dispersion stabilizer in the aqueous medium. As the polymerization initiator, an oil-soluble one suitable for precipitation polymerization may be used.

The polymerization temperature is also preferably in the range of 20 to 100°C, similar to precipitation polymerization. Polymerization initiator is for monomer
It is added in a range of 0.0001 to 0.1 mol%. Since methacrylonitrile is hardly soluble in water, it can be polymerized by a normal suspension polymerization method. The suspension polymerization method is suitable for producing polymer particles of 10 μm or more and 1 mm or less.

Emulsion polymerization of acrylonitrile and methacrylonitrile can be carried out in the same manner as for other hydrophobic monomers. That is, the polymerization may be carried out in an aqueous medium containing an anionic, cationic or nonionic surfactant in the presence of a water-soluble radical generator. In addition, when polymerizing in the coexistence of 0.1 to 2 mol% of sodium or potassium salts such as styrene sulfonic acid, methallyl sulfonic acid, or allyl sulfonic acid, the produced polymer fine particles are Polymerization proceeds in a stably dispersed and suspended state. Examples of preferred polymerization initiators include sodium, potassium or ammonium salts of persulfate, these persulfates, or redox free radical generating systems in combination with sodium chlorate and sodium bisulfite. The concentration of the polymerization initiator is preferably 0.001 to 0.03 mol/1, and the polymerization temperature is preferably 20 to 80°C. The emulsion polymerization method is suitable for producing polymer particles of 0.01 to 1 μm.

Next, when the fine particulate polymer containing acrylonitrile or methacrylonitrile as a main component produced as described above is reduced, amino groups are generated on the surface of the fine particles. Utilizing the reactivity of this amino group, the immunoactive substance can be bonded directly or, if necessary, by converting it into a group that easily reacts with the functional group, depending on the type of functional group the immunoactive substance has, and then using a covalent bond. By doing so, it can be immobilized on the surface of the fine particles.

As is well known, cyanogen groups are converted to amino groups by reduction. Reduction methods suitable for that purpose include, for example, lithium aluminum hydride, aluminum hydride, lithium aluminum trimexyhydride, diborane, 3-methyl-2-
Butylborane, sodium borohydride/aluminum chloride, sodium trisulfide borohydride,
Sodium acetoxyborotrihydride, (trifluoroacetoxy)sodium borohydride,
Examples include reduction with sodium trialkoxyborohydride or sodium hydroxide triborohydride. It is sufficient that the reduction occurs on the surface of the fine particles, and there is no need to extend it to the inside. In addition, if the polymer contains acrylic esters, methacrylic esters, vinyl esters, vinylene carbonate, maleic anhydride, etc. as copolymerized components, their ester bonds, anhydride bonds, etc. are cleaved by reduction and become hydroxyl. Converted to base.

Immunologically active substances have various functional groups depending on their type, but most of them are proteins or contain polypeptide bonds, and therefore have amino groups. To utilize the reactivity of amino groups in immunoactive substances, the amino groups generated by reducing the surface of (meth)acrylonitrile-based fine particles are reacted with dialdehyde, and then the above immunoactive substance is reacted. It is preferable to use dialdehyde as a mediator for immobilization.

The most suitable dialdehyde to react with the amino group is glutaraldehyde. Other aliphatic dialdehydes with different carbon numbers such as succinic aldehyde and adipine aldehyde, aromatic dialdehydes such as terephthalaldehyde, and polymeric aldehydes such as starch aldehyde can also be used. The dialdehyde treatment of the surface-reduced fine particles is preferably carried out at a temperature of 0° C. or higher and 60° C. or lower, using an aqueous solution with a dialdehyde concentration of 0.1 to 25%, with a pH in the range of 5 to 11. The fine particles treated with dialdehyde are thoroughly washed with water and used for the next immobilization of an immunoactive substance. Proteins can also be immobilized on particles by condensing the amino groups of the microparticles and the carboxyl groups of the protein with carbodiimide.
Alternatively, ε-aminocaproic acid or the like may be used as a spacer and bonded to the carboxyl group of the particles using carbodiimide, and then the protein may be bonded to the spacer using carbodiimide.

If an immunologically active substance or physiologically active substance is chemically immobilized on carrier particles in this way, the bond is usually strong, and the immobilized immunologically active substance or physiologically active substance will not be released from the carrier particles. . However, when immobilization is performed by glutaraldehyde activation, the stability of the bond may be somewhat insufficient, but in such cases, sodium borohydride or cyanide hydride may be used to further strengthen the bond. Treatment with sodium boron or dimethylamine borane is effective. However, such secondary processing is rarely necessary.

In the present invention, the term "immune active substance" refers not only to antigens and antibodies, but also to substances that are involved in humoral or cellular immune reactions and specifically bind to a certain substance, such as complement, Fc receptors, and C3 receptors. shall be taken as a thing. Some specific examples include Trebonema pallidum antigen, hepatitis B surface antigen (HBS antigen), antibody to hepatitis B surface antigen (anti-HBS antibody), rubella antigen, toxoplasma antigen, streptolysin O, anti-streptolysin O antibody, Mycoplasma antigen, human chorionic gonadotropin (HCG), anti-HCG antibody, heat-agglutinated human IgG, rheumatoid factor, nuclear protein, DNA, anti-DNA antibody, C-reactive protein (CRP), anti-CRP antibody, anti-estrogen antibody, α- Fetoprotein (α-FP), anti-α-
FP antibody, carcinoembryonic antigen (CEA), anti-CEA antibody,
Clq, anti-Clq antibody, C3, anti-C3 antibody, anti-C3b antibody,
anti-C3bi antibody, C4, anti-C4 antibody, protein-A,
These include conglutinin and immunoconglutinin.

When binding an immunologically active substance to the surface of fine particles, it is preferable to dissolve the immunoactive substance in an aqueous medium and allow the reaction to occur. The speed of the immunoactive substance in the reaction solution needs to be increased or decreased depending on the properties of the individual immunoactive substance and cannot be determined uniformly, but the pH is between 6 and 6.
9. The temperature is preferably in the range of 0 to 40°C.

If there is a possibility that the functional groups will not be completely consumed and remain active due to the reaction with the immunologically active substance, use hydrophilic proteins such as serum albumin and gelatin that do not interfere with the target immunological test as functional groups. By reacting with the functional group, the reactivity of the functional group can be lost. In this case, the hydrophilic protein for functional group elimination may be mixed with the immunoactive substance to be immobilized and reacted simultaneously, or the immunoactive substance may be reacted alone first and then reacted. It is also possible to use amino acids such as glycine and alanine in place of the hydrophilic proteins such as albumin and gelatin. After the immunoactive substance is immobilized in this manner, what is exposed on the surface of the polymer fine particles without being covered with any bond is the hydrophilic moiety derived from the water-soluble monomer. Since proteins are difficult to adsorb to hydrophilic polymers in aqueous media, the immunoactive substance-immobilized microparticles according to the present invention are stable to sample body fluids, do not easily cause non-specific aggregation, and do not adhere non-specifically to cells. There is no.

Furthermore, if the fine particles are appropriately colored with a dye or pigment, or provided with fluorescence, it is advantageous for both the purpose of aggregation reaction and cell labeling. Although the shape of the fine particles is often spherical, it is not essential that the particles be spherical and may have an irregular shape. The diameter of irregularly shaped particles is 1/2 of the sum of the maximum and minimum diameters. The average diameters described in the examples represent those defined by formula (1).

= _N 〓 ⁱ⁼¹ di/N (1) where di is the diameter of the i-th particle and N is the total number of particles. Empirically, it is easy to determine the agglutination reaction when the average diameter is 0.1 μm or more and 10 μm or less.
In addition, for the purpose of cell labeling, the average diameter is 0.03 μm or more.
A range of 5 μm or less is preferable.

Next, the present invention will be explained with reference to some examples.

Example 1 Acrylonitrile in 10g of ethyl propionate
Dissolve 4.95g and add 2,2'azobis(4-methoxy-2,4-dimethylvaleronitrile)
Add 0.12g and dissolve at 30 °C under argon atmosphere.
It was left to stand for 16 hours. . The precipitated polymer was separated, dried and weighed, and the yield was 4.61 g, with a polymerization rate of 85%. Furthermore, as a result of observing the polyacrylonitrile fine particles obtained here using a scanning electron microscope, the diameter was in the range of 0.5 to 0.6 μm and was almost uniform. This polyacrylonitrile fine particle
Disperse 0.5 g in 50 ml of dry ethyl ether and add 0.2 g of lithium aluminum hydride under argon atmosphere.
was added and stirred at room temperature for 3 hours. Next, while stirring, ethyl acetate was gradually added until the heat generation and foaming ceased (approximately 5 ml), and 20 ml of 1N hydrochloric acid was added dropwise. When left to stand, it separated into two layers, so remove the transparent upper layer and centrifuge the grayish white lower layer (PH1-2).
The precipitate was washed twice with 6N hydrochloric acid and twice with distilled water by centrifugation.

Further, when washed twice with 1 mol/1 sodium carbonate and twice-distilled water, the polymer particles became white. The polyacrylonitrile fine particle reduction product thus prepared was dispersed at 1% in distilled water,
Conductivity titration with 1/100N hydrochloric acid revealed that the amino group content was 0.07 mg equivalent/g.

Add 5 ml of a 0.5% dispersion of the above reduced polyacrylonitrile fine particles in distilled water to 25% of the electron microscopy grade.
Add 0.75ml of glutaraldehyde aqueous solution, and
After adjusting the pH to 10 with a 0.25N aqueous sodium hydroxide solution, the mixture was stirred at 30°C for 2 hours under a nitrogen atmosphere.
The reaction mixture was then centrifuged and washed three times with distilled water.
Phosphate buffered saline containing 0.1% sodium azide (disodium hydrogen phosphate + monopotassium hydrogen phosphate 0.01 mol/1, sodium chloride 0.14 mol/1)
1. After washing once with PH7.2 (hereinafter abbreviated as PBS),
Microparticles were dispersed in 0.5 ml of PBS. 10 of the dispersion and bovine serum albumin (hereinafter abbreviated as BSA)
Mix with 0.5 ml of PBS solution with a concentration of mg/ml at 30°C.
The mixture was stirred for 3 hours. Then centrifuge and remove the microparticles in PBS.
After washing three times with human serum albumin at 10 mg/ml.
It was dispersed in 2 ml of PBS solution containing a concentration of . 10 μl of BSA-immobilized fine particle dispersion prepared in this way
and 10 μl of a 100-fold dilution of anti-BSA antiserum (rabbit) in PBS were mixed on a glass plate, and agglutination was observed with the naked eye. Anti-BSA as a control experiment
Similar experiments were conducted using normal rabbit serum instead of antiserum (rabbit), and no agglutination was observed. Therefore, the agglutination reaction described above is due to the antigen-antibody reaction between BSA immobilized on the microparticles and the anti-BSA antibody in the antiserum.

Example 2 The same polyacrylonitrile microparticles used in Example 1 were similarly treated with glutaraldehyde. Separately, the syphilis pathogen Treponema pallidum (hereinafter referred to as
(abbreviated as TP) Nichols strain cells in PBS
The dispersion liquid dispersed at a rate of 10 ⁹ cells/ml was treated with 10 KHz ultrasound for 20 minutes while cooling with ice water to destroy the bacterial cells, and this was used as a TP antigen solution. 1 ml of this TP antigen solution and 1 ml of a dispersion of 12.5 mg of the glutaraldehyde-treated polymer particles dispersed in 1 ml of PBS were mixed and stirred at 30° C. for 2 hours. Next, fine particles were separated by centrifugation, washed three times with PBS by centrifugation, and then washed with bovine serum albumin (hereinafter abbreviated as BSA).
was dispersed in 1 ml of PBS supplemented with 1%. This TP antigen-immobilized microparticle dispersion was left in a refrigerator at 4° C. for 3 days, and then the activity was assayed in the same manner as in Example 1. Agglutination occurred in samples in which syphilis-positive serum with a TPHA titer of 640 was diluted 2-fold and 4-fold with PBS, but no agglutination was observed in the 10-fold dilution. No agglutination was observed in syphilis-negative serum at any of the above dilutions. In this way, it was confirmed that the TP antigen was immobilized on the microparticles while retaining its activity.

Example 3 28.5 parts of methacrylonitrile (parts by weight, hereinafter the same) and 2.9 parts of divinylbenzene were mixed (molar ratio 95:5). This monomer mixture was dissolved in 100 parts of ethyl propionate, and 2,
0.3 part of 2'-azobis(2,4-dimethyl-4-methoxyvaleronitrile) was added, and the mixture was allowed to stand at 40°C for 24 hours under an argon atmosphere. The cloudy polymer solution was centrifuged to separate polymer particles, washed with ethyl acetate, and then dried under reduced pressure. The yield of polymer fine particles was 3.0 parts. Also, most of the polymer particles have a diameter of 2
It was in the range of ~3μm. Next, separate the dry polymer and disperse 1.2 g of cobalt chloride hexahydrate in 15 ml of methanol, and add the above polymethacrylonitrile fine particles.
When 0.16 g of sodium borohydride was added gradually while stirring at 20°C, hydrogen gas was generated and a black precipitate was formed. After the addition of sodium borohydride was completed, stirring was continued for an additional hour at room temperature. Next, 10 ml of 3N hydrochloric acid was added and stirred to dissolve the black precipitate, and then the polymer particles were centrifuged and washed with methanol. The reduced polymer particles were dispersed in 20 ml of distilled water, 3 g of 25% glutaraldehyde aqueous solution was added, stirred at 30° C. for 2 hours, washed with water, and dispersed in 10 ml of PBS.

Separately from adult bovine serum the method of Lachman et al. (DM
“Handbook of Experimental” edited by Weir
"Immunology" Volume 1. "Immunochemistry" 3rd Edition, Blackwell Scientific Publications,
Oxford (1978), Chapter 5, p. 6) was dissolved in CFD at a concentration of 0.45 mg/ml. However, CFD is a buffer solution prepared as described below. That is, dissolve 0.824 g of veronal sodium salt and 8.5 g of sodium chloride in 700 ml of distilled water, add a small amount of 1/10 N hydrochloric acid, and adjust the pH.
Adjusted to 7.2. Separately, prepare a solution by dissolving 0.36 g of magnesium chloride hexahydrate and 0.037 g of calcium chloride dihydrate in 200 ml of distilled water, and mix it with the veronal solution above to obtain 0.2 g of sodium azide.
and 1.0 g of gelatin were added, and distilled water was further added to bring the total to 1.

10 ml of this conglutinin CFD solution was mixed with 10 ml of the PBS dispersion of the glutaraldehyde-activated surface-reduced polymer particles, incubated at 30° C. for 2 hours, and then thoroughly washed with PBS. This conglutinin-immobilized fine particle is a complement component.
The ability to bind to C3-bound immune complexes was demonstrated as follows.

Dissolve 12 mg of human IgG in 2 ml of physiological saline and incubate at 63°C.
was heated for 20 minutes to thermally aggregate the IgG. thermal agglomeration
IgG (immune complex model substance, hereinafter abbreviated as AHG)
The insolubilized portion was removed by centrifugation at 1500XG for 15 minutes, and the supernatant was used as the AHG solution. The above AHG-containing supernatant was diluted 20 times with PBS and mixed with the same volume of fresh human serum to obtain complement.
Incubate at 37°C for 15 minutes to bind AHG. Next, when 10 μl of this incubated AHG serum mixture and 10 μl of the conglutinin-immobilized fine particle dispersion were mixed on a glass plate, they agglomerated violently. When human serum inactivated at 56°C for 30 minutes and AHG were incubated instead of fresh human serum and mixed with the conglutinin-immobilized microparticle dispersion, no aggregation occurred.

Example 4 10 parts of acrylonitrile, methacrylonitrile
12.6 parts of divinylbenzene, 2.5 parts of divinylbenzene, and 0.2 parts of 2,2'-azobis(2.4-dimethyl-4-methoxyvaleronitrile) were dissolved in 100 parts of ethyl propionate, and the mixture was left standing at 40°C for 7 hours under a nitrogen atmosphere ( The molar ratio of acrylonitrile to methacrylonitrile is 1:1). The cloudy polymer solution was centrifuged to separate polymer particles, washed with ethyl acetate, and then dried under reduced pressure. The yield of polymer fine particles was 3.75 parts. Moreover, the average diameter of the polymer fine particles was 1.0 μm. Next, the polymer fine particles were subjected to reduction and glutaraldehyde activation treatment in the same manner as in Example 1, and then the TP antigen was immobilized in the same manner as in Example 2. this
When the TP antigen-immobilized polymer fine particles were reacted with a syphilis-positive serum having an antibody titer of 640 determined by the TPHA method on a glass plate in the same manner as in Example 2, the results were the same as in Example 2.

Claims (1)

[Scope of Claims] 1. An immunoactive substance immobilized product in which an immunoactive substance is bound to the surface of a solid carrier, characterized in that the solid carrier is a surface reduction product of acrylonitrile or methacrylonitrile polymer fine particles. Substance immobilized fine particles. 2. Immune activity characterized by reducing the surface of acrylonitrile or methachronitrile polymer fine particles and bonding an immunoactive substance to the functional groups generated by the reduction, either directly or after bonding with other functional groups. Method for producing substance-immobilized fine particles.