JPS58128326A - Fine particle having immobilized immunologically active substance and preparation thereof - Google Patents

Abstract

PURPOSE:To obtain the titled fine particles useful as an immunological assay reagent, by reducing the surfaces of polymeric fine particles e.g. acrylonitrile, binding the resultant functional groups directly or to other functional groups and then to an immunologically active substance. CONSTITUTION:The surfaces of polymeric fine particles, e.g. acrylonitrile or methacrylonitrile, are reduced, and the reactivity of the resultant functional groups is used and directly or bound to other functional groups and then to an immunologically active substance, e.g. a luetic treponema antigen, hepatitis B surface antigen or rubella antigen, to give the aimed fine particles containing the immobilized immunological active substance. A crosslinking monomer having at least two carbon-carbon double bonds in the molecule in an amount of 30mol% or less based on the total monomer may be added to crosslink the polymer. Divinylbenzene, divinyltoluene, etc. may be cited as the crosslinking agent.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to immunoactive microparticles that are effective as reagents for immunological testing, and in particular to immunoactive microparticles that are effective as immunological test reagents. Quality o□$, -
The present invention relates to improvements in immunoactive fine particles that are effective as immunological test reagents for detecting or measuring components or identifying cells in body fluids of animals or animals using immunoactive particles.

When immunologically detecting or quantifying one of an antigen and an antibody using a reaction, the substance that binds to the substance to be measured is immobilized on a particulate carrier, and then the particle A highly sensitive measurement method that utilizes the phenomenon of aggregation in the presence of a substance to be measured has become an important means for immunological clinical testing. Conversely, when the substance to be determined 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 occur in the sample solution. A method of detecting or detecting a substance to be measured, which is determined by the presence of the substance to be measured, is 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 Y- bind to the cells is often used as a means of immunological testing. It has been adopted.

Conventionally, the particulate carrier in immunological blueprint reagents using immunologically active particles has been used.

Red blood cells of mammals including humans and birds, particles of inorganic substances such as kaolin and carbon, and latexes of organic polymer compounds such as natural rubber latex and polystyrene 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.Since they are synthetic polymer compounds, they can be of constant quality and are stable in themselves.Polystyrene is hydrophobic Since it has the property of adsorbing various proteins, antigens or antibodies are usually immobilized on poly-7 ethylene 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), and therefore the corresponding antibody (or antibody), which is the target substance of measurement, exists. A competitive reaction occurs between the antigen (or antibody) immobilized on particles and the free antigen (or antibody), and this competitive reaction acts to suppress agglutination. As a result, a lack of sensitivity and stability has been noted in many cases.

Naturally, 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 (o T 2゜2, e4941g) in which a styrene-methacrylic acid copolymer latex nine-human chorionic gonadotropin is bound using carbodiimide, a carboxylated styrene-butaimer, Methacrylic acid polymer, Acnitrile-butadiene-sterene copolymer, Polyacetic acid 3
- Particle size 001-09 obtained by condensing human chorionic conatropin, human serum albumin, or modified gamma globulin with various latex polymers such as vinyl acrylate, polyvinyl pyridine, and vinyl chloride-acrylate copolymers using carbodiimide as a condensing agent via an amide bond. Reagent consisting of micron particles (Special Publication No. 53-12966)
A reagent in which Tombonema antigen is bound to a methyl methacrylate latex containing hydroxyl and carboxyl groups produced by copolymerizing methacrylic acid, 2-hydroxyethyl methacrylate, and methyl methacrylate by anogen bromide or carbodiimide method (Clinical Pathology 27) ,
Supplement, p. 522 (19?8)), Free epoxy latex made of polystyrene as a core and coated with a coating of glycidyl metathalene tocopolymer. There are reagents in which antigens or antibodies are bound to carriers through covalent bonds, such as reagents in which cells are bound to latex by reacting human chorionic gonadotropin or insulin with human chorionic gonadotropin or insulin (Japanese Patent Publication No. 5-5-110118). Proposed.

In these prior art techniques, 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 immunoactive substance. By using cyanogen bromide, 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 an immunoactive substance 'a, the method of reacting a protein or polypeptide with a free epoxy group introduced into a polymer 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, the body fluids of humans and #'i animals contain protein from many types of noodles, and in particular, plasma or serum Vce is high.
It is contained in. If proteins from the sample body fluid are adsorbed by the carrier particles, they may interfere with the target immunological reactions such as antigen-antibody reactions, leading to a decrease in the selectivity and sensitivity of the agglutination reaction. .

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

That is, the present invention uses a surface reduction product of acrylonitrile or methacrylonitrile polymer fine particles as a solid support in a solidified immunoactive substance in which an immunoactive substance is bonded to the surface of a solid body. Such an immunoactive substance immobilized product is prepared 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 after bonding other functional groups to this, Manufactured 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,
In producing such a particulate polymer, acrylonitrile or methacrylate trile may be polymerized individually, or they may be mixed and polymerized in any ratio. In either case, a chargeable 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, methacrylate. Acrylic acid vinyl, triallylisocyanurate, divinyl In the present invention, acrylonitrile, methacrylonitrile, or a polymerizable monomer other than a crosslinkable monomer having two or more carbon-carbon double bonds in the molecule is added and copolymerized. It is also possible. Preferred as such a copolymerization component is a hydrophilic unsaturated monomer (I-1) or a compound that is simultaneously reduced and converted into a wood-philic monomer when the nitrile is reduced after the VC4 monomer is formed in the polymer by copolymerization. be. Examples of preferred compounds as hydrophilic monomers include 2-oxyethyl acrylate, 2-oxyethyl acrylate, 2-oxidipropyl acrylate, 2-oxypropyl methacrylate, and polyethylene glycol monoalkyl having a polymerization degree of 2 to 00. acrylic or methacrylic esters of ethers,
Acrylic 7mid.

These include methacrylamide, N-vinylpyrrolidone, and glyceryl methacrylate. Examples of monomers useful for introducing hydrophilic groups into the polymer by reduction after one-way polymerization include methyl, ethyl acrylic acid or methacrylic acid,
Lower alkyl esters such as n-propyl or propyl; lower alkyl esters such as acetic acid or propionic acid; Two or more of these hydrophilic monomers or hydrophilic monomers may be used in combination. The ratio of the sum of the hydrophilic monomer and the monomer that can be converted to hydrophilic 1: to the sum of acrylonitrile and methacrylate nitrile is the molar ratio ′□
It is possible to change the range between 95:5 and 5:95.
11:1゜ Also acrylonitrile and methacrylonitrile 1.
:;1 77Ni! 'm)t, al 00 : O″l, -+L
O: 100 (%“ □, :,.

J+s”)ffzbaMt, “° □β,
1 The polymer fine particles of the present invention can be prepared, for example, by the following method.
1゜:・1! 1ttTi L<hfxbh;E>・216" angle 0
"h・1□1.

At the same time as polymerization, the polymer becomes particulate and precipitates.
:・I,)-C main departure. , target, suitable, good. Shen Fengchong. teeth,
theory, polymerization in which 11-mer A dissolves but is produced by polymerization.
□1 A method in which polymerization is carried out in a medium that does not dissolve the monomer, and the average diameter of the produced polymer is adjusted to fall within the range of VC from OL to 10 μm by selecting the combination of the monomer and the polymerization medium. It is relatively easy to do so, and the particle size distribution is also relatively narrow. One advantage of precipitation polymerization, unlike emulsion polymerization and 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 ethyl acetate, n-propyl acetate, isopropyl acetate, butyl acetate isomers, and esters such as the above-mentioned corresponding esters of propionic acid, methyl ethyl ketone, methyl n-propyl ketone, methyl isopropyl ketone. , ketones such as methyl butyl ketone isomers, benzene, toluene, 0-xylene, m-xylene, p-xylene, carbon tetrachloride, and water.

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

As the polymerization initiator, common radical polymerization initiators such as 2,2-azobisisobutyronitrile, 2,2-azobis(su-4-dimethylvaleronitrile), λ2-azobis(2,4-dimethyl-4- methoxyvaleronitrile),
Peroxides such as benzoyl oxide, 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 about 0001 to 003 mol/l. 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 concentration of 11-weight compound is less than 5% by weight, the number of polymer fine particles obtained will decrease, resulting in lower productivity. Incidentally, the polymerization is preferably carried out in a static atmosphere with substitution of an inert gas such as nitrogen or argon.

Emulsion polymerization and suspension polymerization are usually carried out using % solubility in water.
The nine polymerization method is suitable for polymerizing low llt polymers in an aqueous medium, but since acronitrile is approximately 7% soluble in water at room temperature, when applying the suspension polymerization method among these polymerization methods, 1-1 [I will keep it a secret from my husband. In other words, when performing 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 due to the salting-out effect and fractionate it as oil droplets, generating oil-soluble radicals. The polymerization agent is used as a polymerization initiator and polymerization is carried out with sufficient stirring. At that time, use polyvinyl alcohol, polyacrylic acid, poly! as a dispersion stabilizer. It is difficult to add and dissolve a water-soluble polymer compound such as q-vinylpyrrolidone in an aqueous medium. Although the polymerization initiator is suitable for precipitation polymerization, it is not necessary to use an oil-soluble one.

12- As with precipitation polymerization, the polymerization temperature is preferably in the range of 20 to 100°C. Polymerization initiator 1-titi polymerization and O, O
It is added in a range of 001 to 01 mol%. Since methacrylonitrile is hardly soluble in water, it can be polymerized by the commonly known M layer polymerization method. @Deep type legal is 10μm
It is suitable for producing polymer particles having a tonnage of 1 or less.

Emulsion polymerization of acrylonitrile and methacrylonitrile can be carried out in the same manner as for other hydrophobic monomers.

That is, it is polymerized in an aqueous medium containing an anionic, cationic, or nonionic surfactant in the presence of a water-soluble radical generator. Also styrene sulfonic acid.

When polymerizing in the coexistence of 01 to 2 mol% of sodium or potassium salts such as methallylsulfonic acid or allylsulfonic acid, the resulting polymer fine particles become stable even without the addition of a surfactant. Polymerization proceeds in a dispersed, suspended state. Preferred examples of polymerization initiators include sodium, potassium or ammonium salts of AHIW, persulfates thereof, and #-i chloric acids)
This is a redox free radical generating system that combines IJium and sodium bisulfite. The concentration of polymerization initiator is 0001
The preferable range of the polymerization temperature is 20 to 80°C. Emulsion polymerization method: 001 to 1 μm
It is suitable for producing polymer fine particles of.

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 amine group, the immunoactive substance can be covalently bonded directly or, if necessary, after being converted into a group that easily reacts with the functional group that the immunoactive substance has, depending on the type of functional group it has. This can be immobilized on the surface of the microparticles by bonding with the nanoparticles.

As is known in the art, cyanogen groups are converted to amine groups by reduction. Reduction methods suitable for that purpose include, for example, lithium aluminum hydride, aluminum hydride, trimequin lithium aluminum hydride,
.

・ , diborane, 3-methyl-2-butylborane, sodium borohydride/aluminum chloride, sodium trisulfate borohydride, sodium acetoxyborohydride, (
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. can be cut by reduction. and converted into a hydroxyl group.

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 take advantage of the anti-15 transient property 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.

Glutaraldehyde is the most suitable dialdehyde to react with the amine group. Other aliphatic dialdehydes having different carbon numbers such as scunnic aldehyde and adipine aldehyde, aromatic dialdehydes such as terephthalaldehyde, and polymeric aldehydes such as starch aldehyde can also be used. Dialdehyde treatment of surface reducer particles is
Dialdehyde concentration O1~25'M, aqueous solution po 5
It is preferable to carry out the reaction at a temperature in the range of 0° C. or higher and 60 rev below. Fine particles treated with dialdehyde
R- is thoroughly washed with water and used for the next immobilization of an immunoactive substance. Proteins can also be immobilized on particles by VC by condensing the amino groups of the microparticles with the carboxyl groups of the protein using 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.

When an immunologically active substance or physiologically active substance is chemically immobilized on carrier particles in this manner, the bond is usually strong, and the immobilized immunologically active substance or physiologically active substance will not be released from the carrier particles. However, if immobilization is performed by glutaraldehyde activation, the stability of the bond may be somewhat insufficient, but if this is not possible, use borohydride to further strengthen the bond!・It is effective to treat with lithium, sodium chloride/sodium borohydride, dimethylamine bora 7, etc. However, such secondary processing is rarely necessary.

In the present invention, we use not only immunologically active substances, LJ antigens, and antibodies, but also substances that are involved in humoral immune reactions or cellular immune reactions, such as complement, Fc receptors, and C3 receptors, and that bind specifically to certain substances. shall mean. Specific examples include Trebonema pallidum antigen, hepatitis B surface antigen (HB, antigen), antibody to hepatitis B surface antigen (anti-HB, antibody), rubella antigen, tokinplasma antigen, streptolysin 0. Anti-streptolysin 0 antibody, mycoplasma antigen, human chorionic gonadotropin (HCG),
anti-IT CQ antibody, heat aggregation protein (I'gO), rheumatoid factor, nuclear protein, DNA, anti-DNA antibody, C-reactive protein (ORP).

Anti-CRp antibody, anti-estrogen antibody, α-fetoprotein (α-FP), anti-α-FP antibody, carcinoembryonic antigen (
CEA), anti-CEA antibody, C1q, anti-C1q antibody, C
3, anti-C3 antibody, anti-3b antibody, anti-03bi antibody, C4
, anti-C4 antibody, protein-A, conglutinin, immunoconglutinin, etc.

When binding an immunologically active substance to the surface of the fine particles, it is preferable to dissolve the immunoactive substance in an aqueous medium and react with it. The speed of the immunoactive substance in the reaction solution must be increased by taking into account the properties of each individual immunoactive substance, and cannot be determined uniformly, but it is preferable that the pH range is 6 to 9 and the temperature range is 0 to 40°C. It's okay.

If all free amine groups are not consumed due to reaction with immunologically active substances and there is a possibility that active groups may remain, hydrophilic proteins such as serum albumin and gelatin that do not interfere with the target immunological test are released. By reacting with an amine group, the reactivity of the amino group can be lost. In this case, the hydrophilic protein for amine group elimination may be mixed with the immunoactive substance to be immobilized and reacted at the same time, or the immunoactive substance may be reacted with t1i alone first and then reacted. . Furthermore, it is also possible to use amino acids such as green and alanine in place of the above-mentioned 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 by 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 in sample body fluids, resistant to non-specific aggregation, and non-specific to single cells. No adhesion.

It is also advantageous for both agglutination reactions and cell labeling purposes if the fine particles are irradiated with fluorescent light to suitably color the dye or pigment.

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 the irregularly shaped fine particles is h/2, which is the sum of the maximum diameter and the minimum diameter. The average diameter described in the examples represents d defined by equation (1).

d=Σ d1/N (+)1=where dl is the diameter of the first particle and N is the total number of particles. Empirically, it is easier to determine the agglutination reaction when the average diameter is 0.
This is a case of 1 μm or more and 10 μm or less. Further, for the purpose of cell labeling, the average diameter is preferably in the range of 0.03 μm or more and 5 μm or less.

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

Example 1 495 g of acrylonitrile in ethyl propionate lo#
Dissolved, and further added and dissolved 0, ] 2 p of azobis(4-methoxy-milk 4-dimethylvaleronitrile),
The mixture was left undisturbed at 30° C. for 16 hours under an argon atmosphere. When the precipitated polymer was separated by r and dry weighed, the yield was f[#146
The polymerization rate t was 185% at 1 g. Further, as a result of observing the polyacrylonitrile fine particles obtained here using a scanning electron microscope, the diameter was within the range of 05 to 06 μm and was almost uniform. 05g of these polyacrylonitrile fine particles were mixed with 5oI of dry ethyl ether! lithium aluminum hydride under an argon atmosphere.
2 g was added and stirred at room temperature for 3 hours. Next, ethyl acetate was gradually released while stirring.

Add 1 more (approx. 5m1) at the neck where the foaming disappears.
20ml of normal hydrochloric acid was added dropwise. When left to stand, it separated into two layers, so remove the transparent upper layer and remove the gray-white lower layer (T+ 81-2
) was separated by far L11.

The sediment was washed twice with 6N salt spray and twice with distilled water by centrifugation.

Further, when the polymer particles were washed twice with 1 mol/1 sodium carbonate and twice-distilled water, the polymer particles became white. The polyacrylonitrile micro-r/f reduction product prepared by this method was dispersed at 1% in distilled water, and as a result of immersion in l/+0 (1N hydrochloric acid), the amino-based tFio , o y m l a R/ 9 T 6
The above reduced polyacrylonitrile fine particles were dispersed at 0.5% in 1c of distilled water (5ml), and the pH was adjusted to IO with a 0.75ml aqueous solution of 25% glutaraldehyde, grade for electron microscopy, at 30°C under a nitrogen atmosphere. So I fiffed it for 2 hours. The reaction mixture was then centrifuged 1. Washed three times with distilled water and washed with phosphorus I containing 0.1% sodium azide.
Washed once with v-salt buffered saline (sodium/disodium hydrogen oxide + sodium/potassium hydrogen oxide 001 mol/l, [sodium chloride 0.14 mol/1, pN 7.2, hereinafter abbreviated as PBS). , fine particles 0.51! Il pBBK was isolated. The dispersion and bovine internal albumin (hereinafter abbreviated as B8A) were mixed in a 10 J/ml F B 81H solution of 0.5
After stirring at 30C for 3 hours and centrifuging, the e-particles were washed 3 times with PBS and then dispersed in 2ml/ml of PB11 solution containing human serum albumin at 1OmJil/ml. Like this! tillta B8
When 10 μm of the A-immobilized particle dispersion and 10 μm of a 100-fold dilution of anti-B8^ antiserum (rabbit) diluted with PB8GC were mixed on a glass plate, agglutination was heard in the meat@VC. A similar experiment was conducted using normal rabbit serum instead of anti-B8A antiserum (rabbit), and no agglutination was observed. Therefore, the above agglutination reaction is ratiT
This is due to the anti-B8^ antiserum reaction between BSA immobilized on - and the antiserum.

23-Example 2 Example! The same polyacrylonitrile microparticles used in were similarly treated with glutaralde7ide. Separately, syphilis disease condition τr@panema palldu+++c below T
(abbreviated as P) Nlchola strain bacterial cells were placed in pBs [10
・While cooling the dispersion liquid dispersed at the ratio of call/al with ice water, @frti of 10KH1.

The cells were treated with water for 20 minutes to destroy the bacterial cells, and this was used as a TP antigen solution. This TP antigen solution IEL and the dispersion fil1111 obtained by dispersing the glutaraldehyde-treated polymer particles 125@9 in PBS Licl were combined and stirred at 30°C for 2 hours.Then, fine particles were separated by centrifugation. After washing three times with PBB by centrifugation, run serum albumin c or less BS^
PH81 + ml K with 1% added (abbreviated as ) was dispersed.

This TpvL original immobilized fine particle dispersion was cooled at 4°C for 3 days.
After dissipating in far, the activity was assayed in the same manner as in Example 1.

Agglutination occurred in a sample in which a syphilis-positive serum with a TPHf titer of 640 was diluted 2 times and 4 times with PBS, but no agglutination was observed in the 10 times diluted sample. In syphilis-negative sera, no II collection was observed at any of the above dilutions. It was thus confirmed that the 'rpvL origin was immobilized on the t1 microparticles that retained their activity.

Example 3 The molar ratio of a mixture of 28.521 parts by weight of methacrylonitrile (the same applies hereinafter) and 29 g of dipinylbe/ze is SS:
S), Propio 7tI is the 1st body temperature mixture! ethyl 1o
Dissolve Oslc.

0.3 parts of λ2'-a/bis(λ4-dimethyl-4-notok7I8leronitrile) was added as a polymerization initiator, and the mixture was allowed to stand for 24 hours for 40 minutes under an argon atmosphere. Centrifuge the cloudy polymer solution to separate fine polymer particles, and dilute with ethyl acetate.
cIII and then dried under reduced pressure. TL polymer fine particle collection 1ir
It was the Fixo club. The diameter of most of the polymer fine particles was in the range of 2 to 3 μm.Next, 1.2 jJ of flit cobalt hexahydrate was dispersed in methanol IS + nl separately from the dry polymer, and the above polymer was dispersed therein. Add methacrylonitrile imitation grains 7-o, +69'c, and add 1() of sodium borohydride while stirring at 20°C.
When I is gradually added, hydrogen gas is released/1! 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 mixed with distilled water for 20 minutes.
ml, added 3 g of 25% glutaraldehyde aqueous solution, stirred at 30°C for 2 hours, washed with water, and prepared P B 8
10 mlK was dispersed.

Separately from adult bovine serum, the method of Lachman et al. (D, M.

Weir “Handbook of Experiment”
mentalImmunology”Volume 1・”Imm
unochemistry” 3rd edition, Blac, kwe
ll 8oienti, fio Publication
ns+Oxford (1978), Chapter 5, 6N). That is, H distilled water 700 ml VC Veronal Natriu Amo
, s 24 , jil and sodium chloride 8.5
g was dissolved, and a small amount of I/10N hydrochloric acid was added to adjust the pH to 7.2Kf14m. Distilled water 200mJ apart from Soku
A solution of 0.369 Kq magnesium hexahydrate, 0.037 calcium chloride dihydrate, and pif was prepared, mixed with the above veronal solution, and 0.2 J of sodium azide and 1,0 g of gelatin were added. Then, distilled water was added to bring the total volume to 1.

10 ml of this conglutinin CFDrB solution.

The mixture was mixed with 10 ml of a PBS dispersion of the glutaraldehyde-activated surface-reduced polymer fine particles, incubated at 30° C. for 2 hours, and thoroughly washed with PH1. The ability of these funglutinin-immobilized microparticles to bind to immune complexes bound to complement component C3 was demonstrated by the following procedure.

Human 1.9G12m,9 was dissolved in 2 ml of physiological saline and heated to 63°C for 20 minutes to cause 27-thermal aggregation of IgG. Heat aggregation xgG (immune complex model substance,
The insolubilized portion of AHG (hereinafter abbreviated as AHG) was heated at 1.500
Centrifuge for 15 minutes at G and remove the supernatant from A.
The AHG-containing supernatant was diluted 20 times with PH1, mixed with the same volume of fresh human serum, and incubated for 15 minutes at 37°C to bind complement to AHG. Next, AHG after this incubation
When 10 μl of the serum mixed solution and 10 μm of several ton of conglutinin fixed particles were mixed on a glass plate, they agglomerated violently.

When AHG was incubated with human serum that had been inactivated for 30 minutes at 56°C instead of fresh human serum and mixed with the conglutinin-immobilized fine particle dispersion, no aggregation occurred.

Example 4 10 parts of acrylonitrile, 126 parts of methacrylotrile, and 25 parts of divinylbenzene were prepared.
02 parts of 4-dimethyl-4-methoxypaleronitrile) was left standing in propionic solution at 28-40°C for 7 hours (the molar ratio of acrylonitrile and methacrylonitrile was #''t1:1).The cloudy polymer solution was centrifuged. The polymer fine particles were separated, washed with ethyl acetate, and then dried under reduced pressure. Yield of polymer fine particles Fi3.7s
It was a department. Average diameter of single polymer fine particles #'iloμ
It was m. 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 TP
Antibody titer 6 using antigen-immobilized polymer particles and TPHA method
When 40 plum S-positive sera were reacted on a glass plate in the same manner as in Example 2, the results were the same as in Example 2.

Patent applicant: Toshi Co., Ltd.

Claims (2)

[Claims] (1) Immobilization of an immunoactive substance in an immunoactive substance solidified product in which an immunoactive substance is bonded to the surface of a solid carrier, characterized in that the solid body is a surface reduction product of acrylonitrile or methachronitrile polymer fine particles. Fine particles. (2) After reducing the surface of the acrylonitrile or methachronitrile polymer fine particles and bonding the functional groups generated by the reduction directly or with other functional groups,
-1. A method for producing immunoactive substance-immobilized microparticles, the characteristic of which is to bind an immunoactive substance to stone.