JPS61155958A - Immunological diagnostic reagent - Google Patents

Abstract

PURPOSE:To obtain a reagent having good storage stability, by compounding calcium bromide in a mixture of an alkyl acrylate derivative, an acrylic acid derivative, a vinyl monomer having a sulfonic acid group and a polyfunctional internal crosslinking monomer. CONSTITUTION:As monomer components, not only 0.1-30wt% of an acrylic acid derivative having a carboxyl group represented by R<3>CH=CR<4>COOH [wherein R<3> is H, a lower alkyl group or a carboxyl group, R<4> is H or lower alkyl group and, when R<3> is H or a lower alkyl group, R<4> may be a carbo-(lower) alkoxy group] and 0.1-20wt% of a vinyl monomer having a sulfonic acid group but also 1-20wt% of a polyfunctional internal crosslinking monomer are used in addition to 30-98.9wt% of an alkyl acrylate derivative represented by a general formula CHz=CR<1>COOR<2> (wherein R<1> is H or an alkyl group and R<2> is an 1-8C alkyl group). Further, calcium bromide is compounded as an additive to make it possible to perform stable emulsion copolymerization without generating a floc even if no emulsifier is especially used.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an immunological diagnostic reagent, and more specifically, it consists of an aqueous dispersion of water-dispersed polymer particles on which an immunoactive substance is immobilized, and is made of a latex-based dispersion. The present invention relates to an immunological diagnostic reagent that does not cause non-specific agglutination reactions, allows easy determination of agglutination reactions, and has excellent storage stability.

(Prior Art) In recent years, immunological diagnostic methods that utilize the immune activity of physiologically active substances in blood, urine, and other body fluids have been developed for medical diagnosis of pathological or other conditions in humans and animals. Widely used. In this method, either an antigen or an antibody that causes an immunological reaction, or a combination of both, is reacted with a test fluid such as a body fluid, and the relationship between the antigen or antibody and the corresponding antibody or antigen is detected. By a specific reaction, that is, an agglutination reaction or an agglutination inhibition reaction based on an antigen-antibody reaction,
This is a method for measuring the presence of immunoactive components as described above. In this case, in order to facilitate observation with the naked eye, the antigen or antibody is generally supported on a microparticulate carrier, such as latex or red blood cells, as a diagnostic reagent, and the agglutination reaction of such particles is utilized. Then, test components in body fluids such as serum are measured.

For example, to explain the agglutination reaction of a diagnostic reagent whose microparticles are made of latex, when a diagnostic reagent consisting of an aqueous dispersion containing microparticles carrying an antigen or antibody is mixed with a test liquid, an agglutination reaction corresponding to the antigen or antibody is detected. The antibody or antigen in the test solution reacts specifically with the antigen or antibody on the microparticles, resulting in a latex agglutination reaction, that is, a macroscopically observable agglutination reaction of the microparticles. However, if the antibody or antigen to be measured is not present in the test liquid, no macroscopically observable agglutination occurs. In this way, the presence or absence of an antibody or antigen in a test liquid can be determined by the presence or absence of an agglutination reaction of the microparticles carrying the antigen or antibody.

- Such immunological diagnostic reagents have high detection sensitivity that can detect immunoactive substances, i.e., antigens or antibodies, even if they are present in a trace amount in a sample solution, and have the ability to detect the target immunoactive substance. It is required to have high specificity to react only with Furthermore, it is required to maintain high sensitivity and specificity even during long-term storage.

Conventionally, polystyrene latex particles have been widely used as particulate carriers in such immunological diagnostic reagents. In most cases, this polystyrene latex
It is produced by emulsion polymerization of styrene in the presence of an emulsifier and a water-soluble radical polymerization initiator. Here, the emulsifier is generally effective in ensuring polymerization stability during emulsion polymerization and in obtaining a latex containing polymer particles having a small particle size and good dispersion stability. The effect of emulsifiers on increasing the dispersion stability of latexes obtained in this way is not necessarily clear, but in general, some of the emulsifiers are adsorbed to latex particles, and the rest remains free in the latex. Thus, in the latex, an adsorption-desorption equilibrium exists between the emulsifier adsorbed on the polymer particles and the free emulsifier, and as a result of this equilibrium, stabilization of the latex is said to be achieved. There is.

Therefore, when an antigen or antibody is immobilized on polystyrene latex containing an emulsifier as described above,
If the latex contains free emulsifier, the latex particles may aggregate during this immobilization operation. Furthermore, when performing immunological diagnosis using polystyrene latex immobilized with antigens or antibodies, not only positive serum containing the corresponding antibody or antigen but also polystyrene latex particles are hydrophobic, , an agglutination reaction may also occur against negative serum that does not contain the corresponding antibody or antigen. Such an agglutination reaction is called a non-specific agglutination reaction. In addition, immobilization of antigens or antibodies onto polystyrene latex particles simply causes the antigens or antibodies to be adsorbed onto the surface of the polystyrene particles, and some of them may be desorbed from the latex particles and interfere with the above-mentioned specific agglutination reaction. There is. In this way, conventionally used immunological diagnostic reagents using polystyrene latex particles as carriers are
It has the problem of lacking diagnostic accuracy.

For this reason, monomers having functional groups are emulsion copolymerized, and by utilizing the functional groups of the resulting latex particles,
Immunological diagnostic reagents in which antigens or antibodies are covalently immobilized on these particles have already been proposed. For example, a diagnostic reagent in which an immunoactive substance is immobilized on carboxylated polystyrene latex or carboxylated styrene-butadiene copolymer latex using carbodiimide has already been proposed (Japanese Patent Laid-Open No. 16623/1983).

However, this diagnostic reagent is unstable to environmental changes, and as described below, it easily precipitates when additives are added to suppress nonspecific aggregation of the diagnostic reagent, and it is not stable during storage. There are problems with gender.

In addition, for example, using a water-soluble radical polymerization initiator, methacrylic acid alkyl ester can be converted into hydrophilic methacrylic acid,
A method for obtaining a methacrylic acid ester latex by emulsion copolymerization with 2-hydroxyethyl methacrylate and a polyfunctional monomer in the presence of an emulsifier is also already known (Polymar).

Vol, 19. August, 867-871 (1
978)) However, since the latex obtained by this method also contains an emulsifier, it has the same problems as above, and furthermore, this seems to be because the obtained copolymer particles have hydroxyl groups, but in particular, Latex particles tend to aggregate easily during the immobilization stage.

For this reason, in recent years, a method of obtaining acrylic ester latex in the absence of an emulsifier has been proposed (Kagaku Gijutsu Kenkyusho Report Vol. 75, No. 8, p. 341 (1980)).
The latex produced by this method also does not have sufficient dispersion stability,
In particular, it easily aggregates under mechanical shear stress.

Therefore, in order to prevent non-specific agglutination of latex particles, conventionally, when determining the presence or absence of latex agglutination reaction, serum is diluted with a buffer such as glycine, or complement in serum is lost. A deactivation process is being carried out to keep it active. However, depending on such processing,
It is difficult to sufficiently suppress nonspecific aggregation, and there is also the problem that diagnosis is time-consuming due to the labor involved.

In order to solve these problems with immunological diagnostic reagents, it has been common practice to add additives to diagnostic reagents for the purpose of suppressing non-specific agglutination reactions. Known agents include, for example, glycols, proteins such as gelatin and albumin, and polyanions. However, the effects of these additives are generally not sufficient, so in recent years, additives such as sucrose and choline chloride (Japanese Patent Laid-Open No. 54-02
6327), N,N-dialkylamides, di-lower alkyl sulfoxides (Japanese Unexamined Patent Publication No. 160853/1983), and the like have been proposed.

Furthermore, in recent years, various inorganic salts have been proposed as additives having the effect of suppressing non-specific aggregation. For example, JP-A-56-158947 discloses guafuzine, guanidine hydrochloride, guanidinium thiocyanate,
Chaotropic substances, of which urea is a typical example,
alkali metal halides such as lithium chloride, lithium bromide, sodium bromide, potassium bromide, and lithium iodide, and metal halides such as calcium chloride are described as chemiotropic agents, as well as opic) agents. Further, JP-A-57-35754 also similarly describes alkali metal halides.

However, alkali metal halides generally have significant non-specific agglutination with negative serum, and calcium chloride not only has significant non-specific agglutination with negative serum, but also has a tendency to be mixed with negative serum during diagnosis. If the initial degree of agglutination after several minutes is false positive, nonspecific agglutination becomes stronger over time and the degree of agglutination changes over time, making it impossible to make an accurate diagnosis. be. Furthermore, immunological diagnostic reagents containing calcium chloride as an additive may
Precipitation occurs in the diagnostic reagent, and the diagnostic reagent must be thoroughly shaken to make it homogeneous before use, resulting in poor storage stability and usability. As described above, various inorganic salts have been proposed as additives, but they still have problems in that they are less effective in suppressing nonspecific aggregation and the diagnostic reagents have poor storage stability.

(Purpose of the Invention) As a result of intensive research in order to solve the above-mentioned problems in immunological diagnostic reagents, the present inventors found that an aqueous dispersion which itself has much better dispersion stability than conventional microparticle carriers. type high molecular weight polymer particles are found, an immunoactive substance is immobilized on such polymer particles by covalent bonding, and furthermore, when calcium bromide is allowed to coexist in an aqueous dispersion of such polymer particles, calcium bromide has the above-mentioned properties. Compared to conventionally known additives, including inorganic salt additives, it has a much better effect on suppressing non-specific aggregation, so non-specific aggregation reactions are completely suppressed. Having discovered that it is possible to obtain an immunological diagnostic reagent that has high detection sensitivity and high specificity, and also has excellent storage stability,
This led to the present invention.

Therefore, the present invention does not dilute serum, and moreover,
The purpose of the present invention is to provide an immunological diagnostic reagent in which non-specific agglutination is suppressed without any inactivation process, the presence or absence of an agglutination reaction can be easily determined, and the reagent has excellent storage stability. .

(Structure of the Invention) The immunological diagnostic reagent according to the present invention has the following formula: (a) General formula % Formula % (However, R1 represents hydrogen or a lower alkyl group, and R2 represents an alkyl group having 1 to 8 carbon atoms. ) 30 to 98.8% by weight of an acrylic acid alkyl ester derivative represented by
, M) General formula % Formula % (However, R3 represents hydrogen, a lower alkyl group or a carboxyl group, R4 represents hydrogen or a lower alkyl group, and R3
When is hydrogen or a lower alkyl group, R4 may be a carbo-lower alkoxy group. ) 0.1 to 30% by weight of an acrylic acid derivative represented by (C) 0.1 to 20% of a vinyl monomer having a sulfonic acid group
% by weight, and (dl polyfunctional internal crosslinking monomer 1 to 20% by weight) in an aqueous medium by emulsion copolymerization of the water-dispersible polymer particles. An immunological diagnostic reagent in which an immunologically active substance is immobilized and dispersed in an aqueous medium is characterized by containing calcium bromide.

The acrylic acid alkyl ester derivative used in the present invention has the general formula % (where R1 represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, and R2 represents an alkyl group having 1 to 8 carbon atoms). ), for example, methyl (meth)acrylate, ethyl (meth)acrylate, propyl (meth)acrylate,
Examples include butyl (meth)acrylate, hexyl (meth)acrylate, octyl (meth)acrylate, and the like.

In the present invention, in order to obtain an emulsion copolymer emulsion of an acrylic acid alkyl ester derivative having excellent dispersion stability in an aqueous medium, in addition to the above-mentioned acrylic acid alkyl ester derivative, a carboxyl group is added as a monomer component. In addition to using an acrylic acid derivative having a sulfonic acid group and a vinyl monomer having a sulfonic acid group, a polyfunctional internal crosslinking monomer is used. According to the present invention, by using a mixture of such monomer components in a predetermined ratio, emulsion copolymerization can be carried out stably without the generation of aggregates without using an emulsifier, and in an aqueous medium. This makes it possible to obtain an aqueous dispersion of copolymer particles that maintains a stable dispersion state and is non-swellable.

The above acrylic acid derivative used in the present invention has the general formula % (where R3 represents hydrogen, a lower alkyl group or a carboxyl group, preferably hydrogen or a methyl group, and R4 represents hydrogen or a lower alkyl group, preferably hydrogen or represents a methyl group, and when R3 is hydrogen or a lower alkyl group, R4 may be a carbo-lower alkoxy group.) For example, (meth)acrylic acid, itaconic acid, crotonic acid, maleic acid Preferred examples include fumaric acid, monoalkyl maleic acid, monoalkyl fumaric acid, monoalkyl itaconic acid, etc., and in particular, one type or a mixture of two or more of (meth)acrylic acid and itaconic acid is preferred. used.

As described below, such acrylic acid derivatives provide a functional group for covalently immobilizing an immunologically active substance to the polymer particles, and also provide a spacer group for covalently bonding a spacer group to the polymer particles. In addition to providing functional groups for the polymer particles to function, they also impart a negative charge to the polymer particles, increasing the stability of the particles in aqueous dispersions.

Next, in the present invention, examples of the vinyl monomer having a sulfonic acid group include alkylene sulfonic acids such as ethylene sulfonic acid, and general formula % (where R8 is hydrogen or a lower alkyl group, preferably hydrogen or represents a methyl group, R & represents an alkylene group having 1 to 6 carbon atoms, preferably an alkylene group having 2 to 3 carbon atoms,
M represents hydrogen, alkali metal or ammonium. ) Sulfoalkyl acrylates, such as sulfopropyl (meth)acrylate and its alkyl metal salts, general formula (where R7 represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, and M is the same as above) Styrene sulfonic acid, its derivatives, alkyl metal salts thereof, such as sodium styrene sulfonate, represented by the general formula % (where R1 represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group) , R9 represents an alkylene group having 1 to 6 carbon atoms, preferably an alkylene group having 3 to 4 carbon atoms,
M is the same as above. ) 2-acrylamidoalkanesulfonic acid, derivatives thereof, and alkyl metal salts thereof, such as 2-acrylamido-2-methylpropanesulfonic acid, are used.

Monomers having such sulfonic acid groups, like the acrylate ester derivatives, also impart a negative charge to the resulting particles, increasing the stability of the particles in an aqueous dispersion.

In the present invention, the polyfunctional internal crosslinking monomer introduces a crosslinked structure into the polymer, so if it is included in the diagnostic reagent, it suppresses the formation of undesirable water-soluble polymers, and the resulting polymer particles can increase the glass transition temperature of Furthermore, the internal crosslinking agent is effective in making the water-dispersible polymer particles non-swellable and increasing the dispersion stability of the polymer particles in an aqueous medium.

As such a polyfunctional internal crosslinking monomer, for example, poly(meth)acrylate of aliphatic polyhydric alcohol is preferably used. Specific examples include ethylene glycol dimethacrylate, diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, 113-
Butylene glycol dimethacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, and the like are preferably used. Also, divinylbenzene, N,
'-Methylenebisacrylamide and the like can also be used as a polyfunctional internal crosslinking monomer.

When producing an aqueous dispersion of water-dispersible polymer particles used as a microparticle carrier in the present invention, the monomer composition for emulsion copolymerization is preferably 30 to 98.8% by weight of an acrylic acid alkyl ester derivative. 50 to 98% by weight, 0.1 to 30% by weight of acrylic acid derivatives, preferably 0.5 to 20% by weight, and 0.1 to 20% by weight of vinyl monomers having sulfonic acid groups, preferably 0.5% by weight. ~15% by weight
, 1 to 20% by weight, preferably 1 to 15% by weight of the polyfunctional monomer for internal crosslinking.

As described above, the acrylic acid derivative and the vinyl monomer having a sulfonic acid group are used to improve the polymerization stability during emulsion copolymerization of the acrylic acid alkyl ester derivative and the aqueous dispersion of the resulting copolymer particles. These monomers are essential for liquid stability, and in order to effectively express these effects, each of them requires at least 0.1% by weight in the monomer composition.

However, when used in excess as a copolymerized vehicle component,
On the contrary, they impair the polymerization stability and the stability of the resulting dispersion, so they are used in a range of 30% by weight or less and 20% by weight, respectively.

In addition, as described above, the polyfunctional monomer for internal crosslinking is
A monomer necessary for stably proceeding polymerization, maintaining a stable dispersion state of the obtained particles, and making the particles non-swellable.
% by weight is necessary, but using too much is not preferable because it will actually impair the polymerization stability and the stability of the particle dispersion.

Furthermore, in the present invention, in order to further improve the polymerization stability during emulsion copolymerization of the monomer mixture and the water dispersion stability of the resulting particles, (meth)acrylonitrile may be used as a monomer component. can. The preferred amount to be used is up to 40% by weight based on the monomer composition; if it is used in an amount larger than this, the polymerization stability will be adversely affected, and the resulting particles will have poor dispersion stability. . The lower limit of the effective amount is not particularly limited, but is usually 1% by weight based on the monomer composition. A particularly preferred amount used is in the range 5 to 20% by weight.

In addition, other radical copolymerizable monomers, such as hydroxyethyl acrylate, 2- A monomer having a hydroxyl group such as hydroxyethyl acrylate, a monomer having a glycidyl group such as glycidyl methacrylate, an α-olefin monomer such as ethylene or propylene, a vinyl spinning wheel 11 such as vinyl acetate or vinyl chloride, Styrenic monomers such as styrene, methylstyrene, and chlorostyrene, diene monomers such as butadiene and isoprene, and monomers such as acrylamide and methacrylamide can be used as monomer components as necessary. .

In the preparation of the water-dispersed polymer particle aqueous dispersion, the specific types of individual monomers are such that the resulting copolymer particles have a glass transition point of 0° C. or higher, preferably room temperature or higher. are selected as such. When the glass transition point of the particles is lower than 0°C, mutual fusion and aggregation of the particles tend to occur.
This is because the dispersion stability of the dispersion liquid tends to decrease.

In the present invention, a water-insoluble acrylic acid alkyl ester is obtained by emulsion copolymerizing each of the above monomers in an aqueous medium using a water-soluble radical polymerization initiator in a conventional manner. Aqueous dispersions of copolymer particles of the derivative can be obtained. However, when the emulsifier is present in the resulting aqueous dispersion in a free state or in a state adsorbed to polymer particles, it may be harmful, for example, when immobilizing an immunoactive substance to the particles, as mentioned above. Therefore, it is preferable not to use an emulsifier during emulsion copolymerization. According to the above monomer composition according to the present invention, copolymerization can be carried out stably without particularly using an emulsifier, and the dispersed state of the particles can be stably maintained in the resulting aqueous dispersion of polymer particles. It is a great feature. However, as described above, there is no hindrance to using an emulsifier as long as no aggregation or sedimentation of harmful particles occurs during immobilization or diagnosis, and an emulsifier may be used as needed.

In addition, in the emulsion copolymerization according to the present invention, the concentration of the monomer component mixture in the aqueous medium is usually 0.1 to
It is in the range of 40% by weight.

As the polymerization initiator, a water-soluble radical polymerization initiator is used. Usually, persulfates such as potassium persulfate, sodium persulfate, ammonium persulfate, these persulfates and thiosulfates such as sodium thiosulfate, potassium thiosulfate, sodium hydrogen thiosulfate, etc., or sodium sulfite, potassium sulfite. Redox polymerization initiators with sulfite such as sodium bisulfite and the like are preferably used, but are not limited thereto. The amount of these polymerization initiators to be used is preferably in the range of 0.1 to 1% by weight based on the monomer mixture.Although the atmosphere for polymerization is not particularly limited, it is preferably an inert gas atmosphere excluding oxygen. is used. In addition, the polymerization temperature is not particularly limited, but
Usually, the temperature is in the range of 20 to 100°C, preferably 40 to 90°C.

In the immunological diagnostic reagent according to the present invention, the average particle diameter of the water-dispersed polymer particles obtained as described above, which is a carrier for immobilizing an immunoactive substance, is preferably 0.03. ~2 μm, particularly preferably 0.1-1 μm
It is. If the average particle size is too small, it will be difficult to visually observe the aggregation of water-dispersed polymer particles immobilized with an immunoactive substance due to antigen-antibody reactions; on the other hand, if the average particle size is too large, the polymer particles This is because it becomes difficult to maintain a stable dispersion state. Further, the specific gravity of the polymer particles is preferably in the range of 0.9 to 1.5, and further,
As described later, the specific gravity after immobilizing the immunoactive substance is preferably in the range of 1.0 to 1.3. When the polymer particles have a specific gravity smaller than the above range before and after immobilization of the immunoactive substance, the polymer particles float on the surface of the aqueous medium in the aqueous dispersion, resulting in poor dispersion stability. On the other hand, when the particle size is larger than the above range, the particles tend to settle and aggregate in the aqueous medium of the dispersion (this is because the dispersion stability similarly becomes poor).

Furthermore, in the present invention, the particles have 0.00 carboxyl groups.
It is preferred to have a density in the range of 7X10-' to 60X10-' mol/d. When the carboxyl groups of the particles are less than 0.7X10''mol/n, the water dispersion stability is insufficient;
This is because when the amount is larger than 1, the particles tend to have swelling properties especially in an alkaline aqueous solution, resulting in poor water dispersibility.

As described above, according to the present invention, it is possible to perform emulsion copolymerization of an acrylic acid alkyl ester derivative while ensuring polymerization stability without using an emulsifier, and to obtain copolymer particles obtained. is non-swellable and stably maintains its dispersion state in an aqueous medium. Furthermore,
Generally, when an immunologically active substance is immobilized on a microparticle carrier, the charge state on the particle surface changes and the dispersion stability of the particles changes in the direction of instability. According to the combined particles, since the particles have a carboxyl group and a sulfonic acid group, a stable dispersion state can be maintained during and after immobilization of the immunologically active substance to the particles, thus improving dispersion stability and This makes it possible to obtain an immunological diagnostic reagent with excellent storage stability.

In the immunological diagnostic reagent according to the present invention, it is preferable that the immunoactive substance is covalently immobilized on the water-dispersed polymer particles via a spacer group. That is, it is preferable that a spacer group is bonded to the polymer particle by a covalent bond, and an immunoactive substance is immobilized to this spacer group by a covalent bond. This is because when calcium bromide is immobilized on particles and added to the aqueous dispersion, some immunoactive substances may lose almost no activity and may not show an agglutination reaction with positive serum. As described above, the water-dispersed polymer particles used in the present invention are obtained by emulsion copolymerization of a monomer mixture containing an acrylic acid derivative as a monomer component, and therefore, the water-dispersed polymer particles used in the present invention are derived from this acrylic acid derivative. The carboxyl group functions as a functional group to covalently bond the spacer group to the polymer particle.

The compound that can be used as the spacer group is at least a difunctional organic compound, and does not exclude polyfunctional polymers, but particularly difunctional organic compounds having a carbon chain of 1 to 12 carbon atoms. Compounds are preferred. Specific examples of compounds that function as such spacer groups include, for example,
Diamines such as hexamethylene diamine, dodecamethylene diamine, and xylylene diamine, aminoalkyl carboxylic acids such as glycine, β-aminoprobionic acid, T-aminobutyric acid, ε-aminocaproic acid, and ε-aminocaprylic acid, lysine, and glutamic acid. , β-alanine, arginine, glycylglycylglycine, and the like are preferably used, but are not limited to these. This spacer group may be bonded to the polymer particle in advance, and then the spacer group and the immunoactive substance may be bonded,
Alternatively, the spacer group may be bound to the immunoactive substance in advance and this may be bound to the polymer particles. Furthermore, if necessary, spacer groups can be bonded to both the polymer particles and the immunoactive substance in advance to bond them to each other.

However, if the immunoactive substance has the desired activity when a predetermined concentration of calcium bromide is blended into an aqueous dispersion of water-dispersed polymer particles on which the immunoactive substance is directly immobilized by covalent bonds. Needless to say, the immunoactive substance may be directly immobilized on the water-dispersed polymer particles.

An immunoactive substance is directly covalently immobilized on water-dispersed polymer particles having the above-mentioned functional groups, or a spacer group is bonded to the polymer particles, and the immunoactive substance is covalently bonded to the spacer group. The method for immobilization by binding is not particularly limited, and any conventionally known method can be used.

For example, if an aminocarboxylic acid is used as a spacer group, one of the preferred methods is to combine the amino group of the aminocarboxylic acid with the carboxyl of the water-dispersed polymer particles in the presence of a water-soluble carbodiimide. The spacer group is bonded to the polymer particles by reacting with the group to form an amide bond, and then the immunoactive substance is covalently bonded to the carboxyl group of the spacer group using water-soluble carbodiimide. Can be immobilized.

Examples of the water-soluble carbodiimide used in this method include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-
3-(2-morpholinoethyl)carbodiimide-meth-
Examples include p-)luenesulfonate. The use of such water-soluble carbodiimides to immobilize an immunologically active substance onto polymer particles by covalent bonding, directly with or without a spacer group, can be achieved by conventionally known conventional methods. and conditions may apply. For example, if a spacer group is used, an appropriate amount of water-soluble carbodiimide is added to the aqueous dispersion of polymer particles along with the spacer group, for example, 0.01 to 10 mg/ml per unit volume of the aqueous dispersion. The reaction may be carried out under normal conditions, for example, under pi of 4 to 10, at a temperature of about 5 to 60° C. for several minutes to several tens of hours, usually about 1 to 5 hours.

Next, an immunoactive substance may be similarly immobilized on the polymer particles to which the spacer groups are bonded.

The immunologically active substance used in the present invention is not particularly limited, and any of antigens, antibodies, haptens, etc. may be used. For example, human and animal immunoglobulins, modified immunoglobulins, α-fetoprotein, C-reactive protein (
CRP), hepatitis virus-related antigens, various viral antigens such as wind iHA antigens, various bacteria such as Toxoplasma, Mycoplasma, and Trebonema pallidum, microbial antigens such as fungi and toxins, various plasma protein components such as albumin and complement components, and estrogen. , human chorionic gonadotropin (HCG)
In addition, antibodies against these antigenic components can also be used.

In the immunological diagnostic reagent according to the present invention, the immobilized water-dispersed polymer particles are placed in a glycine buffer with an appropriate pi and concentration so that the immobilized immunoactive substance is not deactivated. The polymer particles are dispersed in a buffer solution such as phosphate buffer or borate buffer, and calcium bromide is added to the aqueous dispersion of the polymer particles as an additive to suppress non-specific aggregation of the particles. It becomes.

Calcium bromide is added in an amount of 0.01 to 1 in an aqueous dispersion of water-dispersed polymer particles immobilized with an immunoactive substance.
．． It is contained in a concentration of 5 mol/l, preferably 0.1 to 1.25 mol/l. When the concentration of calcium bromide in the aqueous dispersion is less than 0.01 mol/l, the effect of suppressing nonspecific agglutination of negative serum is poor, while when the concentration exceeds 1.5 mol/l, This is because, on the contrary, specific agglutination of positive serum will be suppressed.

Furthermore, in the immunological diagnostic reagent according to the present invention, the concentration of the buffer solution is usually in the range of 0.005 to 0.2M, preferably in the range of 0.01 to 0.1M. In addition, the pi of the buffer solution is usually determined in consideration of the dispersion stability of the water-dispersed polymer particles and the activity of the antigen-antibody reaction.
It ranges from 6 to 9, preferably from 7 to 8.5. Further, the solid content concentration of the polymer particles in the diagnostic reagent is usually 0.
The range is from 0.01 to 5% by weight, preferably from 0.1 to 3% by weight.
% by weight. Incidentally, a preservative such as sodium azide may be added to the immunological diagnostic reagent according to the present invention in order to impart a preservative effect.

The immunological diagnostic reagent according to the present invention is prepared by dissolving calcium bromide in a buffer solution, adjusting the pH, and then adding and mixing it to an aqueous dispersion of water-dispersible polymer particles on which an immunoactive substance is immobilized. , can be obtained by further adjusting pi as necessary.

In immunological diagnosis using the immunological diagnostic reagent according to the present invention, for example, the diagnostic reagent and the test liquid are mixed in the recesses of a glass plate or plastic plate, on a flat plate, or on a microplate, and then observed with the naked eye or with a microscope. This is done by determining the presence or absence of aggregation of polymer particles. Moreover, the presence or absence of aggregation can also be determined as an optical change.

(Effects of the Invention) As described above, the immunological diagnostic reagent of the present invention has excellent dispersion stability in itself, and also has water that can maintain a stable dispersion state even when an immunologically active substance is immobilized. An immunoactive substance is immobilized on dispersed polymer particles, and calcium bromide is contained in an aqueous dispersion of the particles.As a result, non-specific aggregation of the particles occurs, although the reason is not necessarily clear. It is completely suppressed and has high detection sensitivity and specificity. Therefore, when making a diagnosis, it is possible to make a quick and accurate determination without diluting the serum with a buffer or inactivating it. can. Furthermore, the diagnostic reagent according to the invention comprises:
For example, since it is easy to mix uniformly with serum on a glass plate and has excellent fluidity, it is easy and accurate to determine the presence or absence of aggregation. In addition, because the diagnostic reagent contains calcium bromide, its specific gravity increases and becomes close to that of the particles, but compared to conventional diagnostic reagents, it does not cause any precipitation over a long period of time. , the uniform dispersibility of the polymer particles is maintained, and its storage stability is extremely excellent.

Examples of the present invention will be shown below and specifically explained, but the present invention is not limited to these Examples.

Example 1 (a) Preparation of water-dispersed polymer particles 45% by weight of methyl methacrylate, 45% by weight of isobutyl methacrylate, 5.0% by weight of acrylic acid, 0.5% by weight of sulfopropyl acrylate sodium salt, and 45% by weight of isobutyl methacrylate. 60 g of a monomer mixture consisting of 4.5% by weight of ethylene glycol dimethacrylate was added to 330 g of distilled water, and 0.0 g of potassium persulfate was added.
75% of a polymerization initiator aqueous solution in which 3g was dissolved in 1Os+1 water.
℃ under a nitrogen stream and polymerized for 6 hours while stirring at 120 rpm, with a polymerization rate of 98.0% and an average particle size of 0.
An aqueous dispersion of water-dispersible polymer particles with a diameter of 29 μm was obtained. Polymerization was very stable and there were no aggregates.

The particles were centrifuged, converted into acid form with 0.01N hydrochloric acid, washed with distilled water, and dispersed in distilled water.

This dispersion was subjected to conductivity titration with a 0.05 N aqueous potassium hydroxide solution to determine the amount of carboxyl groups. Separately, the particle size of the particles was measured using an electron microscope, and the amount of carboxyl groups possessed by the particles was determined from these measurements, and it was found to be 12.0×10 −′ mol/rrf.

This particle dispersion was first centrifugally washed four times with distilled water, and then centrifugally washed twice with 0.01M borate buffer (pH 7,5) to remove water-soluble polymers in the aqueous phase. After the polymer particles were purified, the polymer particles were redispersed in a 0.01M boric acid buffer (pH 7.5) so that the solid content was 5% by weight.

(b) Binding of spacer group to polymer particles 100 ml of the aqueous dispersion of the water-dispersed polymer particles obtained above and ε
-Aminocaproic acid aqueous solution (0°02M) 100a
+1 and adjust the pH with IN sodium hydroxide aqueous solution.
1.5. 0.01M borate buffer (pH 7,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide salt-acid salt aqueous solution (25B/
Add 20 ml of n+1) to the above aqueous dispersion and stir at room temperature for 3
The reaction was allowed to take place under stirring for an hour. - After leaving it in the refrigerator at night, it was centrifugally washed three times with 0.01M borate buffer (pH 8,2) to obtain polymer particles bound with spacer groups, which were then washed with 0.01M borate buffer (pH 8,2). Solid content 5 in liquid (pH 8,2)
% by weight.

(5 ml of an aqueous dispersion of water-dispersible polymer particles having a spacer group obtained on the immobilization of C1 rabbit IgG, 0.OIM borate buffer (pH 8)
, 2> 2 ml and 11 ml of distilled water were mixed, and 1
-2 ml of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride te (5 mg/ml) was added, and 10 minutes later, 5 ml of rabbit IgG aqueous solution (5 mg/ml) was added and reacted at 15°C for 2 hours. .

Next, in order to consume excess water-soluble carbodiimide in the reaction mixture, a 10% by weight aqueous L-arginine solution (pH
8,2) was added and incubated for 1 hour. Next, centrifugal washing was performed three times with 0.01M borate buffer (pH 8°2), and then 0.01M borate buffer (pH 8°2) was used.
8,2) and the total volume was adjusted to 10 ml, thus obtaining an aqueous dispersion of water-dispersible polymer particles in which rabbit IgG was covalently immobilized via the spacer group. The amount of immobilization was approximately 60 mg per gram of polymer particles.

(d) Preparation of immunological diagnostic reagents Various amounts of calcium bromide were added to 0. OIM borate buffer (
After dissolving at pH 8.2), IN aqueous sodium hydroxide solution was added to adjust the pH of the buffer solution to 8.2.

These calcium bromide-containing buffers and the rabbit IgG immobilized particle dispersion described above were mixed to contain calcium bromide at various concentrations and to obtain a polymer particle concentration of 1.0% by weight. An immunological diagnostic reagent was prepared.

(el Evaluation of immunological diagnostic reagent) This reagent in which rabbit IgG is immobilized as an immunologically active substance can be used as a rheumatoid factor detection reagent.

This diagnostic reagent and the rheumatoid factor positive serum and negative serum were mixed in equal volumes on a glass plate and stirred, and the state of agglutination was visually determined after 2 minutes. The result is the first
Shown in the table.

Table 1 (Note) Judgment criteria are as follows. The same applies to the following judgments.

-: No aggregation was observed even by microscopic observation (100x magnification).

- No aggregation was observed with the naked eye, but microscopic observation (1
00 times), slight aggregation is observed.

±: Slight aggregation is observed with the naked eye.

+: Aggregation is clearly observed with the naked eye.

++: Slightly strong aggregation is observed.

+++: Severe aggregation is observed.

In the diagnostic reagent according to the invention, no non-specific agglutination reaction occurred, whereas in the control diagnostic reagent that did not contain calcium bromide, non-specific agglutination occurred.

In addition, the calcium bromide concentration was 0.85 mol/l, respectively.
The oral stability of the rheumatoid factor detection and diagnostic reagent having a concentration of 0.1 mol/1 was examined at a temperature of 4°C. The evaluation method is the same as above. The results are shown in Table 2.

It is clear that the diagnostic reagent of the present invention has a uniform appearance even on the 6-edge, does not cause any non-specific agglutination of negative serum, and has excellent storage stability.

Furthermore, according to the diagnostic reagent of the present invention, the degree of aggregation is substantially constant regardless of reaction time. However, with diagnostic reagents that do not contain calcium bromide, precipitation occurs early and nonspecific aggregation is observed.

Furthermore, a control diagnostic reagent containing calcium chloride as an additive not only causes a nonspecific agglutination reaction with negative serum, but also causes early precipitation, resulting in significantly poor storage stability. However, since this precipitate is caused by sedimentation of particles and not by aggregation of particles, it can be used as a diagnostic reagent if it is sufficiently shaken to make it homogeneous. Furthermore, according to diagnostic reagents containing calcium chloride,
The initial degree of agglutination after a few minutes of mixing with negative serum is (±)
That is, when it is mediastinal, nonspecific aggregation becomes stronger as time passes, and for example, after about 10 minutes, the degree of aggregation reaches (++), and the degree of aggregation varies with time. Thus, accurate diagnosis is not possible.

Furthermore, Table 3 shows the results of examining changes in positive activity when the above rheumatoid factor detection diagnostic reagent with a calcium bromide concentration of 0.85 mol/l was stored at a temperature of 4°C. The criteria for determining aggregation are the same as above.

Next, rabbit IgG was directly immobilized according to method (C) without bonding a spacer group to the water-dispersed polymer particles prepared by method (a), and then rabbit IgG was directly immobilized according to method (dl). Control diagnostic reagent A containing calcium bromide at various concentrations was prepared. Control diagnostic reagent B was also prepared which was the same as the diagnostic reagent of the present invention except that it did not contain calcium bromide.
was prepared.

The diagnostic reagents according to the invention and their reference diagnostic reagents (8
Activity was determined according to the method of 1. The results are shown in Table 4. According to the diagnostic reagent according to the present invention, non-specific agglutination does not occur and the detection sensitivity is high, but the control diagnostic reagent A, in which rabbit IgG is not immobilized via a spacer group, is clearly detected even for positive serum. However, according to the control diagnostic reagent B, non-specific agglutination was observed.

Example 2 (al) Preparation of water-dispersed synthetic polymer particles Monomers include 80% by weight of methyl methacrylate and 5% by weight of acrylic acid.
0% by weight, sulfopropyl acrylate sodium salt 0
．． 5% by weight of methacrylomtrile, 1005% by weight of methacrylomtrile, and 4.0% by weight of triethylene glycol dimethafrylate. An aqueous dispersion of 28 μm water-dispersible polymer particles was obtained. The amount of carboxyl groups on the surface of this particle is 4.8X10-'mol/, lj
Met.

This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were dispersed in a 0.01M boric acid buffer solution (ptt7.5) to a solid content concentration of 5% by weight, and then dispersed in water. An aqueous dispersion of type polymer particles was obtained.

(bl) Bonding of spacer groups to polymer particles In the same manner as in Example 1, spacer groups were bonded to the polymer particles obtained above.

(C) In the same manner as in Example 1, rabbit IgG was immobilized on the spacer group-bonded polymer particles obtained in the immobilization of rabbit IgG, and then treated in exactly the same manner as in Example 1. and o
, to obtain a water-dispersed polymer particle dispersion with immobilized rabbit IgG. Polymer particles 1
The amount of rabbit IgG immobilized per g was 65 mg.

(d) Containing calcium bromide at various concentrations in the same manner as in Example 1 using the rabbit IgG-immobilized water-dispersed polymer particle dispersion obtained in the preparation of the immunological diagnostic reagent. A diagnostic reagent according to the invention was prepared.

For comparison, control diagnostic reagents were prepared using sodium bromide, potassium bromide, lithium bromide, or calcium chloride as additives in place of calcium bromide.

(e) Evaluation of immunological diagnostic reagents The various diagnostic reagents obtained were evaluated for activity in the same manner as in Example 1. The results are shown in Table 5. According to the diagnostic reagent according to the invention, non-specific aggregation does not occur at all, but in the case of the control diagnostic reagent, non-specific aggregation is observed.

Example 3 Human IgG was immobilized on the water-dispersed polymer particles having spacer groups prepared in Example 2 using carbodiimide in the same manner as in Example 1, and then 0.75 mol/kg of calcium bromide was added. Diagnostic reagent A according to the invention containing l
was prepared. The amount of Hime gc immobilized per gram of polymer particles was 65 mg.

Next, human IgG was directly immobilized using carbodiimide without bonding a spacer group to the water-dispersed polymer particles prepared in Example 2, and calcium bromide was added to the aqueous dispersion of the polymer particles. 0゜5mol/l? ! Diagnostic reagent B according to the invention was prepared by dissolving at A degree.

The amount of human I=IgG immobilized in this diagnostic reagent is:
It was 70 μ/g of polymer particles.

In this way, a diagnostic reagent with immobilized H. gG can also be used as a rheumatoid factor detection reagent.

These diagnostic reagents A and B according to the present invention were mixed and stirred with various untreated serums, and the agglutination state was determined after 2 minutes. The results are shown in Table 6.

According to the diagnostic reagent of the present invention, there is no non-specific aggregation (and the sensitivity is also high).

For comparison, human 1gG was immobilized directly using carbodiimide without bonding a spacer group to carboxylated polystyrene latex particles with an average particle size of 0.35 μm, and a control diagnostic reagent C to which no calcium bromide was added. A control diagnostic reagent was prepared by dissolving calcium bromide in the dispersion at a concentration of 0.75 mol/l.

The activities of these control diagnostic reagents are also shown in Table 6. In addition, in the case of the control diagnostic reagent, spontaneous aggregation was observed from the beginning of preparation.
Agglutination (±), which was determined to be mediastinal, occurred not only with the negative serum but also with the buffer solution.

Example 4 (a) Preparation of water-dispersed polymer particles Methyl methacrylate 35.8% by weight, isobutyl methacrylate 35%
．． A monomer mixture consisting of 8% by weight, 10.01% methacrylic acid (1%, 0.5% by weight sulfopropyl acrylate sodium salt, 3.7% by weight nonaethylene gelicoldimethacrylate and 14.2% by weight methacrylonitrile) The polymerization rate was 9 in the same manner as in Example 1 except that 60 g was used.
An aqueous dispersion of water-dispersible polymer particles having an average particle diameter of 0.30 IJm at 9% was obtained.

This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were dissolved in 0.01M borate buffer (pH 7.5).
A water-dispersed polymer particle aqueous dispersion was obtained by dispersing the particles in a solid content of 5% by weight.

Coupling of Spacer Group to Polymer Particles In the same manner as in Example 1, ω-aminocaprylic acid or L-glutamic acid was bonded as a spacer group to the polymer particles obtained above by the carbodiimide method.

(C) 5 ml of an aqueous dispersion containing 5% by weight of the spacer group-bonded polymer particles obtained upon immobilization of anti-human chorionic gonadotropin antibody, 0.01 M borate buffer (pH 7);
, 5) Mix 2 ml and 11 ml of distilled water, and add 1
- After adding 2 ml of ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution (5 mg/n+1), anti-human chorionic gonadotropin antibody solution (anti-HC
5 ml of G, 5 mg/ml) was added, and the reaction was carried out at 15° C. for 3 hours. The amount of anti-HCG immobilized per gram of particles was 38 mg when the spacer group was ω-aminocaprylic acid, and 55 mg when the spacer group was L-glutamic acid.

(d) Preparation of immunological diagnostic reagent By the same method as in Example 1, calcium bromide was contained at a concentration of 0.75 mol/l and 0.50 mol/l, respectively, and the polymer particle concentration was 1. An immunological diagnostic reagent according to the invention with a concentration of 5% by weight was obtained.

(el Evaluation of immunological diagnostic reagent) This reagent with immobilized anti-HCG as an immunoactive substance can be used as a pregnancy diagnostic reagent.

Equal amounts of each of the diagnostic reagents and serum obtained above were mixed as stock solutions on a glass plate, and the presence or absence of agglutination was determined after 3 to 5 minutes. If the HCG was at the level of 1 international vehicle, agglutination would occur, and the presence or absence of pregnancy could be easily and accurately determined. No non-specific aggregation occurred.

On the other hand, a control diagnostic reagent that did not contain calcium bromide caused nonspecific agglutination, which was determined to be mediastinal, even in the case of non-pregnant human serum.

Claims (4)

[Claims] (1) (a) General formula CH_2=CR^1COOR^2 (However, R^1 represents hydrogen or a lower alkyl group, and R^
2 represents an alkyl group having 1 to 8 carbon atoms. ) Acrylic acid alkyl ester derivative 30-
98.8% by weight, (b) General formula R^3CH=CR^4COOH (wherein R^3 represents hydrogen, a lower alkyl group, or a carboxyl group, and R^4 represents hydrogen or a lower alkyl group,
When R^3 is hydrogen or a lower alkyl group, R^4 may be a carbo-lower alkoxy group. ) 0.1 to 30% by weight of an acrylic acid derivative represented by (c) 0.1 to 20% of a vinyl monomer having a sulfonic acid group
% by weight, and (d) 1 to 20% by weight of a polyfunctional internal crosslinking monomer, water-dispersible polymer particles obtained by emulsion copolymerizing in an aqueous medium are covalently bonded. An immunological diagnostic reagent comprising an immunologically active substance immobilized thereon and dispersed in an aqueous medium, characterized in that it contains calcium bromide. (2) The immunological diagnostic reagent according to claim 1, wherein the immunologically active substance is covalently immobilized on the water-dispersed polymer particles via a spacer group. (3) The immunological diagnostic reagent according to claim 1, which contains calcium bromide at a concentration in the range of 0.01 to 1.5 mol/l. (4) The immunological diagnostic reagent according to claim 1, which has a pH of 6 to 9.