JP2545503B2 - Immunological diagnostic reagent - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to an immunological diagnostic reagent, and more specifically, it comprises an aqueous dispersion of water-dispersible high-molecular polymer particles on which an immunoactive substance is immobilized. The present invention relates to an immunological diagnostic reagent which has no non-specific agglutination reaction in the agglutination reaction, is easy to determine the agglutination reaction, and has excellent storage stability.

(Prior Art) In recent years, an immunological diagnostic method utilizing an immunological activity of a physiologically active substance in blood, urine, or other bodily fluids has been available for medical diagnosis of a pathological condition or other condition of humans or animals. Widely used. This method involves reacting either or both of an antigen or an antibody that causes an immunological reaction, or a combination of both, with a test liquid such as a body fluid, and between the antigen or the antibody and the corresponding antibody or the antigen. It is a method of measuring the presence of the immunoactive component as described above by a specific reaction, that is, an agglutination reaction or an agglutination inhibition reaction based on an antigen-antibody response. In this case, in order to facilitate observation with the naked eye,
Generally, the antigen or antibody is carried on a fine particle carrier, for example, latex, red blood cells, etc., to be a diagnostic reagent, and usually,
The presence or absence of such an agglutination reaction of particles in an aqueous medium having a pH in the vicinity of neutrality is used to measure a test component in a body fluid such as serum.

For example, explaining the agglutination reaction of a diagnostic reagent in which fine particles consist of latex, when a diagnostic reagent consisting of an aqueous dispersion liquid containing fine particles carrying an antigen or an antibody is mixed with a test liquid, an analyte corresponding to the antigen or antibody is mixed. The antibody or antigen in the test solution specifically reacts with the antigen or antibody on the microparticles, resulting in a latex agglutination reaction, that is, a macroscopically observable agglutination reaction of microparticles. However, if there is no antibody or antigen to be measured in the test liquid, macroscopically observable aggregation does not occur. In this way, the presence of the antibody or antigen in the test liquid can be determined based on the presence or absence of the agglutination reaction of the fine particles carrying the antigen or antibody.

Such an immunological diagnostic reagent is an immunologically active substance, that is, if an antigen or antibody is present in a test liquid even in a trace amount,
It is required to have a high sensitivity capable of detecting this and a high specificity for reacting only with an immunologically active substance of interest.
Furthermore, even for long-term storage, it is required to maintain high detection sensitivity and specificity.

Polystyrene latex particles have been widely used as a particulate carrier in such immunological diagnostic reagents. This polystyrene latex is, in most cases, in the presence of an emulsifier and a water-soluble radical polymerization initiator,
It is manufactured by emulsion polymerization of styrene. Here, the emulsifier is generally effective for ensuring the polymerization stability during emulsion polymerization and for obtaining a latex containing polymer particles having a small particle size and good dispersion stability. The effect of the emulsifier to increase the dispersion stability of the latex thus obtained is not necessarily clear, but in general, a part of the emulsifier is adsorbed on the latex particles, and the rest is in the free state in the latex. It is said that there is an adsorption-desorption equilibrium between the emulsifier adsorbed on the polymer particles and the free emulsifier in the latex, and stabilization of the latex is achieved as a result of such equilibrium. There is.

Therefore, when immobilizing the antigen or antibody as described above on the polystyrene latex containing the emulsifier,
If the latex contains a free emulsifier, the latex particles may agglomerate during this immobilization operation. Furthermore, in carrying out an immunological diagnosis using a polystyrene latex having an antigen or antibody immobilized thereon, not only positive serum containing the corresponding antibody or antigen but also polystyrene latex particles are hydrophobic. On the other hand, an agglutination reaction may occur even with a negative serum containing no corresponding antibody or antigen. Such an agglutination reaction is called a non-specific agglutination reaction and often causes a misdiagnosis. The reason why such non-specific agglutination reaction occurs is not always clear, but it is considered that it is due to factors such as complement contained in serum.

Further, the immobilization of the antigen or antibody to the polystyrene latex particles only adsorbs the antigen or antibody on the surface of the polystyrene particles, part of which is desorbed from the latex particles and interferes with the specific agglutination reaction. Sometimes. As described above, conventionally used immunological diagnostic reagents having polystyrene latex particles as a carrier have a problem of lacking diagnostic accuracy.

Therefore, a monomer having a functional group is emulsion-copolymerized,
An immunological diagnostic reagent has been already proposed in which an antigen or an antibody is covalently immobilized on the particles using the above-mentioned functional groups of the latex particles. For example, a diagnostic reagent has been already proposed in which an immunologically active substance is immobilized on a carboxylated polystyrene latex or a carboxylated styrene-butadiene copolymer latex by using carbodiimide (Japanese Patent Laid-Open No. 47-16623). However, this diagnostic reagent is unstable against changes in the environment,
As described below, when an additive for suppressing non-specific aggregation of diagnostic reagents is added, precipitation easily occurs and there is a problem of poor storage stability.

Further, for example, by using a water-soluble radical polymerization initiator,
A method of obtaining a methacrylic acid ester latex by emulsion copolymerization of a methacrylic acid alkyl ester with hydrophilic methacrylic acid, 2-hydroxyethyl methacrylate and a polyfunctional monomer in the presence of an emulsifier is already known ( Polymer, Vol. 19, August, 867-871 (1978)). However, since the latex obtained by this method also contains an emulsifier, it has the same problems as described above, and it is considered that the obtained copolymer particles have a hydroxyl group. Therefore, the latex particles tend to aggregate easily.

Therefore, in recent years, a method of obtaining an acrylic ester latex in the absence of an emulsifier has been proposed (Chemical Technology Laboratory Report Vol. 75, No. 8, page 341 (1980)), but the latex by this method is also dispersed. It ’s not stable enough,
Aggregates easily under mechanical shear stress.

On the other hand, conventionally, for the purpose of preventing non-specific aggregation of latex particles, when determining the presence or absence of the latex aggregation reaction, the serum is diluted with a buffer such as glycine,
Alternatively, a deactivating treatment for inactivating complement in serum is performed. However, by such a treatment, it is difficult to sufficiently suppress non-specific aggregation,
In addition, there is a problem in that it takes time and labor for diagnosis.

In order to solve such problems in immunological diagnostic reagents, it has been customary to add an additive to a diagnostic reagent for the purpose of suppressing non-specific agglutination reaction. Known agents include glycols, proteins such as gelatin and albumin, and polyanions. However, since the effects of these additives are not generally sufficient, in recent years, as additives, for example, sucrose and choline chloride (Japanese Patent Laid-Open No. 54-0263).
No. 27), N, N-dialkylamides, lower alkyl sulfoxides (JP-A-55-160853) and the like have been proposed.

Further, in recent years, various inorganic salts have been proposed as additives having an effect of suppressing non-specific aggregation. For example, in JP-A-56-158947, guanidine, guanidine hydrochloride, guanidinium thiocyanate, urea and the like are used as representative examples of chaotropic agents, and similar chaeotropic agents are lithium chloride and lithium bromide. Alkali metal halides such as sodium bromide, potassium bromide and lithium iodide, and metal halides such as calcium chloride are described, and in the same manner in JP-A-57-35754. Alkali metal halides are described.

However, the immunological diagnostic reagents containing the above-mentioned additives are still unsatisfactory in the effect of suppressing non-specific aggregation, and in particular, the original serum is used as the immunological diagnostic reagent without diluting the serum. Non-specific aggregation often occurs when mixed. In particular, such a tendency is remarkable when the above-mentioned salts are used as an additive. Furthermore, immunological diagnostic reagents are generally inferior in storage stability at room temperature or higher, and since this tendency is particularly strong when inorganic salts are used as additives, immunological diagnostic reagents are conventionally Often stored at temperatures below 10 ° C. But,
In such a case, in use, since it requires the troublesome labor of returning to room temperature again, it can be stored at room temperature than before, and by the storage at this room temperature, without causing spontaneous aggregation, Moreover, when using it for diagnosis,
There is a strong demand for immunological diagnostic reagents having excellent storage stability so as to retain high detection sensitivity.

As described above, various additives have heretofore been proposed, but there are still problems that the effect of suppressing non-specific aggregation is poor and the storage stability of the diagnostic reagent is poor.

(Object of the Invention) As a result of intensive studies to solve the above-mentioned problems in immunological diagnostic reagents, the present inventors have found that an aqueous dispersion of water-dispersible polymer particles on which an immunoactive substance is immobilized. In the immunological diagnostic reagent consisting of, by dissolving and coexisting a certain kind of aminosulfonic acid or a salt thereof, including the above-mentioned inorganic salt-based additive, as compared with conventionally known additives, The present invention has been found to provide an immunological diagnostic reagent that is remarkably excellent in the effect of suppressing non-specific aggregation, is easy to undergo the presence or absence of an agglutination reaction, and is also excellent in storage stability. It is the one that led to.

Therefore, the present invention suppresses non-specific agglutination reaction without diluting serum and without performing inactivation treatment, and it is easy to determine the presence / absence of agglutination reaction, and only at low temperature. First, it is an object of the present invention to provide an immunological diagnostic reagent excellent in storage stability at room temperature or higher temperature.

(Structure of the Invention) The immunological diagnostic reagent according to the present invention comprises aminosulfonic acid or a water-soluble salt thereof dissolved in an aqueous dispersion of water-dispersible high-molecular polymer particles on which an immunoactive substance is covalently immobilized. ,
In the immunological diagnostic reagent to be blended, the water-dispersible polymer particles are (a) the general formula CH ₂ = CR ¹ COOR ² (where R ¹ represents hydrogen or a lower alkyl group, R ² is an alkyl group having 1 to 8 carbon atoms.) Alkyl acrylate derivative 30 to 9
8.8% by weight, (b) General formula R ³ CH = CR ⁴ COOH (wherein R ³ represents hydrogen, a lower alkyl group or a carboxyl group, R ⁴ represents a hydrogen or lower alkyl group, and R ³ represents hydrogen or a lower alkyl group). When it is an 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% by weight of a vinyl monomer having a sulfonic acid group, And (d) water-dispersible polymer particles obtained by emulsion-copolymerizing a monomer mixture containing 1 to 20% by weight of a polyfunctional monomer for internal cross-linking. Is the general formula (I) (However, X and Y represent hydrogen or an alkyl group which may have a substituent, a cycloalkyl group or a cyclic amino group residue which is bonded to each other, and Z may have a substituent. An alkylene group, provided that X and Y are not hydrogen at the same time.) And an aminosulfonic acid having a secondary or tertiary amino group.

 Hereinafter, the present invention will be described in detail.

Water-dispersed polymer particles as carrier The immunological diagnostic reagent according to the present invention comprises an aqueous-dispersed polymer particle obtained by emulsion polymerization of a monomer mixture having a predetermined composition. Is immobilized and dispersed in an aqueous medium to give an aqueous dispersion, and a specific aminocarboxylic acid or a water-soluble salt thereof is dissolved and contained as an additive in the aqueous dispersion.

First, in the immunological diagnostic reagent according to the present invention, the water-dispersible polymer particles used as a carrier for covalently immobilizing an immunoactive substance will be described.

Water-dispersed high-molecular polymer particles The water-dispersed high-molecular polymer particles used in the present invention include (a) the general formula CH ₂ = CR ¹ COOR ² (wherein 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 to 9
8.8% by weight, (b) General formula R ³ CH = CR ⁴ COOH (wherein R ³ represents hydrogen, a lower alkyl group or a carboxyl group, R ⁴ represents a hydrogen or lower alkyl group, and R ³ represents hydrogen or a lower group) When it is an alkyl group, R ⁴ may be a lower carboalkoxy group.) 0.1 to 30% by weight of an acrylic acid derivative represented by: (c) 0.1 to 20% by weight of a vinyl monomer having a sulfonic acid group, And (d) a water-dispersible polymer particle obtained by emulsion-copolymerizing a monomer mixture comprising 1 to 20% by weight of a polyfunctional monomer for internal crosslinking.

The acrylic acid alkyl ester derivative used for the preparation of the water-dispersed polymer particles is as described above.
General formula CH ₂ = CR ¹ COOR ² (wherein R ¹ represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, and R ² represents an alkyl group having 1 to 8 carbon atoms). , For example, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate,
Examples thereof include butyl (meth) acrylate, hexyl (meth) acrylate, octyl (meth) acrylate, and the like.

In the present invention, in order to obtain emulsion copolymer emulsion particles of an acrylic acid alkyl ester derivative having excellent dispersion stability in an aqueous medium, in addition to the above acrylic acid alkyl ester derivative, a carboxyl group is used as a monomer component. In addition to using an acrylic acid derivative having a group and a vinyl monomer having a sulfonic acid group, a polyfunctional monomer for internal crosslinking is used. According to the present invention, by using a mixture of such monomer components in a predetermined ratio, it is possible to stably emulsion-copolymerize without generating an agglomerate without particularly using an emulsifier, and to obtain a dispersion state in an aqueous medium. It is possible to obtain an aqueous dispersion of the copolymer particles, which is stably held and is non-swellable.

The acrylic acid derivative used for the preparation of the water-dispersible polymer particles has the general formula R ³ CH═CR ⁴ COOH (wherein R ³ is hydrogen, a lower alkyl group or a carboxyl group, preferably hydrogen or a methyl group). R ⁴ represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group,
When R ³ is hydrogen or a lower alkyl group, R ⁴ may be a carbo lower alkoxy group. ), For example, (meth) acrylic acid, itaconic acid, crotonic acid, maleic acid, fumaric acid, monoalkylmaleic acid, monoalkylfumaric acid, monoalkylitaconic acid and the like can be mentioned as preferred examples. Especially, one or a mixture of two or more of (meth) acrylic acid and itaconic acid is preferably used.

As described below, such an acrylic acid derivative provides a functional group for covalently immobilizing an immunoactive substance to the polymer particles, and also a spacer group is covalently bonded to the polymer particles. In addition to providing a functional group to render the polymer particles negatively charged, they increase the stability of the particles in an aqueous dispersion.

Next, as a vinyl monomer having a sulfonic acid group used for the preparation of water-dispersed polymer particles, an alkylene sulfonic acid such as ethylene sulfonic acid or a general formula CH ₂ = CR ⁵ COOR ⁶ SO ₃ M (provided that R ⁵ represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, R ⁶ 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. ) A sulfoalkyl acrylate represented by, for example, sulfopropyl (meth) acrylate or an alkyl metal salt thereof, a general formula (However, R ⁷ represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, and M is the same as the above.), Styrene sulfonic acid represented by the formula: or a derivative thereof, an alkyl metal salt thereof, for example, styrene sulfone. sodium, formula ^{_{CH 2 = CR 8 CONH-R}} 9 -SO 3 M ( where, R ⁸ is hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, R ⁹ is 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 represented by
Derivatives thereof and these alkyl metal salts such as 2-
Acrylamide-2-methylpropanesulfonic acid or the like is used.

Like the acrylic ester derivative, the monomer having such a sulfonic acid group also imparts a negative charge to the obtained particles to increase the stability of the particles in an aqueous dispersion.

In the present invention, the polyfunctional internal cross-linking monomer introduces a cross-linking structure into the polymer, and thus suppresses the formation of an undesired water-soluble polymer if it is contained in the diagnostic reagent, and the obtained polymer particles. The glass transition temperature of can be increased. Further, the internal cross-linking agent is effective in non-swelling the water-dispersible high-molecular polymer particles and enhancing the dispersion stability of the polymer particles in the aqueous medium.

Examples of such a polyfunctional monomer for internal crosslinking include, for example,
A poly (meth) acrylate of an aliphatic polyhydric alcohol is preferably used. Specific examples include, for example, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, dipropylene glycol di (meth) acrylate, 1,3-butylene glycol di (meth) acrylate. , Trimethylolpropane tri (meth) acrylate, tetramethylolmethane tetra (meth) acrylate and the like are preferably used.

Further, divinylbenzene, N, N′-methylenebisacrylamide and the like can also be used as the polyfunctional monomer for internal crosslinking.

In the production of an aqueous dispersion of water-dispersible polymer particles used as a fine particle carrier in the present invention, the monomer composition for emulsion copolymerization is 30 to 98.8% by weight of an alkyl acrylate derivative, preferably 50-98% by weight, acrylic acid derivative 0.1-30% by weight, preferably 0.5-20% by weight, vinyl monomer having sulfonic acid group 0.1-20% by weight, preferably 0.5-15% by weight, internal crosslinking The functional monomer is 1 to 20% by weight, preferably 1 to 15% by weight.

The acrylic acid derivative and the vinyl monomer having a sulfonic acid group are, as described above, both the polymerization stability at the time of emulsion copolymerization of the acrylic acid alkyl ester derivative and the aqueous dispersion of the resulting copolymer particles. It is a monomer essential for stability, and in order to effectively exhibit these effects, at least 0.1% by weight is required in each monomer composition. However, when it is excessively used as a copolymer component, the polymerization stability and the stability of the obtained aqueous dispersion are impaired, and therefore, they are used in the ranges of 30% by weight and 20% by weight or less, respectively.

Further, as described above, the polyfunctional monomer for internal crosslinking is also necessary for stably proceeding the polymerization and maintaining the stable dispersion state of the obtained particles, and for making the particles non-swelling. It is necessary to use at least 1% by weight in the monomer composition. However, excessive use will impair the polymerization stability and the stability of the particle dispersion, so 20% by weight It is used in the following range.

In the present invention, the water-dispersible high-molecular polymer particles preferably have a carboxyl group at a density in the range of 0.7 × 10 ^{−6 to} 60 × 10 ⁻⁶ mol / m ² . When the carboxyl group of the particles is less than 0.7 × 10 ^-6 mol / m ² , the water dispersion stability is not sufficient, while when more than 60 × 10 ^-6 mol / m ² , the particles are particularly This is because it becomes swellable in an alcal aqueous solution and tends to have poor water dispersibility.

Furthermore, in the present invention, in the preparation of water-dispersible polymer particles, in order to further increase the polymerization stability during emulsion copolymerization of the above-mentioned monomer mixture and the water-dispersion stability of the obtained particles, ( (Meth) acrylonitrile can be used as a monomer component. The preferable amount thereof is up to 40% by weight based on the monomer composition, and if it is used in a larger amount than this, the polymerization stability is rather impaired, and the dispersion stability of the obtained particles becomes poor. . 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 of 5 to 20% by weight.

In addition, other radical copolymerizable monomers such as hydroxyethyl acrylate, 2-, within a range that does not impair the polymerization stability during emulsion copolymerization or the dispersion stability of the resulting water-dispersible polymer particles. A monomer having a hydroxyl group such as hydroxymethyl methacrylate, a monomer having a glycidyl group such as glycidyl methacrylate,
Alpha-olefin monomers such as ethylene and propylene, vinyl acetate, vinyl monomers such as vinyl chloride, styrene,
A styrene-based monomer such as methylstyrene or chlorostyrene, a diene-based monomer such as butadiene or isoprene, or a monomer such as acrylamide or methacrylamide may be used as a monomer component, if necessary.

Furthermore, according to the present invention, in the preparation of the aqueous dispersion of the water-dispersible polymer particles, the specific type of each monomer is such that the glass transition point of the obtained copolymer particles is 0 ° C. Above, it is preferably selected to be room temperature or above. This is because when the glass transition point of the particles is lower than 0 ° C., the particles tend to be fused with each other or aggregated, and the dispersion stability of the dispersion liquid tends to decrease.

According to the present invention, each of the above monomers in an aqueous medium, using a water-soluble radical polymerization initiator, by emulsion copolymerization by a conventional method, excellent dispersion stability An aqueous dispersion of water-insoluble water-dispersible polymer copolymer particles can be obtained. However, when the emulsifier is present in the obtained aqueous dispersion in a free state or in a state of being adsorbed on the polymer particles, as described above, for example, a harmful effect on immobilization of the immunoactive substance on the particles. Therefore, it is preferable not to use an emulsifier in the emulsion copolymerization. According to the above-mentioned monomer composition according to the present invention, it is possible to perform stable copolymerization without using an emulsifier, and it is possible to stably maintain the dispersed state of the particles in the resulting polymer particle aqueous dispersion. It has a great feature. However, as described above, use of an emulsifier is not hindered in the range where harmful particles do not aggregate or settle during immobilization or diagnosis, and an emulsifier may be used if necessary.

Further, in the emulsion copolymerization according to the present invention, the concentration of the monomer component mixture in the aqueous medium is also related to the average particle size of the particles in the obtained aqueous dispersion, but is usually 0.1 to 40.
It is in the range of% by weight.

In the immunological diagnostic reagent according to the present invention, which is a carrier for immobilizing an immunoactive substance, the average particle size of the water-dispersible high-molecular polymer particles obtained as described above is preferably 0.03 to 2 μm. , Particularly preferably 0.1 to 1 μm. If the average particle size is too small, it is difficult to visually observe the aggregation due to the antigen-antibody reaction of the water-dispersed high-molecular polymer particles on which the immunoactive substance is immobilized, while if it is too large, the polymer particles are This is because it becomes difficult to maintain a stable dispersion state. The specific gravity of the polymer particles is 0.
It is preferably in the range of 9 to 1.5, and as described later, the specific gravity after immobilization of 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 the immobilization of the immunoactive substance, the polymer particles float on the surface of the aqueous medium in the aqueous dispersion, and the dispersion stability becomes poor, On the other hand, when it is larger than the above range, the particles tend to settle in the aqueous medium of the dispersion liquid and easily aggregate, and the dispersion stability is similarly deteriorated.

As described above, according to the present invention, in particular, an emulsion copolymerization of an acrylic acid derivative, an acrylic acid derivative, an acrylic acid alkyl ester derivative and other monomers while ensuring polymerization stability without using an emulsifier. And the resulting copolymer particles are non-swellable,
Moreover, the dispersed state is stably maintained in the aqueous medium.
Furthermore, in general, when an immunoactive substance is immobilized on a fine particle carrier, the charge state of the particle surface changes and the dispersion stability of the particles changes to the unstable direction. According to the molecular polymer particles, since the particles have a carboxyl group and / or a sulfonic acid group, it is possible to maintain a stable dispersed state during and after immobilization of the immunoactive substance on the particles, and thus, Thus, an immunological diagnostic reagent having excellent dispersion stability and storage stability can be obtained.

Immunologically active substance The immunologically active substance used in the present invention is not particularly limited, and any of an antigen, an antibody, a hapten and the like may be used. For example, various human and animal immunoglobulins, denatured immunoglobulins, α-fetoprotein, C-reactive protein (CRP) and hepatitis virus-related antigens, various viral antigens such as rubella HA antigens, Toxoplasma, mycoplasma, Treponema pallidum, etc. Microbial antigens such as bacteria, fungi and toxins, albumin, various plasma protein components such as complement components,
Examples include various hormones such as estrogen and human chorionic gonadotropin (HCG), and antibodies to these antigen components can also be used.

Immobilization of Immunoactive Substances on Water-Dispersion Polymer Polymer Particles As described above, the water-dispersion polymer particles used in the present invention include a monomer mixture containing an acrylic acid derivative as a monomer component. Since it is obtained by emulsion copolymerization, the carboxyl group derived from this acrylic acid derivative functions as a functional group for directly binding the above immunoactive substance by a covalent bond.

Furthermore, in the immunological diagnostic reagent according to the present invention, when the immunoactive substance is immobilized on the water-dispersed high molecular weight polymer particles by covalent bond, the immunoactive substance is immobilized on the polymer particles of the immunoactive substance as necessary. In order to increase the degree of freedom of the above, the polymer particles and the immunoactive substance can be bound by a covalent bond with a spacer group interposed. This spacer group may be previously bound to the polymer particles and then this spacer group may be bound to the immunoactive substance, or the spacer group may be preliminarily bound to the immunoactive substance and then bound to the polymer particles. May be. Further, if necessary, a spacer group may be previously bound to both the polymer particles and the immunoactive substance, and these may be bound to each other.

The compound that can be used as the spacer group is at least a bifunctional organic compound, and does not exclude a polyfunctional polymer, but in particular, an organic bifunctional group having a carbon chain of 1 to 12 carbon atoms. Compounds are preferred. Specific examples of the compound that functions as such a spacer group include, for example,
Hexamethylenediamine, dodecamethylenediamine, diamines such as xylylenediamine, glycine, β-aminopropionic acid, γ-aminobutyric acid, ε-aminocaproic acid, ε-aminocaprylic acid and other aminoalkylcarboxylic acids, lysine, glutamic acid, Amino acids such as β-alanine, arginine and glycidylglycylglycine are preferably used, but not limited thereto.

The immunoactive substance is directly immobilized on the water-dispersible polymer particles having the above-mentioned functional group by covalent bond, or the spacer group is bound to the polymer particles, and the immunoactive substance is shared on the spacer group. The method for immobilizing by binding is
The method is not particularly limited, and any conventionally known method can be used. For example, as one of the preferred methods,
A method using water-soluble carbodiimide as a crosslinking agent can be mentioned. For example, if an aminoalkylcarboxylic acid is used as the spacer group, the water-soluble carbodiimide is used to bind the aminoalkylcarboxylic acid to the water-dispersed high molecular weight polymer particles and then to the polymer particles. Similarly, an immunoactive substance can be immobilized by a covalent bond using a water-soluble carbodiimide as an aminoalkylcarboxylic acid.

Examples of the water-soluble carbodiimide used in such a method include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-meth-p-toluenesulfonate, and the like. Can be mentioned.
Immobilizing an immunoactive substance on a polymer particle by covalent bond directly or without a spacer group using such a water-soluble carbodiimide has been conventionally known. The method and conditions can be used. For example, in the case of using a spacer group, an appropriate amount together with the spacer group is added to the aqueous dispersion of polymer particles, for example, water-soluble carbodiimide is added so that the amount is 0.01 to 10 mg / ml per unit volume of the aqueous dispersion. , Normal conditions, eg pH 4
The reaction may be carried out at a temperature of about 5 to 60 ° C. for several minutes to several tens of hours, usually about 1 to 5 hours. Then
The immunologically active substance may be similarly immobilized on the polymer particles having the spacer groups bonded thereto.

When the functional group is a hydroxyl group, the cyanogen bromide method is used, and when it is an amino group, it is reacted with a dialdehyde to activate these functional groups, thereby binding a spacer group, and then, The immunoactive substance can be immobilized on the polymer particles by a covalent bond in the same manner as described above.
Alternatively, the immunoactive substance can be directly immobilized on the polymer particles.

Additive The immunological diagnostic reagent according to the present invention is a glycine buffer solution in which a water-dispersible high-molecular polymer particle has an appropriate pH and concentration so that inactivation of the immunoactive substance immobilized as described above does not occur. In addition to being dispersed in a buffer solution such as a phosphate buffer solution or a borate buffer solution, the above-mentioned general formula (I) is used as an additive for suppressing non-specific aggregation of particles in an aqueous dispersion solution of the polymer particles. And an aminosulfonic acid having a secondary or tertiary amino group represented by or a water-soluble salt thereof.

First, in the immunological diagnostic reagent according to the present invention, the concentration of the above-mentioned buffer is usually appropriate in the range of 0.005 to 0.2M, preferably 0.01 to 0.1M.

Next, in the aminosulfonic acid represented by the general formula (I), the amino group needs to be secondary or tertiary, and in the case of primary, even if this is used as an additive, Almost no result of suppressing non-specific aggregation is obtained.

In the general formula (I), X and Y represent hydrogen or an alkyl group which may have a substituent, a cycloalkyl group or a cyclic amino group residue which is bonded to each other, and preferably has a substituent. Optionally substituted alkyl group having 1 to 5 carbon atoms,
A cycloalkylene group having 5 to 8 carbon atoms or a 5- to 6-membered cyclic amino group residue is shown. Therefore, preferable examples of X and Y include a substituent such as a hydroxymethyl group, a hydroxyethyl group, a hydroxypropyl group, a tri (hydroxymethyl) methyl group, a carbamoylmethyl group and a cyclohexyl group. Examples thereof include a good alkyl group, a cycloalkyl group, and a cyclic amino group residue such as a morpholino group shown below. However, X and Y are not hydrogen at the same time.

Z in the general formula (I) represents an alkylene group which may have a substituent, preferably an alkylene group having 1 to 4 carbon atoms which may have a substituent. Examples of the substituent include a hydroxyl group. Preferable specific examples of Z include, for example, ethylene group, propylene group, hydroxypropylene group and the like.

Therefore, as a preferred specific example of aminosulfonic acid,
For example, (1) (HOCH ₂ CH ₂ ) ₂ NCH ₂ CH ₂ SO ₃ H (2) (HOCH ₂ CH ₂ ) ₂ NCH ₂ CH (OH) CH ₂ SO ₃ H (3) (HOCH ₂ CH ₂ ) ₃ CNHCH ₂ CH ₂ SO ₃ H (4) (HOCH ₂ CH ₂ ) ₃ CNHCH ₂ CH ₂ CH ₂ SO ₃ H (5) (HOCH ₂ CH ₂ ) ₃ CNHCH ₂ HC (OH) CH ₂ SO ₃ H (6) H ₂ NCOCH ₂ NHCH ₂ CH ₂ SO ₃ H Etc. can be mentioned.

In the present invention, the aminosulfonic acid can also be used as a water-soluble alkali metal salt such as sodium salt or potassium salt.

Such an aminosulfonic acid or a water-soluble salt thereof is contained in an aqueous dispersion of water-dispersible high-molecular polymer particles having an immunologically active substance immobilized thereon in an amount of 0.01 to 3 mol / preferably 0.1 mol / mol.
It is contained at a concentration of ~ 1.5 mol /. When the concentration of the above-mentioned additive in the aqueous dispersion is less than 0.01 mol /, the effect of suppressing non-specific aggregation on negative serum is poor, while when the concentration exceeds 3 mol /, it is rather specific to positive serum. This is because the agglomeration is suppressed.

Furthermore, the immunological diagnostic reagent according to the present invention has a pH of usually 6 to 9, preferably 7 to 8.5, in consideration of the dispersion stability of the water-dispersible polymer particles and the activity of the antigen-antibody reaction.
In order to adjust to the above range, an inorganic alkali such as sodium hydroxide, potassium hydroxide or ammonia, or an organic amine other than the aminosulfonic acid is dissolved and contained as a pH adjuster. The amount of such a pH adjuster used is not particularly limited as it varies depending on the additive aminosulfonic acid used, but it is usually in the range of 0.1 to 30 times by mole, preferably 0.5 to 2 times by mole. The organic amine is not particularly limited, but tris (hydroxymethylamino) methane (hereinafter simply referred to as tris) or bis (2
-Hydroxyethyl) iminotris (hydroxymethyl) methane (hereinafter simply referred to as bistris) and the like are preferably used.

The concentration of polymer particles in the diagnostic reagent is usually
It is in the range of 0.01 to 5% by weight, preferably 0.1 to 3% by weight, based on the total weight of the diagnostic reagent. In addition, an antiseptic such as sodium azide may be added to the immunological diagnostic reagent according to the present invention in order to provide an antiseptic effect.

The immunological diagnostic reagent according to the present invention, for example, by adding and dissolving an inorganic alkali or an organic amine to an aqueous dispersion of water-dispersible high-molecular polymer particles on which an immunoactive substance is immobilized,
It can be preserved by preparing by adding and mixing the aqueous solution of the aminosulfonic acid or its salt whose pH has been adjusted.
However, the method for preparing the immunological diagnostic reagent according to the present invention is not limited to this.

Furthermore, the immunological diagnostic reagent according to the present invention may contain an additive that has been conventionally known. Examples of such additives include metal salts such as calcium chloride as described above, animal serum such as human, bovine, horse, rabbit and the like, serum albumin, gelatin, saccharides, polyvinylpyrrolidone, polyethylene glycol and the like.
These additives are usually added at 0,0 based on the total weight of diagnostic reagents.
By using the aminocarboxylic acid or aminosulfonic acid according to the present invention in combination in the range of 1 to 2% by weight, the effect of suppressing non-specific aggregation of the immunological diagnostic reagent can be enhanced in many cases, and By suppressing the sedimentation phenomenon, it is possible to easily determine the presence or absence of aggregation during diagnosis.

Immunological diagnosis using the immunological diagnostic reagent according to the present invention is performed by, for example, mixing the diagnostic reagent and a test liquid on a recess of a glass plate or a plastic plate or on a flat plate or in a microplate, and observing with a naked eye or a microscope. Therefore, it is carried out by determining the presence or absence of aggregation of polymer particles. Further, the presence or absence of aggregation can be determined as an optical change.

The method using a microplate is a highly sensitive detection method in which the amount of liquid required for diagnosis is reduced and detection is performed in a short time.
In general, high-concentration serum has a strong tendency to agglomerate fine particles, and therefore the sample serum needs to be highly diluted before use. However, according to the present invention, in the aggregation reaction liquid,
By dissolving and containing pH adjusted aminosulfonic acid, not only non-specific aggregation is suppressed, but also specific aggregation activity is enhanced, so regardless of the concentration of the sample serum,
High detection sensitivity can be obtained.

(Effects of the Invention) As described above, the immunological diagnostic reagent according to the present invention itself has excellent dispersion stability, and water that can maintain a stable dispersion state even when an immunoactive substance is immobilized. An immunologically active substance is immobilized on the dispersion type high molecular weight polymer particles and dispersed in an aqueous medium to prepare an aqueous dispersion, and the specific aminosulfonic acid or the water-soluble salt thereof is added to the aqueous dispersion. As a result, although the reason for this is not always clear, non-specific aggregation of polymer particles is completely blocked, and it has high sensitivity and specificity. It is possible to make a quick and accurate determination without diluting with a buffer solution or inactivating treatment.

Furthermore, since the immunological diagnostic reagent according to the present invention contains a specific aminosulfonic acid or a water-soluble salt thereof, its specific gravity is increased, and it is considered that the specific gravity approaches that of an aqueous medium. Compared with reagents, it has excellent storage stability over a long period of time, and in particular, not only at low temperatures but also at relatively high temperatures such as room temperature or higher, without causing any spontaneous aggregation, the uniform dispersion of polymer particles can be achieved. It is retained, has excellent storage stability, and exhibits high detection sensitivity even when used after such storage.

In addition to the advantages described above, the water-dispersible polymer particles used as the carrier for the immunoactive substance are obtained by emulsion copolymerization of a predetermined monomer mixture, unlike carriers such as red blood cells. , Quality including particle size is uniform,
The immobilization operation is easy because it has no immunoreactivity by itself.

(Examples) Examples of the present invention will be shown and specifically described below.
The present invention is not limited to these examples.

Example 1 (a) Preparation of water-dispersible 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 triethylene glycol dimethacrylate. 60 g of a monomer mixture consisting of 4.5% by weight was added to 330 g of distilled water, and 0.3 g of potassium persulfate was added to 1 part of water.
An aqueous solution of a polymerization initiator dissolved in 0 ml was added under a nitrogen stream at a temperature of 75 ° C., polymerization was carried out for 6 hours while stirring at 120 rpm, and a water-dispersed polymer having an average particle size of 0.29 μm at a polymerization rate of 98.0%. An aqueous dispersion of particles was obtained. The polymerization was very stable and free of aggregates.

After centrifuging the above particles and acidifying with 0.01N hydrochloric acid,
It was washed with distilled water and dispersed in distilled water. Add this dispersion to 0.
The amount of carboxyl groups was determined by conducting conductivity titration with 05N potassium hydroxide aqueous solution. Separately, the particle size of the particles was measured by an electron microscope, and the amount of carboxyl groups contained in the particles was determined from them, and it was 12.0 × 10 ⁻⁶ mol / m ² .

First, this particle dispersion liquid is centrifugally washed four times with distilled water,
Next, the polymer particles were purified by removing the water-soluble polymer in the aqueous phase by centrifuging and washing twice with 0.01 M borate buffer (pH 7.5), and then removing the polymer particles with 0.01 M borate buffer. Acid buffer (pH 7.
It was redispersed in 5) so that the solid content was 5% by weight.

(B) Bonding of Spacer Group to Polymer Particles Aqueous dispersion of water-dispersible high-molecular polymer particles obtained above 100 m
l and 100 ml of an ε-aminocaproic acid aqueous solution (0.02M) were mixed, and the pH was adjusted to 7.5 with a 1N sodium hydroxide aqueous solution. 20 ml of an aqueous solution of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (25 mg / ml) dissolved in 0.01 M borate buffer (pH 7.5) was added to the above aqueous dispersion, and the mixture was allowed to stand at room temperature for 3 hours. , Reacted under stirring. After leaving it in the refrigerator overnight, it was centrifugally washed three times with 0.01 M borate buffer (pH 8.2) to obtain polymer particles having a spacer group bonded,
This was redispersed in 0.01 M borate buffer (pH 8.2) so that the solid content was 5% by weight.

(C) Immobilization of rabbit IgG 5 ml of an aqueous dispersion of water-dispersible high-molecular polymer particles having a spacer group obtained above, 2 ml of 0.1 M borate buffer (pH 7.5) and 11 ml of distilled water were mixed, 1-ethyl-3- (3-
Dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (5 mg / ml) (2 ml) was added, and 10 minutes later, rabbit IgG aqueous solution (5 mg / ml) was added.
5 ml) was added, and the mixture was reacted at 15 ° C. for 2 hours. Next, in order to consume the excess water-soluble carbodiimide in the reaction mixture, 5 ml of 10 wt% L-arginine aqueous solution (pH 8.2) was added and incubated for 1 hour. Then, after centrifuging and washing with 0.01M borate buffer (pH8.2) three times,
The total amount was adjusted to 10 ml by dispersing in a borate buffer solution (pH 8.2), and thus, an aqueous dispersion of water-dispersible polymer particles in which rabbit IgG was covalently immobilized via the spacer group was prepared. Obtained. The amount of immobilization was about 60 mg per 1 g of polymer particles.

(D) Preparation of immunological diagnostic reagent An aqueous solution containing the aminosulfonic acid (1) at various temperatures and having a pH adjusted to 8.0 with sodium hydroxide, and a water-dispersed type in which the rabbit IgG is immobilized. An immunological diagnostic reagent according to the present invention having a polymer particle concentration of 1.25% by weight containing the above aminosulfone powder was prepared by mixing with an aqueous dispersion of high-molecular polymer particles.

(E) Evaluation of Immunological Diagnostic Reagent This reagent having rabbit IgG immobilized as an immunoactive substance can be used as a rheumatoid factor detection reagent.

The diagnostic reagent and the rheumatoid arthritis factor-positive serum and the negative serum were directly mixed with each other on a glass plate in equal volumes while stirring, and the aggregated state was visually judged after 2 minutes. The results are shown in Table 1.

Non-specific agglutination did not occur in the diagnostic reagent according to the present invention, whereas non-specific agglutination occurred in the control diagnostic reagent.

In addition, regarding the above rheumatoid factor detection reagent,
The stability over time was examined at a temperature of 40 ° C. The evaluation method is the same as above. The results are shown in Table 2.

According to the diagnostic reagent of the present invention, even after 20 weeks, it does not cause non-specific aggregation that would hinder practical use even after 20 weeks, and is not only stable at low temperatures but also immediately at storage stability at high temperatures. It is clear that On the other hand, the control diagnostic reagent is significantly non-specifically aggregated and lacks storage stability at high temperatures. In addition, according to the control diagnostic reagent containing calcium chloride as an additive, the initial characteristics are excellent, but the stability is remarkably reduced by storage, and the non-specific characteristics Heterogeneous aggregation occurs.

Furthermore, changes in positive activity were examined when the above rheumatoid factor detection reagent was stored at temperatures of 4 ° C and 40 ° C. The results are shown in Table 3. The criteria for aggregation are the same as above.

Next, after immobilizing the rabbit IgG directly according to the method of (c) without attaching a spacer group to the water-dispersed polymer particles prepared by the method of (a), (d) Control diagnostic reagent A containing aminosulfone powder (1) at various concentrations was prepared according to the method of 1. Also, a control diagnostic reagent B was prepared which was the same as the diagnostic reagent of the present invention except that it did not contain aminosulfone powder (1).

The activity of the diagnostic reagents according to the invention and these control diagnostic reagents was determined according to the method of (e). The results are shown in Table 4. According to the diagnostic reagent of the present invention, non-specific aggregation does not occur and the detection sensitivity is high, but the control diagnostic reagent A in which rabbit IgG is not immobilized via the spacer group is clear even in negative serum. Shows no agglutination reaction, For example, non-specific aggregation is observed.

Example 2 (a) Preparation of water-dispersible synthetic polymer particles 80% by weight of methyl methacrylate as a monomer, 5.0% by weight of acrylic acid, 0.5% by weight of sodium sulfopropylacrylate, 10.5% by weight of methacrylonitrile and In the same manner as in Example 1 except that 60 g of a mixture consisting of 4.0% by weight of triethylene glycol dimethacrylate was used.
An aqueous dispersion of water-dispersed high-molecular polymer particles having an average particle size of 0.28 μm was obtained at a polymerization rate of 98%. The amount of carboxyl groups on the surface of the particles was 4.8 × 10 ⁻⁶ mol / m ² .

This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were dispersed in 0.01 M borate buffer (pH 7.5) to a solid concentration of 5% by weight to prepare a water dispersion type. An aqueous dispersion of polymer particles was obtained.

(B) Bonding of Spacer Group to Polymer Particles In the same manner as in Example 1, a spacer group was bonded to the polymer particles obtained above.

(C) Immobilization of Rabbit IgG Example 1 was applied to the polymer particle having the spacer group bonded thereto obtained above.
Rabbit IgG was covalently immobilized in the same manner as above, and then treated exactly as in Example 1 to give polymer particles of 0.
It was dispersed in 01M borate buffer (pH 8.2) to a total volume of 10 ml to obtain a water-dispersible high-molecular polymer particle dispersion in which rabbit IgG was immobilized. The amount of immobilized rabbit IgG was 65 mg per 1 g of the polymer particles.

(D) Preparation of immunological diagnostic reagent Using the rabbit IgG-immobilized water-dispersed high-molecular polymer particle dispersion obtained above, the amino acids shown in Table 5 were prepared at various concentrations in the same manner as in Example 1. An immunological diagnostic reagent according to the invention containing sulfonic acid was prepared.

For comparison, in place of the aminosulfonic acid defined in the present invention, an additive acid (HOOCCH ₂ ) ₂ NCH ₂ CH ₂ N (CH ₂ COOH) ₂ or glycine represented by the following formula was used as an additive to obtain a control. Immunological diagnostic reagents Prepared.

(E) Evaluation of immunological diagnostic reagents Regarding various immunological diagnostic reagents obtained above, Example 1
The activity was determined in the same manner as in. The results are shown in Table 5. According to the diagnostic reagent of the present invention, non-specific aggregation does not occur at all, but in the case of the control diagnostic reagent, non-specific aggregation is noticeable.

Example 3 Human IgG was immobilized on the water-dispersible polymer particles having a spacer group prepared in Example 2 using carbodiimide in the same manner as in Example 1, and then pH was adjusted with Tris.
Aminosulphonic acid (10) adjusted to 8.0 of 0.75 mol /
A diagnostic reagent A according to the present invention was prepared containing a concentration.
The amount of immobilized human IgG was 60 mg per 1 g of polymer particles.

Next, without binding a spacer group to the water-dispersible polymer particles prepared in Example 2, human IgG was directly immobilized using carbodiimide, and aminosulfonic acid ( 10) was dissolved at 0.5 mol / concentration to prepare a diagnostic reagent B according to the present invention.
The amount of human IgG immobilized in this diagnostic reagent was 70 mg per 1 g of polymer particles.

Thus, a diagnostic reagent on which human IgG is immobilized 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 sera that had not been treated at all, and the aggregation 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 high.

For comparison, a control diagnostic reagent C to which the above aminosulfonic acid (10) was not added and a carboxyl group having an average particle size of 0.35 μm were not bound to a spacer group, and human IgG was directly used by using carbodiimide. Immobilize and add 0.75 mol / aminosulfonic acid (10) to the dispersion.
A control diagnostic reagent D was prepared by dissolving at a concentration.

The activities of these control diagnostic reagents are also shown in Table 6. In the case of the control diagnostic reagent D, spontaneous agglutination was observed from the beginning of the preparation, and not only against the negative serum but also in the buffer. Aggregation (±) that is determined to be coagulation negative in almost the same way for liquid
Occurred.

Example 4 (a) Preparation of water-dispersible high-molecular polymer particles Methyl methacrylate 35.8% by weight, isobutyl methacrylate 35.8% by weight, methacrylic acid 10.0% by weight, sulfopropyl acrylate sodium salt 0.5% by weight, nonaethylene glycol dimethacrylate Water-dispersible polymer particles having a polymerization rate of 99% and an average particle size of 0.30 μm in the same manner as in Example 1 except that 60 g of a monomer mixture consisting of 3.7% by weight and 14.2% by weight of methacrylonitrile was used. An aqueous dispersion of was obtained.

This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were dispersed in 0.01 M borate buffer (pH 7.5) to a solid concentration of 5% by weight to prepare a water dispersion type. An aqueous dispersion of polymer particles was obtained.

(B) Bonding 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) Immobilization of anti-human chorionic gonadotropin antibody 5 ml of an aqueous dispersion containing 5% by weight of the polymer particles having a spacer group bonded thereto, 2 ml of 0.05M borate buffer (pH 7.5) and distillation 11 ml of water was mixed, and 1-ethyl-3- (3-
After adding 2 ml of dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (5 mg / ml), 5 ml of anti-human chorionic gonadotropin antibody solution (anti-HCG, 5 mg / ml) was added, and the mixture was mixed at 15 ° C for 3 days.
I made a time reaction. The amount of anti-HCG immobilized is 1 g of particles,
It 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 In the same manner as in Example 1, sodium hydroxide was used.
Aminosulfonic acid (1) adjusted to pH 8.0 respectively
An immunological diagnostic reagent according to the present invention containing 0.75 mol / and 0.50 mol / concentration and having a polymer particle concentration of 1.5% by weight was obtained.

(E) Evaluation of Immunological Diagnostic Reagent The present reagent having anti-HCG immobilized as an immunoactive substance can be used as a pregnancy diagnostic reagent.

Each diagnostic reagent and serum obtained above were mixed in equal amounts on a glass plate as undiluted solution, and the presence or absence of agglutination was determined after 3 to 5 minutes. With 1 international unit of HCG, aggregation occurred, and it was possible to easily and accurately determine the presence or absence of pregnancy. No non-specific aggregation occurred.

On the other hand, according to the control diagnostic reagent containing no aminosulfonic acid (1), non-specific agglutination determined to be positive coagulation also occurred in the case of non-pregnant human serum.

Front page continuation (72) Inventor Kazuo Minami 2-5-1, Mishima, Settsu-shi, Shionogi Pharmaceutical Co., Ltd. (72) Inventor Hitoshi Tanaka 2-5-1, Mishima, Settsu-shi, Shionogi Pharmaceutical Co., Ltd. (72) Inventor Yasuo Kihara 1-2-2 Shimohozumi, Ibaraki City Nitto Electric Industry Co., Ltd. (72) Inventor Takashi Kawasaki 1-21-2 Shimohozumi Ibaraki City Nitto Electric Industry Co., Ltd. (72) Invention Kenjiro Mori 1-2-2 Shimohozumi, Ibaraki City Nitto Electric Industrial Co., Ltd. (72) Keiichi Ushiyama 1-2-1 Shimohozumi Ibaraki City Nitto Electric Industrial Co., Ltd.

Claims (5)

(57) [Claims] 1. An immunological diagnostic reagent prepared by dissolving and blending aminosulfonic acid or a water-soluble salt thereof in an aqueous dispersion of water-dispersible high-molecular polymer particles having an immunologically active substance immobilized by a covalent bond. The above-mentioned water-dispersed polymer particles are (a) having the general formula CH ₂ = CR ¹ COOR ² (wherein R ¹ represents hydrogen or a lower alkyl group, and R ² has a carbon number of 1 to 8). Acrylic acid alkyl ester derivative represented by
8.8% by weight, (b) General formula R ³ CH = CR ⁴ COOH (wherein R ³ represents hydrogen, a lower alkyl group or a carboxyl group, R ⁴ represents a hydrogen or lower alkyl group, and R ³ represents hydrogen or a lower alkyl group). When it is an 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% by weight of a vinyl monomer having a sulfonic acid group, And (d) water-dispersible polymer particles obtained by emulsion-copolymerizing a monomer mixture containing 1 to 20% by weight of a polyfunctional monomer for internal cross-linking. Is the general formula (I) (However, X and Y represent hydrogen or an alkyl group which may have a substituent, a cycloalkyl group or a cyclic amino group residue which is bonded to each other, and Z may have a substituent. An alkylene group, wherein X and Y are not hydrogen at the same time) and an aminosulfonic acid having a secondary or tertiary amino group. 2. Aminosulfonic acid or a water-soluble salt thereof is 0.01
The immunological diagnostic reagent according to claim 1, wherein the immunological diagnostic reagent is compounded at a concentration in the range of 3 mol / mol. 3. The immunological diagnostic reagent according to claim 1, wherein an inorganic alkali or an organic amine is mixed as a pH adjusting agent for the aqueous dispersion. 4. The immunological diagnostic reagent according to claim 1, wherein the pH is 6-9. 5. The immunological diagnosis according to claim 1, wherein the water-dispersible high-molecular polymer particles are immobilized by covalent bond with an immunoactive substance through a spacer group. reagent.