JPH07229900A - Immunological diagnostic reagent - Google Patents

Abstract

PURPOSE:To obtain a reagent easily judging the presence of agglutination reaction and excellent in storage stability by compounding aminosulfonic acid or a water-soluble salt thereof with an aq. dispersion of water dispersible polymer particles obtained by the emulsion polymerization of a monomer mixture having a specific compsn. CONSTITUTION:About 60-99.8wt.% of a fluoroalkyl acrylate derivative having a compsn. represented by formula I (wherein R<1> is hydrogen or a lower alkyl group, R<2> is a group represented by formula II, A is hydrogen, fluorine or CF3 and m, n are an integer of 0-12, X+Y= m-1 and R<3> is hydrogen or an acetyl group), about 0.1-20wt.% of an acrylic derivative represented by formula III (wherein each of R<4> and R<5> is hydrogen or a lower alkyl group) and about 0.1-20wt.% of a polyfunctional internal crosslinkable monomer are subjected to emulsion polymerization in an aq. medium to obtain water dispersible polymer particles. An immunological active substance is immobilized on the polymer particles to be dispersed in an aq. medium and aminosulfonlc acid or a water-soluble salt is added to the obtained dispersion.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention 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 immunoactive substances are immobilized, and is used for nonspecific agglutination in the latex agglutination reaction. The present invention relates to an immunological diagnostic reagent that has no reaction, is easy to determine an agglutination reaction, and has excellent storage stability.

[0002]

2. Description of the Related Art In recent years, immunological diagnostic methods utilizing the immunological activity of physiologically active substances in blood, urine and other body fluids have been developed for medical diagnosis of pathological conditions or other conditions in humans and 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 immunologically active component as described above by a specific reaction, that is, an agglutination reaction or an agglutination inhibition reaction based on an antigen-antibody reaction. 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 an aqueous medium having a pH in the vicinity of neutral. The presence or absence of such an agglutination reaction of particles is used to measure the test component in the body fluid such as serum.

For example, the agglutination reaction of a diagnostic reagent in which fine particles are made of latex is explained. When a diagnostic reagent made of an aqueous dispersion liquid containing fine particles carrying an antigen or an antibody is mixed with a test liquid, the above-mentioned antigen or antibody is formed. The corresponding antibody or antigen in the test liquid specifically reacts with the antigen or antibody on the microparticles to cause 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 has a high sensitivity for detecting an immunologically active substance, that is, an antigen or an antibody even if a trace amount thereof is present in a test liquid, and an immunologically active substance of interest. It is required to have high specificity to react only with. Furthermore, even for long-term storage, it is required to maintain high detection sensitivity and specificity.

Polystyrene latex particles have hitherto been widely used as a fine particle carrier in such immunological diagnostic reagents. This polystyrene latex is
In most cases, it is produced by emulsion-polymerizing styrene in the presence of an emulsifier and a water-soluble radical polymerization initiator. 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 the antigen or antibody is immobilized on the polystyrene latex containing the emulsifier as described above and the latex contains the free emulsifier,
Latex particles may aggregate 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 on the polystyrene latex particles merely adsorbs the antigen or antibody on the surface of the polystyrene particles, and a part of them is desorbed from the latex particles to cause the above specific agglutination reaction. May interfere with. As described above, conventionally used immunological diagnostic reagents having polystyrene latex particles as a carrier have a problem of lacking diagnostic accuracy.

Therefore, an immunology in which a monomer having a functional group is emulsion-copolymerized and an antigen or an antibody is covalently immobilized on the particle by utilizing the above-mentioned functional group of the latex particles obtained. Diagnostic reagents have already been proposed. 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 Application Laid-Open No. 47-16623).
Issue). However, this diagnostic reagent is unstable with respect to changes in the environment, and as described below, precipitation easily occurs when an additive for suppressing non-specific aggregation of the diagnostic reagent is added, and storage stability is stable. There is a problem of poor sex.

Further, for example, an methacrylic acid alkyl ester is emulsion-copolymerized with a hydrophilic methacrylic acid, 2-hydroxyethyl methacrylate and a polyfunctional monomer in the presence of an emulsifier using a water-soluble radical polymerization initiator. Thus, a method for obtaining methacrylate ester latex is already known (Polymer, Vol. 19, August, 867-871 (19
78)). 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 for obtaining acrylic ester latex in the absence of an emulsifier has been proposed (Chemical Technology Laboratory Report Vol. 75, No. 8, page 341 (19).
80)), the latex obtained by this method also has insufficient dispersion stability, and in particular, it easily aggregates under mechanical shear stress.

On the other hand, for the purpose of preventing non-specific agglutination of latex particles, the serum was diluted with a buffer such as glycine or supplemented in the serum when determining the presence or absence of the latex agglutination reaction. It is performed to inactivate the body. However, such a treatment has a problem that it is difficult to sufficiently suppress non-specific aggregation, labor is required, and diagnosis takes time.

In order to solve such problems in immunological diagnostic reagents, it has been customary to add an additive to diagnostic reagents for the purpose of suppressing non-specific agglutination reaction. As such additives, for example, glycols, proteins such as gelatin and albumin, and polyanions are known. But,
In general, the effects of these additives are not sufficient, and therefore, as additives, for example, sucrose and choline chloride (Japanese Patent Laid-Open No. 54-026327), N, N-dialkylamides and di-lower alkyl sulfoxides (specially added) have recently been used. Kaisho 55-160
No. 853) is 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 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. 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. In particular, the original serum is immunologically treated without diluting the serum. Non-specific aggregation often occurs when mixed with diagnostic reagents. 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 this tendency is strong especially when inorganic salts are used as an additive. Often stored at temperatures below 10 ° C. However, in such a case, since it requires a troublesome work of returning the temperature to room temperature again, it can be stored at room temperature as compared with the conventional method, and the storage at this room temperature causes spontaneous aggregation. In addition, as it retains high detection sensitivity when used for diagnosis,
There is a strong demand for immunological diagnostic reagents with excellent storage stability.

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

[0016]

DISCLOSURE OF THE INVENTION As a result of intensive studies to solve the above problems in immunological diagnostic reagents, the present inventors have found that water-dispersible high-molecular polymer particles on which immunoactive substances are immobilized are In an immunological diagnostic reagent consisting of an aqueous dispersion, by dissolving and coexisting with a certain kind of aminosulfonic acid or a salt thereof, it is possible to obtain a conventionally known additive including the aforementioned inorganic salt-based additive. In comparison, it was found that it is possible to obtain an immunological diagnostic reagent which is remarkably excellent in the effect of suppressing non-specific aggregation, is easy to undergo the agglutination reaction, and is also excellent in storage stability. Thus, the present invention has been achieved.

Therefore, according to the present invention, the non-specific agglutination reaction is suppressed and the presence or absence of the agglutination reaction is easily determined without diluting the serum and without inactivating the serum. It is an object of the present invention to provide an immunological diagnostic reagent having excellent storage stability not only at low temperatures but also at room temperature or higher temperatures.

[0018]

The immunological diagnostic reagent according to the present invention comprises an aminosulfonic acid or an aqueous solution thereof, which is added to an aqueous dispersion of water-dispersible polymer particles having an immunoactive substance immobilized by a covalent bond. An immunological diagnostic reagent prepared by dissolving and blending a water-soluble salt, wherein the water-dispersible polymer particles are (a) the general formula CH ₂ = CR ¹ COOR ² (CF ₂ ) _n CFA ₂ (however, R ¹ represents hydrogen or a lower alkyl group, R ² represents

[0019]

[Chemical 4]

(However, m represents an integer of 0 to 12, and x +
y = m−1, and R ³ represents hydrogen or an acetyl group. ), A is independently hydrogen, fluorine or C
Shows the F _3, n is an integer of 0 to 12. ) Acrylic acid fluoroalkyl ester derivative 60-99.
8% by weight, (b) the general formula R ⁴ CH = CR ⁵ COOH (wherein R ⁴ represents hydrogen, a lower alkyl group or a carboxyl group, R ⁵ represents a lower alkyl group, and R ⁴ represents hydrogen or a lower group). When it is an alkyl group, R ⁵ may be a carbo lower alkoxy group.) 0.1 to 20% by weight of an acrylic acid derivative, and (c) a polyfunctional monomer for internal crosslinking 0.1 To 20% by weight of a monomer mixture are emulsion-copolymerized in an aqueous medium to obtain water-dispersible polymer particles, wherein the aminosulfonic acid has the general formula (I).

[0021]

[Chemical 5]

(Wherein X and Y represent hydrogen or an alkyl group which may have a substituent, a cycloalkyl group or a cyclic amino group residue bonded to each other, and Z has a substituent. An alkylene group which may be present, provided that X and Y are not hydrogen at the same time.)
It is characterized by being an aminosulfonic acid having a primary amino group.

The present invention will be described in detail below. (Water-dispersed polymer particles as carrier) The immunological diagnostic reagent according to the present invention immunizes water-dispersed polymer particles obtained by emulsion copolymerization of a monomer mixture having a predetermined composition. An active substance is immobilized and dispersed in an aqueous medium to give an aqueous dispersion, and a specific aminosulfonic acid or a water-soluble salt thereof is dissolved and contained in the aqueous dispersion as an additive. is there.

First, the water-dispersible high-molecular polymer particles used as a carrier for covalently immobilizing an immunoactive substance in the immunological diagnostic reagent according to the present invention will be described.
The water-dispersible polymer particles used in the present invention include (a) the general formula CH ₂ = CR ¹ COOR ² (CF ₂ ) _n CFA ₂ (wherein R ¹ represents hydrogen or a lower alkyl group, and R ² represents

[0025]

[Chemical 6]

(However, m represents an integer of 0 to 12, and x +
y = m−1, and R ³ represents hydrogen or an acetyl group. ) Is shown. In addition, each A independently represents hydrogen, fluorine or CF ₃ , and n represents an integer of 0-12. ) Acrylic acid fluoroalkyl ester derivative 60
To 99.8% by weight, (b) the general formula R ⁴ CH = CR ⁵ COOH (wherein R ⁴ represents hydrogen, a lower alkyl group or a carboxyl group, R ⁵ represents hydrogen or a lower alkyl group, R ⁴
When is hydrogen or a lower alkyl group, R ⁵ may be a carbo lower alkoxy group. 0.1% to 20% by weight of the acrylic acid derivative represented by the formula (1), and (c) 0.1 to 20% by weight of the polyfunctional monomer for internal crosslinking, and an emulsion copolymerization in an aqueous medium. The resulting water-dispersed high-molecular polymer particles.

The fluoroalkyl alkyl acrylate derivative used for the preparation of such water-dispersible polymer particles has the general formula CH ₂ = CR ¹ COOR ² (CF ₂ ) _n CFA ₂ (where R ¹ is Hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, and R ² is

[0028]

[Chemical 7]

(However, m represents an integer of 0 to 12, and x +
y = m−1, and R ³ represents hydrogen or an acetyl group. ), A is independently hydrogen, fluorine or CF
₃ is shown and n shows the integer of 0-12. ),
Preferably, the general formula

[0030]

[Chemical 8]

(Wherein R ¹ is hydrogen or a lower alkyl group,
Preferably, it represents a hydrogen atom or a methyl group, R ³ represents a hydrogen atom or an acetyl group, and m represents an integer of 0-12. ), And accordingly, particularly, as specific examples of the fluoroalkyl alkyl acrylate derivative which can be preferably used in the present invention, for example,

[0032]

[Chemical 9]

The following can be exemplified. Emulsion copolymer having such acrylic acid fluoroalkyl ester derivative as a monomer component, the fluoroalkyl ester group reduces the surface tension of the particles, for example, when immobilizing an immunoactive substance on the particles, as described later. In addition, since the adsorption of the immunoactive substance on the particles is prevented, the immobilization is facilitated and the particles after immobilization are made highly sensitive and highly specific.

In the present invention, in the preparation of the water-dispersible polymer particles, an aqueous dispersion of the above-mentioned emulsion copolymer particles of a fluoroalkyl acrylate derivative having excellent dispersion stability in an aqueous medium is prepared. To get the liquid
As the monomer component, in addition to the acrylic acid fluoroalkyl ester derivative, an acrylic acid derivative having a carboxyl group is used, and a polyfunctional internal crosslinking monomer is used. According to the present invention, by emulsion-copolymerizing a mixture of such monomer components in a predetermined ratio, it is possible to stably emulsion-copolymerize without the formation of aggregates even without using an emulsifier, and to perform the same in an aqueous medium. In this way, it is possible to obtain an aqueous dispersion liquid of the copolymer particles in which the dispersion state is stably maintained and which is non-swelling.

The above-mentioned acrylic acid derivative used for the preparation of the water-dispersible polymer particles has the general formula R ⁴ CH = CR ⁵ COOH (wherein R ⁴ represents hydrogen, a lower alkyl group or a carboxyl group, and R ⁵ represents hydrogen or a lower alkyl group, R ⁴
When is hydrogen or a lower alkyl group, R ⁵ 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 preferable examples, but (meth) acrylic acid is particularly preferable. One or a mixture of two or more of itaconic acid is preferably used.

As will be described later, such an acrylic acid derivative provides a functional group for immobilizing an immunoactive substance on a polymer particle by a covalent bond, and a spacer on the polymer particle by a covalent bond. Not only does it provide a functional group for attaching groups, but it also imparts a negative charge to the polymer particles, increasing the stability of the particles in aqueous dispersion.

In the present invention, in the production of an aqueous dispersion of water-dispersible polymer particles, the monomer composition for emulsion copolymerization is 60 to 99.8% by weight of a fluoroalkyl ester derivative of acrylic acid, Preferably 70-95% by weight, acrylic acid derivative 0.1-20% by weight, preferably 1
˜20% by weight, polyfunctional monomer for internal crosslinking 0.1 to 20% by weight, preferably 1 to 10% by weight.

In addition to the above, the acrylic acid derivative is
It is an essential monomer to give negative charge to the particles by the carboxyl group, which is a negative group, to stabilize the polymerization of the fluoroalkyl ester acrylic acid derivative during emulsion copolymerization and the stability of the emulsion obtained. is there. Therefore, the acrylic acid derivative requires at least 0.1% by weight in the monomer composition in order to effectively exhibit these effects. However, when it is excessively used as a copolymerization monomer component, it rather deteriorates the polymerization stability and the stability of the aqueous dispersion of the obtained copolymer particles, so that it is used in an amount of 20% by weight or less.

In the present invention, the polyfunctional internal cross-linking monomer introduces a cross-linking structure into the polymer. Therefore, if it is contained in the diagnostic reagent, the formation of an undesired water-soluble polymer can be suppressed and obtained. The glass transition temperature of the polymer particles 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.

As such a polyfunctional monomer for internal crosslinking, for example, 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.

Such a polyfunctional monomer for internal crosslinking is also
As described above, the polymerization is allowed to proceed stably, and while maintaining a stable dispersed state of the obtained copolymer particles, it is a monomer necessary for making the particles non-swelling, At least 0.1% by weight is required in the composition, but when it is used in excess, it will rather impair the polymerization stability and the stability of the particle dispersion, so it is used in the range of 20% by weight or less.

Furthermore, in the present invention, in the preparation of water-dispersible high-molecular polymer particles, the polymerization stability during emulsion copolymerization of the above-mentioned monomer mixture and the water-dispersion stability of the obtained copolymer particles are further improved. In order to increase the amount, the acrylic acid alkyl ester derivative can be used as a monomer component in place of part of the acrylic acid fluoroalkyl ester derivative. The preferred amount used is 90% by weight or less based on the weight of the mixture of the acrylic acid fluoroalkyl ester derivative and the acrylic acid alkyl ester derivative, and when used in a larger amount, the polymerization stability is not impaired. Further, the obtained particles have poor water dispersion stability. The lower limit of the effective amount is not particularly limited, but is usually 1% by weight or more based on the weight of the acrylic acid fluoroalkyl ester derivative and the mixture of the acrylic acid alkyl ester derivative. Particularly, it is preferably in the range of 10 to 90% by weight.

The alkyl acrylate derivative has the general formula CH ₂ = CR ⁶ COOR ⁷ (wherein R ⁶ represents hydrogen or a lower alkyl group, preferably hydrogen or a methyl group, and R ⁷ has 1 to 8 carbon atoms). Represents an alkyl group of), and includes, for example, methyl (meth) acrylate, ethyl (meth) acrylate, propyl (meth) acrylate, butyl (meth) acrylate, hexyl (meth) acrylate, ( Examples thereof include octyl (meth) acrylate.

Furthermore, according to the present invention, in the preparation of the water-dispersible polymer particles, the polymerization stability during the emulsion copolymerization of the above-mentioned monomer mixture and the resulting water-dispersible polymer particles are obtained. Other radical copolymerizable monomers, for example, a monomer having a hydroxyl group such as hydroxyethyl acrylate and 2-hydroxymethyl methacrylate, and a glycidyl group such as glycidyl methacrylate as long as the dispersion stability is not impaired. Monomers, α-olefin type monomers such as ethylene and propylene, vinyl type monomers such as vinyl acetate and vinyl chloride, styrene type monomers such as styrene, methylstyrene and chlorostyrene, butadiene and isoprene, etc. Diene monomers, acrylonitrile, methacrylonitrile, acrylamide, methacrylamide, etc. It can be used as a monomer component in accordance with the requirements.

Further, according to the present invention, in the preparation of the water-dispersible high-molecular polymer particles, the specific types of the individual monomers are such that the copolymer particles obtained have a glass transition point of 0 ° C. or higher,
It is preferably selected to be room temperature or higher. When the glass transition point of the particles is lower than 0 ° C., mutual fusion and aggregation of the particles are likely to occur, and the dispersion stability of the aqueous dispersion of the water-dispersible polymer particles tends to decrease. is there.

According to the present invention, dispersion stability can be obtained by emulsion-copolymerizing each of the above-described monomers in an aqueous medium with a water-soluble radical polymerization initiator by a usual method. An excellent 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 to the polymer particles, as described above,
For example, it is preferable not to use an emulsifier in the emulsion copolymerization, since a harmful effect may appear when the immunoactive substance is immobilized on the particles. 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.

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

In the immunological diagnostic reagent according to the present invention,
As 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.
˜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 preferably in the range of 0.9 to 1.5,
Furthermore, 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, an acrylic acid derivative, an acrylic acid fluoroalkyl ester derivative (and an acrylic acid alkyl ester derivative) and a poly (acrylic acid alkyl ester derivative) and a poly (acrylic acid alkyl ester derivative) and a poly (acrylic acid alkyl ester) derivative can be used while ensuring the polymerization stability without using an emulsifier. Emulsion copolymerization of the functional internal crosslinking monomer can be carried out, and the resulting copolymer particles are non-swelling,
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, a stable dispersed state can be maintained during and after immobilization of the immunoactive substance on the particles, and thus, dispersion stability and storage stability. An immunological diagnostic reagent having excellent sex can be obtained.

(Immunoactive 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, human and animal immunoglobulins, denatured immunoglobulins, α-fetoprotein, C-reactive protein (CRP), hepatitis virus-related antigens, various viral antigens such as rubella HA antigens, Toxoplasma, Mycoplasma, Treponema pallidum, etc. Examples include microbial antigens such as bacteria, fungi and toxins, albumin, various plasma protein components such as complement components, estrogen, various hormones such as human chorionic gonadotropin (HCG), and antibodies against these antigen components. Can be used.

(Immobilization of Immunoactive Substance on Water-Dispersion Polymer Polymer Particles) As described above, the water-dispersion polymer particles used in the present invention contain an acrylic acid derivative as a monomer component. Since it is obtained by emulsion-copolymerizing the monomer mixture, 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.

Further, in the immunological diagnostic reagent according to the present invention, when the immunoactive substance is immobilized on the water-dispersible high molecular weight polymer particles by covalent bond, a polymer of the immunoactive substance is optionally used. In order to increase the degree of freedom on the particles,
The polymer particles and the immunoactive substance can be bound by a covalent bond with a spacer group interposed. The spacer group may be bound to the polymer particle in advance and then the spacer group may be bound to the immunoactive substance, or the spacer group may be bound to the immunoactive substance in advance and bound to the polymer particle. You may let me. 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 usable as the spacer group is
It is at least a difunctional organic compound and does not exclude a polyfunctional polymer, but particularly has 1 to 12 carbon atoms.
A bifunctional organic compound having a carbon chain of is preferred. Specific examples of the compound functioning as such a spacer group include, for example, hexamethylenediamine, dodecamethylenediamine, diamines such as xylylenediamine, glycine, β-aminopropionic acid, γ-aminobutyric acid, ε-aminocaproic acid, Aminoalkylcarboxylic acids such as ε-aminocaprylic acid, amino acids such as lysine, glutamic acid, β-alanine, arginine, and glycylglycylglycine are preferably used, but not limited thereto.

An immunoactive substance is directly immobilized on the water-dispersible polymer particles having the above-mentioned functional group by a covalent bond, or a spacer group is bonded to the polymer particles, and the spacer group is also immunoactive. The method for immobilizing a substance by covalent bond is not particularly limited, and any conventionally known method can be used. For example, one of the preferable methods is a method using a water-soluble carbodiimide as a crosslinking agent. 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 and 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide-meth-p-. Toluene sulfonate etc. can be mentioned. With such a water-soluble carbodiimide,
To immobilize the immunoactive agent on the polymer particles by covalent bonding, with or without a spacer group, directly:
It is possible to use the conventional methods and conditions known in the art. For example, when a spacer group is used, the water-soluble carbodiimide is added to the aqueous dispersion of polymer particles together with the spacer group in an appropriate amount, for example, 0.01 to 10 mg / ml per unit volume of the aqueous dispersion. It may be added, and the reaction may be carried out under ordinary conditions, for example, maintaining the pH at 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. 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 the functional group is an amino group, it is reacted with a dialdehyde to activate the functional group, thereby binding the spacer group. Then, the immunoactive substance can be covalently immobilized on the polymer particles in the same manner as described above. Alternatively, the immunoactive substance can be directly immobilized on the polymer particles.

(Additive) In the immunological diagnostic reagent according to the present invention, the water-dispersible polymer particles are adjusted to a suitable pH so that the immunoactive substance immobilized as described above is not deactivated.
And a concentration of glycine buffer, phosphate buffer, and dispersed in a buffer solution such as borate buffer, as an additive for suppressing non-specific aggregation of particles in the aqueous dispersion of polymer particles. , The general formula (I)

[0058]

[Chemical 10]

It contains 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 suitable in the range of 0.005-0.2M, preferably in the range of 0.01-0.1M. Is. 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 effect 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,
Preferably an alkyl group having 1 to 5 carbon atoms which may have a substituent, a cycloalkylene group having 5 to 8 carbon atoms or 5 to
A cyclic amino group residue of 6-membered ring is shown. Therefore, as preferable specific examples of X and Y, for example, a hydroxymethyl group,
Cyclic groups such as hydroxyethyl group, hydroxypropyl group, tri (hydroxymethyl) methyl group, carbamoylmethyl group and cyclohexyl group, which may have a substituent, cycloalkyl group and morpholino group as shown below. An amino group residue can be mentioned. However, X
And Y are not hydrogen at the same time.

[0062]

[Chemical 11]

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.

Accordingly, preferred specific examples of aminosulfonic acid include, for example,

[0065]

[Chemical 12]

And the like. 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, in an aqueous dispersion of water-dispersible high-molecular polymer particles on which an immunoactive substance is immobilized, is 0.01 to 3 mol / l, preferably 0.1 to 1 It is contained at a concentration of 0.5 mol / l. When the concentration of the above additives in the aqueous dispersion is less than 0.01 mol / l, the effect of suppressing non-specific aggregation on negative serum is poor, while when the concentration exceeds 3 mol / l, This is because the specific aggregation with respect to the positive serum will be suppressed.

Furthermore, the immunological diagnostic reagent according to the present invention comprises
In consideration of the dispersion stability of the water-dispersed high-molecular polymer particles and the activity of the antigen-antibody reaction, sodium hydroxide is usually used in order to adjust the pH to the range of 6 to 9, preferably 7 to 8.5. An inorganic alkali such as 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
Since it depends on the additive aminosulfonic acid used,
Although not particularly limited, it is usually in the range of 0.1 to 30 times by mole, preferably 0.5 to 2 times by mole. Examples of such organic amines include, but are not limited to, tris (hydroxymethylamino) methane (hereinafter simply referred to as tris) and bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (hereinafter , Simply referred to as Bistris), etc. are preferably used.

The concentration of polymer particles in the diagnostic reagent is usually in the range of 0.01 to 5% by weight, preferably in the range of 0.1 to 3% by weight, based on the total weight of the diagnostic reagent. is there. 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 is adjusted in pH, 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 stored by adding and mixing the above-mentioned aqueous solution of the aminosulfonic acid or its salt. 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 comprises
It is also possible to include conventionally known additives. 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. The use of these additives, usually in the range of 0.1 to 2% by weight, based on the total weight of the diagnostic reagent, with the aminosulfonic acid according to the invention or its water-soluble salts often results in immunology. The effect of suppressing non-specific aggregation of the diagnostic reagent can be enhanced, and the sedimentation phenomenon can be suppressed to facilitate determination of the presence or absence of aggregation during diagnosis.

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

The method using a microplate is a high-sensitivity detection method in which the amount of liquid required for diagnosis is reduced and detection is carried out in a short time. Generally, high concentration serum has a strong tendency to aggregate fine particles. Serum needs to be highly diluted before use. However, according to the present invention, by dissolving and containing a pH-adjusted aminosulfonic acid or a water-soluble salt thereof in the aggregating reaction solution, not only nonspecific aggregation is suppressed but also specific aggregating activity is enhanced. So
High detection sensitivity can be obtained regardless of the concentration of the sample serum.

[0073]

INDUSTRIAL APPLICABILITY 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, the immunological diagnostic reagent according to the present invention comprises
It seems that the specific gravity increases due to the inclusion of a specific amino sulfonic acid or its water-soluble salt, which is close to the specific gravity of the aqueous medium. Excellent, in particular, not only at low temperature, even at a relatively high temperature of room temperature or higher, without causing any spontaneous aggregation, the uniform dispersibility of the polymer particles is maintained, excellent storage stability, and, It exhibits high detection sensitivity even when used after such storage.

In addition to the above-mentioned advantages, the water-dispersible polymer particles used as a carrier for the immunoactive substance are different from carriers such as erythrocytes by the emulsion copolymerization of a predetermined monomer mixture. As a result, the quality including the particle size is uniform, and since it has no immunoreactivity itself, the immobilization operation is easy.

[0076]

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

Example 1 (a) Preparation of water-dispersible high-molecular polymer particles 2,2,2-trifluoroethyl methacrylate represented by the above formula (v) 90.0% by weight, acrylic acid 5.0% by weight And triethylene glycol dimethacrylate 5.0% by weight
60 g of a monomer mixture consisting of
An aqueous solution of a polymerization initiator prepared by dissolving 0.3 g of potassium persulfate in 10 ml of water was added under a nitrogen stream at a temperature of 75 ° C., and 120 rpm
Polymerization was carried out for 7 hours with stirring to obtain an aqueous dispersion of copolymer particles having a polymerization rate of 99.0% and an average particle size of 0.33 μm.
The polymerization was very stable and free of aggregates. Incidentally, the measurement of the agglomerates is carried out by cooling the produced resin emulsion to 30 ° C. after completion of the polymerization, filtering with a filter cloth, drying the agglomerates left on the filter cloth, and measuring the weight thereof. I asked for it.

This particle dispersion was first washed by centrifugation 4 times with distilled water, and then with 0.01M borate buffer (pH 7.5).
The polymer particles are purified by removing the water-soluble polymer in the aqueous phase by centrifuging and washing twice with.
It was redispersed in M borate buffer (pH 7.5) so that the solid content was 5% by weight.

(B) Bonding of Spacer Group to Polymer Particles Aqueous dispersion 100 of water-dispersible high-molecular polymer particles obtained above
ml and ε-aminocaproic acid aqueous solution (0.02M) 100 ml
And are mixed, and pH is adjusted to 7.5 with 1N sodium hydroxide aqueous solution.
Was prepared. 20 ml of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (25 mg / ml) dissolved in 0.01 M borate buffer (pH 7.5)
Was added to the above aqueous dispersion and reacted at room temperature for 3 hours with stirring. After leaving it in the refrigerator overnight, it was washed with 0.01M borate buffer (pH 8.2) three times by centrifugation to obtain polymer particles having a spacer group bonded, which was then added to 0.01M borate buffer. It was redispersed in (pH 8.2) so that the solid content was 5% by weight. (c) Immobilization of rabbit IgG

5 ml of an aqueous dispersion of the water-dispersible polymer particles having a spacer group obtained above, 2 ml of 0.01M borate buffer solution (pH 8.2) and 11 ml of distilled water were mixed, and 1
2 ml of aqueous solution of -ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (5 mg / ml) was added, and 10 minutes later, 5 ml of aqueous solution of rabbit IgG (5 mg / ml) was added.
The reaction was carried out at 0 ° C for 2 hours. Then, in order to consume the excess water-soluble carbodiimide in the reaction mixture, 10 wt% L-
5 ml of an aqueous arginine solution (pH 8.2) was added and the mixture was incubated for 1 hour. Then, 0.01M borate buffer (pH
After performing centrifugal washing 3 times in 8.2), disperse in 0.01 M borate buffer (pH 8.2) to adjust the total volume to 10 ml,
Thus, an aqueous dispersion of water-dispersible high-molecular polymer particles in which rabbit IgG was covalently immobilized via the spacer group was obtained. The amount of immobilization was 55 mg per 1 g of polymer particles.

(D) Preparation of Immunological Diagnostic Reagent Containing the aminosulfonic acid (1) at various concentrations,
An aqueous solution whose pH was adjusted to 8.0 with Tris and the rabbit
An immunology according to the present invention having a concentration of 1.25% by weight of polymer particles containing the above aminosulfonic acid in various concentrations by mixing with an aqueous dispersion of water-dispersible high-molecular polymer particles on which IgG is immobilized. The diagnostic reagents were prepared.

(E) Evaluation of Immunological Diagnostic Reagents This reagent on which rabbit IgG is immobilized as an immunologically active substance is
It can be used as a rheumatoid factor detection reagent.

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

[0084]

[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, the above-mentioned rheumatoid factor detection reagent was examined for stability over time at temperatures of 4 ° C and 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, non-specific agglutination causing no practical hindrance to the negative serum does not occur, and the storage stability at high temperature as well as at low temperature is stable. It is clear that it has excellent sex. On the other hand, the control diagnostic reagent is significantly non-specifically aggregated and lacks storage stability at high temperatures. In addition, a control diagnostic reagent containing calcium chloride as an additive has excellent initial properties, but its storage remarkably decreases in stability and non-specific aggregation occurs.

Furthermore, the above aminosulfonic acid 0.5 mol / l
The rheumatoid factor-detecting reagent containing the above was examined for changes in positive activity when 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.

[0088]

[Table 2]

[0089]

[Table 3]

Next, the water-dispersible high-molecular polymer particles prepared by the method (a) above were directly bonded without binding a spacer group.
After immobilizing rabbit IgG according to the method of (c), a control diagnostic reagent A containing aminosulfonic acid (1) at various concentrations was prepared according to the method of (d). 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 aminosulfonic acid (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 used.
Does not show a clear agglutination reaction against positive serum, and
According to the control diagnostic reagent B, non-specific aggregation is observed.

[0092]

[Table 4]

Example 2 (a) Preparation of water-dispersible synthetic high-molecular polymer particles 1H, 1H, 5H-octafluoropentyl methacrylate represented by the above formula (vii) 13.9% by weight, methacrylic acid As in Example 1 but using 60 g of a mixture consisting of 38.7% by weight of methyl, 38.7% by weight of isobutyl methacrylate, 5.0% by weight of acrylic acid and 3.7% by weight of triethylene glycol dimethacrylate. Thus, an aqueous dispersion of water-dispersed high-molecular polymer particles having a polymerization rate of 99% and an average particle diameter of 0.31 μm was obtained.

This dispersion was purified in the same manner as in Example 1, and the resulting polymer particles were mixed with 0.01M borate buffer (pH
7.5), so that the solid content concentration is 5% by weight,
An aqueous dispersion of water-dispersed polymer particles was obtained.

(B) Attachment of Spacer Group to Polymer Particles Spacer groups were attached to the polymer particles obtained above in the same manner as in Example 1.

(C) Immobilization of Rabbit IgG Rabbit IgG was covalently immobilized on the polymer particles having the spacer group bonded thereto obtained in the same manner as in Example 1 and, thereafter, as in Example 1 Polymer particles were treated in the same manner to 0.0
Disperse in 1M borate buffer (pH 8.2) to obtain a total volume of 10
to give a water-dispersible polymer particle dispersion in which rabbit IgG was immobilized. Rabbit Ig per gram of polymer particles
The amount of G immobilized was 50 mg.

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

For comparison, in place of the aminosulfonic acid specified in the present invention, an editi represented by the following formula as an additive is used.
Control immunological diagnostic reagents were prepared using c acid (HOOCCH ₂ ) ₂ NCH ₂ CH ₂ N (CH ₂ COOH) ₂ or glycine.

(E) Evaluation of Immunological Diagnostic Reagents Regarding the various immunological diagnostic reagents obtained above, the activity was determined in the same manner as in Example 1. 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.

[0100]

[Table 5]

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

Next, without binding a spacer group to the water-dispersible high-molecular polymer particles prepared in Example 2, human IgG was directly immobilized using carbodiimide, and an aqueous dispersion of the polymer particles was prepared. Aminosulfonic acid (10) 0.5
The immunological diagnostic reagent B according to the present invention was prepared by containing it at a mol / l concentration. Human Ig in this diagnostic reagent
The immobilized amount of G was 70 mg per 1 g of the polymer particles.

This diagnostic reagent on which human IgG is immobilized can also be used for the detection of rheumatoid factor. For comparison, a control diagnostic reagent C to which aminosulfonic acid (10) was not added was used, and a carboxyl group of carboxylated polystyrene latex particles having an average particle size of 0.35 μm was not bound with a spacer group, and human IgG was directly used with carbodiimide. A control diagnostic reagent D was prepared by immobilizing and dissolving aminosulfonic acid (10) in the dispersion at a concentration of 0.75 mol / l.

The activity of these control diagnostic reagents is 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 agglutination (±) which was judged to be falsely positive occurred not only in the negative serum but also in the buffer solution. .

[0105]

[Table 6]

Example 4 (a) Preparation of water-dispersible high-molecular polymer particles 1H, 1H, 5H-octafluoropentyl methacrylate represented by the above formula (vii) (20.0% by weight) and methyl methacrylate (31.9%) %, Isobutyl methacrylate 31.9% by weight, methacrylic acid 15.0% by weight and triethylene glycol dimethacrylate 1.2% by weight, except that 60 g of a monomer mixture was used. Rate 9
An aqueous dispersion of water-dispersible high-molecular polymer particles having an average particle size of 0.28 μm was obtained at 9.5%.

This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were mixed with 0.01 M borate buffer (pH
7.5), so that the solid content concentration is 5% by weight,
An aqueous dispersion of water-dispersed polymer particles was obtained.

(B) Bonding of Spacer Group to Polymer Particles In the same manner as in Example 1, ω-aminocaprylic 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, 0.01 M borate buffer (pH 7.5) )
2 ml and distilled water 11 ml are mixed, and 1-ethyl-3 is mixed with this.
-(3-Dimethylaminopropyl) carbodiimide hydrochloride aqueous solution (5 mg / ml) was added to 2 ml, and then anti-human chorionic gonadotropin antibody solution (anti-HCG, 5 mg / ml) was added to 5 ml.
After the addition, the reaction was carried out at 15 ° C for 3 hours. The immobilized amount of anti-HCG was 40 mg per 1 g of particles.

(D) Preparation of Immunological Diagnostic Reagent In the same manner as in Example 1, p with sodium hydroxide was used.
Aminosulfonic acid (1) with H adjusted to 8.0
An immunological diagnostic reagent according to the invention was obtained, which contained 0.75 mol / l and 0.50 mol / l concentrations and had a polymer particle concentration of 1.5% by weight.

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

The respective diagnostic reagents obtained above and the serum were mixed in equal amounts on the glass plate as undiluted solution, and the presence or absence of aggregation was judged after 3 to 5 minutes. If there is 1 international unit of HCG in 1 ml, aggregation will occur,
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 false positive also occurred in the case of non-pregnant human serum.

Example 5 Using the following four types of monomer mixtures for control and for the diagnostic reagent of the present invention, an aqueous dispersion of water-dispersible polymer particles was prepared in the same manner as in Example 1. Prepared. Monomer mixture A (control) Methyl methacrylate 45.0% by weight Isobutyl methacrylate 45.0% by weight Acrylic acid 5.0% by weight Triethylene glycol dimethacrylate 5.0% by weight Monomer mixture B (control) Methacrylic Methyl acid 90.0% by weight Acrylic acid 5.0% by weight Triethylene glycol dimethacrylate 5.0% by weight

Monomer Mixture C (Control) 1H, 1H, 5H-Octafluoropentyl Methacrylate 15.0 wt% Methyl Methacrylate 80.0 wt% Acrylic Acid 5.0 wt% Monomer Mixture D 1H, 1H, 5H-octafluoropentyl methacrylate 15.0% by weight Methyl methacrylate 79.0% by weight Acrylic acid 5.0% by weight Triethylene glycol dimethacrylate 1.0% by weight

Next, a spacer group was bound to each of the thus obtained water-dispersible high-molecular polymer particles in the same manner as in Example 1 to immobilize rabbit IgG, and then 0.75 mol /
A control immunological diagnostic reagent containing amino sulfonic acid (8) at 1 concentration was prepared.

Table 7 shows the polymerization rate, average particle size, and rabbit Ig per 1 g of particles in the preparation of each water-dispersible high-molecular polymer particle.
The amount of G immobilized is shown.

[0117]

[Table 7]

The activity of these immunological diagnostic reagents was determined in the same manner as in Example 5. In the case of the control diagnostic reagents A and B, rabbit IgG was immobilized on the water-dispersible polymer particles. When this was done, the particles spontaneously aggregated, and even after the subsequent washing, the particles did not return to a completely uniform state, and (±) aggregation was always observed, which made it difficult to use as a diagnostic reagent.

Further, the diagnostic reagent D of the present invention and the control diagnostic reagent C
Was examined for changes in activity after long-term storage. The results are shown in Table 8. The immunological diagnostic reagent according to the present invention comprises 1
Monomer mixture C which is homogeneous after 2 months and does not cause any non-specific agglutination against negative serum but does not contain triethylene glycol dimethacrylate, which is a polyfunctional monomer for internal crosslinking. In the case of the control diagnostic reagent C using the water-dispersed high-molecular polymer particles obtained from
After 2 months, it was homogeneous as in the diagnostic reagent according to the present invention, but its sensitivity and specificity decreased over time, and only a degree of aggregation (±) that was judged to be false positive for positive serum was observed. Nakatsuta.

[0120]

[Table 8]

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical display location C08L 33/04 LHV (72) Inventor Kazuo Minamide 2-5-1 Mishima, Settsu City, Osaka Prefecture Shionogi Pharmaceutical Co., Ltd. (72) Inventor, Hitoshi Tanaka, 2-5-1 Mishima, Settsu City, Osaka Prefecture Shionogi Pharmaceutical Co., Ltd. (72), Inventor, Yasuo Kihara 1-2-1, Shimohozumi, Ibaraki-shi, Osaka Nitto Electric Industry Co., Ltd. (72) Takashi Kawasaki, 1-2 Shimohozumi 1-2-2, Ibaraki City, Osaka Prefecture Nitto Electric Industry Co., Ltd. (72) Kenjiro Mori 1-2-1, Shimohozumi, Ibaraki City, Osaka Nitto Electric Industry Co., Ltd. (72) Inventor Keiichi Ushiyama 1-2-1, Shimohozumi, Ibaraki City, Osaka Prefecture Nitto Electric Industry Co., Ltd.

Claims (6)

[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 water-dispersible polymer particles are (a) having the general formula CH ₂ = CR ¹ COOR ² (CF ₂ ) _n CFA ₂ (wherein R ¹ represents hydrogen or a lower alkyl group, and R ² is [Chemical 1] (However, m represents an integer of 0 to 12, x + y = m-1, and R ³ represents hydrogen or an acetyl group), and A
Each independently represent hydrogen, fluorine or CF ₃ , and n is 0.
Indicates an integer of -12. ) Acrylic acid fluoroalkyl ester derivative 60 to 99.8% by weight, (b) the general formula R ⁴ CH = CR ⁵ COOH (wherein R ⁴ is hydrogen, a lower alkyl group or a carboxyl group, and R ⁵ is Hydrogen represents a lower alkyl group, and when R ⁴ is hydrogen or a lower alkyl group, R ⁵ may be a carbo lower alkoxy group) 0.1 to 20% by weight, and ( c) Water-dispersible polymer particles obtained by emulsion-copolymerizing a monomer mixture containing 0.1 to 20% by weight of a polyfunctional monomer for internal crosslinking. Is represented by 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 is an aminosulfonic acid having a secondary or tertiary amino group. 2. Aminosulfonic acid or a water-soluble salt thereof is 0.0
The immunological diagnostic reagent according to claim 1, wherein the reagent is mixed at a concentration of 1 to 3 mol / l. 3. The immunological diagnostic reagent according to claim 1, wherein an inorganic alkali or an organic amine is blended as a pH adjusting agent for the aqueous dispersion. 4. The immunological diagnostic reagent according to claim 1, which has a pH of 6-9. 5. The immunological diagnostic reagent according to claim 1, wherein the immunologically active substance is immobilized on the water-dispersible polymer particles by a covalent bond via a spacer group. 6. The water-dispersible polymer particles are (a) represented by the general formula CH ₂ = CR ¹ COOR ² (CF ₂ ) _n CFA ₂ (wherein R ¹ represents hydrogen or a lower alkyl group, and R ² represents [Chemical 3] (However, m represents an integer of 0 to 12, x + y = m-1, and R ³ represents hydrogen or an acetyl group), and A
Each independently represent hydrogen, fluorine or CF ₃ , and n is 0.
Indicates an integer of -12. ) And an acrylic acid fluoroalkyl ester derivative represented by the general formula CH ₂ = CR ⁶ COOR ⁷ (wherein R ⁶ represents hydrogen or a lower alkyl group, and R ⁷ represents an alkyl group having 1 to 8 carbon atoms). ) A mixture with an alkyl acrylate derivative represented by
60 to 99.8% by weight of a mixture in which the acrylic acid alkyl ester derivative is 90% by weight or less based on this mixture, (b) the general formula R ⁴ CH = CR ⁵ COOH (where R ⁴ is hydrogen or a lower alkyl group). Or a carboxyl group, R ⁵ represents hydrogen or a lower alkyl group, R ⁴
When is hydrogen or a lower alkyl group, R ⁵ may be a carbo lower alkoxy group. 0.1% to 20% by weight of an acrylic acid derivative represented by the formula (1), and (c) 0.1 to 20% by weight of a polyfunctional monomer for internal crosslinking, an emulsion copolymerization in an aqueous medium. The immunological diagnostic reagent according to claim 1, wherein