JPH0564744B2 - - Google Patents

Description

[Detailed description of the invention]

(Industrial Application Field) The present invention relates to an immunological diagnostic reagent, and in detail,
It is composed of an aqueous dispersion of water-dispersed polymer particles on which an immunoactive substance is immobilized, and there is no non-specific agglutination reaction in the latex agglutination reaction, and it is easy to judge the agglutination reaction and has good storage stability. This article relates to excellent immunological diagnostic reagents. (Prior Art) In recent years, immunological diagnostic methods that utilize the immune activity of physiologically active substances in blood, urine, and other body fluids have been developed for medical diagnosis of pathological or other conditions in humans and animals. Widely used. In this method, either an antigen or an antibody that causes an immunological reaction, or a combination of both, is reacted with a test fluid such as a body fluid, and the relationship between the antigen or antibody and the corresponding antibody or antigen is detected. This is a method of measuring the presence of an immunoactive 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, the antigen or antibody is generally supported on a microparticulate carrier, such as latex or red blood cells, as a diagnostic reagent, and the agglutination reaction of such particles is utilized. , test components in body fluids such as serum are measured. For example, to explain the agglutination reaction of a diagnostic reagent whose microparticles are latex, when a diagnostic reagent consisting of an aqueous dispersion containing microparticles carrying an antigen or an antibody is mixed with a test liquid, a sample corresponding to the antigen or antibody is detected. The antibody or antigen in the test solution reacts specifically with the antigen or antibody on the microparticles, resulting in a latex agglutination reaction, that is, a macroscopically observable microparticle agglutination reaction. However, if the antibody or antigen to be measured is not present in the test liquid, no macroscopically observable agglutination occurs. In this way, the presence of the antibody or antigen in the test liquid can be determined based on the presence or absence of an agglutination reaction of the microparticles carrying the antigen or antibody. Such immunological diagnostic reagents are highly sensitive and can detect immunoactive substances, i.e., antigens or antibodies, even if they are present in a trace amount in a sample solution, and they react only with the target immunoactive substance. It is required to have high specificity. Furthermore, it is required to maintain high detection sensitivity and specificity even during long-term storage. Conventionally, such immunological diagnostic reagents include
Diagnostic reagents are made by immobilizing antigens and antibodies on the surface of polystyrene latex particles by physical adsorption, and carbodiimide and carboxylated latex particles are used.
Diagnostic reagents and the like that are immobilized by covalent bonds using dialdehyde and the like have been proposed. However, when such conventional diagnostic reagents are reacted with serum, they cause an agglutination reaction not only with positive serum containing the corresponding antibody or antigen but also with negative serum without the corresponding antibody or antigen. Sometimes.
Such an agglutination reaction is called a nonspecific agglutination reaction, and can often mislead the diagnosis.
The reason why such a non-specific agglutination reaction occurs is not necessarily clear, but it is thought that one reason is due to factors such as complement contained in serum. Therefore, in order to prevent non-specific agglutination of latex particles, conventionally, when determining the presence or absence of latex agglutination reaction, serum is diluted with a buffer solution such as glycine, or complement in serum is Inactivation treatment is carried out to deactivate it. However, such treatment has the problem that it is difficult to sufficiently suppress nonspecific aggregation, and that it is laborious and time-consuming for diagnosis. In order to solve these problems with immunological diagnostic reagents, it has been common practice to add additives to diagnostic reagents for the purpose of suppressing non-specific agglutination reactions. Examples of agents include glycols, gelatin,
Proteins such as albumin, polyanions, etc. are known. However, the effects of these additives are generally not sufficient, so in recent years, additives such as sucrose and choline chloride (Japanese Patent Application Laid-Open No. 1983-1996) have been used as additives.
026327), N,N-dialkylamides, di-lower alkyl sulfoxides (Japanese Unexamined Patent Publication No. 160853/1983), and the like have been proposed. Furthermore, in recent years, various inorganic salts have been proposed as additives having the effect of suppressing non-specific aggregation. For example, JP-A-56-158947 describes chaotropic agents such as guanidine, guanidine hydrochloride, guanidinium thiocyanate, and urea, as well as lithium chloride and lithium bromide as similar chaiotropic agents. , alkali metal halides such as sodium bromide, potassium bromide, and lithium iodide, and metal halides such as calcium chloride, and JP-A-57-35754 also describes halogen Alkali metals are listed. However, alkali metal halides generally have significant non-specific agglutination with negative serum, and calcium chloride not only has significant non-specific agglutination with negative serum, but also has a tendency to be mixed with negative serum during diagnosis. If the initial degree of agglutination after several minutes is false positive, nonspecific agglutination becomes stronger over time and the degree of agglutination changes over time, making it difficult to make an accurate diagnosis. It is possible. Furthermore, immunological diagnostic reagents containing calcium chloride as an additive. When stored for a long period of time, a precipitate forms in the diagnostic reagent, and the diagnostic reagent must be thoroughly shaken to make it uniform before use, resulting in poor storage stability and usability. in this way,
Conventionally, various inorganic salts have been proposed as additives, but they still have problems in that they are less effective in suppressing nonspecific aggregation and the diagnostic reagents have poor storage stability. (Purpose of the Invention) As a result of intensive research to solve the above-mentioned problems in immunological diagnostic reagents, the present inventors have discovered that water-dispersible polymer particles having an immunologically active substance immobilized thereon by covalent bonds can be used in an aqueous solution. In an immunological diagnostic reagent consisting of a dispersion, by coexisting calcium bromide, non-specific aggregation is reduced compared to conventionally known additives, including the above-mentioned inorganic salt additives. The present invention was achieved based on the discovery that it is possible to obtain an immunological diagnostic reagent that has a significantly superior effect in suppressing the reaction, is easy to test for the presence or absence of agglutination reactions, and has a significantly superior storage stability. It is. Therefore, the present invention allows serum to be diluted without diluting it.
Furthermore, the purpose is to provide an immunological diagnostic reagent that suppresses non-specific agglutination reactions without performing inactivation treatment, makes it easy to determine the presence or absence of agglutination reactions, and has excellent storage stability. shall be. (Structure of the Invention) The immunological diagnostic reagent according to the present invention is an immunological diagnostic reagent comprising an aqueous dispersion of water-dispersed polymer particles on which an immunoactive substance is covalently immobilized. It is characterized by containing calcium bromide in the dispersion. In the immunological diagnostic reagent according to the present invention, the average particle diameter of the water-dispersed polymer particles that are the carrier for immobilizing the immunoactive substance is preferably 0.03
~2 μm, particularly preferably 0.1 to 1 μm. If the average particle size is too small, it will be difficult to visually observe the aggregation of the water-dispersed polymer particles immobilized with the immunoactive substance due to the antigen-antibody reaction;
This is because if it is too large, it becomes difficult to maintain a stable dispersion state of the polymer particles. Also,
The specific gravity of the polymer particles is preferably in the range of 0.9 to 1.5, and as described later, the specific gravity after immobilizing the immunoactive substance is preferably in the range of 1.0 to 1.3. When the polymer particles have a specific gravity smaller than the above range before and after immobilization of the immunoactive substance, the polymer particles float on the surface of the aqueous medium in the aqueous dispersion, resulting in poor dispersion stability. On the other hand, if the size is larger than the above range, the particles tend to settle and aggregate in the aqueous medium of the dispersion, resulting in similarly poor dispersion stability. The water-dispersed polymer particles used in the present invention need to have a functional group to which an immunoactive substance can be covalently bonded, as described below. Furthermore, when an immunologically active substance is immobilized on a polymer particle via a so-called spacer group, the polymer particle needs to have a functional group. Preferred examples of such functional groups include carboxyl groups, amino groups, hydroxyl groups, glycidyl groups, and hydrazide groups. Therefore, for example, an aqueous dispersion of water-dispersible polymer particles having carboxyl groups may contain acrylic acid, methacrylic acid, itaconic acid, crotonic acid, maleic acid, fumaric acid, monoalkylmaleic acid, monoalkylfumaric acid. If necessary, vinyl monomers having a carboxyl group such as monobasic acids such as monoalkyl itaconic acid or polybasic acids are co-emulsified with other monomers having radical copolymerizability. It can be obtained by polymerization. Further, for example, an aqueous dispersion of water-dispersible polymer particles having a hydroxyl group or a glycidyl group can be, for example, hydroxyethyl acrylate,
By emulsion copolymerizing a monomer having a hydroxyl group such as 2-hydroxymethyl methacrylate or a monomer having a glycidyl group such as glycidyl methacrylate with other radical copolymerizable monomers as necessary. You can get it. Examples of the above-mentioned radical copolymerizable monomers include α-olefin monomers such as ethylene and propylene, vinyl monomers such as vinyl acetate and vinyl chloride, and styrenes such as styrene, methylstyrene, and chlorostyrene. acrylic ester monomers such as methyl acrylate, methacrylic ester monomers such as methyl methacrylate, diene monomers such as butadiene and isoprene, acrylonitrile, methacrylonitrile, acrylamide, Monomers such as methacrylamide can be mentioned. Further, after polymerizing the required monomer components, a functional group can be introduced into the obtained water-dispersed polymer particles. As a method for this purpose, for example, polymer particles having carboxyl groups can be obtained by hydrolyzing polymer particles obtained by polymerizing an acrylic acid ester as a monomer component. In addition, in order to prepare water-dispersed polymer particles having an amino group or a hydrazide group, for example, a monomer having an amide group such as acrylamide,
Alternatively, a monomer having a methyl ester group such as methyl acrylate is emulsion copolymerized with other monomers, and the amide group in the resulting copolymer is subjected to Hofmann degradation, or the methyl ester group is copolymerized with hydrazine. It can be obtained by reaction. Furthermore, crosslinked water-dispersed polymer particles can also be obtained by emulsion copolymerizing a polyfunctional monomer as a monomer component with the above monomer as an internal crosslinking agent. Since the internal crosslinking agent introduces a crosslinked structure into the polymer, when included in the diagnostic reagent, it can suppress the formation of undesirable water-soluble polymers and increase the glass transition temperature of the resulting polymer particles. Furthermore, the internal crosslinking agent is effective in making the water-dispersible polymer particles non-swellable and increasing the dispersion stability of the polymer particles in an aqueous medium. As such a polyfunctional monomer for internal crosslinking, for example, poly(meth)acrylate of aliphatic polyhydric alcohol is preferably used. As a specific example,
For example, ethylene glycol dimethacrylate,
Diethylene glycol dimethacrylate, triethylene glycol dimethacrylate, dipropylene glycol dimethacrylate, 1,3-butylene glycol dimethacrylate, triethylene glycol diacrylate, trimethylolpropane trimethacrylate, trimethylolpropane triacrylate, tetramethylolmethanetetraacrylate, etc. Preferably used. Also,
Divinylbenzene, N,N-methylenebisacrylamide, and the like can also be used as internal crosslinking agents. The specific types of individual monomers should be selected to ensure that the resulting water-dispersed polymer particles do not fuse or aggregate during storage as diagnostic reagents with immobilized immunoactive substances. Selected to have a glass transition temperature. The glass transition point of the polymer particles is preferably 10° C. or higher, particularly room temperature or higher, taking into account the storage temperature and use temperature of the diagnostic reagent. In the immunological diagnostic reagent according to the present invention, it is preferable that the immunoactive substance is covalently immobilized on the water-dispersed polymer particles via a spacer group. That is, it is preferable that a spacer group is bonded to the polymer particle by a covalent bond, and an immunoactive substance is immobilized to this spacer group by a covalent bond. When an immunoactive substance is directly immobilized on polymer particles without using a spacer group, and calcium bromide is added to this aqueous dispersion, some immunoactive substances will hardly lose their activity and will be positive. This is because they may not show an agglutination reaction even with serum. The compound that can be used as the spacer group is at least a difunctional organic compound, and does not exclude polyfunctional polymers. Organic compounds are preferred. Specific examples of compounds that function as such spacer groups include diamines such as hexamethylene diamine, dodecamethylene diamine, and xylylene diamine, 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 the present invention is not limited to these. This spacer group may be bonded to the polymer particle in advance, and then the spacer group and the immunoactive substance may be bonded,
Alternatively, the spacer group may be bound to the immunoactive substance in advance and this may be bound to the polymer particles. Furthermore, if necessary, spacer groups can be bonded to both the polymer particles and the immunoactive substance in advance to bond them to each other. However, if the immunoactive substance has the desired activity when a predetermined concentration of calcium bromide is blended into an aqueous dispersion of water-dispersed polymer particles on which the immunoactive substance is directly immobilized by covalent bonds. Needless to say, the immunoactive substance may be directly immobilized on the water-dispersed polymer particles. An immunoactive substance is directly covalently immobilized on water-dispersed polymer particles having the above-mentioned functional groups, or a spacer group is bonded to the polymer particles, and the immunoactive substance is covalently bonded to the spacer group. The method for immobilization by binding is not particularly limited, and any conventionally known method can be used. For example, one preferred method is to use water-soluble carbodiimide as a crosslinking agent. For example, when diamine is used as a spacer group, the amino group of the diamine and the carboxyl group of the water-dispersed polymer particles are reacted in the presence of water-soluble carbodiimide to form an amide bond. A spacer group is bonded to a polymer particle using the method, and then an immunologically active substance can be covalently immobilized on the amino group of this spacer group using a water-soluble carbodiimide. Examples of the water-soluble carbodiimide used in such a method include 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride,
Examples include 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide-meth-p-toluenesulfonate. In order to immobilize an immunologically active substance to polymer particles by covalent bonding directly with or without a spacer group using such a water-soluble carbodiimide, conventionally known conventional methods can be used. The method and conditions may be used. For example, if a spacer group is used, an appropriate amount is added to the aqueous dispersion of polymer particles together with the spacer group, for example, per unit volume of the aqueous dispersion.
Water-soluble carbodiimide is added at a concentration of 0.01 to 10 mg/ml, and incubated under normal conditions, for example, while 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 1 to 10. It is enough to react for about 5 hours. Next, an immunoactive substance may be similarly immobilized on the polymer particles to which the spacer groups are bonded. Further, when the functional group is a hydroxyl group, the spacer group is bonded by the cyanogen bromide method, or by reacting with dialdehyde when it is an amino group to activate these functional groups, and then, Immunoactive substances can be covalently immobilized on polymer particles in the same manner as described above. Alternatively, the immunologically active substance can be immobilized directly onto the polymer particles. The immunoactive substances used in the present invention include:
There are no particular limitations, and any antigen, antibody, hapten, etc. may be used. For example, human and animal immunoglobulin, modified immunoglobulin, α-fetoprotein, C-reactive protein (CRP), hepatitis virus-related antigen, various virus antigens such as rubella HA antigen, various virus antigens such as Toxoplasma, Mycoplasma, Treponema pallidum, etc. Examples include microbial antigens such as bacteria, fungi, and toxins, various plasma protein components such as albumin and complement components, various hormones such as estrogen and human chorionic gonadotropin (HCG), and antibodies against these antigen components. can be used. In the immunological diagnostic reagent according to the present invention, the water-dispersed polymer particles are mixed with a glycine buffer or a phosphate buffer with an appropriate pH and concentration so that the immobilized immunoactive substance is not deactivated as described above. The polymer particles are dispersed in a buffer such as a boric acid buffer, and calcium bromide is added to the aqueous dispersion of the polymer particles as an additive to suppress non-specific aggregation of the particles. Calcium bromide is contained at a concentration of 0.01 to 1.5 mol/, preferably 0.1 to 1.25 mol/, in an aqueous dispersion of water-dispersed polymer particles on which an immunoactive substance is immobilized. When the concentration of calcium bromide in the aqueous dispersion is less than 0.01 mol/mol, the effect of suppressing non-specific agglutination against negative serum is poor, whereas when the concentration exceeds 1.5 mol/mol, the effect of suppressing non-specific agglutination on positive serum is rather poor. This is because the aggregation of particles will be suppressed. Furthermore, in the immunological diagnostic reagent according to the present invention, the concentration of the buffer solution is normally in the range of 0.005 to 0.2M, preferably in the range of 0.01 to 0.1M. In addition, the pH of the buffer solution is usually 6 to 9, preferably 7 to 8.5, considering the dispersion stability of the water-dispersed polymer particles and the activity of antigen-antibody reaction.
is within the range of Further, 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. still,
A preservative such as sodium azide may be added to the immunological diagnostic reagent according to the present invention to provide a preservative effect. The immunological diagnostic reagent according to the present invention contains calcium bromide dissolved in a buffer solution, and after adjusting the pH,
It can be obtained by adding and mixing an aqueous dispersion of water-dispersible polymer particles on which an immunoactive substance is immobilized, and further adjusting the pH as necessary. In immunological diagnosis using the immunological diagnostic reagent according to the present invention, for example, the diagnostic reagent and the test solution are mixed on a hollow or flat plate of a glass plate or plastic plate, or on a microplate, and then observed with the naked eye or with a microscope. Therefore, the determination is made by determining the presence or absence of aggregation of polymer particles. Furthermore, the presence or absence of aggregation can also be determined as an optical change. (Effects of the invention) As described above, the immunological diagnostic reagent of the present invention has
It consists of an aqueous dispersion of water-dispersed polymer particles on which an immunoactive substance is immobilized, and also contains calcium bromide, resulting in non-specific aggregation of the polymer particles, although the reason is not always clear. Since the serum is completely inhibited, it is possible to make a quick and accurate diagnosis without diluting the serum with a buffer or inactivating it. Further, the diagnostic reagent according to the present invention easily mixes uniformly with serum, for example, on a glass plate, and has excellent fluidity, making it easy and accurate to determine the presence or absence of agglutination. In addition, since the diagnostic reagent contains calcium bromide, its specific gravity increases and becomes close to that of particles, but compared to conventional diagnostic reagents, no precipitation occurs over a long period of time. The uniform dispersibility of the polymer particles is maintained without any problems, and the storage stability is extremely excellent. In addition, the water-dispersed polymer particles used as carriers for immunoactive substances are uniform in quality, including particle size, and do not have immunoactivity themselves, so they are easy to immobilize. The immobilized particles have high detection sensitivity and high specificity, thus providing a diagnostic reagent that allows diagnosis with high accuracy. (Examples) Examples of the present invention will be shown below and specifically explained, but the present invention is not limited to these Examples. In the following, after immobilizing the immunoactive substance on the polymer particles, filter paper (No.
5), and additives were added to the obtained dispersion to prepare a diagnostic reagent. Example 1 (a) Preparation of water-dispersed synthetic polymer particles 8.0 g of methyl methacrylate, 5.0 g of isobutyl methacrylate, 1.0 g of acrylic acid, 3.0 g of methacrylonitrile, and 0.7 g of triethylene glycol dimethacrylate were mixed with distilled water. 370g plus distilled water
After adding an aqueous polymerization initiator solution in which 0.1 g of potassium persulfate was dissolved in 10 g, polymerization was carried out for 8 hours under a nitrogen stream at a temperature of 75° C. and stirring at a stirring speed of 190 rpm. An aqueous dispersion containing water-dispersed polymer particles with an average particle diameter of 0.30 μm was obtained at a polymerization rate of 99%. This aqueous dispersion of polymer particles was first centrifugally washed four times with distilled water, and then centrifugally washed twice with 0.01M borate buffer (PH7.5) to increase the water solubility in the aqueous phase. After removing molecules and purifying the polymer particles, the polymer particles were redispersed in 0.01 M borate buffer (PH 7.5) to a solid content of 5% by weight. (b) Binding of spacer group to polymer particles 100 ml of the aqueous dispersion of the water-dispersed polymer particles obtained above and an aqueous solution of ε-aminocaproic acid (0.02M)
100 ml and adjusted to pH 7.5 with 1N aqueous sodium hydroxide solution. 0.01M borate buffer (PH7.5)
An aqueous solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (25
mg/ml) to the above aqueous dispersion, and
The reaction was allowed to take place under stirring for an hour. After leaving it in the refrigerator overnight, it was centrifugally washed three times with 0.01M borate buffer (PH8.2) to obtain polymer particles with spacer groups attached. .2) was redispersed to a solid content of 5% by weight. (c) Immobilization of rabbit IgG Mix 5 ml of the aqueous dispersion of the water-dispersed polymer particles having spacer groups obtained above, 2 ml of 0.01M borate buffer (PH8.2), and 11 ml of distilled water. 1 for this
-ethyl-3-(3-dimethylaminopropyl)
2 ml of carbodiimide hydrochloride aqueous solution (5 mg/ml) was added, and 10 minutes later, 5 ml of rabbit IgG aqueous solution (5 mg/ml) was added, followed by reaction at 15° C. for 2 hours. Next, in order to consume the excess water-soluble carbodiimide in the reaction mixture, a 10 wt% L-arginine aqueous solution (PH
7.5) 5 ml was added and incubated for 1 hour.
Next, centrifugal washing was performed three times with 0.01M borate buffer (PH8.2), and then 0.01M borate buffer (PH8.2) was used.
8.2) and adjust the total volume to 10ml, thus,
An aqueous dispersion of water-dispersible polymer particles in which rabbit IgG was covalently immobilized via the spacer group was obtained. The amount of immobilization is approximately 50 per gram of polymer particles.
It was mg. (d) Preparation of immunological diagnostic reagent After dissolving various amounts of calcium bromide in 0.01M borate buffer (PH8.2), it was added to 1N aqueous sodium hydroxide solution to bring the pH of the buffer to approx. Prepared in 8.2. These calcium bromide-containing buffers and the rabbit IgG-immobilized particle dispersion described above were mixed to contain calcium bromide at various concentrations and immunochemicals at a polymer particle concentration of 1.0% by weight. A diagnostic reagent was prepared. (e) Evaluation of immunological diagnostic reagent This reagent in which rabbit IgG is immobilized as an immunoactive substance can be used as a rheumatoid factor detection reagent. This diagnostic reagent and the rheumatoid arthritis factor positive serum and negative serum were mixed in equal volumes on a glass plate as stock solutions and stirred, and the state of agglutination was visually determined after 2 minutes. The results are shown in Table 1.

【table】

[Table] In the diagnostic reagent according to the present invention, no non-specific agglutination reaction occurred, but in the control diagnostic reagent not containing calcium bromide, non-specific agglutination occurred. Furthermore, the stability over time was investigated at a temperature of 4° C. for the rheumatoid factor detection reagents having calcium bromide concentrations of 0.85 mol/ and 0.1 mol/, respectively. The evaluation method is the same as above. The results are shown in Table 2. It is clear that the diagnostic reagent of the present invention has a uniform appearance even after 6 months, does not cause any non-specific agglutination with negative serum, and has excellent storage stability. Furthermore, according to the diagnostic reagent of the present invention, the degree of aggregation is substantially constant regardless of reaction time. However, with diagnostic reagents that do not contain calcium bromide, precipitation occurs early and

[Table] In addition, non-specific agglutination was observed. Furthermore, a control diagnostic reagent containing calcium chloride as an additive not only causes a nonspecific agglutination reaction with negative serum, but also causes early precipitation, resulting in significantly poor storage stability. However, since this precipitate is caused by sedimentation of particles and not by agglomeration of particles, it can be used as a diagnostic reagent if it is sufficiently shaken to make it homogeneous. Moreover, according to diagnostic reagents containing calcium chloride, when the initial degree of agglutination after a few minutes of mixing with negative serum is (±), i.e., a false positive, nonspecific agglutination occurs over time. become stronger, e.g. approx.
After 10 minutes, the degree of aggregation was about (++),
The degree of aggregation varies over time, thus making accurate diagnosis impossible. Furthermore, changes in positive activity when the above rheumatoid factor detection reagent was stored at a temperature of 4°C were investigated. The results are shown in Table 3. The criteria for determining aggregation are the same as above. Next, the water-dispersed polymer prepared by the method (a) above

[Table] Directly without bonding a spacer group to the child polymer particles.
After immobilizing rabbit IgG according to the method in (c), (d)
Control diagnostic reagent A containing calcium bromide at various concentrations was prepared according to the method of . In addition, a control diagnostic reagent B, which was the same as the diagnostic reagent of the present invention except that it did not contain calcium bromide, was prepared. The activity of the diagnostic reagent according to the present invention and these control diagnostic reagents was determined according to method (e). The results are shown in Table 4. According to the diagnostic reagent according to the present invention, non-specific agglutination does not occur and the detection sensitivity is high, but the control diagnostic reagent A, in which rabbit IgG is not immobilized via a spacer group, is clearly detected even for positive serum. Furthermore, control diagnostic reagent B shows significant non-specific agglutination. Example 2 (a) Preparation of water-dispersed synthetic polymer particles Same as Example 1 except that 15.0 g of methyl methacrylate, 1.0 g of acrylic acid, and 0.7 g of triethylene glycol dimethacrylate were used as monomers. The average particle size is 0.36μ at a polymerization rate of 98%.

[Table] An aqueous dispersion of water-dispersible polymer particles of m was obtained. This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were added to a 0.01M borate buffer (PH7.5).
Disperse it to a solid content concentration of 5% by weight,
An aqueous dispersion of water-dispersible polymer particles was obtained. (b) Bonding of spacer groups to polymer particles In the same manner as in Example 1, spacer groups were bonded to the polymer particles obtained above. (c) Immobilization of rabbit IgG Rabbit IgG was covalently immobilized on the spacer group-bound polymer particles obtained above in the same manner as in Example 1, and then treated in exactly the same manner as in Example 1. The polymer particles were soaked in 0.01M borate buffer (PH8.2).
The total volume was adjusted to 10 ml to obtain a water-dispersed polymer particle dispersion in which rabbit IgG was immobilized. The amount of rabbit IgG immobilized was 46 mg per gram of polymer particles. (d) Preparation of immunological diagnostic reagent Using the rabbit IgG-immobilized water-dispersed polymer particle dispersion obtained above, the procedure was carried out in the same manner as in Example 1.
Immunological diagnostic reagents according to the invention containing calcium bromide at various concentrations were prepared. For comparison, control immunodiagnostic reagents were prepared using sodium bromide, calcium bromide, lithium bromide, calcium chloride, or calcium acetate as additives in place of calcium bromide. (e) Evaluation of immunological diagnostic reagents The various immunological diagnostic reagents obtained above were evaluated for activity in the same manner as in Example 1. The results are shown in Table 5. According to the diagnostic reagent according to the present invention, non-specific aggregation does not occur at all, whereas in the case of the control diagnostic reagent, non-specific aggregation is significantly observed. Example 3 Human IgG was immobilized using carbodiimide in the same manner as in Example 1 to the water-dispersed polymer particles having spacer groups prepared in Example 2, and then calcium bromide was added to the water-dispersed polymer particles containing 0.75 mol of calcium bromide. An immunological diagnostic reagent A according to the present invention was prepared. heavy

【table】

[Table] The amount of human IgG immobilized per 1 g of combined particles was 55 mg. Next, human IgG was directly immobilized using carbodiimide without bonding a spacer group to the water-dispersed polymer particles prepared in Example 2, and calcium bromide was added to the aqueous dispersion of the polymer particles.
The immunological diagnostic reagent B according to the present invention was prepared by dissolving it at a concentration of 0.5 mol/concentration. The amount of human IgG immobilized in this diagnostic reagent is 1
It was 62 mg per g. In this way, even when human IgG is immobilized, it can be used as a rheumatoid factor detection reagent. These diagnostic reagents A and B according to the present invention are mixed and stirred with various untreated serums, and 2
Table 6 shows the results of determining the state of aggregation after 5 minutes.
Results for a control diagnostic reagent that does not contain calcium bromide are also shown. The diagnostic reagent of the present invention is free from non-specific agglutination and has high detection sensitivity. Example 4 (a) Preparation of water-dispersed synthetic polymer particles 50 g of methyl methacrylate, acrylonitrile
The average particle size was determined in the same manner as in Example 1 except that 8.0 g and 2.5 g of methacrylic acid were used at a polymerization rate of 99.5%.
An aqueous dispersion of water-dispersible polymer particles of 0.28 μm was obtained. This dispersion was purified in the same manner as in Example 1, and the obtained polymer particles were added to a 0.01M borate buffer (PH7.5).
Disperse it to a solid content concentration of 5% by weight,
An aqueous dispersion of water-dispersible polymer particles was obtained. (b) Binding of spacer group to polymer particles Using glutamic acid as a spacer group compound,
A spacer group was bonded to the polymer particles obtained above using the carbodiimide method in the same manner as in Example 1. (c) Immobilization of anti-human chorionic gonadotropin antibody 5 ml of the aqueous dispersion containing 5% by weight of the spacer group-bonded polymer particles obtained above, 2 ml of 0.1 M borate buffer (PH7.5) and distilled Mix 11ml of water and add 1
-ethyl-3-(3-dimethylaminopropyl)
After adding 2 ml of carbodiimide hydrochloride aqueous solution (5 mg/ml), 5 ml of anti-human chorionic gonadotropin antibody solution (anti-HCG, 5 mg/ml) was added, and reaction was carried out at 15° C. for 3 hours. Thereafter, the polymer particles were treated in exactly the same manner as in Example 1, and the polymer particles were dispersed in 0.01M boric acid buffer (PH8.2), the total volume was adjusted to 10 ml, and the anti-HCG immobilized water-dispersed polymer was prepared. A particle dispersion was obtained. The amount of anti-HCG immobilized was 35 mg per gram of polymer particles. (d) Preparation of immunological diagnostic reagents According to the present invention containing calcium bromide at a concentration of 0.75 mol/and 0.50 mol/, respectively, and a polymer particle concentration of 1.5% by weight by the same method as in Example 1. An immunological diagnostic reagent was obtained. (e) Evaluation of immunological diagnostic reagent This reagent with immobilized anti-HCG as an immunoactive substance can be used as a pregnancy diagnostic reagent. Equal amounts of this diagnostic reagent and serum were mixed as undiluted solutions on a glass plate, and the presence or absence of agglutination was determined after 3 to 5 minutes.If there was 1 international unit of HCG in 1 ml of serum, agglutination would occur. The presence or absence of pregnancy could be determined easily and accurately. No non-specific aggregation occurred. On the other hand, according to a control diagnostic reagent that does not contain calcium bromide, even in the case of non-pregnant human serum,
Nonspecific agglutination occurred, which was determined to be a false positive. Example 5 (a) Preparation of water-dispersed synthetic polymer particles 10 g of glycidyl methacrylate, 50 g of styrene
g, methyl methacrylate 18g, divinylbenzene
After adding 2.0g of acrylonitrile and 20g of acrylonitrile to 230g of distilled water, and adding an aqueous polymerization initiator solution in which 0.3g of potassium persulfate was dissolved in 10g of distilled water, the mixture was heated to 70°C under a nitrogen stream while maintaining the pH at 7.0. Polymerize for 8 hours while stirring at a stirring speed of 120 rpm at
An aqueous dispersion containing water-dispersible polymer particles having a solid content of about 30% and an average particle size of 0.25 μm was obtained. The amount of aggregates was 0.03%. (b) Binding of spacer group to polymer particles The water-dispersed polymer particles obtained above were centrifuged, redispersed in 100 ml of 0.05M sodium acetate-sodium hydroxide aqueous solution (PH10), and - 2 g of xylylene diamine was added and reacted at 30°C for 24 hours. This is followed by centrifugation to remove unreacted m-xylylenediamine, and then the spacer group bound polymer particles are separated in this way.
Solid content 5% by weight in 0.01M borate buffer (PH8.2)
It was redispersed so that (c) Immobilization of rabbit IgG Rabbit IgG was immobilized on the polymer particles obtained above in the same manner as in Example 1. The amount of immobilization per gram of polymer particles was 35 mg. (d) Preparation of immunological diagnostic reagent Calcium bromide was added to the aqueous dispersion of the rabbit IgG-immobilized polymer particles obtained above in the same manner as in Example 1, and the immunological diagnostic reagent according to the present invention was prepared. Prepared. (e) Evaluation of immunological diagnostic reagent This diagnostic reagent and rheumatoid factor-positive serum and negative serum were mixed in equal volumes on a glass plate as stock solutions and were stirred, and the agglutination state was determined after 2 minutes. The results are shown in Table 7.

[Table] The results for a control immunological diagnostic reagent without calcium bromide are also shown. According to the diagnostic reagent according to the present invention, no non-specific agglutination reaction occurs, but with the control diagnostic reagent which does not contain calcium bromide, non-specific agglutination is significant.

Claims (1)

[Scope of Claims] 1. An immunological diagnostic reagent comprising an aqueous dispersion of water-dispersed polymer particles on which an immunoactive substance is covalently immobilized, wherein calcium bromide is blended into the aqueous dispersion. An immunological diagnostic reagent characterized by: 2. The immunological diagnostic reagent according to claim 1, wherein the immunologically active substance is covalently immobilized on the water-dispersed polymer particles via a spacer group. 3. The immunological diagnostic reagent according to claim 1, which contains calcium bromide at a concentration in the range of 0.01 to 1.5 mol/. 4. The immunological diagnostic reagent according to claim 1, which has a pH of 6 to 9.