JPH0372261A - Immunoassy - Google Patents

Abstract

PURPOSE:To simply perform measurement with high sensitivity and high accuracy within a short time by using solid phase particles obtained by bonding an immunologically active substance to polymer particles having a sulfonic acid group and a carboxyl group by covalent bond as a solid phase. CONSTITUTION:A solution to be examined is added to an aqueous dispersion of solid phase particles composed of an immunologically active substance obtained by bonding the immunologically active substance to polymer particles with a particle size of 0.5 - 500mum having a sulfonic acid group and a carboxyl group through the carboxyl group on the surfaces thereof by a covalent bond and the substance to be examined in the solution to be examined is specifically bonded to the immunologically active substance. Next, a labelled substance specifically bonded to said substance to be examined is specifically bonded to said substance. The dispersion of solid phase particles obtained as a result is filtered by the first filter material having a mean holding particle size twice or more the particle size of said solid phase particles and the filtrate is filtered by the second filter material having a mean holding particle size of 0.2mum - the particle size of the solid phase particles and the label bonded to the solid phase particles on the filter material is detected to make it possible to simply perform measurement within a short time with high sensitivity and high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an immunoassay method that utilizes an antigen-antibody reaction. The present invention relates to an immunoassay method that can easily detect a predetermined immunoactive substance contained in a test liquid with high sensitivity and precision.

Conventional methods for measuring a given test substance contained in body fluids using immunological techniques have been to use red blood cells or polystyrene particles carrying an antigen or antibody specific to the test substance and the test substance. A method is known in which the agglutination reaction is measured visually, optically, or mechanically by bringing substances into contact with each other. Since it is difficult to detect, enzyme immunoassay (EIA) and radioimmunoassay (RIA) have been used as more sensitive methods.

In these methods, solid-phase methods using a solid phase in which a predetermined immunoactive substance that specifically reacts with the test substance is bound to a water-insoluble carrier are preferably used, and in particular, the Sand-Deutsch method and the Competition law is well known.

For example, in the Sand-Deutsch method, when the test substance is a predetermined antigen, the predetermined antibody is bound to an inert solid phase carrier to form a solid-phase antibody, and the antigen, which is the test substance, is added to the immobilized antibody. Perform antibody reaction (-next reaction). Then,
An excess of labeled antibody is added to the solid phase, and the labeled antibody is further specifically bound to the antigen bound to the solid phase antibody in an antigen-antibody reaction (secondary reaction). In this way, the antigen, which is the test substance, is bound in a sandwich between the immobilized antibody and the labeled antibody.

Therefore, after removing the excess labeled antibody that did not participate in the above reaction from the solid phase by washing or other means, (
B/F separation), the antigen, which is the test substance, is quantified by detecting the labeled antibody on the solid phase.

In the Sand-Deutsch method, in the case of EIA, an enzyme-labeled antibody is used as the labeled antibody. Therefore, in order to quantify such an enzyme-labeled antibody, an enzyme as a labeling substance is reacted with a substrate, and the reaction result of the enzyme reaction is determined, for example, by colorimetry.

As described above, when a solid phase method is employed in an immunoassay, it is particularly important to separate the labeled antibodies bound to the solid phase from the labeled antibodies not bound to the solid phase, that is, to separate the labeled antibodies bound to the solid phase. Complicated operations are required to separate F, and therefore, a long time is required for measurement.

Therefore, for example, in JP-A-62-98258 and JP-A-62-98259, antibodies are applied to polymer particles with a particle size of 0.02 μm to 1 μm. A solid-phase aqueous dispersion of bound polymer particles is prepared, and after reacting with excess labeled antibody, B/F separation is performed using a porous membrane. Methods for detecting solid phase labels have been proposed.

Although this method simplifies B/F separation,
Since the particulate solid phase used is similar to antibody-immobilized latex that is generally used in latex immunoagglutination, the solid phase particles aggregate when reacting with the test substance, and the solid phase on the membrane surface is Non-specific reactions may occur that are thought to be due to heterogeneity, and as a result, there are still some problems with the quantitativeness and reproducibility of measurements, and furthermore, the quantification range is relatively limited. There is also the problem of

The present invention was made to solve the above-mentioned problems in conventional immunoassay methods, and is particularly applicable to EIA and RIA using solid-phase methods, which can be easily performed in a short time with high sensitivity and high precision. The purpose of the present invention is to provide an immunoassay method capable of detecting a predetermined test substance.

The immunoassay method according to the present invention for the prevention of ``Shin'' involves the use of polymer particles with a particle diameter of 0.5 to 500 tIm having sulfonic acid groups and carboxyl groups on the surface, and an immunologically active substance shared through the carboxyl groups. A first step in which a test liquid is added to an aqueous dispersion of immobilized particles of an immunoactive substance bound by binding, and a predetermined test substance in the test liquid is specifically bound to the immunoactive substance. and a second step of specifically binding a labeled substance that specifically binds to the test substance to the test substance, and then the dispersion of solid-phase particles obtained in this way is A third step of filtering with a first filter medium having an average retention particle diameter of twice or more the particle diameter of the solidified particles, and
A fourth step of filtering with a second filter medium having an average retained particle size of 0.2 μm or more, and a fifth step of detecting the label bound to the solid-phase particles captured on the second filter medium. It is characterized by

In the immunoassay method according to the present invention, particles having a sulfonic acid group and a carboxyl group on the surface have a diameter of 0.5 to 50.
0 pm (D) Solid-phase particles formed by covalently bonding an immunoactive substance to polymer particles via the carboxyl group are used as the solid phase.

The above polymer particles are produced by copolymerizing a monomer having a sulfonic acid group and a monomer having a carboxyl group, or a monomer having both a sulfonic acid group and a carboxyl group, with these monomers as necessary. It can be obtained by emulsion (co)polymerization together with the vinyl monomer to be obtained. Various types of monomers having sulfonic acid groups, monomers having carboxyl groups, and vinyl monomers that can be copolymerized with these monomers are already known. Emulsion (co)polymerization is also already known.

Examples of the above-mentioned monomer having a sulfonic acid group include -
Format %Formula %: (In the formula, R' represents hydrogen or an alkyl group, R2 represents an alkylene group, and M represents hydrogen or an alkali metal.) Sulfoalkyl acrylate or its alkali metal salt represented by -formula (In the formula, R3 is an alkyl group, M is hydrogen or an alkali metal.) Styrene sulfonic acid derivatives or alkali metal salts thereof, -formula % formula % (In the formula, R4 is an alkyl group, R5 is an alkylene group) and M represents hydrogen or an alkali metal) and its alkali metal salts.

Therefore, specific examples of the vinyl monomer having a sulfonic acid group include sodium styrene sulfonate, sodium sulfopropyl methacrylate, and the like.

Such a vinyl monomer having a sulfonic acid group increases the dispersion stability of copolymer particles produced by emulsion copolymerization in the absence of an emulsifier.

In addition, as a vinyl monomer having a carboxyl group,
Preferably, an acrylic acid derivative represented by the formula % (in the formula, R6 and R7 each independently represents an alkyl group) is used. Specific examples include acrylic acid and methacrylic acid. Furthermore, vinyl acetic acid, fumaric acid, maleic acid, etc. can also be used as the vinyl monomer having a carboxyl group.

Such an acrylic acid derivative is essential for providing a carboxyl group, which is a functional group for covalently immobilizing an immunologically active substance, to the obtained emulsion copolymer particles, and also It also has the effect of increasing dispersion stability.

Vinyl monomers that are copolymerizable with monomers having sulfonic acid groups or carboxyl groups are not particularly limited, but include aromatic vinyl monomers such as styrene and vinyltoluene, meth) methyl acrylate,
Examples include (meth)acrylic acid alkyl esters such as ethyl (meth)acrylate, propyl (meth)acrylate, and hydroxypropyl (meth)acrylate, vinyl acetate, butadiene, isoprene, and (meth)acrylonitrile.

Furthermore, as the above-mentioned copolymerizable vinyl monomer, it is preferable to use a polyfunctional monomer at the same time. By using such a polyfunctional monomer, the glass transition point of the resulting copolymer particles can be increased by internal crosslinking. Furthermore, when emulsion copolymerizing monomers having sulfonic acid groups or carboxyl groups as described above, undesirable water-soluble polymers may be produced as by-products. By using these polymers together, it is possible to suppress the by-product of undesirable water-soluble polymers.

Examples of such polyfunctional monomers include ethylene glycol diacrylate, ethylene glycol dimethacrylate, propylene glycol diacrylate, propylene glycol dimethacrylate, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, divinylbenzene, and glycerin triacrylate. etc. can be mentioned.

Such polymer particles used in the present invention have a particle size of 0.
．． It is necessary that the thickness be in the range of 5 to 500 μm. When the particle diameter of the polymer particles is smaller than 0.5 μm, it is difficult to capture the polymer particles on the filter medium during filtration in the fourth step described below. Furthermore, even if a filter medium capable of capturing such polymer particles is used, the filtration is extremely slow and the labeled substance becomes difficult to filter, which may cause a non-specific reaction, which is not preferable.

On the other hand, when the particle diameter of the polymer particles is larger than 500 μm, the total surface area of the particles per unit volume is small and the sensitivity is low. Furthermore, the particles settle during storage and are difficult to disperse uniformly, resulting in poor storage stability and measurement reproducibility.

In the present invention, the emulsion copolymer particles obtained in this way have carboxyl groups on the surface of 0.3 to 30 μm.
It is preferable to have it in the range of mol/nf, especially 1 to
It is preferable to have it in the range of 20 μmol/bo. When the number of carboxyl groups on the surface of the emulsion copolymer particles is less than 0.3 μmol/m2, the amount of antibody that can bind thereto is too small, resulting in low sensitivity. When added, aggregation occurs in the solidified particles, reducing measurement accuracy. On the other hand, when the number of carboxyl groups on the surface of the emulsion copolymer particles is more than 30 μmol/bo, the solid phased particles will have poor dispersion stability, and the reproducibility of measurement will be impaired.

The polymer particles used in the present invention preferably have sulfonic acid groups on their surfaces in an amount of 0.001 to 10 μmol/bo, particularly in a range of 0.01 to 1.0 μmol/bo. It is preferable to have When the amount of sulfonic acid groups possessed by the polymer particles is less than the above, the dispersion stability of the polymer particles is poor and aggregation is likely to occur in the first and second steps. On the other hand, when the amount is more than the above, the particles tend to swell, resulting in poor storage stability and not having sufficiently high reactivity in the first and second steps.

The solid-phase particles used in the present invention thus have carboxyl groups and sulfonic acid groups on their surfaces, so for example, antibodies can be immobilized on the particles using water-soluble carbodiimide using the carboxyl groups of the particles. Since the sulfonic acid groups remain free during the reaction, antibodies can be bound to the particles at high density while maintaining the dispersion stability of the solid-phase particles. Examples of the carbodiimide include l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, 1-cyclohexyl-
3-(2-monopholinoethyl) carboxydometo P-)luenesulfonate and the like are preferably used.

In the method of the present invention, as a first step, an appropriate test liquid such as urine containing a predetermined test substance is added to the aqueous dispersion of the solid-phase particles, and the reaction is carried out for several minutes to several tens of minutes. Let me,
The above-mentioned test substance is specifically bound to the above-mentioned solid-phase particles, and then, as a second step, a labeled immunoactive substance that specifically reacts with the above-mentioned test substance is added. The labeled immunoactive substance is allowed to specifically bind to the test substance by reacting for several minutes.

Next, as a third step, the aqueous dispersion of the solidified particles after the reaction is filtered through a first filter medium having an average retained particle diameter that is at least twice the particle diameter of the solidified particles. but,
According to the invention, the first and second steps may be performed substantially simultaneously. That is, even if a test substance and a labeling substance are added almost simultaneously to an aqueous dispersion of solid-phase particles and reacted for several minutes to several tens of minutes, the dispersion is filtered through the first filter medium. good.

In the present invention, the average retained particle size refers to the minimum particle size that is not filtered by preparing a diluted polystyrene particle suspension, filtering it, and examining its degree of leakage using a microscopic measurement method. say.

In the present invention, the first filter medium needs to have an average retention particle diameter that is at least twice the particle diameter of the solid-phase particles, and an average retention particle diameter that is at least 10 times the particle diameter of the solid-phase particles so that the first filter medium can pass through the solid-phase particles. It is preferable to have a particle size. This is because when the average retained particle diameter of the first filter medium is smaller than twice the particle diameter of the solidified particles, the amount of retained particles increases. However, 2
When it exceeds 0 times, the effect of filtering particulate matter, colloidal matter, etc. coexisting in the test substance will be reduced, as will be described later.

According to the present invention, non-specific reactions can be significantly reduced by such a third step. In particular, there is no false positive in a negative sample that does not contain a predetermined immunologically active substance in the test substance, and the blank value can be made extremely low. The reason for this is not necessarily clear, but it may be that particulate matter or colloidal substances coexisting in the test substance are filtered out, or that labeled antibodies exist between microaggregated solid phase particles. This seems to be because microagglomerates are removed by the first filter medium.

Since the first filter medium must not nonspecifically adsorb proteins such as antigens and antibodies in the test liquid, it is preferably made of a hydrophilic material such as regenerated cellulose, filter paper, glass fiber filter, etc.

Next, according to the method of the present invention, in the fourth step, the obtained filtrate is filtered through a second filter medium to remove the solidified particles from the second filter medium.
captured on the filter media. Therefore, it is necessary for the second filter medium to have an average retained particle diameter that is equal to or less than the particle diameter of the solidified particles and is equal to or greater than 0.2 μm. When the average retained particle diameter of the second filter medium is smaller than 0.2 μm, non-specific reactions increase or filtration takes an unnecessarily long time.

Further, when the average retained particle size of the second filter medium is larger than the particle size of the solidified particles, the solidified particles pass through the filter medium.

The average retained particle size of the second filter medium is preferably 80% or less of the particle size of the solidified particles.

Like the first filter medium, the second filter medium must not non-specifically adsorb proteins such as antigens and antibodies in the test solution, so it should be made of a hydrophilic material such as regenerated cellulose, filter paper, glass fiber, etc. Preferably, it consists of a filter or the like.

Next, a case in which a test substance is measured by the Sanderch method using the method of the present invention will be specifically explained as an example.

First, an antigen or antibody specific to a test substance is prepared and immobilized on polymer particles. For example, if the test substance is CR
When P, an anti-CRP antibody as an antibody specific to CRP is bonded to the polymer particle via its carboxyl group by a covalent bonding method. The amount of immunoactive substances such as antigens or antibodies specific to the test substance bound to polymer particles varies depending on the immunoactive substance, but is usually in the range of 1 to 500 μg per 1 g of polymer particles. . in this way,
The polymer particles on which the immunoactive substance is immobilized are dispersed in an appropriate buffer solution at a concentration of 0.5 to 20% by weight, preferably 1 to 10% by weight so that the immunoactive substance is not deactivated. .

As the buffer solution, a glycine buffer solution, a borate buffer solution, a phosphate buffer solution, etc., which are adjusted to an appropriate pH and concentration, are usually used. For example, when the anti-CRP antibody is bound to polymer particles, the polymer particles are preferably dispersed in a glycine buffer.

Next, a test solution containing CRP is added to the dispersion containing the anti-CRP antibody immobilized particles, and the reaction is usually carried out for 1 to 60 minutes, so that the CRP in the test solution specifically binds to the anti-CRP antibody. let Next, an excess amount of anti-CRP antibody labeled with alkaline phosphatase etc. is added and further reacted.
The labeled anti-CRP antibody is specifically bound to the CRP bound to the solid phase.

This reaction system is filtered through the first filter medium, and the resulting filtrate is further filtered through a second filter medium and washed to perform B/F separation. Here, since the second filter medium has sufficient filterability through natural filtration, forced filtration such as suction filtration is not necessary, and rather, forced filtration causes clogging and agglomeration, resulting in non-use. This is not preferable because it may induce a specific reaction or increase the blank value.

In this manner, CRPi can be determined by detecting the label of the solid-phase particles remaining on the second filter medium. For example, when using an anti-CRP antibody labeled with alkaline phosphatase, nitroblue tetrazolium, TNBS, which is a substrate of alkaline phosphatase, is used.
(5-bromo-4-chloro-3-indolylphosphate-p-t-luidine salt) and magnesium chloride-containing jetanolamine solution is dropped onto the solidified particles on the second filter medium, After about 3 minutes, the solid-phase particles turn brown, so the label can be detected visually or by measuring the reflected light.

Labels can be similarly detected using competitive methods.

As described above, according to the method of the present invention, since the polymer particles used are fine particles, the surface area capable of binding antigens or antibodies is very large, and moreover, the dispersion of solid-phase particles is Since it has good properties and does not inhibit the antigen-antibody reaction, the antigen-antibody reaction is performed in a nearly homogeneous state. Furthermore, even if a chromatic agglutination reaction occurs, the aggregates are filtered out by the first filter medium, and the excess labeled antibody is reliably separated into B/F by the second filter medium. Therefore, compared to the conventional solid-phase EIA method, it is possible to perform immunological analysis in a short time, with simple operation, with high sensitivity and high precision, and with high reproducibility while suppressing non-specific reactions. Measurements can be taken.

EXAMPLES The present invention will be explained below with reference to Examples, but the present invention is not limited to these Examples in any way.

(a) Preparation of latex 1 part by weight of styrene, 2.0 parts by weight of sodium styrene sulfonate, 5.0 parts by weight of acrylic acid and 4 parts by weight of distilled water
After charging 10 parts by weight into a reaction vessel and thoroughly purging with nitrogen gas, the temperature was raised to 70°C and stirred at 300 rpm for 30 minutes.

Next, 0.5 parts by weight of ammonium persulfate was added to 20 parts by weight of distilled water.
An aqueous solution of a polymerization initiator dissolved in parts by weight was added to the above monomer mixture, and polymerization was carried out at 70°C for 8 hours with stirring.

Using distilled water, the latex obtained in this way is
After purification by centrifugation three times, the solid content concentration was 1.
It was adjusted to 0% by weight. The average particle size of this latex is 0
．． It was 15 μm.

Next, 15.0 parts by weight of the purified latex obtained above, 100 parts by weight of styrene, 4.0 parts by weight of acrylic acid, 0.2 parts by weight of divinylbenzene, and 395 parts by weight of distilled water were charged into a reaction vessel, and nitrogen gas After sufficient replacement at 70°
The temperature was raised to C and stirred at 300 rpm for 30 minutes.

After this, add 0.5 parts by weight of ammonium persulfate to 20 parts by weight of distilled water.
An aqueous solution of a polymerization initiator dissolved in parts by weight was added to the mixture containing the latex and monomer, and the mixture was heated at 70°C for 12 hours with stirring.
Polymerized for hours.

The thus obtained sulfonated carboxylated polystyrene latex was dissolved in borate buffer (pH 8, 2, 0,
After purification by centrifugation five times using 01 mol/l), the solid content concentration was adjusted to 5% by weight. The average particle size of this latex is 0.62 μm, the amount of carboxyl groups on the surface of each particle is 9.2 μmol/bo, and the amount of sulfonic acid groups is 0.1
It was 3 μmol/bo.

(b) Preparation of antibody-bound latex Add boric acid buffer (pH 8,
Mix 2 ml of 2,0,1 mol/fi) and 11 ml of distilled water, and add 1-ethyl-3-(3-dimethylacetinopropyl)carbodiimide hydrochloride aqueous solution (2 mg/ml).
Add 2 ml of hCG antibody (4 mg/ml) and add hCG antibody (4 mg/ml) 10 minutes later.
), and the mixture was reacted at 10°C for 24 hours.

Next, 5 ml of a 10% by weight arginine aqueous solution (pH 8,2) was added to the obtained reaction mixture to consume the excess carbodiimide remaining in the reaction mixture, and the mixture was incubated for 1 hour. After this, Tris buffer (pi(8)
, 2, 0, 01 mol/ff1) three times, and then redispersed in the same buffer to obtain an anti-hCG antibody-bound latex in a total volume of 5 ml. In this latex, the amount of antibody bound was 35 mg/g.

(C) Preparation of enzyme-labeled antibody Peroxidase (manufactured by Sigma, Type Vl) 40■
Dissolve it in 10ml of distilled water, and add sodium metaperiodate water to this. 8 liquid (40mg/ml) 0.5ml
was added and stirred at room temperature for 10 minutes.

This solution was mixed with Sephadex G-
25, the peroxidase solution was fractionated, and the pH was adjusted to 9.5 with sodium hydroxide. To this was added 10 ml of anti-hCG antibody (manufactured by Dako, rabbit IgG, 10 μ/n+1) which had been dialyzed in carbonate buffer (p) 19.5.0.01 mol/ff1), and the mixture was heated at 4°C. After leaving it for 24 hours, add sodium borohydride (5
/ml) was added and allowed to stand at 4°C for 2 hours. Then, Tris buffer (pH 8, 2, 0, 01 mol/l)
Dialyzed with Sephacryl S
-200, and the first peak was fractionated to obtain an enzyme-labeled antibody fraction. Since unreacted antibody with peroxidase remains in the first peak, it is purified by affinity chromatography using Concanavalin A-Sepharose (manufactured by Pharmacia) to obtain a peroxidase-labeled anti-HCC antibody. Ta.

(d) Measurement of hCG 450 ul of hCG standard product (control) diluted with healthy male urine was added to 50 μ2 of anti-hCG antibody-bound sulfonated carboxylated polystyrene latex (5% by weight) solution and mixed for 2 minutes at room temperature. Next, peroxidase-labeled anti-hCG antibody (0.1% bovine serum albumin,
Contains 0.9% salt, 0.01 mol/(l Tris buffer (
Diluted 5000 times at pH 8.2). )500
μ2 was added and mixed for 1 minute at room temperature.

Filter from the bottom layer! (Non-asbestos filter plate made by Atvanchik), membrane filter (same as above, nitrocellulose, pore size 0.45 μm), and membrane filter (same as above, nitrocellulose, pore size 3 μm) are stacked in three layers in this order, and this is attached to a holder. The reaction solution was dropped into the center of the filter using a dropper.

After the reaction solution permeated, the filter with a pore size of 3 μm was removed and a Tris buffer solution (0.2% bovine serum albumin, 0.2% bovine serum albumin, 0.2% bovine serum albumin, 0.2% bovine serum albumin,
Washed with 500 μl of ρ)18.2) containing 9% sodium chloride.

Next, 200 μl of enzyme substrate solution (1 m Mo-phenylenediamine, 0.05% hydrogen peroxide, 0.1 mol/°C phosphate-citrate buffer, pH 6.0) was added dropwise, and the mixture was allowed to stand for 2 minutes. The presence or absence of color that appeared at that time was determined with the naked eye.

Comparative Example 1 (a) Measurement of hCG Using the anti-hCG antibody-bound latex prepared in Example 1 and the peroxidase-labeled anti-hCG antibody, hCG was measured.

Measurement was carried out in the same manner as in Example 1, except that the filter medium was changed to a filter plate in the lower layer and a membrane filter (pore diameter 0.45 μm) in the upper layer, and the membrane filter with a pore diameter of 3 μm was not used.

Comparative Example 2 (a) Preparation of latex 100 parts by weight of styrene, 5.0 parts by weight of acrylic acid, and 410 parts by weight of distilled water were placed in a reaction vessel, and after being sufficiently replaced with nitrogen gas, the temperature was raised to 70''C. The mixture was stirred at 300 rpm for 30 minutes.

Next, 0.5 parts by weight of ammonium persulfate was added to 20 parts by weight of distilled water.
An aqueous solution of a polymerization initiator dissolved in parts by weight was added to the above monomer mixture, and the mixture was polymerized at 70°C for 12 hours with stirring.

Using distilled water, the latex obtained in this way is
After purification by centrifugation three times, the solid content concentration was 1.
It was adjusted to 0% by weight. The average particle size of this latex is 0
．． It was 31 μm.

Next, 100 parts by weight of the purified latex obtained above, 85 parts by weight of styrene, 1 part by weight of acrylic acid, 0.2 parts by weight of divinylbenzene, and 325 parts by weight of distilled water were charged into a reaction vessel, and the mixture was sufficiently heated with nitrogen gas. After replacing the mixture, the temperature was raised to 70°C and stirred at 30Qrpm for 1 hour.

After this, add 0.5 parts by weight of ammonium persulfate to 20 parts by weight of distilled water.
An aqueous solution of a polymerization initiator dissolved in parts by weight was added to the mixture containing the latex and monomer, and the mixture was heated at 70°C for 12 hours with stirring.
Polymerized for hours.

The thus obtained carboxylated polystyrene latex was soaked in a borate buffer (pH 8, 2, 0, 1 mol/O).
After purification by centrifugation was performed five times using 1), the solid content concentration was adjusted to 5% by weight.

The average particle diameter of this latex was 0.68 μm, and the amount of carboxyl groups on the particle surface was 5.3 μmol/rTf.

(b) Preparation of antibody-bound latex An anti-hCG antibody-bound carboxylated latex was prepared in the same manner as in Example 1. The amount of antibody bound was 39μ/g.

(c) Measurement of hCG using anti-hCG antibody-bound carboxylated latex,
hCG was measured in the same manner as in Example 1.

Example 2 (a) Preparation of antibody-conjugated vector The sulfonated carboxylated polystyrene latex prepared in Example 1 was bonded with an anti-human CRP antibody (manufactured by Dako) in the same manner as in Example 1.

The final dispersion medium was a glycine buffer (pH 8, 2, 0, 1 mol/poor). The amount of antibody bound was 38 mg/g.

0) Preparation of enzyme-labeled antibody Alkaline phosphatase (manufactured by Sigma) 0°2■ in phosphate buffer (pH 6, 8, 0, 1 mol/l,) 0°5
ml, and further add anti-Human CRP antibody (manufactured by Dako Co., Ltd., adjusted to 5 / m + with the above buffer solution) 0
．． 1 ml was mixed. Add the above buffer to the mixture at 0°1
0.1 ml of glutaraldehyde adjusted to % was slowly added under stirring, and the mixture was allowed to stand at room temperature for 3 hours. Next, it was developed on Sephacryl S-200 to obtain an alkaline phosphatase-labeled anti-human CRP antibody.

(c) Measurement of CRP Anti-human CRP antibody conjugated to 50 μl of sulfonated carboxylated polystyrene latex solution (5% by weight), 10 μl of test serum and glycine buffer (pH 8, 2, 0, 2%)
400 μl of bovine serum albumin (containing 0.9% sodium chloride) was added and mixed for 3 minutes at room temperature.

Next, alkaline phosphatase-labeled anti-human CRP antibody (diluted 100 times with the above buffer) 500
μl was added and mixed for 2 minutes at room temperature.

As an enzyme substrate coloring solution, 2mM concentration of 5-bromo-4-
Chloro-3-indolyl phosphate ρ-toluidine salt was added to jetanolamine buffer (pH 9,0,0, 1 mol/l)
Measurement was carried out in the same manner as in Example 1, except that a solution dissolved in .

Comparative Example 3 An anti-human CRP antibody was bound to the carboxylated polystyrene latex prepared in Comparative Example 2 in the same manner as in Comparative Example 2. The final dispersion medium is glycine buffer (pH 8, 2, 0
, 1 mol/l). The amount of antibody bound was 40 μ/g.

(d) Measurement of CRP CRP was measured in the same manner as in Example 2 using the above anti-human CRP antibody-bound carboxyl polystyrene latex.

The above results are shown in Tables 1 and 2. In Comparative Example 1, non-specific aggregation occurred due to the absence of the first filtration step,
In Comparative Examples 2 and 3, since the latex particles did not have sulfonic acid groups, the sensitivity decreased due to aggregation of the latex.

Claims (2)

[Claims] (1) Immunoactive substance solid-phase particles formed by covalently bonding an immunoactive substance to polymer particles having a particle diameter of 0.5 to 500 μm and having a sulfonic acid group and a carboxyl group on the surface through the carboxyl group. A first step of adding a test solution to an aqueous dispersion of the sample and causing a predetermined test substance in the test solution to specifically bind to the immunoactive substance, and a label that specifically binds to the test substance. a second step of specifically binding the substance to be tested to the test substance, and then retaining the thus obtained dispersion of solid-phase particles in an average size of at least twice the particle diameter of the solid-phase particles. a third step of filtering with a first filter medium having a particle size;
A fourth step of filtering with a second filter medium having an average retained particle size of 0.2 μm or more, and a fifth step of detecting the label bound to the solid-phase particles captured on the second filter medium. An immunoassay method characterized by: (2) Polymer particles have 0.3 to 3 carboxyl groups on the surface
The immunoassay method according to claim 1, characterized in that it has a concentration in the range of 0 μmol/m^2.