JP2000221196A - Immunological assay - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable accurate detection of a target component by using protein used for the blocking of an antibody reaction system or insoluble carrier particles, in which an alteration of the protein is fixed at the time of measuring antigens and antibodies in a sample by agglutinations due to antigen-antibody reactions. SOLUTION: Nonspecific agglutinations are brought about by various mechanisms in immunoreactions. For overcoming nonspecific reactions which occur to protein to be fixed to the surfaces of insoluble carrier particles as a blocking agent for insoluble carrier particles obtained by sensitizing antigens or antibodies, the same protein used for blocking as an absorbent of nonspecific reactions or its alteration is fixed to other insoluble carrier particles, and they are made coexistent at the time of immuno agglutinations. Physical adsorption, chemical combination, or the like is used as a method for fixing protein used as a blocking agent or its alteration to insoluble carrier particles without particular limitation, however physical adsorption is preferable in the simplicity of operation, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an immunoassay method for detecting and quantifying a trace substance in a living body.

[0002]

2. Description of the Related Art An immunoassay method for measuring a specific component in a specimen such as a biological sample utilizing an antigen-antibody reaction is widely used in the field of clinical examination. For example, one-way immunodiffusion (SRID), immunoturbidimetry (TIA), immunoassay (HA), latex agglutination (LA), enzyme immunoassay (EIA), radioimmunoassay (RIA), etc. And
They are used depending on the purpose of use in terms of operability, sensitivity, accuracy, and the like. Among these various immunoassays, a highly sensitive and simple method is to immobilize an antigen or antibody on insoluble carrier particles such as sheep erythrocytes or polymer latex and cause an agglutination reaction with the corresponding antibody or antigen in the sample. It has been known.

As a method for immobilizing an antigen or an antibody on an insoluble carrier, there are methods such as physical adsorption and chemical covalent bonding. However, in general, physical adsorption is simple and impairs antigen activity or antibody activity. Widely used because there is no. Since the hydrophobic interaction between the antigen or antibody to be adsorbed and the water-insoluble carrier plays a large role in physical adsorption, a polystyrene-based synthetic latex having a high surface hydrophobicity is used as the insoluble carrier particles. Polystyrene latex particles are more stable than other carriers because they are synthetic,
Many immunoreactive reagents, especially agglutination reactions, are excellent in that they can strongly adsorb and immobilize physiologically active substances such as proteins and lipids such as antigens and antibodies, and can stably retain the immobilized antigens or antibodies for a long period of time. It is commonly used as a carrier for reagents.

However, polystyrene-based synthetic latex is liable to cause spontaneous aggregation between latex particles during storage and aggregation due to reaction with a substance other than the antigen-antibody reaction to be measured, so-called non-specific aggregation reaction. In order to solve this problem, a blocking operation is performed by adding an immunologically inactive protein such as bovine serum albumin, casein, and gelatin to a suspension of latex particles to which an antigen or antibody has been sensitized and immobilized. Thus, a latex reagent for immunological reaction, which covers the hydrophobic surface of latex particles and prevents non-specific agglutination, has been proposed. (JP-A-58-144748). Furthermore, as a non-specific adsorption inhibitor, skim milk powder dissolved in water or Tris buffer (Gene Anal. Techno)
l. , 1, 3-8 (1984), Proc. Nat
l. Acad. Sci. , 82, 6741-6744 (1985), Cell Engineering, 5, 264-270 (19).
86)) and a non-specific adsorption inhibitor containing a blood protein or a plant protein as an active ingredient (JP-A-7-2704).
13) Various methods of blocking agents have been devised such as a method of coating particles with a rabbit blood component or a denatured product thereof (JP-A-5-322895). However, there are cases where the non-specific reaction cannot be sufficiently prevented even with these conventional blocking agents, for example, there is a case where the protein reacts with the blocked protein itself.

[0005]

The above-mentioned method of preventing the non-specific agglutination reaction with the blocking agent by adding the protein used for blocking at the time of the reaction to prevent the non-specific agglutination reaction is not sufficiently effective. When the protein is added to the concentration, not only the viscosity of the measurement solution increases and the operability becomes poor, but also the quantitativeness of the measurement may be affected. Therefore, an effective method for preventing non-specific reactions without affecting the operability and quantitativeness of measurement is desired. Therefore, an object of the present invention is to provide a reagent that accurately and precisely detects a target component in a sample sample and has good quantification by simply and effectively suppressing a non-specific reaction accompanying measurement by an immunoagglutination reaction. It is in.

[0006]

Means for Solving the Problems The present invention has been completed as a result of solving the above-mentioned conventional problems and studying a method for suppressing non-specific reactions without complicated operation. That is, the present invention provides (1) using an insoluble carrier particle in which an antigen or an antibody is immobilized and blocked with a protein,
When measuring an antigen or an antibody in a sample by an agglutination reaction by an antigen-antibody reaction, the antigen-antibody reaction system uses another insoluble carrier particle in which the same protein as the protein used for blocking or a denatured product thereof is immobilized. (2) the insoluble carrier particles having the same protein as the protein used for blocking or a denatured product thereof immobilized thereon have a particle size of 0.01
(3) two or more types of insoluble carrier particles having the same protein or a denatured product thereof immobilized thereon as the immunoassay method according to the above (1), wherein The immunoassay according to the above (1) or (2), comprising insoluble carrier particles of

(4) The immunoassay according to the above (1), (2) or (3), wherein the protein is a normal animal serum or an albumin fraction thereof, or a denatured product thereof.
(5) Using the insoluble carrier particles obtained by performing a heat treatment simultaneously with immobilizing the same protein as the protein used as the blocking agent on the insoluble carrier particles as described in (1) to (1) above.
(4) The immunoassay according to any of (1) to (6), wherein the insoluble carrier particles are polystyrene latex.
(7) the immunoassay method according to any of (5), (7) blocking before the antigen-antibody reaction between the insoluble carrier particles on which the antigen or antibody is immobilized and blocked with the protein and the antigen or antibody in the sample is performed. Treating the specimen with another insoluble carrier particle in which the same protein as the used protein or a denatured product thereof is immobilized,
(6) An immunoassay method according to any of (6).

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. The non-specific agglutination reaction in the immune reaction is caused by various mechanisms, but the present invention overcomes the non-specific reaction that occurs to the protein immobilized on the particle surface as a blocking agent for insoluble carrier particles sensitized with antigen or antibody. Therefore, as an absorbent for the non-specific reaction, the same protein or a denatured product thereof as the protein used for blocking is immobilized on another insoluble carrier particle, and is coexisted in the immunoagglutination reaction.

In the present invention, the method of immobilizing the protein used as the blocking agent or its denatured product on the insoluble carrier particles is not particularly limited, such as physical adsorption or chemical bonding. It is preferable from the viewpoint of properties and the like. The preparation can be carried out according to the same method as that for sensitizing a normal antigen or antibody. For example, a protein is dissolved in a buffer, and then the insoluble carrier is suspended in the solution and mixed by stirring for a certain period of time to allow the protein to be sufficiently adsorbed on the surface of the insoluble carrier, and then used as it is or in a buffer. It can be prepared by centrifugation and washing. The buffer used is not particularly limited, and examples thereof include a phosphate buffer, a glycine-sodium hydroxide buffer, a Tris-hydrochloride buffer, an ammonium chloride-ammonia buffer, and a Good's buffer. The concentration and pH can be used without any particular limitation.
A range of 4 to 10 is preferred. When the protein or its denatured product used as the blocking agent in the present invention is immobilized on insoluble carrier particles, the concentration of the protein or its denatured product in the buffer is usually 0.1% (W / V) to 1%.
0% (W / V), preferably 0.5% (W / V) to 5%
(W / V) is used. The concentration of the insoluble carrier particles in the buffer at that time is usually 0.1% (W / V) to 10 (W / V).
/ V), preferably 0.5% (W / V) to 5% (W / V).
V) is used.

When a denatured protein used as a blocking agent in the present invention is used, the denaturation may be performed by denaturation with heat, denaturation with pH, or an oxidizing agent.
Modification by a chemical denaturing agent such as a reducing agent, a cross-linking agent, or a chemical modification reactant is possible, but denaturation by heat is simple, effective and preferable. The method of applying heat depends on the protein used, but generally heating at 35 to 70 ° C. for about 10 to 60 minutes is sufficient. At this time, an optimum one may be selected depending on the type, pH and concentration of the buffer for dissolving the protein. Further, the present invention includes denaturation of the protein by applying heat after immobilizing the protein on the insoluble carrier particles, and simultaneous denaturation and immobilization of the protein by immobilization by heating. .

As the protein to be immobilized, the same protein as that used for the blocking agent or a denatured product thereof is used. As the protein, for example, normal animal serum or its albumin fraction, casein, gelatin and the like are preferable. Bovine serum albumin as the albumin fraction,
Porcine serum albumin, goat serum albumin, rabbit serum albumin and the like can be used.

The insoluble carrier particles are particles substantially insoluble in a liquid medium used for immobilization, storage and measurement, and are generally particles insoluble in a buffer solution, that is, water, and have an average particle diameter of Is preferably 0.01 μm or more and 1 μm or less.

As these particles, various fine particles used for an antigen-antibody reaction are not particularly limited and can be used. For example, it can be obtained by an emulsion polymerization method such as polystyrene, styrene-divinylbenzene copolymer, styrene-butadiene copolymer, styrene-methacrylic acid copolymer, styrene-glycidyl methacrylate copolymer, polyglycidyl methacrylate, and polyacrolein. Organic polymer particles such as organic polymer latex, or inorganic oxide particles such as silica, silica-alumina and alumina, or magnetic particles such as various iron oxides, or silane coupling treatment to these inorganic oxides There are inorganic fine particles with functional groups introduced by operation, and composite fine particles in which inorganic fine particles are coated with an organic polymer.
Biologically-derived particles include biologically-derived particles such as human O-type red blood cells, sheep red blood cells, and chicken red blood cells.

Particularly preferred insoluble carrier particles include polystyrene latex particles. Further, it may be a copolymer of styrene and a copolymerizable monomer, and may form a cross-linking bond. The measuring method by the immunoagglutination reaction used in the present invention is not particularly limited as long as it is an agglutination reaction, and any known method can be used as long as the degree of agglutination can be determined. For example, there is a method of visual judgment called a glass plate method, a method of viewing a sedimentation image of a microplate called a microtiter method, a method of optically measuring the change in aggregation, or a method of electrically measuring the degree of aggregation. is there. A method of optically measuring is preferable, and a method of optically measuring a change in turbidity, absorbance, or scattered light is more preferable.

The method of adding the insoluble carrier particles, on which the protein or its denatured product used in the blocking agent according to the present invention is immobilized, to the immunoassay system is not particularly limited. Effectively, the sample is preferably reacted with the protein-immobilized insoluble carrier particles of the present invention prior to the immunoagglutination reaction as a pretreatment reaction. Specifically, it is preferably added to a sample diluent (pretreatment liquid). The concentration of the additive varies depending on the type of the specimen, but is usually 0.005 to 0.2% by weight, particularly preferably 0.01 to 0.1% by weight, as a solid content of the protein-immobilized insoluble carrier during the immunoagglutination reaction. 0.00
If the amount is less than 5% by weight, the effect of preventing non-specific reaction is difficult to be obtained. Accuracy may be degraded. Further, the optimum amount of addition varies depending on the particle size of the protein-immobilized insoluble carrier particles to be added. That is, the larger the particle size, the smaller the optimal addition amount, and the smaller the particle size, the larger the addition amount.

The protein-immobilized insoluble carrier particles according to the present invention have different nonspecific prevention effects depending on the particle size. That is, as the nonspecific prevention effect, insoluble carrier particles having a larger particle size, preferably those having the same particle size as the particles used for the sensitized particles on which the antigen or antibody is immobilized are preferably used. However, insoluble carrier particles having a particle size that is too large increase the turbidity of the reagent itself, which may reduce accuracy when detecting an increase in optical characteristics due to aggregation, and is currently used in an automated analyzer. May cause a decrease in measurement accuracy.

The particle size of the insoluble carrier particles on which the protein or its denatured product used in the blocking agent of the present invention is immobilized is usually 1 to the particle size of the sensitized insoluble carrier particles on which the antigen or antibody used for the agglutination reaction is immobilized. / 1-1 / 5
0, preferably 1/2 to 1/20. Furthermore,
By using two or more types of protein-immobilized insoluble carrier particles having different particle diameters, it is possible to enhance the effect of preventing nonspecific reaction without increasing turbidity of the reagent itself. That is, when two types of protein-immobilized insoluble carrier particles having different particle diameters are used, the particle size of the sensitized insoluble carrier particles immobilized with the antigen or antibody used for the agglutination reaction is 1%.
Particles having a particle diameter of 1/1 to 1/5 and particles having a particle diameter of 1/5 to 1/20 (except when both particles have the same particle diameter) are 2: 1 to 1:10, preferably 1: The protein used as a blocking agent for the sensitized insoluble carrier particles or a denatured product thereof is used as immobilized insoluble carrier particles at a ratio of 2-1: 5. Specifically, as an example according to the present invention, an antibody was sensitized to polystyrene latex having an average particle size of 0.5 μm, and blocked with 1% bovine serum albumin to obtain agglutinated particles. On the other hand, the average particle size of 0.2 μm and 0.0
A pretreatment reaction solution is obtained by immobilizing 1% bovine serum albumin on 5 μm polystyrene latex in the same manner as the antibody-sensitized particles.

The antigen or antibody immobilized on the insoluble carrier particles in the present invention is not particularly limited, and any known antigen or antibody can be used. To show a typical example,
For example, alpha fetoprotein (AFP), anti-AF
P antibody, oncofetal protein (CEA), anti-CEA antibody, hepatitis B surface antigen (HBs), anti-HBs antibody, human reactive protein (CRP), anti-CRP antibody, streptolysin O, anti-streptolysin O antibody, Albumin, anti-albumin antibody, immunoglobulin (Ig) G, anti-IgG antibody, Ig
A, anti-IgA antibody, IgM, anti-IgM antibody, complement third component (C3), anti-C3 antibody, C4, anti-C4 antibody, denatured gamma globulin, rheumatoid factor, human placental lactogen (h
PL), anti-hPL antibody, human chorionic gonadotropin (h
Known antigens or antibodies such as CG), anti-hCG antibody, insulin, anti-insulin antibody, treponema pallidum antigen, rubella antigen, anti-rotavirus antibody and rotavirus antigen.

Samples of the present invention include serum, plasma, urine,
Body fluid such as cerebrospinal fluid can be used, but is not limited thereto. Examples of the insoluble carrier particles used for the sensitized insoluble carrier particles in the present invention include the insoluble carrier particles used for the protein-immobilized insoluble carrier particles, and may be the same as those used for the protein-immobilized insoluble carrier particles. Alternatively, different ones may be used. The immobilization and blocking of the antigen or antibody on the insoluble carrier particles can be performed by a known method.

[0020]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

(1) Preparation of non-specific absorption latex A 9 mL of 0.05 M Tris-HCl buffer (pH 7.4)
Was dissolved 0.09 g of bovine serum albumin (BSA) in
A protein solution was prepared. 10% (W / V) polystyrene latex suspension (average particle size: 0.12 μm)
mL was added and stirred at room temperature for 1.5 hours. afterwards,
Centrifuge at 15,000 rpm for 1 hour at 10 ° C.
The obtained precipitate was washed with a buffer containing 1% BSA containing 0.02% nonionic surfactant, and diluted with the liquid to finally give a latex concentration of 1%.

(2) Preparation of non-specific absorption latex B 9 mL of 0.01 M Tris-HCl buffer (pH 7.4)
Was dissolved in 0.09 g of BSA to prepare a protein solution.
To this, 1 mL of a 10% (W / V) polystyrene latex suspension (average particle size: 0.03 μm) was added, and stirred at room temperature for 1.5 hours to prepare.

(3) Preparation of nonspecific absorption latex C (thermal denaturation) 9 mL of 0.05 M Tris-HCl buffer (pH 7.4)
Was dissolved in 0.09 g of BSA to prepare a protein solution.
1 mL of a 10% (W / V) polystyrene latex suspension (average particle size: 0.12 μm) was added thereto, and the mixture was stirred at 60 ° C. for 1.5 hours. Then, at 10 ° C, 15,000
After centrifugation at 1 rpm for 1 hour, the resulting precipitate was centrifuged at 0.
The plate was washed with a buffer containing BSA containing 02% nonionic surfactant, and then diluted with the buffer to a final latex concentration of 1%.

(4) Preparation of nonspecific absorption latex D (reduction treatment) 10 mM diethanolamine buffer (pH 11) 10 m
BSA was dissolved in 1 to 1% by weight. continue,
Add 15.6 μL of 2-mercaptoethanol and add 3
The reaction is carried out at 7 ° C. for 2 hours with stirring. Thereafter, 25 mg of N-ethylmaleimide powder is added to the reaction solution, and the mixture is stirred at room temperature for 30 minutes. Upon completion of the reaction, the resulting reduced denatured bovine serum albumin was dialyzed overnight against 0.05 M Tris-HCl buffer (pH 7.4),
Buffer replacement was performed. The concentration of reduced denatured bovine serum albumin is 1% by weight as BSA. afterwards,
To 9 mL of this 1% reduced denatured bovine serum albumin, add 10%
(W / V) polystyrene latex suspension (average particle size;
0.12 μm), and stirred at room temperature for 1.5 hours. Thereafter, centrifugation was performed at 15,000 rpm for 1 hour at 10 ° C., and the obtained precipitate was washed with a buffer containing BSA containing 0.02% nonionic surfactant, and finally the latex concentration was 1%. The solution was diluted so that

Example 1 Measurement of anti-Treponemal anti-Syphilis antibody using non-specific absorption latex (1) Preparation of latex sensitized to T. pallidum antigen sensitized to polystyrene latex particles having an average particle size of 0.4 μm
The crushed syphilis cells were sensitized and reacted for 1 hour. Next, 0.1 M Tris-HCl buffer (pH 7.4) containing 1% bovine serum albumin was added, and the mixture was reacted for 1.5 hours to perform blocking. Thereafter, washing and replacement were performed with a BSA-containing buffer solution containing a nonionic surfactant to prepare a sensitized latex suspension such that the solid content of the latex was 0.1 (W / V)% (R-2). (2) Preparation of sample diluting buffer As sample diluting solution, 0.1 M Tris-HCl buffer (p
H7.4) with 3% by weight dextran (average molecular weight 25
10,000) 1% by weight BSA, 1.8% by weight of NaCl and non-specific absorption latex A as a solid content of 0.03 (W / V)%
(R-1). (3) Reagent for measurement of anti-Treponemal antibody against T. pallidum The reagent for measurement of anti-Treponemal anti-Syphilis is R-2 composed of the latex sensitized to T. pallidum antigen of the above (1) and R-1 composed of the buffer for serum dilution of the above (2) And a two-reagent reagent. The measurement parameters when using the 7150-type automatic analyzer (manufactured by Hitachi, Ltd.) are shown below.

Sample 15 μL Sample dilution buffer (R-1) 250 μL Latex suspension (R-2) 80 μL Measurement wavelength 750 nm Photometry point 2-point end (35
In the 7150-type automatic analyzer, the sample diluting buffer (R-1) is added immediately after dispensing the sample, and the sample is diluted and mixed. Five minutes later, the latex suspension (R-2) was added, and the turbidity change from 35 points to 50 points was determined, and this was defined as the reaction amount. Among the reagents used this time, those showing a turbidity change larger than the 30 U / mL standard solution are judged to be positive.

Example 2 In the preparation of the sample diluting buffer of Example 1 (2), the non-specific absorption latex B was used as a solid content of 0.1 (W / V).
%, Except that (R-1) was used in the same manner as in Example 1.

Example 3 In the preparation of the buffer for diluting a sample in Example 1 (2), the non-specific absorption latex C was used in an amount of 0.03 (W /
V) The same procedure as in Example 1 was carried out except that (R-1) added so as to give%.

Example 4 In the preparation of the sample diluting buffer of Example 1 (2), the non-specific absorption latex D was used as a solid content of 0.03 (W / W
V) The same procedure as in Example 1 was carried out except that (R-1) added so as to give%.

Example 5 In the preparation of the buffer for diluting a sample in Example 1 (2), the non-specific absorption latex A was treated as 0.03 (W / W
V)%, and (R-1) to which non-specific absorption latex B was added so as to have a solid content of 0.1 (W / V)%, was carried out in the same manner as in Example 1.

Comparative Example 1 The procedure of Example 1 was repeated, except that (R-1) was used in the preparation of the buffer for diluting the specimen, except that the non-specific absorption latex was removed.

Results The turbidity change amounts of the samples obtained in Examples 1 to 5 and Comparative Example 1 are summarized below. Each of them shows an average value of n = 2.

Turbidity Change (ΔmAbs / 10) Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Healthy Sample 12 18 13 24 8 32 30 U / ml 256 283 225 204 223 287 Non-Specific Sample 1 89 228 35 59 13 546 Non-specific sample 2 198 98 150 145 24 1058 Non-specific sample 3 11 32 38 41 9 357 Non-specific sample 4 7 124 4 23 3 850 Non-specific sample 5 120 437 95 105 88 3090 In the measurement reagents to which the non-specific absorption latex was added (Examples 1 to 5), all non-specific reactions could be suppressed for non-specific samples. On the other hand, the non-added measurement reagent (Comparative Example 1) could not suppress the nonspecific reaction of the syphilis negative specimen.

Example 6 Measurement of human alpha-fetoprotein (AFP) using non-specific absorption latex (1) Preparation of anti-AFP antibody-sensitized latex Polystyrene latex particles having an average particle size of 0.6 μm
750 μL of an anti-human AFP antibody (goat) IgG fraction 1 mg / mL product was sensitized and reacted for 1 hour. Next, 0.1 M Tris-HCl buffer (pH 7.4) containing 1% bovine serum albumin was added, and the mixture was reacted for 1.5 hours to perform blocking. Thereafter, washing and replacement were performed with a BSA-containing buffer solution containing a nonionic surfactant to prepare a sensitized latex suspension such that the solid content of the latex was 0.1 (W / V)% (R-2). (2) Preparation of sample diluting buffer As sample diluting solution, 0.1 M Tris-HCl buffer (p
H7.4) with 3% by weight dextran (average molecular weight 25
10,000) 1% by weight of BSA, 0.5% by weight of NaCl and non-specific absorption latex A as solid content of 0.03 (W / V)%
(R-1). (3) AFP measurement reagent The AFP measurement reagent is composed of R-2 consisting of the anti-human AFP antibody-sensitized latex of the above (1) and R-1 consisting of the serum dilution buffer of the above (2). This is a two-reagent system reagent. The measurement parameters when using a Kobasmira automatic analyzer (manufactured by Roche Diagnostics Co., Ltd.) are shown below.

Sample 20 μL Sample dilution buffer (R-1) 250 μL Latex suspension (R-2) 100 μL Measurement wavelength 750 nm Photometry point 2-point end (15
In the automatic analyzer, the sample diluting buffer (R-1) is added immediately after dispensing the sample, and the sample is diluted and mixed. Five minutes later, the latex suspension (R-2) was added and 1
Find the turbidity change from 5 points to 50 points,
This was defined as the reaction amount. 20 ng /
Those showing a turbidity change larger than the mL standard solution are judged as positive.

Example 7 In the preparation of the sample diluting buffer of Example 6 (2), the non-specific absorption latex B was used as a solid content of 0.1 (W / V).
%, Except that (R-1) was used in the same manner as in Example 6.

Example 8 In the preparation of the buffer for sample dilution in Example 6 (2), the non-specific absorption latex C was treated as 0.03 (W / W
V) The same procedure as in Example 6 was carried out except that (R-1) was used so as to be%.

Example 9 In the preparation of the buffer for diluting a sample in Example 6 (2), the non-specific absorption latex D was treated as 0.03 (W /
V) The same procedure as in Example 6 was carried out except that (R-1) was used so as to be%.

Example 10 In the preparation of the buffer for diluting a sample in Example 6 (2), the non-specific absorption latex A was treated as 0.03 (W / W
V)%, and (R-1) to which 0.1% (W / V)% of the non-specific absorption latex B was added as a solid content was used.

Comparative Example 2 In the preparation of the buffer for diluting a sample in Example 6 (2), (R-1) from which non-specific absorption latex was removed was used.
The same was done.

Results The turbidity change amounts of the samples obtained in Examples 6 to 10 and Comparative Example 2 are summarized below. Each of them shows an average value of n = 2.

Turbidity Change (ΔmAbs / 10) Example 6 Example 7 Example 8 Example 9 Example 10 Comparative Example 2 Healthy Sample 6 23 19 24 16 38 20 ng / ml 400 410 295 259 288 440 Non-specific Sample 1 77 105 42 21 5 746 Non-specific sample 2 5 99 1 55 8 798 Non-specific sample 3 128 348 100 120 90 90 2800 As described above, in the measurement reagent to which the non-specific absorption latex is added (Examples 6 to 10) Non-specific reactions were all suppressed for non-specific samples. On the other hand, the non-added measurement reagent (Comparative Example 2) could not suppress the non-specific reaction of the AFP negative sample.

[0043]

According to the present invention, non-specific reactions can be efficiently prevented in the measurement of immunoagglutination, and the accuracy of the immunoagglutination reaction based on the antigen-antibody reaction can be improved without changing the simplicity, speed, and high sensitivity. The measurement can be performed well, and the diagnosis accuracy of the disease can be improved.

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Claims (7)

[Claims] 1. An antigen or antibody is immobilized, and when the antigen or antibody in a sample is measured by an agglutination reaction by an antigen-antibody reaction using insoluble carrier particles blocked with a protein, the antigen-antibody reaction system is blocked. An immunoassay method, comprising using another insoluble carrier particle on which the same protein or a denatured product thereof is immobilized as the protein used in (1). 2. The immunoassay according to claim 1, wherein the particle size of the insoluble carrier particles immobilized with the same protein as the protein used for blocking or a denatured product thereof is 0.01 μm to 0.5 μm. 3. The immunoassay according to claim 1, wherein the insoluble carrier particles on which the same protein as the protein used for blocking or a denatured product thereof is immobilized comprise two or more types of insoluble carrier particles having different particle diameters. . 4. The immunoassay according to claim 1, wherein the protein is serum of a normal animal, an albumin fraction thereof, or a denatured product thereof. 5. The method according to claim 1, wherein the same protein as the protein used as the blocking agent is immobilized on the insoluble carrier particles, and the heat treatment is carried out at the same time.
5. The immunoassay according to any one of 4. 6. The method according to claim 1, wherein the insoluble carrier particles are polystyrene latex. 7. The same protein as the protein used for the blocking or a denatured product thereof before immobilizing the antigen or antibody and performing an antigen-antibody reaction between the insoluble carrier particles blocked with the protein and the antigen or antibody in the sample. The immunoassay according to any one of claims 1 to 6, wherein the specimen is treated with another immobilized insoluble carrier particle.