JP2018173334A - Method for immunological measurement and reagent kit used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for an immunological measurement and a reagent kit used for the method.SOLUTION: There is provided a carrier aggregation immunological measuring method for measuring the antigen in a sample, which includes a first step of mixing the sample with a first reagent and a second step of mixing the mixed solution obtained in the first step with a second reagent. The first reagent includes a first particle, which collects one of the epitopes of the antigen, and the second reagent includes a second particle, which is a particle that collects at least two of the epitopes of the antigen or is the mixture of two or more types of particles which each collect one of the epitopes of the antigen, the epitope collected by the first particle being different from those collected by the second particle.SELECTED DRAWING: None

Description

  The present invention relates to an immunological measurement method and a reagent kit used therefor, and in particular, has high reactivity to an antigen in a carrier agglutination immunoassay, thereby enabling highly sensitive measurement. The present invention relates to an immunological measurement method and a reagent kit used therefor.

Among the carrier agglutination immunoassays, the latex agglutination immunoassay is generalized by applying a biochemical automatic analyzer and is widely used for measurement of many clinical laboratory items.
Here, in the standard latex agglutination immunoassay method, a first reagent (R1) mainly composed of a buffer solution is added to a reaction cell, a test sample (S) is added thereto, mixed, and allowed to stand for a certain time. Next, the absorbance (turbidity) in the process of adding and mixing the second reagent (R2) mainly composed of latex particles carrying an antibody that recognizes the target antigen molecule, and mixing the mixture for a certain period of time. ) The amount of change is measured optically.

The latex agglutination immunoassay method using the biochemical automatic analyzer as described above can measure a large amount of specimens in a short time, and is therefore frequently used in central laboratories of large hospitals such as university hospitals and contract clinical laboratories. high.
However, since the detection sensitivity of latex agglutination immunoassay is about 1.0 ng / mL for general protein antigens, there is a limit to the measurement of samples with a more dilute antigen concentration. For measurement of items that cannot be covered by the method, it is necessary to use dedicated equipment and reagents such as chemiluminescent enzyme immunoassay (CLIA).

  Here, the configuration of an immunoassay reagent kit for latex agglutination immunoassay for a biochemical automatic analyzer is usually composed of a first reagent (R1) and a second reagent (R2). The main component of is a buffer solution, and the main component of the second reagent is latex particles carrying an antibody that recognizes the measurement object (antigen molecule). The main role of the first reagent is to correct the difference between samples (including neutralization of non-specific reactions) and to homogenize the reaction, and to enhance detection sensitivity with a sensitizer. On the other hand, the role of the second reagent is to aggregate latex particles according to the number (concentration) of antigen molecules present in the test sample by reacting the antibody immobilized on the latex particles with the measurement target (antigen molecule). It is to form a lump and induce a concentration-dependent change in absorbance (turbidity).

Until now, several proposals have been made to increase the sensitivity of a latex agglutination immunoassay using an automatic biochemical analyzer. For example, Japanese Patent Application Laid-Open No. 2001-091516 (Patent Document 1) removes the Fc portion of human α-fetoprotein antibody as a highly sensitive and rapid quantification method of human α-fetoprotein (AFP), and F (ab ′) _2. A method for quantitative determination of AFP using latex particles carrying only antibodies is proposed, and Japanese Patent Application Laid-Open No. 2003-294653 (Patent Document 2) realizes high measurement sensitivity and quantitative performance, particularly in latex agglutination method. As an immunoassay that can be performed, an immunoassay using an agglutination sensitizer containing at least one kind of alginic acid has been proposed.

JP 2001-091516 A JP 2003-294753 A

However, the methods described in Patent Documents 1 and 2 unfortunately cannot sufficiently achieve the purpose of increasing the sensitivity of the latex agglutination immunoassay method to which the biochemical automatic analyzer is applied.
Accordingly, an object of the present invention is to provide an immunological measurement method and a reagent kit used therefor that have high reactivity to an antigen and thereby enable highly sensitive measurement in a carrier agglutination immunoassay. .

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that in a carrier agglutination immunoassay method in which a first reagent and a second reagent containing antibody-carrying particles are sequentially added to and mixed with a specimen, an antigen is used as the first reagent. When particles that capture one of the epitopes possessed by the antibody are added, when the second reagent is added and mixed, the antibody-carrying particles contained in the second reagent exhibit high reactivity with the antigen, and the antigen in the specimen As a result, it was found that the antigen can be measured with high sensitivity, and the present invention has been completed.

That is, the present invention
[1] A method for measuring an antigen in a sample, comprising: a first step of mixing a sample with a first reagent; and a second step of mixing the mixed solution obtained in the first step with a second reagent A carrier agglutination immunoassay method comprising:
The first reagent includes a first particle that captures one of the epitopes of the antigen, the second reagent includes a second particle, and the second particle includes two or more of the epitopes of the antigen. Or a mixture of two or more particles that capture one of the epitopes of the antigen, wherein each particle is a particle that captures a different epitope of the antigen. Measuring method,
[2] The measurement method according to [1], wherein the first particle is a particle carrying a polyclonal antibody having affinity for one of the epitopes of the antigen.
[3] The measurement method according to [1], wherein the first particle is a particle carrying a monoclonal antibody having affinity for one of the epitopes of the antigen.
[4] The measurement method according to any one of [1] to [3], wherein the second particle is a particle carrying a polyclonal antibody having affinity for two or more epitopes of the antigen. ,
[5] The second particle is a particle carrying two or more kinds of monoclonal antibodies having affinity for one of the epitopes of the antigen, and various monoclonal antibodies have affinity for different epitopes of the antigen. The measurement method according to any one of [1] to [3], wherein the antibody has an antibody.
[6] The second particle is a mixture of two or more particles carrying a monoclonal antibody having affinity for one of the epitopes of the antigen, and each particle has an affinity for a different epitope of the antigen. The measurement method according to any one of [1] to [3] above, which is a particle carrying a monoclonal antibody having properties.
[7] An immunoassay reagent kit comprising a first reagent and a second reagent for use in a carrier agglutination immunoassay method for measuring an antigen in a specimen,
The first reagent includes the first particle according to [2] or [3], and the second reagent is the second particle according to any one of [4] to [6]. A reagent kit for immunoassay, comprising:
[8] The present invention relates to the reagent kit for immunoassay according to [7], further including a third reagent that does not include particles carrying an antibody.

The present invention provides an immunological measurement method that has a high reactivity to an antigen in a carrier agglutination immunoassay, thereby enabling highly sensitive measurement.
The present invention also provides a reagent kit for use in the above highly sensitive immunological measurement method.
By adding a particle that captures one of the epitopes of the antigen to the first reagent, the antibody-carrying particles contained in the second reagent show high reactivity with the antigen, and the concentration of the antigen in the sample is dilute. However, the reason for aggregation is not always clear, but the particles contained in the first reagent capture one of the epitopes of the antigen, so that the other epitope of the antigen contains the surface of the particles contained in the first reagent. The antibody-carrying particles contained in the second reagent can efficiently capture the presented epitope, and as a result, even if the concentration of the antigen in the sample is dilute, it is high with the antigen. It is thought that it showed reactivity.

The present invention will be described in more detail.
The present invention includes a first step of mixing a sample with a first reagent, and a second step of mixing the mixed solution obtained in the first step with a second reagent, for measuring an antigen in the sample. A method for measuring carrier agglutination immunity of
The first reagent includes a first particle that captures one of the epitopes of the antigen, the second reagent includes a second particle, and the second particle includes two or more of the epitopes of the antigen. Or a mixture of two or more particles that capture one of the epitopes of the antigen, wherein each particle is a particle that captures a different epitope of the antigen. It relates to a measuring method.
In addition, although the epitope in this invention intends the antigenic determinant of an antigen (antigen molecule), regarding the small molecule in which an antigenic determinant is not clear, the amino acid sequence of one part of the amino acid sequence which this molecule has is intended.

  The antigen (antigen molecule) that can be contained in the specimen is not particularly limited, and generally includes all the biologically active substances and antigens of pathogens (viruses, bacteria, etc.) that can be measured using an antigen-antibody reaction. Specifically, examples of the physiologically active substance include various in vivo receptors, enzymes, blood proteins and the like existing in the living body. Specific examples of the physiologically active substance include myoglobin, D-dimer, and α-fetoprotein. Specific pathogen antigens include, for example, filaria antigen, hepatitis B virus antigen, hepatitis C virus antigen, syphilis cell-derived antigen, HBS antigen, HCV antigen, HIV antigen, ATLA antigen, chlamydia antigen, herpes antigen, Examples include Helicobacter pylori antigen.

The 1st reagent which can be used for this invention contains the 1st particle | grains which capture | acquire one of the epitope which said antigen has.
Since the first particle captures only one of the epitopes possessed by the antigen, aggregation of the particles is not induced even when the epitope is captured by mixing with the antigen.

The first particle may be a particle carrying a polyclonal antibody having affinity for one of the epitopes of the antigen.
The polyclonal antibody as described above can be obtained, for example, by designing and synthesizing a peptide antigen corresponding to a specific epitope from the result of epitope mapping analysis, and preparing a polyclonal antibody using the peptide antigen as an immunogen.
Further, the first particle may be a particle carrying a monoclonal antibody having an affinity for one of the epitopes possessed by the antigen.

The particles for supporting the antibody that can be used for the first particles are not particularly limited as long as they can support the antibody, and examples thereof include organic polymer powders, inorganic substance powders, microorganisms, blood cells, and cell membrane fragments. It is done. Examples of the organic polymer powder include insoluble agarose, cellulose, and insoluble dextran, and a latex suspension is preferably used. Examples of latex particles used in the latex suspension include polystyrene, seletin-styrene sulfonate polymer, acrylonitrile-butadiene-styrene copolymer, vinyl chloride-acrylate copolymer, and polyvinyl acetate acrylate. . The average particle diameter of the latex particles used is appropriately selected within the range of 0.05 to 1.0 μm. Examples of the inorganic substance carrier include silica, alumina, carbon powder, or metal pieces such as gold, titanium, iron, and nickel.
The particles for supporting the antibody that can be used for the first particles are preferably latex particles, and polystyrene latex particles are particularly preferable.

Monoclonal antibodies and polyclonal antibodies that can be used for the first particles can be obtained by methods known in the art, and commercially available antibodies can also be used.
In addition, the method of supporting the antibody on the particles can also be performed by a method known in the art, and examples thereof include a physical adsorption method and a chemical bonding method.
The amount of the first particles contained in the first reagent is usually 0.001 to 0.1% (w / v), preferably 0.003 to 0, as the mass with respect to the total volume of the first reagent. 0.07% (w / v).
In principle, the first reagent does not include particles carrying antibodies other than the first particles.

The first reagent can include an aqueous medium in addition to the first particles.
The aqueous medium is not particularly limited, and examples thereof include phosphate buffer, glycine buffer, Tris-HCl buffer, Good buffer, and the like. The pH of the medium is preferably 5.5 to 8.5.

The first reagent may further dissolve bovine serum albumin, sucrose, sodium chloride or the like for adjusting the salt concentration.
In addition, a sensitizer can be added to the first reagent in order to improve measurement sensitivity and promote antigen-antibody reaction. Specific examples of the sensitizer include polyethylene glycol, methyl cellulose, ethyl cellulose, pullulan, polyvinyl pyrrolidone, and alginic acid.

The second reagent includes second particles.
The second particles that can be used in the present invention are not particularly limited as long as they are particles that cause aggregation of the particles when mixed with an antigen, and may be particles carrying the same antibody or different. It may be a mixture of two or more particles carrying an antibody.
That is, the second particle only needs to capture two or more epitopes of the antigen as a whole, and when capturing the epitope by mixing with the antigen by capturing two or more epitopes of the antigen. In addition, aggregation of particles is caused.
The second reagent is, as the second particle, a particle that captures two or more epitopes possessed by an antigen, or a mixture of two or more particles that capture one epitope possessed by the antigen, Various types of particles include particles that capture different epitopes of the antigen.
Here, at least one of the two or more epitopes of the antigen captured by the second particle is an epitope different from the epitope of the antigen captured by the first particle.
That is, one of the two or more epitopes of the antigen captured by the second particle may be the same as the epitope of the antigen captured by the first particle. Further, all of the two or more epitopes of the antigen captured by the second particle may be different from the epitope of the antigen captured by the first particle.

The second particle may be a particle carrying a polyclonal antibody having affinity for two or more epitopes of the antigen.
The particles carrying the polyclonal antibody can be particles carrying the same polyclonal antibody, but can also be a mixture of particles carrying different polyclonal antibodies.
The second particles may be particles carrying two or more monoclonal antibodies having affinity for one of the epitopes of the antigen. In this case, the various monoclonal antibodies have affinity for different epitopes of the antigen. Antibody.
The particles carrying two or more of the monoclonal antibodies can be particles carrying the same two or more monoclonal antibodies, but can also be a mixture of particles carrying two or more different monoclonal antibodies.
In addition, the second particle may be a mixture of two or more kinds of particles carrying a monoclonal antibody having affinity for one of the epitopes of the antigen. In this case, the various particles have affinity for different epitopes of the antigen. It becomes the particle | grains which carry | supported the monoclonal antibody which has.
In addition, the particles contained in the second reagent are only required to cause aggregation of the particles by mixing with the antigen. Therefore, the second reagent includes particles carrying other antibodies in addition to the second particles. obtain.
For example, the second reagent is
A mixture of the above-described polyclonal antibody-supported particles with the above-mentioned two or more types of monoclonal antibody-supported particles;
A mixture of the above-described polyclonal antibody-supported particles and the above-mentioned one type of monoclonal antibody-supported particles,
A mixture of particles carrying two or more types of monoclonal antibodies described above and particles carrying one type of monoclonal antibody described above;
A mixture of the above-described polyclonal antibody-supported particles with the above-mentioned two or more types of monoclonal antibody-supported particles and the above-mentioned one type of monoclonal antibody-supported particles;
Can be included.

As the particles for supporting the antibody that can be used for the second particles, the same particles as those used for the first particles can be used.
The particles for supporting the antibody used for the first particles and the particles for supporting the antibody used for the second particles may be the same, but may be different.
The particles for supporting the antibody that can be used for the second particles are preferably latex particles, and polystyrene latex particles are particularly preferable.

Monoclonal antibodies and polyclonal antibodies that can be used for the second particles can be performed by methods known in the art, and commercially available antibodies can also be used.
In addition, the method of supporting the antibody on the particles can also be performed by a method known in the art, and examples thereof include a physical adsorption method and a chemical bonding method.
The amount of the second particles contained in the second reagent is usually 0.01 to 0.15% (w / v) as a mass relative to the total volume of the second reagent, preferably 0.05 to 0. .1% (w / v).

The second reagent can include an aqueous medium in addition to the second particles.
The aqueous medium is not particularly limited, and examples thereof include phosphate buffer, glycine buffer, Tris-HCl buffer, Good buffer, and the like. The pH of the medium is preferably 5.5 to 8.5.

  The second reagent may further dissolve bovine serum albumin, sucrose, sodium chloride and the like as appropriate for adjusting the salt concentration.

The carrier agglutination immunoassay method of the present invention includes a first step of mixing a specimen with a first reagent, and a second step of mixing the mixed solution obtained in the first step with a second reagent.
The first step of mixing the sample with the first reagent is performed by adding the first reagent to the sample and mixing, but it is preferably performed at a constant temperature, preferably at 25 to 40 ° C., and 30 to 38. ℃
More preferably, The mixing is performed for 5 seconds to 30 minutes, more preferably for 5 seconds to 15 minutes. The second step of mixing the liquid mixture obtained in the first step subsequent to this with the second reagent is performed by adding the second reagent to the obtained liquid mixture and mixing, but it is performed at a constant temperature. Preferably, it is carried out at 25 to 40 ° C, more preferably 30 to 38 ° C. The mixing is performed for 5 seconds to 30 minutes, more preferably for 5 seconds to 15 minutes.

The antigen in the specimen is measured by measuring the amount of change in absorbance of the mixture obtained by performing the first step and the second step. The absorbance change amount is an increase in absorbance with the progress of particle aggregation.
The measurement wavelength at the time of measuring the amount of change in absorbance is usually 340 to 1000 nm, preferably an appropriate wavelength selected from the range of 500 to 800 nm. The measuring device used for measuring the amount of change in absorbance is not particularly limited as long as it can measure the absorbance of the solution over time, and examples thereof include a general-purpose biochemical automatic analyzer. The measurement wavelength, sample amount, reagent amount and the like can be appropriately selected according to the measurement apparatus.

The present invention also relates to an immunoassay reagent kit containing a first reagent and a second reagent for use in a carrier agglutination immunoassay method for measuring an antigen in a specimen.
As the first reagent that can be used in the immunoassay reagent kit, the above-mentioned first reagent can be used, and the above-mentioned second reagent can be used as the second reagent. And includes the second particles described above.

The present invention further relates to a reagent kit for immunoassay comprising a third reagent that does not contain particles carrying antibodies.
When using the above-described immunoassay reagent kit containing the three types of reagents, for example, the first step of mixing the specimen with the third reagent, and the first step of mixing the mixed solution obtained in the first step with the first reagent And a third step of mixing the liquid mixture obtained in the second step and the second step with the second reagent.
The third reagent is a reagent mainly composed of a buffer solution, such as a phosphate buffer solution, a glycine buffer solution, a Tris-HCl buffer solution, a Good buffer solution, bovine serum albumin, sucrose, sodium chloride, a sensitizer and the like. Can be included.
Examples of the sensitizer include polyethylene glycol, methyl cellulose, ethyl cellulose, pullulan, polyvinyl pyrrolidone, and alginic acid.
When employing an immunoassay reagent kit containing the above three types of reagents, the first reagent preferably does not contain a sensitizer from the viewpoint of storage stability.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.
Preparation Example 1: Preparation of suspension of anti-human myoglobin / monoclonal antibody-carrying particles (A1) Polystyrene particles having an average particle size of 220 nm were diluted with 0.1 molar glycine saline buffer (GSB) pH 8.2, 1 mL of a 5% (w / v) suspension was prepared, and 1 mL of a commercially available anti-human myoglobin monoclonal antibody (Mikuri Institute for Immunology: Lot8) (0.7 mg / mL) was added to the 1 mL suspension. , Mixed.
After mixing at 37 ° C. for 2 hours, the supernatant was discarded by centrifugation, and the precipitated particles were added to a 0.1 molar glycine saline buffer (GSB) supplemented with 0.05% bovine serum albumin (BSA). A suspension of particles (latex suspension) (A1) was prepared by suspending in 2 mL of pH 8.2.

Preparation Example 2: Preparation of suspension of anti-human myoglobin / monoclonal antibody-carrying particles (A2) Monoclonal antibody was replaced with a commercially available anti-human myoglobin / monoclonal antibody (Mikuri Institute for Immunology: Lot 44) (0.7 mg / mL). Except for the above, the same operation as in Preparation Example 1 was performed to prepare a suspension of particles (latex suspension) (A2).

Preparation Example 3: Preparation of suspension of anti-human myoglobin / rabbit polyclonal antibody-carrying particles (A3) Human myoglobin was administered into rabbit skin with Freund's complete adjuvant and immunized to obtain anti-antigen obtained from the rabbit Serum was subjected to a protein-G column method, and IgG antibody (anti-human myoglobin / rabbit polyclonal antibody) was fractionated.
Polystyrene particles having an average particle size of 220 nm were diluted with 0.1 molar glycine saline buffer (GSB) pH 8.2 to prepare 1 mL of a 5% (w / v) suspension. 1 mL of the IgG antibody solution (0.7 mg / mL) obtained above was added to the suspension and mixed.
After mixing at 37 ° C. for 2 hours, the supernatant was discarded by centrifugation, and the precipitated particles were added to a 0.1 molar glycine saline buffer (GSB) supplemented with 0.05% bovine serum albumin (BSA). A suspension of particles (latex suspension) (A3) was prepared by suspending in 2 mL of pH 8.2.

Example 1
Standard myoglobin dilution series solutions (0, 25, 50, 100, 200, 400 ng / mL) were prepared.
The suspension of the particles (latex suspension) (A1) prepared in Preparation Example 1 is added to the reaction buffer reagent, and the suspension (second latex) containing 0.07% (w / v) particles (latex particles) is added. 1 reagent), and 150 μL of the suspension is added to 5 μL of the standard myoglobin dilution row solution prepared above, and mixed by stirring. After about 5 minutes, the mixture is mixed with the particles prepared in Preparation Example 3. The suspension (latex suspension) (A3) was added with 100 μL of suspension (second reagent) diluted and adjusted so that the particle (latex particle) content was 0.075% (w / v), and stirred. After mixing, the amount of change in absorbance for about 5 minutes was measured at a wavelength of 660 nm using a Hitachi 7180 automatic analyzer (manufactured by Hitachi High-Technologies Corporation).

Comparative Example 1
The same operation as in Example 1 was performed except that a reaction buffer reagent (not containing particles (latex particles)) was used as the first reagent.

Comparative Example 2
As a first reagent, a reaction buffer reagent (not including particles (latex particles)) is used, and as a second reagent, a suspension of particles (latex suspension) (A1) prepared in Preparation Example 1 and a preparation example. The suspension of the particle suspension (latex suspension) (A2) prepared in step 2 was mixed with an equal amount, and the dilution was adjusted so that the particle (latex particle) content was 0.075% (w / v). The same operation as in Example 1 was performed except that the suspended liquid was used.

結果：第１試薬にＡ１の粒子（ラテックス粒子）を添加した実施例１は、第１試薬に粒子（ラテックス粒子）を添加しなかった比較例１及び比較例２に比して、大きな吸光度変化量（高感度）を示した。 Table 1 summarizes the amount of change in absorbance in the standard myoglobin dilution series solution (0, 25, 50, 100, 200, 400 ng / mL) in Example 1, Comparative Example 1, and Comparative Example 2.

Results: Example 1 in which A1 particles (latex particles) were added to the first reagent had a greater absorbance change than Comparative Examples 1 and 2 in which particles (latex particles) were not added to the first reagent. Amount (high sensitivity) was shown.

Preparation Example 4: Preparation of suspension of anti-filaria monoclonal antibody-carrying particles (B1) Antigens of anti-filaria monoclonal antibody were obtained by immunizing mice with antigenic components extracted from filaria adults with physiological saline.
Polystyrene particles having an average particle size of 200 nm were diluted with 0.1 molar glycine saline buffer (GSB) pH 8.2 to prepare 1 mL of a 5% (w / v) suspension. To the suspension, 1 mL of the anti-filaria monoclonal antibody (0.7 mg / mL) obtained above was added and mixed.
After mixing at 37 ° C. for 2 hours, the supernatant was discarded by centrifugation, and the precipitated particles were added to a 0.1 molar glycine saline buffer (GSB) supplemented with 0.05% bovine serum albumin (BSA). A suspension of particles (latex suspension) (B1) was prepared by suspending in 2 mL of pH 8.2.

Preparation Example 5 Preparation of Rabbit Antifilar Antigen IgG Antibody (Polyclonal Antibody) -Supported Particle Suspension (B2) Antigen Rabbit is immunized with an antigen component extracted from filaria adults with physiological saline by a conventional method. Filaria antigen IgG antibody (polyclonal antibody) was obtained.
Polystyrene particles having an average particle size of 317 nm were diluted with 0.1 molar glycine saline buffer (GSB) pH 8.2 to prepare 1 mL of a 5% (w / v) suspension. To the suspension, 1 mL of the rabbit antifilaria antigen IgG antibody (polyclonal antibody) (0.7 mg / mL) obtained above was added and mixed.
After mixing at 37 ° C. for 2 hours, the supernatant was discarded by centrifugation, and the precipitated particles were added to a 0.1 molar glycine saline buffer (GSB) supplemented with 0.05% bovine serum albumin (BSA). The suspension was suspended in 2 mL of pH 8.2 to prepare a suspension of particles (latex suspension) (B2).

Example 2
Filaria standard antigen dilution train solutions (0, 8, 32, 130 ng / mL) were prepared.
The suspension of the particles prepared in Preparation Example 4 (latex suspension) (B1) is added to the reaction buffer reagent, and the suspension (first solution) containing 0.0125% (w / v) particles (latex particles) is added. 1 reagent), and 60 μL of the suspension is added to 10 μL of a diluted solution of Filaria standard antigen dilution series solution prepared above with 10 μL of a diluted saline solution of 120 μL, and mixed by stirring. After about 5 minutes, The mixture was diluted and adjusted so that the particle suspension (latex suspension) (B2) prepared in Preparation Example 5 had a content of particles (latex particles) of 0.075% (w / v). After adding 55 μL of the suspension (second reagent) and mixing with stirring, the amount of change in absorbance for about 5 minutes was measured at a wavelength of 571 nm using BM2250 (manufactured by JEOL Ltd.).

Example 3
As the first reagent, except that the suspended liquid (latex suspended liquid) (B1) prepared in Preparation Example 4 was added and the suspended liquid containing 0.00625% (w / v) of particles (latex particles) was used. The same operation as in Example 2 was performed.

Comparative Example 3
The same operation as in Example 2 was performed except that a reaction buffer reagent (not containing particles (latex particles)) was used as the first reagent.

結果：第１試薬にＢ１の粒子（ラテックス粒子）を添加した実施例２及び実施例３は、第１試薬に粒子（ラテックス粒子）を添加しなかった比較例３に比して、大きな吸光度変化量（高感度）を示した。 Table 2 summarizes the amount of change in absorbance in the filarial standard antigen dilution train solution (0, 8, 32, 130 ng / mL) in Example 2, Example 3, and Comparative Example 3.

Results: Example 2 and Example 3 in which B1 particles (latex particles) were added to the first reagent had a larger absorbance change than Comparative Example 3 in which particles (latex particles) were not added to the first reagent. Amount (high sensitivity) was shown.

Preparation Example 6: Preparation of suspension (C1) of anti-human α-fetoprotein (AFP) / monoclonal antibody-carrying particles Polystyrene particles having an average particle size of 220 nm were mixed with 0.1 molar glycine saline buffer (GSB) pH 8.2. 1 ml of a 0.45% (w / v) suspension was prepared, and a commercially available anti-human α-fetoprotein monoclonal antibody (Mikuri Immuno Laboratory: clone6G2) ( 0.235 mg / mL) 1 mL was added and mixed.
After mixing at 37 ° C. for 2 hours, the supernatant was discarded by centrifugation, and the precipitated particles were added to a 0.1 molar glycine saline buffer (GSB) supplemented with 0.05% bovine serum albumin (BSA). The suspension was suspended in 2 mL of pH 8.2 to prepare a suspension of particles (latex suspension) (C1).

Preparation Example 7: Preparation of suspension of anti-human α-fetoprotein / monoclonal antibody-carrying particles (C2) The particle suspension (latex suspension) (C2) was prepared by performing the same operation as in Preparation Example 6 except that it was replaced with ().

Preparation Example 8: Preparation of suspension of anti-human α-fetoprotein / rabbit polyclonal antibody-carrying particles (C3) Human α-fetoprotein (AFP) was immunized by administering it into the skin of rabbits with Freund's complete adjuvant, The antiserum obtained from the rabbit was subjected to a protein-G column method to obtain an IgG antibody (anti-human α-fetoprotein / rabbit polyclonal antibody).
Polystyrene particles having an average particle size of 220 nm were diluted with 0.1 molar glycine saline buffer (GSB) pH 8.2 to prepare 1 mL of a 0.45% (w / v) suspension. 1 mL of the above-obtained IgG antibody solution (0.235 mg / mL) was added and mixed.
After mixing at 37 ° C. for 2 hours, the supernatant was discarded by centrifugation, and the precipitated particles were added to a 0.1 molar glycine saline buffer (GSB) supplemented with 0.05% bovine serum albumin (BSA). The suspension was suspended in 2 mL of pH 8.2 to prepare a suspension of particles (latex suspension) (C3).

Example 4
A standard human α-fetoprotein (AFP) dilution series solution (0, 35, 144, 619, 2557 ng / mL) was prepared.
The suspension of the particles prepared in Preparation Example 6 (latex suspension) (C1) is added to the reaction buffer reagent, and the suspension (second latex / particle) containing 0.025% (w / v) 1 reagent), 180 μL of the suspension is added to 15 μL of the standard myoglobin dilution row solution prepared above, and mixed by stirring. After about 5 minutes, the mixture is mixed with the particles prepared in Preparation Example 8. Add 90 μL of suspended liquid (latex suspended liquid) (C3) to the suspension liquid (second reagent) diluted so that the particle (latex particle) content is 0.075% (w / v), and stir. After mixing, the amount of change in absorbance for about 5 minutes was measured at a wavelength of 700 nm using a Hitachi 7170 automatic analyzer (manufactured by Hitachi High-Technologies Corporation).

Example 5
As the first reagent, except that the suspended liquid (latex suspended liquid) (C1) prepared in Preparation Example 6 was added and the suspended liquid containing 0.0125% (w / v) particles (latex particles) was used. The same operation as in Example 4 was performed.

Example 6
As the first reagent, except that the suspended liquid (latex suspended liquid) (C1) prepared in Preparation Example 6 was added and the suspended liquid containing 0.00625% (w / v) particles (latex particles) was used. The same operation as in Example 4 was performed.

Example 7
As the first reagent, except that the suspension of particles (latex suspension) (C1) prepared in Preparation Example 6 was added and a suspension containing 0.003125% (w / v) of particles (latex particles) was used. The same operation as in Example 4 was performed.

Example 8
As a first reagent, the suspension of particles (latex suspension) (C1) prepared in Preparation Example 6 was added, and the suspension was prepared by replacing the suspension of particles (latex particles) containing 0.025% (w / v). Same as Example 4 except that the suspension of particles (latex suspension) (C2) prepared in Example 7 was added and the suspension containing 0.025% (w / v) of particles (latex particles) was used. Was performed.
Example 9
As the first reagent, except that the suspended liquid (latex suspended liquid) (C2) prepared in Preparation Example 7 was added and the suspended liquid containing 0.0125% (w / v) particles (latex particles) was used. The same operation as in Example 8 was performed.

Example 10
As the first reagent, except that the suspended liquid (latex suspended liquid) (C2) prepared in Preparation Example 7 was added and the suspended liquid containing 0.00625% (w / v) particles (latex particles) was used. The same operation as in Example 8 was performed.

Example 11
As the first reagent, except that the suspended liquid (latex suspended liquid) (C2) prepared in Preparation Example 7 was added and the suspended liquid containing 0.003125% (w / v) particles (latex particles) was used. The same operation as in Example 8 was performed.

Comparative Example 4
The same operation as in Example 4 was performed except that a reaction buffer reagent (not containing particles (latex particles)) was used as the first reagent.

結果：第１試薬にＣ１の粒子（ラテックス粒子）を添加した実施例４乃至実施例７並びに第１試薬にＣ２の粒子（ラテックス粒子）を添加した実施例８乃至実施例１１は、第１試薬に粒子（ラテックス粒子）を添加しなかった比較例４に比して、大きな吸光度変化量（
高感度）を示した。
また、第１試薬にＣ１の粒子（ラテックス粒子）を添加した実施例４乃至実施例７では、ＡＦＰの低濃度領域での感度向上の程度が大きかったのに対して、第１試薬にＣ２の粒子（ラテックス粒子）を添加した実施例８乃至実施例１１では、ＡＦＰの高濃度領域での感度向上の程度が大きく、使用する抗体の種類により反応性が異なっていた。
また、何れの抗体を用いた場合においても、粒子（ラテックス粒子）の非常に低い濃度において、感度の向上が観られており、抗原に対する高い反応性を示したことが分る。 Table 4 summarizes the amount of change in absorbance in standard human α-fetoprotein (AFP) dilution series solutions (0, 35, 144, 619, 2557 ng / mL) in Examples 4 to 7 and Comparative Example 4. Table 4 summarizes the amount of change in absorbance in standard human α-fetoprotein (AFP) dilution series solutions (0, 35, 144, 619, 2557 ng / mL) in Example 11 and Comparative Example 4.

Results: Examples 4 to 7 in which C1 particles (latex particles) were added to the first reagent and Examples 8 to 11 in which C2 particles (latex particles) were added to the first reagent Compared with Comparative Example 4 in which no particles (latex particles) were added, the absorbance change amount (
High sensitivity).
In Examples 4 to 7 in which C1 particles (latex particles) were added to the first reagent, the degree of sensitivity improvement was large in the low concentration region of AFP, whereas C2 was added to the first reagent. In Examples 8 to 11 to which particles (latex particles) were added, the degree of sensitivity improvement in the high concentration region of AFP was large, and the reactivity was different depending on the type of antibody used.
Moreover, in any case of using any antibody, an improvement in sensitivity was observed at a very low concentration of particles (latex particles), indicating that the antibody showed high reactivity with antigens.

Claims (8)

Carrier agglutination immunization for measuring an antigen in the sample, comprising: a first step of mixing the sample with the first reagent; and a second step of mixing the liquid mixture obtained in the first step with the second reagent. A measuring method,
The first reagent includes a first particle that captures one of the epitopes of the antigen, the second reagent includes a second particle, and the second particle includes two or more of the epitopes of the antigen. Or a mixture of two or more particles that capture one of the epitopes of the antigen, wherein each particle is a particle that captures a different epitope of the antigen. Measuring method to do. The measurement method according to claim 1, wherein the first particle is a particle carrying a polyclonal antibody having affinity for one of epitopes of the antigen. The measurement method according to claim 1, wherein the first particle is a particle carrying a monoclonal antibody having affinity for one of epitopes of the antigen. The measurement method according to any one of claims 1 to 3, wherein the second particle is a particle carrying a polyclonal antibody having affinity for two or more epitopes of the antigen. The second particles are particles carrying two or more kinds of monoclonal antibodies having affinity for one of the epitopes of the antigen, and various monoclonal antibodies are antibodies having affinity for different epitopes of the antigen. The measurement method according to any one of claims 1 to 3, wherein: The second particle is a mixture of two or more kinds of particles carrying a monoclonal antibody having affinity for one of the epitopes of the antigen, and each type of particle has an affinity for a different epitope of the antigen. The measurement method according to any one of claims 1 to 3, which is a particle carrying a monoclonal antibody. A reagent kit for immunoassay comprising a first reagent and a second reagent for use in a carrier agglutination immunoassay method for measuring an antigen in a specimen,
The first reagent includes the first particles according to claim 2 or claim 3, and the second reagent includes the second particles according to any one of claims 4 to 6. A reagent kit for immunoassay characterized. Furthermore, the reagent kit for immunoassay of Claim 7 containing the 3rd reagent which does not contain the particle | grains which carry | supported the antibody.