JP2001074742A - Latex immunity nephelometry measurement method and kit - Google Patents

Abstract

PROBLEM TO BE SOLVED: To measure the concentration of a substance to be inspected widely by copolymerizing a specific weight of monomer derived from (meta)acrylate, non- saturation carboxylic acid, and vinyl monomer, and adding an agglutination reaction accelerator to a mixing system that is subjected to an antigen/antibody reaction. SOLUTION: A monomer derived from (meta)acrylate with a fatty hydrocarbon group with 1-20 carbons is copolymerized by approximately 20-100 wt.%, monomer derived from non-saturation carboxylic acid is copolymerized by approximately 1-10 wt.%, and a monomer derived from a vinyl monomer is copolymerized by approximately 0-80 wt.%. When the average particle diameter of the particle is set to dnm and the absorbance with wavelength of nm of water dispersion liquid of a solid content concentration of approximately w/v% is set to A, conditions indicated by an expression are satisfied (M0=0.012573, M1=0.0020732, M2=6.333×3-5, M3=8.7935×e-8, and M4=3.529×e-11). A agglutination reaction accelerator is included in a mixing system where antigen/antibody reaction is made, and the agglutination state of a particle where a specimen and an immunoreaction substance have been sensitized is measured, thus determining the substance to be inspected in the specimen.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a latex immunoturbidimetry and a measurement kit applied thereto.

[0002]

2. Description of the Related Art In the field of clinical tests, immunoassays utilizing antigen-antibody reactions are widely used as methods for quantifying trace substances in a specimen. Among them, the latex immunoturbidimetric method using particles is simple and has a short measurement time, and thus has been widely used in response to recent demands for rapid tests. Quantification of a test substance (antigen or antibody) in a specimen by latex immunoturbidimetry is performed by optically detecting a change in absorbance due to aggregation of particles. This change in absorbance is based on a change in the apparent particle size due to aggregation of the particles.

Conventionally, in latex immunoturbidimetry,
Polystyrene-based particles such as polystyrene particles and carboxylic acid-modified styrene-based particles are widely used because they can easily immobilize antigens and antibodies. Here, examples of the carboxylic acid used in a small amount to obtain the carboxylic acid-modified styrene-based particles include methacrylic acid, acrylic acid, itaconic acid, and fumaric acid.

However, in the case where the turbidity due to the aggregation of particles is measured by measuring the absorbance, the absorbance of the conventionally known polystyrene particles in a non-aggregated state (concentration of the test substance = 0) is large. Even in a region where the concentration of the test substance is low, there is a problem that the absorbance exceeds the measurement limit of the test device. Therefore, if the concentration of the particles is reduced to reduce the absorbance in the non-aggregated state, there is a problem that a so-called prozone phenomenon occurs in a region where the concentration of the test substance is high, and the clinical test requires dilution of the sample. A great deal of time and effort is required on site. Further, if the particle diameter is reduced in order to reduce the absorbance in the non-aggregated state, the sensitivity (low value sensitivity) in a region where the concentration of the test substance is low is undesirably reduced.

As a means for preventing the occurrence of the prozone phenomenon without causing the absorbance after agglutination to exceed the measurement limit of a test instrument, a technique of applying a fluorine-containing latex material having a lower refractive index than polystyrene-based particles has been proposed. (For example, JP-A-61-247966, JP-A-63-200605, and JP-A-63-2
No. 78913, JP-A-1-266111).

However, fluorine-containing latex materials have a problem in terms of manufacturing costs, such as the necessity of a pressure-resistant container in order to manufacture them. The specific gravity of the particles is 1.
Since it is as large as 5 or more, the dispersion stability is inferior, and there is a problem from the viewpoint of storage stability. In addition, the glass transition temperature (Tg) is low, so that redispersion after centrifugation is difficult, and it is also difficult to fix the antigen or antibody by physical adsorption. Furthermore, fluorine-containing particles having a low refractive index have a small increase in absorbance due to aggregation, and do not have sufficiently high low-value sensitivity.

[0007]

SUMMARY OF THE INVENTION The present invention has been made based on the above circumstances. A first object of the present invention is to provide a latex immunoassay capable of measuring the concentration of a test substance in a sample in a wide concentration range and not causing a prozone phenomenon even in a region where the test substance concentration is high. An object of the present invention is to provide a turbidimetric method. A second object of the present invention is to provide a latex immunoturbidimetric assay that can accurately quantify even a low concentration of a test substance. A third object of the present invention is to provide a measurement kit capable of measuring the concentration of a test substance in a sample in a wide concentration range.

[0008]

According to the latex immunoturbidimetry of the present invention, a suspension containing a specimen containing a test substance and particles sensitized with a substance (immunoreactive substance) which reacts with the test substance by an antigen-antibody reaction is prepared. A latex immunoturbidimetry to measure the state of aggregation of the particles due to the antigen-antibody reaction, and to quantify the test substance in the sample,
(A) 20 to 100% by weight of a structural unit (hereinafter referred to as “structural unit A”) derived from a (meth) acrylate having an aliphatic hydrocarbon group having 1 to 20 carbon atoms, and (B) an unsaturated carboxylic acid. 0 to 10% by weight of a structural unit (hereinafter referred to as “structural unit B”) derived from a vinyl monomer copolymerizable with the (meth) acrylate and the unsaturated carboxylic acid (hereinafter referred to as “structural unit B”) , Referred to as “structural unit C”). The particles are composed of a polymer (hereinafter, referred to as “specific (co) polymer”) composed of 0 to 80% by weight, and the average particle diameter of the particles is d (nm). ), When the absorbance at a wavelength of 600 nm measured with respect to an aqueous dispersion having a solid content concentration of 0.05 w / v% of the particles is A, a mixture satisfying the condition represented by the following formula 1 and performing the antigen-antibody reaction is satisfied. Addicted to the system Characterized in that the presence of a reaction accelerator.

[0009]

[Number 2] Formula 1: A <f (d) = (M 0 -M 1 d + M 2 d 2 -M 3 d 3 + M 4 d 4) ( _{wherein, M 0 = 0.012573, M 1} = 0. 0020
732, M ₂ = 6.333 × e ⁻⁵ , M ₃ = 8.7935
× e ^-8 and M ₄ = 3.529 × e ^-11 . )

[0010] The measurement kit of the present invention mixes a specimen containing a test substance with a suspension of particles sensitized with a substance that reacts with the test substance with an antigen-antibody, and agglutinates the particles by the antigen-antibody reaction. In a measurement kit applied to latex immunoturbidimetry for measuring the condition and quantifying the test substance in the sample, the particles (specifically (identified) ) A polymer as a component,
A suspension of particles satisfying the condition shown in the above formula 1,
An agglutination reaction accelerator is contained in this suspension or another reagent constituting the measurement kit.

In the present invention, "average particle diameter d (n
"m)" means a number average particle diameter obtained by taking an electron micrograph of particles with a transmission electron microscope and measuring the particle diameter of 200 or more randomly selected particles.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. <Particles> The particles used in the latex immunoturbidimetry of the present invention contain a specific (co) polymer (a specific copolymer or a specific polymer) as a component. Specific examples of (meth) acrylate (hereinafter, referred to as "monomer (A)") used to obtain structural unit (A) constituting the specific (co) polymer include methyl acrylate and ethyl acrylate. , N-
Propyl acrylate, isopropyl acrylate, methyl methacrylate, ethyl methacrylate, n-propyl methacrylate, isopropyl methacrylate,
-Ethylhexyl acrylate, 2-ethylhexyl methacrylate, n-butyl acrylate, n-butyl methacrylate, t-butyl acrylate, t-butyl methacrylate, isobutyl acrylate, isobutyl methacrylate, pentyl acrylate, pentyl methacrylate, stearyl acrylate, stearyl methacrylate, lauryl acrylate, Alkyl (meth) acrylate having a chain alkyl group such as lauryl methacrylate; alkyl (meth) acrylate having a cyclic aliphatic group such as cyclohexyl acrylate, cyclohexyl methacrylate, cyclohexylethylene glycol methacrylate, cyclohexyl dipropylene glycol methacrylate; methyl-substituted cyclohexyl acrylate Such as Substituted cyclohexyl (meth) acrylates and cyclohexenedi (meth) acrylates, in which some of the hydrogen atoms of the clohexyl group are substituted with an alkyl group having 1 to 4 carbon atoms, may be mentioned, and these may be used alone or in combination of two or more. They can be used in combination.

The content of the structural unit (A) in the specific (co) polymer is usually 20 to 100% by weight,
Preferably, it is 65 to 100% by weight. When the content ratio of the structural unit (A) is 20% by weight or more,
Particles satisfying the conditions shown in the above formula 1 can be formed.

The unsaturated carboxylic acid (hereinafter referred to as "monomer (B)") used to obtain the structural unit (B) constituting the specific copolymer contains a radical polymerizable unsaturated bond and It is a polymerizable monomer having a carboxyl group in the molecule. Specific examples of the monomer (B) include acrylic acid, methacrylic acid, crotonic acid, itaconic acid, fumaric acid, and maleic acid. These may be used alone or in combination of two or more. be able to. The content ratio of the structural unit (B) in the specific copolymer is usually 0 to 10% by weight, preferably 0 to 5% by weight. Particles containing the specific copolymer into which the structural unit (B) has been introduced are mainly subjected to sensitization treatment by chemical bonding, and particles not having the structural unit (B) have been mainly subjected to sensitization by physical adsorption. Provided for crop processing.

Specific examples of the vinyl monomer (hereinafter referred to as "monomer (C)") used to obtain the structural unit (C) constituting the specific copolymer include styrene and α-.
Methyl styrene, vinyl toluene, divinyl benzene,
Aromatic vinyl compounds such as styrene sulfonic acid; vinyl ester compounds such as vinyl acetate; and the like, and these can be used alone or in combination of two or more. The content ratio of the structural unit (C) in the specific copolymer is generally 0 to 80% by weight, preferably 0 to 35% by weight.

The particles used in the latex immunoturbidimetry of the present invention may be particles composed only of a specific (co) polymer, or may be particles on the surface of core particles made of a material other than the specific (co) polymer. Although a composite particle in which a layer of the specific (co) polymer is formed continuously or intermittently may be used, it is a particle composed of only the specific (co) polymer because of easy production. Preferably, there is. In addition, examples of the constituent material of the core particles include polystyrene, carboxyl group-modified polystyrene, and silica.

The diameter of the particles used in the latex immunoturbidimetry of the present invention is not particularly limited, but the average particle diameter d (number average particle diameter) is preferably in the range of 30 to 1,000 nm, more preferably. Is 50 to 1,000 nm.
If the average particle size d is too small, the measurement sensitivity (low value sensitivity) in the low concentration range of the test substance tends to be insufficient. On the other hand, when the average particle diameter d is too large, the sensitivity tends to be too high, and the sedimentation of the particles tends to be too fast.

The average particle size of the particles used in the latex immunoturbidimetric immunoassay of the present invention is d (nm), and the wavelength of the particles measured with respect to an aqueous dispersion having a solid concentration of 0.05 w / v% is 6
When the absorbance at 00 nm is A, it is necessary to satisfy the condition shown in the above formula 1, and it is preferable to satisfy the condition shown in the following formula 2. According to the particles satisfying the condition of the above formula 1, the absorbance in the non-aggregated state is lower than that of the polystyrene-based particles. As a result, the absorbance is measured by a test instrument in a wide concentration range (range of the concentration of the test substance). be able to. According to conventionally known polystyrene particles, the absorbance A measured under the same conditions
Is always greater than or equal to f (d).

[0019]

Formula 2: A <k · f (d) (where k is less than 1, preferably 0.95 or less,
More preferably, it is 0.90 or less.)

<Test substance / immunoreactive substance> The test substance (antibody or antigen) to be quantified by the latex immunoturbidimetry of the present invention and the immunoreactive substance (antigen or antibody) reacting with the test substance (antigen or antibody) include: There is no particular limitation as long as it is a substance generally contained in a sample and a substance that can react with the substance. For example, an anti-antiplasmin antibody for an antiplasmin test, an anti-D-dimer antibody for a D-dimer test, and an anti-DDP antibody for an FDP test. FDP antibody, anti-tPA for tPA test
Antigens or antibodies for coagulation / fibrinolysis-related tests such as antibodies, anti-thrombin-antithrombin complex antibodies for TAT tests, anti-FPA antibodies for FPA tests; anti-BFP antibodies for BFP tests, C
Anti-CEA antibody for EA test, anti-AFP antibody for AFP test,
Anti-ferritin antibody for ferritin test, antigen or antibody for tumor-related test such as anti-CA19-9 antibody for CA19-9 test; anti-apolipoprotein antibody for apolipoprotein test;
For serum protein-related tests, such as anti-β2-microglobulin antibody for β2-microblobrin test, anti-α1-microglobulin antibody for α1-microglobulin test, anti-immunoglobulin antibody for immunoglobulin test, and anti-CRP antibody for CRP test Antigen or antibody; antigen or antibody for endocrine function test such as anti-HCG antibody for HCG test; anti-H for HBs antigen test
Bs antibody, HBs antigen for HBs antibody test, HCV antigen for HCV antibody test, HIV-1 antigen for HIV-1 antibody, H
HIV-2 antigen for IV-2 antibody test, HTLV-1 antigen for HTLV-1 test, mycoplasma antigen for mycoplasmosis test, toxoplasma antigen for toxoplasma test, streptolysin O antigen for ASO test, etc. Antibody; DNA antigen for anti-DNA antibody test, antigen or antibody for autoimmune-related test such as heat denatured human IgG for RF test; Anti-digoxin antibody for digoxin test;
Examples include, but are not limited to, antigens or antibodies for drug analysis such as anti-lidocaine antibodies for lidocaine testing. As the antibody, either a polyclonal antibody or a monoclonal antibody may be used.

The method for sensitizing the particles used in the latex immunoturbidimetry of the present invention to an immunoreactive substance (biochemical substance such as antibody, antigen or nucleic acid) is not particularly limited. Here, a sensitization treatment method mainly by a chemical bond can be adopted for particles containing the specific copolymer into which the structural unit (B) is introduced, and the structural unit (B) is introduced. The sensitization treatment method mainly by physical adsorption can be adopted for particles that are not used.

<Agglutination Reaction Accelerator> The latex immunoturbidimetry of the present invention uses a mixed system in which an antigen-antibody reaction of a test substance and an immunoreactive substance is performed, that is, a sample containing the test substance,
It is characterized in that an agglutination reaction promoter is present in a mixed system with a suspension of particles sensitized with an immunoreactive substance. Examples of such an agglutination reaction accelerator include water-soluble polymers having a molecular weight of 500 to 2,000,000, preferably 500 to 1,000,000.

Specific examples of the agglutination accelerator used in the latex immunoturbidimetry of the present invention include, for example, polyvinylpyrrolidone, polyethyleneimine, polyethylene glycol, CHAPS (3-[(3-coramidopropyl)
Dimethylammonio] -1-propanesulfonic acid), B
IGCHAP (N, N-bis (3-D-gluconamidopropyl) cholamide), digitonin, saponin, pullulan, polyglycosylethyl methacrylate, polyglycosylethyl methacrylate, and the like. Of these, polyethylene glycol, digitonin Is preferred. These compounds can be used alone or in combination of two or more.

<Other Additives> In the present invention, a non-specific agglutination inhibitor may be further present when mixing a specimen containing a test substance with particles sensitized with an immunoreactive substance. Non-specific agglutination inhibitors include proteins such as gelatin and albumin; glycols; polyanions; N, N-dialkylamides; lower alkyl sulfoxides; potassium nitrate, sodium nitrate, lithium nitrate;
Nitrates such as barium nitrate, calcium nitrate and ammonium nitrate; potassium thiocyanate, sodium thiocyanate, lithium thiocyanate, barium thiocyanate,
Thiocyanates such as calcium thiocyanate and ammonium thiocyanate can be mentioned. Also,
It is also possible to add a quaternary ammonium salt such as choline chloride, EDTA or the like in order to specifically aggregate the particles sensitized with the immunoreactive substance.

<Measurement Kit> The measurement kit of the present invention comprises a suspension of sensitized particles comprising a specific (co) polymer as a component and sensitizing an immunoreactive substance to particles satisfying the condition shown in the above formula 1. A liquid, wherein the suspension or the other reagent constituting the measurement kit contains an agglutination reaction accelerator. The assay kit applied to the latex immunoturbidimetry consists of two liquid preparations, a suspension of particles sensitized with a substance that reacts with the test substance and the antigen and an antibody, and a buffer solution that is a medium for the antigen and antibody reaction. Generally, in this case, the agglutination reaction accelerator may be added to the suspension of the sensitized particles or may be added to the buffer. Further, the agglutination reaction accelerator may be added to other reagents (reagents other than the suspension and the buffer) constituting the measurement kit. Each reagent constituting the measurement kit is housed in a container or the like, and is appropriately diluted when used for the latex immunoturbidimetry. When the non-specific agglutination reaction inhibitor is added, it may be added to either the suspension of the sensitized particles or the buffer, but is usually added to the buffer.

The concentration of the test substance is measured by performing an agglutination reaction of the particles using the reagents described above and optically observing the degree of the generated agglutination using an analyzer.
Aggregation of particles is measured using an optical instrument for measuring scattered light intensity, transmittance or transmitted light intensity. Measurement wavelength is 300
It is preferably from 2 to 400 nm. The measurement is performed by setting the particle diameter, concentration, and reaction time, and measuring the increase or decrease of the scattered light intensity, the transmittance or the transmitted light intensity. As the medium for the antigen-antibody reaction, various buffers are appropriately selected depending on the test substance. This buffer is
There is no particular limitation as long as it has a pH or ionic strength that does not affect the test substance or the antigen-antibody reaction. For example, phosphate buffer, Tris buffer, glycine buffer, HEPES
A buffer solution or the like is used. The reaction is carried out at pH 5-10, especially at 6
It is preferable to be carried out in the vicinity of about 8 to neutrality. The reaction temperature is preferably from 0 to 50C, particularly preferably from 20 to 40C. The reaction time is appropriately determined.

The particles used in the latex immunoturbidimetry of the present invention have an absorbance (initial absorbance) in an unaggregated state (test substance concentration = 0) as compared with polystyrene-based particles.
Is small. Therefore, even if the concentration of the particles is set high,
The absorbance can be measured by an inspection instrument (the measurement limit is not exceeded). Since the concentration of the particles can be set high, the prozone phenomenon does not occur even in a region where the concentration of the test substance is high. In addition, in the latex immunoturbidimetry of the present invention, since the agglutination reaction promoter is present in the mixed system in which the antigen-antibody reaction is performed, the low value sensitivity is high, and even a low concentration of the test substance can be accurately quantified. And the analyte concentration can be measured in a wide concentration range.

[0028]

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples. [Preparation Example 1A (Preparation of Particles)] A glass with a stirrer having a capacity of 5 liters comprising 100 parts by weight of cyclohexyl methacrylate, 500 parts by weight of water, 1 part by weight of potassium persulfate, and 0.2 part by weight of sodium dodecylbenzenesulfonate. The average particle diameter (d) was 105 by charging the flask and conducting a polymerization reaction at a temperature of 80 ° C. for 6 hours under a nitrogen atmosphere.
Thus, particles having a particle size of nm (hereinafter, referred to as “particle 1”) were obtained.
Here, the polymerization conversion rate was 98% or more. Next, the particles 1 were purified by dialysis to prepare a suspension having a solid concentration of 10 w / v%. The solid content concentration of the particles 1 is 0.0
When the absorbance A at a wavelength of 600 nm was measured for the 5 w / v% aqueous dispersion, the absorbance A was 0.215,
f (105) = 0.396 [A <f (d)].

[Preparation Example 1B (Preparation of Sensitized Particles)] A suspension of particles 1 (solid content concentration: 10 w / v%) obtained in Preparation Example 1A was subjected to a 50 mM Tris buffer (pH 7.0).
It was diluted 10-fold in 4). On the other hand, 10 mg / ml anti-human CRP antibody was diluted 10-fold with 50 mM Tris buffer (pH 7.4). Mixing 1 part by volume of the diluent of the particles 1 and 1 part by volume of the diluent of the anti-human CRP antibody, 37
After stirring for 1 hour at 50 ° C., 50 mM Tris buffer (pH
It was centrifugally washed twice in 7.4). Subsequently, a 50 mM Tris buffer (pH 7.4) containing 1% BSA was added to dilute the solution to give an anti-human CRP antibody-sensitized latex having a solid concentration of 0.15 w / v% (hereinafter, “sensitized latex”). 1 ") was prepared.

[Preparation Example 2A (Preparation of Particles)] Styrene 1
00 parts by weight, water 500 parts by weight, potassium persulfate 1 part by weight, sodium dodecylbenzenesulfonate 0.2%
Parts by weight and charged in a glass flask equipped with a stirrer having a capacity of 5 liters, and subjected to a polymerization reaction under a nitrogen atmosphere at a temperature of 80 ° C. for 6 hours to obtain comparative particles having an average particle diameter (d) of 103 nm (hereinafter, referred to as “particles”). "Particle 2").
Here, the polymerization conversion rate was 98% or more. Next, the particles 2 were purified by dialysis to prepare a suspension having a solid content of 10 w / v%. The solid content concentration of the particles 2 is 0.0
When the absorbance A at a wavelength of 600 nm was measured for the 5 w / v% aqueous dispersion, the absorbance A was 0.378, f
(103) = 0.378 [A = f (d)].

[Preparation Example 2B (Preparation of Sensitized Particles)] A suspension (solid content concentration: 10 w / v%) of the particles 2 obtained in the above Preparation Example 2A was treated with 50 mM Tris buffer (pH 7.0).
It was diluted 10-fold in 4). On the other hand, 10 mg / ml anti-human CRP antibody was diluted 10-fold with 50 mM Tris buffer (pH 7.4). Mixing 1 part by volume of the diluent of the particles 2 and 1 part by volume of the diluent of the anti-human CRP antibody, 37
After stirring for 1 hour at 50 ° C., 50 mM Tris buffer (pH
It was centrifugally washed twice in 7.4). Subsequently, a 50 mM Tris buffer (pH 7.4) containing 1% BSA was added to dilute the solution to give an anti-human CRP antibody-sensitized latex (hereinafter referred to as “sensitized latex”) having a solid concentration of 0.30 w / v%. 2 ").

[Preparation Example 3A (Preparation of Particles)] 90 parts by weight of cyclohexyl methacrylate, 5 parts by weight of methacrylic acid, 5 parts by weight of styrene, 500 parts by weight of water, 1 part by weight of potassium persulfate, and dodecylbenzenesulfone 0.2 parts by weight of sodium acid were charged into a 5 liter glass flask equipped with a stirrer, and a polymerization reaction was carried out at 80 ° C. for 6 hours in a nitrogen atmosphere to obtain an average particle diameter (d) of 1%.
Particles having a diameter of 02 nm (hereinafter, referred to as “particle 3”) were obtained. Here, the polymerization conversion rate was 98% or more. Next, the particles 3 are purified by dialysis, and the solid content concentration is 10 w / v.
% Suspension was prepared. The aqueous dispersion having a solid content concentration of the particles 3 of 0.05 w / v% has a wavelength of 600 nm.
When the absorbance A was measured, the absorbance A was 0.2
30, f (102) = 0.370 [A <f (d)].

[Preparation Example 3B (Preparation of Sensitized Particles)] A suspension (particles concentration: 10 w / v%) of particles 3 obtained in Preparation Example 3A described above was dissolved in 1 volume part of 100 mg / ml water-soluble. After adding 1 part by volume of carbodiimide and reacting at room temperature for 20 minutes, the mixture was centrifugally washed once with 50 mM Tris buffer (pH 7.4), and the solid content concentration was adjusted to 1 w / v% with 50 mM Tris buffer (pH 7.4). Prepared. On the other hand, 10 mg / ml
Of anti-human CRP antibody in 50 mM Tris buffer (pH 7.
It was diluted 10-fold in 4). One part by volume of the diluent of the particles 3 and 1 part by volume of the diluent of the anti-human CRP antibody were mixed, stirred at 37 ° C. for 1 hour, and then centrifuged twice with 50 mM Tris buffer (pH 7.4). did. Then 1% B
An anti-human CRP antibody-sensitized latex having a solid concentration of 0.3 w / v% (hereinafter referred to as “sensitized latex 3”) is obtained by adding and diluting a 50 mM Tris buffer solution (pH 7.4) containing SA. Was prepared.

[Preparation Example of Sample Diluent] A sample diluent (R1) was prepared by adding each of the substances shown in Table 1 to 50 mM Tris buffer (pH 7.4) containing 1% BSA.
1) to (R12) were prepared. A 50 mM Tris buffer containing 1% BSA was used as a sample diluent (R13).

[0035]

[Table 1] * HLB (Hydrophile-Lipophil)
e Balance)

<Examples 1 and 2 and Comparative Example 1>
Using the sensitized latex and the sample diluent shown in
A calibration curve was measured using the standard solution as described below.

(Measurement conditions)-Measuring device: Hitachi 7020 automatic analyzer-Wavelength used: 600 nm-Measuring temperature: 37 ° C-Specimen: [0-60 mg / dl CRP (test substance) standard solution]: 3 µl Sample diluent: as shown in Table 2 below-Sensitized latex: as shown in Table 2 below

Measurement Example 1 (Endpoint Method) After starting the mixing and stirring of the sample (3 μl), the sample diluent (300 μl) and the sensitized latex (300 μl),
The calibration curve was measured by measuring the absorbance after 11 seconds. The results are shown in FIG.

Measurement Example 2 (Late Assay) In order to evaluate low value sensitivity, a sample (3 μl), a sample diluent (300 μl) and a sensitized latex (300 μl)
Turbidity after 36 seconds from the start of mixing and stirring
A calibration curve was measured by a rate assay method that measures the difference (change amount) from the turbidity at the elapse of seconds. The results are shown in FIG.

[0040]

[Table 2]

Example 3 A calibration curve was measured in the same manner as in Example 1 except that sensitizing latex 3 was used instead of sensitizing latex 1 (use amount: 300 μl). The results are shown in FIG. 3 (measurement example 1) and FIG. 4 (measurement example 2).

As shown in FIG. 1 and FIG.
According to the latex immunoturbidimetric method of Example 3, the analyte concentration can be measured in a wide concentration range without the absorbance exceeding the measurement limit of the measuring device, and in the region where the analyte concentration is high. Did not cause the prozone phenomenon. As shown in FIGS. 2 and 4,
According to the latex immunoturbidimetry of Examples 1 to 3,
The low value sensitivity was high, and it was possible to accurately measure even a low concentration of the test substance.

[0043]

According to the latex immunoturbidimetric method of the present invention, the concentration of a test substance in a sample can be measured in a wide concentration range, and even in a region where the concentration of the test substance is high. No zone phenomenon occurs. According to the latex immunoturbidimetry of the present invention,
Even a low concentration of a test substance can be accurately quantified. ADVANTAGE OF THE INVENTION According to the measurement kit of this invention, the density | concentration of a test substance in a test substance can be measured accurately in a wide concentration range by simple and quick operation.

[Brief description of the drawings]

FIG. 1 shows that CRP was used in Examples 1 and 2 and Comparative Example 1.
It is a calibration curve which measured concentration by the end point method.

FIG. 2 shows CRPs in Examples 1-2 and Comparative Example 1.
It is a calibration curve which measured the concentration by the late assay method.

FIG. 3 is a calibration curve obtained by measuring a CRP concentration by an end point method in Example 3.

FIG. 4 is a calibration curve obtained by measuring a CRP concentration by a late assay method in Example 3.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/543 587 G01N 33/543 587 // (C08F 220/18 212: 04 220: 04) (C08F 220 / 18 212: 04 222: 02) (72) Inventor Toru Masukawa 2-11-24 Tsukiji, Chuo-ku, Tokyo Inside JSR Corporation (72) Inventor Satoshi Katayose 2-11-24 Tsukiji, Chuo-ku, Tokyo Within JSR Co., Ltd. (72) Inventor Sumi Kasai 2--11-24 Tsukiji, Chuo-ku, Tokyo JSR Co., Ltd. F-term (reference) 4J100 AB02Q AB03Q AB04Q AB07Q AB16Q AJ01R AJ02R AJ08R AJ09R AL03P AL04P AL05P AL08P BA03 BA CA05 DA65 EA07 EA09 JA53

Claims (2)

[Claims] A sample containing a test substance is mixed with a suspension of particles sensitized with a substance that reacts with the test substance with an antigen-antibody, and the aggregation state of the particles due to the antigen-antibody reaction is measured. In a latex immunoturbidimetric method for quantifying the test substance in the sample, the particles are (A) having 1 carbon atom.
20 to 100% by weight of a structural unit derived from a (meth) acrylate having from 20 to 20 aliphatic hydrocarbon groups; (B) 0 to 10% by weight of a structural unit derived from an unsaturated carboxylic acid; A) particles comprising a polymer consisting of 0 to 80% by weight of structural units derived from an acrylate and a vinyl monomer copolymerizable with the unsaturated carboxylic acid, wherein the average particle diameter of the particles is d (nm); When the absorbance at a wavelength of 600 nm measured for an aqueous dispersion having a solid concentration of particles of 0.05 w / v% is A,
A latex immunoturbidimetric method, which satisfies the following condition, and wherein an agglutination reaction promoter is present in a mixed system in which the antigen-antibody reaction is performed. Equation 1 Equation 1: A <f (d) = (M 0 -M 1 d + M 2 d 2 -M 3 d 3 + M 4 d 4) ( _{wherein, M 0 = 0.012573, M 1} = 0. 0020
732, M ₂ = 6.333 × e ⁻⁵ , M ₃ = 8.7935
× e ^-8 and M ₄ = 3.529 × e ^-11 . ) 2. A sample containing a test substance is mixed with a suspension of particles sensitized with a substance that reacts with the test substance with an antigen-antibody, and the aggregation state of the particles due to the antigen-antibody reaction is measured. The suspension of particles according to claim 1, wherein the measurement kit applied to latex immunoturbidimetry for quantifying the test substance in the sample sensitizes a substance that reacts with the test substance with an antigen-antibody. Wherein the agglutination reaction promoter is contained in the suspension or in another reagent constituting the measurement kit.