JPH10239316A - Carrier for immunodiagnosis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To control the amount of an antibody or antigen related to an agglutination reaction at a surface to be optimum and obtain a highly sensitive immunodiagnosis carrier, by using latex particles of a specific particle size and a specific degree of dispersion of particle sizes, etc. SOLUTION: This is an immunodiagnosis carrier using latex particles of a particle size of 0.05-2μm and a degree of dispersion of particle sizes of 10% or lower. The latex particles are polymerized or surface-treated so that a carboxyl group is in a range of 3-40% at a surface layer within 50Å from a surface of the latex particles. The latex particles are constituted with the use of a copolymer of styrene and a polymerizable unsaturated carboxylic acid. Furthermore, the carrier is colored with a colorant. The latex particles used are polymerized by a suitable method, e.g. dispersion polymerization, suspension polymerization, or emulsion polymerization. The emulsion polymerization is preferred.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a carrier for immunodiagnosis using latex, and more particularly, to a carrier for immunodiagnosis provided with a carboxyl group on the surface of a carrier for binding an antigen or an antibody to the surface of the carrier. Carrier.

[0002]

2. Description of the Related Art An immunoassay for detecting a substance to be detected comprising a specific antigen or antibody by utilizing a specific reaction with an antigen-antibody has been well known. In particular, an immunoassay using a carrier for immunodiagnosis can be used for any of a test for detecting a substance to be detected by simple visual inspection and a quantitative analysis method using a measuring device. It has become mainstream.

As an immunoassay using the above-mentioned immunodiagnostic carrier, for example, a latex agglutination method and an immunochromatography method in which an antibody or an antigen is bound to latex particles and used for detecting the antigen or the antibody, respectively, are known. Have been. In either method, the binding of the antibody or antigen to the latex particles is typically performed by simply adsorbing the antibody or antigen to the surface of the latex particles.

[0004] However, the passive adsorption as described above has a problem that the reproducibility, stability and measurement sensitivity are reduced because the antibody or antigen is easily desorbed. That is, although immunoassays using immunodiagnostic carrier particles and sensitized fine particle reagents are known, they did not satisfy the recently required characteristics of high sensitivity, good reproducibility and storage stability.

Various improvements have been made to solve the above problems. In order to enhance the dispersibility of latex particles in an aqueous solvent, a method of subjecting the surface of the latex particles to a hydrophilic treatment or introducing a carboxyl group to the surface of the latex particles has been proposed. By these treatments, the surface of the latex particles has a negative charge, the particles repel each other in water or an aqueous solvent, the dispersibility of the particles is increased, and non-specific aggregation can be reduced.

On the other hand, a carboxyl group is a functional group suitable for covalently bonding an antibody or antigen to latex particles, and has the advantage that the antibody or antigen is hard to desorb from the latex particles.

[0007] However, as the dispersibility of latex particles is enhanced, the repulsion of the electrostatic charge increases, and even if the antigen or antibody as a detection substance is adsorbed on the antibody or antigen adsorbed on the latex particles, the latex does not. It is difficult to get close to the distance where the particles can bind, and as a result,
In some cases, latex particles did not aggregate even when the detection substance was adsorbed. For this reason, it sometimes becomes impossible to detect the antigen or antibody as the detection substance.

In order to suppress the decrease in detection sensitivity due to the above-mentioned electrostatic repulsion, a method has been proposed in which a salt is added to an aqueous solvent to suppress the electrostatic repulsion or to control the amount of a carboxyl group. (JP-A-7-301632 and JP-A-58-215403).

For example, in Japanese Patent Application Laid-Open No. Hei 7-301632, the carboxyl group content of latex particles is determined by titration, and the content is divided by the total surface area of the latex particles to obtain a carboxyl group occupying region (particle surface). Is calculated.) However, in this titration, the carboxyl groups present inside the latex particles are also measured, so if the carboxyl group content is controlled by this calculation, there will be more carboxyl groups on the particle surface than the divided value. It is not unusual for the actual amount of carboxyl groups on the surface to be several times or more different from the target amount of carboxyl groups on the surface depending on the size of the latex particles.

On the other hand, Japanese Patent Application Laid-Open No. 58-215403 employs a neutralization titration method similar to the above prior art, but disperses latex particles in an aqueous solvent, adds a basic substance, and heats the dispersion. As a result, a carboxyl group, which is an acidic electrolytic group, is transferred to the particle surface, and thereafter, a neutralization titration is performed. Therefore, the carboxyl group which is an acidic electrolytic group on the surface of the latex particles can be determined.

However, also in this method, the transfer state of the acidic electrolytic group changes depending on the particle size of the latex particles, and it is difficult to transfer 100% of the carboxyl groups inside the particles to the particle surface.

[0012] In addition, in the case of colored latex particles containing a colorant or a dye therein, when a basic substance is added and heated, the colorant or the dye may be transferred to the surface of the latex particle. However, it was practically impossible to accurately measure only the acidic electrolytic groups to be quantified present on the surface.

In addition, in a system containing a large amount of an initiator, when the terminal of the initiator contains a functional group to be subjected to neutralization titration, in the above-mentioned conventional method, the amount of the acidic electrolytic group to be quantified is smaller than the actual amount. Was also measured a lot. As a result, the acidic electrolytic group actually present on the surface is considerably separated from the intended amount of the acidic electrolytic group.

[0014]

SUMMARY OF THE INVENTION An object of the present invention is to provide a carrier for immunodiagnosis using latex particles used in a latex agglutination method or an immunochromatography method. Accordingly, it is an object of the present invention to provide a highly sensitive immunodiagnostic carrier capable of optimally controlling the amount of an antibody or an antigen involved in an agglutination reaction on the surface.

[0015]

The carrier for immunodiagnosis according to the first aspect of the present invention uses latex particles having a particle diameter of 0.05 to 2 μm and a degree of dispersion of the particle diameter of 10% or less. A carrier for immunodiagnosis, wherein the latex particles are polymerized or surface-treated such that the carboxyl group is within a range of 3 to 40% in a surface layer within a range of 50 ° from the surface of the latex particles. I do.

According to a second aspect of the present invention, there is provided the carrier for immunodiagnosis according to the first aspect of the present invention, wherein the latex particles comprise a copolymer of styrene and a polymerizable unsaturated carboxylic acid. It is characterized by comprising.

According to a third aspect of the present invention, in the immunodiagnostic carrier according to the first or second aspect, the carrier is colored by a coloring agent. Hereinafter, details of the present invention will be described.

The latex particles used in the present invention are polymerized by an appropriate polymerization method such as dispersion polymerization, suspension polymerization or emulsion polymerization, preferably by emulsion polymerization. The monomer component for constituting the latex particles is not particularly limited, but may be, for example, at least one polymerizable unsaturated aromatic compound such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene. Species and at least one of polymerizable unsaturated carboxylic acids such as methacrylic acid, acrylic acid and itaconic acid.
It can be obtained by polymerizing or copolymerizing a monomer composition consisting of seeds. Above all, as described in claim 2, a copolymer composed of styrene and at least one kind of polymerizable unsaturated carboxylic acid is particularly preferably used.

The preferred compounding ratio of the polymerizable unsaturated aromatics and the polymerizable unsaturated carboxylic acids is 0.1 to 150 parts by weight of the polymerizable unsaturated carboxylic acids per 100 parts by weight of the polymerizable unsaturated aromatics. Parts, more preferably 0.5 to
It is in the range of 80 parts by weight. If the blending ratio of the polymerizable unsaturated carboxylic acids is less than 0.1 part by weight, the amount of the carboxyl group becomes insufficient, and if it exceeds 150 parts by weight, the degree of dispersion of the particle diameter may increase.

The particle size of the above latex particles is usually 0.05 to 2.0 μm, preferably 0.05 to 2.0 μm, because it is easy to handle for a diagnostic reagent.
０．８0.8 μm. If copolymerized in accordance with the above mixing ratio, spherical latex particles having a particle diameter in the range of 0.05 to 2.0 μm can be easily obtained.

The degree of dispersion of the latex particle diameter is as follows:
10% or less. If the degree of dispersion exceeds 10%, the accuracy of the obtained reagent will decrease. Further, in the present invention,
A surface layer within 50 ° from the surface of the latex particles,
The latex particles are polymerized as described above so that the carboxyl groups are present in the range of 3 to 40%. By treating the surface of the latex particles, the content ratio of the carboxyl groups in the surface layer is 3 to 40. % May be used.

As a method of introducing a carboxyl group by surface treatment, a method of irradiating a latex particle having the above particle diameter, for example, a polystyrene-based latex particle with ultraviolet rays can be mentioned. In this case, in the case of polystyrene latex particles dispersed in an aqueous solvent, since water molecules shield ultraviolet rays, it is preferable to irradiate the ultraviolet rays in a state where the concentration of the latex particles is high or in a thin layer state.

Further, in the invention described in claim 1,
The content ratio of carboxyl groups in the surface layer can also be within the above specific range by seed polymerization of unsaturated carboxylic acids on the aromatic latex particles.

The measurement of the carboxyl group content in the surface layer was carried out in the present specification by the following method. That is, the functional groups on the surface of the produced latex particles were analyzed by a time-of-flight secondary ion mass spectrometer (Charles
Evans TFS Surface Analyzer: T
Abbreviated as OF-SIMS. ) Was used to perform the following spectrum measurement.

Preparation of sample: A latex dispersion was dropped on a silicon wafer and dried at room temperature to obtain a sample. Measurement conditions: primary ion: 69 Ga ⁺ ; ion voltage: 1
5 kV; ion current: 2 μA; analysis time: 10 minutes; charge prevention: neutralization of electron irradiation and random raster scan by inserting a mesh on the sample surface.

The analysis conditions are as follows. That is, a polystyrene latex containing no carboxylic acids was measured as described above, and as shown in the formula (1), the total count of the polystyrene latex containing a carboxyl group (m5
0 to 200), the ratio of m91 (carbostyrene count; styrene count in polystyrene latex containing carboxyl groups) to m50 to 20.
It was divided by the count ratio near m91 (m90.8 to m91.4), which is a peak unique to polystyrene in the total count of 0, subtracted from 1 and expressed as a percentage to obtain the carboxyl group content C (%).

[0027]

(Equation 1)

By using latex particles whose C (%) is in the range of 3 to 40%, more preferably 5 to 30%, as will be apparent from the examples described below, a very useful latex diagnostic agent can be obtained. Can be made.

When the C (%) is lower than 3%, the amount of the antibody or the antigen that can be immobilized on the surface of the latex particles becomes unnecessarily small. The change in turbidity due to aggregation is small, the sensitivity is reduced, and the measurement time is prolonged.

Conversely, when C (%) is higher than 40%,
The amount of the antibody or antigen that can be immobilized on the surface of the latex particles becomes too large, exceeds the amount that can participate in aggregation even when bound to the reacting antigen or antibody, and aggregation that reflects the antigen or antigen amount does not occur. Further, since the antibody or the antigen is used more than necessary, the cost is increased.

In the present invention, the latex particles may be colored with a coloring agent according to the use as described in claim 3. In this case, the coloring method using a coloring agent can be performed by blending a coloring agent at the stage of preparing latex particles, but can be preferably performed by a method disclosed in Japanese Patent Application No. 8-288234.

That is, the following method can be employed for coloring latex particles with a coloring agent. First, a dye that is soluble in a solvent in which latex particles are insoluble is selected, and the dye is dissolved in the solvent to near the saturation solubility. Examples of such a solvent include ethers such as ethyl ether and isopropyl ether; alcohols such as methanol and ethanol; methylene chloride; ethylene dichloride; chloroform; carbon tetrachloride; ethyl acetate; methyl acetate; methyl ethyl ketone; Cyclopentane; tetrahydrofuran; toluene; hexane; heptane; water and the like are used, and are used as one or more mixed solutions depending on the type of dye.

Next, latex particles are added to the dye solution and dispersed. Next, the dye solution containing the latex particles is heated with stirring. Then, the dye is gradually contained in the latex particles, and the dye concentration in the solution gradually decreases. When the dye concentration in the solution decreases, the dye tends to equilibrate in the latex particles and the solution, so that the dye concentration in the solution decreases by evaporating the solvent or gradually adding new dye. Not to be. Such a method promotes the transfer of the dye from the solution to the latex particles.

The colorant is not particularly limited as long as it can color latex particles.
Dyes and pigments. The dye is appropriately selected depending on the color tone, the type of the solvent in which the dye is dissolved, the type of the latex, and the like, but is preferably an oil-soluble dye, a direct dye, an acid dye,
Examples thereof include basic dyes, azoic dyes, aqueous dyes, reactive dyes, and the like, and oil-soluble dyes are particularly preferable. Specific examples of the oil-soluble dye include Solvent Blue, Solvent Red, Solvent Orange, and Solvent Green. Dyes include food dyes, vital dyes, cosmetic dyes, pharmaceutical dyes, and the like.

In the carrier for immunodiagnosis according to the present invention, a preferable method for covalently bonding the above-mentioned latex particles to an antibody or an antigen includes a method using a carbodiimide condensation reagent. The use of a carbodiimide condensing reagent for performing condensation between a carboxyl group and an amine is well known in the immunodiagnostic industry, and is a particularly suitable carbodiimide condensing reagent for condensing a carboxyl group on the surface of latex particles with an antibody or antigen. Include dicyclohexylcarbodiimide (DCC), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide (CMC), and the like. Among them, EDC is particularly preferable in consideration of high solubility, excellent effectiveness under mild conditions, and ease of reaction control.

The immunodiagnostic carrier according to the invention described in the action claim 1, uses a latex particles having the specific particle diameter and degree of dispersion, the carboxyl in the surface layer in the range of 50Å from the surface of the latex particles Since the base amount is in the range of 3 to 40% of the whole, the amount of the antibody or antigen that can be immobilized on the latex particle surface as described above becomes a sufficient amount,
And because the amount of the antibody or antigen that can be immobilized is not excessive, the reaction with the antigen or antibody in the sample that reacts with the antibody or antigen is sufficiently performed, and the latex particles result in the reaction, and the antigen or antibody in the sample is reacted. Aggregate reliably according to amount. Therefore, the amount of antigen or antibody in the sample can be detected with high accuracy.

[0037]

The present invention will now be described by way of examples.
The present invention will be elucidated. It should be noted that the following examples are non-limiting and, for example, the type of antibody or antigen can be arbitrarily selected.

(Preparation of carboxylated latex) A raw material for emulsion polymerization consisting of 240 g of styrene, 30 g of acrylic acid, 50 g of a 5% by weight aqueous solution of potassium persulfate and 2250 g of water was stirred at a rate of 180 rpm under a nitrogen stream.
Emulsion polymerization was performed at 8 ° C. for 8 hours to obtain a styrene-acrylic acid copolymer latex having an average particle diameter of 0.30 μm and a solid content of 8% by weight. Based on the production conditions, the composition and the polymerization temperature were changed so that the average particle size was 0.10 to 0.30 ± 0.05 μm.
m and carboxyl group content is 0.06-0.87me
A plurality of q / g carboxyl group-containing latexes were produced. These latexes were all suspended in pure water and prepared as a 5% by weight suspension.

(Measurement of Charge by Reverse Titration Method) 3 g of a 5% by weight latex suspension was sampled, and then N / 10 hydrochloric acid was added and stirred, and N / 100 sodium hydroxide was added at 150 sec / ml.
And the conductivity was measured over time. A curve showing the relationship between the conductivity and the sodium hydroxide titer was determined from the above measurement results, and the carboxyl group content a was determined by the following equation.

[0040]

(Equation 2)

V _ab = amount of sodium hydroxide N = normality of sodium hydroxide W = weight of latex suspension S = latex suspension solids (% by weight) That is, W (3 g) × S (0.05) = 0.15
(G).

(Measurement of Surface Carboxyl Group Amount by TOF-SIMS) As described above, the carboxyl group amount according to the formula (1) was measured using the TOF-SIMS described above.

(Reagentization of latex) Carboxylated latex is suspended in MES solution (pH = 6.1).
1 ml of 10 mg / ml EDC in 10 ml of a weight% suspension
And the hCG antibody was added to ME at a concentration of 1 mg / ml.
HCG antibody solution 10 added to solution S (pH = 6.1)
ml was added and stirred at room temperature for 3 hours. Next, the mixture was centrifuged at 4 ° C. under the conditions of 15000 rpm for 20 minutes, the supernatant was removed, washed three times with MES solution, and suspended in a blocking buffer containing 1% serum albumin.
An hCG-sensitized latex suspension having a concentration of 0.1% by weight was prepared.

(Evaluation) Reactivity with anti-hCG antiserum was compared for each sensitized carboxylated latex. Anti-h
CG antiserum is given in 10 steps between 100-20,000-fold, ie 100, 200, 400, 800, 100
0, 2000, 4000, 8000, 12000 and 1
After dilution to 6000 times each concentration, the maximum dilution at which aggregation was observed was evaluated. The results are shown in Table 1 below.

[0045]

[Table 1]

As is clear from Table 1, the TOF-SIM
In Comparative Example 1 in which the C (%) measured in S was as high as 63.9%, the antiserum dilution factor that could be detected was as low as 200, indicating that the measurement sensitivity was low.

Similarly, also in Comparative Example 2 in which C (%) is as low as 2.6%, the antiserum dilution factor is as low as 400, indicating that the measurement sensitivity is not sufficient. On the other hand, C (%)
However, in Examples 1 to 3 in the range of 10.1 to 30.5, the detected antiserum dilution factor was as high as 8000 times or more, and particularly in Examples 1 and 3, it was as high as 12000 times. It turns out that it is excellent.

[0048]

As described above, according to the first aspect of the present invention, in the immunodiagnostic carrier using the latex particles having the specific particle size and the degree of dispersion, the surface within a range of 50 ° from the surface of the latex particles is used. Since the structure is such that the carboxyl group in the layer is in the range of 3 to 40%, the measurement sensitivity at the time of diagnosis utilizing an immune reaction can be reliably increased. In addition, since the correlation between the sensitivity when the latex is converted into a reagent and the amount of the carboxyl group is better than the conventional titration method, an immunodiagnostic carrier more suitable as an immunodiagnostic reagent can be provided. You can see that.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Yuji Kaneko 2-1 Hyakuyama, Shimamoto-cho, Mishima-gun, Osaka Sekisui Chemical Co., Ltd.

Claims (3)

[Claims] 1. An immunodiagnostic carrier using latex particles having a particle diameter of 0.05 to 2 μm and a degree of dispersion of the particle diameter of 10% or less, wherein the carrier is within 50 ° from the surface of the latex particles. Carboxyl group in surface layer is 3 ~
A carrier for immunodiagnosis, wherein latex particles are polymerized or surface-treated so as to fall within a range of 40%. 2. The carrier for immunodiagnosis according to claim 1, wherein the latex particles are constituted by using a copolymer composed of styrene and a polymerizable unsaturated carboxylic acid. 3. The immunodiagnostic carrier according to claim 1, wherein the carrier is colored by a coloring agent.