JP3478939B2 - Carrier for immunodiagnosis - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a carrier for immunodiagnosis using a latex, and more particularly to an immunodiagnosis carrier having a carboxyl group on the surface of a carrier for binding an antigen or an antibody to the surface of the carrier. Regarding the carrier.

[0002]

2. Description of the Related Art An immunoassay method for detecting a substance to be detected consisting of a specific antigen or antibody by utilizing a specific reaction between an antigen and an antibody is well known in the art. In particular, the immunoassay using the immunodiagnostic carrier 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 is the mainstream.

Immunoassays using the above immunodiagnostic carriers include, for example, a latex agglutination method and an immunochromatography method in which an antibody or an antigen is bound to latex particles and used to detect the antigen or the antibody, respectively. Has 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 on the surface of the latex particles.

However, the passive adsorption as described above has a problem that the reproducibility, stability and measurement sensitivity are lowered because the antibody or the antigen is easily desorbed. That is, although immunoassays using carrier particles for immunodiagnosis and sensitized fine particle reagents are known, they have not satisfied the required characteristics of high sensitivity, good reproducibility and storage stability that have been recently demanded.

Various improvements have been made in order to solve the above problems. In order to improve the dispersibility of latex particles in an aqueous solvent, methods have been proposed in which the surface of the latex particles is subjected to hydrophilic treatment, or a carboxyl group is introduced into the surfaces of the latex particles. 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 enhanced, and non-specific aggregation can be reduced.

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

[0007] However, as the dispersibility of the latex particles is increased, conversely, electrostatic repulsion is increased, and even if the antibody or antigen adsorbed on the latex particles is adsorbed by the antigen or antibody as the detection substance, It becomes difficult for particles to come close to each other, and as a result,
Even if the detection substance was adsorbed, the latex particles sometimes did not aggregate. Therefore, the detection of the antigen or antibody that is the detection substance may not be possible.

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

For example, in Japanese Unexamined Patent Publication No. 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) on the surface. The amount of the carboxyl group of) is calculated. However, this titration also measures the carboxyl groups present inside the latex particles, so if the carboxyl group content is controlled by this calculation, there will be more carboxyl groups than the divided value on the particle surface. It was not uncommon for the actual amount of carboxyl groups on the surface to differ by several times or more from the target amount of carboxyl groups on the surface, depending on the size of the latex particles.

On the other hand, in Japanese Patent Laid-Open No. 215403/1983, a neutralization titration method is adopted as in the above-mentioned prior art, but latex particles are dispersed in an aqueous solvent, and a basic substance is added and heated. Thus, the carboxyl group, which is an acidic electrolytic group, is transferred to the surface of the particle, and then neutralization titration is performed. Therefore, it is possible to quantify the carboxyl group which is the acidic electrolytic group on the surface of the latex particles.

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

In addition, in the case of colored latex particles containing a coloring agent or dye inside, when a basic substance is added and heated, the coloring agent or dye may also migrate to the latex particle surface. However, it was virtually impossible to accurately measure only the acidic electrolytic groups present on the surface to be quantified.

Further, in a system in which a large amount of an initiator is mixed, when the end of the initiator contains a functional group to be subjected to neutralization titration, in the above-mentioned conventional method, the acidic electrolytic group to be quantified is more than the actual amount. Was also often measured. As a result, the amount of acidic electrolytic groups actually present on the surface and the target amount of acidic electrolytic groups were considerably separated.

[0014]

An object of the present invention is to provide a carrier for immunodiagnosis using latex particles used in latex agglutination method, immunochromatography method, etc., wherein carboxyl groups on the surface of latex particles can be accurately quantified. By doing so, it is possible to provide a highly sensitive carrier for immunodiagnosis, which can optimally control the amount of the antibody or antigen involved in the agglutination reaction on the surface.

[0015]

The carrier for immunodiagnosis according to the invention of claim 1 uses latex particles having a particle size of 0.05 to 2 μm and a particle size dispersity of 10% or less. The carrier for immunodiagnosis, which has a flight time of 5 nm from the surface of the latex particle
The following formula measured using a secondary ion mass spectrometer
The carboxyl group content ratio represented by C (%) in (1) is
It is characterized in that the latex particles are polymerized or surface-treated so as to be in the range of 10.1 to 30.5 %.

[Equation 2] (In the formula, CalboStyreneCount (90.
8-91.4) is a polystyrene containing a carboxyl group.
Count of styrene in latex (m90.8-m9
1.4) to CalboLatexTotalCount
t (50 to 200) is a polyester containing a carboxyl group
Ren latex total count (m50-200)
In the StyreneCount (90.8-91.4)
Is a polystyrene latex containing no carboxylic acids.
Count of styrene in the glass (m90.8 to m91.4)
LatexTotalCount (50-200)
Is a polystyrene latex containing no carboxylic acids.
Table of total count (m50 to 200)
You )

According to a second aspect of the present invention, in the immunodiagnostic carrier according to the first aspect of the present invention, the latex particle is a copolymer of styrene and a polymerizable unsaturated carboxylic acid. It is characterized by being configured.

The invention according to claim 3 is the immunodiagnostic carrier according to claim 1 or 2, characterized in that the carrier is colored with a coloring agent. Hereinafter, the 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 emulsion polymerization. The monomer component for forming the latex particles is not particularly limited, but is, for example, at least one of polymerizable unsaturated aromatics such as styrene, chlorostyrene, α-methylstyrene, divinylbenzene, and vinyltoluene. At least one of a seed and a polymerizable unsaturated carboxylic acid such as methacrylic acid, acrylic acid and itaconic acid
It can be obtained by polymerizing or copolymerizing a monomer composition comprising a seed. Among them, 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 preferable mixing 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 to 100 parts by weight of the polymerizable unsaturated aromatics. Part range, more preferably 0.5 to
It is in the range of 80 parts by weight. When the compounding ratio of the polymerizable unsaturated carboxylic acid is less than 0.1 parts by weight, the amount of the carboxyl group becomes insufficient, and when it exceeds 150 parts by weight, the dispersity of the particle size may be increased.

The particle size of the latex particles is usually 0.05 to 2.0 μm, preferably 0.05, because it is easy to handle as a diagnostic reagent.
Is about 0.8 μm. If the copolymerization is carried out according to the above blending 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 size is 10% or less. When the dispersity exceeds 10%, the accuracy of the obtained reagent decreases. Further, in the present invention, the latex particles are polymerized as described above so that the carboxyl groups are present in the range of 10.1 to 30.5 % in the surface layer within the range of 5 nm from the surface of the latex particles. However, the content ratio of the carboxyl group in the surface layer is 10.1 by surface-treating the latex particles.
It may be 30.5 %.

Examples of the method of introducing a carboxyl group by surface treatment include a method of irradiating the latex particles having the above particle diameter, for example, polystyrene latex particles, with ultraviolet rays. In this case, in the case of polystyrene-based latex particles dispersed in a water solvent, since water molecules shield ultraviolet rays, it is preferable to irradiate ultraviolet rays in a state in which the concentration of latex particles is increased or in a thin layer state.

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

The carboxyl group content in the surface layer was measured by the following method in this specification. That is, the functional groups on the surface of the prepared 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: The 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 ... Insert mesh on sample surface, electron irradiation neutralization and random raster scan.

The analysis conditions are as follows.
That is, the polystyrene latex containing no carboxylic acids was measured as described above, and as shown in the formula (1), in order to eliminate the influence of the preservatives, etc., the total count of the polystyrene latex containing the carboxyl group (m
50-200), the count (carbostyrene count; count of styrene in polystyrene latex containing a carboxyl group) near m91 was calculated as m50-2.
It was divided by the count ratio in the vicinity of m91 (m90.8 to m91.4), which is a peak peculiar to polystyrene in the total count of 00, subtracted from 1, and expressed as a percentage to give a carboxyl group content ratio C (%).

[0027]

[Equation 1]

The above C (%) is 10.1 to 30.5 %
By using latex particles in the range described above, a very useful latex diagnostic agent can be prepared, as will be apparent from the examples described below.

When C (%) becomes lower than 10.1 %,
The amount of antibody or antigen that can be immobilized on the latex particle surface becomes unnecessarily small, and even if there is a sufficient amount of reacting antigen or antibody in the sample, it will not be able to react and the turbidity change due to aggregation will be small, and the sensitivity will be low. It will decrease or the measurement time will increase.

On the other hand, when C (%) is higher than 30.5 %, the amount of antibody or antigen that can be immobilized on the latex particle surface becomes too large, and the amount that can be involved in aggregation even if it binds to the reacting antigen or antibody. And the aggregation that reflects the antigen or the amount of the antigen does not occur. Moreover, since the antibody or the antigen is used more than necessary, the cost becomes high.

In the present invention, the latex particles may be colored with a coloring agent depending on the application as described in claim 3. In this case, the method of coloring with a colorant may be carried out by blending a colorant at the stage of producing latex particles, but preferably the method disclosed in Japanese Patent Application No. 8-288234.

That is, as a method of coloring the latex particles with a coloring agent, the following method can be adopted. 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 up to a 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; cyclohexane; Cyclopentane; Tetrahydrofuran; Toluene; Hexane; Heptane; water, etc., and one or more mixed solutions are used depending on the type of dye.

Next, latex particles are added to and dispersed in this dye solution. Next, the dye solution containing this latex particle group is heated with stirring. Then, the dye is gradually contained in the latex particles, and the concentration of the dye in the solution gradually decreases. When the dye concentration in the solution decreases, the dye tends to reach an equilibrium state in the latex particles and in the solution, so the solvent concentration may be reduced or a new dye may be gradually added to lower the dye concentration in the solution. Try not to. By such a method, transfer of the dye from the solution to the latex particles is promoted.

The colorant is not particularly limited as long as it can color the latex particles.
Examples include dyes and pigments. The dye is appropriately selected depending on the color tone, the type of solvent that dissolves the dye, the type of latex, and the like. Oil-soluble dyes, direct dyes, acid dyes,
Examples thereof include basic dyes, azoic dyes, aqueous dyes, reactive dyes, etc., and oil-soluble dyes are particularly preferable. Specific examples of the oil-soluble dye include solvent blue, solvent red, solvent orange, solvent green and the like. Examples of the dye include food dyes, vital dyes, cosmetic dyes, and pharmaceutical dyes.

In the carrier for immunodiagnosis according to the present invention, a preferred method for covalently bonding the latex particles and the antibody or antigen is to use a carbodiimide condensation reagent. The use of a carbodiimide condensing reagent for condensing a carboxyl group with an amine is well known in the immunodiagnostic industry, and a particularly suitable carbodiimide condensing reagent for condensing a carboxyl group on the latex particle surface with an antibody or an 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 view of high solubility, excellent effectiveness under mild conditions, and easy 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 in the surface layer in the range of 5nm from the surface of the latex particles Carboxyl group content represented by C (%)
Since the ratio is within the range of 10.1 to 30.5 %, the amount of the antibody or antigen that can be immobilized on the latex particle surface is sufficient as described above, and the amount of the antibody or antigen that can be immobilized. Is not excessive, the reaction with the antigen or the antibody in the sample that reacts with the antibody or the antigen is sufficiently performed, and the latex particles are surely aggregated according to the amount of the antigen or the antibody in the sample as a result of the reaction. Therefore,
The amount of antigen or antibody in a sample can be detected with high accuracy.

[0037]

EXAMPLES Hereinafter, by giving examples of the present invention,
The present invention will be clarified. It should be pointed out that the following examples are non-limiting, and the type of antibody or antigen can be arbitrarily selected.

(Production 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 wt% potassium persulfate aqueous solution and 2250 g of water was stirred at a speed of 180 rpm under a nitrogen stream 6
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 these production conditions, the composition and polymerization temperature were changed, and the average particle size was 0.10 to 0.30 ± 0.05μ.
m and carboxyl group content is 0.06-0.87me
Plural kinds of q / g carboxyl group-containing latex were prepared. All of these latexes were suspended in pure water to prepare a 5 wt% suspension.

(Measurement of Charge by Back Titration Method) 3 g of a 5 wt% latex suspension was sampled, N / 10 hydrochloric acid was added and the mixture was stirred, and N / 100 sodium hydroxide was added for 150 seconds / ml.
Then, the conductivity was measured with 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 amount a was determined by the following equation.

[0040]

[Equation 2]

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

(Measurement of Surface Carboxyl Group Content Ratio by TOF-SIMS) As described above, the carboxyl group content according to the formula (1) is contained.
The ratio was measured using the TOF-SIMS described above.

(Reaction of latex) Carboxylated latex is suspended in MES solution (pH = 6.1) 1
1 ml of 10 mg / ml EDC in 10 ml of suspension by weight
ME with hCG antibody to a concentration of 1 mg / ml.
HCG antibody solution 10 added to S solution (pH = 6.1)
ml was added, and the mixture was stirred at room temperature for 3 hours. Next, the mixture was centrifuged at 4 ° C. and 15,000 rpm for 20 minutes, the supernatant was removed, washed three times with MES solution, and then suspended in a blocking buffer containing 1% serum albumin,
A hCG-sensitized latex suspension having a concentration of 0.1% by weight was prepared.

(Evaluation) The reactivity with anti-hCG antiserum was compared in each sensitized carboxylated latex. Anti h
The CG antiserum has 100 steps, that is, 100, 200, 400, 800, and 100 steps in 100 to 20000 times.
0, 2000, 4000, 8000, 12000 and 1
It was diluted to each concentration of 6000 times, and the maximum dilution number at which aggregation was observed was evaluated. The results are shown in Table 1 below.

[0045]

[Table 1]

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

Similarly, in Comparative Example 2 in which the 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 antiserum dilution factor that could be detected was as high as 8000 times or more, and particularly in Examples 1 and 3, it was as high as 12000 times, and the detection sensitivity was high. It turns out to be excellent.

[0048]

As described above, according to the invention of claim 1, in the immunodiagnostic carrier using the latex particles having the above-mentioned specific particle size and dispersity, the surface within 5 nm from the surface of the latex particles. In the C (%) in the layer
Carboxyl group content represented the 10.1 to 30.5
Since it is configured to be within the range of%, it is possible to surely increase the measurement sensitivity in the diagnosis using the immune reaction. In addition, compared to conventional titration, and sensitivity in the case of a reagent the latex, carboxyl group-containing
It can be seen that the carrier for immunodiagnosis, which is more suitable as a reagent for immunodiagnosis, can be provided because the correlation with a certain proportion is good.

─────────────────────────────────────────────────── ─── Continuation of the front page (56) References JP-A-57-168163 (JP, A) JP-A-7-301632 (JP, A) JP-A-58-215403 (JP, A) (58) Field (Int.Cl. ⁷ , DB name) G01N 33/53-33/579

Claims (3)

(57) [Claims] 1. An immunodiagnostic carrier using latex particles having a particle size of 0.05 to 2 μm and a particle size dispersity of 10% or less, wherein
In the surface layer within the range of 5 nm, the time-of-flight secondary ion is
C of the following formula (1) measured using a mass spectrometer
The carboxyl group content ratio represented by (%) is 10.1 to
An immunodiagnostic carrier characterized in that latex particles are polymerized or surface-treated so as to be within the range of 30.5 %. [Equation 1] (In the formula, CalboStyreneCount (90.
8-91.4) is a polystyrene containing a carboxyl group.
Count of styrene in latex (m90.8-m9
1.4) to CalboLatexTotalCount (50-2 )
00) is a polystyrene latex containing a carboxyl group
The total count (m50 to 200) of the space , the StyreneCount (90.8 to 91.4) is
In polystyrene latex containing no carboxylic acids
Styrene count (m90.8-m91.4) of LatexTotalCount (50-200) is
Of polystyrene latex containing no carboxylic acids
Table of total counts (m50 to 200)
You ) 2. The carrier for immunodiagnosis according to claim 1, wherein the latex particles are composed of a copolymer of styrene and polymerizable unsaturated carboxylic acids. 3. The carrier for immunodiagnosis according to claim 1, which is colored with a coloring agent.