JP2000081436A - Diagnostic agent and polymer grain for it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an excellent turbidity calibration curve with a long proportional area by comprising polymer grain and a dispersion medium and setting a relationship represented by a specific formula between density of them. SOLUTION: A diagnostic agent is a polymer dispersion product consisting of polymer grain and a dispersion medium, and a relationship between density of polymer grain and that of the dispersion medium is represented by the following expression: (polymer grain density)-(dispersion medium density)=-0.02-+0.02 g/ml (at about 25 deg.C). Outside this range, an inclination of a calibration curve is lowered on the high concentration side of a tested material. A using quantity of the polymer grain is usually 0.001-1 wt.%, and density of the polymer grain at about 25 deg.C is 0.983-1.023 approximately. Outside this density range, a proportional area of the turbidity calibration curve is narrowed, and particularly, an inclination is lowered on the high concentration side. As the dispersion medium, physiological saline is used usually, and physiological saline, to which a sensitiveness regulating agent, a pH buffer, an antiseptic agent, or the like is added, can be also used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a diagnostic agent and a polymer particle for a diagnostic agent used in the measurement of biochemical substances, particularly in medical examinations. In particular, the present invention relates to an aqueous dispersion of polymer particles whose surface has been sensitized with an antigen, an antibody or a nucleic acid, and a substance that specifically binds to the aqueous dispersion (hereinafter referred to as a “substance to be tested”). The present invention relates to a diagnostic agent used for a test for quantifying the amount of a corresponding antibody or antigen by measuring a change in turbidity due to aggregation and polymer particles for the diagnostic agent.

[0002]

2. Description of the Related Art It is an important means of a clinical test to carry an immunoreactive substance such as an antibody or an antigen or a nucleic acid on a carrier particle and detect a corresponding test substance such as an antigen, an antibody or a nucleic acid by a specific reaction. It is. For example, a dispersion in which an antibody is carried on the surface of polymer particles by physical adsorption or chemical bonding is prepared, and a test solution containing the corresponding antigen is added. Then, a change in the surface state of the particles due to the specific binding of the antigen / antibody on the particle surface or a bridge between the particles occurs, and the particles aggregate. This change can be visually observed on a suitable plate, but the change in turbidity of the sample, usually caused by aggregation, is measured with an instrument. In many current measurement systems, the rate of change in turbidity of a sample after the addition of a test solution is measured, and a calibration curve (hereinafter, referred to as a "turbidity calibration curve") for the amount of a test substance obtained in advance is measured. Quantification of the test substance is performed using In some measurement systems, a turbidity calibration curve is created based on the quasi-equilibrium value of the turbidity after the addition of the test solution for a certain period of time. In these turbidity calibration curves, the slope is usually small on the low concentration side (early insensitive region), gradually becomes a constant gradient (proportional region), and becomes higher at higher concentrations (late insensitive region), and in some cases, the concentration becomes even higher. In some cases, a region with a reverse gradient (prozone region) may occur. As a latex diagnostic agent, it is desired that the proportional region of the turbidity calibration curve starts at a low concentration and is as long as possible up to a high concentration. In addition, the prozone phenomenon impairs the reliability of the inspection and must be avoided as much as possible. Conventionally, as the diagnostic agent carrier polymer particles, polystyrene particles or small amounts of methacrylic acid, acrylic acid, itaconic acid, carboxylic acid-modified styrene-based particles obtained by copolymerizing styrene with carboxylic acid monomers such as fumaric acid have been used, The antibody antigen or the like is sensitized to this, and the medium of the diagnostic agent is adjusted mainly with physiological saline and some additives to adjust the performance of the diagnostic agent. In the technology of latex diagnostics so far, the selection of antibodies to be used, sensitization method, sensitization conditions, optimization of dispersion composition, etc. are performed to widen the proportional range of the turbidity calibration curve and eliminate prozone. That was to be adjusted. However, it is difficult to sufficiently prepare prozone by the conventional method, and a technique for further increasing the proportional region of the turbidity calibration curve has been desired.

[0003]

SUMMARY OF THE INVENTION The present invention provides a diagnostic agent and a polymer particle for the diagnostic agent which can provide a good turbidity calibration curve having a long proportional region even under standard sensitization conditions.

[0004]

The present invention comprises polymer particles and a dispersion medium, and the relationship between the density of the polymer particles and the density of the dispersion medium is expressed as (polymer particle density) − (dispersion medium density) = − The diagnostic agent according to claim 1, wherein the diagnostic agent has a density of 0.02 to +0.02 g / ml and a density at 25C of 0.983 to 1.023 g / ml, preferably 0.993 to 1.013 g / ml. It is intended to provide medicinal polymer particles.

Hereinafter, the present invention will be described more specifically. <Polymer particles> In the present invention, the composition of the polymer particles is not particularly limited. For example, 45 to 95% by weight of 2-ethylhexyl acrylate, 55 to 5% by weight of styrene, methacrylic acid, acrylic acid, itaconic acid, fumaric acid Polymer particles obtained by copolymerizing a monomer comprising 0 to 5% by weight of unsaturated carboxylic acid such as 2-ethyl methacrylate 7
Polymer particles obtained by copolymerizing a monomer comprising 0 to 100% by weight, 0 to 30% by weight of styrene, 0 to 5% by weight of unsaturated carboxylic acid such as methacrylic acid, acrylic acid, itaconic acid, fumaric acid, isobutyl acrylate 70-100% by weight, styrene 0-30% by weight, unsaturated carboxylic acids such as methacrylic acid, acrylic acid, itaconic acid, fumaric acid, etc.
Polymer particles obtained by copolymerizing a monomer consisting of 5% by weight are exemplified. Of these, polymer particles mainly using 2-ethylhexyl acrylate are preferred because of their wide range of density adjustment. In the polymer composition, particles without unsaturated carboxylic acid are mainly used for sensitization with antigen and antibody by physical adsorption, and particles obtained by copolymerizing unsaturated carboxylic acid are mainly used for antigen, antibody and nucleic acid by chemical bonding. Suitable for use in sensitization. Furthermore, since the dispersion medium used for ordinary diagnostic agents is mainly composed of physiological saline, the polymer particles in the present invention have a density at 25 ° C of 0.983 to 1.02.
3 g / ml, preferably 0.993 to 1.013 g
/ Ml is preferred. If the density of the polymer particles at 25 ° C is less than 0.983 g / ml or more than 1.023 g / ml, the proportional range of the turbidity calibration curve is narrow in the conventional standard diagnostic agents, and the gradient especially at the high concentration side is low. Lower.

[0006] In the present invention, the density of the diagnostic agent medium is measured only when it is floating. The measurement of the density of the polymer particles is
A particle aggregate formed by adding a small amount of a 5% by weight aqueous solution of calcium chloride to the polymer particle dispersion is put into a density standard solution (0.002 g / ml increments) whose density has been measured in advance using a float scale, and the mixture is added at 25 ° C. The particles are left standing all day long to determine the density at which the particle aggregate does not settle or float. In the present invention, the average particle size of the polymer particles is usually from 0.03 to 10 μm, preferably from 0.05 to 3 μm. In the diagnostic agent of the present invention,
The sensitizing method for sensitizing the polymer particles with a biochemical substance such as an antibody / antigen or nucleic acid is not particularly limited, and a conventional physical adsorption method or chemical bonding method can be employed.

<Dispersion Medium> In the present invention, a physiological saline is usually used as the dispersion medium. In the present invention, the dispersion medium may be a physiological saline solution to which a sensitivity adjuster, a pH buffer, a preservative, an antifreezing agent, and the like are added, and in this case, the density of the dispersion medium is determined by adding these additives. Is a density in a state where is dissolved. Note that the density of the dispersion medium can be measured with a float scale. <Diagnostic Agent> The diagnostic agent of the present invention is a polymer dispersion composed of polymer particles and a dispersion medium, and the relationship between the density of the polymer particles and the density of the dispersion medium is (density of polymer particles) − (dispersion medium). And the density of the density) = -0.02 to + 0.02g
/ Ml, preferably -0.015 to +0.015 g / ml, more preferably -0.01 to +0.01 g / ml. In the present invention, the densities of the polymer particles and the dispersion medium are values measured at 25 ° C. (Density of polymer particles) − (density of dispersion medium) <− 0.02 g / ml or (density of polymer particles) −
If (density of the dispersion medium)> + 0.02 g / ml, the slope becomes small in the turbidity calibration curve, especially on the high concentration side of the test substance. In the diagnostic agent of the present invention, the amount of the polymer particles used is usually
It is 0.001 to 1% by weight, preferably 0.01 to 0.5% by weight.

<Test Method> In the test using the diagnostic agent of the present invention, a conventional tester for measuring turbidity can be used. As a method for measuring the turbidity of a test solution, a rate assay method that measures the rate of change in turbidity after the addition of the test substance is preferable, but an endpoint method that measures quasi-equilibrium turbidity after a certain period of time has elapsed. Measurement is also possible.

Hereinafter, the present invention will be described in more detail with reference to Examples. In the following, parts are parts by weight. Examples 1-4 and Comparative Examples 1-3 100 parts of a monomer having the composition shown in Table 1, 500 parts of water, 1 part of potassium persulfate, 0.1 part of sodium dodecylbenzenesulfonate were polymerized at a polymerization temperature of 80 ° C. Polymerized at a polymerization conversion of 95% or more for 6 hours, and polymer particles 1 to 1 shown in Table 1
7 was obtained.

[0010]

[Table 1] Note 2-EHA: 2-ethylhexyl acrylate MAA: methacrylic acid, IBA: isobutyl acrylate

The obtained polymer particles were mixed with a 1:15 M phosphate buffer (pH 7.2) and a 1: 3 volume ratio mixture of physiological saline (hereinafter referred to as PBS) at a concentration of 1%.
%, And anti-CRP antibody (rabbit)
1 mg / ml solution was added in an equal amount and kept at 56 ° C. for 30 minutes.
I was sensitized. After the sensitization, unsensitized antibodies are removed by dialysis and gel filtration, and a diluent (PBS containing 0.1% bovine serum albumin) is added so that the polymer particle concentration becomes 0.13%. The anti-CRP antibody sensitized diagnostic agent was obtained. For this diagnostic agent, a turbidity calibration curve was prepared using a CRP antigen standard solution to evaluate the performance of the latex reagent. Apparatus: Hitachi 7020 automatic analyzer Applicable wavelength: 570 nm, measurement temperature 37 ° C Test substance (0-100 mg / dl CRP standard solution): 3 μl First reagent (PBS containing 0.1% bovine serum albumin): 200 μl Second Reagent (latex diagnostic agent): 200μl For the measurement, after stirring the test substance, the first reagent and the second reagent,
A calibration curve was prepared by a rate assay method that measures the difference (change amount) between the turbidity at the 60th and 180th seconds. In the calibration curve, the concentration of the test substance at which the proportional region starts is C1 (mg / d
l) The concentration of the test substance at which the proportional area ends is C2 (mg / dl)
The results are shown in Table 2. In addition, the thing which has a pro zone area | region was described to that effect.

[0012]

[Table 2] * Indicates prozone phenomenon

[0013]

According to the present invention, by examining the difference between the carrier particle density and the medium density, a diagnostic agent having a calibration curve characteristic having a wide proportional area has been found. (Polymer particle density)-
When (dispersion medium density) = − 0.02 g to + 0.02 / ml, a good diagnostic agent can be obtained because the proportional region of the calibration curve is wide. In addition, since the polymer particles of the present invention have a small difference in density from a commonly used dispersing medium, there is no sedimentation of dry particles and aggregated particles on the container wall which cannot be avoided during storage of the latex diagnostic agent.

Claims (2)

[Claims] 1. A method comprising: polymer particles and a dispersion medium;
The relation between the density of the polymer particles and the density of the dispersion medium is (density of the polymer particles) − (density of the dispersion medium) = − 0.02 to +0.
A diagnostic agent characterized by being 02 g / ml. 2. The polymer particles for a diagnostic agent according to claim 1, wherein the density at 25 ° C. is 0.983 to 1.023 g / ml.