JP2003004745A - Polymer latex for detecting target substance - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting a target substance in a sample to be tested which is simple to operate and has a high measurement sensitivity in a wide concentration range from a low concentration to a high concentration, and a polymer latex for executing the detection method. SOLUTION: The polymer latex for detecting the target substance in which carrier particles with a substance having affinity to the target substance in the sample immobilized are minutely aggregated, and the method for detecting the target substance in the sample to be tested are provided. In the method, the polymer latex for detecting the target substance and the sample to be tested are mixed, and an optical physical-properties value of the mixed liquid is measured.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel polymer latex for detecting a target substance and a method for detecting a target substance using the same.

[0002]

2. Description of the Related Art In recent years,
At hospitals, test centers, etc., with the reduction of personnel and costs, clinical tests are being automated, simplified, and the measurement time is shortened. An immunological assay suitable for a commonly used general-purpose automatic analyzer includes an agglutination method in which an antigen or antibody is qualitatively or quantified using an insoluble carrier, particularly a latex immunoaggregation assay method.

This is because, for example, an antigen substance or an antibody or fragment thereof that specifically reacts with the antigen substance is directly carried on the latex, and a test sample is allowed to react with the carried antigen substance or antibody to prevent the latex from aggregating. The substance to be measured is detected and quantified by optically measuring the degree. Here, a polyclonal antibody or a monoclonal antibody is usually used as an antibody used when measuring an antigenic substance in a test sample by the latex immunoaggregation method. Examples of the latex include polystyrene or a latex made of styrene and a copolymer of styrene and styrenesulfonic acid, acrylic acid, acrylic acid ester, methacrylic acid ester, acetonitrile or the like (JP-A-54-890195). Fluoropolymer latex (Japanese Patent Laid-Open No. 1-266111) and the like are known. These polymer latices are inexpensive and can be obtained in large quantities in the same quality, and are therefore widely used in immunoassays.

However, the measurement sensitivity in the immunoaggregation method in which the antigen substance or antibody is directly carried on the latex is
It is hard to say that it is high enough. In particular, the sensitivity is low for the measurement of trace antigens or antibodies, and improvement thereof has been desired.

Further, in order to increase the measurement sensitivity of the latex agglutination immunoassay, a method is known in which an antibody against an antigenic substance in a test sample is not directly carried on the latex but is carried on the latex via a second antibody against the antibody. However, this method has a problem that the operation is complicated.

Therefore, the present invention provides a method for detecting a target substance in a test sample which is easy to operate and has high measurement sensitivity in a wide concentration range from low concentration to high concentration, and a polymer latex for carrying out such detection method. The purpose is to provide.

[0007]

Means for Solving the Problems As a result of intensive studies aimed at achieving the above object, the inventors of the present invention microaggregated carrier particles having a substance having an affinity for a target substance in a test sample immobilized thereon. It was found that the target substance can be measured with high sensitivity in a wide concentration range from low concentration to high concentration and the operation is simple, and the present invention has been completed.

[0008] That is, the present invention provides a polymer latex for detecting a target substance, which is characterized in that carrier particles, to which a substance having an affinity for the target substance in a test sample is immobilized, are finely aggregated. Is. The present invention is also a method for detecting a target substance in a test sample, wherein the target substance detection polymer latex is mixed with the test sample, and the optical property value of the mixed solution is measured. The present invention provides a method for detecting a target substance therein.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As the carrier particles used in the present invention, as long as the carrier particles have a relatively constant particle size and can be mass-produced with industrially constant quality and performance, There is no particular limitation. The type of polymer is not particularly limited, and examples thereof include homopolymers and copolymers of vinyl monomers such as styrene, vinyl chloride, acrylonitrile, vinyl acetate, acrylic acid ester, and methacrylic acid ester; styrene-butadiene copolymer, methyl methacrylate. -Butadiene-based copolymers such as butadiene copolymer and the like can be mentioned. Of these, polystyrene-based polymer particles are preferable from the viewpoints of excellent adsorbability of substances such as antibodies, and long-term retention of biological activity. The particle size of the carrier particles is preferably 0.01 to 10 μm
Is preferable, and 0.03 to 1 μm is particularly preferable.

In the present invention, the target substance to be detected is not particularly limited, but examples thereof include causative substances of various diseases and allergies, pathogenic bacteria, physiologically active substances, antibodies thereof and the like. Specifically, immunoglobulins (eg, human and animal immunoglobulins, denatured immunoglobulins), various viral antigens (eg, hepatitis virus-related antigens, rubella HA antigens, etc.), microbial antigens such as various bacteria, fungi, toxins ( For example, Toxoplasma, Treponema pallidum, etc.), various plasma protein components (eg, α-fetoprotein, C-reactive protein (CRP), albumin complement component, etc.), various hormones (eg, estrogen, human chorionic gonadotropin, (HCG)) Etc.), antibodies (eg anti-CEA (ca
rcinoembryonic antigen) antibody, anti-reactive protein antibody, anti-fibrinogen antibody, anti-γ-globulin antibody etc.), hormone receptor (eg thyroid stimulating hormone receptor, HCG receptor etc.), enzyme (eg alkaline phosphatase, cholinesterase etc.), Substrates (eg, cholesterol, uric acid, etc.), hemoglobin derivatives, sugar derivatives (eg, sugar compounds, glycoproteins, etc.) can be mentioned.

In the present invention, the "substance having an affinity for a target substance" includes a substance having an affinity for the above-mentioned target substance by an antigen-antibody reaction, an enzyme-substrate reaction, a ligand-receptor reaction or the like. .

In order to support the substance having an affinity for the target substance on the polymer particles, they may be simply mixed when they are physically adsorbed. Further, in the case of chemically bonding, the functional group existing on the surface of the polymer particle or the substance having an affinity for the target substance can be utilized, or the spacer can be bonded through a spacer having an appropriate functional group. .

The method for detecting a target substance according to the present invention is to mix finely aggregated polymer latex with a test sample and measure the optical properties of the mixed solution. By using the finely aggregated polymer latex, the target substance can be measured with high sensitivity in a wide concentration range from low concentration to high concentration. Although it is not always clear why the measurement can be performed with high sensitivity by microaggregation, it is considered that the polymer latex scatters light well, that is, the scattering intensity increases. That is, it is considered that the turbidity of the system more sensitively responds to the aggregation of the polymer latex.

The size of the fine agglomeration of the carrier particles in the present invention is such that the target substance can be measured with high sensitivity in a wide concentration range from a low concentration to a high concentration. The diameter is preferably 0.01 to 10 μm, and particularly preferably 0.03 to 1 μm. If it is less than 0.01 μm or exceeds 10 μm, the scattering intensity is not sufficiently improved, and the measurement sensitivity cannot be improved.

As means for finely agglomerating carrier particles, when the substance having an affinity for the target substance is a protein,
For example, a method of changing pH, ionic strength, or the like, and stirring the mixture, if necessary, is also possible. In this method, the carrier particles are finely aggregated, and the fine aggregation can be relatively easily generated by controlling the amount of change in pH, ionic strength, etc., the stirring strength, and the like. However, in the above method, it is necessary to add an acid, an alkali, a salt, or the like, and the presence of these compounds may hinder the subsequent detection of the target substance. Further, the operation is complicated to neutralize or remove them. Therefore, in the present invention, the following method is more preferable.

The target substance is allowed to act on carrier particles carrying a substance having an affinity for the target substance. As a result, the target substance is bound to the surface of the carrier particles via the substance having an affinity for the target substance. Further, in some cases, the target substance and the substance having an affinity for the target substance crosslink between the carrier particles. As a result, fine agglomeration of carrier particles occurs. According to such a method, since only the target substance is used to generate the microaggregates, a complicated operation as in the above method is not required. The size of the micro-aggregates can be controlled by the amount of the target substance to act and, if necessary, the temperature, time and the like when the substance acts. The polymer latex obtained by finely agglomerating such carrier particles can be effectively used for the latex agglutination method which is generally widely used, particularly for diagnostic reagents.

The carrier particles may carry a plurality of types of substances. That is, first, one substance is caused to react with a substance that has an affinity for it and is carried by the carrier particles to generate fine aggregates. Then, by reacting the target substance with a substance that has an affinity for the target substance and is similarly supported by the carrier particles, a substance different from the substance used for the microaggregation can be detected.

Next, the method for detecting the target substance of the present invention will be described. First, the polymer latex that has been finely aggregated is mixed with a test sample and appropriately stirred. Aggregation occurs when the target substance is present. The conditions of the agglutination reaction are not particularly limited as long as the agglutination reaction can occur, but the temperature is preferably a constant temperature of 25 to 37 ° C., and the reaction time is preferably 5 seconds to 15 minutes. The reaction solution is not particularly limited, and examples thereof include a phosphate buffer solution, a glycine buffer solution, a Tris hydrochloric acid buffer solution, and Good's buffer solution. The pH of the medium is preferably 5.
It is 5 to 8.5, and particularly preferably 6.5 to 8.0. The concentration of the polymer latex after mixing depends on the properties of the target substance,
It can be appropriately determined in consideration of the expected abundance, the intensity of scattered light to be obtained, etc., but generally it is preferably 0.01 to 10% by weight in terms of latex particles, and 0.1 to 10% by weight.
1% by weight is particularly preferred.

In the reaction solution, bovine serum albumin, sucrose and the like as stabilizers; water-soluble polysaccharides such as polyethylene glycol and dextran as measuring sensitivity improving agents; sodium azide as an antiseptic; chloride as a salt concentration adjusting agent. Additives such as sodium can be added as appropriate.

Next, the optical properties of the mixed solution are measured. The specific optical measurement method for detecting agglutination is not particularly limited, and examples thereof include nephelometry and nephelometry. Using such a method, the concentration of the target substance in the test sample can be easily detected from the calibration curve prepared in advance. Examples of test samples include blood, serum, body fluid, urine, feces and the like.

[0021]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

Example 1 (1) Preparation of anti-human CRP goat polyclonal antibody sensitized particles Anti-human CRP goat polyclonal antibody (Interna)
regional Immunology Corp. Made by:
Hereinafter, "CRP antibody") was dissolved in 10 mM Tris-HCl buffer (pH 7.2) at a concentration of 2.0 mg / mL to 10.0 mL of a solution, and polystyrene particles having an average particle size of 55 nm (Fujikura Chemical Co., Ltd. ): Solid content 1% by weight) 10.
0 mL was added and stirred at 4 ° C. for 60 minutes. Next, bovine serum albumin (hereinafter referred to as "BSA") is added to this solution.
0.1M phosphate buffer solution (pH 7.
0: hereinafter, referred to as “PBS”) was added, and the mixture was stirred at 4 ° C. for 60 minutes and then centrifuged at 20000 rpm for 60 minutes for washing. The washing operation was performed 3 times. To the obtained precipitate, 10 mL of PBS containing 1% by weight of BSA was added to suspend the sensitized particles, followed by dispersion treatment with an ultrasonic crusher to obtain a solid content of 0.1% (W / V). CRP antibody-sensitized particles were prepared. (2) Preparation of latex reagent 1.0 μg of human CRP (manufactured by Oriental Yeast Co., Ltd.) was added to 10.0 mL of the CRP antibody-sensitized particles prepared in (1), and the mixture was stirred at 4 ° C. for 60 minutes. After that, the solution was allowed to stand for one day to prepare a latex test solution. (3) Preparation of sample A sample was prepared by dissolving human CRP in human pool serum to a predetermined concentration. (4) 300 μL of measurement PBS and 3 μ of the sample prepared in (3) above
After mixing with L and preheating at 37 ° C. for 5 minutes, the latex reagent solution 100 prepared in (2) above was further added.
The reaction was started by adding μL, and the amount of change in absorbance for 5 minutes immediately after the addition of the latex reagent solution was measured. The measuring device is an automatic general-purpose measuring machine Hitachi 7020 type (Hitachi Ltd.)
(Manufactured by Mitsui Chemicals Co., Ltd.) was used at a measurement wavelength of 570 nm and a measurement temperature of 37 ° C. The results are shown in Fig. 1.

Comparative Example 1 (2) In the preparation of the latex reagent solution (2) in Example 1, CR was used.
The measurement was performed in the same manner as in Example 1 except that human CRP was not added to the P antibody-sensitized particles and the particles were used as a latex reagent solution as it was. The results are shown in Figure 1.

As is clear from FIG. 1, when CRP is allowed to act on the CRP antibody-sensitized particles to use the latex reagent solution which is finely aggregated, it becomes possible to increase the measurement sensitivity in a low concentration range.

[0025]

EFFECTS OF THE INVENTION By using the polymer latex for detecting a target substance of the present invention, it has sufficient measurement sensitivity depending on the concentration of the substance to be measured, and has a low concentration range to a high concentration range as compared with the conventional method. The concentration can be measured with high accuracy in a wide measurement range over the range.

[Brief description of drawings]

FIG. 1 is a diagram showing the absorbance with respect to the CRP antigen concentration in Examples and Comparative Examples.

Continued front page    (72) Inventor Hideki Goto             5-13-1 Sakurada, Washinomiya-cho, Kitakatsushika-gun, Saitama Prefecture               Fujikura Kasei Co., Ltd. (72) Inventor Hideyuki Kuroda             5-13-1 Sakurada, Washinomiya-cho, Kitakatsushika-gun, Saitama Prefecture               Fujikura Kasei Co., Ltd. F-term (reference) 2G054 AA06 AB04 BA03 BA04 BB13                       CA21 CD04 EA04 EA09 EB01                       EB05 GA03 GB01

Claims (6)

[Claims] 1. A polymer latex for detecting a target substance, comprising carrier particles, to which a substance having an affinity for the target substance in a test sample is immobilized, which are finely aggregated. 2. The polymer latex for detecting a target substance according to claim 1, wherein carrier particles are finely aggregated by causing the target substance to act on a substance having an affinity for the target substance. 3. The substance according to claim 1 or 2, wherein the substance having an affinity for the target substance has an affinity for the target substance through an antigen-antibody reaction, an enzyme-substrate reaction, or a ligand-receptor reaction. Polymer latex for target substance detection. 4. The polymer latex for detecting a target substance according to claim 1, which is used for a diagnostic reagent. 5. The polymer latex for detecting a target substance according to claim 1, wherein the aggregate size is 0.01 to 10 μm. 6. A method for detecting a target substance in a test sample, which comprises mixing the polymer latex for detecting a target substance according to any one of claims 1 to 5 with a test sample to obtain optical properties of the mixed solution. A method for detecting a target substance in a test sample, which comprises measuring a value.