JPH10132819A - Method for measuring substance to be inspected by use of particle and measuring appliance for this method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measuring method for a substance to be inspected in a sample and a measuring appliances to be used for the method capable of judging whether there is a substance in the sample easily, even if the concentration of the substance is low, and accordingly capable of measuring the substance in the sample with high sensitivity. SOLUTION: This concerns a measuring method for a substance in a sample and measuring appliances for this method, contains a process of bringing a sample, and particles of singular combined substances against the substance, to which singular combined substances the same as or different from a singular combined substance fixed to a carrier are fixed, into contact with a surface, coated with a singular combined substance against a substance to be inspected in a sample, of the above-mentioned carrier to which the singular combined substance is fixed, and a process of inclining the above-mentioned carrier so that the above-mentioned particles may move along the above-mentioned surface, and is characterized in judging whether there is any substance, from the distributed state of the particles on the above-mentioned surface.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for measuring a test substance in a sample using particles to which a specific binding substance to the test substance is immobilized, and a measuring instrument used for the method, and particularly to a method for measuring a test substance in a sample. The present invention relates to a method for measuring a test substance which can easily and easily measure even a small amount of a test substance contained therein without being affected by an interfering substance, and a measuring instrument used for the method. INDUSTRIAL APPLICABILITY The present invention is useful in the life science field such as a clinical test field.

[0002]

2. Description of the Related Art A trace amount of a sample contained in a sample utilizing a reaction between a substance having a specific affinity such as an antigen and an antibody, a sugar and a lectin, a nucleotide chain and its complementary nucleotide chain, and a ligand and a receptor. Various types of measuring methods or measuring instruments for analytes are known.

[0003] Among them, immunological measurement methods utilizing an antigen-antibody reaction (immune reaction) between an antigen and an antibody are widely practiced. Among these immunological measurement methods, the indirect agglutination measurement method is widely used because it is a simple and inexpensive method.

In this indirect agglutination measurement method, particles (polymer particles such as latex particles or gelatin particles) or red blood cells to which an antibody against a test substance (an antigen can be used if the test substance is an antibody) are bound. ) And a sample presumed to contain the test substance are mixed in a measurement container having a U-shaped or V-shaped bottom surface such as a microtiter plate,
The presence or absence of the test substance in the sample is determined by observing the aggregation image between the particles via the test substance generated by the reaction.

FIG. 9 shows an agglutination image when a test substance in a sample is measured by the indirect agglutination reaction measurement method. If the test substance does not exist in the sample (negative), the particles bound with the antibody (or antigen) do not cause aggregation, so they settle down by gravity, and fall on the inner wall surface of the U-shaped or V-shaped measurement container. It rolls down (slids down) and gathers in the center of the bottom surface of the measurement container. Therefore, in the case of a negative case, when viewed from above the measurement container, an image in which particles converge at the center of the bottom surface of the measurement container is observed (A in FIG. 9). on the other hand,
When the test substance is present (positive) in the sample, the particles to which the antibody (or antigen) is bound undergo three-dimensional aggregation via the test substance to form an aggregate. This agglomerate is less likely to roll on the inner wall surface of the measurement container (harder to slide down) and has a lower rolling speed (sliding speed) than particles that do not cause aggregation, so that the agglomerate spreads on the inner wall surface of the measurement container. Stop. Therefore, in the case of positive, when viewed from above the measurement container, a state in which the particles are spread on the bottom surface of the measurement container, that is, a “button” -like aggregation image can be observed (FIG. 9B), and the test substance is present in the sample. Can be confirmed.

However, in this indirect agglutination measurement method, the presence or absence of a test substance in a sample, that is, whether it is positive or negative, is determined based on the size of a circular image formed by particles on the bottom surface of a measurement container. Therefore, in the case of a sample having a low concentration of the test substance, both the particles aggregated via the test substance and the particles not bound to the test substance are mixed in the center of the bottom surface of the measurement container, and Since the circle of the image was small, it was difficult to distinguish from the negative case. And, because of the difficulty of the determination in the case of a sample having a low concentration of the test substance, it is not possible to measure the low-concentration test substance, and it is necessary to take a long time to sufficiently form an agglutination image and then make a determination. Therefore, the time required for the measurement was long, and the measurement time was usually one hour or more.

[0007] In the indirect agglutination measurement method, particles bound to an antibody (or antigen) are aggregated or particles bound to an antibody (or antigen) are passed through components in the sample other than the test substance. In some cases, the sample bound to the inner wall of the measurement container and showed an agglutination image as if the test substance was present (positive) despite the absence of the test substance in the sample (negative) (non-specific Agglutination reaction). The possibility that a negative sample may be erroneously determined to be positive has been a serious problem in diagnosing a disease by a clinical test.

On the other hand, JP-A-64-69954 discloses that
A sample in which an antibody or antigen corresponding to an antigen or an antibody as a test substance in a sample is fixed to a measurement container having an inner wall immobilized thereon, and the sample is fixed to the measurement container simultaneously or subsequently without washing. Insoluble carrier particles having the same antibody or antigen or specific binding analog immobilized thereon are added to the measurement container, and the presence or absence of the expressed agglutination reaction is used to determine the presence or absence of the test substance antigen or antibody in the sample sample An immunoassay has been disclosed. In this measurement method, the state in the measurement container when the antigen (or antibody) as the test substance is not present (negative) in the sample sample is viewed from above,
An image similar to the aggregation image shown in FIG. 9A is observed. Further, when an antigen (or antibody) as a test substance is present (positive) in the specimen sample, when the state in the measurement container is viewed from above, an image similar to the agglutination image shown in FIG. 9B is observed. Is done.

The measuring method described in Japanese Patent Application Laid-Open No. 64-69954 is a method capable of measuring a low concentration of a test substance, suppressing a zone phenomenon, and obtaining a clear aggregated image in a short time. However, since the presence or absence of the test substance in the sample is determined by the size of the circular image due to the particles on the bottom of the measurement container, if the sample has a very low concentration of the test substance, The problem that it is difficult to judge is inevitable.

Japanese Unexamined Patent Publication (Kokai) No. 2-124464 discloses a magnetic material in which a substance (antibody or antigen, etc.) which specifically binds to or competes with a substance to be measured (test substance) is immobilized on magnetic particles. The marker particles and the sample solution (sample) are mixed, and the reaction solution is acted on by a magnet arranged outside the predetermined wall region of the measurement container, whereby the distribution of the magnetic marker particles collected in the predetermined wall region is increased. An immunological measurement method for measuring a substance to be measured in a sample based on a state is disclosed. In this measurement method, when the substance to be measured does not exist in the sample (negative), when the state in the measurement container is viewed from above, an image similar to the agglutination image shown in FIG. When the state inside the measurement container is viewed from above when the measurement substance is present (positive), an image similar to the aggregation image shown in FIG. 9B is observed.

According to the measuring method described in Japanese Patent Application Laid-Open No. 2-24464, the magnetic particles are attracted to the bottom surface of the measuring container by a magnet to increase the sedimentation speed of the magnetic particles, thereby forming an aggregation image on the bottom surface of the measuring container. Is performed in a short time, and the time required for the determination can be reduced. However, since the presence or absence of the test substance in the sample is also determined based on the size of the circular image formed by the particles on the bottom of the measurement container, the case of a sample having a low test substance concentration is considered to be negative. There was no change in that it was difficult to determine.

[0012]

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a method for measuring a test substance in a sample visually or by using an instrument by utilizing a specific binding reaction. It is an object of the present invention to provide a measurement method capable of determining the presence or absence of a test substance in a short time regardless of the concentration, and a highly reliable measurement method and a measurement instrument used in the method. .

[0013]

According to the present invention, there is provided a sample having a surface on which a specific binding substance to a test substance in a sample is immobilized and coated with the specific binding substance, A step of contacting the specific binding substance fixed to the carrier with the specific binding substance that is the same or different from the specific binding substance fixed to the carrier, and the particle moves along the surface. The method of tilting the carrier as described above, and determining the presence or absence of a test substance from the distribution state of the particles on the surface (first measurement method) It is. Also,
In the present invention, a test substance and / or an analog thereof are brought into contact with a surface of a carrier having a surface to which a specific binding substance to a test substance in a sample is fixed and coated with the specific binding substance. Binding the test substance and / or its analog to the specific binding substance immobilized on the surface, wherein a sample and a specific binding substance for the test substance immobilized on the carrier, Contacting the same or different specific binding substance with the immobilized particles, and tilting the carrier so that the particles move along the plane, comprising the steps of: A method for measuring a test substance in a sample (second measurement method), characterized by determining the presence or absence of the test substance from the distribution state. Furthermore,
The present invention provides a carrier having a surface on which a specific binding substance to a test substance in a sample is immobilized and coated with the specific binding substance, and a specific binding substance to the test substance, which is immobilized on the carrier. And a particle on which the same or different specific binding substance is immobilized, and a measuring instrument (first measuring instrument) for a test substance in a sample. Further, the present invention provides a carrier having a surface on which a specific binding substance to a test substance in a sample is immobilized and coated with the specific binding substance, a test substance and / or an analog thereof, and a test substance. And a specific binding substance immobilized on the carrier and a particle on which the same or different specific binding substance is immobilized. ).

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION The test substance to be measured in the present invention may be any biologically relevant substance such as an organic substance such as protein, carbohydrate, lipid and nucleic acid, and an inorganic substance. Specifically, HBs antigen, anti-HBs antibody,
Virus-related antigens or antibodies such as HBe antigen, anti-HBe antibody, anti-HBc antibody, anti-HCV antibody, anti-HIV antibody, anti-ATLV antibody; anti-Treponemal pallidum (TP) antibody, anti-mycoplasma antibody, anti-streptolysin O antibody ( Bacterial antigens or antibodies such as ASO); immunoglobulins such as immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM) or immunoglobulin E (IgE); C-reactive protein (CRP ), Inflammatory markers such as α1-acid glycoprotein, haptoglobin, complement C3, complement C4, rheumatoid factor; α-fetoprotein;
Tumor markers such as CEA and CA19-9; hormones such as human placental chorionic gonadotropin; allergens or allergen-related antigens or antibodies such as allergen-specific IgE antibodies; blood coagulation-related substances such as antithrombin III (ATIII); Decomposition products (FDP), fibrinolytic related substances such as D-dimer;
Blood group-related antigens or antibodies such as ABO blood group antibodies and irregular antibodies; viral DNA or RNA; bacterial DNA
Or RNA; DNA or R of animal or plant such as human
NA; substances or drugs related to other diseases such as lipoprotein (a).

In the present invention, the term "sample" refers to a liquid substance to which the above-mentioned test substance may be present and which is to be used for confirming the presence or absence of the test substance or for quantification in some cases. For example, human or animal blood, serum, plasma, urine, semen, cerebrospinal fluid, saliva, sweat, tears, ascites, amniotic fluid, and other body fluids; human or animal brain and other organs, hair, skin, nails, muscles, and nerves Extracts of tissues, etc .; extracts of human or animal feces; extracts of cells or bacterial cells; extracts of plants, and the like.

In the present invention, a specific binding substance to a test substance (hereinafter, simply referred to as a specific binding substance) refers to a substance having an affinity for the test substance, and a specific interaction with the test substance. A substance that stably binds non-covalently to the test substance by action. For example, a specific binding substance is
If the test substance is an antigen, the antibody is the antibody.If the test substance is an antibody, the antibody is an antibody against the antigen or the antibody.If the test substance is a nucleotide chain, the nucleotide is a complementary nucleotide chain. is there. When the test substance is a ligand, it is its receptor. The specific binding substance immobilized on the particles and the specific binding substance immobilized on the carrier may be the same or different, respectively, as long as the specific binding substance can be bound via the test substance.

The particles used in the present invention may be particles generally used for an indirect aggregation reaction. For example, organic polymer particles such as liposomes, latex particles, gelatin particles, polyacrylamide particles, microcapsules, emulsions, etc .; inorganic polymer particles such as glass beads, silica beads, and benite; other artificial particles; and erythrocytes. And the like. Further, the particles may be colored by coating with a dye or dispersing or enclosing the dye in the particles.

The particle diameter of the particles is preferably 0.01 to 100 μm.
m, particularly preferably 0.5 to 10 μm. Also,
The specific gravity of the particles may be any specific gravity that sediments in the dispersion medium,
For example, those having a specific gravity of 1 to 10 are preferable.

In the present invention, particles having a specific binding substance immobilized thereon are used. In order to fix the specific binding substance to the particles, the specific binding substance is bonded to the surface of the particles by hydrophobic bonding,
It can be performed by a physical adsorption method such as hydrophilic adsorption, a chemical bonding method such as covalent bonding, or a combination of these methods.

When the specific binding substance is fixed to the particles by a physical adsorption method, the specific binding substance and the particles are mixed and contacted in a solution such as a buffer according to a known method. be able to. For example, the specific binding substance and the particles are brought into contact with each other by mixing and stirring in a solution such as a buffer solution, and the adsorbing reaction is performed at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day. The particles may be washed with a buffer or the like.

When the specific binding substance is fixed to the particles by a chemical bonding method using a cross-linking reagent, a special edition of the Special Issue on Clinical Pathology, No. 53, Special Issue on Clinical Pathology, edited by the Japanese Society of Clinical Pathology, is available. Application, "Clinical Pathology Publishing Association, 1983, The Society of Biochemistry, Japan
Protein IV '', Tokyo Chemical Dojin, 1991, etc., according to a known method, mix the specific binding substance and particles with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide, etc. The functional group such as amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, etc. of the target binding substance and the particles can be fixed by reacting with a crosslinking reagent. For example, a divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide is added to a buffer solution containing particles, and the mixture is stirred and reacted. Next, a specific binding substance is added thereto, and the mixture is stirred and reacted. In some cases, the reaction is then terminated by removing the crosslinking reagent by a treatment such as dialysis or gel filtration, or by adding a reaction terminator for the crosslinking reaction. Then, the obtained particles may be washed with a buffer solution or the like.

By changing the amount of the specific binding substance fixed to the particles, the sensitivity can be easily changed according to the level of the concentration of the test substance in the sample. For example, when a specific binding substance is immobilized on particles, the use of a high concentration of a specific binding substance increases the amount of immobilization, thereby increasing sensitivity.

If necessary, in order to suppress nonspecific reactions, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, skim milk powder, etc. are brought into contact with the particles on which specific binding substances are immobilized. The particles may be masked by the known method described above. For example,
Masking is performed by adding the particles having the specific binding substance immobilized thereto to a buffer solution containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, etc., and allowing the particles to stand, and coating the surface of the particles with various proteins. It can be done by doing.

In the present invention, a carrier having a surface coated with the specific binding substance by immobilizing the specific binding substance is used as the carrier. The carrier has a surface coated with a specific binding substance, and may have any shape and structure as long as a solution (liquid) such as a sample can be brought into contact with the surface, such as a container or a plate. Body and the like. When a container is used as the carrier, the shape of the concave portion serving as the solution storage portion may be any shape such as a hemispherical shape, a cylindrical shape, or a rectangular parallelepiped shape, and the inner wall surface of the concave portion may be covered with a specific binding substance. The concave shape is cylindrical,
In the case of a shape having a flat bottom surface such as a rectangular parallelepiped shape, it is preferable that the flat bottom surface is covered with a specific binding substance. The container may have a plurality of recesses. The container is a container provided with at least one concave portion having a flat bottom surface, and the flat bottom surface is preferably coated with a specific binding substance. Examples of the container provided with at least one concave portion having a flat bottom include a flat bottom microplate and a tray. When a plate is used as a carrier, the surface of the plate may be any shape such as a circle, a rectangle, and a square, and its surface may be coated with a specific binding substance. Also, by forming a groove on the surface of the plate-like body and introducing a solution such as a sample into the groove, the solution can be prevented from flowing down from the surface.

The material of the carrier is not particularly limited as long as the specific binding substance can be physically or chemically immobilized. Examples thereof include glass, polystyrene, polyvinyl chloride, polyacrylate, polypropylene, nylon, polyethylene and polycarbonate. , Polymethacrylate and the like.

In the present invention, when the specific binding substance is immobilized on the surface of the carrier, it is preferable that the surface is partially immobilized with the specific binding substance. This is because, when the test substance is present in the sample, that is, when the test substance is positive, the judgment of positive is made clearer by comparing the images of the part coated with the specific binding substance and the part not coated with the specific binding substance. This is because it is easy to determine whether or not the sample is negative, that is, whether or not the test substance is present in the sample. Where "partial"
"It means that the specific binding substance is unevenly distributed and is not covered with the specific binding substance over the entire surface.
The shape and area of the portion coated with the specific binding substance are not particularly limited as long as the presence or absence of the test substance in the sample can be determined. Examples of the partial coating include a case where one half of the surface of the carrier is coated, and a case where the surface of the carrier is coated in a strip shape.

When the surface of the carrier is partially covered with the specific binding substance, a plurality of the covering parts may be present. For example, in order to measure a plurality of test substances in a sample, the carrier may be coated. Can be provided with a portion coated with a specific binding substance for each test substance.

FIG. 1 shows a flat-bottom microplate used as a carrier in the present invention, in which one half of the flat bottom of each well is coated with a specific binding substance. FIG. 1A is a perspective view of a flat-bottom microplate (the hatched portion is a portion covered with the specific binding substance), and FIG. 1B is a plan view of the flat-bottom microplate (the hatched portion is the above-mentioned portion). This is the part coated with the specific binding substance.).

FIG. 2 shows a rectangular parallelepiped transparent container having a rectangular bottom surface and having a bottom surface having a portion covered with a specific binding substance in a band shape (hereinafter referred to as a band-shaped coating portion). It is. FIG. 2A is a perspective view of the container (the hatched portion is a portion covered with the specific binding substance), and FIG. 2B is a plan view of the container (the hatched portion is the specific binding material). This is the part covered by the binding substance.)

FIG. 3 shows a rectangular plate-shaped body having a surface having a band-shaped covering portion made of two different types of specific binding substances. FIG. 3A is a perspective view of the plate-like body (shaded portions are portions covered with different specific binding substances).
FIG. 3B is a plan view of the plate-like body (shaded portions are portions covered with different specific binding substances, respectively).

FIG. 4 shows a rectangular plate-like body having a rectangular cross section on its surface, and a band-shaped covering portion made of two different kinds of specific binding substances on the bottom surface of the groove. It is a thing which formed what was formed. A of FIG.
FIG. 4B is a perspective view of such a plate-like body (shaded portions are portions covered with different specific binding substances), and FIG. 4B is a plan view of such a plate-like body (hatched line). The parts are parts coated with different specific binding substances.) Also, if the plate is covered by placing a plate on the band-covered portion side of the groove (see FIG. 4A), the sample dropped in the groove on the uncovered side moves in the direction of the band-covered portion by capillary action. preferable.

The immobilization of the specific binding substance on the surface of the carrier is as follows:
It can be performed by a physical adsorption method or a chemical bonding method using a crosslinking reagent. In the case of the physical adsorption method, it can be carried out by bringing a specific binding substance dissolved in a buffer solution or the like into contact with the surface of the carrier according to a known method. For example, when the carrier is a container, a specific binding substance dissolved in a buffer solution or the like is brought into contact with the container by allowing it to stand in a concave portion of the container and allowed to stand at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day. After the reaction, the liquid in the concave portion may be removed by suction and washed with a buffer solution or the like.

In the case of using a chemical bonding method with a cross-linking reagent, the Japanese Society of Clinical Pathology, “Special Issue on Clinical Pathology Extraordinary Issue No. 53, Immunoassay for Clinical Examination-Technology and Application-”, Clinical Pathology Publishing Association, 1983 19th edition, The Biochemical Society of Japan, “Chemical Experiment Lecture 1 Protein IV”, Tokyo Kagaku Dojin, 19
According to a known method described in, for example, 1991, a specific binding substance and a carrier for a test substance are mixed with a divalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, and maleimide, and contacted with the specific binding substance. The carrier can be immobilized by reacting with an amino group, a carboxyl group, a thiol group, an aldehyde group, a hydroxyl group or the like of the carrier. For example, when the carrier is a container, a divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, or maleimide is added to the recess of the container, and the mixture is left to react. Next, a specific binding substance is added thereto, and the mixture is left to stand for reaction. In some cases, the reaction is then stopped by adding a reaction terminator for the crosslinking reaction thereto. Then, washing is performed with a buffer solution or the like.

In the present invention, as a method of partially covering the surface of the carrier, for example, the specific binding substance and / or the cross-linking reagent may be dropped only on the portion of the surface to be coated with the specific binding substance. A method of immobilizing according to the method, enclosing a portion of the surface to be coated with the specific binding substance with a plastic plate or the like, and dropping the specific binding substance and / or the crosslinking reagent onto the enclosed portion to perform the above-described immobilization. Or a method in which a specific binding substance and / or a cross-linking reagent is absorbed by an absorbent such as a sponge, and the above-mentioned immobilization is carried out by placing on a portion of the surface to be covered with the specific binding substance and contacting or applying the same. Is mentioned.

Further, by changing the amount of the specific binding substance fixed on the surface of the carrier, the sensitivity can be easily changed according to the level of the concentration of the test substance in the sample. For example, when a specific binding substance is immobilized on a carrier, the use of a high-concentration specific binding substance increases the amount of immobilization and increases the sensitivity.

Further, if necessary, in order to suppress non-specific reactions, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, skim milk powder and the like are brought into contact with a carrier on which a specific binding substance is immobilized. The carrier on which the specific binding substance is immobilized may be masked by a known method such as the above. For example, when the carrier is a container, a buffer containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and the like are added to the concave portion of the container in which the specific binding substance is immobilized, and the mixture is allowed to stand. Then, after coating the surface of the concave portion of the container in which the specific binding substance is immobilized with various proteins or the like, the liquid in the concave portion can be removed by suction.

In the first measuring method according to the present invention,
First, a sample suspected of containing a test substance, and a particle having the specific binding substance immobilized thereon, on which the specific binding substance is immobilized as described above and the carrier having a surface coated with the specific binding substance, Contact. When a plurality of test substances in a sample are measured, the particles in which a specific binding substance to each test substance is immobilized are brought into contact.

The sample can be diluted with, for example, a diluent and brought into contact with a carrier. Further, the particles can be dispersed in an appropriate dispersion medium and brought into contact with a carrier, for example. As the diluent and the dispersion medium of the particles used above, respectively,
Various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, and phosphate buffered saline, and physiological saline can be used. The pH of this buffer
Is preferably in the range of pH 4 to 12.

The sample diluent and the dispersion medium of the particles include various proteins such as bovine serum albumin, human serum albumin, casein or its salt, various salts such as sodium chloride, various sugars, skim milk powder, normal rabbit Various animal serum such as serum, various preservatives such as sodium azide, various surfactants such as nonionic surfactant, amphoteric surfactant, anionic surfactant, etc. Can be used. And the concentration when adding these additives is not particularly limited,
0.001 to 10% (w / v) is preferable, and particularly 0.01 to 5% (w / v)
Is preferred. As the surfactant, sorbitan fatty acid ester, glycerin fatty acid ester, decaglycerin fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene phytosterol , Non-ionic surfactants such as phytostanol, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, polyoxyethylene lanolin, amphoteric surfactants such as betaine acetate or polyoxyethylene alkyl ether sulfate Alternatively, an anionic surfactant such as polyoxyethylene alkyl ether acetate may be used.

When the carrier is, for example, a container as described above, the sample and the particles can be brought into contact with the inner wall surface of the container by adding the sample and the particles to the concave portion of the container. In this case, particles may be added to the concave portion of the container after adding the sample, or conversely, the sample may be added after adding the particles. Further, after mixing the sample and the particles in advance, the mixture may be added to the concave portion of the container. When the carrier is, for example, a plate-like body as described above, the sample is dropped after the particles are dispersed on the surface of the plate-like body, or the particles are dispersed after the sample is dropped, And the particles may be preliminarily mixed, and the mixture may be contacted by dropping the mixture.

After the sample and the particles are brought into contact with the surface of the carrier as described above, the carrier is tilted so that the particles move along the surface coated with the specific binding substance. The angle of tilting the carrier may be any angle at which the particles move along the surface of the carrier coated with the specific binding substance by gravity, and an angle of 90 ° or less may be appropriately selected.
Angles between ゜ and 90 ° are preferred, especially angles between 45 ° and 65 °. Further, the carrier may be tilted before or while the sample and the particles are in contact with the surface of the carrier, as long as a positive or negative determination can be made based on the distribution state of the particles on the surface of the carrier.

When the entire surface of the carrier is covered with the specific binding substance, the carrier can be tilted in any direction. When the surface of the carrier is partially coated with the specific binding substance, the carrier is tilted in such a direction that the particles move on the portion of the carrier covered with the specific binding substance, What is necessary is just to move a particle by gravity. For example, the carrier may be arranged with the direction to be moved downward. More specifically, for example, when using a carrier in which one half of the surface is coated with a specific binding substance, the direction from the portion of the surface not coated with the specific binding substance to the direction of the coated portion (FIG. 1) The carrier is tilted so that the particles move in the direction of the arrow (in the direction of the arrow) (in this case, the carrier may be tilted in the direction of the arrow in FIG. 1). When a carrier whose surface is coated in a band with a specific binding substance is used, a portion of the surface not covered with the specific binding substance is coated with the specific binding substance through the band-coated portion. The carrier is tilted so that the particles move in the direction of the missing portion (the direction of the arrow in FIGS. 2 to 4) (in this case, the carrier may be tilted in the direction of the arrow in FIGS. 2 to 4).

When the test substance is present in the sample, the test substance binds to the specific binding substance fixed on the particles. In this case, when the carrier is tilted so that the particles move along the surface covered with the specific binding substance of the carrier, the particles move downward along the surface of the carrier due to gravity. When the particles encounter the specific binding substance immobilized on the surface at the above, the particles bind to the specific binding substance immobilized on the surface via the test substance and stop the movement, or the movement is significantly slowed down. In areas of the surface where the specific binding agent is not immobilized, the movement of the particles depends on the interaction between the particles and the surface and the angle at which the carrier is tilted, and the particles move quickly downward in the tilt. On the other hand, in a region of the surface where the specific binding substance is fixed, there is a large difference in the moving speed of the particle between the case where the test substance is bound to the particle and the case where the test substance is not bound. That is, when the test substance is not present in the sample, the particles do not bind to the test substance, so that the particles do not show an affinity with the specific binding substance fixed on the surface, and the area where the specific binding substance is not fixed. Moves quickly downward as well as. Thus, if no analyte is present in the sample, the particles will collect at the lower end of the carrier. On the other hand, if the test substance is present in the sample, the particles bind the test substance,
The particles bind to the specific binding substance immobilized on the surface via the test substance, and the movement stops or becomes extremely slow. Therefore,
If the test substance is present in the sample, the particles collect on the surface of the sample covered with the specific binding substance.

The distribution of particles on the surface of the carrier, ie, the image, is
It can be easily confirmed by the naked eye or by an optical reading device such as a microplate reader by absorbance measurement or pattern recognition.

FIG. 1C is a diagram showing a well viewed from above when a sample containing a test substance (positive sample) is measured using a flat bottom microplate.
D is a view of a well when a sample containing no test substance (negative sample) was measured using a flat-bottomed microplate as viewed from above. If positive, particles that have migrated from the portion of the flat bottom of the well that is not coated with the specific binding substance are trapped in the specific binding substance coated section.

FIG. 2C shows a sample (positive sample) in which a test substance is present using a rectangular parallelepiped transparent container having a rectangular bottom surface.
FIG. 2D is a view of the container when the measurement is performed from above, and FIG. 2D shows the measurement of a sample (negative sample) in which a test substance does not exist using a rectangular parallelepiped transparent container having a rectangular bottom surface. It is the figure which looked at the container concerned from the upper part when performing. In the case of positive, particles that have migrated from the part of the bottom surface that is not coated with the specific binding substance are trapped in the band-shaped coated part.

FIG. 3C shows a plate-like body obtained by measuring a sample containing two kinds of test substances (a sample in which each test substance is positive) using a rectangular plate-like body. FIG. 3D is a view of the plate when viewed from above when a measurement is performed on a sample (negative sample) in which no test substance is present using a rectangular plate. is there. If positive, particles that have migrated from portions of the surface that are not coated with the specific binding substance are trapped in the strip-shaped portions corresponding to the respective test substances.

FIG. 4C shows a plate-like body obtained by measuring a sample (positive sample) in which two types of test substances are present using a rectangular plate-like body provided with grooves on the surface. FIG. 4D is a diagram of the plate-like body viewed from above when the measurement is performed on a sample (a negative sample) in which no test substance is present using such a plate-like body. is there.
If positive, particles that have migrated from the bottom of the groove that are not coated with the specific binding substance are trapped in the strip-shaped coating corresponding to each test substance.

When the carrier is tilted to move the particles along the specific binding substance-coated surface of the carrier, the interaction between the particles to which the test substance is not bound and the surface is weak, and the moving speed is inclined. Almost depends on the angle. Therefore, when a high moving speed is required, that is, when a measurement result is obtained in a short time, the angle of tilting the carrier may be increased. Further, the angle at which the carrier is inclined may be changed over time. Further, when a plate-shaped carrier is used, the speed at which particles move may be changed by bending the plate-shaped body and changing the angle of the plate-shaped body.

In the second measuring method according to the present invention,
Before bringing the sample suspected of containing the test substance and the particles immobilized with the specific binding substance into contact with the surface of the carrier having the surface coated with the specific binding substance with the specific binding substance fixed thereon, The test substance and / or its analog are brought into contact with the specific binding substance immobilized on the surface by contacting the test substance and / or its analog in advance. According to this second measurement method, when the test substance is not present in the sample, that is, when the sample is negative, the particles do not bind to the test substance, so that the particles are fixed to the specific binding substance fixed on the surface in advance. The binding to the specific binding substance immobilized on the surface via the test substance and / or its analogs has stopped or significantly slowed down the movement of the particles, so that the particles become bound to the specific binding substance-coated portion of the surface. Will gather. On the other hand, when the test substance is present in the sample, that is, when the test substance is positive, the particles bind to the test substance in the sample, so that the test substance previously bound to the specific binding substance fixed on the surface is used. And / or immediately move downwards without being affected by its analogs and gather at the lower end of the carrier. Therefore, according to the second measurement method, a result opposite to the distribution state of the positive or negative particles obtained by the first measurement method is obtained.

Here, the analogs of the test substance include a part of the test substance, a substance in which another substance is bound to the test substance, a substance in which a part of the structure of the test substance is substituted, and the like. A substance that has a structure that binds to a specific binding substance of a test substance and can bind to the specific binding substance. For example, when the test substance is an antigen, a substance containing an antigenic determinant of the antigen can be mentioned as an analog of the test substance.

In order to bind the test substance and / or its analog to the specific binding substance immobilized on the surface of the carrier, the test substance and / or its analog is converted into a tris (hydroxymethyl) aminomethane buffer, What is necessary is just to disperse or dissolve in various buffers such as a phosphate buffer and a phosphate buffered saline, or a physiological saline, and contact the surface. The pH of the buffer is preferably in the range of pH 4 to 12.

In the conventional indirect agglutination measurement method, when the sample is blood (a whole blood sample), the red blood cells also roll down (slide down) on the inner wall surface of the measurement container together with the particles by gravity.
Therefore, when measuring the test substance in the blood sample, there is a problem that the distribution state of the particles cannot be confirmed because it is blocked by the red blood cells contained in the sample, making it difficult to determine positive or negative. did. On the other hand, in the present invention, by tilting the carrier, the red blood cells also move by gravity downward and gather at the lower end of the carrier, so that the distribution state of the particles is blocked by the red blood cells. Is reduced, and the determination becomes easier.

Further, when the height of the liquid containing the sample and the particles on the portion coated with the specific binding substance on the surface of the carrier is reduced, the number of red blood cells on the coated portion is further reduced, so that the number of red blood cells on the coated portion is reduced. The distribution state of the particles can be more clearly confirmed. In this case, the height of the liquid containing the sample and the particles on the portion coated with the specific binding substance on the surface of the carrier is preferably 1 mm or less. In order to reduce the height of the liquid containing the sample and the particles on the portion coated with the specific binding substance on the surface of the carrier, for example, when a container as shown in FIG. 1 or FIG. The depth of the container may be reduced, and when a plate-like body having a groove on the surface as shown in FIG. 4 is used as a carrier, the depth of the groove may be reduced.

When the concentration of the test substance in the sample is low, it is necessary to use a large amount of the sample. This is because an image is not formed in the measurement unless a certain number of test substances are present. Therefore, when the concentration of the test substance in the blood sample is low, it is preferable to use, as the carrier, a carrier that has a sufficiently small height on the portion coated with the specific binding substance but has a large capacity for containing liquid. In order to make the carrier having a sufficiently small height on the portion coated with the specific binding substance but a large capacity of the liquid to be accommodated, for example, the portion not covered with the specific binding substance is higher than the portion covered with the specific binding substance. May be increased, or the width and the depth may be increased separately from the height.

Specifically, although not limited thereto, the carriers exemplified in FIGS. 5 to 8 are suitable for measurement of a test substance in a blood sample having a low concentration of the test substance. ing. 5 and 6 are perspective views of containers each having a small height on a portion covered with the specific binding substance and a height not covered with the specific binding substance being greater than the height on the coated portion. It is. FIG. 7 is a perspective view of a container in which the height of the portion covered with the specific binding substance is small and the height of the portion not covered with the specific binding substance is small but the width and depth are large. Further, FIG. 8 shows that a groove having a small height on a portion covered with the specific binding substance and a small height but a large width and depth of a portion not covered with the specific binding substance is provided on the surface. It is a perspective view of a plate-shaped object. In addition, like this,
When a carrier having a sufficiently small height on the portion coated with the specific binding substance but having a large volume of liquid to be contained is used as the carrier, the number of test substances on the portion is reduced as in the case of red blood cells, but the carrier is tilted. As a result, the test substance bound to the particles moves from the other part to the above-mentioned coated part, so that there is no problem in the measurement.

In the present invention, a blood sample (a whole blood sample) is brought into contact with the surface of the carrier coated with the specific binding substance, and then the blood sample on this surface is removed. The surface is washed with a particle dispersion medium or a buffer, etc., and thereafter, a particle having a specific binding substance fixed thereto is brought into contact with the surface, and the carrier is tilted so that the particle moves along the surface,
Thereafter, by determining the presence or absence of the test substance from the distribution state of the particles on this surface, the test substance in the blood sample can be measured. In this case, although the red blood cells in the blood sample are removed from the portion covered with the specific binding substance on the surface of the carrier by removing the blood sample, the test substance in the blood sample is bound to the specific binding substance fixed on the surface of the carrier. Binds to substance. Further, since the particles to which the specific binding substance is fixed are bound to the bound test substance, the distribution state of the bound particles can be clearly confirmed without being blocked by the red blood cells. To remove the blood sample on the surface of the carrier coated with the specific binding substance, a sample absorber made of a water-absorbing material is brought into contact with the blood sample on the surface of the carrier to absorb the blood sample, or to remove the blood sample. By sucking the blood sample on the surface of the carrier or turning the carrier upside down and dropping the blood sample. As a sample absorber made of this water-absorbing material, a material made of a material having a property of absorbing liquid such as paper such as filter paper, paper towel, tissue paper, chemical fiber such as rayon and polyester, cloth, non-woven fabric or cotton is used. Can be used. When removing a blood sample using a plate-like body provided with grooves on the surface as shown in FIG. 4 as a carrier and using a sample absorber, the surface of the carrier covered with a specific binding substance is removed. The height of the liquid containing the sample and the particles is preferably 5 mm or less.

Further, in the present invention, when the sample is blood (whole blood sample), the blood sample is diluted with a diluent of the above-mentioned sample to lower the concentration of red blood cells, thereby allowing the carrier to be coated with the specific binding substance. Since the number of upper red blood cells is reduced, the distribution state of particles can be more clearly confirmed without being blocked by red blood cells.

[0059]

[Example 1]

(Measurement of HBs antigen) (1) Preparation of anti-HBs antibody-immobilized microplate As shown in FIG. 1, the flat bottom of a well of a flat-bottom microplate (Nunc, well diameter: 7 mm, well depth: 8 mm) 25 μl of an anti-HBs antibody solution (5 μg / ml, dissolved in 10 mM phosphate buffer at pH 7) was placed on one side of the sample with a pipette, and allowed to come in contact with the solution, and allowed to stand at 37 ° C. for 3 hours. did. Next, the solution in the well was removed by suction with a pipette, and 0.3 ml of 0.5% (w / v) casein-containing Tris buffer (pH 7.5, 50 mM) (hereinafter referred to as diluent A) was added. It was left at 4 ° C. overnight, and this was aspirated off with a pipette. Thus, a microplate in which the anti-HBs antibody was immobilized on the left half of the flat bottom surface of the well was prepared.

(2) Preparation of Anti-HBs Antibody Immobilized Particles Particles (Dynabeads M-450 uncoated, manufactured by Dynal, particle size: 4.5 μm, CV 5% or less of particle size, specific gravity 1.5, concentration 3% (w / v)) and 1 ml of an anti-HBs antibody solution (Sinotest, dissolved in 10 mM phosphate buffer at pH 7.0 at a concentration of 0.1 mg / ml) were mixed and reacted at 37 ° C. for 30 minutes. About 20 times the amount of the diluting solution A was added thereto to perform masking.
Next, the obtained particles were washed with the diluent A, and the particles were redispersed in the diluent A so as to have a concentration of about 0.1% (w / v). Thus, an anti-HBs antibody fixed particle dispersion was prepared.

(3) Measurement of HBs Antigen 25 μl of the sample HBs antigen-positive serum was added to the wells of the anti-HBs antibody-fixed microplate prepared in (1) above.
Was added, and then 25 μl of the anti-HBs antibody-fixed particle dispersion prepared in the above (2) was added thereto, followed by stirring for 1 minute. The anti-HBs antibody-immobilized particles move from the flat bottom of the well where the anti-HBs antibody is not fixed to the partial bottom where the anti-HBs antibody is fixed (the direction of the arrow in FIG. 1).
Anti-HBs so that the direction of the arrow in FIG.
The antibody-immobilized microplate was tilted. Within 2 minutes after the tilt, an image of particles as shown in FIG. 1C was observed. In each well of the anti-HBs antibody-fixed microplate prepared in the above (1), sample HBs antigen solution of various concentrations (Meiji Dairy Co., Ltd., 50 mM Tris containing 0.5% (w / v) casein and 0.1 M sodium chloride). 25 μl each of which was dissolved in a hydrochloric acid buffer solution (pH 8.0) at a concentration of 0, 1, 2, 10, 100, or 1000 ng / ml), and the anti-HBs antibody-fixed particles prepared in (2) above were further dispersed. Each of the liquids was added in an amount of 25 μl and stirred for 1 minute. The anti-HBs on the flat bottom of the well
The anti-HBs is directed from the portion where the antibody is not fixed to the portion where the anti-HBs antibody is fixed on the flat bottom (the direction of the arrow in FIG. 1).
As shown in Fig. 1 at an angle of about 60 °,
The microplate immobilized with the anti-HBs antibody was tilted so that the arrow direction of was downward. Within 3 minutes after the slanting, particle images such as those shown in FIG. 1C were observed for all but the antigen concentration of 0 ng / ml.

Example 2 (Measurement of Anti-HBs Antibody) (1) Preparation of HBs Antigen Immobilized Microplate As shown in FIG. 1, a flat bottom microplate (manufactured by Nunc, well diameter: 7 mm, well depth) : 8 mm) 25 μl of HBs antigen solution (5 μg / ml, dissolved in 10 mM phosphate buffer at pH 7 at a concentration of 5 μg / ml) was placed on one side half of the flat bottom of the well using a pipette and contacted. , 3
The mixture was allowed to stand at 7 ° C. for 3 hours. Then, after the solution in the well was removed by suction with a pipette, 0.3 ml of the above diluent A was added, and
It was left overnight at 0 ° C. and this was aspirated off with a pipette. Thus, a microplate in which the HBs antigen was fixed to the left half of the flat bottom surface of the well was prepared.

(2) Preparation of HBs antigen-immobilized particles Particles (Dynabeads M-450 uncoated, manufactured by Dynal, particle size: 4.5 μm, CV 5% or less of particle size, specific gravity 1.5, concentration 3% (w / v)) 1 ml and HBs antigen solution (Meiji Dairies,
1 ml of a 10 mM phosphate buffer of pH 7.2 dissolved at a concentration of 40 μg / ml) was mixed and reacted at 37 ° C. for 30 minutes. About 20 times the amount of the diluting solution A was added thereto to perform masking. Next, the obtained particles were washed with the diluent A, and the particles were redispersed in the diluent A so as to have a concentration of about 0.1% (w / v). Thus, an HBs antigen-fixed particle dispersion was prepared.

(3) Measurement of Anti-HBs Antibody 25 μl of serum containing anti-HBs antibody as a sample was added to the wells of the HBs antigen-fixed microplate prepared in (1) above.
Then, 25 μl of the HBs antigen-fixed particle dispersion prepared in the above (2) was added and stirred for 1 minute. The HBs antigen-immobilized particles are moved by about 60 ° from the portion of the flat bottom of the well where the anti-HBs antibody is not fixed toward the portion of the flat bottom where the HBs antigen is fixed (the direction of the arrow in FIG. 1). The HBs antigen-immobilized microplate was tilted so that the direction of the arrow in FIG. Within 2 minutes after the tilt, an image of particles as shown in FIG. 1C was observed.

Example 3 (Measurement of Anti-HBc Antibody) (1) Preparation of HBc Antigen Immobilized Microplate As shown in FIG. 1, a flat-bottomed microplate (manufactured by Nunc Corporation, well diameter: 7 mm, well depth) 25 μl of HBc antigen solution (manufactured by Sinotest, dissolved in 10 mM phosphate buffer at pH 7 at a concentration of 5 μg / ml) was placed on one half of the flat bottom of the well using a pipette, and brought into contact therewith. It was left at 37 ° C. for 3 hours. Next, the solution in the well was removed by suction with a pipette, and then a glycine buffer (pH 9, 100 mM) containing 0.5% (w / v) casein (hereinafter referred to as diluent B)
That. ) Was added thereto, and the mixture was allowed to stand at 37 ° C overnight, and this was removed by suction with a pipette. Thus, a microplate in which the HBc antigen was fixed to the left half of the flat bottom surface of the well was prepared.

(2) Preparation of HBc antigen-immobilized particles Particles (Dynabeads M-450 uncoated, manufactured by Dynal Co., Ltd., particle size: 4.5 μm, CV 5% or less of particle size, specific gravity 1.5, concentration 3% (w / v)) 1 ml of an HBc antigen solution (Sinotest, dissolved in 10 mM phosphate buffer at pH 7.2 at a concentration of 40 μg / ml) was mixed and reacted at 37 ° C. for 30 minutes.
About 20 times the amount of the diluent B was added thereto, and masking was performed at 37 ° C. for 3 days. Next, the obtained particles were washed with the diluent B, and the particles were redispersed in the diluent B so as to have a concentration of about 0.1% (w / v). Thus, HBc antigen-immobilized particles were prepared.

(3) Measurement of anti-HBc antibody To the wells of the HBc antigen-fixed microplate prepared in (1) above, 37.5 μl of the HBc antigen-fixed particle dispersion prepared in (2) was added, and then the anti-HBc antibody as a sample was added. 12.5 μl of serum containing HBc antibody was added and stirred for 1 minute. The HBc antigen-immobilized particles are moved from the portion where the anti-HBc antigen is not fixed on the flat bottom of the well to the portion where the HBc antigen is fixed on the flat bottom (the direction of the arrow in FIG. 1).
The HBc antigen-immobilized microplate was tilted so that the direction of the arrow in FIG. Within 2 minutes after the tilt, an image of particles as shown in FIG. 1C was observed.

[Example 4] (Measurement of HBs antigen using plate-shaped carrier) (1) Preparation of anti-HBs antibody-immobilized plate-shaped plate Extruded acrylic resin (25 mm in width, 40 mm in depth, 1 mm in thickness) [ [Acrylic Sunday Co., Ltd.]], two acrylic extruded plates (width 10 mm, depth 40 mm, thickness 0.5 mm) [Acrylic Sunday Co., Ltd.] were adhered with a solvent at an interval of 5 mm, and shown in FIG. A plate-like body having grooves (width 5 mm, depth 40 mm, height 0.5 mm) provided on the surface thus prepared. 20 μl of an anti-HBs antibody solution (manufactured by Sinotest and dissolved in a 10 mM phosphate buffer at a pH of 7 at a concentration of 5 μg / ml) was pipetted into a portion of the bottom of the groove of the plate-like body at a distance of 5 mm to 10 mm from the left end using a pipette. The plate was placed on the plate and brought into contact, and allowed to stand at 37 ° C. for 3 hours. Next, after these solutions were removed by suction with a pipette, 0.3 ml of the above-mentioned diluent A was added thereto, left at 4 ° C. overnight, and removed by suction with a pipette. Then, an acrylic resin extruded plate (width 25 mm, depth 25 mm, thickness 1 mm) is placed on the left end of the plate.
[Acrylic Sunday Co., Ltd.] was adhered with a solvent to cover the plate-like body as shown in FIG. In this way, a plate-like body having a band-shaped covering portion formed with a specific binding substance (anti-HBs antibody) on the bottom surface of the groove was prepared.

(2) Measurement of HBs antigen A sample HBs antigen solution (Meiji Dairy Products, pH 7.2
(100 ng / ml dissolved in mM phosphate buffer)
50 μl of the anti-HBs antibody-fixed particle dispersion prepared in (2) of Example 1 was mixed and stirred in a test tube. 100 μl of this mixture was placed using a pipette near the boundary between the covered portion and the uncovered portion of the groove of the anti-HBs antibody-fixed plate prepared in (1) above, and was brought into contact with the band-shaped covering portion. . The anti-HBs antibody on the bottom surface of the groove of the plate is not fixed, with the side of the plate having the band-shaped covering portion formed facing down (the direction of the arrow in FIG. 10 is pointing down). The anti-HBs antibody-fixed plate is tilted at an angle of about 60 ° so that the anti-HBs antibody-immobilized particles move from the portion toward the portion where the anti-HBs antibody is immobilized on the bottom surface of the groove (the direction of the arrow in FIG. 10). Was. Within 3 minutes after the tilt, an image of particles as shown in FIG. 10C was observed.

Example 5 (Simultaneous Measurement of HBs Antigen and Hemoglobin) (1) Preparation of Anti-HBs Antibody / Anti-Hemoglobin Antibody Immobilized Plate Acrylic resin extruded plate (25 mm in width, 40 mm in depth, 1 mm in thickness) [acrylic Sandy] [made by Acrylic Sunday] with two acrylic extruded plates (width 10 mm, depth 40 mm, thickness 0.5 mm) [solvent] at a distance of 5 mm, as shown in FIG. A plate-like body provided with grooves (width 5 mm, depth 40 mm, height 0.5 mm) on the surface was prepared. 20 μl of an anti-HBs antibody solution (manufactured by Sinotest and dissolved in a 10 mM phosphate buffer at a pH of 7 at a concentration of 5 μg / ml) was pipetted into a portion of 5 to 10 mm from the left end of the bottom of the groove of the plate using a pipette. And placed in contact. Further, from the left end of the bottom surface of the groove of this plate-like body,
An anti-hemoglobin antibody solution (manufactured by Nippon Biotest Laboratory Co., Ltd., at a concentration of 5 μg / ml and pH
7) (dissolved in 10 mM phosphate buffer) was placed in contact with a pipette using a pipette, and allowed to stand at 37 ° C for 3 hours.
Next, after these solutions were removed by suction with a pipette, 0.3 ml of the above-mentioned diluent A was added thereto, left at 4 ° C. overnight, and removed by suction with a pipette. Then, an acrylic resin extruded plate (width: 25 mm, depth: 25 mm, thickness: 1 mm) [manufactured by Acrylic Sandy Co., Ltd.] is bonded on the left end of the plate-like body with a solvent, thereby forming a plate-like plate as shown in FIG. I covered my body. In this way, a plate-like body having a band-shaped covering portion formed with two different types of specific binding substances (anti-HBs antibody and anti-hemoglobin antibody) on the bottom surface of the groove was produced.

(2) Preparation of anti-hemoglobin antibody-immobilized particles Particles (Dynabeads M-450 uncoated, manufactured by Dynal Co., Ltd., particle size: 4.5 μm, CV 5% or less of particle size, specific gravity 1.5, concentration 3% (w / v)) 1 ml and an anti-hemoglobin antibody solution (manufactured by Nippon Biotest Laboratory Co., Ltd., dissolved in 10 mM phosphate buffer at pH 7.0 at a concentration of 0.1 mg / ml) were mixed and reacted at 37 ° C for 30 minutes. I let it. About 20 times the amount of the diluent A was added thereto, and masking was performed at 37 ° C. for 3 days. Next, the obtained particles were washed with the diluent A, and the particles were redispersed in the diluent A so as to have a concentration of about 0.1% (w / v). Thus, an anti-hemoglobin antibody fixed particle dispersion was prepared.

(3) Simultaneous measurement of HBs antigen and hemoglobin HBs antigen solution (Meiji Dairy Products, pH 7.2, 10 m
Dissolved in M phosphate buffer at a concentration of 100 ng / ml) 50
In a test tube, 50 µl of 1 µg / ml of human hemoglobin (manufactured by Sinotest) was mixed and stirred in a test tube.
50 μl of the anti-hemoglobin antibody-fixed particle dispersion prepared in the above and 50 μl of the anti-HBs antibody-fixed particle dispersion prepared in Example 1, (2) were added thereto and stirred. 150μ of this mixture
1 was dropped with a pipette near the boundary between the covered portion and the uncovered portion of the groove of the anti-HBs antibody / anti-hemoglobin antibody immobilized plate-like body prepared in (1) above. The anti-HBs antibody and the anti-hemoglobin antibody on the bottom surface of the groove of the plate-like body are set such that the side of the plate-like body where the band-shaped covering portion is formed is directed downward (the direction of the arrow in FIG. 4 is down). The anti-HBs antibody-immobilized particles and the anti-hemoglobin antibody-immobilized particles are moved from the non-immobilized portion toward the portion where the anti-HBs antibody and the anti-hemoglobin antibody are immobilized on the bottom surface of the groove (the direction of the arrow in FIG. 4). The plate fixed with the anti-HBs antibody / anti-hemoglobin antibody was tilted at an angle of 60 °. An image of two particles as shown in FIG. 4C was observed within 3 minutes after the tilt.

[Example 6] (Measurement of HBs antigen in blood sample) HBs antigen positive blood
100 μl of the anti-HBs antibody-immobilized plate-like body prepared in Example 4 (1) was placed using a pipette near the boundary between the covered and uncovered grooves, and was brought into contact with the band-shaped covered portion. Tissue paper (Product name: Kim Wipe,
A sample absorber prepared by folding 3 × 3 cm × 4 mm (thickness) of Jujo Kimberly Co., Ltd. was folded to form an end portion on the side of the anti-HBs antibody-fixed plate-like body on which the band-shaped covering portion was formed (the plate in FIG. 10A). HBs antigen-positive blood in contact with the strip-shaped coated portion was removed by absorption. Next, the anti-HBs antibody-fixed particle dispersion prepared in (1) of Example 1 was pipetted near the boundary between the covered and uncovered grooves of the anti-HBs antibody-fixed plate. And placed in contact with the strip-shaped coating.
Thereafter, the anti-HBs antibody on the bottom surface of the groove of the plate is fixed with the side of the plate having the band-shaped covering portion formed facing downward (the direction of the arrow in FIG. 10 pointing down). The anti-HBs antibody-fixed plate at an angle of about 60 ° so that the anti-HBs antibody-fixed particles move from the non-portion to the portion of the bottom of the groove where the anti-HBs antibody is fixed (the direction of the arrow in FIG. 10). Tilted. Within 3 minutes after the tilt, an image of particles as shown in FIG. 10C was observed. No particle image was observed when the measurement was performed using negative blood as a sample.

[Example 7] (Measurement of anti-HBs antibody in blood sample) (1) Preparation of HBs antigen-fixed plate Acrylic resin extruded plate (width 25 mm, depth 40 mm, thickness 1 mm) [Acrylic Sunday Co., Ltd.] ], Two acrylic extruded plates (width 10 mm, depth 40 mm, thickness 0.5 mm) [manufactured by Acrylic Sunday Co., Ltd.] are adhered at intervals of 5 mm with a solvent, and the surface shown in FIG. A plate-like body provided with grooves (width 5 mm, depth 40 mm, height 0.5 mm) was produced. Using a pipette, 20 μl of HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., dissolved in 10 mM phosphate buffer at a pH of 7 at a concentration of 5 μg / ml) was placed in a portion of 5 to 10 mm from the left end of the bottom of the groove of the plate-like body using a pipette. The plate was placed on the plate and brought into contact, and allowed to stand at 37 ° C for 3 hours. Next, after these solutions were removed by suction with a pipette, 0.3 ml of the above-mentioned diluent A was added thereto, left at 4 ° C. overnight, and removed by suction with a pipette. Then, an acrylic resin extruded plate (width: 25 mm, depth: 25 mm, thickness: 1 mm) [manufactured by Acrylic Sunday Co., Ltd.] is adhered on the left end of the plate with a solvent to form the plate as shown in FIG. Covered. In this way, a plate-like body having a band-shaped covering portion formed with a specific binding substance (HBs antigen) on the bottom surface of the groove was prepared.

(2) Measurement of Anti-HBs Antibody 100 μl of anti-HBs antibody-positive blood was prepared from H prepared in (1) above.
The Bs antigen-fixed plate was placed with a pipette near the boundary between the covered portion and the uncovered portion of the groove, and was brought into contact with the band-shaped covering portion. Tissue paper (Product name:
Kim wipe, Jujo Kimberly) 3cm x 3cm x
A sample absorber made by folding to 4 mm (thickness) is HB
The s antigen-fixed plate is brought into contact with the end on the side where the band-shaped portion is formed (the left end side of the plate in FIG. 10A) to absorb the anti-HBs antibody-positive blood contacted with the band-shaped portion. And removed. Next, the HBs antigen-fixed particle dispersion prepared in (2) of Example 2 was placed using a pipette near the boundary between the covered portion and the uncovered portion of the groove of the HBs antigen-fixed plate. It was brought into contact with the strip-shaped covering part. Then, the HBs antigen on the bottom surface of the groove of the plate is fixed with the side of the plate having the band-shaped covering portion formed facing downward (the direction of the arrow in FIG. 10 pointing downward). The HBs antigen-immobilized plate was inclined at an angle of about 60 ° so that the HBs antigen-immobilized particles moved from the portion where no HBs antigen was immobilized on the bottom surface of the groove (the direction of the arrow in FIG. 10). Within 3 minutes after the tilt, an image of particles as shown in FIG. 10C was observed. No particle image was observed when the measurement was performed using negative blood as a sample.

[0076]

According to the first measurement method and the second measurement method of the present invention, the presence or absence of a test substance in a sample is determined by the size of a circular image of particles collected on the bottom of a container as in the prior art. Not
It is easy to determine from whether the particles are collected at the end of the surface of the carrier or at the portion of the surface of the carrier coated with the specific binding substance, so that the determination is easy and even when the concentration of the test substance is low. The determination is easy. Therefore, a test substance in a sample can be measured with high sensitivity. Moreover, no erroneous judgment is given even when a non-specific agglutination reaction occurs. In addition, according to the first measurement method of the present invention, the sensitivity can be easily changed by changing the amount of the specific binding substance fixed to the surface of the particles and / or the carrier, so that the efficiency is high. Therefore, even when the concentration of the test substance is low, the presence or absence of the test substance can be easily determined. Furthermore, according to the first measurement method and the second measurement method of the present invention, in a short time, specifically, depending on the type of the test substance, usually about 20 seconds to 10 minutes, The presence or absence of a test substance can be determined. For example, in the case of HBs antigen, measurement can be performed within 3 minutes even at a concentration of about 1 ng / ml. Furthermore, according to the first measurement method and the second measurement method of the present invention, it is possible to simultaneously measure a plurality of test substances in a sample. According to the first measurement method and the second measurement method of the present invention, even when the sample is blood, by tilting the carrier, the red blood cells in the sample move by gravity downward in the tilt, and the Since the particles gather at the lower end, the degree of the particle distribution state blocked by the red blood cells is reduced, and the determination becomes easier. Further, the first measuring instrument and the second measuring instrument according to the present invention can be suitably used for the first measuring method and the second measuring method, respectively.

[Brief description of the drawings]

FIG. 1 is a diagram showing a flat-bottom microplate in which one half of the flat bottom of each well is coated with a specific binding substance.

FIG. 2 is a view showing a rectangular parallelepiped transparent container whose bottom surface is covered with a specific binding substance in a band shape.

FIG. 3 is a view showing a plate-like body whose surface is covered in a band shape with a specific binding substance.

FIG. 4 is a diagram showing a plate-like body having a groove provided on a surface, and a bottom surface of the groove covered with a specific binding substance in a band shape.

FIG. 5 is a perspective view of a container in which the height on the portion coated with the specific binding substance is small, and the height of the portion not coated with the specific binding substance is larger than the height on the coated portion.

FIG. 6 is a perspective view of a container in which the height on the portion coated with the specific binding substance is small and the height of the portion not coated with the specific binding substance is larger than the height on the coated portion.

FIG. 7 is a perspective view of a container having a small height on a portion coated with a specific binding substance and a small height but a large width and depth of a portion not coated with the specific binding substance.

FIG. 8 is a diagram showing a plate-like structure having a small height on a portion covered with a specific binding substance, and a small groove not covered with the specific binding substance but having a large width and depth. It is a perspective view of a body.

FIG. 9 is a diagram showing an agglutination image when a test substance in a sample is measured by an indirect agglutination reaction measurement method.

FIG. 10 is a view showing a plate-like body in which a groove is provided on a surface, and a bottom surface of the groove is covered with a specific binding substance in a band shape.

Claims (11)

[Claims] 1. A sample having a specific binding substance to a test substance in a sample immobilized thereon and having a surface coated with the specific binding substance, the sample and a specific binding substance to the test substance being Contacting the specific binding substance and the same or different specific binding substance immobilized on the carrier with particles immobilized thereon, and tilting the carrier such that the particles move along the surface. A method for measuring a test substance in a sample, the method comprising determining the presence or absence of the test substance from the distribution state of the particles on the surface. 2. A test substance and / or an analog thereof are brought into contact with a surface of a carrier having a surface on which a specific binding substance to a test substance in a sample is fixed and coated with the specific binding substance. Binding the test substance and / or its analog to the specific binding substance immobilized on the surface, wherein the sample and the specific binding substance to the test substance are immobilized on the carrier. Contacting the specific binding substance and the same or different specific binding substance with particles immobilized thereon, and tilting the carrier so that the particles move along the plane, wherein the particles on the plane Determining the presence or absence of a test substance from the distribution state of the test substance. 3. The method for measuring a test substance in a sample according to claim 1, wherein the surface is partially coated with the specific binding substance. 4. The container according to claim 1, wherein the carrier is provided with at least one concave portion having a flat bottom, wherein the flat bottom is coated with a specific binding substance. A method for measuring a test substance in a sample. 5. The method for measuring a test substance in a sample according to claim 1, wherein the carrier is a plate-like body, and the surface is coated with a specific binding substance. 6. The method for measuring a test substance in a sample according to claim 1, wherein the test substance is an antigen, and the specific binding substance is an antibody. 7. The method for measuring a test substance in a sample according to claim 1, wherein the test substance is an antibody, and the specific binding substance is an antigen. 8. A carrier having a surface on which a specific binding substance to a test substance in a sample is immobilized and coated with the specific binding substance, and a specific binding substance to the test substance, the carrier being immobilized on the carrier. An instrument for measuring a test substance in a sample, comprising: a specified specific binding substance; and a particle on which the same or different specific binding substance is immobilized. 9. A carrier on which a specific binding substance to a test substance in a sample is immobilized and has a surface coated with the specific binding substance, the test substance and / or its analog, An instrument for measuring a test substance in a sample, comprising a specific binding substance, the specific binding substance immobilized on the carrier, and particles having the same or different specific binding substance immobilized thereon. 10. The instrument for measuring a test substance in a sample according to claim 8, wherein the test substance is an antigen, and the specific binding substance is an antibody. 11. The measuring instrument for a test substance in a sample according to claim 8, wherein the test substance is an antibody, and the specific binding substance is an antigen.