JP3749959B2 - Measuring method of test substance using magnetic particles and measuring instrument used for the method - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring a test substance in a sample using magnetic particles in which a specific binding substance for the test substance is fixed, and a measuring instrument used in the method, and particularly to a trace amount of test contained in the sample. The present invention relates to a test substance measuring method capable of measuring a substance in a short time and easily without being affected by an interfering substance, and a measuring instrument used for the method. The present invention is useful in the field of life science such as a clinical examination field.
[0002]
[Prior art]
A method for measuring a small amount of test substance contained in a sample using a reaction between substances having specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, and ligand and receptor. Alternatively, various measuring instruments are known.
[0003]
Among these, immunological measurement methods using 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.
[0004]
This indirect agglutination measurement method uses particles (polymer particles such as latex particles or gelatin particles or erythrocytes) bound with an antibody against the test substance (if the test substance is an antibody, an antigen can also be used). A sample presumed to contain a test substance is mixed in a measurement container with a U-shaped or V-shaped bottom such as a microtiter plate, reacted, and particles generated via the test substance. The presence / absence of the test substance in the sample is determined by observing the aggregated image.
[0005]
FIG. 9 shows an agglomerated image when the test substance in the sample is measured by this indirect agglutination measurement method. When the test substance does not exist in the sample (negative), the antibody (or antigen) -bound particles do not aggregate, so they settle as they are by gravity and are deposited on the inner wall of the U-shaped or V-shaped measuring container. Roll along (slide down) and gather at the center of the bottom of the measuring container. Therefore, in the case of negative, when viewed from above the measurement container, an image in which the 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 exists in the sample (positive), the particles to which the antibody (or antigen) is bound cause three-dimensional aggregation via the test substance to generate an aggregate. This agglomerate is less likely to roll on the inner wall surface of the measurement container (harder to slip off) than the non-aggregated particles and has a lower rolling speed (sliding speed). Stop. Therefore, in the case of positive, when viewed from above the measurement container, a state in which the particles spread on the bottom surface of the measurement container, that is, a “button” -shaped aggregated image can be observed (B in FIG. 9), and the test substance exists in the sample. It can be confirmed.
[0006]
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 by the size of a circular image formed by particles on the bottom surface of the measurement container.
Therefore, in the case of a sample with 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 central part of the bottom of the measurement container, and the aggregate Since the circle of the image was small, it was difficult to distinguish from the negative case.
Because of the difficulty of determination in the case of a sample with a low concentration of the test substance, it is impossible to measure a low concentration of the test substance. Therefore, the time required for the measurement is long, and the normal measurement time is 1 hour or more.
[0007]
In addition, in the indirect agglutination measurement method, particles bound to antibodies (or antigens) are aggregated via components in a sample other than the test substance, or particles bound to antibodies (or antigens) are bound to the measurement container. In some cases, the sample binds to the inner wall surface and shows an aggregated image as if the test substance is present (positive) even though the test substance is not present in the sample (negative) (non-specific agglutination reaction) . Such a possibility that a negative sample may be erroneously determined as positive has been a serious problem in diagnosis of diseases by clinical examination.
[0008]
In contrast, JP-A-64-69954 discloses that a specimen sample is added to a measurement container in which an antibody or antigen corresponding to an antigen or an antibody as a test substance in a specimen sample is fixed to the inner wall, and simultaneously or subsequently. An insoluble carrier particle fixed with the same antibody or antigen or specific binding analog fixed to the measurement container without washing is added to the measurement container, and the sample sample is determined depending on the presence or absence of an agglutination reaction. An immunological measurement method for determining the presence or absence of an antigen or antibody as a test substance is disclosed. In this measurement method, when the state in the measurement container when the antigen (or antibody) as the test substance does not exist (negative) in the specimen sample is viewed from above, an image similar to the aggregated image shown in FIG. Is observed. In addition, when an antigen (or antibody), which is a test substance, is present in the sample sample (positive), when the state in the measurement container is viewed from above, an image similar to the aggregated image shown in FIG. 9B is observed. Is done.
[0009]
The measuring method described in JP-A-64-69954 is a method that can measure a low concentration of a test substance, suppress a zone phenomenon, and obtain a clear aggregated image in a short time. Since the presence or absence of the test substance in the sample is determined by the size of the circular image formed by the particles on the bottom of the measurement container, it is determined that the test substance is negative when the test substance concentration is extremely low The problem of being difficult was inevitable.
[0010]
Japanese Patent Application Laid-Open No. 2-124464 discloses magnetic marker particles in which substances (antibodies or antigens) that specifically bind to or compete with a substance to be measured (test substance) are immobilized on magnetic particles; Based on the distribution state of the magnetic marker particles collected on the predetermined wall surface area by mixing the sample solution (sample) and applying a magnet arranged outside the predetermined wall surface area of the measurement container to the reaction solution. An immunological measurement method for measuring a substance to be measured in a sample 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 aggregated image shown in FIG. When the measurement substance is present (positive), when the state in the measurement container is viewed from above, an image similar to the aggregated image shown in FIG. 9B is observed.
[0011]
According to the measuring method described in Japanese Patent Laid-Open No. 2-124464, the formation of an agglomerated image on the bottom surface of the measurement container can be performed in a short time by attracting the magnetic particles to the bottom surface of the measurement container with a magnet to increase the sedimentation rate of the magnetic particles. The time required for determination can be shortened. However, since the presence or absence of the test substance in the sample is still determined by the size of the circular image formed by the particles on the bottom of the measurement container, it is said that the sample is negative when the test substance concentration is low. It was unchanged in that it was difficult to distinguish.
[0012]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to measure a test substance in a sample visually or using an instrument using a specific binding reaction, in a short time regardless of the concentration of the test substance in the sample. An object of the present invention is to provide a measurement method capable of determining the presence or absence of a test substance and having high reliability of the determination, and a carrier and a measurement instrument used in the method.
[0013]
[Means for Solving the Problems]
  The present invention provides a surface in which a specific binding substance for a test substance in a sample is fixed and partially covered with the specific binding substance.That is, the surface that has both the part not covered with the specific binding substance and the part coveredThe sample is brought into contact with the surface of the carrier having a magnetic particle having a specific binding substance for the test substance and a specific binding substance that is the same as or different from the specific binding substance immobilized on the carrier. A step, and the magnetic particlesFrom the part not coated with the specific binding substance ofSaid surface comprising the step of acting a magnet to moveParts that are not coated with specific binding substances and parts that are coatedThe method for measuring a test substance in a sample (first measurement method) is characterized in that the presence or absence of a test substance is determined from the distribution state of magnetic particles in the sample. The present invention also provides a surface in which a specific binding substance for a test substance in a sample is fixed and partially covered with the specific binding substance.That is, the surface that has both the part not covered with the specific binding substance and the part coveredContacting the test substance and / or its analog with the surface of the carrier having the above-mentioned, and binding the test substance and / or its analog to the specific binding substance immobilized on the surface; Contacting the sample with magnetic particles that are specific binding substances for the test substance and fixed with specific binding substances that are the same as or different from the specific binding substances fixed to the carrier; and surfaceFrom the part not coated with the specific binding substance ofSaid surface comprising the step of acting a magnet to moveParts that are not coated with specific binding substances and parts that are coatedIt is a measuring method (2nd measuring method) of the to-be-tested substance in a sample characterized by determining the presence or absence of a to-be-tested substance from the distribution state of the magnetic particle in (2).
Furthermore, the present invention is a container in which a carrier used for measurement of a test substance in a sample is provided with at least one recess having a flat bottom surface, and a specific binding substance for the test substance is fixed and partially A carrier having a flat bottom surface coated with a specific binding substance, and a magnetic substance in which a specific binding substance that is the same as or different from the specific binding substance fixed to the carrier is fixed to the test substance An instrument for measuring a test substance in a sample, including particles.
According to the present invention, the carrier used for measuring the test substance in the sample is a plate-like body, and the specific binding substance for the test substance is fixed and partially covered with the specific binding substance. A test in a sample, comprising: a carrier having a surface; and a magnetic substance having a specific binding substance to the test substance and a specific binding substance fixed or the same as the specific binding substance fixed to the support. It is a measuring instrument for substances.
Furthermore, the present invention is a container in which a carrier used for measurement of a test substance in a sample is provided with at least one recess having a flat bottom surface, and a specific binding substance for the test substance is fixed and partially A carrier having a flat bottom surface coated with a specific binding substance, a test substance and / or its analog, and a specific binding substance for the test substance, which is the same as the specific binding substance immobilized on the carrier Or it is a measuring instrument of the test substance in a sample containing the magnetic particle to which the different specific binding substance was fixed.
According to the present invention, the carrier used for measuring the test substance in the sample is a plate-like body, and the specific binding substance for the test substance is fixed and partially covered with the specific binding substance. A carrier having a surface, a test substance and / or its analog, a specific binding substance for the test substance, which is the same or different from the specific binding substance fixed to the carrier, is immobilized. A measuring instrument for a test substance in a sample, including magnetic particles.
[0014]
DETAILED DESCRIPTION OF THE INVENTION
The test substance to be measured in the present invention may be any biological substance such as an organic substance such as protein, carbohydrate, lipid and nucleic acid, or an inorganic substance.
Specifically, virus-related antigens or antibodies such as HBs antigen, anti-HBs antibody, HBe antigen, anti-HBe antibody, anti-HBc antibody, anti-HCV antibody, anti-HIV antibody, anti-ATLV antibody; anti-treponema paridum (TP) Bacteria-related antigens or antibodies such as antibodies, anti-mycoplasma antibodies, anti-streptridine O antibodies (ASO); immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), or immunoglobulin E (IgE) ); Immunoglobulins such as C-reactive protein (CRP), α1-acid glycoprotein, haptoglobin, complement C3, complement C4, rheumatoid factor, etc .; tumor markers such as α-fetoprotein, CEA, CA19-9 Hormones such as human placental chorionic gonadotropin; allergens, allergen-related antigens or antibodies such as allergen-specific IgE antibodies; Blood coagulation related substances such as thrombin (AT); fibrin degradation products (FDP), fibrinolytic related substances such as D dimer; blood group related antigens or antibodies such as ABO blood group antibodies and irregular antibodies; Examples include substances or drugs related to other diseases such as DNA or RNA; bacterial DNA or RNA; animal or plant DNA or RNA such as humans; lipoprotein (a).
[0015]
In the present invention, the sample refers to a liquid sample in which the above-mentioned test substance may exist and the presence or absence of the test substance is to be confirmed or quantified.
For example, human or animal blood, serum, plasma, urine, semen, spinal fluid, saliva, sweat, tears, ascites, amniotic fluid, etc .; human or animal brain or other organs, hair, skin, nails, muscles, nerves Extracts of tissues and the like; human or animal fecal extracts; cells or fungal extracts; plant extracts and the like.
[0016]
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 the specific binding substance is caused by specific interaction with the test substance. A substance that stably binds to a test substance in a non-covalent manner.
For example, the specific binding substance is the antibody when the test substance is an antigen, the antigen when the test substance is an antibody, and the complementary substance when the test substance is a nucleotide chain. It is a nucleotide chain.
Further, when the test substance is a ligand, it is the receptor.
The specific binding substance immobilized on the magnetic particles and the specific binding substance immobilized on the carrier may be the same or different as long as the binding through the test substance is possible.
[0017]
In the present invention, the magnetic particles may be particles that are magnetized at least while a magnet is applied from the outside. Examples of the magnetic particles include ferromagnetic metals such as iron, cobalt, and nickel, alloys containing these ferromagnetic metals, those containing a ferromagnetic metal or an alloy containing a ferromagnetic metal in a non-magnetic material, and ferromagnetic Particles obtained by molding a ferromagnetic material such as one containing a non-magnetic material in a metal or an alloy containing a ferromagnetic metal into a single particle, the surface of the ferromagnetic material is polystyrene, silica gel, gelatin, polyacrylamide Particles coated with a high molecular weight material such as polystyrene, silica gel, gelatin, acrylamide, etc., and particles coated with a ferromagnetic material as a core, red blood cells, liposomes, microcapsules, etc. Examples thereof include particles encapsulating a ferromagnetic material.
In addition, it is particularly preferable that the magnetic particles have a property of being magnetized while a magnet is applied from the outside and demagnetizing quickly by blocking the magnet from the outside. For example, Dynabeads M-450 uncoated (trade name, manufactured by Dynal Co., Ltd.), which is a magnetic particle in which iron (III) oxide (Fe2O3), which is a ferromagnetic material, is dispersed in a particle.
Furthermore, as the magnetic particles, those colored by coating a pigment or dispersing or encapsulating the pigment in the particles may be used.
[0018]
The particle diameter of the magnetic particles is preferably 0.01 to 100 μm, particularly preferably 0.5 to 10 μm. The magnetic particles preferably have a specific gravity of 1 to 10.
[0019]
As the magnetic particles in the present invention, as described above, particles already used as immunomagnetic separation carriers in EIA, RIA, FIA, chemiluminescence immunoassay, etc. or used as cell magnetic separation carriers The particles used can be used without any limitation.
[0020]
In the present invention, magnetic particles to which a specific binding substance is immobilized are used. In order to immobilize a specific binding substance on the magnetic particle, the specific binding substance is attached to the surface of the magnetic particle by hydrophobic bonding, hydrophilic adsorption, or the like. It can be carried out by a physical adsorption method, a chemical bonding method such as a covalent bond, or a combination of these methods.
[0021]
When the specific binding substance is immobilized on the magnetic particles by the physical adsorption method, the specific binding substance and the magnetic particles are mixed and brought into contact with each other in a buffer solution or the like according to a known method. Can do.
For example, the specific binding substance and magnetic particles are mixed in a solution such as a buffer solution and brought into contact with each other by stirring, and the adsorption reaction is carried out at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day. The obtained magnetic particles may be washed with a buffer solution or the like.
[0022]
In addition, when the specific binding substance is immobilized on the magnetic particles by a chemical binding method using a cross-linking reagent, the Clinical Pathology Special Edition No. 53 Special Issue on Clinical Pathology Immunoassay for Clinical Examination -Technology and Applications- ”, Clinical Pathology Publications, 1983, Japan Biochemical Society,“ Neochemistry Experiment Course 1 Protein IV ”, Tokyo Kagaku Dojin, 1991, etc. Mixing and contacting with bivalent cross-linking reagents such as carbodiimide, imide ester, maleimide, etc., cross-linking reagents for specific groups and functional groups such as amino groups, carboxyl groups, thiol groups, aldehyde groups, and hydroxyl groups of magnetic particles. It can fix by making it react.
For example, a divalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide or the like is added to a buffer solution containing magnetic particles, and the mixture is stirred and reacted. Next, a specific binding substance is added thereto, and the mixture is allowed to react with stirring. In some cases, the reaction is then stopped by removing the cross-linking reagent by adding dialysis, gel filtration, or the like, or adding a reaction terminator for the cross-linking reaction. Then, the obtained magnetic particles may be washed with a buffer solution or the like.
[0023]
Further, by changing the amount of the specific binding substance immobilized on the magnetic particles, the sensitivity can be easily changed according to the concentration of the test substance in the sample.
For example, when a specific binding substance is immobilized on magnetic particles, if a high concentration of a specific binding substance is used, the amount of immobilization increases and the sensitivity can be increased.
[0024]
In order to suppress non-specific reactions, if necessary, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and nonfat dry milk are brought into contact with magnetic particles to which a specific binding substance is immobilized. The magnetic particles may be masked by this method.
For example, in the masking, magnetic particles on which a specific binding substance is immobilized are added to a buffer solution containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and the surface of the magnetic particles is allowed to stand. It can carry out by coating with etc.
[0025]
  In the present invention, the carrier is a surface partially coated with the specific binding substance by immobilizing the specific binding substance.That is, the surface that has both the part not covered with the specific binding substance and the part coveredA carrier having is used. The carrier has a surface partially covered with a specific binding substance, and may have any shape and structure as long as the surface can be contacted with a solution such as a sample. Examples include the body. When a container is used as the carrier, the shape of the concave portion that is 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 partially covered with a specific binding substance. When the shape of the concave portion is a shape having a flat bottom surface such as a cylindrical shape or a rectangular parallelepiped shape, the flat bottom surface is preferably partially covered with a specific binding substance. The container may have a plurality of recesses. The container is preferably a container having at least one recess having a flat bottom surface, and the flat bottom surface is partially covered with a specific binding substance. Examples of the container having at least one recess having a flat bottom include a flat bottom microplate and a tray. When a plate-like body is used as the carrier, the surface shape of the plate-like body may be any shape such as a circle, a rectangle, and a square, and the surface may be partially covered with a specific binding substance. Moreover, it is possible to prevent the solution from flowing down from the surface by forming a groove on the surface of the plate-like body and introducing a solution such as a sample into the groove.
[0026]
The material of the carrier is not limited as long as the specific binding substance can be physically or chemically fixed. For example, glass, polystyrene, polyvinyl chloride, polyacrylate, polypropylene, nylon, polyethylene, polycarbonate, polymethacrylate Etc.
[0027]
  In the present invention, when a specific binding substance is immobilized on the surface of the carrier, the surface is fixed so that the surface is partially covered with the specific binding substance.Do. This is because the specific binding substance is present in the sample, that is, when it is positive, the judgment of being positive by comparing the image of the part coated with the specific binding substance and the uncoated part becomes clearer, This is because it becomes easy to discriminate between positive and negative, that is, the presence or absence of the test substance in the sample. Here, “partial” 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 covered 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 one side of the surface of the carrier is coated or a belt is coated on the surface of the carrier.
[0028]
  Also, the surface of the carrierofDepending on the specific binding substanceRuThere may be a plurality of coating portions. For example, in order to measure a plurality of test substances in a sample, a coating portion made of a specific binding substance for each test substance can be provided on the surface of the carrier.
[0029]
FIG. 1 shows a flat bottom microplate used as a carrier in the present invention, in which one half of the flat bottom surface of each well is coated with a specific binding substance.
1A is a perspective view of a flat-bottom microplate (the hatched portion is a portion coated with the specific binding substance), and FIG. 1B is a plan view of the flat-bottomed microplate (the hatched portion is the above-described portion). This is the part coated with a specific binding substance.)
[0030]
FIG. 2 shows a rectangular parallelepiped transparent container having a bottom surface and having a bottom surface having a portion coated with a specific binding substance in a band shape (hereinafter referred to as a band-shaped coating portion).
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 portion). The part covered with the binding substance.)
[0031]
FIG. 3 shows a plate-like body having a rectangular surface shape and having a surface having a band-shaped covering portion made of two different types of specific binding substances.
3A is a perspective view of the plate-like body (shaded portions are portions coated with different specific binding substances), and FIG. 3B is a plan view of the plate-like body. (The shaded area is a portion covered with different specific binding substances.)
[0032]
FIG. 4 shows a plate-like body having a rectangular surface shape, and a groove having a rectangular cross-sectional shape is provided on the surface, and a band-shaped covering portion made of two different types of specific binding substances is formed on the bottom surface of the groove. It shows things.
FIG. 4A is a perspective view of such a plate-like body (shaded portions are portions coated with different specific binding substances), and FIG. 4B is a view of such a plate-like body. It is a plan view (hatched portions are portions covered with different specific binding substances).
Further, if the cover is covered by placing a plate on the side of the belt-like covering portion of the groove (see A in FIG. 4), the sample dropped in the groove on the uncovered side moves in the direction of the belt-like covering portion by capillary action. preferable.
[0033]
The specific binding substance can be immobilized on the surface of the support by a physical adsorption method or a chemical binding method using a crosslinking reagent.
The physical adsorption method can be performed 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 placed in the concave portion of the container and allowed to stand and adsorbed at about 2 ° C. to about 40 ° C. for about 10 minutes to about 1 day. After the reaction is performed, the liquid in the concave portion may be removed by suction and washed with a buffer solution or the like.
[0034]
In addition, when the chemical bonding method using a cross-linking reagent is performed, the Japanese Society of Clinical Pathology “Special Issue on Extraordinary Clinical Pathology No. 53 Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publishing Society, 1983, Japan According to the known method described in the Biochemical Society edited by "Shinsei Kagaku Kenkyu 1 Lecture IV", Tokyo Kagaku Dojin, 1991, etc., the specific binding substance and carrier for the test substance are glutaraldehyde, carbodiimide, imide ester, maleimide, etc. It can be fixed by mixing and bringing into contact with a divalent cross-linking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, etc. of the specific binding substance and the carrier.
For example, when the carrier is a container, a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide or the like is added to the concave portion of the container and left to react. Next, a specific binding substance is added to this and allowed to stand to react. In some cases, the reaction is stopped after that by adding a reaction stopper for the crosslinking reaction. Then, washing is performed with a buffer solution or the like.
[0035]
In the present invention, as a method for partially covering the surface of the carrier, for example, a specific binding substance and / or a cross-linking reagent is dropped only on the part to be coated with the specific binding substance on the surface and fixed by the above method. A method for carrying out the immobilization, a method for carrying out the above-mentioned immobilization by surrounding a portion to be coated with a specific binding substance on the surface with a plastic plate or the like and dropping a specific binding substance and / or a crosslinking reagent in the enclosed part, Alternatively, a method of immobilizing a specific binding substance and / or cross-linking reagent by absorbing it in an absorbent body such as a sponge and placing it on the surface to be coated with a specific binding substance on the surface, or applying it is mentioned. .
[0036]
Further, by changing the amount of the specific binding substance immobilized on the carrier surface, the sensitivity can be easily changed according to the concentration of the test substance in the sample.
For example, when a specific binding substance is immobilized on a carrier, if a high concentration of a specific binding substance is used, the amount of immobilization increases and the sensitivity can be increased.
[0037]
Furthermore, if necessary, in order to suppress non-specific reactions, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and nonfat dry milk 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.
For example, when the carrier is a container, a buffer solution containing various proteins such as bovine serum albumin, human serum albumin, casein, or a salt thereof is added to the concave portion of the container to which the specific binding substance is fixed, and left still. And after coating the surface of the recessed part of the container which fixed the specific binding substance with various proteins etc., it can carry out by sucking and removing the liquid of a recessed part.
[0038]
  In the first measurement method according to the present invention, first, a surface that is specifically coated with a specific binding substance and partially covered with the specific binding substance as described above.That is, the surface that has both the part not covered with the specific binding substance and the part coveredA sample suspected of the presence of the test substance is brought into contact with the surface of the carrier having a magnetic particle fixed with a specific binding substance. When measuring a plurality of test substances in a sample, magnetic particles fixed with a specific binding substance for each test substance are brought into contact with each other. The sample can be diluted with, for example, a diluent and brought into contact with the carrier. The magnetic particles can be dispersed in an appropriate dispersion medium and brought into contact with the carrier, for example. As the above-described diluent and dispersion medium for magnetic particles, various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, phosphate buffered saline, or physiological saline, respectively, may be used. Can do. The pH of this buffer is preferably in the range of pH 4-12.
[0039]
Sample diluent and magnetic particle dispersion medium include various proteins such as bovine serum albumin, human serum albumin, casein or salts thereof, various salts such as sodium chloride, various sugars, skim milk powder, normal rabbit serum, etc. Various additives such as various serums, various preservatives such as sodium azide, nonionic surfactants, amphoteric surfactants, anionic surfactants, etc. Can do.
The concentration when these additives are added is not particularly limited, but is preferably 0.001 to 10% (w / v), and particularly preferably 0.01 to 5% (w / v).
Moreover, as surfactant, sorbitan fatty acid ester, glycerol fatty acid ester, decaglycerol fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyethylene glycol fatty acid ester, polyoxyethylene alkyl ether, polyoxyethylene phytosterol , Phytostanol, polyoxyethylene alkylphenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, nonionic surfactant such as polyoxyethylene lanolin, amphoteric surfactant such as betaine acetate or polyoxyethylene alkyl ether sulfate Alternatively, an anionic surfactant such as polyoxyethylene alkyl ether acetate can be used.
[0040]
In the case where the carrier is a container as described above, for example, the sample and magnetic particles can be brought into contact with each other by adding the sample and magnetic particles to the recess of the container.
In this case, the magnetic particles may be added to the concave portion of the container after adding the sample, and conversely, the sample may be added after adding the magnetic particles.
Furthermore, after mixing a sample and magnetic particles in advance, the mixture may be added to the concave portion of the container.
Further, when the carrier is a plate-like body as described above, for example, the sample is dropped after dispersing the magnetic particles on the surface of the plate-like body, or the magnetic particles are dispersed after dropping the sample. The sample and magnetic particles may be mixed in advance and then the mixture may be dropped to make contact.
[0041]
  As described above, after bringing the sample and magnetic particles into contact with the surface of the carrier, the magnet is operated so that the magnetic particles move along the surface partially covered with the specific binding substance. As long as positive or negative determination can be made based on the distribution state of the magnetic particles on the surface of the carrier, a magnet is applied before or while the sample and magnetic particles are brought into contact with the surface of the carrier. May be. Further, when the surface of the carrier is partially coated with a specific binding substance, the magnet may be arranged anywhere as long as the magnetic particles can be moved in the desired direction, What is necessary is just to arrange | position to the direction to move, and its periphery. More specifically, for example, when a carrier in which one half of a surface is coated with a specific binding substance is used, a portion coated from a portion that is not coated with a specific binding substance on the surface.Minute (FigureIt is preferable to arrange the magnet so that the magnetic particles move in the direction of the arrow 1). In addition, when a carrier whose surface is coated in a band with a specific binding substance is used, the surface is covered with a specific binding substance from the part not covered with the specific binding substance through the band-shaped coating part. No partMinute (FigureIt is preferable to arrange the magnet so that the magnetic particles move in the directions of the arrows 2-4.
[0042]
Any magnet may be used as long as it generates a magnetic field and magnetizes the magnetic particles, and a permanent magnet, an electromagnet, or the like may be used.
The magnetic flux density is usually 5 to 100 gauss, although it depends on the interaction between the magnetic particles used and the surface of the carrier.
[0043]
When a test substance is present in the sample, the test substance binds to a specific binding substance immobilized on the magnetic particles.
In this case, when a magnet is applied to the magnetic particles to which the test substance is bound, the magnetic particles are attracted by the magnet and move in the direction of the magnet along the surface of the carrier. When a specific binding substance is encountered, the magnetic particles bind to the specific binding substance fixed on the surface of the magnetic substance via the test substance and stop moving, or the movement becomes significantly slow.
In the region where the specific binding substance on the surface is not fixed, the movement of the magnetic particle moves promptly in the direction of the magnet depending on the interaction between the particle and the surface and the strength of the magnetic field.
On the other hand, in the region where the specific binding substance on the surface is fixed, there is a large difference in the moving speed of the magnetic particle between the case where the test substance is bound to the magnetic particle and the case where it is not bound.
That is, when the test substance is not present in the sample, the magnetic particles do not bind the test substance, so that the specific binding substance immobilized on the surface does not show affinity and the specific binding substance is not fixed. It moves in the direction of the magnet as quickly as the area. Therefore, when the test substance is not present in the sample, the magnetic particles gather at a position close to the magnet, that is, at the end of the surface of the carrier.
On the other hand, when the test substance is present in the sample, the magnetic particles bind the test substance, so that the magnetic particles bind to the specific binding substance fixed on the surface via the test substance and stop moving. Or it will be significantly slower. Therefore, when the test substance is present in the sample, the magnetic particles gather on the part of the surface covered with the specific binding substance.
[0044]
The distribution state, that is, the image of the magnetic particles on the surface of the carrier 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.
[0045]
C in FIG. 1 is a view of the well when the sample (positive sample) in which the test substance exists is measured using the flat bottom microplate from above, and FIG. 1D shows the flat bottom microplate. It is the figure which looked at the well at the time of measuring about the sample (negative sample) which used and does not have a test substance.
In the case of positive, magnetic particles that have migrated from the portion of the flat bottom surface of the well that is not coated with the specific binding substance are trapped in the specific binding substance coating portion.
[0046]
C in FIG. 2 is a view of the container when the sample (positive sample) in which the test substance exists is measured from above using a rectangular parallelepiped transparent container having a rectangular bottom surface. D is a view of the container as viewed from above when a sample (negative sample) in which a test substance does not exist is measured using a rectangular parallelepiped transparent container having a rectangular bottom surface.
In the case of positive, the magnetic particles that have migrated from the portion not covered with the specific binding substance on the bottom surface are trapped in the band-shaped covering portion.
[0047]
FIG. 3C shows a rectangular plate-like body when the plate-like body in the case where measurement is performed on a sample in which two kinds of test substances are present (a sample in which each test substance is positive) is viewed from above. FIG. 3D is a view of the plate-like body as viewed from above when a sample (negative sample) in which a test substance does not exist is measured using a rectangular plate-like body.
In the case of positive, the magnetic particles that have migrated from the portion that is not coated with the specific binding substance on the surface are trapped in the band-shaped covering portions corresponding to the respective test substances.
[0048]
FIG. 4C shows the plate-like body in the case where measurement is performed on a sample (positive sample) in which two kinds of test substances are present using a rectangular plate-like body having a groove on the surface. FIG. 4D is a diagram of the plate-like body viewed from above when measurement is performed on a sample (negative sample) in which the test substance does not exist using the plate-like body.
In the case of positive, the magnetic particles that have migrated from the portion not covered with the specific binding substance on the bottom surface of the groove are trapped in the band-like covering portions corresponding to the respective test substances.
[0049]
Further, when a container is used as the carrier, the magnetic particles mixed with the sample settle down below the concave portion of the container due to its specific gravity. At this time, the sedimentation of the magnetic particles may be promoted by applying a magnet from the top of the container to the bottom.
[0050]
When a magnet is made to act on magnetic particles so that it moves along the specific binding substance-coated surface of the carrier, the interaction between the magnetic particles to which the test substance is not bound and the surface is weak, and the moving speed is almost the same as the magnet. Depends on. Therefore, when a high moving speed is required, that is, when a measurement result is obtained in a short time, a magnet that generates a strong magnetic field may be used. It is also possible to perform measurement while adjusting the strength of the magnetic field using an electromagnet.
[0051]
  In the second measuring method according to the present invention, a surface that is specifically coated with a specific binding substance and partially covered with the specific binding substanceThat is, the surface that has both the part not covered with the specific binding substance and the part coveredBefore contacting a sample suspected of the presence of a test substance and magnetic particles with a specific binding substance to the surface of the carrier having a test substance, the test substance and / or its analogs are contacted and fixed to the surface. A test substance and / or its analog is previously bound to the specific binding substance. According to the second measurement method, when the test substance does not exist in the sample, that is, when the test substance is negative, the magnetic particles do not bind the test substance, so that the specific binding substance fixed on the surface is fixed in advance. Bound to the specific binding substance immobilized on the surface via the test substance and / or its analog, the movement stopped or significantly slowed down, and the magnetic particles were coated with the specific binding substance on the surface It will be gathered in the part. On the other hand, when the test substance exists in the sample, that is, when the sample is positive, the magnetic particles bind to the test substance in the sample, so that the magnetic particles bind to the specific binding substance immobilized on the surface in advance. It moves immediately in the direction of the magnet without being affected by the test substance and gathers at a position close to the magnet, that is, at the end of the surface of the carrier. Therefore, according to the second measurement method, a result opposite to the distribution state of the positive or negative magnetic particles obtained by the first measurement method can be obtained.
[0052]
Here, the analog of the test substance is a part of the test substance, a substance bound to the test substance, a part of the structure of the test substance substituted, etc. It is a substance that has a structure of a portion that binds to a specific binding substance and can bind to a specific binding substance.
For example, when the test substance is an antigen, a substance containing the antigenic determinant of this antigen can be cited as an analog of this test substance.
[0053]
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 bound to tris (hydroxymethyl) aminomethane buffer, phosphate buffer. What is necessary is just to disperse | distribute or melt | dissolve in various buffer solutions, such as a liquid, phosphate buffered saline, or physiological saline, and to make it contact with this surface. The pH of this buffer is preferably in the range of pH 4-12.
[0054]
In addition, when the sample is blood (whole blood sample), if the test substance in the blood sample is measured, it is blocked by the red blood cells contained in the sample, and the distribution state of the magnetic particles cannot be confirmed. , It becomes difficult to judge negative.
On the other hand, if the height of the liquid containing the sample and the magnetic particles on the coated portion by the specific binding substance on the surface of the carrier is reduced, the number of red blood cells on the coated portion decreases, so that the magnetic particles are not obstructed by the red blood cells. The distribution state of can be confirmed.
[0055]
When blood is used as the sample, the height of the sample and the liquid containing magnetic particles on the coated part of 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 magnetic particles on the coated portion by the specific binding substance on the surface of the carrier, for example, when a container as shown in FIG. 1 or 2 is used as the carrier What is necessary is just to make the depth small, and when using the plate-shaped body in which the groove | channel was provided in the surface as shown in FIG. 4 as a support | carrier, what is necessary is just to make the depth of the groove | channel small.
[0056]
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 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 a carrier that has a sufficiently small height on the coating portion of the specific binding substance but a large volume of liquid that can be accommodated.
In order to use a carrier having a sufficiently small liquid volume that can be accommodated by the specific binding substance but the height of the liquid that can be accommodated is higher than the part that is not coated with the specific binding substance, for example, Alternatively, the width and depth may be increased separately from the height.
Specifically, although not limited to these, a carrier as illustrated in FIGS. 5 to 8 is suitable for measuring the test substance in a blood sample having a low test substance concentration.
FIGS. 5 and 6 are perspective views of a container in which the height on the portion covered with the specific binding substance is small and the height of the portion not covered with the specific binding substance is larger than the height on the covering portion. It is.
FIG. 7 is a perspective view of a container having a small height on the portion covered with the specific binding substance and a small height but a large width and depth on the portion not covered with the specific binding substance.
Furthermore, FIG. 8 shows that a groove on the surface is provided with a small height on the portion covered with the specific binding substance and a small height but a large width and depth on the portion not covered with the specific binding substance. It is a perspective view of a plate-shaped object.
[0057]
As described above, when a carrier with a sufficiently small liquid volume that can be accommodated is used as a carrier, the number of test substances on the part is the same as that of red blood cells. However, since the test substance is bonded to the magnetic particles and moves from the other part to the coated part by the action of the magnet, there is no problem in the measurement.
[0058]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
[Example 1]
(Measurement of HBs antigen)
(1) Preparation of anti-HBs antibody fixed microplate
As shown in FIG. 1, an anti-HBs antibody solution (manufactured by Sinotest, 5 μg / ml) was placed on one half of the flat bottom of a flat bottom microplate (Nunk, well diameter: 7 mm, well depth: 8 mm). The solution was dissolved in a 10 mM phosphate buffer solution at pH 7 at a concentration of 25 μl) and placed using a pipette and allowed to stand at 37 ° C. for 3 hours. Next, after removing the solution in the well by suction, 0.3 ml of Tris buffer solution (pH 7.5, 50 mM) containing 0.5% (w / v) casein (hereinafter referred to as Diluent A) was added, and 4 ° C. Left overnight and removed by suction. In this way, a microplate was prepared in which the anti-HBs antibody was fixed to the left half of the flat bottom surface of the well.
[0059]
(2) Preparation of anti-HBs antibody fixed magnetic particles
1 ml of magnetic particles (Dynabeads M-450 uncoated, manufactured by Dynal, particle size: 4.5 μm, 3% (w / v)) and an anti-HBs antibody solution (manufactured by Shinotest, pH 7.0, 10 mM phosphate buffer with a concentration of 0.1) 1 ml) was mixed and reacted at 37 ° C. for 30 minutes. Masking was performed by adding about 20 times the amount of Diluent A thereto. Next, the obtained magnetic particles were washed with Diluent A, and the magnetic particles were redispersed in Diluent A so that the concentration was about 0.1% (w / v). In this way, an anti-HBs antibody-fixed magnetic particle dispersion was prepared.
[0060]
(3) Measurement of HBs antigen
(1) Add 25 μl of the sample HBs antigen-positive serum to the well of the anti-HBs antibody-fixed microplate prepared in (1) above, and then add 25 μl of the anti-HBs antibody-fixed magnetic particle dispersion prepared in (2) above. And stirred for 1 minute. This is left to stand, and a magnet is installed in the side surface direction of the microplate, so that the anti-HBs antibody on the flat bottom surface is fixed from the portion where the anti-HBs antibody on the flat bottom surface is fixed (in FIG. 1). A magnetic field with a magnetic flux density of 40-60 gauss was generated in the direction of the arrow. An image of magnetic particles as shown in FIG. 1C was observed within 2 minutes after the magnetic field was generated.
(2) To each well of the anti-HBs antibody-fixed microplate prepared in (1) above, various concentrations of HBs antigen solution (0.5% (w / v) casein and 0.1 M sodium chloride, manufactured by Meiji Dairies) Anti-HBs antibody prepared in (2) above, and 25 μl each of 0, 1, 2, 10, 100 and 1000 ng / ml dissolved in 50 mM Tris-HCl buffer (pH 8.0). 25 μl each of the fixed magnetic particle dispersion was added and stirred for 1 minute. This is left to stand, and a magnet is installed in the side surface direction of the microplate, so that the anti-HBs antibody on the flat bottom surface is fixed from the portion where the anti-HBs antibody on the flat bottom surface is fixed (in FIG. 1). A magnetic field with a magnetic flux density of 40-60 gauss was generated in the direction of the arrow. Within 3 minutes, an image of magnetic particles as shown in FIG. 1C was observed for all but the antigen concentration 0 ng / ml.
[0061]
[Example 2]
(Measurement of anti-HBs antibody)
(1) Preparation of HBs antigen-fixed microplate
As shown in FIG. 1, an HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., 5 μg / ml) is placed on one half of the flat bottom of a flat bottom microplate (Nunk, well diameter: 7 mm, well depth: 8 mm). The solution was dissolved in a 10 mM phosphate buffer solution at pH 7 at a concentration of 25 μl) and placed using a pipette and allowed to stand at 37 ° C. for 3 hours. Next, after removing the solution in the well by suction, 0.3 ml of the diluent A was added and left at 4 ° C. overnight to remove it by suction. In this way, a microplate was prepared in which the HBs antigen was fixed to the left half of the flat bottom surface of the well.
[0062]
(2) Preparation of HBs antigen-fixed magnetic particles
1 ml of magnetic particles (Dynabeads M-450 uncoated, manufactured by Dynal Co., Ltd., particle size: 4.5 μm, 3% (w / v)) and HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., 40 μg / pH in 10 mM phosphate buffer solution, pH 7.2) 1 ml) was mixed and reacted at 37 ° C. for 30 minutes. Dilution liquid A was added here and masking was performed. Next, the obtained magnetic particles were washed with Diluent A, and the magnetic particles were redispersed in Diluent A so that the concentration was about 0.1% (w / v). In this way, an HBs antigen-fixed magnetic particle dispersion was prepared.
[0063]
(3) Measurement of anti-HBs antibody
25 μl of serum containing anti-HBs antibody as a sample is added to the well of the HBs antigen-fixed microplate prepared in (1) above, and then 25 μl of the HBs antigen-fixed magnetic particle dispersion prepared in (2) above is added for 1 minute. Stir. This is left still, and a magnet is installed in the side surface direction of the microplate, so that the HBs antigen on the flat bottom surface is fixed from the portion where the HBs antigen on the flat bottom surface is not fixed (the direction of the arrow in FIG. 1). ) Generated a magnetic field with a magnetic flux density of 40-60 gauss. An image of magnetic particles as shown in FIG. 1C was observed within 2 minutes after the magnetic field was generated.
[0064]
Example 3
(Measurement of anti-HBc antibody)
(1) Preparation of HBc antigen-fixed microplate
As shown in FIG. 1, an HBc antigen solution (5 μg / ml, manufactured by Meiji Dairies) was applied to one half of the flat bottom of a flat bottom microplate (Nunk, well diameter: 7 mm, well depth: 8 mm). The solution was dissolved in a 10 mM phosphate buffer solution at pH 7 at a concentration of 25 μl) and placed using a pipette and allowed to stand at 37 ° C. for 3 hours. Next, after removing the solution in the well by suction, 0.3 ml of glycine buffer solution (pH 9, 100 mM) containing 0.5% (w / v) casein (hereinafter referred to as Diluent B) was added, and the solution was added at 37 ° C. It was left overnight and removed by suction. In this way, a microplate was prepared in which the HBc antigen was fixed to the left half of the flat bottom surface of the well.
[0065]
(2) Preparation of HBc antigen-fixed magnetic particles
1 ml of magnetic particles (Dynabeads M-450 uncoated, Dynal, particle size: 4.5 μm, 3% (w / v)) and HBc antigen solution (Meiji Dairies, 40 μg / pH in 10 mM phosphate buffer 1 ml) was mixed and reacted at 37 ° C. for 30 minutes. Diluent B was added thereto and masked at 37 ° C. for 3 days. Next, the obtained magnetic particles were washed with Diluent B, and the magnetic particles were redispersed in Diluent B so that the concentration was about 0.1% (w / v). In this way, an HBc antigen-immobilized magnetic particle dispersion was prepared.
[0066]
(3) Measurement of anti-HBc antibody
Add 37.5 μl of the HBc antigen-immobilized magnetic particle dispersion prepared in (2) above to the well of the HBc antigen-fixed microplate prepared in (1) above, and then add 12.5 μl of the serum containing the anti-HBc antibody as a sample. Stir for 1 minute. This is left still, and a magnet is installed in the side surface direction of the microplate, so that the HBc antigen on the flat bottom surface is fixed from the portion where the HBc antigen on the flat bottom surface is fixed (the direction of the arrow in FIG. 1). ) Generated a magnetic field with a magnetic flux density of 40-60 gauss. An image of magnetic particles as shown in FIG. 1C was observed within 2 minutes after the magnetic field was generated.
[0067]
Example 4
(Simultaneous measurement of HBs antigen and hemoglobin)
(1) Preparation of anti-HBs antibody / anti-hemoglobin antibody fixed plate
Acrylic extruded plate (width 25 mm, depth 40 mm, thickness 1 mm) (made by Acrylic Sunday) 2 acrylic extruded plates (width 10 mm, depth 40 mm, thickness 0.5 mm) (made by Acrylic Sunday) 5 mm A plate-like body in which grooves (width 5 mm, depth 40 mm, height 0.5 mm) were formed on the surface shown in FIG.
Using a pipette, 20 μl of an anti-HBs antibody solution (manufactured by Shinotest Co., dissolved in 10 mM phosphate buffer at pH 7 at a concentration of 5 μg / ml) is applied to a portion from 5 mm to 10 mm from the left end of the bottom of the groove of the plate-like body. Placed and contacted. Furthermore, an anti-hemoglobin antibody solution (made by Nippon Biotest Laboratories, Inc., dissolved in 10 mM phosphate buffer at pH 7 at a concentration of 5 μg / ml) from the left end of the bottom surface of the groove of the plate-like body to a portion of 15 mm to 20 mm. 20 μl was placed in contact with a pipette and allowed to stand at 37 ° C. for 3 hours.
Then, after removing these solutions by suction, 0.3 ml of the above-mentioned diluent A was added thereto and left at 4 ° C. overnight to remove them by suction.
Then, an acrylic extruded plate (width 25 mm, depth 25 mm, thickness 1 mm) [manufactured by Acrylic Sunday Co., Ltd.] is pasted on the left end of this plate-like body with a solvent, thereby forming a plate-like body as shown in FIG. Covered.
In this manner, a plate-like body having a band-shaped covering portion formed of two different types of specific binding substances (anti-HBs antibody and anti-hemoglobin antibody) on the bottom surface of the groove was produced.
[0068]
(2) Preparation of anti-hemoglobin antibody fixed magnetic particles
1 ml of magnetic particles (Dynabeads M-450 uncoated, Dynal, particle size: 4.5 μm, 3% (w / v)) and anti-hemoglobin antibody solution (Nippon Biotest Laboratories, pH 7.0, 10 mM phosphate buffer) 1 ml of a solution dissolved at a concentration of 0.1 mg / ml) was mixed and reacted at 37 ° C. for 30 minutes. Masking was performed by adding about 20 times the amount of Diluent A thereto.
Next, the obtained magnetic particles were washed with Diluent A, and the magnetic particles were redispersed in Diluent A so that the concentration was about 0.1% (w / v). In this way, an anti-hemoglobin antibody-fixed magnetic particle dispersion was prepared.
[0069]
(3) Simultaneous measurement of HBs antigen and hemoglobin
Sample HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., dissolved in 10 mM phosphate buffer at pH 7.2 at a concentration of 100 ng / ml) 50 μl and 1 μg / ml human hemoglobin (manufactured by Sinotest) 50 μl in a test tube Then, 50 μl of the anti-hemoglobin antibody-immobilized magnetic particle dispersion prepared in (2) above and 50 μl of the anti-HBs antibody-immobilized magnetic particle dispersion prepared in (1) of Example 1 were added and stirred here. .
150 μl of this mixed solution is placed with a pipette near the boundary between the covered and uncovered portions of the anti-HBs antibody / anti-hemoglobin antibody-fixed plate prepared in (1) above, using a pipette. Contact.
This is left standing, and a magnet is installed in the side surface direction where the band-shaped covering portion of the plate-like body is formed, so that the groove from the portion where the anti-HBs antibody and anti-hemoglobin antibody are not fixed on the bottom surface of the groove of the plate-like body. A magnetic field with a magnetic flux density of 40 to 60 gauss was generated in the partial direction (in the direction of the arrow in FIG. 4) where the anti-HBs antibody and anti-hemoglobin antibody on the bottom surface of the plate were fixed. Within 3 minutes after the magnetic field was generated, an image of two magnetic particles as shown in FIG. 4C was observed.
[0070]
Example 5
(Measurement of HBs antigen in blood)
10 μl of HBs antigen-positive blood was added to the anti-HBs antibody-fixed flat-bottomed microplate prepared in Example 1 (1), and then 50 mM Tris-HCl buffer (pH 8) containing 0.5% (w / v) casein and 0.1 M sodium chloride. 0.0) was added, and 25 μl of the anti-HBs antibody-fixed magnetic particle dispersion prepared in (2) of Example 1 was further added and stirred for 1 minute, and then allowed to stand.
A glass rod having a diameter of 5 mm is placed in the well of this microplate so that it floats 1 mm from the bottom, and the height of the liquid containing the sample and magnetic particles on the coated portion with the specific binding substance (anti-HBs antibody) is 1 mm. .
By placing a magnet on the side surface of the microplate, the magnetic flux density extends from the portion where the anti-HBs antibody on the flat bottom surface of the well is not fixed to the portion where the anti-HBs antibody on the flat bottom surface is fixed (arrow direction in FIG. 1). A magnetic field of 40-60 gauss was generated.
Within 2 minutes after the magnetic field was generated, an image of magnetic particles as shown in FIG. 1C was observed without being blocked by red blood cells.
When negative blood was used, no magnetic particle image was observed.
[0071]
Example 6
(Measurement of anti-HBs antibody in blood)
10 μl of anti-HBs antibody-positive blood was added to the HBs antigen-fixed flat-bottomed microplate prepared in (2) of Example 2, and then 50 mM Tris-HCl buffer (pH 8) containing 0.5% (w / v) casein and 0.1 M sodium chloride. 0.0) 5 μl was added, and 25 μl of the HBs antigen-immobilized magnetic particle dispersion prepared in (2) of Example 2 was further added, stirred for 1 minute, and allowed to stand.
A glass rod having a diameter of 5 mm was placed in the well of this microplate so that it floated 1 mm from the bottom, and the height of the liquid containing the sample and magnetic particles on the portion coated with the specific binding substance (HBs antigen) was 1 mm.
By installing a magnet in the lateral direction of the microplate, a magnetic flux density of 40 to 40 in the direction (in the direction of the arrow in FIG. 1) where the HBs antigen on the flat bottom surface is fixed from the portion where the HBs antigen on the flat bottom surface is not fixed. A magnetic field of 60 gauss was generated.
Within 2 minutes after the magnetic field was generated, an image of magnetic particles as shown in FIG. 1C was observed without being blocked by red blood cells.
When negative blood was used, no magnetic particle image was observed.
[0072]
【The invention's effect】
According to the first measurement method and the second measurement method of the present invention, the presence or absence of the test substance in the sample is not the size of the circular image of the particles collected on the bottom of the container as in the prior art. Judgment is easy because it is determined based on the position close to the magnet, i.e., at the end of the surface or at the portion coated with the specific binding substance on the surface of the carrier, and the test substance concentration is low. However, the determination is easy. Therefore, the test substance in the sample can be measured with high sensitivity. Moreover, no erroneous determination is given even when a nonspecific agglutination occurs.
Further, 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 immobilized on the surface of the magnetic particles and / or the carrier, so that the efficiency is high. Therefore, the presence or absence of the test substance can be easily determined even when the concentration of the test substance is low.
Furthermore, according to the first measurement method and the second measurement method of the present invention, it usually takes 20 seconds to 10 minutes in the sample in a short time, specifically depending on the type of the test substance. The presence or absence of the 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 present invention, a plurality of test substances in a sample can be measured simultaneously.
Moreover, the 1st measuring instrument and the 2nd measuring instrument by this invention can be used suitably for the 1st measuring method and the 2nd measuring method, respectively.
[Brief description of the drawings]
FIG. 1 is a view showing a flat bottom microplate in which one half of a flat bottom surface of each well is coated with a specific binding substance.
FIG. 2 is a diagram showing a rectangular parallelepiped transparent container whose bottom surface is coated in a band shape with a specific binding substance.
FIG. 3 is a view showing a plate-like body whose surface is coated with a specific binding substance in a band shape.
FIG. 4 is a view showing a plate-like body in which a groove is provided on the surface and the bottom surface of the groove is 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 covering portion with the specific binding substance is small and the height of the portion not covered with the specific binding substance is larger than the height on the covering portion.
FIG. 6 is a perspective view of a container in which the height on the portion covered with the specific binding substance is small and the height of the portion not covered with the specific binding substance is larger than the height on the covering 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 on a portion not covered with a specific binding substance.
FIG. 8 is a plate shape in which a groove on the surface is provided with a small height on the surface covered with the specific binding substance and a small height but a large width and depth of the part not covered with the specific binding substance. It is a perspective view of a body.
FIG. 9 is a diagram showing an agglomerated image when a test substance in a sample is measured by an indirect agglutination measurement method.

Claims (12)

A surface in which a specific binding substance to a test substance in a sample is fixed and partially covered with the specific binding substance, that is, a face having both a portion not covered with a specific binding substance and a covered portion. A step of bringing a sample and a magnetic particle, which is a specific binding substance for a test substance and has the same or different specific binding substance immobilized on the carrier, on the surface of the carrier having the carrier And a step of acting a magnet so that the magnetic particles move from a portion of the surface not coated with the specific binding material to a portion coated with the surface , and coated with the surface specific binding material. A method for measuring a test substance in a sample, characterized in that the presence or absence of a test substance is determined from the distribution state of magnetic particles in a non-covered part and a covered part . A surface in which a specific binding substance to a test substance in a sample is fixed and partially covered with the specific binding substance, that is, a face having both a portion not covered with a specific binding substance and a covered portion. Contacting the test substance and / or its analog with the surface of the carrier having the test substance and / or its analog bound to the specific binding substance immobilized on the surface; And a step of contacting a magnetic substance having a specific binding substance that is the same or different from the specific binding substance fixed to the carrier, the specific binding substance for the test substance, and specific binding substances by comprises the step of reacting the magnet to move to the portion covered by the portion not covered in the specific binding substances by the uncoated portion of the surface to be It is characterized by determining the presence or absence of the test substance from the distribution of the magnetic particles in the portion are, the measurement method of the analyte in the sample.   The test substance in the sample according to claim 1 or 2, wherein the carrier is a container having at least one recess having a flat bottom surface, and the flat bottom surface is partially covered with a specific binding substance. Measuring method.   The method for measuring a test substance in a sample according to claim 1 or 2, wherein the carrier is a plate-like body, and the surface thereof is partially coated with a specific binding substance.   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.   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. A carrier used for measurement of a test substance in a sample is a container having at least one recess having a flat bottom surface, and a specific binding substance for the test substance is fixed and partially covered with the specific binding substance. A sample having a flat bottom surface, and a magnetic particle having a specific binding substance to a test substance and a specific binding substance fixed or the same as the specific binding substance fixed to the support. Measuring instrument for test substance inside. Carrier used in the measurement of the analyte in the sample is a plate-like body, a support having a front surface which a specific binding substance for the analyte is covered by fixed partially specific binding substances , and a magnetic particle specific binding identical specific binding substance fixed to the carrier a substance or different specific binding substance is fixed with respect to the test substance, measuring instruments of the analyte in the sample. A carrier used for measurement of a test substance in a sample is a container provided with at least one recess having a flat bottom surface, and a specific binding substance for the test substance is fixed and partially covered with the specific binding substance. A carrier having a flat bottom surface, a test substance and / or its analog, and a specific binding substance which is the same or different from the specific binding substance fixed to the carrier, which is a specific binding substance to the test substance An instrument for measuring a test substance in a sample, comprising magnetic particles to which is fixed. A carrier used for measuring a test substance in a sample is a plate-like body, and a carrier having a surface on which a specific binding substance for the test substance is fixed and partially covered with the specific binding substance; A test substance and / or an analog thereof, and a magnetic particle having a specific binding substance to the test substance and a specific binding substance fixed or the same as or different from the specific binding substance fixed to the carrier, A measuring instrument for a test substance in a sample.   The instrument for measuring a test substance in a sample according to any one of claims 7 to 10, wherein the test substance is an antigen and the specific binding substance is an antibody.   The measurement instrument for a test substance in a sample according to any one of claims 7 to 10, wherein the test substance is an antibody and the specific binding substance is an antigen.

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