JP4284431B2 - Measuring instrument and measuring method for test substance using particles - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a measuring instrument and a measuring method for measuring a test substance in a sample using particles in which a specific binding substance to the test substance is fixed, and in particular, a trace amount of a test substance contained in a sample such as a blood sample. In particular, the present invention relates to a measuring instrument and a measuring method for a test substance that can be measured in a short time and easily without being affected by an interfering substance.
The present invention is useful in the field of life science such as a clinical examination field.
[0002]
[Prior art]
Instrument for measuring a small amount of analyte contained in a sample using reaction between substances having specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, ligand and receptor, etc. Various methods 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. 7 shows an agglomerated image when the test substance in the sample is measured by the 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 negative case, 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. 6). 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 aggregate image can be observed (B in FIG. 7), 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 test substance antigen (or antibody) 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) 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 aggregated image shown in FIG. 7B 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. 7B 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]
In addition, the conventional indirect agglutination reaction measurement method, the measurement method described in JP-A No. 64-69954, and the measurement method described in JP-A No. 2-124464 all have the presence or absence of a test substance in the sample. Therefore, if the sample is blood (whole blood sample) or feces, contaminants such as red blood cells and food residues in the sample are also collected by gravity together with the particles. When the test substance in the sample rolls down (slides down) on the inner wall of the measurement container and the sample is measured, the distribution of particles is caused by impurities such as red blood cells and food residues contained in the sample. There was a problem that it could not be confirmed because it was blocked, making it difficult to determine positive or negative.
[0013]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a blood sample containing red blood cells that are substances that interfere with determination when measuring a test substance in a sample visually or using an instrument using a specific binding reaction such as an antigen-antibody reaction. Even for samples such as feces containing contaminants such as food residues, the determination of the presence or absence of the test substance can be performed without obstruction without performing the absorption and removal operation of samples that are complicated and dangerous for infection. It is an object of the present invention to provide a measuring instrument and a measuring method that can be performed in time and have high reliability in the determination.
[0014]
[Means for Solving the Problems]
  The present invention relates to 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, a part not covered with a specific binding substance and a part covered with the specific binding substance. Comprising a carrier having a surface having both a surface and a specific binding substance for a test substance, wherein the specific binding substance is the same or different from the specific binding substance immobilized on the carrier. In the test substance measuring instrument, an absorption part for absorbing and removing the sample brought into contact with the surface is provided., The downstream end in the direction in which the sample moves from the part not coated with the specific binding substance on the surface of the carrier to the part coatedIt is a measuring instrument characterized by being arrange | positioned.
[0015]
  In the present invention, a specific binding substance for a test substance in a sample is fixed and coated with a surface partially covered with the specific binding substance, that is, a part not covered with the specific binding substance. A step of bringing a sample into contact with the surface of a carrier having a surface having a portion having a portion, and a sample brought into contact with the surface, The downstream end in the direction in which the sample moves from the part not coated with the specific binding substance on the surface of the carrier to the part coatedA step of absorbing and removing in the absorption part disposed on the surface, particles that are specific binding substances for the test substance and that have the same or different specific binding substance immobilized on the carrier A portion of the surface that is not coated with the specific binding substance, and a portion of the surface that is not coated with the specific binding substance. And determining the presence / absence of the test substance from the distribution state of the particles in the coated part. (First measurement method)
[0016]
   Furthermore, the present invention is such that a specific binding substance for a test substance in a sample is fixed and coated with a surface partially covered with the specific binding substance, that is, a part not covered with the specific binding substance. A particle and a sample on which a specific binding substance that is specific to the test substance and that is the same or different from the specific binding substance immobilized on the carrier is immobilized on the surface of the carrier having a surface that has a portion having a certain portion. Contacting the mixture after mixing, the mixture of particles and sample brought into contact with the surface;A downstream end in a direction in which the mixture of the particles and the sample moves from the part not coated with the specific binding substance on the surface of the carrier to the part coatedA step of absorbing and removing in the absorption part arranged in the step, and a particle having a specific binding substance to the test substance, the specific binding substance being the same or different from the specific binding substance fixed to the carrier being fixed Contacting the surface and moving the particles from a portion of the surface not coated with the specific binding material to a portion coated with the surface, the surface not coated with the surface specific binding material. A method for measuring a test substance in a sample (second measurement method), wherein the presence or absence of the test substance is determined from the distribution state of particles in the part and the covered part.
[0017]
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; E. coli O157 antigen, anti-treponema Bacteria-related antigens or antibodies such as Paridam (TP) antibody, anti-mycoplasma antibody, anti-streptridine O antibody (ASO); immunoglobulin G (IgG), immunoglobulin A (IgA), immunoglobulin M (IgM), or immunity Immunoglobulins such as globulin E (IgE); C-reactive protein (CRP), α1-acid glycoprotein, haptoglobin, complement C3, complement C4, inflammation markers such as rheumatoid factor; α-fetoprotein, CEA, CA19-9 Tumor markers such as human placental chorionic gonadotropin; allergens such as allergens and allergen-specific IgE antibodies A series of antigens or antibodies; blood coagulation-related substances such as antithrombin III (ATIII); fibrin degradation products (FDP), fibrinolytic substances such as D-dimer; blood types such as ABO blood group antibodies and irregular antibodies Related antigens or antibodies; viral DNA or RNA; bacterial DNA or RNA; animal or plant DNA or RNA such as humans; lipoprotein (a), substances or drugs related to other diseases such as ferritin can do.
[0018]
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, or other body fluids; human or animal brain or other organs, hair, skin, nails, muscles, or Extracts such as neural tissue; human or animal stool extracts or suspensions; cell or fungal extracts; plant extracts and the like.
[0019]
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 or the antibody against the antibody when the test substance is an antibody, and the test substance is a nucleotide chain. Is a complementary nucleotide chain. Here, an antibody is a fragment made from an antibody, such as Fab and F (ab ').₂Etc. are also included.
Further, when the test substance is a ligand, it is the receptor.
The specific binding substance immobilized on the particle and the specific binding substance immobilized on the carrier may be the same as or different from each other as long as the binding through the test substance is possible.
[0020]
The particles used in the present invention may be particles generally used for indirect aggregation reaction. For example, organic polymer particles such as liposome, latex particles, gelatin particles, polyacrylamide particles, microcapsules, emulsions, inorganic polymer particles such as glass beads, silica beads, bentonite, other artificial particles, and red blood cells Etc.
[0021]
Also, magnetic particles can be used as the particles. 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 polymer materials such as polystyrene, silica gel, gelatin, polyacrylamide and other particles coated with ferromagnetic materials, erythrocytes, liposomes, microcapsules, etc. And 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. As such magnetic particles, Examples thereof include “Dynabeads M-450 uncoated (trade name) (manufactured by Dynal)”, which is a magnetic particle in which iron (III) oxide (Fe 2 O 3), which is a ferromagnetic material, is dispersed in the particle.
[0022]
Furthermore, as the particles, particles colored by coating a pigment or dispersing or encapsulating the pigment in the particles may be used.
[0023]
The particle diameter of the particles is preferably 0.01 to 100 μm, particularly preferably 0.5 to 10 μm. Moreover, the specific gravity of particle | grains should just be specific gravity settled in a dispersion medium, for example, the thing of specific gravity 1-10 is preferable.
[0024]
In the present invention, particles to which a specific binding substance is immobilized are used. In order to immobilize a specific binding substance on the particle, the specific binding substance is placed on the surface of the particle, such as a hydrophobic bond or a hydrophilic adsorption. It can be carried out by an adsorption method, a chemical bonding method such as a covalent bond, or a combination of these methods.
[0025]
When the specific binding substance is immobilized on the particles by a physical adsorption method, the specific binding substance and the particles can be mixed and brought into contact with each other in a buffer solution or the like according to a known method. .
For example, the specific binding substance and the particles are mixed in a solution such as a buffer and brought into contact with stirring, and the adsorption 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 solution or the like.
[0026]
In addition, when the specific binding substance is immobilized on the particle by a chemical binding method using a cross-linking reagent, the Japanese Society of Clinical Pathology “Special Issue on Clinical Pathology Special Issue 53 Immunoassay for Clinical Examination—Technology and Applications—” , Clinical Pathology Publications, 1983, Japan Biochemical Society, “Shinsei Chemistry Laboratory Lecture 1 Protein IV”, Tokyo Kagaku Dojin, 1991, etc., in accordance with a known method, glutaraldehyde, carbodiimide, Mixing and bringing into contact with a divalent cross-linking reagent such as imide ester or maleimide, and reacting the functional group such as amino group, carboxyl group, thiol group, aldehyde group and hydroxyl group of the specific binding substance and particles with the cross-linking reagent. Can be fixed.
For example, a bivalent crosslinking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide or the like 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 allowed to react with stirring. In some cases, the reaction is then stopped by removing the cross-linking reagent or adding a reaction stopper for the cross-linking reaction by dialysis, gel filtration, or the like. Then, the obtained particles may be washed with a buffer solution or the like.
[0027]
Further, by changing the amount of the specific binding substance immobilized on the 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 the particles, if a high concentration of the specific binding substance is used, the amount of immobilization increases and the sensitivity can be increased.
[0028]
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 non-fat dry milk are brought into contact with particles to which a specific binding substance is immobilized. The particles may be masked by a method.
For example, in the masking, particles having a specific binding substance immobilized thereon are added to a buffer solution or the like containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and the surface of the particles is coated with various proteins. This can be done by coating.
[0029]
  In the present invention, as the carrier, the specific binding substance is immobilized by immobilizing the specific binding substance.PartiallyCoated surfaceThat is, the surface that has both the part not covered with the specific binding substance and the part coveredAnd a carrier on which an absorption part for absorbing and removing the sample brought into contact with the surface is disposed. Depending on the specific binding substance as the carrierPartiallyIt has a coated surface 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. Examples thereof include a container and a plate-like body.
[0030]
  When a container is used as a carrier, the shape of the recess that is the solution storage portion may be any shape such as a hemispherical shape, a cylindrical shape, a rectangular parallelepiped shape, and the inner wall surface of the recess is made of a specific binding substance.PartiallyWhat is necessary is just to coat. When the shape of the recess is a shape having a flat bottom surface such as a cylindrical shape or a rectangular parallelepiped shape, the flat bottom surface is made of a specific binding substance.PartiallyIt is preferable to coat. The container may have a plurality of recesses.
  The container is a container having at least one recess having a flat bottom surface, and the flat bottom surface is made of a specific binding substance.PartiallyIt is preferably coated. Examples of the container having at least one recess having a flat bottom include a flat bottom microplate and a tray.
[0031]
  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, a square, etc., and the surface is made of a specific binding substance.PartiallyWhat is necessary is just to coat.
  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.
[0032]
In the present invention, as the absorption part for absorbing and removing the sample that is fixed on the surface of the carrier coated with the specific binding substance with a specific binding substance immobilized on the test substance in the sample, for example, filter paper, paper towel Or papers such as tissue paper, glass fiber filters, polymer absorbers, porous materials such as sponges, chemical fibers such as rayon or polyester, cloths, non-woven fabrics, or materials having a property of absorbing liquids such as cotton Anything can be used. There is no particular limitation on the shape and size of the absorption part for absorbing and removing the sample.
[0033]
In the present invention, the position at which the absorption part for absorbing and removing the sample is arranged on the carrier is a test substance on the surface coated with the specific binding substance with a specific binding substance fixed to the test substance in the sample. After contacting the sample containing, the sample may be placed anywhere as long as the sample is absorbed and removed from the surface of the carrier.
For example, when a container having a flat bottom surface is used as a carrier, the absorption part for absorbing and removing the sample is formed into a strip shape, and the flat bottom surface is stood along the side surface of the container where the specific binding substance is not fixed. You may arrange in.
When a plate-like body is used as the carrier, for example, an absorption part that absorbs and removes the sample may be disposed at the end of the surface coated with the specific binding substance of the carrier or at a position adjacent to the surface. Good.
For example, the sample is absorbed from the direction in which the sample moves, that is, the horizontal direction of the surface coated with the specific binding substance so that the sample can smoothly move on the surface coated with the specific binding substance. It is preferable to arrange an absorption part for absorbing and removing the sample at a position where the sample can be absorbed.
[0034]
In addition, you may arrange | position the absorption part which absorbs and removes a sample, so that it can remove after contacting a sample. Furthermore, the absorbent part may be configured to be removable or the form of a cartridge or the like so that the absorbent part can be replaced many times.
[0035]
Moreover, the absorption part which removes a sample can also be made into space. In this case, a certain amount of sample can be stored in this space by making the absorption part a space.
In the present invention, when the absorption part for absorbing and removing the sample is made into a space, the specific binding substance for the test substance in the sample is fixed and coated with the specific binding substance. The sample may be placed anywhere as long as the sample is absorbed and removed from the surface of the carrier after the sample containing the test substance is brought into contact with the surface.
For example, an absorption part that absorbs and removes the sample may be disposed at the end of the surface coated with the specific binding substance of the carrier or at a position adjacent to the surface.
For example, when a container having a flat bottom surface is used as the carrier, the absorption part for absorbing and removing the sample may be formed in a box shape and disposed on a portion where the specific binding substance on the flat bottom surface is not fixed.
When using a plate-like body as a carrier, for example, the direction in which the sample moves, that is, the specific binding substance is coated so that the sample can smoothly move on the surface coated with the specific binding substance. It is preferable to arrange an absorption part for absorbing and removing the sample at a position where the sample can be absorbed from the horizontal direction of the surface.
Furthermore, when a plate-like body is used as the carrier, it is desirable to provide an enclosure around the absorption portion so that the moving sample does not leak.
For example, an absorption part molded in a box shape or the like may be disposed at the end of the surface of the carrier covered with the specific binding substance. Further, in the case of a plate-like body having a groove, an absorption part having a surrounding may be arranged at the end of the surface covered with the specific binding substance of the carrier.
[0036]
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.
[0037]
  In the present invention, when a specific binding substance is immobilized on the surface of the carrier, the surface is immobilized so that the surface is partially covered with the specific binding substance.The
  This is because when the test substance is present in the sample, that is, when it is positive, the judgment of the positive is clearer by comparing the image of the part coated with the specific binding substance and the part not covered, positive This is because it becomes easy to distinguish between negative 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.
[0038]
  Also, the surface of the carrierofDepending on the specific binding substanceRuThere may be a plurality of coated portions. For example, in order to measure a plurality of types of test substances in a sample, a coating portion with a specific binding substance for each test substance can be provided on the surface of the carrier. .
[0039]
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. This shows a flat bottom surface where a specific binding substance is not fixed and arranged so as to stand along the side of the container. 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.)
[0040]
FIG. 2 shows a rectangular parallelepiped transparent container having a bottom surface and 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) and molded into a strip shape. The absorption part 1 is arranged so as to lean against the part of the bottom surface not coated with the specific binding substance along the side surface of the container.
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.)
[0041]
FIG. 3 shows a plate-like body having a rectangular surface shape, which has a surface having a band-shaped coating portion made of two different types of specific binding substances, and the surface of the carrier coated with the specific binding substance. It shows the one in which the absorption part 1 for absorbing and removing the sample is disposed at the end.
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.)
[0042]
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. FIG. 2 shows an arrangement in which an absorption part 1 for absorbing and removing a sample is arranged at the end of the surface of the plate-like body covered with a specific binding substance.
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.
[0043]
The specific binding substance can be immobilized on the surface of the carrier by a physical adsorption method such as hydrophobic bonding or hydrophilic adsorption, or a chemical bonding 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.
[0044]
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. 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.
[0045]
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. .
[0046]
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.
[0047]
Furthermore, in order to suppress non-specific reactions if necessary, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, skim milk powder, etc. are brought into contact with a carrier on which a specific binding substance is fixed, etc. 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.
[0048]
  In the first measurement method according to the present invention, first, a specific binding substance is immobilized as described above, and the specific binding substance is used.PartiallyCoated surfaceThat is, the surface that has both the part not covered with the specific binding substance and the part coveredAnd a sample suspected of the presence of the test substance is brought into contact with the surface of the carrier on which the absorption part for absorbing and removing the sample brought into contact with the surface is disposed.
[0049]
When the carrier is, for example, a container as described above, the sample can be brought into contact with the inner wall surface of the container by adding the sample to the concave portion of the container.
Further, when the carrier is a plate-like body as described above, for example, the sample may be contacted by dropping a sample on the surface of the plate-like body.
[0050]
The sample can be diluted with, for example, a diluent and brought into contact with the carrier.
As a diluted solution of the sample, various buffers such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, phosphate buffered saline, or physiological saline can be used. The pH of the buffer is preferably in the range of pH 4-12.
[0051]
Sample dilutions include various proteins such as bovine serum albumin, human serum albumin, casein, or salts thereof, various salts such as sodium chloride, various sugars, various animal sera such as skim milk powder, normal rabbit serum, Additives such as various preservatives such as sodium hydride, various surfactants such as nonionic surfactant, amphoteric surfactant and anionic surfactant can be added as appropriate.
[0052]
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).
[0053]
Further, surfactants include 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. , Polyoxyethylene phytostanol, polyoxyethylene alkyl phenyl ether, polyoxyethylene castor oil, hydrogenated castor oil, nonionic surfactant such as polyoxyethylene lanolin, amphoteric surfactant such as betaine acetate or polyoxyethylene alkyl An anionic surfactant such as ether sulfate or polyoxyethylene alkyl ether acetate can be used.
[0054]
As described above, after the sample is brought into contact with the surface of the carrier, the sample brought into contact with the surface is removed by being absorbed by the absorption portion arranged on the carrier.
[0055]
In addition, when the absorption part arrange | positioned at the support | carrier is space, a sample can be moved to an absorption part also by inclining a support | carrier, and can be absorbed and removed.
The angle at which the carrier is tilted may be an angle that allows the sample to move to the absorption part of the carrier by gravity, and an angle of 90 ° or less may be selected as appropriate, but an angle between 25 ° and 90 ° may be selected. An angle between 45 ° and 65 ° is particularly preferred.
[0056]
Further, when a carrier having a cover (A in FIG. 6) in the groove on the surface of the plate-like body and having a space in the absorption part is used as a carrier, the sample moves in the direction of movement, that is, covered with a specific binding substance. In the case where the absorbing portion is disposed at a position where the sample is absorbed and removed from the horizontal direction of the surface, in order to smoothly move the sample into the absorbing portion, a small hole (in FIG. 7) should be left open. Moreover, if a small hole is previously made in the cover in this way, the sample can pass through the inside of the groove uniformly.
The position where the small hole is made in the cover may be anywhere as long as the position corresponds to the tip of the absorbing portion of the plate-like body.
The size of the small holes is not particularly limited, but is preferably 0.1 to 2.0 mm, particularly preferably 0.5 to 1.0 mm.
Furthermore, the sample can be more smoothly guided to the absorbing portion of the plate-like body by reducing the thickness of the absorbing portion or reducing the width thereof.
[0057]
If the absorption part that absorbs the sample is placed on the carrier in advance, in order to remove the sample, the sample absorber made of a water-absorbing material is brought into contact with the sample on the surface of the carrier to absorb the sample, pipette, etc. Thus, it is not necessary to perform operations such as sucking out the sample on the surface of the carrier or dropping the sample by turning the carrier upside down.
Moreover, since the sample can be removed from the surface of the carrier without directly touching the sample, it is possible to avoid the risk of infection due to the sample.
[0058]
In addition, when the concentration of the test substance in the sample is low, an image is not formed in the measurement unless there is a certain number of test substances, so it is necessary to use a large amount of the sample.
Therefore, when the concentration of the test substance in the sample is low, the measurement using a large amount of sample can be made possible by increasing the capacity of the absorption part arranged on the carrier.
[0059]
In addition, when the sample brought into contact with the surface is removed in this way, when the sample is a blood sample (whole blood sample), red blood cells in the blood sample are removed from the surface of the carrier covered with the specific binding substance by the removal. The In contrast, the test substance in the blood sample remains bound to the specific binding substance immobilized on the surface of the carrier. Furthermore, since the particles to which the specific binding substance is immobilized bind to the bound test substance, the distribution state of the bound particles can be clearly confirmed without being blocked by red blood cells.
Also, when the sample is a stool sample, the contaminants such as food residues in the stool sample are removed from the surface of the carrier covered with the specific binding substance. In contrast, the test substance in the stool sample remains bound to the specific binding substance immobilized on the surface of the carrier. Furthermore, since the particles to which the specific binding substance is fixed bind to the bound test substance, the distribution state of the bound particles can be clearly confirmed without being obstructed by impurities such as food residues. It is.
[0060]
In addition, after the sample brought into contact with the surface is removed by the absorbing portion disposed on the carrier, the surface of the carrier may be washed with the above-described sample dilution or buffer solution.
In the case of cleaning, for example, the capacity of the absorption part is increased so that the absorption part arranged on the carrier does not become saturated with the sample, or a water-absorbing material having a large water absorption capacity is used for the absorption part. Or you may add an absorption part newly. Moreover, after removing the absorption part which absorbed the sample which contacted the surface, you may wash | clean by making a cleaning liquid contact after attaching an absorption part newly.
[0061]
As described above, after the sample brought into contact with the surface is removed by the absorption part arranged on the carrier, it is the same or different from the specific binding substance immobilized on the carrier, which is a specific binding substance to the test substance. The particles on which the specific binding substance is immobilized are brought into contact with the surface of the carrier from which the sample has been removed.
When measuring a plurality of types of test substances in a sample, particles fixed with specific binding substances for each test substance are brought into contact with each other.
[0062]
Further, instead of the particles to which the specific binding substance is fixed, particles to which the test substance or its analog is fixed may be used. 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 the specific binding substance and can bind to the 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.
[0063]
The particles can be dispersed in, for example, a suitable dispersion medium and brought into contact with the carrier.
As the particle dispersion medium, various buffer solutions such as tris (hydroxymethyl) aminomethane buffer, phosphate buffer, phosphate buffered saline, or physiological saline can be used. The pH of the buffer is preferably in the range of pH 4-12.
[0064]
In addition, the additives described in the section of the sample diluent can be appropriately added to the dispersion medium for the particles.
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).
[0065]
The surface of the carrier from which the sample has been removed may be washed by excessively contacting a particle dispersion in which particles are dispersed in an appropriate dispersion medium. The excess amount here may be, for example, an amount larger than the capacity inside the space surrounded by the groove on the surface of the plate-like body and the cover. Specifically, 2 to 10 times the amount of the above-mentioned particle dispersion can be used.
When cleaning is performed by contacting an excessive amount of the particle dispersion in which the particles are dispersed, for example, the absorption unit disposed on the carrier is not saturated with the sample and the particle dispersion. You may enlarge a capacity | capacitance, may use a water absorptive material with a big water absorption power for this absorption part, or you may add an absorption part newly. In addition, after removing the sample brought into contact with the surface and the absorbing part from which the particle dispersion has been absorbed and removed, it may be cleaned by excessively bringing the particle dispersion into contact with the surface after newly attaching the absorbing part. .
[0066]
When a carrier having a cover (A in FIG. 4) is used as a carrier on the surface of the plate-like body, the sample is taken from the direction in which the sample moves, that is, from the horizontal direction of the surface coated with the specific binding substance. If the absorption part is placed at a position where it is absorbed and removed, the absorption part is saturated with the sample, and there is no escape route for the air inside the space surrounded by the groove and cover on the surface of the plate-like body. In some cases, the particles to which the substance is fixed do not smoothly enter the space surrounded by the groove and cover on the surface of the plate-like body. In this case, it is preferable to make a small hole in advance in the cover as an air escape path.
The position where the small hole is made in the cover may be anywhere as long as the position corresponds to the upper part of the groove covered with the specific binding substance of the plate-like body.
The size of the small holes is not particularly limited, but is preferably 0.1 to 2.0 mm, particularly preferably 0.5 to 1.0 mm.
Moreover, after removing the absorption part saturated with the sample, the particles to which the specific binding substance is immobilized may be brought into contact.
[0067]
  As described above, after contacting the particle to the surface of the carrier, the particle is bound by the specific binding substance of the carrier.PartiallyCoated surfaceFrom the part not coated with the specific binding substance ofMove.
  This sideFrom the part not coated with the specific binding substance ofThe particles can be moved by applying a magnet or tilting the carrier.
[0068]
  When the particles are moved by the action of a magnet, the above-described magnetic particles are used as the particles. And this magnetic particle is caused by a specific binding substance.PartiallyCoated surfaceFrom the part not coated with the specific binding substance ofActuate the magnet to move.
[0069]
In addition, 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, the magnet may be acted before or while the magnetic particles are in contact with the surface of the carrier.
[0071]
  BurdenWhen the surface of the body is partially covered with a specific binding substance, the magnet may be placed anywhere as long as the magnetic particles can be moved in the direction in which they are to be moved. It can be placed in the desired direction or around it. 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.Minutes (It is preferable to arrange the magnet so that the magnetic particles move in the direction of the arrow in FIG.
[0072]
  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 partMinutes (It is preferable to arrange the magnet so that the magnetic particles move in the direction of the arrows in FIGS.
[0073]
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.
[0074]
When a test substance exists in the sample, the test substance binds to a specific binding substance immobilized on the carrier.
In this case, when a magnet is applied to the magnetic particles, the magnetic particles are attracted by the magnet and move in the direction of the magnet along the surface of the carrier. In the process, the magnetic particles are bound to a specific binding substance immobilized on the surface. When a test substance is encountered, the magnetic particles bind to the carrier via the test substance and the specific binding substance bound thereto, and stop moving, or the movement is significantly slowed down.
[0075]
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, the movement speed of the magnetic particle is different depending on whether or not the test substance is bound to the specific binding substance fixed on the face. Make a big difference.
[0076]
That is, when there is no test substance in the sample, the specific binding substance immobilized on the surface does not bind the test substance, so the magnetic particles do 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.
[0077]
On the other hand, when a test substance is present in the sample, the specific binding substance immobilized on the surface binds the test substance, so that the magnetic particles bind to the carrier via the test substance and the specific binding substance. Movement stops or becomes extremely slow. 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.
[0078]
When a container is used as the carrier, the magnetic particles added to the container 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.
[0079]
  Specific binding substance of carrierFrom the uncoated part to the covered partWhen a magnet is made to act on the magnetic particles so as to move, the interaction between the magnetic particles and the surface to which the test substance is not bonded is weak, and the moving speed almost depends on the strength of the magnetic field. 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.
[0080]
  When moving by tilting the support, the particles are bound by the specific binding substance of the support.PartiallyCoated surfaceFrom the part not coated with the specific binding substance ofTilt the carrier to move. The angle at which the carrier is tilted is the surface on which the particles are coated with the specific binding substance of the carrier by gravityFrom the part not coated with the specific binding substance ofThe angle may be an angle that moves, and an angle of 90 ° or less may be selected as appropriate, but an angle between 25 ° and 90 ° is preferable, and an angle between 45 ° and 65 ° is particularly preferable.
[0081]
In addition, the carrier may be tilted before or while the particles are brought into contact with the surface of the carrier, as long as positive or negative determination can be made according to the distribution state of the particles on the surface of the carrier.
[0083]
  BurdenIf the surface of the body is partly coated with a specific binding substance, the carrier is tilted in such a direction that the particles move over the part of the carrier covered with the specific binding substance and What is necessary is just to move a particle. For example, the carrier may be arranged with the direction to be moved down. 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 (FigureThe carrier is tilted so that the particles move in the direction of the arrow 1 (in this case, it may be tilted in the direction of the arrow in FIG. 1).
[0084]
  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 (FigureThe carrier is tilted so that the particles move in the direction of arrows 2 to 4 (in this case, it may be tilted in the direction of arrows in FIGS.
[0085]
When a test substance exists in the sample, the test substance binds to a specific binding substance immobilized on the carrier.
In this case, when the carrier is tilted so that the particles move along the surface of the carrier coated with the specific binding substance, the particles move downward along the surface of the carrier by gravity. When a test substance bound to a specific binding substance immobilized on the surface is encountered, the particle binds to the carrier via the test substance and the specific binding substance bound to the test substance and stops moving. Or the movement is significantly slowed down.
[0086]
In the region where the specific binding substance on the surface is not fixed, the movement of the particle depends on the interaction between the particle and the surface and the angle of tilting the carrier, and the particle moves quickly downward in the tilt.
On the other hand, in the region where the specific binding substance on the surface is fixed, the movement speed of the particles depends on whether the test substance is bound to the specific binding substance fixed on the face or not. Make a big difference.
[0087]
That is, when there is no test substance in the sample, the specific binding substance immobilized on the surface does not bind the test substance, so that the particles do not show affinity and the specific binding substance is not fixed. As with, immediately move downward in the tilt direction. Therefore, when the test substance does not exist in the sample, the particles gather at the lower end of the carrier.
On the other hand, when the test substance is present in the sample, the specific binding substance immobilized on the surface binds the test substance, so that the particles bind to the carrier via the test substance and the specific binding substance. Movement stops or becomes significantly slower. Therefore, when the test substance is present in the sample, the particles collect on the part of the surface covered with the specific binding substance.
[0088]
  Tilt the carrier to make the particles specifically bind to the carrierFrom the uncoated part to the covered partWhen moving, the interaction between the particle and the surface to which the test substance is not bonded is weak, and the moving speed almost depends on the angle at which the carrier is tilted. Therefore, when a large moving speed is required, that is, when a measurement result is obtained in a short time, the angle at which the carrier is inclined may be increased. Further, the angle at which the carrier is tilted may be changed over time. Further, when a plate-like carrier is used, the speed at which the particles move may be changed by curving the plate-like body and changing the angle of the plate-like body.
[0089]
In the case of using a carrier having a cover (A in FIG. 4) in the groove on the surface of the plate-like body as the carrier, the absorption part is located at a position where the sample is absorbed and removed from the horizontal direction of the surface coated with the specific binding substance If the capacity is such that the absorption part is saturated with the sample when it is placed, the particles dispersed in a suitable dispersion medium are brought into contact with the surface of the carrier from which the sample has been removed, and the particles are brought into the absorption part. Without being absorbed and removed, it can be retained in the space surrounded by the groove on the surface of the plate-like body and the cover. It is preferable that the particles can be retained because the specific binding substance immobilized on the particles can sufficiently react with the test substance in the sample.
[0090]
  As described above, the particles are coated with a specific binding substance of the carrier.From the uncovered part to the covered partAfter the movement, the presence or absence of the test substance is determined from the particle distribution state on this surface. The distribution state of the particles on the surface of the carrier, that is, the image 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.
[0091]
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, 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.
[0092]
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, 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.
[0093]
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 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 portion corresponding to each test substance.
[0094]
FIG. 4C shows a case where measurement is performed on a sample in which two kinds of test substances are present (samples in which each test substance is positive) using a rectangular plate having a groove on the surface. FIG. 4D is a view of the plate-like body as viewed from above, and FIG. 4D shows the plate-like body when the sample (negative sample) is measured using the plate-like body from above. FIG.
In the case of positive, 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 portion corresponding to each test substance.
[0095]
In addition, when using a particle to which a test substance or its analog is immobilized instead of a specific binding substance, a result opposite to the distribution state of positive or negative particles obtained by the first measurement method is obtained.
[0096]
  In the second measurement method according to the present invention, a surface that is specifically coated with a specific binding substance and is partially covered with the specific binding substance, that is, a portion that is not covered with the specific binding substance and a portion that is covered with the specific binding substance are used. The mixture after mixing the particles having the specific binding substance immobilized thereon and the sample is brought into contact with the surface of the carrier having the surface having both surfaces. Then, the particle and sample mixture in contact with this surfaceA downstream end in a direction in which the mixture of the particles and the sample moves from the part not coated with the specific binding substance on the surface of the carrier to the part coatedIt removes with the absorption part arrange | positioned. Then, particles having a specific binding substance immobilized thereon are brought into contact with this surface. Next, the particles are moved from the portion not coated with the specific binding substance on the surface to the coated portion, and the presence or absence of the test substance is determined from the distribution state of the particles on the surface.
[0097]
According to the second measurement method, when the test substance is present in the sample, that is, when the test substance is positive, the particle binds to the test substance in the sample, so that the mixture of the particle and the sample is brought into contact with the surface of the carrier. When this is done, the particles bind to the specific binding substance immobilized on this surface via the test substance. Then, when the mixture of particles and sample is removed from this surface by the absorption part arranged on the carrier, and further the particles are brought into contact with this surface and moved along the surface, the particles are fixed to the surface of the carrier first. The movement is stopped or remarkably slowed by the particles that have been trapped, and the particles collect on the part of the surface covered with the specific binding substance.
[0098]
On the other hand, when the test substance is not present in the sample, that is, when the test substance is negative, the particle does not bind the test substance. It does not bind to the specific binding substance. Thus, even if the mixture of particles and sample is removed from this surface by the absorber located on the carrier, and the particles are moved along the surface in contact with this surface, the particles will pass over this surface. As a result, it quickly moves downward in the direction of the magnet or the inclination, and gathers at a position close to the magnet, that is, at the end of the surface of the carrier or at the lower portion of the carrier.
Therefore, according to the second measurement method, the same result as the positive or negative particle distribution obtained by the first measurement method can be obtained.
[0099]
This measurement method can be used for a confirmation test. The confirmation test is a method for confirming whether a positive image is actually obtained by measurement, whether it is due to the presence of a test substance or non-specific agglutination reaction.
[0100]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
[0101]
[Example 1]
(Measurement of HBs antigen in blood sample: using filter paper as absorption part)
(1) Preparation of anti-HBs antibody fixed plate
Acrylic resin extrusion plate (25 mm in width, 60 mm in depth, 1 mm in thickness) (made by Acrysanday) Two acrylic resin extrusion plates (10 mm in width, 40 mm in depth, 0.5 mm in thickness) (made by Acrysanday) in 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.
An anti-HBs antibody solution (manufactured by Shinotest, pH 7 at a concentration of 5 μg / ml and a pH of 7 10 mM phosphate buffer) is placed on the portion from 5 mm to 10 mm from the left end of the groove shown in FIG. 5 surrounded by two acrylic plates. 20 μl of the solution dissolved in the solution was placed using a pipette and brought into contact, and allowed to stand at 37 ° C. for 3 hours.
Next, this solution was removed by suction with a pipette, and 0.3 ml of Tris buffer solution (pH 7.5, 50 mM) (hereinafter referred to as Diluent A) containing 0.5% (W / V) casein was added thereto. It was left overnight at 0 ° C., and this was removed by suction with a pipette.
Then, an acrylic resin extruded plate (width 25 mm, depth 50 mm, thickness 1 mm) [manufactured by Acrysanday Co., Ltd.] is bonded onto the left end of this plate-like body with a solvent, thereby forming a plate-like body as shown in FIG. Covered.
In this way, a plate-like body was produced in which a band-shaped coating portion made of a specific binding substance (anti-HBs antibody) was formed on the bottom surface of the groove.
Qualitative filter paper No2 (manufactured by Advantech Toyo Co., Ltd.) as an absorbing portion in the space formed by the two upper and lower plates shown in FIG. 5 of this plate (the left end side surface of the plate in FIG. 5A). Was cut to a width of 25 mm and a depth of 20 mm.
[0102]
(2) Preparation of anti-HBs antibody fixed particles
Particles (magnetic particles) [Dynabeads M-450 uncoated, manufactured by Dynal, particle size: 4.5 μm, C.D. V. 5% or less, specific gravity 1.5, concentration 3% (w / v)] 1 ml and anti-HBs antibody solution (manufactured by Shinotest, pH 7.0, dissolved in 10 mM phosphate buffer at a concentration of 0.1 mg / ml) 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 particles were washed with the diluent A, and the particles were redispersed in the diluent A so that the concentration was about 0.1% (w / v). Thus, an anti-HBs antibody fixed particle dispersion was prepared.
[0103]
(3) Measurement of HBs antigen
HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., dissolved in 10 mM phosphate buffer, pH 7.2 at a concentration of 40 μg / ml) is adjusted to 1 ng / ml in blood that is negative for both HBs antigen and HBs antibody. Add 100 μl of the sample prepared by adding it using a pipette near the boundary between the covered and uncovered portions of the anti-HBs antibody-fixed plate prepared in (1) above, and put it on the belt-shaped covered portion. Made contact.
Thereafter, the HBs antigen-positive blood brought into contact with the belt-like covering portion is absorbed by the absorption portion arranged on the plate-like body, and then the anti-HBs antibody-immobilized particle dispersion prepared in (2) is used as the anti-HBs antibody immobilization solution. The plate-like body was placed in the vicinity of the boundary between the covered portion and the uncovered portion of the groove by using a pipette and brought into contact with the belt-like covering portion. (Since the sample penetrated and was saturated in the absorption part, the anti-HBs antibody fixed particle dispersion did not penetrate into the absorption part.)
Thereafter, the anti-HBs antibody on the bottom surface of the groove of the plate-like body is not fixed so that the anti-HBs antibody-fixed particles move by disposing the magnet in the side surface direction where the band-shaped covering portion of the plate-like body is formed. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated from the portion in the partial direction in which the anti-HBs antibody on the bottom surface of the groove was fixed (the direction in which the absorption portion was disposed).
Within 3 minutes after the magnetic field was generated, an image of particles as shown in FIG. 5C was observed.
When the negative blood was used as a sample, no particle image was observed.
[0104]
[Example 2]
(Measurement of HBs antigen in blood sample: using porous material as absorption part)
(1) Preparation of anti-HBs antibody fixed plate
An anti-HBs antibody-immobilized plate was prepared in the same manner as (1) of Example 1 except that the absorption part was prepared by cutting Berglin (manufactured by Kanebo Co., Ltd., thickness 2 mm) into a width of 5 mm and a depth of 8 mm. did.
[0105]
(2) Preparation of anti-HBs antibody fixed particles
An anti-HBs antibody-fixed particle dispersion was prepared in the same manner as in Example 1 (2).
[0106]
(3) Measurement of HBs antigen
HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., dissolved in 10 mM phosphate buffer, pH 7.2 at a concentration of 40 μg / ml) is adjusted to 1 ng / ml in blood that is negative for both HBs antigen and HBs antibody. Add 100 μl of the sample prepared by adding it using a pipette near the boundary between the covered and uncovered portions of the anti-HBs antibody-fixed plate prepared in (1) above, and put it on the belt-shaped covered portion. Made contact.
Thereafter, the HBs antigen-positive blood brought into contact with the belt-like covering portion is absorbed by the absorption portion arranged on the plate-like body, and then the anti-HBs antibody-immobilized particle dispersion prepared in (2) is used as the anti-HBs antibody immobilization solution. The plate-like body was placed in the vicinity of the boundary between the covered portion and the uncovered portion of the groove by using a pipette and brought into contact with the belt-like covering portion. (Since the sample penetrated and was saturated in the absorption part, the anti-HBs antibody fixed particle dispersion did not penetrate into the absorption part.)
Thereafter, the anti-HBs antibody on the bottom surface of the groove of the plate-like body is not fixed so that the anti-HBs antibody-fixed particles move by disposing the magnet in the side surface direction where the band-shaped covering portion of the plate-like body is formed. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated from the portion in the partial direction in which the anti-HBs antibody on the bottom surface of the groove was fixed (the direction in which the absorption portion was disposed).
Within 3 minutes after the magnetic field was generated, an image of particles as shown in FIG. 5C was observed.
When the negative blood was used as a sample, no particle image was observed.
[0107]
Example 3
(Measurement of HBs antigen in blood sample: absorption part made into space)
(1) Preparation of anti-HBs antibody fixed plate
Two acrylic resin extrusion plates 3 (width 10 mm, depth 40 mm, thickness 0.5 mm) on the acrylic resin extrusion plate 2 (width 25 mm, depth 52.5 mm, thickness 1 mm) [manufactured by Acrysanday Co., Ltd.] The product is bonded with a solvent at an interval of 5 mm to produce a groove on the surface of the plate-like body, and two acrylic resin extruded plates 4 (width 2.5 mm, depth 12.5 mm, thickness are formed on the left side of the groove) 0.5 mm) is pasted with a solvent at an interval of 20 mm, and an acrylic resin extruded plate 5 (width 20 mm, depth 2.5 mm, thickness 0.5 mm) is pasted to the left end of the plate with a solvent, 6 is provided with grooves (width 5 mm, depth 40 mm, height 0.5 mm), and an absorption part (width 20 mm, depth 10 mm, height 0) surrounded by the acrylic resin extrusion plates 4 and 5. .5mm) One plate-like body was produced. An anti-HBs antibody solution (manufactured by Shinotest Co., pH 7 at a concentration of 5 μg / ml, 10 mM phosphoric acid is applied to a portion 10 mm to 5 mm from the left end of the groove shown in FIG. 6 surrounded by two acrylic plates 3 of this plate-like body. 20 μl of the sample dissolved in the buffer solution was placed using a pipette, brought into contact, and allowed to stand at 37 ° C. for 3 hours.
The solution was then removed by suction with a pipette, 0.3 ml of Diluent A was added thereto and left at 4 ° C. overnight, and this was removed by suction with a pipette.
Then, an acrylic resin extruded plate 6 (width 25 mm, depth 42.5 mm, thickness 1 mm) [made by Acrysanday Co., Ltd.] having a small hole 7 formed on the left end of the plate-like body is bonded with a solvent to obtain FIG. The plate was covered as shown in.
In this way, a plate-like body was produced in which a band-shaped coating portion made of a specific binding substance (anti-HBs antibody) was formed on the bottom surface of the groove.
[0108]
(2) Preparation of anti-HBs antibody fixed particles
An anti-HBs antibody-fixed particle dispersion was prepared in the same manner as in Example 1 (2).
[0109]
(3) Measurement of HBs antigen
HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., dissolved in 10 mM phosphate buffer, pH 7.2 at a concentration of 40 μg / ml) is adjusted to 1 ng / ml in blood that is negative for both HBs antigen and HBs antibody. Add 100 μl of the sample prepared by adding it using a pipette near the boundary between the covered and uncovered portions of the anti-HBs antibody-fixed plate prepared in (1) above, and put it on the belt-shaped covered portion. Made contact.
Thereafter, the HBs antigen-positive blood brought into contact with the band-shaped covering portion moves so that the sample is moved in the direction in which the absorbing portion is disposed so that the direction in which the absorbing portion of the plate-like body is disposed is downward. The plate was tilted at an angle of about 60 °.
After the sample is absorbed by the absorption part, the plate-like body is returned to the horizontal position, and the anti-HBs antibody-fixed particle dispersion prepared in (2) above is covered with the groove covered portion of the anti-HBs antibody-fixed plate body. It was placed in the vicinity of the boundary of the uncovered portion with a pipette and brought into contact with the strip-shaped covering portion. (Since the absorption part was completely filled with the sample, the anti-HBs antibody-fixed particle dispersion did not penetrate into the absorption part.)
Thereafter, the anti-HBs antibody on the bottom surface of the groove of the plate-like body is not fixed so that the anti-HBs antibody-fixed particles move by disposing the magnet in the side surface direction where the band-shaped covering portion of the plate-like body is formed. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated from the portion in the partial direction in which the anti-HBs antibody on the bottom surface of the groove was fixed (the direction in which the absorption portion was disposed).
A particle image as shown in FIG. 6C was observed within 3 minutes after the magnetic field was generated.
When the negative blood was used as a sample, no particle image was observed.
[0110]
Example 4
(Measurement of anti-HBs antibody in blood sample)
(1) Preparation of HBs antigen fixed plate
Acrylic resin extrusion plate (25 mm in width, 60 mm in depth, 1 mm in thickness) (made by Acrysanday) Two acrylic resin extrusion plates (10 mm in width, 40 mm in depth, 0.5 mm in thickness) (made by Acrysanday) in 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.
An HBs antigen solution (manufactured by Meiji Dairies Co., Ltd., 10 mM phosphate buffer having a pH of 7 at a concentration of 5 μg / ml) is placed on a portion from 5 mm to 10 mm from the left end of the groove shown in FIG. 5 surrounded by two acrylic plates of this plate-like body. 20 μl of the solution dissolved in the solution was placed using a pipette and brought into contact, and allowed to stand at 37 ° C. for 3 hours.
Then, after the solution was removed by suction with a pipette, 0.3 ml of the diluent A was added thereto and left at 4 ° C. overnight, and this was removed by suction with a pipette.
Then, an acrylic resin extruded plate (width 25 mm, depth 50 mm, thickness 1 mm) [manufactured by Acrysanday Co., Ltd.] is bonded onto the left end of this plate-like body with a solvent, thereby forming a plate-like body as shown in FIG. Covered.
In this way, a plate-like body was produced in which a band-shaped covering portion made of a specific binding substance (HBs antigen) was formed on the bottom of the groove.
Width of qualitative filter paper No2 (manufactured by Advantech Toyo Co., Ltd.) as an absorbing portion in the space formed by the two upper and lower plates shown in FIG. 5 of this plate (the left side surface of the plate in A of FIG. 5). What was cut | disconnected to 25 mm and depth 20mm was arrange | positioned.
[0111]
(2) Preparation of HBs antigen fixed particles
1 ml of particles (Dynabeads M-450 uncoated, manufactured by Dynal Co., Ltd., particle size: 4.5 μm, CV less than 5%, specific gravity 1.5, concentration 3% (w / v)) and HBs antigen solution (Meiji Dairies) 1 ml of 10 mM phosphate buffer, pH 7.2, dissolved at a concentration of 40 μg / 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 particles were washed with the diluent A, and the particles were redispersed in the diluent A so that the concentration was about 0.1% (w / v). In this way, an HBs antigen-immobilized particle dispersion was prepared.
[0112]
(3) Measurement of anti-HBs antibody
Place 100 μl of anti-HBs antibody-positive blood using a pipette near the border between the covered and uncovered portions of the groove of the HBs antigen-fixing plate produced in (1) above, and contact the strip-shaped covered portion I let you.
Thereafter, the anti-HBs antibody-positive blood brought into contact with the band-shaped covering portion is absorbed by the absorption part arranged on the plate-like body, and the HBs antigen-immobilized particle dispersion prepared in (2) above is used as the HBs antigen-immobilized plate. The strip was covered with a pipette in the vicinity of the boundary between the covered portion and the uncovered portion of the groove and brought into contact with the strip-shaped covering portion. (Since the sample penetrated and was saturated in the absorption part, the HBs antigen-fixed particle dispersion did not penetrate into the absorption part.)
Thereafter, the HBs antigen on the bottom surface of the groove of the plate-like body is placed with the side surface direction in which the band-shaped covering portion of the plate-like body is formed facing down (so that the direction in which the absorption part is disposed is down). HBs antigen-immobilized plate at an angle of about 60 ° so that the HBs antigen-immobilized particles move from the non-immobilized part to the partial direction where the HBs antigen on the bottom surface of the groove is immobilized (the direction in which the absorption part is disposed). Tilt the body.
Within 3 minutes after tilting, an image of particles as shown in FIG. 5C was observed. When the negative blood was used as a sample, no particle image was observed.
[0113]
Example 5
(Measurement of E. coli O157: H7)
(1) Preparation of anti-E. Coli O157: H7 antibody fixed plate
Acrylic resin extrusion plate (25 mm in width, 60 mm in depth, 1 mm in thickness) (made by Acrysanday) Two acrylic resin extrusion plates (10 mm in width, 40 mm in depth, 0.5 mm in thickness) (made by Acrysanday) in 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.
Anti-Escherichia coli O157: H7 antibody solution [manufactured by Kirkegaard & Perry Laboratories, at a concentration of 5 μg / ml, is placed between 5 mm and 10 mm from the left end of the groove shown in FIG. 5 surrounded by two acrylic plates of this plate. [Dissolved in 10 mM phosphate buffer at pH 7] 20 μl was placed using a pipette and contacted, and allowed to stand at 37 ° C. for 3 hours.
Then, after the solution was removed by suction with a pipette, 0.3 ml of the diluent A was added thereto and left at 4 ° C. overnight, and this was removed by suction with a pipette.
Then, an acrylic resin extruded plate (25 mm in width, 25 mm in depth, 1 mm in thickness) (manufactured by Acrysanday Co.) 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 way, a plate-like body was produced in which a band-shaped coating portion made of a specific binding substance (anti-E. Coli O157: H7 antibody) was formed on the bottom surface of the groove.
Width of qualitative filter paper No2 (manufactured by Advantech Toyo Co., Ltd.) as an absorbing portion in the space formed by the two upper and lower plates shown in FIG. 5 of this plate (the left side surface of the plate in A of FIG. 5). What was cut | disconnected to 25 mm and depth 20mm was arrange | positioned.
[0114]
(2) Preparation of anti-E. Coli O157: H7 antibody fixed particles
1 ml of particles (magnetic particles) (Dynabeads M-450 uncoated, manufactured by Dynal Co., Ltd., particle size: 4.5 μm, CV less than 5%, specific gravity 1.5, concentration 3% (w / v)) and anti-E. Coli O157 : 1 ml of an H7 antibody solution (manufactured by Kirkegaard & Perry Laboratories, dissolved in 10 mM phosphate buffer of pH 7.0 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 particles were washed with the diluent A, and the particles were redispersed in the diluent A so that the concentration was about 0.1% (w / v).
Thus, an anti-E. Coli O157: H7 antibody fixed particle dispersion was prepared.
[0115]
(3) Measurement of E. coli O157: H7
E. coli O157: H7 (Kirkegaard & Perry Laboratories) 10^Three100 μl of a sample prepared by adding to the fecal suspension of a healthy person at a rate of 1 / ml, and the covered portion of the groove of the anti-E. Coli O157: H7 antibody-fixed plate prepared in (1) above It was placed in the vicinity of the boundary of the uncovered portion with a pipette and brought into contact with the strip-shaped covering portion.
Then, after the sample brought into contact with the belt-like covering portion is absorbed by the absorption portion arranged on the plate-like body, the anti-E. Coli O157: H7 antibody fixed particle dispersion prepared in (2) above is used as the anti-E. Coli O157. : The H7 antibody-fixed plate was placed using a pipette near the boundary between the covered portion and the uncovered portion of the groove and brought into contact with the belt-shaped covering portion. (Since the sample penetrated and was saturated in the absorption part, the anti-E. Coli O157: H7 antibody fixed particle dispersion did not penetrate into the absorption part.)
Thereafter, by installing a magnet in the side surface direction where the band-shaped covering portion of the plate-like body is formed, the anti-E. Coli O157: at the bottom of the groove of the plate-like body is moved so that the anti-E. Coli O157: H7 antibody fixed particles move. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated from the portion where the H7 antibody was not fixed in the direction of the portion where the anti-E. Coli O157: H7 antibody was fixed at the bottom of the groove (the direction in which the absorption portion was arranged).
Within 3 minutes after the magnetic field was generated, an image of particles as shown in FIG. 5C was observed.
When measurement was performed using a stool suspension to which E. coli O157: H7 had not been added as a sample, no particle image was observed.
[0116]
Example 6
(Confirmation test for HBs antigen in blood samples (1))
Place 100 μl of HBs antigen-positive blood using a pipette in the vicinity of the boundary between the covered and uncovered portions of the anti-HBs antibody-fixed plate produced in (1) of Example 1 on the band-shaped covered portion. Made contact.
Then, after the HBs antigen positive blood brought into contact with the belt-shaped covering part is absorbed by the absorption part arranged in the plate-like body, the absorption part in which this HBs antigen positive blood is absorbed is removed, and as a new absorption part, A qualitative filter paper No. 2 (manufactured by Advantech Toyo Co., Ltd.) cut to a width of 25 mm and a depth of 20 mm was placed on a plate-like body as before.
Next, an anti-HBs antibody solution (manufactured by Sinotest, dissolved in diluent A at a concentration of 0.1 mg / ml) was covered with the groove of the anti-HBs antibody-fixed plate from which the HBs antigen-positive blood was absorbed and removed. It was placed in the vicinity of the boundary between the part and the uncovered part using a pipette and brought into contact with the strip-shaped covering part.
Then, after the anti-HBs antibody solution brought into contact with the belt-like covering portion is absorbed by the absorption part newly arranged on the plate-like body, the anti-HBs antibody-immobilized particle dispersion prepared in (2) of Example 1 is used as the dispersion. The anti-HBs antibody-fixed plate was placed using a pipette near the boundary between the covered part and the uncovered part of the groove, and was brought into contact with the belt-like covering part. (The anti-HBs antibody fixed particle dispersion did not penetrate into the absorption part because the anti-HBs antibody solution penetrated into the absorption part and was saturated.)
Thereafter, the anti-HBs antibody on the bottom surface of the groove of the plate-like body is not fixed so that the anti-HBs antibody-fixed particles are moved by installing a magnet in the side surface direction where the band-shaped covering portion of the plate-like body is formed. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated from the portion in the direction of the portion where the anti-HBs antibody on the bottom of the groove was fixed (in the direction of the arrow in FIG. 5).
Three minutes after the generation of the magnetic field, the distribution state of the particles on the surface of the plate-like body was confirmed, but no particle image as shown in FIG. 5C was observed. This confirmed that the HBs antigen was truly present in the sample.
[0117]
Example 7
(Confirmation test for HBs antigen in blood samples (2))
Place 100 μl of HBs antigen-positive blood using a pipette in the vicinity of the boundary between the covered and uncovered portions of the anti-HBs antibody-fixed plate produced in (1) of Example 1 on the band-shaped covered portion. Made contact.
Thereafter, the HBs antigen-positive blood brought into contact with the belt-like covering portion is absorbed by the absorption portion arranged on the plate-like body, and then the anti-HBs antibody-fixed particle dispersion prepared in (2) of Example 1 is added to the anti-HBs. The antibody was added to 0.1 mg / ml to prepare an anti-HBs antibody-added anti-HBs antibody-fixed particle dispersion. This dispersion is placed with a pipette near the boundary between the covered and uncovered portions of the anti-HBs antibody-fixed plate from which the HBs antigen-positive blood has been absorbed and removed, and brought into contact with the strip-shaped covering portion. It was. (Since the sample penetrated and was saturated in the absorption part, the anti-HBs antibody-added anti-HBs antibody fixed particle dispersion did not penetrate into the absorption part.)
Thereafter, the anti-HBs antibody on the bottom surface of the groove of the plate-like body is not fixed so that the anti-HBs antibody-fixed particles are moved by installing a magnet in the side surface direction where the band-shaped covering portion of the plate-like body is formed. A magnetic field having a magnetic flux density of 40 to 60 gauss was generated from the portion in the direction of the portion where the anti-HBs antibody on the bottom of the groove was fixed (in the direction of the arrow in FIG. 5).
Three minutes after the magnetic field was generated, the distribution state of the particles on the surface of the plate-like body was confirmed, but no particle image as shown in FIG. 5C was observed. This confirmed that the HBs antigen was truly present in the sample.
[0118]
【The invention's effect】
According to the measuring instrument and the measuring method of the present invention, red blood cells that are substances that hinder the determination when measuring a test substance in a sample visually or using an instrument using a specific binding reaction such as an antigen-antibody reaction Even in a stool sample containing contaminants such as blood samples and food residues, the presence or absence of the test substance can be reliably determined without interference.
In addition, according to the measuring instrument and the measuring method of the present invention, it is not necessary for the measurer to perform an absorption operation of a sample that is complicated and has a risk of infection or the like.
Further, according to the measuring instrument and the measuring 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 surface of the container as in the prior art, but the particles are not the end of the surface of the carrier. Since the determination is made based on whether the sample is collected at the portion or the portion coated with the specific binding substance on the surface of the carrier, the determination is easy even when the test substance concentration is low. 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.
According to the measuring instrument and the measuring 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 particles and / or the carrier, which is efficient. 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 measuring instrument and the measuring method of the present invention, in a short time, specifically depending on the type of the test substance, the presence or absence of the test substance in the sample is usually about 20 seconds to 10 minutes. For example, in the case of HBs antigen, measurement can be performed within 3 minutes even at a concentration of about 1 ng / ml. In the case of E. coli O157, 10^Three Measurement is possible within 3 minutes even at a concentration of about 1 / ml.
Furthermore, according to the measuring instrument and measuring method of the present invention, it is possible to simultaneously measure a plurality of types of test substances in a sample.
Moreover, according to the measuring instrument and measuring method of this invention, a confirmation test can be performed simply.
[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 view 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 in a band shape with a specific binding substance.
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 view showing a plate-like body in which a groove is provided on the surface and the bottom surface of the groove is coated in a band shape with a specific binding substance.
FIG. 6 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 coated in a band shape with a specific binding substance.
FIG. 7 is a diagram showing an agglomerated image when a test substance in a sample is measured by an indirect agglutination measurement method.
[Explanation of symbols]
1: Absorbing part, 2: Acrylic resin extrusion plate, 3: Acrylic resin extrusion plate,
4: Acrylic resin extrusion plate, 5: Acrylic resin extrusion plate, 6: Acrylic resin extrusion plate
7: Small hole

Claims (11)

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. Measurement of a test substance in a sample, comprising a carrier having a specific binding substance for the test substance and particles having the same or different specific binding substance as the specific binding substance fixed to the support. In the apparatus, the absorption part for absorbing and removing the sample brought into contact with the surface is downstream in the direction in which the sample moves from the part not coated with the specific binding substance on the surface of the carrier to the part coated with the surface. A measuring instrument for a test substance in a sample, characterized in that it is arranged at the end of the sample.   The measuring instrument for a test substance in a sample according to claim 1, 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.   The measurement device for a test substance in a sample according to claim 1, wherein the carrier is a plate-like body, and the surface thereof is partially coated with a specific binding substance.   The instrument for measuring a test substance in a sample according to any one of claims 1 to 3, wherein the test substance is an antigen and the specific binding substance is an antibody.   The instrument for measuring a test substance in a sample according to any one of claims 1 to 3, wherein the test substance is an antibody, and the specific binding substance is an antigen or an antibody against the antibody. 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 the sample into contact with the surface of the carrier having the sample; the sample brought into contact with the surface is moved from a portion of the surface of the carrier not coated with the specific binding substance to a portion coated with the sample; A step of absorbing and removing it in the absorption part arranged at the downstream end of the direction, a specific binding substance for the test substance, which is the same or different from the specific binding substance fixed to the carrier Contacting said surface with said surface; and moving said particle from a portion of said surface not coated with a specific binding substance to a portion coated with said surface. Binding substances and judging the presence or absence of the test substance from the distribution of the particles in the portion covered with the portion not covered by the measuring method of the analyte in the sample. 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 mixture after mixing particles and a sample, which are specific binding substances for a test substance and have the same or different specific binding substance immobilized on the carrier, on the surface of the carrier. Contacting, moving the mixture of particles and sample in contact with the surface from a portion of the support surface where the particles and sample are not coated with a specific binding substance to a portion coated with the surface. removing is absorbed into the absorbent portion disposed downstream of the end of the direction and the specific binding substance and a same or different and immobilized specific binding agent to the carrier for the test substance Contacting the surface with particles having a foreign binding substance immobilized thereon, and moving the particles from a portion of the surface that is not coated with the specific binding material to a portion that is coated with the surface, A method for measuring a test substance in a sample, wherein the presence or absence of the test substance is determined from the distribution state of particles in a part not covered with a specific binding substance on the surface and a part covered with the surface.   The test substance in the sample according to claim 6 or 7, 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 6 or 7, 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 any one of claims 6 to 9, 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 any one of claims 6 to 9, wherein the test substance is an antibody, and the specific binding substance is an antigen or an antibody against the antibody.

Images