JP4045314B2 - Method for measuring analytes using particles - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for measuring a test substance in a sample using particles in which a specific binding substance to the test substance is immobilized, and particularly to a trace amount of a test substance contained in a sample such as a blood sample. It is related with the measuring method of the to-be-tested substance which can be measured in a short time and simply, without being influenced by.
The present invention is useful in the field of life science such as a clinical examination field.
[0002]
[Prior art]
A method for measuring a small amount of test substance contained in a sample using a reaction between substances having specific affinity such as antigen and antibody, sugar and lectin, nucleotide chain and complementary nucleotide chain, and ligand and receptor. Various types 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. 6 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 aggregated image can be observed (B in FIG. 6), and the test substance exists in the sample. It can be confirmed.
[0006]
However, in this indirect agglutination measurement method, the presence or absence of a test substance in a sample, that is, whether it is positive or negative, is determined by the size of a circular image formed by particles on the bottom surface of the measurement container.
Therefore, in the case of a sample with a low concentration of the test substance, both the particles aggregated via the test substance and the particles not bound to the test substance are mixed in the central part of the bottom of the measurement container, and the aggregate Since the circle of the image was small, it was difficult to distinguish from the negative case.
Because of the difficulty of determination in the case of a sample with a low concentration of the test substance, it is impossible to measure a low concentration of the test substance. Therefore, the time required for the measurement is long, and the normal measurement time is 1 hour or more.
[0007]
In addition, in the indirect agglutination measurement method, particles bound to antibodies (or antigens) are aggregated via components in a sample other than the test substance, or particles bound to antibodies (or antigens) are bound to the measurement container. In some cases, the sample binds to the inner wall surface and shows an aggregated image as if the test substance is present (positive) even though the test substance is not present in the sample (negative) (non-specific agglutination reaction) . Such a possibility that a negative sample may be erroneously determined as positive has been a serious problem in diagnosis of diseases by clinical examination.
[0008]
In contrast, JP-A-64-69954 discloses that a specimen sample is added to a measurement container in which an antibody or antigen corresponding to an antigen or an antibody as a test substance in a specimen sample is fixed to the inner wall, and simultaneously or subsequently. An insoluble carrier particle fixed with the same antibody or antigen or specific binding analog fixed to the measurement container without washing is added to the measurement container, and the sample sample is determined depending on the presence or absence of an agglutination reaction. An immunological measurement method for determining the presence or absence of an antigen or antibody as a test substance is disclosed. In this measurement method, when the state in the measurement container when the antigen (or antibody) as the test substance does not exist (negative) in the specimen sample is viewed from above, an image similar to the aggregated image shown in FIG. Is observed. In addition, when an antigen (or antibody) 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. 6B 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 is not present 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. 6B 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, when the sample is blood (whole blood sample), red blood cells in the blood sample roll on the inner wall of the measurement container by gravity together with the particles. When the test substance in the blood sample is measured, the particle distribution is blocked by the red blood cells contained in the sample, making it difficult to determine positive or negative. There was a problem of becoming.
[0013]
[Problems to be solved by the invention]
Accordingly, an object of the present invention is to provide a sample such as a blood sample or stool containing a substance that hinders determination of red blood cells or the like when measuring a test substance in the sample visually or using an instrument using a specific binding reaction. However, it is also possible to provide a measurement method in which the determination of the presence / absence of the test substance can be performed in a short time without interference, and the determination is highly reliable.
[0014]
[Means for Solving the Problems]
In the present invention, a specific binding substance to a test substance in a sample is immobilized, and the specific binding substance is used. Partially Coated surface That is, the surface that has both the part not covered with the specific binding substance and the part covered A step of bringing a sample into contact with the surface of the carrier having Move over the part of the surface covered by the specific binding substance A step of removing, a step of bringing a particle having a specific binding substance fixed to the carrier, which is a specific binding substance to the test substance, the same or different from the specific binding substance into contact with the surface from which the sample has been removed, And the particles on the surface From the part not coated with the specific binding substance of Comprising the step of moving the surface Parts that are not coated with specific binding substances and parts that are coated The method for measuring a test substance in a sample (first measurement method) is characterized in that the presence or absence of a test substance is determined from the distribution state of particles in the sample.
[0015]
In the present invention, a specific binding substance to a test substance in a sample is immobilized and the specific binding substance is used. Partially Coated surface That is, the surface that has both the part not covered with the specific binding substance and the part covered 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 as the specific binding substance immobilized on the carrier, on the surface of the carrier having Contacting the surface with a mixture of particles and sample in contact with the surface Move over the part of the surface covered by the specific binding substance A step of removing, a step of bringing a particle that is specifically bound to a test substance and having a specific binding substance that is the same or different from the specific binding substance immobilized on the carrier into contact with the surface, and the particle The surface From the part not coated with the specific binding substance of Comprising the step of moving the surface Parts that are not coated with specific binding substances and parts that are coated It is a measuring method (2nd measuring method) of the to-be-tested substance in a sample characterized by determining the presence or absence of the to-be-tested substance from the particle distribution state in (2).
[0016]
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; DNA or RNA of viruses; DNA or RNA of bacteria; DNA or RNA of animals or plants such as humans; Substances or drugs related to other diseases such as lipoproteins (a) Can do.
[0017]
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.
[0018]
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.
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.
[0019]
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.
[0020]
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.
[0021]
Furthermore, as the particles, particles colored by coating a pigment or dispersing or encapsulating the pigment in the particles may be used.
[0022]
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.
[0023]
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.
[0024]
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.
[0025]
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 by adding dialysis, gel filtration, or the like, or adding a reaction terminator for the cross-linking reaction. Then, the obtained particles may be washed with a buffer solution or the like.
[0026]
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.
[0027]
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 containing various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and left to stand, and the surface of the particles is coated with various proteins. This can be done by coating.
[0028]
In the present invention, as the carrier, the specific binding substance is immobilized by immobilizing the specific binding substance. Partially Coated surface That is, the surface that has both the part not covered with the specific binding substance and the part covered A carrier having is used. Depending on the specific binding substance as the carrier Partially It 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.
[0029]
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. Partially What is necessary is just to coat. When the shape of the concave portion is a shape having a flat bottom surface such as a cylindrical shape or a rectangular parallelepiped shape, the flat bottom surface is made of a specific binding substance. Partially It 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. Partially It is preferably coated. Examples of the container having at least one recess having a flat bottom include a flat bottom microplate and a tray.
[0030]
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. Partially What 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.
[0031]
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.
[0032]
In the present invention, when a specific binding substance is immobilized on the surface of the carrier, the surface is fixed so that the surface is partially covered with the specific binding substance. Do . This is because 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.
[0033]
Also, the surface of the carrier of Depending on the specific binding substance Ru There may be a plurality of coating portions. For example, in order to measure a plurality of test substances in a sample, a coating portion made of a specific binding substance for each test substance can be provided on the surface of the carrier.
[0034]
FIG. 1 shows a flat bottom microplate used as a carrier in the present invention, in which one half of the flat bottom surface of each well is coated with a specific binding substance.
1A is a perspective view of a flat-bottom microplate (the hatched portion is a portion coated with the specific binding substance), and FIG. 1B is a plan view of the flat-bottomed microplate (the hatched portion is the above-described portion). This is the part coated with a specific binding substance.)
[0035]
FIG. 2 shows a rectangular parallelepiped transparent container having a bottom surface and having a bottom surface having a portion coated with a specific binding substance in a band shape (hereinafter referred to as a band-shaped coating portion).
2A is a perspective view of the container (the hatched portion is a portion covered with the specific binding substance), and FIG. 2B is a plan view of the container (the hatched portion is the specific portion). The part covered with the binding substance.)
[0036]
FIG. 3 shows a plate-like body having a rectangular surface shape and having a surface having a band-shaped covering portion made of two different types of specific binding substances.
3A is a perspective view of the plate-like body (shaded portions are portions coated with different specific binding substances), and FIG. 3B is a plan view of the plate-like body. (The shaded area is a portion covered with different specific binding substances.)
[0037]
FIG. 4 shows a plate-like body having a rectangular surface shape, and a groove having a rectangular cross-sectional shape is provided on the surface, and a band-shaped covering portion made of two different types of specific binding substances is formed on the bottom surface of the groove. It shows things.
FIG. 4A is a perspective view of such a plate-like body (shaded portions are portions coated with different specific binding substances), and FIG. 4B is a view of such a plate-like body. It is a plan view (hatched portions are portions covered with different specific binding substances).
Further, if the cover is covered by placing a plate on the side of the belt-like covering portion of the groove (see A in FIG. 4), the sample dropped in the groove on the uncovered side moves in the direction of the belt-like covering portion by capillary action. preferable.
[0038]
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.
[0039]
In the case of chemical bonding using a crosslinking reagent, the Japanese Society of Clinical Pathology “Special Issue on Clinical Pathology No. 53, Immunoassay for Clinical Examination—Technology and Applications”, Clinical Pathology Publications, 1983, Japan According to the known method described in the Biochemical Society edited by "Shinsei Kagaku Kogaku Kenkyu 1 Protein IV", Tokyo Kagaku Dojin, 1991, etc., the specific binding substance and carrier for the test substance are glutaraldehyde, carbodiimide, imide ester, maleimide, etc. It can be fixed by mixing and bringing into contact with a divalent cross-linking reagent and reacting with the amino group, carboxyl group, thiol group, aldehyde group, hydroxyl group, etc. of the specific binding substance and the carrier.
For example, when the carrier is a container, a bivalent cross-linking reagent such as glutaraldehyde, carbodiimide, imide ester, maleimide or the like is added to the concave portion of the container and left to react. Next, a specific binding substance is added to this and allowed to stand to react. In some cases, the reaction is stopped after that by adding a reaction stopper for the crosslinking reaction. Then, washing is performed with a buffer solution or the like.
[0040]
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. .
[0041]
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.
[0042]
Furthermore, if necessary, in order to suppress non-specific reactions, various proteins such as bovine serum albumin, human serum albumin, casein or a salt thereof, and nonfat dry milk are brought into contact with a carrier on which a specific binding substance is immobilized. The carrier on which the specific binding substance is immobilized may be masked by a known method.
For example, when the carrier is a container, a buffer solution containing various proteins such as bovine serum albumin, human serum albumin, casein, or a salt thereof is added to the concave portion of the container to which the specific binding substance is fixed, and left still. And after coating the surface of the recessed part of the container which fixed the specific binding substance with various proteins etc., it can carry out by sucking and removing the liquid of a recessed part.
[0043]
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. Partially Coated surface That is, the surface that has both the part not covered with the specific binding substance and the part covered A sample suspected of the presence of the test substance is brought into contact with the surface of the carrier having
[0044]
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.
[0045]
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.
[0046]
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 surfactants, amphoteric surfactants and anionic surfactants can be appropriately added and used.
[0047]
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).
[0048]
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.
[0049]
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.
The removal of the sample brought into contact with the surface of the carrier coated with the specific binding substance can be performed by bringing the sample absorber made of a water-absorbing material into contact with the sample on the surface of the carrier to absorb the sample, or using a pipette or the like. This can be done by sucking out the sample on the surface or by turning the carrier upside down and dropping the sample down.
[0050]
When a 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, or when the sample on the surface of the carrier is sucked with a pipette or the like and removed. In this case, it is preferable that the sample is absorbed or sucked from the horizontal direction of the portion of the surface coated with the specific binding substance so that the sample moves on the portion coated with the specific binding substance. In such a case, the test substance can be measured with higher sensitivity.
[0051]
Sample absorbents made of this water-absorbing material include papers such as filter paper, paper towels or tissue papers, chemical fibers such as rayon or polyester, cloths, non-woven fabrics, or materials having a property of absorbing liquids such as cotton. Can be used. In addition, there is no restriction | limiting in particular about the shape of this sample absorber, and a magnitude | size. When the sample brought into contact with the surface is removed as described above, 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. 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.
[0052]
In addition, after removing the sample brought into contact with the surface, the surface of the carrier may be washed with the above-described sample diluent or buffer solution.
[0053]
As described above, after removing the sample brought into contact with the surface, particles that are specific binding substances for the test substance and that have the same or different specific binding substance as the specific binding substance fixed to the carrier. Is brought into contact with the surface of the carrier from which the sample has been removed.
When measuring a plurality of test substances in a sample, particles fixed with specific binding substances for each test substance are brought into contact with each other.
[0054]
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.
[0055]
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.
[0056]
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).
[0057]
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. Partially Coated surface From the part not coated with the specific binding substance of Move. This side From the part not coated with the specific binding substance of The particles can be moved by applying a magnet or tilting the carrier.
[0058]
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. Partially Coated surface From the part not coated with the specific binding substance of Actuate the magnet to move.
[0059]
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.
[0061]
Burden When 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.
[0062]
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 part Minutes ( It is preferable to arrange the magnet so that the magnetic particles move in the direction of the arrows in FIGS.
[0063]
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.
[0064]
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.
[0065]
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.
[0066]
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.
[0067]
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.
[0068]
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.
[0069]
Specific binding substance of carrier From the uncoated part to the covered part When 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.
[0070]
When moving by tilting the support, the particles are bound by the specific binding substance of the support. Partially Coated surface From the part not coated with the specific binding substance of Tilt 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 gravity. From the part not coated with the specific binding substance of The angle may be an angle that moves, and an angle of 90 ° or less may be appropriately selected. However, an angle between 25 ° and 90 ° is preferable, and an angle between 45 ° and 65 ° is particularly preferable.
[0071]
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.
[0073]
Burden If 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 (Figure The 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).
[0074]
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 part Minute (Figure The 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 directions of arrows in FIGS. 2 to 4).
[0075]
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.
[0076]
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.
[0077]
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.
[0078]
Tilt the carrier to make the particles specifically bind to the carrier From the uncoated part to the covered part When 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.
[0079]
As described above, the particles are coated with a specific binding substance of the carrier. From the uncovered part to the covered part After 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.
[0080]
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.
[0081]
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.
[0082]
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 portions corresponding to the respective test substances.
[0083]
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.
[0084]
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.
[0085]
In the second measurement method according to the present invention, a specific binding substance is immobilized and the specific binding substance is used. Partially Coated surface That is, the surface that has both the part not covered with the specific binding substance and the part covered 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 carrier. Then, the particle and sample mixture in contact with this surface Move over the part of the surface covered by the specific binding substance Remove. Then, particles having a specific binding substance immobilized thereon are brought into contact with this surface. Next, this particle From the part not coated with the specific binding substance of It is moved, and the presence or absence of the test substance is determined from the distribution state of the particles on this surface.
[0086]
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, and further the particles are brought into contact with this surface and moved along the surface, the particles are first bonded to the surface of the carrier via the test substance. As a result, the movement is stopped or significantly slowed down, and the particles collect on the part of the surface covered with the specific binding substance.
[0087]
On the other hand, when the test substance does not exist in the sample, that is, when the test substance is negative, the particle does not bind the test substance, so that when the mixture of the particle and the sample is brought into contact with the surface of the carrier, the particle is fixed to this surface in advance. It does not bind to the specific binding substance. Therefore, even if the mixture of particles and sample is removed from this surface and the particles are brought into contact with this surface and moved along the surface, the particles will pass over this surface and the direction or inclination of the magnet will be quickly It moves downward and gathers at a position close to the magnet, that is, at the end of the surface of the carrier or at the lower carrier 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.
[0088]
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.
[0089]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
[0090]
[Example 1]
(Measurement of HBs antigen in blood sample)
(1) Preparation of anti-HBs antibody fixed plate
Acrylic resin extrusion plate (width 25 mm, depth 40 mm, thickness 1 mm) [made by Acrysanday] two acrylic extrusion plates (width 10 mm, depth 40 mm, thickness 0.5 mm) [Acrysandy] made 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. 5 was bonded with a solvent at intervals.
Using a pipette, 20 μl of an anti-HBs antibody solution (manufactured by Shinotest Co., dissolved in 10 mM phosphate buffer at pH 7 at a concentration of 5 μg / ml) is applied to a portion from 5 mm to 10 mm from the left end of the bottom of the groove of the plate-like body. Placed and contacted, left 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 (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-HBs antibody) was formed on the bottom surface of the groove.
[0091]
(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.
[0092]
(3) Measurement of HBs antigen
100 μl of HBs antigen-positive blood was placed using a pipette near the border between the groove-covered part and the non-covered part of the anti-HBs antibody-fixed plate prepared in (1) above, and was brought into contact with the belt-like covering part. .
The end on the side where the band-shaped covering portion of the anti-HBs antibody-fixing plate was formed from the sample absorbent prepared by folding tissue paper (trade name: Kimwipe, manufactured by Tokuma Kimberley) into 3 cm x 3 cm x 4 mm (thickness) The HBs antigen positive blood brought into contact with the part (the left end side surface of the plate-like body in FIG. 5A) and brought into contact with the band-shaped covering part was absorbed and removed.
Next, the anti-HBs antibody-immobilized particle dispersion prepared in (2) above is placed with a pipette in the vicinity of the boundary between the covered and uncovered portions of the anti-HBs antibody-immobilized plate. The coated part was brought into contact.
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).
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.
[0093]
[Example 2]
(Measurement of anti-HBs antibody in blood sample)
(1) Preparation of HBs antigen fixed plate
Acrylic resin extrusion plate (width 25 mm, depth 40 mm, thickness 1 mm) [made by Acrysanday] two acrylic extrusion plates (width 10 mm, depth 40 mm, thickness 0.5 mm) [Acrysandy] made 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. 5 was bonded with a solvent at intervals.
Pipet 20 μl of HBs antigen solution (made by Meiji Dairies Co., Ltd., dissolved in 10 mM phosphate buffer at pH 7 at a concentration of 5 μg / ml) from the left end of the bottom of the groove of this plate-like body to 10 mm. Placed and contacted, left 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 covering portion made of a specific binding substance (HBs antigen) was formed on the bottom surface of the groove.
[0094]
(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.
[0095]
(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.
The end of the side where the band-shaped covering portion of the HBs antigen-fixing plate was formed from the sample absorbent prepared by folding tissue paper (trade name: Kimwipe, manufactured by Jujo Kimberley) into 3 cm x 3 cm x 4 mm (thickness) The anti-HBs antibody positive blood brought into contact with (the left end side surface of the plate-like body in FIG. 5A) and brought into contact with the band-shaped covering portion was absorbed and removed.
Next, the HBs antigen-immobilized particle dispersion prepared in (2) above is placed using a pipette in the vicinity of the boundary between the covered portion and the uncovered portion of the HBs antigen-immobilized plate, and the belt-like covered portion Contact.
Thereafter, the HBs antigen on the bottom surface of the groove of the plate-like body is fixed with the side surface direction in which the band-shaped covering portion of the plate-like body is formed facing downward (the arrow direction in FIG. 5 is down). The HBs antigen-immobilized plate was tilted at an angle of about 60 ° so that the HBs antigen-immobilized particles moved from the non-exposed portion to the partial direction where the HBs antigen on the bottom surface of the groove was immobilized (the arrow direction in FIG. 5).
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.
[0096]
Example 3
(Measurement of E. coli O157)
(1) Preparation of anti-E. Coli O157 antibody fixed plate
Acrylic resin extrusion plate (width 25 mm, depth 40 mm, thickness 1 mm) [made by Acrysanday] two acrylic extrusion plates (width 10 mm, depth 40 mm, thickness 0.5 mm) [Acrysandy] made 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. 5 was bonded with a solvent at intervals.
Anti-E. Coli O157: H7 antibody solution (made by Kirkegaard & Perry Laboratories, dissolved in 10 mM phosphate buffer pH 7 at a concentration of 5 μg / ml) from the left end of the bottom of the groove of this plate-like body to 10 mm to 10 mm 20 μl was brought into contact with a pipette 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.
[0097]
(2) Preparation of anti-E. Coli O157 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 antibody fixed particle dispersion was prepared.
[0098]
(3) Measurement of E. coli O157
100 μl of a sample prepared by adding E. coli O157: H7 (manufactured by Kirkegaard & Perry Laboratories) to the stool suspension covers and covers the groove of the anti-E. Coli O157 antibody-fixed plate prepared in (1) above. -A pipette was placed near the border of the unexposed part to bring it into contact with the strip-shaped covering part.
A sample absorbent prepared by folding tissue paper (trade name: Kimwipe, manufactured by Jujo Kimberley Co., Ltd.) into 3 cm × 3 cm × 4 mm (thickness) is formed on the side where the band-shaped coating portion of the anti-E. Coli O157 antibody-fixed plate is formed. The end sample (the left end side surface of the plate-like body in FIG. 5A) was brought into contact, and the sample brought into contact with the strip-shaped covering portion was absorbed and removed.
Next, the anti-E. Coli O157 antibody-immobilized particle dispersion prepared in (2) above is placed using a pipette near the boundary between the covered portion and the uncovered portion of the anti-E. Coli O157 antibody-fixed plate. And contacted with the belt-shaped covering portion.
Thereafter, the anti-E. Coli O157 antibody on the bottom surface of the plate-like groove is fixed so that the anti-E. Coli O157 antibody-fixed particles move 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 non-exposed portion in the direction of the portion where the anti-E. Coli O157 antibody on the bottom of the groove was fixed (arrow direction in FIG. 5).
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.
[0099]
Example 4
(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.
The end on the side where the band-shaped covering portion of the anti-HBs antibody-fixing plate was formed from the sample absorbent prepared by folding tissue paper (trade name: Kimwipe, manufactured by Tokuma Kimberley) into 3 cm x 3 cm x 4 mm (thickness) The HBs antigen positive blood brought into contact with the part (the left end side surface of the plate-like body in FIG. 5A) and brought into contact with the band-shaped covering part was absorbed and removed.
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.
The end on the side where the band-shaped covering portion of the anti-HBs antibody-fixing plate was formed from the sample absorbent prepared by folding tissue paper (trade name: Kimwipe, manufactured by Tokuma Kimberley) into 3 cm x 3 cm x 4 mm (thickness) And the anti-HBs antibody solution brought into contact with the band-shaped coating part was absorbed and removed.
Further, the anti-HBs antibody-immobilized particle dispersion prepared in (2) of Example 1 is placed using a pipette near the boundary between the covered portion and the uncovered portion of the anti-HBs antibody-fixed plate. And contacted with the belt-shaped covering portion.
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 particle distribution state on the surface of the plate-like body was confirmed. As a result, no particle image as shown in FIG. 5C was observed. This confirmed that the HBs antigen was truly present in the sample.
[0100]
Example 5
(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.
The end on the side where the band-shaped covering portion of the anti-HBs antibody-fixing plate was formed from the sample absorbent prepared by folding tissue paper (trade name: Kimwipe, manufactured by Tokuma Kimberley) into 3 cm x 3 cm x 4 mm (thickness) The HBs antigen positive blood brought into contact with the part (the left end side surface of the plate-like body in FIG. 5A) and brought into contact with the band-shaped covering part was absorbed and removed.
Next, an anti-HBs antibody is added to the anti-HBs antibody-fixed particle dispersion prepared in (2) of Example 1 so as to be 0.1 mg / ml, and the anti-HBs antibody-added anti-HBs antibody-fixed particle dispersion is added to the anti-HBs antibody-fixed particle dispersion. The anti-HBs antibody-fixed plate from which HBs antigen-positive blood was absorbed and removed was placed using a pipette in the vicinity of the boundary between the covered portion and the uncovered portion of the plate and brought into contact with the strip-shaped covered portion.
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 particle distribution state on the surface of the plate-like body was confirmed. As a result, no particle image as shown in FIG. 5C was observed. This confirmed that the HBs antigen was truly present in the sample.
[0101]
【The invention's effect】
According to the measurement method of the present invention, when a test substance in a sample is measured visually or using an instrument using a specific binding reaction, in a sample such as a blood sample containing a substance that hinders determination of red blood cells and the like. In addition, the presence or absence of the test substance can be reliably determined without being disturbed.
Further, according to the measurement method of the present invention, the presence or absence of the test substance in the sample is not determined based on 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 gather at the end of the surface of the carrier. Alternatively, since the determination is made based on whether the particles are collected on the portion of the carrier surface coated with the specific binding substance, 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 measurement method of the present invention, the sensitivity can be easily changed by changing the amount of the specific binding substance immobilized on the surface of the particles and / or the carrier, so that the efficiency is high. Therefore, even when the concentration of the test substance is low, the presence or absence of the test substance can be easily determined.
Furthermore, according to the measurement method of the present invention, the presence or absence of the test substance in the sample is usually determined in a short time, specifically about 20 seconds to 10 minutes, depending on the type of the test substance. For example, in the case of HBs antigen, measurement is possible within 3 minutes even at a concentration of about 1 ng / ml.
Furthermore, according to the measurement method of the present invention, it is possible to simultaneously measure a plurality of test substances in a sample.
Moreover, according to the 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 diagram showing an agglomerated image when a test substance in a sample is measured by an indirect agglutination measurement method.

Claims (9)

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, a step of removing the sample brought into contact with the surface so as to move over a portion of the surface covered with the specific binding substance, and a specific to the test substance specific binding steps to the same specific binding substance fixed to the carrier a binding agent or a different specific binding substance is immobilized particles into contact with the surface to remove the sample, and the particles said surface comprises the step of moving the portion covered by the portion not covered by the material, put a portion covered with the portion not covered by a specific binding agent of the surface And judging the presence or absence of the test substance from the distribution of the particle, 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. A step of contacting, a step of removing the mixture of particles and sample brought into contact with the surface so as to move over a portion of the surface coated with the specific binding material, and a specific binding material for the test substance. step causes the specific binding substance and the same or different specific binding substance fixed to the carrier there is secured the particles into contact with the surface, and the particles on the specific binding substance of said surface Ri comprises a step of moving from the uncoated portion to the portion covered, from distribution of the particles in specific binding substances by portion covered with the portion not covered the surface of the test substance A method for measuring a test substance in a sample, characterized by determining the presence or absence. The test substance in the sample according to claim 1 or 2 , wherein the carrier is a container having at least one recess having a flat bottom surface, and the flat bottom surface is partially covered with a specific binding substance. Measuring method. The method for measuring a test substance in a sample according to claim 1 or 2 , wherein the carrier is a plate-like body, and the surface thereof is partially coated with a specific binding substance. The test substance is an antigen, the specific binding substance is an antibody, the measurement method of the analyte in the sample according to any one of claims 1-4. The method for measuring a test substance in a sample according to any one of claims 1 to 4 , wherein the test substance is an antibody, and the specific binding substance is an antigen or an antibody against the antibody. Samples absorber made of a water-absorbing material is contacted with the sample in contact with the surface to remove the sample, the measurement method of the analyte in the sample according to any one of claims 1-6. Moving the portion covered with the particles by the action of the magnet from the specific binding agent by uncoated portions of the surface, the test of the sample according to any one of claims 1-7 Method for measuring substances. Moving the particles by tilting the carrier portion covered by specific binding substances by uncoated portions of said surface, analyte in a sample according to any one of claims 1-7 Measuring method.

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