JP6531307B2 - Solid phase reaction vessel and measuring method using the same - Google Patents

Description

  The present invention relates to a solid phase reaction vessel and a measurement method using the same.

  Conventionally, there is known a method for capturing an analyte contained in a sample using a specific binding reaction such as an antigen-antibody reaction, a sugar (chain) -lectin reaction, or a biotin-avidin reaction, among which measurement From the viewpoint of simplicity and the like, a method of completing the above reaction on a solid support is preferably used.

  For example, Patent Document 1 discloses that, in an analysis method using a porous matrix as a solid phase, a first substance that specifically reacts with a substance to be measured is bound to a filter made of glass fibers having a specific range of fiber diameter. A method for measuring a solid phase biological specific reaction in which the obtained porous matrix is used as a solid phase is described.

  Also, for example, Patent Document 2 uses a fibrous porous filter as a solid phase carrier, and the measurement sensitivity at the nanogram level per 1 ml is very short in the time required for conventional immunoassays. A method of measuring a biologically active sample substance based on the immunological assay that can be obtained is described.

  In the prior art cited as the above prior art document, a porous matrix (filter) is cut out in a circular shape having a diameter of about 5 mm, set on the opening side of a container having an opening, and olefin non-woven fabric, cellulose Etc. are sequentially laminated, and a reaction vessel provided with a bottom plate as needed is used.

  The first substance that specifically binds to the measurement target is immobilized on the porous matrix through the opening, and from the top of the porous matrix, a sample containing the measurement target, a detectable signal generating substance Measurement that remained on the porous matrix by sequentially supplying a second substance that specifically reacts with the measurement target or the first substance, and the washing solution, and measuring the signal emitted from the signal generation substance. It determines the quantity of the object.

Unexamined-Japanese-Patent No. 4-318462 JP, 2010-44083, A

  However, in the conventional reaction container, the diameter of the opening is relatively large at about 5 mm, and the amount of sample liquid and washing solution supplied through the opening needs at least 20 μl to 100 μl. As described above, when the amount of the liquid supplied through the opening is large, it is not possible to proceed to the next operation until the supply liquid is completely absorbed by the absorption layer, so the measurement object remaining on the porous matrix It takes time to determine the quantity of goods. In addition, when the liquid volume of the sample liquid containing the measurement target is large, the physical burden on the subject who provides it also becomes large.

  In addition, for example, when an allergen that is a causative agent of an allergic disease is specified, the number of objects to be measured (the number of samples) inevitably increases. In fact, in medical institutions where a subject suspected of allergic disease is diagnosed, it is required to provide a prompt diagnostic material using a very small amount of sample liquid in consideration of the physical burden, temporal burden, etc. of the subject. The prior art has not been satisfactory.

  The present invention has been made in view of such circumstances, and it is an object of the present invention to make it possible to use a very small amount of sample solution necessary for measurement, and to obtain a solid phase reaction capable of rapidly obtaining measurement results. It is providing a container and a measurement method using the same.

In order to solve the above-mentioned subject, the solid reaction vessel concerning the present invention is formed as a taper shape in the approximate center part of a top face part, and has an upper lid member which has an opening of diameter 1.5 mm or less , and the above-mentioned upper lid member A lower lid member which is formed to be fittable and has a ventilating portion at the bottom, an absorbing member housed in an internal space formed by fitting the upper lid member and the lower lid member, and the absorbing member And a filter member provided on the end face on the side of the upper lid member, wherein the exposed surface of the filter member through the opening is a solid phase reaction field, and the upper lid member and the lower lid member are fitted The outer shape formed by fitting is characterized by being a shallow cylindrical shape .

In the measurement method using the solid phase reaction container according to the present invention, the upper lid member having an opening with a diameter of 1.5 mm or less and fitted in the upper lid member is formed in a tapered shape substantially at the center of the top surface portion. A lower cover member having a ventilating portion at the bottom, an absorption member accommodated in an internal space formed by fitting the upper cover member and the lower cover member, and the absorption member Using a solid-phase reaction vessel comprising a filter member provided on the end face on the upper lid member side , wherein the outer shape formed by fitting the upper lid member and the lower lid member is a shallow-bottomed cylindrical shape, A first binding step of binding a substance to be measured on the exposed surface of the filter member through an opening, and a labeled substance having a specific binding ability to the substance to be measured and labeled with a signal generating substance The substance to be measured A second coupling step of coupling, is characterized in that it comprises a measuring step of measuring the signal generated by the signal generating agents.

  ADVANTAGE OF THE INVENTION According to this invention, the sample liquid required for measurement can be made very small quantity, and the solid-phase reaction container which can obtain a measurement result rapidly can be provided, and the measurement method using the same.

It is a perspective view explaining the external appearance of the solid phase reaction container concerning this embodiment. They are a top view (a) and a bottom view (b) of a solid reaction vessel. It is sectional drawing for demonstrating each member which comprises a solid-phase reaction container. It is sectional drawing for demonstrating the internal structure of a solid-phase reaction container. It is sectional drawing explaining the state which laminated | stacked the solid phase transport container to the vertical direction. It is a flowchart explaining the process of fixing a ligand capture | acquisition substance to a solid-phase reaction container. It is a flowchart explaining the specific measuring method using the solid-phase reaction container 100 which fixed the ligand capture | acquisition substance. It is the figure which showed the measurement result at the time of selecting a standard solution (a) and cedar (b) as an allergen. It is a figure explaining the other form of a solid-phase reaction container. It is a figure explaining the other form of a solid-phase reaction container.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The present invention is not limited to the following description, and can be appropriately modified without departing from the scope of the present invention. Further, the drawings are schematic, and the ratio of each dimension may be different from the actual one. Specific dimensions and the like should be determined in consideration of the following description. Further, it is needless to say that parts having different dimensional relationships and ratios among the drawings are included.

  The solid-phase reaction container according to the present invention comprises an upper lid member having a tapered opening at a substantially central portion of a top surface portion, and a lower lid member formed to be engageable with the upper lid member and having a vent portion at the bottom. A filter member provided with an absorbing member accommodated in an internal space formed by fitting the upper lid member and the lower lid member, and a filter member provided on the upper lid member side end face of the absorbing member, and a filter member through an opening Is the reaction surface of the solid phase reaction. Details will be described below.

  FIG. 1 is a perspective view for explaining the appearance of a solid phase reaction container 100 according to the present embodiment. FIG. 2 shows a top view (a) and a bottom view (b) of the solid reaction vessel 100. The solid-phase reaction container 100 is formed into a nested shape, such as an upper lid 10 as an upper lid member formed in a shallow-bottomed substantially cylindrical shape, and a lower lid having an aperture slightly smaller than the aperture of the upper lid 10. It is comprised by fitting with the lower cover 20 as a member. Then, as shown in FIG. 2A, a tapered opening 13 is formed in the annular portion 12 provided substantially at the center of the upper lid 10, and the measurement target is provided via the opening 13. A liquid such as a sample liquid containing a substance or a washing liquid can be introduced into the container. Further, as shown in FIG. 2B, the ventilation portion 22 is formed in the bottom portion 21 of the lower lid 20 to facilitate the flow of the liquid introduced through the opening portion 13 into the absorbing member. .

  FIG. 3 is a cross-sectional view for explaining each member constituting the solid-phase reaction container 100, and FIG. 4 is a cross-sectional view for explaining an internal configuration of the solid-phase reaction container 100. As described above, when the upper lid 10 and the lower lid 20 are fitted, the outer shape of the solid reaction vessel is formed. An engaging portion 16 engageable with an engaged portion 24 formed on the outer peripheral wall surface 23 side of the lower lid 20 is formed on the inner peripheral wall surface 15 side of the upper lid 10, and the engaged portion 16 is engaged with the engaged portion 16. The upper lid 10 and the lower lid 20 are fitted by engagement with the portion 24. The outer circumferential wall surface 23 of the lower lid 20 is formed extending from the pedestal 25 forming the bottom portion 21 in the fitting direction to the upper lid 10, and the outer circumferential wall surface 23 is an upper lid 10 when it is joined with the upper lid 10. Contact with the inner circumferential wall surface 15 of the The outer peripheral wall surface 14 of the upper lid 10 and the outer peripheral wall surface 26 of the pedestal 25 of the lower lid 20 are arranged to be the same wall surface when fitted, and the shape of the solid phase reaction container 100 after fitting is shown in FIG. It has a cylindrical shape with a shallow bottom as shown.

  Here, the dimensions of the solid-phase reaction vessel 100 shown in FIGS. 3 and 4 are preferably about 15 mm in diameter, about 5 mm in height, and 1.5 mm or less in diameter 13 a of the opening 13. In addition, the actual dimension of the solid-phase reaction container 100 is not limited to this, It can change suitably according to a use.

  By the way, the material used to manufacture the upper lid 10 and the lower lid 20 is not particularly limited as long as it does not allow the liquid to permeate and has non-adsorbability to proteins etc. For example, polyethylene, polycarbonate, Plastics such as polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, tetrafluoroethylene, polypropylene, unsaturated polyester, epoxy and the like can be used.

  Then, the absorbing member 40 is accommodated in a crushed state in the internal space 30 formed by the upper cover 10 and the lower cover 20 being fitted. Further, the filter member 50 is accommodated at the end face on the upper lid 10 side of the absorbing member 40 so that a part is exposed as the exposed surface 50a through the opening 13, and the exposed surface 50a is used as a reaction field for solid phase reaction. Be

  The material of the absorbent member 40 is, for example, polyester fibers such as polyethylene terephthalate and polyethylene telebutyrate, polyolefin fibers such as polyethylene and polypropylene, or composite fibers obtained by combining them, pulp fibers, cotton fibers, hemp fibers, etc. Fibers such as regenerated fibers such as vegetable fibers, silk fibers and rayon fibers, woven fabrics, non-woven fabrics, paper and the like can be used. In particular, it is preferable that the porous matrix is made of cellulose such as pulp fiber or rayon fiber as a main component, or polysaccharide such as acetate (acetylcellulose) produced by reacting wood pulp with acetic acid.

  The dimension of the absorbing member 40 is not particularly limited as long as it has a volume slightly larger than the volume of the internal space 30 formed of the upper lid 10 and the lower lid 20, and the shape thereof is also accommodated in the internal space 30 As long as it can maintain a crushed state (a state of being in contact with the inner peripheral wall surfaces of the upper lid 10 and the lower lid 20), it may not be cylindrical but rectangular.

  The material of the filter member 50 is not particularly limited as long as liquid can be passed through and the ligand capture substance is immobilized in the solid phase reaction and the subsequent measurement steps can be performed on the same solid phase. For example, a porous organic filter obtained by sinter molding of a uniform plastic powder such as low density polyethylene, high density polyethylene, polypropylene, polymethyl acrylate or polytetrafluoroethylene, or a metal such as stainless steel, nickel or aluminum Porous metal filters, porous inorganic filters formed by molding alumina, zirconia, silicon carbide, etc., membrane filters such as nitrocellulose, PVDF, cellulose acetates, nylons, etc. Filter paper made of cellulose fibers, glass fibers, etc. Can be used. Among these materials, it is preferable to use a glass fiber filter.

  The dimension of the filter member 50 is not particularly limited as long as the exposed surface 50a can be exposed through the opening 13. However, in order to equalize the pressure contact force received from the absorbing member 40 in a crushed state, It is preferable to make it circular shape which has an area substantially the same as the area of the bottom part 21 of the lower lid 20.

  The solid-phase reaction vessel 100 having the above-described configuration has a shallow cylindrical shape with a diameter of about 15 mm and a height of about 5 mm, and the diameter 13 a of the opening 13 is 1.5 mm or less. Then, the filter member 50 is accommodated in the internal space 30 in a state where the absorbing member 40 is crushed, and the end surface on the upper lid 10 side of the absorbing member 40 is partially exposed as the exposed surface 50 a through the opening 13. It is housed. The amount of liquid such as the sample liquid and the washing liquid to be supplied to the solid phase reaction vessel 100 through the opening 13 may be about 10 μl at the maximum, and the supplied liquid is absorbed quickly by the absorbing member 40 after passing through the filter member 50 As a result, processing relating to each step of the solid phase reaction can be performed rapidly.

  Furthermore, as shown in FIG. 3, in the pedestal portion 25 of the lower lid 20, a groove portion 27 capable of accommodating the opening 13 (annular portion 12) of the other solid phase reaction vessel 100 is formed. As a result, as shown in FIG. 5, a plurality of solid-phase reaction vessels 100 can be set in a chamber such as an automatic dispenser or a luminescence meter in a state where they are vertically stacked. It is possible to speed up. In addition, since the dimensions of the solid-phase reaction container 100 itself are very small, about 15 mm in diameter, the device dimensions of an automatic dispenser, a luminescence meter, etc. can be miniaturized, and even in a narrow space such as medical sites These devices can be installed.

  Next, a measurement method using the solid phase reaction container 100 according to the present embodiment will be described. In this measurement method, the solid-phase reaction vessel 100 is used to have a first binding step of binding the substance to be measured on the exposed surface of the filter member through the opening, and specific binding ability to the substance to be measured. And a second binding step of binding a labeled substance labeled with a signal generating substance to the substance to be measured, and a measuring step of measuring a signal generated by the signal generating substance.

  Then, in the first binding step, a ligand capture substance for capturing a ligand is immobilized on the exposed surface of the filter member through the opening, a target substance to be measured, and the target substance to be measured. The method includes a mixing step of forming a complex by mixing with a first substance having a specific binding ability and having a ligand introduced thereto, and a step of binding the complex by capture of the ligand via the ligand capture substance. The second binding step preferably includes the step of binding a labeled substance having a specific binding ability to a measurement target substance or a first substance and labeled with a signal generating substance.

  Here, the ligand is not particularly limited, but for example, peptides, polypeptides, proteins (enzymes, antibodies, antigenic proteins, glycoproteins, lipoproteins, avidin etc.), hormones Immune system modulators, vitamins, steroids, carbohydrates (eg, saccharides), glycolipids, nucleic acids (including single-stranded and double-stranded oligonucleotides), haptens, lectins, biotin, etc. Can. Among these, it is preferable to use biotin.

  The ligand capture substance may be any substance as long as it can capture the ligand introduced into the first substance, and can be appropriately selected according to the ligand. Examples of combinations with ligand-ligand capture agents include antigen-antibody, hapten-antibody, sugar-lectin, antibody-protein A / G, biotin-anti-biotin antibody and the like. And when using biotin as a ligand, it is preferable to use an anti-biotin antibody, avidin, streptavidin etc. as a ligand capture | acquisition substance.

  The ligand capture substance can be immobilized on the exposed surface 50 a of the solid reaction vessel 100 by physical adsorption or chemical bonding generally used. In this case, the ligand capture substance may be directly immobilized on the exposed surface 50a, or an antibody or the like that specifically binds to the ligand capture substance is immobilized on the exposed surface 50a as a spacer substance, and then the ligand is bound via the spacer substance. The capture substance may be immobilized.

  Examples of the signal generating substance include alkaline phosphatase, β-galactosidase, glucose oxidase, urease, creatine kinase, uricase, glucose-6-phosphate dehydrogenase, peroxidase and the like. Among these, it is preferable to use horseradish peroxidase. And, when the signal generating substance is a peroxidase such as horseradish peroxidase, the generated signal can be obtained as a colorimetric signal or a chemiluminescent signal depending on the substrate. As a substrate used for generating a colorimetric signal, for example, tetramethyl benzidine and its derivatives, o-phenylenediamine, triarylmethanes, imidazole leuco dyes and the like can be used. In addition, examples of generation of a chemiluminescence signal include acridinium salts, dioxetanes, luciferin, lucigenin, oxalyl chloride and the like. The signal generating substance is not limited to the above-mentioned substances used in the so-called enzyme antibody method, for example, radioactive isotopes used in autoradiography, gold colloids used in the gold colloid method, fluorescence It is also possible to use fluorochromes of FITC, RITC, CY, and Alexe type used for antibody method as a signal generating substance.

  Next, regarding the measurement method according to the present embodiment, an example in which the substance to be measured is allergen specific IgE will be specifically described. The allergen is not particularly limited. For example, house dust 1 (2), house dust mite, cypress, cypress, cypress, persimmon (genus), birch (genus), moth, ragweed, mugwort, alternaria, aspergillus, malacetia (Genus), cat (dandruff), dog (dandruff), cockroach, moth, inhalant system and other allergens such as latex, milk, egg white, opamucoid, rice, wheat (fruit), buckwheat, soybean, peanut, apple, kiwi Any allergen can be selected as long as it can be examined as an allergen test item at a medical institution or the like, such as food-based allergens such as sesame, beef, chicken, shrimp, crab, mackerel, salmon and tuna.

  FIG. 6 is a flow chart for explaining the process of immobilizing an anti-biotin antibody (goat) as a ligand capture substance on the solid-phase reaction container 100 (exposed surface 50a). First, in step S200, for example, 10 μl of a wetting solution such as a citrate buffer solution is supplied through the opening 13 of the unsensitized solid phase reaction container 100.

  After 10 μl of the wetting solution is absorbed by the absorbing member 40, 5 μl of anti-goat IgG antibody (donkey) solution is supplied as a spacer material, and the anti-goat IgG antibody is immobilized on the exposed surface 50a of the filter member 50 (step S201) .

  Next, 5 μl of an anti-biotin antibody (goat) solution as a ligand capture substance is supplied, and the anti-biotin antibody is immobilized via the anti-goat IgG antibody (step S202).

  Then, for example, after passing 10 μl of a protective solution such as a phosphate buffer containing bovine serum albumin or the like (step S203), the ligand capture substance is immobilized by drying by air drying or the like for about 1 hour.

  Immobilization of the ligand capture substance described in FIG. 6 may be performed immediately before the measurement of the measurement target, or the filter member 50 on which the ligand capture substance is immobilized is cut out and set in the solid phase reaction apparatus 100. It is also good.

  FIG. 7 is a flow chart for explaining a specific measurement method using the solid phase reaction container 100 on which an anti-biotin antibody is immobilized as a ligand capture substance. First, 5 μl of a sample (specimen) collected from a subject and 5 μl of a biotinylated anti-human IgE antibody or a biotinylated allergen solution as a first substance are subjected to liquid phase reaction for 3 minutes to form a complex (step S300).

  Next, 5 μl of the mixed solution prepared in step S300 is dispensed and supplied through the opening 13 of the solid reaction vessel 100 to perform a solid reaction for 1 minute (step S301).

  In step S302, solid-phase reaction is performed by supplying 5 μl of anti-human IgE antibody (mouse) solution as a labeling substance labeled with horseradish peroxidase (HRP) through the opening 13 of the solid-phase reaction container 100 I do.

  Then, for example, after washing three times with 5 μl of a washing solution containing a surfactant such as tween 20 (step S303), a substrate such as tetramethyl benzidine (TMB) is added, and the absorbance at 600 to 660 nm is measured. The amount of allergen-specific IgE contained is measured (step S304).

  As described above, assuming that the time required to measure the absorbance is, for example, one minute, according to the present embodiment, all the measurement operations are completed in about six minutes, so that the measurement result can be obtained quickly.

  FIG. 8 is a view showing measurement results when cedar was selected as the standard solution and the allergen in the measurement method according to the present embodiment. As shown in FIG. 8 (a), as the concentration of the standard solution changes (0 IU / ml to 100 IU / ml), a difference is observed in the color development of TMB, confirming that this measurement method is an effective measurement system. It was done. In addition, as shown in the measurement results when cedar is selected as the allergen in FIG. 8 (b), differences in the color development of TMB can be seen according to the amount of allergen (cedar) specific IgE contained in the sample, It was confirmed that this measurement method is an effective measurement system.

  As a mixing container for forming a complex by liquid phase reaction of the sample (specimen) collected from the subject and the first substance, for example, as shown in FIG. It is also possible to use one in which the containers 101 are juxtaposed.

  Further, as shown in FIG. 10, in order to realize the continuous processing of multiple samples, a plurality of solid phase reaction vessels 100 'according to the present embodiment are provided on the plate 102 continuously and configured like a microtiter plate shape. It is also possible. In this case, it is also possible to provide a cleaning solution well 103 for storing the cleaning solution, a protective solution well 104 for storing the protective solution, and the like on the plate.

  As described above, according to the present embodiment, it is possible to make the amount of sample liquid necessary for measurement a very small amount, and to provide a solid-phase reaction container capable of rapidly obtaining measurement results and a measurement method using the same. can do.

  In the description of the present embodiment, an example of allergen-specific IgE has been described as an object to be measured, but the present invention is not limited to this. For example, as an object to be measured, peptides, polypeptides, proteins (enzymes, antibodies other than IgE, antigenic proteins, glycoproteins, lipoproteins, avidin etc.), hormones, immune system modulators, It is also possible to use vitamins, steroids, carbohydrates (eg, saccharides), glycolipids, nucleic acids (including single-stranded and double-stranded oligonucleotides).

  By targeting the above-mentioned measurement target, for example, esophagus cancer, lung cancer, squamous cell carcinoma, small cell cancer, hepatocellular carcinoma, biliary tract cancer, prostate cancer, neuroblastoma, thyroid marrow Tumor markers such as cancer, breast cancer, stomach cancer, pancreatic cancer, colon cancer, cervical cancer, endometrial cancer, ovarian cancer, amebic dysentery, hepatitis E, influenza, West Nile fever, HIV infection・ AIDS, hepatitis A, echinococcosis, Ebola hemorrhagic fever, Yersinia infection, yellow fever, onchocerciasis, relapsing fever, scabies, Campylobacter infection, Q fever, rabies, bovine spongy encephalopathy, helminthiasis, cryptosporidiosis, Crimea Congo hemorrhagic fever, tuberculosis, coccidioidosis, cholera, dysentery, cyclosporidosis, salmonella infection, SARS, giardiasis, hepatitis C, diphtheria , Schistosomiasis, schistosomiasis, castorosis, renal symptomatic hemorrhagic fever, chicken pox, meningococcal meningitis, sexually transmitted disease, swallowing disease, tick-borne encephalitis, anthrax, chikungunya fever, Enteritis vibrio, enterohemorrhagic E. coli infection, typhoid fever, hand-foot-and-mouth disease, dengue fever, eczema, toxoplasmosis, avian influenza, trypanosomiasis, South American hemorrhagic fever, Japanese encephalitis, cystosis, norovirus infection, pneumococcal disease, tetanus , Bartonella related diseases, Hantavirus lung syndrome, hepatitis B, histoplasmosis, human papillomavirus infection, cutaneous larva transgression, filariasis, rubella, brucellosis, fecal worm disease, plague, polio, Marburg disease, measles, Malaria, Lyme disease, Lassa fever, Leishmaniasis, rickettsial infection, Rift Valley fever, Nasal fin, Legionellosis, Leptospirosis, Rotau Infection such as heart failure, myocardial necrosis / injury marker, myocardial stress marker, plaque destabilization marker, myocardial marker such as inflammation marker, or autoimmune hemolytic anemia, bullous pemphigoid, Goodpasture syndrome, Graves' disease, It can also be used as a simple diagnostic tool for autoimmune diseases such as Hashimoto's thyroiditis, multiple sclerosis, myasthenia gravis, pemphigus, malignant anemia, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, vasculitis and the like.

  Further, the solid-phase reaction container according to the present invention is excellent not only in ease of measurement but also in portability, so it can be used not only as a diagnostic tool for human diseases described above, but also sheep, pigs, goats, cattle, chickens , Ducks, turkeys, geese, pigtails, pheasants, etc. Edible livestock such as dogs, cats, hamsters, guinea pigs, parrots, parrots, tropical animals such as tropical fish, horses, camels, water buffalo, mules, donkeys, ponies, etc. The present invention is also applicable to simple outdoor inspections of labor livestock in Japan, and various pathogens owned by other wildlife.

  Although the so-called sandwich method has been described in the description of the present embodiment, the present invention is not limited to this, and for example, a direct adsorption method in which an anti-human IgE antibody or allergen is directly immobilized on a filter member It is needless to say that the present invention is also applicable to the present invention.

DESCRIPTION OF SYMBOLS 10 top cover 11 top surface part 12 ring part 13 opening part 13a aperture 14 outer peripheral wall surface 15 inner peripheral wall surface 16 engagement part 20 lower cover 21 bottom part 22 vent 23 outer peripheral wall surface 24 engaged part 25 pedestal 26 outer peripheral wall surface 27 groove 30 Internal space 40 Absorbing member 50 Filter member 50a Exposed surface 100, 100 'Solid phase reaction vessel 101 Mixing vessel 102 Plate 103 Cleaning solution well 104 Protective solution well

Claims (16)

An upper lid member formed as a tapered shape at a substantially central portion of the top surface portion and having an opening having a diameter of 1.5 mm or less ;
A lower lid member formed to be engageable with the upper lid member and having a vent at the bottom thereof;
An absorbing member housed in an internal space formed by fitting the upper lid member and the lower lid member;
And a filter member provided on the end face on the upper lid member side of the absorbing member,
The exposed surface of the filter member through the opening is used as a solid reaction field ,
A solid-phase reaction vessel characterized in that an outer shape formed by fitting the upper cover member and the lower cover member is a shallow bottomed cylindrical shape . The solid-phase reaction vessel according to claim 1, wherein the absorbing member is accommodated in the inner space in a crushed state. The filter member is glass fiber
The solid-phase reaction container according to claim 1 or 2, characterized in that An engaged portion engageable with an engaging portion formed on the inner peripheral wall of the upper cover member is provided on the outer peripheral wall of the lower cover member, and the engaged portion and the engaged portion engage with each other. That the upper lid member and the lower lid member fit together
The solid-phase reaction vessel according to any one of claims 1 to 3, characterized in that The outer peripheral wall of the lower lid member is formed to extend from a pedestal forming the bottom.
The solid-phase reaction vessel according to any one of claims 1 to 4, characterized in that The pedestal may include a groove capable of receiving the opening of the upper lid member.
The solid-phase reaction vessel according to claim 5, characterized in that When the upper lid member and the lower lid member are fitted, the outer peripheral wall surface of the upper lid member and the outer peripheral wall surface of the pedestal portion are disposed on the same wall surface
The solid reaction vessel according to claim 5 or 6, characterized in that The absorbent member is a porous matrix comprising polysaccharides
The solid-phase reaction container according to any one of claims 1 to 7, characterized in that The polysaccharide is acetate
The solid-phase reaction vessel according to claim 8, characterized in that An upper lid member formed as a tapered shape at a substantially central portion of the top surface portion and having an opening having a diameter of 1.5 mm or less;
A lower lid member formed to be engageable with the upper lid member and having a vent at the bottom thereof;
An absorbing member housed in an internal space formed by fitting the upper lid member and the lower lid member;
And a filter member provided on the end face on the upper lid member side of the absorbing member,
Using a solid-phase reaction vessel whose outer shape formed by fitting the upper lid member and the lower lid member is a shallow-bottomed cylindrical shape,
Bonding a substance to be measured on the exposed surface of the filter member through the opening;
A second binding step of binding a labeled substance having a specific binding ability to the substance to be measured and labeled with a signal generating substance to the substance to be measured;
And measuring the signal generated by the signal generating substance.
Measuring method characterized by An immobilization step of immobilizing a ligand capture substance that captures a ligand on the exposed surface of the filter member through the opening;
A mixing step of forming a complex by mixing the substance to be measured and a first substance having a specific binding ability to the substance to be measured and to which the ligand is introduced;
Binding the complex by capture of the ligand via the ligand capture agent.
The second binding step includes the step of binding a labeled substance having a specific binding ability to a substance to be measured or the first substance and labeled with a signal generating substance.
The measurement method according to claim 10, characterized in that The combination of ligand-ligand capture agent is any of antigen-antibody, hapten-antibody, sugar-lectin, antibody-protein A / G, biotin-anti-biotin antibody, biotin-avidin, or biotin-streptavidin
The measurement method according to claim 11, characterized in that The first substance is an allergen into which biotin is introduced as the ligand
The measurement method according to claim 12, characterized in that The first substance is an anti-IgE antibody into which biotin is introduced as the ligand
The measurement method according to claim 12, characterized in that The labeling substance is an antibody against the substance to be measured or the first substance
The measurement method according to any one of claims 11 to 14, characterized in that The signal generating substance has peroxidase activity
The measurement method according to any one of claims 10 to 15, characterized by