WO2011111108A1 - Instrument pour le dosage immunologique lié à une enzyme et dosage immunologique lié à une enzyme - Google Patents

Instrument pour le dosage immunologique lié à une enzyme et dosage immunologique lié à une enzyme Download PDF

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Publication number
WO2011111108A1
WO2011111108A1 PCT/JP2010/001757 JP2010001757W WO2011111108A1 WO 2011111108 A1 WO2011111108 A1 WO 2011111108A1 JP 2010001757 W JP2010001757 W JP 2010001757W WO 2011111108 A1 WO2011111108 A1 WO 2011111108A1
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WIPO (PCT)
Prior art keywords
region
substrate
developing solution
enzyme
developing
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PCT/JP2010/001757
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English (en)
Japanese (ja)
Inventor
岸岡洋
深澤眞
小林行治
高山勝好
Original Assignee
Ｄｉｃプラスチック株式会社
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Priority to PCT/JP2010/001757 priority Critical patent/WO2011111108A1/fr
Publication of WO2011111108A1 publication Critical patent/WO2011111108A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • the present invention relates to an enzyme immunoassay instrument and a measurement method for simply measuring a test substance in a sample such as a biological component or a drug in a specimen.
  • an instrument or method for easily examining and measuring whether or not antigens such as proteins, biological components, drugs, etc. are contained in a sample using an immune reaction it is sent by capillary action using an enzyme as a labeling substance.
  • an enzyme as a labeling substance.
  • a measurement method in which a specimen, an enzyme labeling reagent, and a substrate are reacted on a liquid-based development substrate.
  • a developing solution supply zone is provided on the upstream side of a developing device that can be infused by capillary action, and the developing solution supply zone includes an enzyme inhibitor.
  • the developing solution can be supplied from a developing solution tank that stores the developing solution to which is added.
  • a substrate zone having a dry substrate is provided at the lower end of the developing solution supply zone.
  • a specimen spotting zone for spotting a specimen on the downstream side of the substrate zone in the developing device, and in the vicinity of the specimen spotting zone, the antigen to be detected in the specimen or the antibody or antigen that reacts with the antibody, etc.
  • an enzyme-labeled reagent zone in which an immunoreactive substance is enzyme-labeled and spotted with an enzyme reagent.
  • a detection zone is provided on the downstream side of the enzyme labeling reagent zone, and an absorption pad for absorbing a developing solution that has not been trapped in the detection zone is provided on the downstream side thereof.
  • the specimen is added to the specimen spotting zone, the developing solution is supplied from the developing solution tank to the developing solution supply zone, and the substrate is dissolved in the substrate zone in the developing solution supply zone.
  • the added specimen reacts with the reagent in the enzyme labeling reagent zone, and the specimen that has reacted with the reagent is flowed to the developing solution containing the substrate flowing from the developing solution supply zone and reaches the detection zone.
  • the antigen or antibody in the sample is trapped by the antigen or antibody immobilized on the detection zone, the antigen or antibody in the sample is bound to the enzyme labeling reagent. stay.
  • the enzyme labeled reagent that binds to the antigen or antibody trapped in the detection zone reacts with the substrate in the developing solution to develop color. To do. Therefore, whether or not the antigen or antibody to be detected is contained in the sample can be determined by whether or not color is developed in the detection zone. Moreover, it is disclosed that the enzyme reaction is inhibited by the enzyme inhibitor contained in the developing solution, and color development due to the reaction stops after a predetermined time.
  • the specimen is dropped on the specimen spotting zone of the developing device of the test piece, the enzyme-labeled reagent and the specimen are reacted, and after a certain time has elapsed since the reaction has progressed.
  • the developing solution needs to be transferred to the detection zone in the developing substrate.
  • the reaction solution of the specimen and the enzyme labeling reagent is transferred not only downstream but also upstream by capillary action of the developing device. For this reason, before supplying the developing solution, the reaction solution transferred to the upstream side reacts with the substrate attached to the pad of the substrate zone provided at the downstream end of the developing solution supply zone.
  • the present invention aims to provide an enzyme immunoassay instrument and an enzyme immunoassay method that can reliably perform measurement by reaction without the need for time management after addition of a sample in enzyme immunoassay. To do.
  • the present invention provides the enzyme immunoassay device of the following first aspect and the enzyme immunoassay method of the second aspect.
  • the first aspect of the present invention is A development base material that can be infused by capillary action, a development liquid region that is separated so as to be in contact with the development base material and includes a substrate, and a development liquid that can be supplied to the development liquid region,
  • the development substrate is provided with a sample addition region for adding a sample and an enzyme labeling reagent region, and a detection region provided on the downstream side of the sample addition region and the enzyme labeling reagent region,
  • An enzyme immunoassay instrument comprising the developing solution region and the developing solution provided upstream of the specimen addition region and the enzyme labeling reagent region of the developing substrate.
  • the enzyme immunoassay device according to (1) it is preferable that a substrate is provided in a downstream portion of the developing solution region.
  • the developing solution does not contain an enzyme inhibitor.
  • the developing solution region has a region containing the substrate and a region containing an enzyme inhibitor upstream thereof.
  • the enzyme immunoassay device according to (1) to (4) above includes a developing solution storage tank that stores a developing solution in the vicinity of the developing solution region, and the developing solution storage tank transfers the developing solution region to the developing solution region. It is preferable to supply a developing solution.
  • the developing solution reservoir is provided with a sealing film for sealing the developing solution, and the developing solution is opened in the developing solution region by opening the sealing film. It is preferable that an operation unit for supplying the liquid is further provided.
  • the enzyme immunoassay device according to (6) is provided with an operation unit for bringing the developing solution region into contact with the developing substrate in conjunction with opening of the sealing film of the developing solution storing unit by the operation unit. It is preferable that
  • the enzyme immunoassay method according to the present invention is the enzyme immunoassay method using a development base material that can be infused by capillary action.
  • a development liquid region containing a substrate is provided separately from the development base material. Is added onto the developing substrate to react with the enzyme labeling reagent in the enzyme labeling reagent region, and then a developing solution containing a substrate is supplied to the developing substrate through the developing solution region.
  • the second aspect of the present invention is (8) In an enzyme immunoassay method using a development substrate that can be infused by capillary action, a sample addition region and an enzyme labeling reagent region to which a sample is added, and provided downstream of these sample addition region and enzyme labeling reagent region A step of preparing a development base material capable of being infused by capillary action, provided with a detection region, a step of providing a development liquid region containing the substrate separately from the development base material, and adding a specimen to the specimen of the development base material And a step of reacting with the enzyme labeling reagent in the enzyme labeling reagent region, and after the reaction, a portion located on the upstream side of the specimen addition region and the enzyme labeling reagent region of the developing substrate and a developing solution region. And a step of supplying a developing solution containing a substrate to a developing substrate through a developing solution region.
  • the developing solution region containing the substrate is maintained in a state separated from the developing substrate. For this reason, it is possible to prevent the reaction solution of the sample and the enzyme labeling reagent from reacting with the substrate upstream of the development substrate before the substrate and the enzyme trapped in the detection region react with each other. It is possible to reliably perform the reaction. Therefore, in the enzyme immunoassay, it is not necessary to strictly control the time for the developing solution to develop so that the reaction solution does not develop upstream after the sample is added, and the measurement is simple and easy.
  • FIG. 2 is a perspective view of the lower housing excluding the development base material. It is a top view of an upper case.
  • FIG. 3B is a central longitudinal sectional view taken along line AA of the upper housing in FIG. 3A. It is a perspective view which shows the back surface of an upper housing
  • Fig. 4 shows protrusions according to examples. It is a figure which shows the modification of a protrusion. It is a figure which shows the modification of a protrusion. It is the disassembled perspective view which isolate
  • the present invention is not limited to these examples.
  • a sample is added to the sample addition region and reacts with the enzyme labeling reagent in the enzyme labeling reagent region, and this reaction solution is not only on the downstream side but also on the upstream side of the development substrate due to capillary action. Flowing.
  • the reaction solution developed upstream does not react with the substrate.
  • the developing solution region is provided with a substrate at a downstream portion thereof.
  • the developing solution is supplied to the developing solution region, the substrate contained on the downstream side of the developing solution region is dissolved in the developing solution and transferred to the developing substrate.
  • a specimen is added to the development substrate and reacts with the enzyme labeling reagent in the enzyme labeling reagent region, and this reaction solution is not only on the downstream side of the development substrate but also on the upstream side by capillary action. Also expand.
  • the developing solution region is not in contact with the developing substrate, the reaction solution does not react with the substrate.
  • the developing solution is supplied to the developing substrate through the developing solution region containing the substrate. Accordingly, the substrate is supplied to the detection region, and the enzyme labeling reagent trapped in the detection region reacts with the substrate.
  • the substrate develops color.
  • the developing solution may not contain an enzyme inhibitor. Since the enzyme immunoassay by the enzyme immunoassay instrument and the enzyme immunoassay method of the present invention can be processed, measured and observed in a short time, there is no problem even if the color development is not stopped by the enzyme inhibitor.
  • the developing solution region may have an area containing the substrate and an area containing the enzyme inhibitor upstream of the area containing the substrate. The sample is added onto the developing substrate and reacted with the enzyme labeling reagent in the enzyme labeling region, and then the developing solution is supplied to the developing solution region containing the substrate and the developing solution containing the substrate is supplied to the developing substrate.
  • the supplied developing solution develops downstream including the substrate.
  • the supplied developing solution flows to the upstream side of the developing substrate to dissolve the enzyme inhibitor in the area including the enzyme inhibitor, and as a result, the developing solution containing the enzyme inhibitor flows to the downstream side.
  • the development solution containing the enzyme inhibitor flows in to stop the development of the substrate.
  • the enzyme immunoassay instrument may include a developing solution storage tank that stores the developing solution in the vicinity of the developing solution region, and supply the developing solution from the developing solution storage tank to the developing solution region.
  • a developing solution storage tank that stores the developing solution in the vicinity of the developing solution region, and supply the developing solution from the developing solution storage tank to the developing solution region.
  • the developing solution storage tank may be provided with a sealing film for sealing the developing solution, and further provided with an operation unit for supplying the developing solution to the developing solution region by opening the sealing film. Also good.
  • the sealing film of the developing solution reservoir is broken and the developing solution can flow out to the developing solution region.
  • an operation unit that brings the developing solution region into contact with the developing substrate may be provided in conjunction with the opening of the sealing film of the developing solution storing unit by the operation unit.
  • the developing solution region can be brought into contact with the developing substrate in conjunction with the opening operation of the operation unit, so that the developing solution flowing out from the developing solution storage tank dissolves the substrate by passing through the developing solution region, and the developing solution region From the above, the developing solution containing the substrate can be transferred to the aforementioned developing substrate.
  • FIG. 1 is an exploded perspective view of an enzyme immunoassay instrument according to an embodiment of the present invention separated into an upper housing and a lower housing
  • FIG. 2 is a perspective view of the lower housing
  • FIGS. 3A and 3B are plan views of the upper housing.
  • FIG. 4 is a perspective view showing the rear surface of the upper casing
  • FIG. 5 is an exploded view of the developing liquid storage tank in the upper casing shown in FIG. 4, and FIG. FIG.
  • FIG. 7 is a central longitudinal sectional view showing the enzyme immunoassay instrument in a state before operating the operating section
  • FIG. 8 is a central longitudinal sectional view of the operating section pressed.
  • 9A and 9B are longitudinal sectional views in the width direction showing states before and after operating the operation unit.
  • the enzyme immunoassay instrument 1 binds an antigen or antibody of a specimen to be added to an enzyme labeling reagent, traps the antigen or antibody bound to the enzyme labeling reagent with the antibody or antigen in the detection zone, Furthermore, the bound enzyme labeling reagent is reacted with the substrate, and the presence or absence of coloration of the substrate is confirmed in the detection zone to inspect and measure whether or not the antigen or antibody to be detected is contained in the sample.
  • It is an instrument. For example, it is used in a measurement method by immunochromatography.
  • an enzyme immunoassay instrument 1 is formed by fitting an upper housing 2 and a lower housing 3 that are made of, for example, a synthetic resin.
  • the upper housing 2 is formed by, for example, a substantially rectangular plate material having a step, and an operation unit 5 for cantilever operation is provided at one end of the housing.
  • an operation unit 5 for cantilever operation is provided at one end of the housing.
  • a loading hole 6 which is a sample loading portion
  • a detection window 7 for observing the reaction of the sample
  • an air hole 8 for evaporating excess developing liquid.
  • the operation unit 5 side is referred to as the upstream side and the other end in the longitudinal direction is referred to as the downstream side, depending on the infusion direction of the developing liquid 26 described later when viewed in the longitudinal direction. .
  • the lower housing 3 has, for example, a substantially rectangular shape that can be fitted into the upper housing 2 as shown in FIG.
  • a step portion 3a that is one step higher than the other regions is formed, and the inclined portions 10a and 10a are inclined from both ends in the width direction toward the center.
  • a groove portion 11 in which a plurality of grooves are formed is provided at the center lower portion. The groove of the groove portion 11 is formed so as to be inclined so that the developing liquid flowing in from the inclined portion 10a flows in the other end direction (downstream direction) of the lower housing 3 by capillary action.
  • a pedestal 12 is provided along the longitudinal direction at the bottom of the lower housing 3 adjacent to the downstream side of the groove 11, and a belt-shaped development base material 14 made of a pad member is installed on the pedestal 12 (see FIG. 1). ).
  • the developing substrate 14 may be made of any material that allows the developing solution to flow by capillary action. For example, nitrocellulose, sponge, water-absorbing nonwoven fabric, or the like is used. Note that cylindrical recesses 15 are planted on the peripheral wall of the lower housing 3 at predetermined intervals.
  • a developing solution pad 16 containing the substrate in a dry state is provided on the groove 11 as a developing solution region.
  • a part of the developing liquid pad 16 may overlap with a part of the developing base material 14 when viewed from above, that is, the free end of the developing liquid pad 16 is not in contact with the developing base material 14 from the step portion 3a. It extends.
  • the developing solution pad 16 preferably includes a substrate in a dry state on the downstream side (free end side) in the longitudinal direction. When the developing solution is introduced as described later, the developing solution contains the dissolved substrate. In a state of being included, it is adsorbed to the developing substrate 14.
  • the developing base material 14 is provided with a specimen addition region 17 below the specimen insertion hole 6 in the upper housing 2.
  • a labeling reagent pad 18 containing a large amount of a labeling reagent labeled with an enzyme or an immunoreactive substance such as an antigen that reacts with an antigen to be detected or an antibody is used as the enzyme labeling reagent region.
  • the labeling reagent pad 18 may be any material that absorbs water, for example, a sponge formed of a porous natural or synthetic polymer compound such as nitrocellulose, cellulose, and polyvinyl alcohol, a water-absorbing nonwoven fabric, and Filter paper or the like can be used.
  • an enzyme used as a labeling reagent to be attached can be arbitrarily selected, and any enzyme used for a known labeling can be used. Specific examples include peroxidase, alkaline phosphatase, ⁇ -D-galactosidase, glucose oxidase, luciferase, esterase, and ⁇ -D-glucuronidase. Preferred are peroxidase, alkaline phosphatase, and ⁇ -D-galactosidase, which can achieve more sensitive and stable detection. Such enzymes can be used alone or in admixture of two or more.
  • the substrate may be any substrate as long as it can react with the enzyme used for the labeling reagent and develop color.
  • chromogenic chemical substances such as chromogenic substrates, fluorescent substrates, and luminescent substrates can be used.
  • the enzyme is alkaline phosphatase, p-nitrophenyl phosphate, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), AS-TR phosphate and the like can be mentioned.
  • the enzyme is peroxidase, 2,2′-azino-bis (3-ethylbenzthiazoline) -6-sulfonic acid, o-phenylenediamine, 3,3 ′, 5,5′-tetramethylbenzidine (TMB) O-dianididine, 5-aminosalicyclic acid, 3,3′-diaminobenzidine, 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol and the like.
  • the application amount of the chromogenic chemical substance as the substrate is preferably about 0.1 to 10 mg per 1 cm 2 of the coating part from the viewpoint of clarifying the color development when BCIP is taken as an example. If the amount of the chromogenic substrate is low, the chromogenic intensity cannot be obtained. On the other hand, if the amount of the chromogenic substrate is large, the background becomes high and detection sensitivity cannot be obtained.
  • a detection area 19 is provided on the downstream side of the specimen addition area 17.
  • an antigen to be detected in the specimen or an antibody that reacts with the antibody, or an immunoreactive substance such as an antigen is held on the development substrate 14 by physical adsorption or chemical bonding.
  • the antigen or antibody in the specimen is trapped and retained by the antibody or antigen immobilized on the detection region 19, and the bound enzyme labeling reagent is also detected because the antigen or antibody in the specimen is bound to the enzyme labeling reagent.
  • the enzyme of the enzyme labeling reagent trapped in the detection region 19 reacts with the substrate in the developing solution.
  • an absorption pad 20 is disposed on the downstream side of the detection region 19, and things that are not trapped in the detection region 19, for example, an excess developing solution, a specimen, a labeling reagent, and the like are absorbed. Further, when the antibody or antigen to be detected is not contained in the specimen, trapping does not occur, and the moved labeling reagent, developing solution, etc. are absorbed by the absorption pad 20.
  • the developing solution stored in the absorbent pad 20 is dried through the air holes 8.
  • the operation portion 5 is cantilevered at one end 5a on a frame 22 formed on the main body of the upper housing 2 when not in use, and the other end is a thin rib 5b that can be easily broken. It is connected to the main body via. Both sides of the operation unit 5 are not connected to the frame 22. Therefore, when the operation unit 5 is pressed, the rib 5b is divided and inclined down onto the developing liquid pad 16 in the lower housing 3, and the state is maintained by the configuration described later.
  • FIG. 4 shows the back surface of the upper housing 2 that is obtained by turning the upper housing 2 upside down.
  • a reservoir 24 of a developing liquid 26 having a side wall 24a surrounding the operation unit 5 is formed on the back surface of the operation unit 5, and a substantially triangular pyramid-shaped protrusion 25 is provided in a protruding manner at the center as a slit. ing.
  • the operation portion 5 has a curved shape that is convex upward in the non-pressed state and a thin shape around the pressed portion that has the protrusion 25 on the back surface so that it can be curved downwardly depressed in the pressed state. (See FIGS. 8, 9A, and 9B).
  • the developing liquid 26 is stored in the storage tank 24 in an appropriate amount arbitrarily selected, for example, 200 to 400 microliters.
  • An aluminum seal or a polyethylene film sheet or the like is provided in a tension state, and the developing solution 26 is sealed in a liquid-tight manner.
  • the sealing method can be arbitrarily selected, and for example, thermal welding, ultrasonic welding, adhesion with an adhesive, and fitting with a molded product are used. Due to the presence of the sealing film 27, the developing liquid 26 does not leak downward even when the upper housing 2 holds the storage tank 24 downward.
  • the sealing film 27 is not limited to an aluminum seal or a polyethylene film sheet, and any material can be used as long as it is liquid-tight and can form a structure that can be easily broken.
  • various buffer solutions such as acetate buffer solution, borate buffer solution, Tris buffer solution, and ethanolamine buffer solution are used.
  • an operation piece 28 for pressing the free end of the developing solution pad 16 from above at a distance from the side wall 24 a on the developing substrate 14 side of the storage tank 24 of the upper housing 2, that is, the front side. Is provided.
  • the operating portion 5 When the operating portion 5 is pushed to divide the rib 5b and the operating portion 5 is inclined, the protrusion 25 tears the sealing film 27, and as a result, the developing solution pad 16 in which the developing solution 26 in the storage tank 24 is located below. Move up. Further, due to the inclination of the operation unit 5, the operating piece 28 pushes the tip of the developing solution pad 16 downward, and the tip of the developing solution pad 16 comes into contact with the developing substrate 14 (see FIGS. 6 and 8).
  • the stepped portion 3a is provided with a tapered portion 30, and the tapered portion 30 abuts against and locks the side wall 24a in a state where the storage tank 24 is inclined, and the user releases the finger from the operation portion 5. This prevents the storage tank 24 from returning to its original position (see FIGS. 1 and 2).
  • the protrusion 25 of the upper housing 2 is retracted above the lower end of the side wall 24a of the storage tank 24, and the operating piece 28 is from the lower end of the side wall 24a. Projects downward.
  • the operation piece 28 stops at the position where the developing liquid pad 16 is pressed against the developing base material 14 by the pressing of the operation unit 5, and the side wall 24 a of the storage tank 24 is also stopped and held. It is easy to tear the stop film 27. Further, pin portions 29 are planted at predetermined intervals along the peripheral wall on the back surface of the upper housing 2.
  • the enzyme immunoassay instrument 1 can be assembled by fitting the pin portions 29 of the upper housing 2 into the cylindrical recesses 15 of the lower housing 3.
  • the enzyme immunoassay instrument 1 of the present invention can be summarized as follows: the sample addition region 17 for adding the sample to the development base material 14 that can be infused by capillary action provided in the lower housing 3, and the sample addition
  • the structure has an enzyme labeling reagent pad 18 provided on the region 17 or upstream or downstream thereof, and a detection region 19 provided on the downstream side thereof.
  • a developing solution pad 16 containing a substrate separated from the developing substrate 14 is provided on the upstream side of the developing substrate 14.
  • a developing liquid storage tank and protrusions are provided on the back surface of the operation unit 5 provided in the upper casing 2, and an operating piece for bringing the developing liquid pad 16 into contact with the developing base material 14 is provided adjacent to the storage tank.
  • the developing liquid pad is brought into contact with the developing base material by the operating piece, the sealing film of the storage tank is ruptured by the protrusion, and the developing liquid is supplied to the developing liquid pad 16, and the developing including the substrate is performed. It is possible to cause the liquid to flow through the developing substrate 14.
  • the developing solution 26 does not contain an enzyme inhibitor, but may be included to stop color development. You may employ
  • Enzyme inhibitors are antagonistic enzyme inhibitors that bind to the substrate binding site of the enzyme instead of the substrate and reversibly inhibit the enzyme activity, or the structure of the enzyme molecule by binding to the enzyme at a site different from the substrate binding site. It is a non-antagonistic enzyme inhibitor that inhibits by altering.
  • examples of enzyme inhibitors include phosphate, phosphate (sodium phosphate, potassium phosphate, etc.), phosphate monoester, naphthol phosphate, glycerol phosphate, phenyl phosphate. And phosphoric acid compounds such as phosphoethanolamine, phosphorylcholine, phenanthroline, and glycholic acid, and chelating compounds such as ethylenediaminetetraacetic acid and ethyleneglycose bistetraacetic acid.
  • examples of the enzyme inhibitor include reducing agents such as ascorbic acid and azide compounds such as sodium azide and potassium azide.
  • ⁇ -D galactosidase When ⁇ -D galactosidase is used as an enzyme, lactose and the like can be mentioned as an enzyme inhibitor.
  • an acid or a base can be used as an enzyme inhibitor in order to shift the optimum pH of the enzyme in the detection zone after a predetermined time.
  • the enzyme immunoassay instrument 1 has the above-described configuration. Next, an enzyme immunoassay method using the enzyme immunoassay instrument 1 will be described.
  • the enzyme immunoassay instrument 1 is not used, as shown in FIGS. 7 and 9A, the developing solution 26 is sealed in the storage tank 24 on the back surface of the operation unit 5, and the developing solution pad 16 impregnated with the substrate is used. And are separated. The developing liquid pad 16 is also separated from the developing base material 14. From this state, the specimen is dropped from the introduction hole 6 onto the specimen addition region 17 of the development substrate 14.
  • the sample undergoes an antigen-antibody reaction with the enzyme labeling reagent of the labeling reagent pad 18 containing the enzyme labeling reagent, and the reaction solution of the sample and the enzyme labeling reagent is transferred from the sample addition region 17 to the developing substrate 14.
  • the reaction solution of the sample and the enzyme labeling reagent is transferred from the sample addition region 17 to the developing substrate 14.
  • Flows toward the detection region 19 on the downstream side by the capillary phenomenon and also flows to the upstream side and diffuses.
  • the enzyme in the reaction solution does not react with the substrate.
  • the operation part 5 of the enzyme immunoassay instrument 1 is pushed when a predetermined time elapses after the sample is dropped.
  • the time from dropping to pressing the operation unit can be determined in consideration of conditions and the like.
  • FIGS. 8 and 9B by pushing the operation portion 5, the rib 5b of the operation portion 5 is easily divided, and the operation portion 5 is inclined and pushed toward the developing solution pad 16 side with the one end portion 5a as a fulcrum. It is.
  • the operating piece 28 provided on the back surface of the operation unit 5 pushes the free end of the developing solution pad 16 to bring the developing substrate 14 into contact with the end.
  • the storage tank 24 on the back surface approaches or comes into contact with the inclined portions 10a and 10a, a part of the side wall 24a of the storage tank 24 is sandwiched and fixed between the pair of taper portions 30 and 30, and the operation unit 5 is Stop.
  • the center of the back surface of the pressed operation portion 5 is recessed inside the container, so that the downward convex portion 25 breaks through the sealing film 27 thermally welded to the peripheral wall 24a of the storage tank 24.
  • the developing solution 26 in the storage tank 24 flows out and is sucked into the developing solution pad 16. Even when a part of the developing solution 26 flows out from the storage tank 24 to both sides of the developing solution pad 16 at a position upstream of the stepped portion 3a, it is absorbed by the developing solution pad 16 by the inclined portion 10a and the groove portion 11.
  • the substrate contained in the developing solution pad 16 is dissolved in the developing solution 26 and sucked together with the developing solution 26 by the developing substrate 14. Then, the reaction solution of the sample and the enzyme labeling reagent reacted on the labeling reagent pad 18 reaches the downstream detection region 19 by the developing solution 26 supplied from the upstream side.
  • the antigen or antibody in the specimen is trapped by the antibody or antigen fixed and held in advance in the detection region. Since the antigen or antibody in the sample has reacted with the enzyme labeling reagent, the enzyme labeling reagent that has reacted with the antigen or antibody also remains in the detection region 19.
  • the enzyme trapped in the detection region 19 reacts with the substrate in the developing solution 26, and the substrate develops color.
  • the presence or absence of color development in the detection region 19 can be observed through the detection window 7 of the upper housing 2.
  • the developing liquid 26, the reaction liquid not trapped, and the like are absorbed by the absorption pad 20 on the downstream side.
  • the enzyme-labeled reagent is not trapped in the detection region 19, so that color development is not confirmed in the detection region 19, and the antigen, antibody, or enzyme-labeled reagent is not Absorbed by the absorbent pad 20.
  • the developing solution pad 16 containing the substrate is separated from the developing substrate 14.
  • the substrate is flowed together with the developing solution 26 to the developing substrate 14 via the developing solution pad 16, and the substrate is added to the specimen addition region 17, the enzyme labeling pad 18, and the detection region 19 of the developing substrate 14.
  • the developing liquid 26 containing is transferred.
  • the protrusion 25 provided on the back surface of the operation unit 5 has a pyramid shape such as a triangular pyramid as shown in FIG. 10A and has a structure in which the sealing film 27 is easily broken, but the protrusion 25 has such a shape. It is not limited to. Any shape can be applied as long as the sealing film such as the sealing film 27 can be easily torn by pressing force. For example, as shown in FIG. 10A
  • a protrusion 25A provided with a pointed head 25a that protrudes further sharply at the apex of the pyramid may be provided, or a protrusion 25B that intersects a triangular plate as shown in FIG. 10C. It may be a shape such as providing.
  • the enzyme labeling pad 18 in the development substrate 14 is provided on the specimen addition region 17. However, it may be provided upstream or downstream of the sample addition region 17 as long as it is upstream of the detection region 19 and downstream of the contact portion between the development liquid pad 16 and the development substrate 14. Further, in the above-described embodiment, the protrusion 25 that breaks the sealing film 27 of the developing liquid storage tank 24 and the like on the back surface of the operation unit 5 provided in the upper casing 2 and the developing liquid pad 16 are provided on the developing base material 14.
  • the operating piece 28 to be brought into contact with the actuator is interlocked by the tilting of the operation unit 5. However, you may make it operate
  • the enzyme immunoassay instrument 1 contains various measuring members such as the developing base material 14 and the developing solution pad 16 in a container composed of the upper housing 2 and the lower housing 3.
  • the upper housing 2 and the lower housing 3 are not necessarily provided. In this case, for example, as shown in FIG.
  • the developing base material 14 and the developing solution pad 16 are provided in a non-contact manner on a sheet-like substrate corresponding to the base 12 of the bottom plate of the lower housing 3, It may have a configuration in which the operation section 5 and the operation piece 28 for breaking the developing solution storage tank 24 are disposed.
  • an enzyme inhibitor that stops the color development by inhibiting the enzyme reaction in the detection region 19 is not used in order to check the presence or absence of the color development reaction in a short time.
  • the enzyme immunoassay instrument 1 may be stopped in a colored state using an enzyme inhibitor.
  • an enzyme inhibitor may be mixed in the developing solution 26.
  • the enzyme inhibitor may be contained in a portion upstream of the specimen addition region and / or the enzyme labeling reagent region in the development substrate and upstream of the portion of the development solution region containing the substrate. .
  • FIG. 11 a configuration in which a substrate and an enzyme inhibitor are added to the developing solution pad 16 will be described with reference to FIG. 11 as a modified example.
  • the same reference numerals are used for the same or similar members and parts as those in the above-described embodiment, and the description thereof is omitted.
  • a region 31 containing, for example, a dry substrate is provided in a part of the developing solution pad 16, for example, a central portion or a downstream portion in the longitudinal direction. Except for this area 31, the developing solution pad 16 contains no substrate.
  • the developing solution pad 16 is further provided with an area 32 containing, for example, a dry enzyme inhibitor, on the upstream side away from the area 31 containing the substrate.
  • the protrusion 25 is located at a position facing the area 31 containing the substrate or in the vicinity thereof.
  • the protrusion 25 that breaks the sealing film 27 is a region containing the substrate of the developing solution pad 16. While abutting at or near the region 31, it is spaced from the region 32 containing the enzyme inhibitor. Therefore, the developing solution flowing out from the hole of the broken sealing film 27 does not directly contact the area 32 containing the enzyme inhibitor. Therefore, the developing solution flowing out from the hole in which the sealing film 27 is broken flows downstream including the substrate dissolved in the region 31 including the substrate of the developing solution pad 16 and is moved by the working piece 28.
  • the developing solution that has flowed out to the developing solution pad 16 gradually develops to the upstream side by capillary action, the developing solution penetrates into the region 32 containing the enzyme inhibitor and the developing solution dissolves the enzyme inhibitor. Flows downstream with inhibitor. Therefore, after the sample, the enzyme label, and the substrate react and the substrate develops color, the developing solution containing the enzyme inhibitor flows with a delay and the color development is fixed. In this case, it is not necessary to consider the timing of supplying the developing solution to the developing solution pad 16, and the reaction of the substrate is stopped in a timely manner even if the enzyme immunoassay instrument 1 is left for a long time after pressing the operation unit 5. It is possible to make an accurate determination.

Abstract

L'invention concerne un instrument destiné à être utilisé dans un dosage immunologique lié à une enzyme, lequel instrument comprenant : un matériau de base de développement dans lequel un matériau fluide peut être infusé au moyen d'une action capillaire; une zone de solution de développement qui est suffisamment séparée du matériau de base de développement d'une manière accessible et qui contient un substrat; et une solution de développement qui peut être apportée à la zone de solution de développement. L'instrument est caractérisé en ce que sur le matériau de base de développement est formé une zone d'addition d'échantillon sur laquelle un échantillon peut être ajouté, une zone de réactif marqué par une enzyme et une zone de détection qui est située en aval de la zone d'addition d'échantillon et de la zone de réactif marqué par une enzyme, et la zone de solution de développement et la solution de développement sont disposées en amont de la zone d'addition d'échantillon et de la zone de réactif marqué par une enzyme dans le matériau de base de développement.
PCT/JP2010/001757 2010-03-11 2010-03-11 Instrument pour le dosage immunologique lié à une enzyme et dosage immunologique lié à une enzyme WO2011111108A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016536590A (ja) * 2013-11-12 2016-11-24 ボディテックメド インコーポレイテッドＢｏｄｉｔｅｃｈｍｅｄ． Ｉｎｃ 一体化された反応及び検出手段を備えるマルチウェルキュベット
WO2017104143A1 (fr) * 2015-12-18 2017-06-22 富士フイルム株式会社 Kit d'immunochromatographie
JPWO2016114122A1 (ja) * 2015-01-16 2017-10-26 富士フイルム株式会社 イムノクロマトグラフキット
US10384300B2 (en) 2015-12-18 2019-08-20 Fujifilm Corporation Method of manufacturing an immunochromatographic kit

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Publication number Priority date Publication date Assignee Title
JPH02138961A (ja) * 1988-05-19 1990-05-28 Chemtrak Inc 浸漬棒測定装置
JPH10300750A (ja) * 1997-04-23 1998-11-13 Fujirebio Inc 酵素免疫測定方法及び試験片
WO2007105680A1 (fr) * 2006-03-13 2007-09-20 Fujirebio Inc. Garniture d'absorption pour immunoassay, bande pour immunoassay et appareil d'immunoassay
JP2008539426A (ja) * 2005-04-29 2008-11-13 キンバリー クラーク ワールドワイド インコーポレイテッド アッセイ装置のための流量制御技術
WO2009119722A1 (fr) * 2008-03-28 2009-10-01 国立大学法人北海道大学 Anticorps monoclonal anti-(hémagglutinine du sous-type h5 du virus de la grippe de type a)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02138961A (ja) * 1988-05-19 1990-05-28 Chemtrak Inc 浸漬棒測定装置
JPH10300750A (ja) * 1997-04-23 1998-11-13 Fujirebio Inc 酵素免疫測定方法及び試験片
JP2008539426A (ja) * 2005-04-29 2008-11-13 キンバリー クラーク ワールドワイド インコーポレイテッド アッセイ装置のための流量制御技術
WO2007105680A1 (fr) * 2006-03-13 2007-09-20 Fujirebio Inc. Garniture d'absorption pour immunoassay, bande pour immunoassay et appareil d'immunoassay
WO2009119722A1 (fr) * 2008-03-28 2009-10-01 国立大学法人北海道大学 Anticorps monoclonal anti-(hémagglutinine du sous-type h5 du virus de la grippe de type a)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016536590A (ja) * 2013-11-12 2016-11-24 ボディテックメド インコーポレイテッドＢｏｄｉｔｅｃｈｍｅｄ． Ｉｎｃ 一体化された反応及び検出手段を備えるマルチウェルキュベット
JPWO2016114122A1 (ja) * 2015-01-16 2017-10-26 富士フイルム株式会社 イムノクロマトグラフキット
EP3246704A4 (fr) * 2015-01-16 2017-12-06 Fujifilm Corporation Kit d'immunochromatographie
WO2017104143A1 (fr) * 2015-12-18 2017-06-22 富士フイルム株式会社 Kit d'immunochromatographie
JPWO2017104143A1 (ja) * 2015-12-18 2018-09-27 富士フイルム株式会社 イムノクロマトグラフキット
US10384300B2 (en) 2015-12-18 2019-08-20 Fujifilm Corporation Method of manufacturing an immunochromatographic kit
US10520497B2 (en) 2015-12-18 2019-12-31 Fujifilm Corporation Immunochromatographic kit

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