WO2021025066A1 - 固相反応容器及びこれを用いた測定方法 - Google Patents

固相反応容器及びこれを用いた測定方法 Download PDF

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Publication number
WO2021025066A1
WO2021025066A1 PCT/JP2020/030018 JP2020030018W WO2021025066A1 WO 2021025066 A1 WO2021025066 A1 WO 2021025066A1 JP 2020030018 W JP2020030018 W JP 2020030018W WO 2021025066 A1 WO2021025066 A1 WO 2021025066A1
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Prior art keywords
substance
solid
phase reaction
liquid
reaction vessel
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PCT/JP2020/030018
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English (en)
French (fr)
Japanese (ja)
Inventor
昭雄 中原
Original Assignee
株式会社パートナーファーム
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Priority to CN202080055831.2A priority Critical patent/CN114207409A/zh
Priority to KR1020227005444A priority patent/KR20220041854A/ko
Priority to JP2021537350A priority patent/JPWO2021025066A1/ja
Publication of WO2021025066A1 publication Critical patent/WO2021025066A1/ja

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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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6402—Atomic fluorescence; Laser induced fluorescence
    • 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
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G01N33/5304—Reaction vessels, e.g. agglutination plates
    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0681—Filter
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/069—Absorbents; Gels to retain a fluid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0803—Disc shape
    • B01L2300/0806—Standardised forms, e.g. compact disc [CD] format
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/12—Specific details about materials

Definitions

  • the present invention relates to a solid phase reaction vessel and a measurement method using the same.
  • a method of capturing a substance to be detected contained in a sample on a porous matrix using a biologically specific reaction such as an antigen-antibody reaction, a sugar (chain) -lectin reaction, or a biotin-avidin reaction has been known.
  • a method of completing the above reaction on a solid phase carrier is preferably used from the viewpoint of ease of measurement and the like.
  • the measurement method using a solid phase carrier such as a porous matrix, combined with the concentration effect on the solid phase carrier, makes it possible to detect substances to be detected that are contained only in ultra-low concentrations at the nanogram level in the sample solution.
  • a solid phase carrier such as a porous matrix
  • a measurement procedure a complex consisting of a first physiologically active substance to which a ligand is bound, a measurement sample substance, and an enzyme-labeled second physiologically active substance is previously formed in a solution.
  • a technique is described in which the enzyme activity is measured by adding it to a porous filter to which a ligand trapping substance is bound.
  • the diameter of the opening formed as a solid-phase reaction field on the porous matrix is relatively large, about 5 mm, and the opening At least about 20 to 100 ⁇ l of the sample solution supplied via the above was required.
  • a very small amount of sample solution is taken into consideration in consideration of the physical burden and time burden of the subject.
  • the prior art has not been satisfactory, as the prompt provision of diagnostic materials using the above is required.
  • An upper lid member formed in a tapered shape at a substantially central portion of the surface portion and having an opening having a diameter of 1.5 mm or less, a lower lid member formed so as to be matable with the upper lid member, and having a ventilation portion at the bottom, and an upper lid member.
  • a filter member provided with a liquid absorbing member accommodated in an internal space formed by fitting the lower lid member and a lower lid member and a filter member provided on the upper lid member side end surface of the liquid absorbing member, and a filter member via an opening.
  • Patent Document 3 The exposed surface of the above is used as a solid phase reaction field, and the outer shape formed by fitting the upper lid member and the lower lid member is a shallow-bottomed cylindrical shape.
  • the present invention is an improved invention relating to the solid phase reaction vessel and the measurement method using the same solid reaction vessel proposed so far, and the subject of the present invention is the solid phase reaction vessel utilizing a biologically specific reaction and the measurement using the same.
  • the present invention is to provide a solid phase reaction vessel capable of providing stable measurement accuracy while achieving a minimum amount of sample solution and speeding up measurement, and a measurement method using the same.
  • the solid phase reaction vessel according to the present invention is formed so as to be able to be fitted with an upper lid member having a tapered opening at a substantially central portion of a top surface portion and the upper lid member, and at the bottom portion.
  • the lower lid member having a ventilation portion, the first filter member that captures the substance to be detected contained in the sample liquid supplied through the opening, and the upper lid member and the lower lid member are fitted to each other.
  • It is provided with a liquid absorbing member that is accommodated in the internal space formed by the above and absorbs the sample liquid that has passed through the first filter, and the sucking member is between the first filter member and the liquid absorbing member.
  • a second filter member having a surface having a wettability different from that of the liquid member surface is provided.
  • the measurement method using the solid phase reaction vessel according to the present invention has a specific binding ability to the substance to be detected and a ligand capable of binding to the ligand trapping substance immobilized on the surface of the first filter member.
  • a capture step of capturing the substance to be detected via the ligand-introducing substance into which the substance has been introduced, and a labeling substance having a specific binding ability to the substance to be detected and labeled with the signal-producing substance are used. It is characterized by comprising a measurement step for measuring a signal emitted by the labeling substance bound to the detection substance.
  • the solid-phase reaction vessel includes an upper lid member having a tapered opening at a substantially central portion of a top surface portion, a lower lid member formed so as to be matable with the upper lid member, and having a ventilation portion at the bottom.
  • the first filter member that captures the substance to be detected contained in the sample liquid supplied through the opening is housed in the internal space formed by fitting the upper lid member and the lower lid member, and the first one.
  • a second filter member is provided with a liquid absorbing member that absorbs the sample liquid that has passed through the filter, and has a surface different in wettability from the surface of the liquid absorbing member between the first filter member and the liquid absorbing member. It is characterized in that it is provided. This will be described in detail below.
  • FIG. 1 is a perspective view illustrating the appearance of the solid-phase reaction vessel 100 according to the present invention.
  • FIG. 2 is a top view (a) and a bottom view (b) of the solid phase reaction vessel 100.
  • the solid-phase reaction vessel 100 has an upper lid 10 as an upper lid member formed in a substantially cylindrical shape with a shallow bottom so as to be nested, and a lower lid formed so as to have a diameter slightly smaller than the diameter of the upper lid 10. It is configured by fitting the lower lid 20 as a member. Then, as shown in FIG. 2A, a tapered opening 13 is formed in the annular portion 12 provided at the substantially central portion of the upper lid 10, and is detected through the opening 13.
  • a sample liquid containing a substance, a cleaning liquid or other liquid can be introduced into the container. Further, as shown in FIG. 2B, a ventilation portion 22 is formed in the bottom portion 21 of the lower lid 20, so that the liquid introduced through the opening 13 can be easily passed through the liquid absorbing member. To do.
  • FIG. 3 is a cross-sectional view for explaining each member constituting the solid-phase reaction vessel 100
  • FIG. 4 is a cross-sectional view for explaining the internal configuration of the solid-phase reaction vessel 100.
  • the outer shape of the solid-phase reaction vessel is formed by fitting the upper lid 10 and the lower lid 20.
  • an engaging portion 16 that can be engaged with the engaged portion 24 formed on the outer peripheral wall surface 23 side of the lower lid 20 is formed, and is engaged with the engaging portion 16.
  • the upper lid 10 and the lower lid 20 are fitted by engaging with the portion 24.
  • the outer peripheral wall surface 23 of the lower lid 20 is formed so as to extend in the fitting direction from the pedestal portion 25 forming the bottom portion 21 to the upper lid 10, and the outer peripheral wall surface 23 is formed when the upper lid 10 is combined with the upper lid 10. It will face the inner peripheral wall surface 15 of.
  • the outer peripheral wall surface 14 of the upper lid 10 and the outer peripheral wall surface 26 of the pedestal portion 25 of the lower lid 20 are arranged so as to be the same wall surface at the time of fitting, and the shape of the solid phase reaction vessel 100 after fitting is shown in FIG. It has a shallow cylindrical shape as shown.
  • the pedestal portion 25 of the lower lid 20 is formed with a groove portion 27 capable of accommodating the opening portion 13 (annular portion 12) of the other solid-phase reaction vessel 100.
  • a plurality of solid-phase reaction vessels 100 can be vertically stacked and set in a chamber such as an automatic dispenser or a luminescence measuring device, so that it is possible to speed up the processing of multiple samples. Is.
  • the solid-phase reaction vessel 100 itself has a very small diameter of about 15 mm, the dimensions of devices such as an automatic dispenser and a light emission measuring device can be reduced, and even in a small space such as a medical site. These devices can be installed.
  • the dimensions of the solid-phase reaction vessel 100 shown in FIGS. 3 and 4 are such that the diameter is about 15 mm, the height is about 5 mm, and the diameter 13a of the opening 13 is 1.5 to 3.5 mm, which is 1.5 mm.
  • the following are preferred.
  • the actual size of the solid-phase reaction vessel 100 is not limited to this, and can be appropriately changed depending on the application.
  • the material used for producing the upper lid 10 and the lower lid 20 is not particularly limited as long as it does not allow liquid to permeate and has non-adsorption property to proteins and the like.
  • polyethylene polyethylene, polycarbonate, etc.
  • Plastics such as polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, ethylene tetrafluoride, polypropylene, unsaturated polyester, and epoxy can be used.
  • the liquid absorbing member 40 is housed in a crushed state in the internal space 70 formed by fitting the upper lid 10 and the lower lid 20. Further, the upper surface 60a of the second filter member 60 accommodates the first filter member 50 so that a part of the second filter member 60 is exposed as an exposed surface 50a through the opening 13, and the exposed surface 50a is a solid phase reaction. Used as a reaction field.
  • the material of the liquid absorbing member 40 includes, for example, polyester fibers such as polyethylene terephthalate and polyethylene terebutyrate, polyolefin fibers such as polyethylene and polypropylene, composite fibers obtained by combining these, pulp fibers, cotton fibers, and linen fibers. And other plant fibers, silk fibers, recycled fibers such as rayon fibers, woven fabrics, non-woven fabrics, paper and the like can be used.
  • the porous matrix is composed of cellulose-based materials such as pulp fibers and rayon fibers, and polysaccharides such as acetate (acetyl cellulose) produced by reacting wood pulp with acetic acid.
  • the size of the liquid absorbing member 40 is not particularly limited as long as it has a volume slightly larger than the volume of the internal space 70 formed from the upper lid 10 and the lower lid 20, and the shape thereof is also accommodated in the internal space 70. As long as it can be maintained in 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 be a rectangular parallelepiped shape instead of a cylindrical shape.
  • the material of the first filter member 50 is particularly limited as long as it can pass a liquid, immobilize the ligand trapping substance in the solid phase reaction, and perform the subsequent measurement steps on the same solid phase.
  • a porous organic filter obtained by sintering and molding a uniform plastic powder such as low-density polyethylene, high-density polyethylene, polypropylene, polymethylacrylate, and polyvinylidene fluoride, and metals such as stainless steel, nickel, and aluminum.
  • a porous metal filter formed of a porous inorganic filter formed of alumina, zirconia, silicon carbide, etc., a membrane filter of nitrocellulose, PVDF, cellulose acetates, nylons, etc., cellulose fibers, glass fibers, etc. Filter papers and the like can be used. Among these materials, it is preferable to use a glass fiber filter.
  • the dimensions of the first filter member 50 are not particularly limited as long as the exposed surface 50a can be exposed through the opening 13, but the liquid absorbing member 40 in the crushed state and the second filter 60
  • the material of the second filter member 60 is not particularly limited as long as it has a different wettability from the surface 40a of the liquid absorbing member 40, and is the same as that of the first filter member 50 or the liquid absorbing member 40. Equivalent or identical materials can be used.
  • the wettability referred to in the present invention means that when a sample liquid, a cleaning liquid or other liquid is applied to the exposed surface 50a of the first filter member 50 in contact with the surface 60a of the second filter member 60 through the opening 13. , It shows the size of how the liquid spreads.
  • high wettability means that when a liquid is applied to the exposed surface 50a, the liquid spreads over the entire exposed surface 50a (so-called hydrophilicity), and conversely, low wettability means that the exposed surface 50a
  • low wettability means that the exposed surface 50a
  • the wettability can also be expressed by the contact angle ⁇ between the liquid and the exposed surface 50a. When the contact angle ⁇ is less than 90 °, the wettability is high, and when the contact angle ⁇ is 90 ° or more. It is also possible to express a certain case as having low wettability.
  • it is preferable that the surface 60a of the second filter member 60 has a higher wettability than the surface 40a of the liquid absorbing member 40.
  • the second filter member 60 having a surface 60a having a wettability larger than the surface 40a of the liquid absorbing member between the first filter member 50 and the liquid absorbing member 40, a conventional (first) filter is provided. It is possible to promote the absorption of liquid from the surface 50a of the first filter member 50 as compared with the configuration in which the liquid absorbing (absorbing) member is provided directly below the member. From the viewpoint of liquid absorption efficiency other than wettability, the configuration of the second filter member 6 may be defined by using the porosity and the average pore diameter.
  • the porosity of the liquid absorbing member 40 is 60 to 80%
  • the porosity of the second filter member 60 is set to be less than that of the liquid absorbing member 40 (less than 60%), so that the first filter member It is possible to promote the absorption of liquid from the exposed surface 50a of 50.
  • the average pore diameter of the second filter member 50 is set to 10 ⁇ m or less, it is possible to increase the liquid absorption rate due to the capillary phenomenon, and it is also possible to suppress the reversion of the liquid.
  • the second filter member 60 can use the same material as the first filter member 50 or the liquid absorbing member 40.
  • the surface 60a of the second filter member 60 may be processed in consideration of the hydrophilicity or hydrophobicity of the material to be used.
  • the surface 60a of the second filter member 60 has a surface roughness larger than that of the flat surface. It is possible to improve the wettability by using the one processed so as to be.
  • the second filter member 60 is composed of the polyolefin fiber which is the same material as the liquid absorbing member 40 and is hydrophobic, the surface 60a of the second filter member 60 is further smoothed. It is also possible to improve the wettability by using a material.
  • the size of the second filter member 60 is not particularly limited as long as it can be sandwiched between the first filter member 50 and the liquid absorbing member 40, but is fixed to the liquid absorbing member 40 in the crushed state.
  • the surface 60a of the second filter member 60 is preferably one having a higher wettability than the surface 40a of the liquid absorbing member 40. It should not be interpreted that the structure in which the surface 60a of the filter member 60 is less wettable than the surface 40a of the liquid absorbing member 40 is excluded. That is, when the material of the selected liquid absorbing member 40 has a higher wettability than the base (excellent in hydrophilicity), the liquid absorption from the exposed surface 50a of the first filter member 50 becomes faster depending on the combination. It may be too much and it may be difficult to carry out a sufficient solid phase reaction. In this case, the solid phase reaction time on the exposed surface 50a can be adjusted by making the surface 60a of the second filter member 60 less wettable than the surface 40a of the liquid absorbing member 40.
  • the solid-phase reaction vessel 100 having the above configuration has a shallow cylindrical shape with a diameter of about 15 mm and a height of about 5 mm, and the diameter 13a of the opening 13 is 1.0 to 3.0 mm.
  • the liquid absorbing member 40 is housed in the internal space 70 in a crushed state, and the first filter member 50 and the liquid absorbing member 50 that capture the substance to be detected contained in the sample liquid supplied through the opening 13 and sucking the liquid absorbing member 40.
  • a second filter member 60 having a surface 60a that is more wettable than the surface 40a of the liquid absorbing member 40 is provided between the liquid member 40 and the liquid member 40.
  • the maximum amount of the cleaning liquid or the like supplied to the solid-phase reaction vessel 100 through the opening 13 is about 25 ⁇ l, and the sample liquid is about 5 to 20 ⁇ l.
  • the supplied liquid passes through the first filter member 50 and then passes through the first filter member 50. Since it is rapidly absorbed by the liquid absorbing member 40 via the second filter member 60, the processing related to each step of the solid phase reaction can be performed quickly and accurately.
  • the tapered portion 13b of the opening 13 may be coated with a matte coating.
  • FIG. 5A is a perspective view illustrating the appearance of the solid-phase reaction vessel 101 in which the tapered portion 13b of the opening 13 is coated with a matte coating
  • FIG. 5B is a top view thereof.
  • the measurement light emitted from the light source at the time of measurement is absorbed and the measurement accuracy is improved. be able to.
  • the form is not limited to the tapered portion 13b of the opening 13, and a matte coating may be applied to all portions other than the exposed surface 50a.
  • FIG. 6 is a perspective view illustrating the appearance of the solid-phase reaction vessel 102 in which the mixing vessel portion 30 is provided in the solid-phase reaction vessel 101, and FIG. 6B is a cross-sectional view thereof.
  • the mixing container portion 30 has a tray-like shape (box shape) having a liquid reservoir empty portion 31 formed by erecting a side wall portion 33 from the peripheral edge of the bottom portion 32 continuous from the outer peripheral wall surface 14 of the upper lid 10'. Is formed in.
  • a flange portion 34 extending toward the outside of the liquid storage empty portion 31 is formed on the upper end side of the side wall portion 33, and the length of the side wall portion 33 including the flange portion 34 in the longitudinal direction is the upper lid 10'.
  • the length is substantially the same as the diameter, and the length in the height direction of the side wall portion 33 including the flange portion 34 is substantially the same as the length in the height direction of the upper lid 10'.
  • the shape of the opening 31a of the liquid reservoir 31 is rectangular, but the shape of the opening is not particularly limited, and for example, a round shape, a triangular shape, or the like may be adopted. If there is no effect on the liquid absorption / waste liquid operation by the pipettor, such as a premix of the substance to be detected contained in the sample liquid and the substance having a specific binding ability to the substance to be detected, the opening of the liquid reservoir 31
  • the shape of 31a may be any shape.
  • the width of the opening 31a of the liquid storage empty portion 31 of the mixing container portion 30 is preferably about 2 to 4 mm, and more preferably the width of the opening 31a is 3 mm.
  • the liquid reservoir empty portion 31 has a substantially inverted trapezoidal cross section that is recessed downward from the opening portion 31a toward the bottom surface portion 31b.
  • the mixing container portion 30a is formed. It is also possible to provide another mixing container portion 30b at the position of the outer peripheral wall surface 14 facing the formed position, and there is no limit to the number of mixing container portions provided on the upper lid.
  • the mixing vessel portion 30b can be used not only as a place for other premixes, but also for storing a washing liquid such as a washing buffer, for example.
  • an automatic measuring device capable of fully automating the process from sample preparation to measurement and continuously measuring a large number of samples
  • a cleaning solution or the like is often shared in one vial, which may cause contamination.
  • each solid-phase reaction vessel is provided with a plurality of mixing vessels, the independence of measurement in each sample can be ensured, and the effect of suppressing the occurrence of unnecessary contamination can be expected.
  • the flange portions provided in each of the mixing container portions 30a and 30b can be used as a measurement positioning / fixing member for the automatic measuring device. As a result, the horizontal position of the automatic measuring device described later with respect to the detection unit can be accurately maintained, which is expected to contribute to the improvement of measurement accuracy.
  • any of the solid phase reaction vessels 100-103 is used (hereinafter, simply referred to as the solid phase reaction vessel 100), has a specific binding ability to the substance to be detected, and is fixed to the surface of the first filter member. It has a capture step of capturing the substance to be detected via the ligand-introducing substance into which a ligand capable of binding to the modified ligand-capturing substance has been introduced, and has a specific binding ability to the substance to be detected, and is labeled with a signal-producing substance. It is provided with a measurement step of measuring a signal emitted by the labeled substance bound to the substance to be detected by using the labeled substance.
  • the capture step has a specific binding ability to the sample liquid containing the substance to be detected and the ligand trapping substance immobilized on the exposed surface 50a of the first filter member 50.
  • a solution containing a ligand-introducing substance into which a new ligand has been introduced and a solution containing a labeling substance having a specific binding ability to the substance to be detected and labeled with a signal-producing substance are premixed and used as the first. It may be in the form of being applied to the exposed surface 50a of the filter member 50 of 1. In this case, the three may be mixed at the same time, or the sample solution may be added to a mixed solution of the solution containing the ligand-introducing substance and the solution containing the labeling substance.
  • a solution containing the sample solution and the ligand-introducing substance is premixed and subjected to the exposed surface 50a of the first filter member 50, and the complex of the ligand-introducing substance and the detected substance is applied to the ligand-capturing substance. It is also possible to provide a solution containing the labeling substance labeled with the signal-producing substance after binding.
  • the ligand is not particularly limited, but for example, peptides, polypeptides, proteins (enzymes, antibodies, antigenic proteins, glycoproteins, lipoproteins, avidin, etc.), hormones, etc. , Immune system modulators, vitamins, steroids, carbohydrates (eg, sugars), glycolipids, nucleic acids (including single-stranded and double-stranded oligonucleotides), haptens, lectins, biotin, etc. Can be done. Of these, biotin is preferably used.
  • the ligand-capturing substance may be any substance as long as it can capture the ligand introduced into the ligand-introducing substance, and can be appropriately selected according to the ligand.
  • Examples of the combination with the ligand-ligand capture substance include an antigen-antibody, a hapten-antibody, a sugar-lectin, an antibody-protein A / G, and a biotin-antibiotin antibody.
  • biotin it is preferable to use an anti-biotin antibody, avidin, streptavidin, or the like as the ligand-capturing substance.
  • the ligand trapping substance can be immobilized on the exposed surface 50a of the first filter member 50 by a generally used physical adsorption or chemical bond.
  • the ligand-capturing substance may be directly immobilized on the exposed surface 50a, or an antibody or the like that specifically binds to the ligand-capturing substance may be immobilized on the exposed surface 50a as a spacer substance, and then the ligand is interposed via the spacer substance. It may be in the form of immobilizing the trapping substance.
  • examples of the labeling substance having a specific binding ability to the substance to be measured and labeled with the signal-producing substance include alkaline phosphatase, ⁇ -galactosidase, glucose oxidase, urease, creatine kinase, uricase, and glucose.
  • examples thereof include antibodies labeled with -6-phosphate dehydrogenase, peroxidase and the like, proteins such as protein A / G, lectin, avidin and streptavidin, nucleic acids, or fragments thereof. Of these, horseradish peroxidase is preferably used.
  • the generated signal can be obtained as a colorimetric signal or a chemical luminescence signal depending on the substrate.
  • the substrate used for generating the colorimetric signal for example, tetramethylbenzidine and its derivatives, o-phenylenediamine, triarylmethanes, imidazole leuco dyes and the like can be used.
  • examples of the generation of chemical luminescence signals include acridinium salts, dioxetane, luciferin, lucigenin, and oxalyl chloride.
  • labeling substances antibodies labeled with any of radioactive isotopes, lanthanoid elements, free radical derivatives, chemical luminescent substances, and fluorescent luminescent substances, protein A / Proteins, nucleic acids such as G, lectin, avidin, streptavidin, or fragments thereof can be used.
  • the binding of the signal-producing substance to the labeled substance is not particularly limited, and examples thereof include covalent bonds, ionic bonds, hydrogen bonds, coordination bonds, physical adsorption, and chemisorption.
  • a linker composed of an organic molecule such as a polyethylene glycol chain may be provided.
  • the substance to be detected is an IgE antibody
  • a radioisotope is used as the signal generating substance
  • 125 I, 131 I, 3 H and the like can be mentioned, and 125 I is preferably used from the viewpoint of sensitivity and half-life.
  • iodine When iodine is oxidized, it easily binds to tyrosine residue, histidine residue, etc. in an antibody (protein) as a new electronic reagent, and is therefore suitable for labeling.
  • the elements having atomic numbers 57 to 71 (La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu) are used.
  • the chelate of Eu (Europium) having atomic number 63 has a large wavelength difference between excitation light (340 nm) and synchrotron radiation (615 nm), and has a long synchrotron radiation (fluorescence) life, so time-resolved fluorescence measurement is performed. be able to.
  • a free radical derivative is used as the signal-producing substance
  • a free radical derivative for example, piperidine-N-oxide derivative, pyrrolidin-N-oxide derivative, oxazolidine-N-oxide derivative and the like can be mentioned, and labeling labeled with these free radical derivatives can be mentioned.
  • the change in the ESR spectrum of the antibody is used as an index for measurement.
  • chemiluminescent substance for example, luminol derivatives, acridinium salts, dioxetans, luciferin, luciferin, oxalyl chloride, indoxyl derivatives and the like can be mentioned, and labeled antibodies labeled with these chemiluminescent substances Is chemiluminescent and measured, for example, in the presence of enzymes such as microperoxidase and peroxidase, in the presence of H 2 O 2 or in alkaline conditions, and in the presence of H 2 O 2 .
  • enzymes such as microperoxidase and peroxidase
  • any of fluorescent dyes, quantum dots, or fluorescent dyes or fluorescent particles (beads) composed of quantum dots and particles can be mentioned.
  • the fluorescent light emitting material referred to here is of visible light to near infrared light having a wavelength in the range of 400 nm to 1100 nm when excited by ultraviolet light to near infrared light having a wavelength in the range of 200 nm to 700 nm. It indicates radiated light (emission).
  • fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, ALexaFluor (Invigen) dye molecules, BODIPY (Invigen) dye molecules, cascade dye molecules, coumarin dye molecules, and eodin.
  • fluorescent dyes include fluorescein dye molecules, rhodamine dye molecules, ALexaFluor (Invigen) dye molecules, BODIPY (Invigen) dye molecules, cascade dye molecules, coumarin dye molecules, and eodin.
  • dye molecules NBD-based dye molecules, pyrene-based dye molecules, cyanine-based dye molecules, aromatic hydrocarbon-based molecules and the like.
  • Examples include merocyanin, the organic nitrogen compounds aclydin, pyranine, pyrromethene, luciferin, DCM (4- (dicyanomethylene) -2-methyl-6- (4-dimethylaminostyryl) -4H-pyran), and butylene. it can. These fluorescent dyes may be used alone or in combination of two or more.
  • quantum dots containing II-VI group compounds, III-V group compounds, IV group elements and the like can be used, and these compounds may be used alone or in a plurality of types. It may be combined.
  • CdSe, CdS, CdTe, ZnSe, ZnS, ZnTe, InP, InN, InAs, InGaP, GaAs, Si, Ge and the like can be mentioned.
  • these quantum dots are used as cores, and core / shell type quantum dots shelled with other nanomaterials are prepared, and the surface thereof is made of an organic substance having high biocompatibility such as polyethylene glycol or amphipathic polymer.
  • the coated one can also be used in the present invention.
  • fluorescent particles composed of fluorescent dyes or quantum dots and particles include inorganic particles such as silica and alumina or resin particles (resin polymers) composed of organic polymers such as polystyrene and poly (meth) acrylic acid ester. Examples thereof include those in which either a fluorescent dye or a quantum dot is bonded, partially bonded, or encapsulated inside a particle.
  • the method for introducing the fluorescent dye or the like into the resin particles is not particularly limited, and is a method of binding the fluorescent dye to the monomer which is the raw material of the resin and then polymerizing the monomer, or a method of forming the polymer and then adding the fluorescent dye to the polymer.
  • a method of binding, a method of mixing a monomer and a fluorescent dye and simultaneously performing polymerization and binding of the fluorescent dye, a method of imparting resin polymerizable to the fluorescent dye itself, and the like can be adopted.
  • the average particle size of the fluorescent particles is not particularly limited as long as it does not affect the immune reaction and can obtain sufficient brightness, but it is preferably in the range of 1 nm to 100 ⁇ m.
  • An example of fluorescent particles is a combination of a fluorescent substance and particles having the property of emitting radiated light (emission) of visible light to near infrared light when excited by ultraviolet light to near infrared light. If there is, there is no particular limitation, and for example, a combination of the above-mentioned lanthanoid element and resin particles may be used.
  • the fluorescent particles that can be used in the present invention can be produced by a known method, or commercially available fluorescent particles may be used. It is also possible to use the fluorescent particles in which the aggregation-induced luminescent molecule described in Patent Document 2 is incorporated into a swelling polymer.
  • allergen-specific IgE the substance to be detected is allergen-specific IgE.
  • the allergen is not particularly limited, but for example, house dust 1 (2), dandruff mite, sugi, hinoki, hannoki (genus), shirakamba (genus), camogaya, butakusa, yomogi, alternaria, aspergillus, maracetia.
  • Inhalation system / other allergens such as (genus), cat (dandruff), dog (dandruff), cockroach, moth, latex, milk, egg white, opomcoid, rice, wheat (fruit), buckwheat, soybean, peanut, apple, kiwi , Sesame, beef, chicken, shrimp, crab, mackerel, salmon, tuna and other food-based allergens, any allergen that can be examined as an allergen test item at a medical institution or the like can be selected.
  • allergens such as (genus), cat (dandruff), dog (dandruff), cockroach, moth, latex, milk, egg white, opomcoid, rice, wheat (fruit), buckwheat, soybean, peanut, apple, kiwi , Sesame, beef, chicken, shrimp, crab, mackerel, salmon, tuna and other food-based allergens, any allergen that can be examined as an allergen test item at a medical institution or the
  • FIG. 8 is a flowchart illustrating a step of immobilizing an anti-biotin antibody (goat) as a ligand trapping substance on the exposed surface 50a of the solid phase reaction vessel 100-103 (hereinafter, simply referred to as the solid phase reaction vessel 100). .. First, in step S10, 5 ⁇ l of a wetting solution such as a citrate buffer is supplied through the opening 13 of the unsensitized solid-phase reaction vessel 100.
  • a wetting solution such as a citrate buffer
  • step S20 After 5 ⁇ l of the wetting liquid is absorbed by the absorbing member 40, 5 ⁇ l of the anti-goat IgG antibody (donkey) solution is supplied as a spacer substance, and the anti-goat IgG antibody is immobilized on the exposed surface 50a of the filter member 50 (step S20). ..
  • step S30 5 ⁇ l 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 S30).
  • step S40 after passing 5 ⁇ l of a protective solution such as a phosphate buffer solution containing bovine serum albumin or the like (step S40), the ligand trapping substance is immobilized by drying with air drying or the like for about 1 hour (step S50). Is complete.
  • a protective solution such as a phosphate buffer solution containing bovine serum albumin or the like
  • FIG. 9 uses a solid phase reaction vessel 100 in which a biotinylated allergen was used as the ligand-introducing substance, an HRP-labeled anti-IgE antibody was used as the labeling substance, and an anti-biotin antibody (or streptavidin) was immobilized on the exposed surface 50a as the ligand capturing substance.
  • a biotinylated allergen was used as the ligand-introducing substance
  • an HRP-labeled anti-IgE antibody was used as the labeling substance
  • an anti-biotin antibody or streptavidin
  • step S60 of FIG. 9 5 ⁇ l of a sample solution (serum, plasma, nasal juice, tears, saliva, etc.), 5 ⁇ l of a biotinylated allergen solution, and 5 ⁇ l of an HRP-labeled anti-IgE antibody solution are mixed in a liquid reservoir of the mixing container portion 30. It is introduced into 31 and subjected to a liquid phase reaction for 2 minutes by absorbing or effluent with a pipetter or allowing it to stand to form a complex consisting of a substance to be detected-a ligand-introducing substance-a labeling substance (first reaction).
  • a sample solution serum, plasma, nasal juice, tears, saliva, etc.
  • an HRP-labeled anti-IgE antibody solution 5 ⁇ l of an HRP-labeled anti-IgE antibody solution
  • step S70 the sample liquid is sucked from the liquid reservoir 31 by a pipetter, discharged onto the exposed surface 50a through the adjacent opening 13, supplied, and then subjected to a solid phase reaction for 1 minute (second reaction). ).
  • a substrate such as tetramethylbenzidine (TMB) is added, and the absorbance at 1600 to 660 nm is measured to measure the allergen contained in the sample.
  • TMB tetramethylbenzidine
  • the time required for measuring the absorbance is 1 minute
  • all the measurement operations are completed in about 4 minutes, so that the measurement result can be obtained quickly in an extremely short time. It is possible to obtain stable measurement accuracy.
  • the automatic measuring device 200 includes a dispensing unit 201 capable of sucking and discharging a solution from at least a set reagent cartridge, a detection unit 202 including a color sensor, a lens unit, and the like, and an input means such as a touch panel.
  • An operation unit 203 provided with a display means such as an LCD (Liquid Crystal Display) for displaying device information or measurement results, an analysis unit 204 for analyzing a color-developing signal detected by the detection unit 202, and an analysis unit 204.
  • a display means such as an LCD (Liquid Crystal Display) for displaying device information or measurement results
  • an analysis unit 204 for analyzing a color-developing signal detected by the detection unit 202
  • an analysis unit 204 for analyzing a color-developing signal detected by the detection unit 202.
  • a measurement result generation unit 206 that generates measurement results from analysis results and a control unit 205 that controls these in an integrated manner, and the actual operation by the inspector is performed except for setting the reagent cartridge or solid-state reaction chip 100 in the device. It is configured to be unnecessary.
  • the final measurement result can be obtained in about 20 minutes, so that the throughput is high, and the operation can be intuitively performed according to the screen displayed via the operation unit 203. Because it can be done, there is an advantage that no skill is required. Further, since the device itself can be miniaturized, it can be applied as a device for immediate clinical examination.
  • FIG. 11 is a schematic configuration diagram illustrating a configuration example of the detection unit 202 of the automatic measuring device 200 described with reference to FIG.
  • the detection unit 202 is provided in a light source unit 2021 composed of a high-intensity white LED or the like and a light guide unit 2022 provided in the housing 2025 and linearly guides the light emitted from the light source unit 2021 to the exposed surface 50a of the solid phase reaction vessel 100.
  • a lens unit 2023 that collects a color-developing signal from the exposed surface 50a, and a color sensor unit 2024 that acquires an RGB value from the color-developing signal obtained through the lens unit 2023 and outputs the RGB value to the analysis unit 204.
  • the angle ⁇ formed by the optical axis A of the light source unit 2021 and the central axis B of the color sensor unit 2024 via the lens unit 2023 is preferably 45 °.
  • a laser light source that irradiates ultraviolet light to near infrared light having a wavelength in the range of 200 nm to 700 nm is used as the light source unit 2021 instead of the high-intensity white LED. It is also possible.
  • FIG. 12 shows the result of measuring the RGB value of the light emitted by the signal-producing substance of the labeling substance based on the measurement method shown in the flowchart of FIG. Specifically, 5 ⁇ l of a sample solution containing an IgG antibody is preliminarily prepared with 5 ⁇ l of a solution containing an anti-biotin-labeled antibody (anti-IgG) as a ligand-introducing substance and 5 ⁇ l of a solution containing an HRP-labeled antibody (anti-IgG) as a labeling substance for 2 minutes.
  • anti-IgG anti-biotin-labeled antibody
  • FIG. 12A is a result when a solid-phase reaction vessel having no second filter member 60 between the first filter member 50 and the liquid absorbing member 40 is used
  • FIG. b) is a result of using a solid-phase reaction vessel having a second filter member 60 between the first filter member 50 and the liquid absorbing member 40 according to the present invention.
  • the RGB values obtained from the rising edge of the graph are not stable, while the solid-phase reaction having the second filter member 60 is provided.
  • stable RGB values could be obtained from the rising edge of the graph (FIG. 12 (b). Therefore, according to the solid-phase reaction vessel according to the present invention, the amount of the sample solution was minimized and the measurement was rapid. It was confirmed that it is possible to provide a solid-phase reaction vessel capable of providing stable measurement accuracy while achieving a high level of measurement, and a measurement method using the same.
  • the solid-state reaction vessel 104 shown in FIG. 13 has a structure in which the length c in the vertical direction and the length d in the horizontal direction have substantially the same square shape and have a length e in the height direction shorter than these lengths. As illustrated, the vertical length c and the horizontal length d do not necessarily have to be equal, and one of them is longer (for example, the vertical length c> the horizontal length d). It may be in the form.
  • the solid phase reaction vessel 104 may be provided with the above-mentioned mixing vessel portion.
  • FIG. 14 shows an example of a solid-phase reaction vessel 105 in which the solid-phase reaction vessel 104 is provided with two mixing vessel portions 30a and 30b.
  • allergen-specific IgE as a substance to be detected has been described, but the present invention is not limited to this.
  • substances to be detected peptides, polypeptides, proteins (enzymes, antibodies other than IgE, antigenic proteins, glycoproteins, lipoproteins, avidin, etc.), hormones, immune system modulators, etc. It can also be vitamins, steroids, carbohydrates (eg, sugars), glycolipids, nucleic acids (including single-stranded and double-stranded oligonucleotides).
  • esophageal cancer lung cancer, squamous epithelial cancer, small cell cancer, hepatocellular carcinoma, biliary tract cancer, prostate cancer, neuroblastoma, thyroid spinal cord Tumor markers such as sickness, breast cancer, stomach cancer, pancreatic cancer, colon cancer, cervical cancer, uterine body cancer, ovarian cancer, amoeba erythema, hepatitis E, influenza, Westnile fever, HIV infection ⁇ AIDS, Hepatitis A, Echinococcus, Ebola hemorrhagic fever, Ersina infection, Yellow fever, Oncoselka disease, Return fever, Cirrhosis, Campylobacter infection, Q fever, Mad dog disease, Cow spongy encephalopathy, Crimea, Cryptosporidium disease, Crimea Congo hemorrhagic fever, tuberculosis, coccidiosis, cholera, bacterial erythema, cycl
  • the solid-state reaction vessel according to the present invention is excellent in portability as well as easy measurement, it is not only used as a diagnostic tool for the above-mentioned human diseases, but also as a sheep, pig, goose, cow, and chicken. , Duck, turkey, goose, eye duck, pheasant, quail, etc., pet livestock such as dog, cat, hamster, guinea pig, inco, parrot, tropical fish, horse, camel, suikyu, mule, donkey, yak, pony, etc. It can also be applied to simple outdoor inspections of various pathogens carried by domestic domestic animals or other wild animals.
  • the present invention is not limited to this, and for example, a direct adsorption method in which an anti-human IgE antibody or an allergen is directly immobilized on a filter member. Needless to say, it is also applicable.

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JPH03233359A (ja) * 1989-10-19 1991-10-17 Kyowa Medetsukusu Kk 抗原もしくは抗体の定量法およびその固相免疫測定装置
JPH05312809A (ja) * 1992-03-10 1993-11-26 Mochida Pharmaceut Co Ltd 免疫学的簡易測定方法および装置
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JPH05312809A (ja) * 1992-03-10 1993-11-26 Mochida Pharmaceut Co Ltd 免疫学的簡易測定方法および装置
US6057165A (en) * 1997-02-07 2000-05-02 Becton, Dickinson And Company Quality control procedure for membrane flow-through diagnostic assay devices
JP2016200429A (ja) * 2015-04-08 2016-12-01 株式会社パートナーファーム 固相反応容器及びこれを用いた測定方法
JP3215045U (ja) * 2017-12-12 2018-02-22 株式会社パートナーファーム 固相反応容器

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