WO2021095858A1

WO2021095858A1 - Method for measuring physiologically active substance

Info

Publication number: WO2021095858A1
Application number: PCT/JP2020/042470
Authority: WO
Inventors: 義徳鈴木
Original assignee: インターメディック株式会社; 常盤化学工業株式会社; 有限会社ＶｅｎｔｕｒｅＬａｂ
Priority date: 2019-11-14
Filing date: 2020-11-13
Publication date: 2021-05-20
Also published as: JPWO2021095858A1

Abstract

Provided is a method for measuring a physiologically active substance, in which a tripartite complex in which three parts of: an object to be measured in a specimen solution; a first binding substance to which a ligand binds to a first physiologically active substance that specifically binds to the object to be measured; and a second binding substance to which a labeling substance binds to a second physiologically active substance that specifically binds to the object to be measured, are combined, is fixed to a porous filter via a ligand scavenger, and a light emitted by the labeling substance is measured, the method characterized in that the labeling substance contains fluorescent beads.

Description

Method for measuring physiologically active substances

The present invention relates to a method for measuring a physiologically active substance.

A method for measuring a sample substance having physiological activity based on an immunological measurement method has been known conventionally. Patent Documents 1 and 2 also describe methods for measuring a large number of types of measurement objects with one type of supplement-immobilized porous filter. Patent Document 1 describes a measurement method using an enzyme substance as a labeling substance, and Patent Document 2 using an enzyme substance and a fluorescent dye as a labeling substance. However, all of these measuring methods have low measurement sensitivity and cannot be put into practical use, or even if they can be put into practical use, a large amount of sample solution is required, which causes a heavy burden on the patient. Medical personnel are demanding that the amount of sample solution required for measurement be reduced and the burden on patients be reduced. In addition, there is a problem that the reliability is inferior as a measurement method that requires a large number of measurements because the reproducibility of the measured value is inferior. Further, since the measurement takes time, there is a problem that it is necessary to wait for a long time until the measurement result is obtained. There is a strong demand from medical institutions to shorten the time from the collection of sample solution to the presentation of measurement results.

JP-A-2010-44083 Japanese Unexamined Patent Publication No. 2016-200429

An object of the method for measuring a physiologically active substance in a sample solution of the present invention is to improve the sensitivity and accuracy of measurement. As a result, the required amount of the sample solution can be reduced and the burden on the patient can be reduced. It is also to improve the reproducibility of measured values. As a result, the number of measurements required can be reduced, and the burden on patients and medical staff can be reduced. Furthermore, the measurement is speeded up. As a result, the time from sample solution collection to diagnosis can be shortened, the burden on the persons concerned can be reduced, and the cost required for measurement can be reduced. A method for measuring a physiologically active substance using a porous filter having these high sensitivity, high accuracy, high reproducibility, and quickness, and capable of measuring various types of measurement objects with one type of supplement-immobilized porous filter. Is to provide.

The present invention
(1) Label the measurement target in the sample solution, the first binding substance to which the ligand is bound to the physiologically active substance that specifically binds to the measurement target, and the physiologically active substance that specifically binds to the measurement target. The bioactivity of the object to be measured is measured by fixing the ternary complex in which the ternary of the second binding substance to which the substance is bound to a porous filter via a ligand trapping agent and measuring the light emitted by the labeling substance. A method for measuring a bioactive substance, which comprises a labeling substance containing fluorescent beads in a method for measuring a substance.
(2) The method for measuring a physiologically active substance according to Item 1, wherein the tripartite complex is formed in a solution and the solution is added to a porous filter.
(3) The method for measuring a physiologically active substance according to item 2, wherein three kinds of solutions, a sample solution, a solution of a first binding substance, and a solution of a second binding substance, are mixed at the same time.
(4) The physiologically active substance according to item 2, wherein two kinds of solutions, a solution of the first binding substance and a solution of the second binding substance, are mixed, and a sample solution is mixed with the mixed solution. Measuring method.
(5) The method for measuring a physiologically active substance according to Item 2, wherein a tripartite complex is formed in a solution containing at least one of polyethylene glycol, polyvinyl alcohol, and polypyrrolidone.
(6) When the sample liquid is mixed with the solution containing the first binding substance and the solution containing the second binding substance, the volume ratio of the total amount of the solutions of both binding substances to the sample liquid volume is determined. The method for measuring a physiologically active substance according to item 1, wherein the content is 2/1 to 1/20.
,.

By the method for measuring a physiologically active substance in a sample solution of the present invention, the sensitivity and accuracy of measurement can be improved, the amount of sample solution required for measurement can be reduced, and the burden on the patient can be reduced. In addition, the reproducibility of the measured values can be improved, the required number of measurements can be reduced, and the burden on patients and medical staff can be reduced. Furthermore, the measurement can be speeded up, the time from sample solution collection to diagnosis can be shortened, the burden on the persons concerned can be reduced, and the cost required for the measurement can be reduced. Further, conventionally, in order to measure a physiologically active substance in various kinds of sample liquids, it has been necessary to use a porous filter in which a ligand scavenger peculiar to each physiologically active substance is immobilized. However, according to the present invention, any physiologically active substance can be measured with a porous filter on which one kind of ligand supplement is immobilized, and has high workability, easy automation, low cost, and high sensitivity as described above. We were able to provide a method for measuring bioactive substances with high accuracy, high reproducibility, and rapid measurement.

The present inventor has diligently studied the cause of low reproducibility of the measured values of the method for measuring a physiologically active substance using an enzyme substance or a porous filter using a fluorescent dye as a labeling substance. As a result, the enzyme substance and fluorescent dye, which are the labeling substances, penetrate deep into the porous filter, and the light emitted by the labeling substance may be obstructed by the porous filter and may not reach the detector. It was presumed that the variation was the cause of the low reproducibility of the measured values. The size of the compound of the enzyme substance and the fluorescent dye, which are conventionally used labeling substances, is about 3 nm, which is significantly smaller than the size of the mesh of the porous filter, so that the labeling substance is buried in the mesh of the porous filter. It ends up. It was estimated that the reproducibility of the measured values would be low because this burial method would change each time the measurement was performed. Therefore, the reproducibility of the measured values could be remarkably improved by using fluorescent beads having a large size of 20-500 nm as the labeling substance.

This is an example of the method for measuring a physiologically active substance according to the present invention. It is a perspective view of the solid phase reaction vessel of 1st Embodiment. It is a top view of the solid phase reaction vessel shown in FIG. It is a perspective view of the bottom surface side of the solid phase reaction vessel shown in FIG. It is a bottom view of the solid phase reaction vessel shown in FIG. It is sectional drawing of the solid phase reaction vessel shown in FIG. It is a perspective view of the solid phase reaction vessel of 2nd Embodiment. It is a top view of the solid phase reaction vessel shown in FIG. 7. It is a back view of the solid phase reaction vessel shown in FIG. 7. It is a side view of the solid phase reaction vessel shown in FIG. 7. It is a front view of the solid phase reaction vessel shown in FIG. 7. It is a bottom view of the solid phase reaction vessel shown in FIG. 7. It is sectional drawing of the solid phase reaction vessel shown in FIG. It is an exploded view of the solid phase reaction vessel shown in FIG. 7. This is another example of the method for measuring a physiologically active substance according to the present invention.

100, 100B

Solid reaction vessel

10, 10B Top lid member 11 Top

lid body part

12, 12b Top surface part 13

Ring part

14, 14b Opening part 15

Outer wall wall

16, 35b Positioning part 17 Inner peripheral wall 18

Engagement part

20, 20B

Bottom Lid member

21, 21b

Lower lid body

22,

22b Bottom

23, 23b Ventilation 24 Pedestal 25 Outer wall 26 Engagement 27 Pedestal outer wall 28

Groove

30,

30B Mixing container

31, 31b Liquid reservoir 31a Opening Part 310

Liquid reservoir bottom

32,

32b Bottom

33, 33b Side wall 34

Flange

40, 40B

Internal space

50, 50B Porous filter 50a Exposed

surface

60, 60B Absorbent member

The embodiments of the present invention will be described with reference to the drawings as appropriate, but the drawings are schematic and the dimensions and the like may differ from the actual ones.

The ligand of the binding substance L of the present invention that directly immobilizes the physiologically active substance that specifically binds to the object to be measured to the porous filter without the intervention of a ligand such as the binding substance L (first binding substance). It corresponds to the measurement method that does not use. In the case of this measurement method, it is necessary to immobilize the physiologically active substance corresponding to each object to be measured on the porous filter, so that a filter to which a specific scavenger corresponding to the substance is fixed is required for each measurement object. .. That is, if there are 40 types of measurement objects, a filter in which 40 types of different scavengers are fixed is required. However, in the measuring method of the embodiment according to the present invention, a large number of measurement objects can be measured by using one kind of porous filter in which one kind of ligand scavenger corresponding to the ligand of the binding substance L is fixed.

When forming the tripartite complex, I) the sample solution and the solution containing the binding substance L (first binding substance) are mixed, and the solution containing the binding substance F is mixed with the mixed solution. II) The sample solution and the solution containing the binding substance F (second binding substance) are mixed, and the solution containing the binding substance L is mixed with the mixed solution. III) Simultaneously mix three types of solutions: the sample solution, the solution containing the binding substance L, and the solution containing the binding substance F. IV) The solution containing the binding substance L and the solution containing the binding substance F are mixed, and the mixed solution and the sample solution are mixed. Any of these methods I), II), III) and IV) can be used, but the methods III) and IV) are more preferable because the formation of the tripartite complex can be rapidly and the reaction rate can be increased. ) Is the most preferable.
Binding substance L (first binding substance): A substance in which a ligand is bound to a first physiologically active substance that specifically binds to an object to be measured.
Binding substance F (second binding substance): A substance in which a labeling substance is bound to a second physiologically active substance that specifically binds to the object to be measured. The first bioactive substance and the second bioactive substance may be the same or different.
Ligand scavenger: A scavenger that supplements a ligand containing the binding substance L.
Tripartite complex: A complex of three substances, the object to be measured, the binding substance L, and the binding substance F.

Sample fluids include whole blood, serum, plasma, tears, nasal juice, lymph, digestive juice, saliva, gastric juice, bile, pancreatic juice, intestinal juice, sweat, urine, semen, cavity fluid, amniotic fluid, and milk. It can be applied to both human and animal sample solutions.

Any bioactive substance required for human and animal medical treatment and examination can be used as a measurement target. Examples include allergen-specific IgE antibodies, non-specific IgE, hepatitis viruses (A, B, C, D, E types), viral infections (HIV, HTLV, herpes, rota, rubella, etc.), protozoal infections. (Toxoplasma, Spiroheta, etc.), various infectious disease markers such as fungal infections (Chlamydia, Candida, Tricomonas, etc.), tumor markers such as CEA, AFP, PSA, CA19-9, CA125, ferritin, DUPANII, CRP, ASO, RF Infectious markers such as troponin I, troponin T, myoglobin and other myocardial infarction markers, peptides, polypeptides, proteins (enzymes, antibodies other than lgE, antigenic proteins, glycoproteins, lipoproteins, etc. Avidin, etc.), hormones, immune system modulators, vitamins, steroids, carbohydrates (eg, sugars), glycolipids, nucleic acids (including single-stranded and double-stranded oligonucleotides), stimulants, cannabis ( Marijuana), cocaine, LSD, magic mushroom, MDMA power. When the object to be measured is an allergen-specific IgE, the allergen is not particularly limited, but for example, house dust 1 (2), house dust mite, sugi, hinoki, alder (genus), malassezia (genus), and camouflage. , Putaxa, Yomogi, Alternaria, Aspergillus, Malassezia (genus), cat (dandruff), dog (dandruff), cockroach, moth, latex, etc. Medical allergens such as other allergens, milk, egg white, opomcoid, rice, wheat (fruit), buckwheat, soybean, peanut, apple, kiwi, sesame, beef, chicken, shrimp, crab, mackerel, salmon, tuna and other food allergens. Any allergen can be selected as long as it can be examined as an allergen test item at an institution or the like. In addition, the genotype derived from human papillomavirus (HPV) and the genomic DNA probe derived from the binding substance HPV are defined as 16 type, 18 type, 31 type, 33 type, 39 type, 45 type, 51 type, 52 type, 13 types of high risk type such as 56 type, 58 type, 59 type, 68 type, 82 type and 7 types of low risk type such as 6, 11 type, 42 type, 43 type, 53 type, 54 type, 70 type HPV classified into a type can also be a measurement target.

By targeting the above measurement objects, various cancer tumor markers, amebiasis, hepatitis E, influenza, salmonellosis, various infectious diseases such as SARS, myocardial markers, autoimmune diseases, stimulants, cannabis ( It can be used as a simple diagnostic tool for marijuana), cocaine, LSD, magic mushrooms, MDMA, hormones, etc.

As ligands for the binding substance L, peptides, polypeptides, proteins (enzymes, antibodies, antigenic proteins, glycoproteins, lipoproteins, avidin, etc.), hormones, immune system modulators, vitamins, etc. , Steroids, carbohydrates (eg, sugars), glycolipids, nucleic acids (including single-stranded and double-stranded oligonucleotides), haptens, lectins, biotin, etc., but biotin is particularly preferred. preferable.

The ligand scavenger may be any substance that can capture the ligand to be introduced into the binding substance L. Examples of the combination with the ligand-ligand scavenger include an antigen antibody, a hapten antibody, a sugar lectin, an antibody-protein Ano G, and a biotin-antibiotin antibody. When biotin is used as the ligand, it is preferable to use an anti-biotin antibody, avidin, streptavidin or the like as the ligand scavenger.

The labeling substance of the binding substance F contains fluorescent beads. Fluorescent beads include a fluorescent substance that emits fluorescence and a fine particle-like base material that retains the fluorescent substance. Fluorescent substances include fluorescent dyes and rare earth element-containing substances. Fluorophores include merocyanine, perylene, acrydin, perylene, luciferin, pyranine, stilben, rhodamine, coumarin, 4- (dicyanomethylene) -2-methyl-6- (4-dimethylaminostyryl), 4H, pyranine (DCM), Pyrromethene, fluorescein, umbelliferone, and silol are preferable, and silol is more preferable because of its high luminescence intensity. Among the sirors, tetraphenyl siror, 1,1,2,3,4,5, hexaphenyl siror, 2,5 dianicil, 3,4 diphenyl siror are preferable. As the rare earth element, trivalent ions of Sm, Eu, Tb, Dy, Ce, Pr, Nd, Pm, Er, Tm and Yb are preferable, trivalent ions of Sm, Eu, Tb and Dy are more preferable, and the fluorescence intensity is high. Due to its high value, Eu trivalent ions are particularly preferable.

Inorganic compound particles, plastic particles, and latex particles can be used as the base material for holding the fluorescent substance. As the inorganic compound particles, metal oxides such as silica, alumina, and titania are preferable, and silica is more preferable from the viewpoint of surface reactivity and particle size uniformity. As the plastic particles, polystyrene (PS), polymethyl methacrylate (PMMA), and polytetrafluoroethylene (PTFE) are preferable, and PS is more preferable from the viewpoint of surface reactivity and particle size uniformity. It is preferable to have a polar group such as a hydroxyl group, an amino group, or a carboxyl group on the surface of the base material.

The method for producing fluorescent beads is not particularly limited, but there is a method in which a dispersion of latex particles, plastic particles or inorganic compound particles is mixed with a solution of a fluorescent dye or a rare earth element-containing substance, evaporated, and centrifuged. .. There is also a method in which a fluorescent dye and a monomer such as styrene are mixed and the particles of the replastic polymer contain the fluorescent dye by a polymerization reaction of the monomers. Fluorescent beads using a silol compound such as tetraphenyl silol, 1,1,2,3,4,5, hexaphenylcyclol, 2,5 dianicil, 3,4 diphenyl silol contain the silol compound and a styrene monomer. It is obtained by performing emulsion polymerization in an aqueous solution to form polystyrene particles containing the silol compound inside, and chemically modifying the surface. The particle size of the fluorescent beads is preferably 50 to 500 nm, more preferably 100 to 300 nm.

As the fluorescent beads, the following commercially available ones can also be preferably used. Thermo Fisher (Fluoro, MAX, etc.), Luminex (xMAP, etc.), Merck (fluorescent silica nanobeads, etc.), Cosmo Bio (polystyrene fluorescent particles, etc.), Nippon Techno Cruster (fluorescent dyed carboxyl group-modified microspheres, etc.), Spectortech (Fluoroscent Particles, etc.).

A blocking solution can be used to prevent non-specific binding, and as the blocking solution, a solution such as protein blocking, biotin blocking, or endogenous enzyme blocking can be used. For example, serum, proteins such as BSA (bovine serum albumin), casein, skim milk, and water-soluble synthetic polymers can be used. An aqueous solution containing a surfactant can be used as the cleaning liquid, and polyoxyethylene sorbitan, Tv7een20 and the like are preferable as the surfactant.

Immobilization of the ligand scavenger on the porous filter surface can be performed by physical adsorption or chemical bonding. The ligand scavenger may be directly immobilized on the porous filter surface, and after immobilizing on the porous filter surface using an antibody or the like that specifically binds to the ligand scavenger as a spacer, the ligand scavenger may be immobilized on the porous filter surface. However, the method using a spacer is more preferable because of the high certainty of ligand scavenging. When the ligand-capturing agent is an anti-biotin antibody, an antibody against the anti-biotin antibody can be used for the spacer, and when the ligand-capturing agent is streptavidin, an anti-streptavidin antibody can be used for the spacer.

Materials for the porous filter include low-density polyethylene, high-density polyethylene, polypropylene, polymethylacrylate, plastics such as polytetrafluoride ethylene, membrane filters such as PVDF, cellulose acetates, nylons, cellulose fibers, and glass fibers. Metals such as stainless steel, nickel and aluminum, and inorganic compounds such as alumina, zirconia and silicon carbide can be used, but fibers are preferable, cellulose fibers and glass fibers are more preferable, and glass fibers are most preferable.

It is preferable to install an absorbent material in the measuring container to remove unnecessary liquid. As the material of the absorbent material, fibers such as polyester, polyolefin and polyamide, recycled fibers such as sodium polyacrylate, pulp fiber, cellulose fiber, silk fiber and rayon, woven fabric, non-woven fabric, paper and the like can be used. It is important to control the liquid absorption rate of the absorbent material. If the speed is too fast, the reaction on the porous filter becomes insufficient and the measured value becomes smaller than normal, and if the speed is too slow, unnecessary liquid remains on the filter and the measured value becomes large, which is not preferable. The liquid absorption rate can be adjusted by selecting the material of the absorbent material and the structure (porosity) of the absorbent material. The liquid absorption rate can also be adjusted by combining two or more types of absorbent materials. The porosity of the absorbent material is preferably 60 to 75%, more preferably 65 to 70%. If the porosity is too high, the removal of the liquid to be removed on the porous filter will be delayed and the measurement time will be longer, and the labeling substance to be removed will remain on the porous filter and the emission intensity will be higher than the original value. It becomes high and causes erroneous measurement, which is not preferable. If it is too low, the liquid will be removed too quickly and the reaction will not be performed normally, which is not preferable. It is also preferable to control the absorption rate of the liquid on the porous filter by sequentially laminating two or more kinds of absorbent materials having different porosities. For example, by arranging an absorbent material having a slow liquid absorption rate and a high porosity directly under the porous filter and arranging an absorbent material having a high liquid absorption rate and a low porosity under the absorbent material, the absorption speed can be increased. Can be controlled. As for the porosity of the laminated absorbent material, it is preferable that the absorbent material closest to the porous filter has the highest porosity and the porosity gradually decreases. The absorbent material is preferably a synthetic chemical fiber, more preferably polyolefin, polyester, or polyamide, and most preferably polyester, because the porosity can be easily adjusted. The liquid absorption rate of the absorbent material can also be adjusted by selecting the material of the absorbent material. Generally, polyolefins and polyesters with low liquid wettability have a slow absorption rate, and celluloses, polyamides and polyacrylates with high liquid wettability have a high absorption rate. Therefore, by arranging polyolefin and polyester with slow liquid absorption rate directly under the porous filter and arranging cellulose, polyamide and polyacrylic acid salt with high liquid absorption rate under the absorbent material, the absorption speed can be increased. It is preferable to control.

In the measuring method of the embodiment according to the present invention, it is preferable to use a solid-phase reaction vessel containing a porous filter. As shown in FIGS. 2 to 6, a preferred example of the solid-phase reaction vessel 100 is a top lid member 10 having a tapered opening 14 at a substantially central portion of a top surface portion, and a top lid member 10 formed so as to be matable with the top lid member 10. The absorption member 60 and the absorption member 60 housed in the internal space 40 formed by fitting the lower lid member 20 having the ventilation portion 23 on the bottom portion 22 and the upper lid member 10 and the lower lid member 20. It is provided with a porous filter 50 provided on the end surface on the upper lid member side, and the exposed surface 50a of the porous filter 50 via the opening 14 is used as a reaction field for a solid phase reaction. The size of the solid-phase reaction vessel 100 is preferably 8 to 16 mm in length, 6 to 12 mm in width, and 8 to 20 mm in height. The size of the diameter of the narrowest portion of the opening 14 of the tapered structure is preferably 1 to 5 mm, more preferably 2 to 3 mm. As the material of the solid phase reaction vessel 100, plastics such as polyethylene, polycarbonate, polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, ethylene tetrafluoride, polypropylene, polyester and epoxy can be used.

Here, an example of the measurement method of the embodiment according to the present invention and the solid phase container used in the measurement method will be described with reference to FIGS. 1 to 6.
As shown in FIGS. 4 and 5, a ventilation portion 23 is formed on the bottom portion 22 of the lower lid main body portion 21. Since the ventilation portion 23 is formed in the bottom portion 22, for example, as shown in the example of FIG. 1, a liquid such as a sample liquid or a cleaning liquid containing a substance to be measured introduced through the opening 14 of the upper lid member 10 It is possible to facilitate the passage of liquid inside the solid phase reaction vessel 100. For example, the outer peripheral wall 25 of the lower lid main body 21 is formed so as to extend from the pedestal portion 24 forming the bottom portion 22 in the fitting direction to the upper lid member 10, and the outer peripheral wall 25 is formed with the upper lid member 10. At the same time, it faces the inner peripheral wall 17 of the upper lid member 10. The outer peripheral wall 15 of the upper lid member 10 and the outer peripheral wall 27 of the pedestal portion 24 of the lower lid member 20 on the side where the positioning portion 16 is provided are arranged and fitted so as to be located on the same wall surface at the time of fitting. The shape of the solid phase reaction vessel 100 after the joint is a shallow cylindrical shape with no steps on the outer peripheral wall.

As shown in FIGS. 2 to 5, the mixing container 30 has a liquid storage 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 15 of the upper lid main body portion 11. It is formed in a tray shape (box shape) with an open upper part. 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 longitudinal length of the side wall portion 33 including the flange portion 34 is the length of the upper lid member 10. The length is almost the same as the diameter (Fig. 2). The flange portion 34, together with the positioning portion 16, is provided for fixing the solid-phase reaction vessel 100 to a measurement chamber in a measuring device (not shown). As shown in FIGS. 2 and 6, the length of the side wall portion 33 including the flange portion 34 in the height direction is substantially the same as the length of the upper lid main body portion 11 in the height direction.

FIG. 6 is a sectional view taken along line AA'of the solid-phase reaction vessel 100 in FIG. In the example shown in FIG. 6, the absorbing member 60 is housed in the crushed state in the internal space 40 formed by fitting the upper lid member 10 and the lower lid member 20. On the outer peripheral wall 25 of the lower lid main body 21, an engaged portion 26 that can be engaged with the engaging portion 18 formed on the inner peripheral wall 17 side of the upper lid member 10 is formed, and the engaging portion 26 and the engaging portion 18 are formed. By engaging the engaged portion 26, the upper lid member 10 and the lower lid member 20 can be fitted. Further, a porous filter 50 is housed in the upper lid member 10 side end surface of the absorbing member 60 so that a part of the absorbing member 60 is exposed as an exposed surface 50a through the opening 14, and the exposed surface 50a is a reaction field for a solid phase reaction. Used as.

The liquid reservoir empty portion 31 according to the present embodiment has a substantially inverted trapezoidal cross section that dents downward from the opening 31a toward the bottom portion 32. By forming the liquid reservoir 31 in such a shape, these liquids are used as the liquid in the liquid reservoir 31 when premixing the substance to be measured and the substance having a specific binding ability to the substance to be measured. Since it can be collected at the bottom 310, the liquid absorption / waste liquid operation by the pipetter can be surely performed. The shape of the opening 31a of the liquid reservoir 31 is a rectangular shape as shown in FIG. 2, but the shape of the opening is not particularly limited, and for example, a round shape, a triangular shape, or the like is adopted. Is also good.

The size of the absorbing member 60 is not particularly limited as long as it has a volume slightly larger than the volume of the internal space 40 formed by the upper lid member 10 and the lower lid member 20, and the shape thereof is also limited to the internal space 40. As long as it can be maintained in a crushed state when it is housed, it may have a rectangular parallelepiped shape instead of a cylindrical shape.

Further, as shown in FIG. 6, the pedestal portion 24 of the lower lid member 20 may be provided with a groove portion 28 capable of accommodating the opening portion 14 (annular portion 13) of the other solid-phase reaction vessel 100. As a result, 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.

Next, the measurement method using the solid-phase reaction vessel 100 according to the present embodiment will be described. In this measurement method, a ligand-capturing substance that has a specific binding ability to the substance to be measured and captures the ligand-introducing substance into which a predetermined ligand has been introduced is previously provided with respect to the exposed surface 50a exposed through the opening 14. This is done using the immobilized solid phase reaction vessel 100.

The ligand trapping substance can be immobilized on the exposed surface 50a of the solid phase reaction vessel 100 by generally used physical adsorption or chemical bonding. In this case, 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.

Then, in the measurement method using the solid phase reaction vessel 100 according to the present embodiment, in the mixing vessel 30 of the solid phase reaction vessel 100, a sample containing the substance to be measured, a ligand-introducing substance having a specific binding ability to the sample, and the like. And a labeling substance containing fluorescent beads having a specific binding ability to the substance to be measured or the ligand-introducing substance and fluorescing under predetermined conditions is performed. A sample solution containing a complex consisting of a substance to be measured-ligand-introduced substance-labeled substance generated by premix is applied to an exposed surface 50a on which a ligand-capturing substance is immobilized to capture the complex and emit light by fluorescent beads. The concentration of the substance to be measured is measured by measuring the spots.

First, in Step 1 of FIG. 1, several μL of samples (serum, plasma, nasal juice, tears, etc.), several μL of the first binding substance (biotinylated allergen solution), and the number of second binding substances (fluorescent bead-labeled antibody solution). μL is introduced into the liquid reservoir 31 of the mixing vessel 30 and subjected to liquid absorption / waste liquid by a pipetter or a liquid phase reaction by allowing it to stand to form a complex consisting of a substance to be measured-a ligand-introducing substance-a labeling substance ( First reaction).
Next, a blocking agent containing a block ace and a blocking liquid containing a preservative are added to the exposed surface 50a of the porous filter 50 through the opening 14 (Step 2).
Next, in Step 3, the sample liquid is sucked from the liquid reservoir 31 by a pipetter, discharged onto the exposed surface 50a through the adjacent opening 14, supplied, and then subjected to a solid phase reaction (second reaction).

After the solid-phase reaction, wash with a washing solution such as a washing buffer (Step 4). After washing, the concentration of the substance to be measured is measured by measuring the spots emitted by the fluorescent beads (Step 5).

The temperature at the time of measurement by the measuring method of the embodiment according to the present invention is preferably 30 to 45 ° C, more preferably 34 to 42 ° C, and most preferably 36 to 40 ° C. If the temperature is too low, the reaction rate becomes slow and the required reaction time becomes long, or the viscosity of the liquid becomes high and the liquid stays on the porous filter and cannot be removed, which is not preferable. If the temperature is too high, the properties of the liquid such as the sample liquid will change, which is not preferable.

Examples of preferred embodiments related to the ligand of the present invention will be described.
(1) Ligand; Biotin (2) Binding agent L; Biotinylated allergen (3) Binding substance F; Fluorescent bead-labeled antibody (4) Ligand trapping agent; Anti-biotin antibody (5) Ligand trapping agent; Anti-biotin antibody, And spacers; antibodies against anti-biotin antibodies (6) ligand trapping agents; streptavidin (7) ligand trapping agents; streptavidin, and spacers; anti-streptavidin antibodies (8) ligand trapping agents; streptavidin, spacers; anti-streptavidin antibodies Anti-streptavidin antibody is layered on the antibody against

The measuring method of the embodiment according to the present invention can be preferably used for measuring an allergen-specific antibody in a sample solution, and particularly an IgE antibody. In addition, it can be used as a platform capable of measuring autoimmune diseases, cancer markers, infectious diseases, myocardial markers, drugs, hormones, etc., and can automate the measuring device to measure a large amount of sample solution quickly and accurately.

The solid-phase reaction vessel of the embodiment according to the present invention can be used as a platform capable of detecting and measuring autoimmune diseases, cancer markers, infectious diseases, myocardial markers, etc., in addition to the above measurement examples.

Hereinafter, examples of the present invention will be described, but the present invention is not limited to the description of these examples.

(Example 1)
<Preparation of anti-biotin antibody-immobilized porous filter>
5 μL of a wetting agent was added to a glass fiber filter having a diameter of 10 mm, 5 μL of an anti-goat IgG antibody (donkey) solution was added as a spacer substance, 5 μL of an anti-biotin antibody (goat) solution was added as a ligand capture agent, and bovine serum was added. A protective solution such as a phosphate buffer containing albumin was passed through 10 μL, left for 1 hour to dry, and an anti-biotin antibody (goat) was immobilized via an anti-goat IgG antibody (donkey) to obtain a glass fiber filter A. .. A rectangular parallelepiped shape with a length of 13 mm, a width of 9 mm, and a height of 13 mm, with a tapered opening for liquid addition having a diameter of 2.5 mm at the narrowest part, and a solid having a ventilation hole at the bottom of the container. The glass fiber filter A was housed in the phase reaction vessel. A polyester fiber absorber was placed on the back surface of the glass fiber filter A.

<Example of solid-phase reaction vessel used in the measurement method of Examples>
Here, as an example of the solid-phase reaction vessel used in the measurement method of Example 1 of the embodiment according to the present invention, a rectangular parallelepiped solid-phase reaction vessel 100B is shown in FIGS. 7 to 15. FIG. 7 is a perspective view of the solid-phase reaction vessel 100B of the second embodiment. 8 is a top view of the solid-phase reaction vessel 100B shown in FIG. 7, and FIGS. 9 to 12 are a rear view, a side view, a front view, and a bottom view. 13 is a cross-sectional view of the solid-phase reaction vessel 100B shown in FIG. 7, and FIG. 14 is an exploded view of the solid-phase reaction vessel 100B shown in FIG. 7.
The solid-phase reaction vessel 100B is formed so as to be fitted with an upper lid member 10B having a tapered opening 14b at a substantially central portion of a top surface portion 12b and an upper lid member 10B, and a lower lid member having a ventilation portion 23b at a bottom portion 22b. 20B, an absorbent member 60B (for example, a polyester fiber absorbent) accommodated in an internal space formed by fitting the upper lid member 10B and the lower lid member 20B, and an absorption member 60B provided on the upper lid member side end surface. It is provided with a porous filter 50B (for example, the glass fiber filter A of Example 1). With the above configuration, the solid-phase reaction vessel 100B undergoes a solid-phase reaction on the exposed surface of the porous filter 50B through the opening 14b by, for example, the procedure (Steps 1 to 5) as shown in FIG. It is a place.

In the solid phase reaction vessel 100B, a liquid such as a sample solution or a cleaning solution containing a substance to be measured can be introduced into the internal space of the solid phase reaction vessel 100B through the opening 14b. The upper lid member 10B is formed with a mixing container 30B as a sample adjusting portion extending from the side wall portion 33b of the lower lid member 20B. The mixing container 30B is used, for example, as a place for premixing the substance to be measured and the substance having a specific binding ability to the substance to be measured.
As shown in FIG. 13, an internal space 40B is formed inside the lower lid member 20B so as to accommodate the fitting portion of the upper lid member 10B. On the inner peripheral wall of the lower lid main body 21b forming the internal space 40B, an engaged portion that can be engaged with the engaging portion formed on the outer peripheral wall side of the upper lid member 10B is formed, and the engaging portion is formed. The upper lid member 10B and the lower lid member 20B can be fitted to each other by engaging the engaged portion with the engaged portion.
As shown in FIGS. 12 and 13, a ventilation portion 23b is formed on the bottom portion 22b of the lower lid member 20B. Since the ventilation portion 23b is formed in the bottom portion 22b, a liquid such as a sample liquid or a cleaning liquid containing a substance to be measured introduced through the opening 14b of the upper lid member 10B can be passed through the solid phase reaction vessel 100B. Can be easy. The positioning portion 35b, which is a groove provided in the direction from the bottom portion 32b to the liquid reservoir empty portion 31b, is provided for positioning and fixing the solid phase reaction vessel 100B in the measurement chamber in the measuring device (not shown).

The mixing container 30B has a tray-shaped (box-shaped) open upward, which has a liquid storage empty portion 31b formed by erecting a side wall portion 33b from the peripheral edge of the bottom portion 32b continuous from the bottom portion 22b of the lower lid main body portion 21b. ) Is formed.
In the present embodiment, the shape of the opening of the liquid reservoir 31b 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. That is, if there is no influence on the liquid absorption / waste liquid operation by the pipettor or the like such as the premix of the substance to be measured and the substance having a specific binding ability to the substance to be measured, what shape is the opening shape of the liquid reservoir 31b? May be.
As shown in FIG. 9, the liquid reservoir empty portion 31b has a substantially V-shaped cross section that is recessed downward from the opening toward the bottom portion 32b. By forming the liquid reservoir 31b in such a shape, when premixing the substance to be measured and the substance having a specific binding ability to the substance to be measured, these liquids are collected at the bottom of the liquid reservoir 31b. Since it is easy to collect the liquid on the side, it is possible to reliably perform the liquid absorption / drainage operation by the pipetter.
As the material of the solid phase reaction vessel 100B, for example, plastics such as polyethylene, polycarbonate, polyethylene terephthalate, vinyl chloride, polystyrene, ABS resin, polyamide, ethylene tetrafluoride, polypropylene, polyester and epoxy can be used.

(Example 2)
(A) Measurement method A using standard IgE (Example of the present invention)
5 μL of IgE standard solution (concentration is shown in the table) as the object to be measured, 5 μL of biotin-labeled anti-human IgE monoclonal antibody (4 μg / mL) which is the binding substance L, and Fluoro of the fluorescent dose-containing labeled antibody which is the binding substance F. -MAX (manufactured by Thermo Fisher) was mixed with 5 μL of the three substances, and the mixed solution was heated and held at 37 ° C. for 2 minutes. On the other hand, 10 pL each of a blocking agent containing a block ace and a blocking solution containing a preservative was added to the glass fiber filter A in the solid phase reaction vessel. Next, 10 pL of the preheated tripartite mixed solution was added dropwise, heated at 37 ° C. for 1 minute, and buffered physiological saline containing 0.5% Tween 20 in each solid phase reaction vessel. A cleaning solution consisting of 20 μL was supplied three times after the solution supplied immediately before was completely absorbed, and the excess labeling substance remaining on the glass fiber filter A was removed.
The spot where the immobilization part emitted light was measured with a CMOS camera, the emission intensity was obtained, and the measured value was calculated using a standard curve.
(B) Measurement method B using standard IgE (Example of the present invention)
After mixing the binding substance L solution and the binding substance F solution, an IgE standard solution was added to prepare a tripartite mixed solution, which was the same as the measurement method A.
(C) Measurement method C using standard IgE (Example of the present invention)
The measurement method A was the same except that the binding substance L solution and the binding substance F solution were mixed, μL of the PEG solution was added to the mixed solution, and then the IgE standard solution was added to prepare a three-way mixed solution.

(Comparative Example 1)
(X) Measurement method using standard IgE X (enzyme method, comparative example)
The measurement method except that the binding substance F was changed to 30 μL of a peroxidase-labeled anti-IgE monoclonal antibody, 40 μL of a tetramethylbenzidine (TMBZ) solution which is a substrate of horseradish peroxidase was added after removing the labeled substance, and the reaction was carried out at 37 ° C. The measuring method X was obtained in the same manner as in A. The reflectance (ΔK / S) of the blue color tone on the glass fiber filter was measured with an integrating sphere detector using a laser beam of 670 nm as a light source. The measured values of each concentration of the IgE standard solution are shown in Table 1. We also tried to use a fluorescent dye as a labeling substance, but the sensitivity was so low that we could not conduct a meaningful experiment.

(Example 3)
Under the conditions that the concentration of the IgE standard solution was as shown in Table 1, the measurement methods A, B, C, and X were repeated 7 times each. Table 1 shows the coefficient of variation (average value of standard deviation,%). If the coefficient of variation is 10% or less, it can be put into practical use without any problem, but a smaller coefficient is preferable.
<Table 1 Evaluation of coefficient of variation of measured values>

The methods for measuring A, B, and C of the examples of the present invention were preferred because the coefficient of variation was smaller than that of X of the comparative example, and the coefficients of variation were all 10% or less, which was a preferable level without any problem in practical use. It was. The coefficient of variation became smaller in the order of the measuring method A → B → C of the embodiment of the present invention, which was more preferable, and the measuring method C was the most preferred.

(Example 4)
The measurement was repeated 7 times each using each of the measurement methods A, B, C, and X under the conditions that the concentration of the IgE standard solution was as shown in Table 2. The evaluation indicates that ○ could be detected and × could not be detected.
<Table 2 Sensitivity evaluation>

Although the embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. Further, for example, the features of each embodiment may be combined. Furthermore, these embodiments can be implemented in various other embodiments, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments and modifications thereof are included in the scope and gist of the invention as well as in the scope of the invention described in the claims and the equivalent scope thereof.

Claims

The target substance to be measured in the sample solution, the first binding substance to which the ligand is bound to the physiologically active substance that specifically binds to the measurement target, and the labeling substance to be bound to the physiologically active substance that specifically binds to the measurement target. The bioactive substance of the measurement target is measured by fixing the ternary complex in which the three of the second binding substances are bound to a porous filter via a ligand scavenger and measuring the light emitted by the labeling substance. A method for measuring a physiologically active substance, wherein the labeling substance contains fluorescent beads.
The method for measuring a bioactive substance according to claim 1, wherein the three-party complex is formed in a solution, and the solution is added to the porous filter.
The method for measuring a physiologically active substance according to claim 2, wherein three kinds of solutions, the sample solution, the solution of the first binding substance, and the solution of the second binding substance, are mixed at the same time.
The physiologically active substance according to claim 2, wherein two kinds of solutions, a solution of the first binding substance and a solution of the second binding substance, are mixed, and the sample solution is mixed with the mixed solution. Measuring method.
The method for measuring a physiologically active substance according to claim 2, wherein the tripartite complex is formed in a solution containing at least one of polyethylene glycol, polyvinyl alcohol, and polypyrrolidone.
When the sample liquid, the solution containing the first binding substance, and the solution containing the second binding substance are mixed, the volume ratio of the total amount of the solutions of both binding substances to the sample liquid amount is 2. The method for measuring a physiologically active substance according to claim 1, wherein the content is 1/1 to 1/20.