WO2021182558A1 - Procédé et kit pour immunoessai de substance cible dans un échantillon - Google Patents

Procédé et kit pour immunoessai de substance cible dans un échantillon Download PDF

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Publication number
WO2021182558A1
WO2021182558A1 PCT/JP2021/009722 JP2021009722W WO2021182558A1 WO 2021182558 A1 WO2021182558 A1 WO 2021182558A1 JP 2021009722 W JP2021009722 W JP 2021009722W WO 2021182558 A1 WO2021182558 A1 WO 2021182558A1
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Prior art keywords
antibody
complex
target substance
minutes
sample
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PCT/JP2021/009722
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English (en)
Japanese (ja)
Inventor
亨 本多
伊藤　雅浩
一久 中島
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日立化成ダイアグノスティックス・システムズ株式会社
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Priority to JP2022507274A priority Critical patent/JPWO2021182558A1/ja
Publication of WO2021182558A1 publication Critical patent/WO2021182558A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • 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

Definitions

  • the present invention relates to a method and a kit for immunoassaying a target substance in a sample. More specifically, in an immunoassay method using a first antibody that specifically recognizes a target substance and a complex recognition type antibody that specifically recognizes a complex of the target substance and the first antibody, the measurement process.
  • the present invention relates to a method in which a protein denaturant is applied to.
  • Measurement of the concentration of peptide hormones, haptens, etc. contained in biological samples, etc. is carried out for the diagnosis of various diseases.
  • the LC-MS method is used to measure the aldosterone concentration, which is one of the haptens and is used for the differential diagnosis of primary aldosteronism, Bartter syndrome, Liddle's syndrome, hydroxylase deficiency, and selective hypoaldosteronism.
  • immunoassays and the like are used.
  • the immunoassay method include a radioimmunoassay method (RIA method) and an enzyme-linked immunosorbent assay method (EIA method) (see Patent Document 1).
  • Patent Document 2 discloses a method for measuring immunity of a hapten using an anti-hapten antibody that binds to a hapten and an anti-immunocomplex antibody that binds to a hapten-anti-hapten antibody complex. .. Anti-immunocomplex antibodies may also have slight reactivity with free (non-immune complex) anti-hapten antibodies, measured as anti-immunocomplex antibodies binding to free anti-hapten antibodies. It may cause noise (see Patent Document 2, paragraph 0005).
  • Patent Document 2 The technique described in Patent Document 2 is "to allow a hapten-like substance to coexist when reacting a hapten with an anti-hapten antibody and reacting the produced hapten-anti-hapten antibody complex with an anti-immunocomplex antibody" (claim). (See Item 1), it is said that the measurement noise caused by the binding between the anti-immunocomplex antibody and the free anti-hapten antibody can be reduced.
  • a main object of the present invention is to provide a technique for accurately measuring the concentration of a substance such as a peptide hormone or a hapten contained in a sample.
  • the present invention provides the following [1]-[48].
  • [1] Based on the detection of a multiplex complex comprising a target substance, a first antibody that recognizes the target substance, and a complex recognition type antibody that specifically recognizes the complex of the target substance and the first antibody. , A method for measuring immunity of substances in a sample, A step of contacting the first antibody and the complex recognition type antibody in the presence of a protein denaturing agent (denaturing agent treatment step) is included. Method. [2] The method of [1], wherein the denaturant treatment step is performed in the presence and / or absence of the target substance.
  • the chaotropic denaturant is at least one selected from the group consisting of urea, thiourea, guanidine or a salt thereof, and iodide.
  • the surfactants are polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, and poly.
  • the reducing agent is glutathione and / or cysteine.
  • the protein denaturing agent is urea and / or polyoxyethylene sorbitan monolaurate.
  • the target substance is a peptide hormone or a hapten.
  • the peptide hormone is at least one selected from the group consisting of atrial natriuretic pepti (ANP), cerebral natriuretic pepti (BNP) and C-type natriuretic peptide (CNP), [8]. the method of. [10] The method of [8], wherein the hapten is at least one selected from the group consisting of steroid hormones, thyroid hormones, catecholamines and vitamins. [11] The method of [8], wherein the target substance is aldosterone or thyroid hormone.
  • the sample is any one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, spinal fluid, saliva, amniotic fluid, urine, sweat, feces and pancreatic juice [1]. -A method according to any one of [11].
  • the concentration of urea in the solution in which the complex and the complex recognition antibody are reacted is 0.1 M or more and 8 M or less, preferably 0.5 M or more and 6 M or less, and more preferably 2 M or more and 4 M or less.
  • the concentration of polyoxyethylene sorbitan monolaurate in the solution in which the complex and the complex recognition antibody are reacted is 0.0005% or more and 10% or less, preferably 0.001% or more and 5% or less.
  • the amount of the target substance is determined based on the detection signal intensity of the multiple complex and the calibration curve that defines the correlation between the amount of the target substance and the detection signal intensity prepared in advance using a known amount of the target substance.
  • [21] The method according to any one of [1] to [12], wherein the modifier treatment step is performed in the absence of the target substance.
  • (B1a) A step of dissolving the first antibody and the complex recognition type antibody in a solvent containing a protein denaturant to prepare an antibody solution (denaturant treatment step).
  • (B1b) A step of replacing the solvent of the antibody solution with a solvent containing no protein denaturant, and
  • (B2) A step of mixing the antibody solution after solvent replacement and the sample, and contacting the target substance in the sample with the first antibody and the complex recognition type antibody to form a multiple complex.
  • the method of [22], wherein the concentration of urea in the antibody solution is 0.1 M or more and 8 M or less, preferably 0.5 M or more and 6 M or less, and more preferably 2 M or more and 4 M or less.
  • the concentration of polyoxyethylene sorbitan monolaurate in the antibody solution is 0.0005% or more and 10% or less, preferably 0.001% or more and 5% or less, and more preferably 0.0025% or more and 2.5% or less.
  • the amount of the target substance is determined based on the detection signal intensity of the multiple complex and the calibration curve that defines the correlation between the amount of the target substance and the detection signal intensity prepared in advance using a known amount of the target substance.
  • the method of any of [22]-[26] which is a sandwich ELISA method or a latex agglutination method.
  • [28] Based on the detection of a multiplex complex comprising a target substance, a first antibody that recognizes the target substance, and a complex recognition type antibody that specifically recognizes the complex of the target substance and the first antibody. , A method of suppressing the binding between the first antibody and the complex recognition type antibody in the immunoassay of the substance in the sample. A method comprising holding a first antibody and a complex recognition antibody in a solvent containing a protein denaturant. [29] The method of [28], wherein the protein denaturant is at least one selected from the group consisting of chaotropic denaturants, surfactants and reducing agents.
  • the method of [29], wherein the chaotropic denaturant is any one or more selected from the group consisting of urea, thiourea, guanidine or a salt thereof, and iodide.
  • the surfactant is polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, poly.
  • the reducing agent is glutathione and / or cysteine.
  • the protein denaturant is urea and / or polyoxyethylene sorbitan monolaurate.
  • concentration of polyoxyethylene sorbitan monolaurate in the solvent is 0.0005% or more and 10% or less, preferably 0.001% or more and 5% or less, more preferably 0.0025% or more and 2.5% or less.
  • the method of [33] which is particularly preferably 0.25% or more and 0.5% or less.
  • An immunoassay kit for a substance in a sample which comprises a first antibody that recognizes a target substance and a complex recognition type antibody that specifically recognizes a complex of the target substance and the first antibody.
  • (1) Contains a protein denaturant or (2) For diluting a sample containing the first antibody solution, the complex recognition type antibody solution, the first antibody and / or a solvent for redissolving the complex recognition type antibody, and the target substance. It comprises any one or more liquids selected from the group consisting of solvents, and the one or more liquids contain a protein modifier.
  • kit [37] The kit of [36], wherein the protein denaturant is at least one selected from the group consisting of chaotropic denaturants, surfactants and reducing agents.
  • the surfactant is polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, poly.
  • the kit of [37] which is any one or more polyoxyethylene sorbitan fatty acid esters selected from the group consisting of oxyethylene sorbitan monooleate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan tetraoleate.
  • the reducing agent is glutathione and / or cysteine.
  • Urea is prepared to have a concentration of 0.1 M or more and 8 M or less, preferably 0.5 M or more and 6 M or less, and more preferably 2 M or more and 4 M or less in the solution in which the first antibody and the complex recognition type antibody are contacted.
  • kit. In the solution in which the first antibody and the complex recognition type antibody are contacted, the concentration of polyoxyethylene sorbitan monolaurate is 0.0005% or more and 10% or less, preferably 0.001% or more and 5% or less.
  • the kit of [41] which is more preferably prepared to be 0.0025% or more and 2.5% or less, and particularly preferably 0.25% or more and 0.5% or less.
  • kits according to any one of [36]-[43], wherein the target substance is a peptide hormone or a hapten.
  • the peptide hormone is at least one selected from the group consisting of atrial natriuretic pepti (ANP), cerebral natriuretic pepti (BNP) and C-type natriuretic peptide (CNP), [44].
  • Kit. [46] The kit of [44], wherein the hapten is at least one selected from the group consisting of steroid hormones, thyroid hormones, catecholamines and vitamins. [47] The kit of [44], wherein the target substance is aldosterone or thyroid hormone.
  • the sample is any one or more biological samples selected from the group consisting of whole blood, plasma, serum, urine, spinal fluid, saliva, amniotic fluid, urine, sweat, feces and pancreatic juice [36]. -A kit of any of [47].
  • the term "antibody” is used in the broadest sense and is a monoclonal antibody, polyclonal antibody, dimer, multimer, multispecific antibody (eg, double) as long as it exhibits the desired biological activity. It may be a specific antibody), an antibody fragment or an antibody modified product.
  • the antibody may be a mouse antibody, a rabbit antibody, a human antibody, a humanized antibody or a chimeric antibody, or may be an antibody derived from another species.
  • Antibodies can be in any class of immunoglobulin molecules (eg, IgG, IgE, IgM, IgD and IgA), in any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2).
  • the terms "antibody” and "immunoglobulin” are used in a broad sense with compatibility.
  • an “antibody fragment” is a portion of an antibody that comprises a variable domain of an antibody, or at least an antigen-binding region.
  • Antibody fragments include, for example, Fab, Fab', F (ab') 2 , Fv fragment, linear antibody, single chain antibody (scFv), sc (Fv) 2 , Fab 3 , domain antibody (dAb), diabody. , Triabodies, tetrabodies, minibodies, and multispecific antibodies formed from antibody fragments thereof.
  • An “Fv fragment” is the smallest antibody fragment that contains a complete antigen recognition region and antigen binding region.
  • the "antibody modified product” is obtained by chemically modifying an antibody or antibody fragment, and examples thereof include antibodies to which various molecules such as polyethylene glycol (PEG) are bound.
  • PEG polyethylene glycol
  • the molecule that binds to the antibody is not limited.
  • Specifically recognizing the complex of the target substance and the first antibody that recognizes the target substance means that the complex of the target substance and the first antibody has a higher affinity than that of the free target substance. It means that it binds, preferably it binds only to the complex of the target substance and the first antibody, and does not bind to the free target substance.
  • the affinity of the free target substance or the complex of the target substance and the first antibody can be measured by, for example, the ELISA method or a method using the principle of surface plasmon resonance.
  • Antibodies that specifically bind to the complex of the target substance and the first antibody have an affinity for the complex of the target substance and the first antibody 10 times, 20 times, and 30 times the affinity for the free target substance.
  • the “complex recognition type antibody” is synonymous with a so-called anti-metatype antibody, and means an antibody that specifically recognizes a complex of an antigen and an antibody.
  • the complex recognition type antibody means an antibody that binds to a complex of a target substance and a first antibody that recognizes the target substance, and binds to any site of the complex of the target substance and the first antibody. It may be an antibody to be used.
  • the complex recognition type antibody either a polyclonal antibody or a monoclonal antibody can be used, but a monoclonal antibody is preferable.
  • the antibody fragment include the above-mentioned antibody fragment and the like.
  • the present invention provides a technique for accurately measuring the concentration of a target substance such as a peptide hormone or a hapten contained in a sample.
  • the method according to the present invention comprises a target substance, a first antibody that recognizes the target substance, and a complex of the target substance and the first antibody (
  • a method for immunometrically measuring a substance in a sample based on the detection of a multiplex complex including a complex recognition type antibody that specifically recognizes hereinafter, also simply referred to as “complex”
  • the first antibody includes a step of contacting the complex-recognizing antibody with the complex-recognizing antibody in the presence of a protein denaturing agent (hereinafter referred to as "modifying agent treatment step").
  • modifying agent treatment step by retaining the first antibody and the complex recognition type antibody in a solvent containing a protein denaturing agent, non-specific binding between the first antibody and the complex recognition type antibody is suppressed. Can be done.
  • the immunoassay method according to the present invention is generally not particularly limited as long as it is a measurement method using an immune reaction with an antibody.
  • ELISA method radioimmunoassay method (RIA), fluorescence immunoassay method (FIA), latex agglutination.
  • RIA radioimmunoassay method
  • FFA fluorescence immunoassay method
  • latex agglutination examples thereof include a method and an immunochromatography method.
  • the immunoassay method according to the present invention is not limited to the method used, and can be applied to the practice by an automatic analyzer. There are no particular restrictions on the combination of reagents, etc. when measuring using the method or automatic analyzer, depending on the environment and model of the automatic analyzer to be applied, or taking other factors into consideration. A combination of appropriate reagents and the like may be selected and used.
  • the immunoassay method according to the present invention can also be applied to Micro-TAS (Micro-Total Analysis Systems: ⁇ -
  • the first antibody and the complex recognition type antibody may be antibodies that specifically bind to the target substance and the complex, respectively, and their structures are not particularly limited. Commercially available antibodies can be used as the first antibody and the complex recognition type antibody.
  • the first antibody and the complex recognition type antibody fuse spleen B cells and myeloma cells (for example, P3-U1 cells) of an animal (for example, mouse) immunized with the target substance or complex, respectively, to form a hybridoma library. It can also be obtained by preparing, selecting a hybridoma producing the desired antibody from the library, and purifying the antibody produced from the selected hybridoma.
  • the first antibody and the complex recognition type antibody may be in contact with each other in the presence of the target substance (see the first embodiment described later), and may be in contact with each other in the absence of the target substance (described later). It may be contacted in the second embodiment). Further, the first antibody and the complex recognition type antibody may be contacted in the absence of the target substance and then further in the presence of the target substance.
  • the protein denaturant used in the denaturant treatment step can be any one or more selected from the group consisting of chaotropic denaturants, surfactants and reducing agents.
  • chaotropic modifier any one or more selected from the group consisting of urea, thiourea, guanidine or a salt thereof, and iodide can be used.
  • Surfactants include polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan tristearate, and polyoxyethylene sorbitan.
  • Any one or more polyoxyethylene sorbitan fatty acid esters selected from the group consisting of monooleate, polyoxyethylene sorbitan trioleate and polyoxyethylene sorbitan tetraoleate can be used.
  • glutathione and / or cysteine can be used.
  • urea and / or polyoxyethylene sorbitan monolaurate can be preferably used.
  • the concentration of the protein denaturant in the solution containing the first antibody, the complex recognition type antibody and the protein denaturant is, for example, 0.0005% (w / v) or more and 48% (w / v) or less, preferably 0.001. % (W / v) or more and 36% (w / v) or less, more preferably 0.0025% (w / v) or more and 24% (w / v) or less.
  • the concentration of the protein denaturant in the solution is, for example, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M.
  • the concentration of the protein modifier in the solution is, for example, 0.0005%, 0.001%, 0.0025%, 0.005%, 0.01%.
  • Examples thereof include 4%, 4.5%, 5%, 6%, 7%, 8%, 9% and 10%, for example, 0.0005% or more and 10% or less, preferably 0.001% or more and 5% or less. It can be more preferably 0.0025% or more and 2.5% or less, and particularly preferably 0.25% or more and 0.5% or less.
  • the target substance is not particularly limited, but may be a peptide hormone or a hapten.
  • the peptide hormone may be any one or more selected from the group consisting of atrial natriuretic pepti (ANP), cerebral natriuretic pepti (BNP) and type C natriuretic peptide (CNP).
  • the hapten may be any one or more selected from the group consisting of steroid hormones, thyroid hormones, catecholamines and vitamins. Examples of steroid hormones include mineralocorticoids, glucocorticoids and sex hormones. Examples of mineralocorticoids include aldosterone and fludrocortisone.
  • Examples of the glucocorticoid include cortisol, corticosterone and cortisone.
  • sex hormones include testosterone, dehydroepiandrosterone, estradiol, estriol, estrone, progesterone and the like.
  • thyroid hormones include triiodothyronine (T3) and thyroxine (T4).
  • catecholamines include levodopa, dopamine, noradrenaline and adrenaline.
  • vitamins include vitamin D, vitamin D metabolites (25-hydroxyvitamin D), vitamin B12, vitamin E and vitamin K.
  • the target substance can be aldosterone or thyroid hormone in particular.
  • the sample is not particularly limited as long as it may contain the target substance, but is selected from the group consisting of whole blood, plasma, serum, urine, spinal fluid, saliva, sheep water, urine, sweat, feces and pancreatic fluid. It may be any one or more biological samples.
  • the sample is preferably whole blood, plasma, serum or urine.
  • the “target substance in the sample” means the target substance present in the sample.
  • the target substance is not particularly limited as long as it can bind to an antibody, and may be, for example, a free form of the substance alone or a complex form with another substance (for example, protein). good.
  • Embodiment 1-1 The method according to this embodiment includes the following steps.
  • A1-1) A step of contacting a target substance in a sample with a first antibody to form a complex.
  • A2-1 A step of contacting a complex and a complex-recognizing antibody in the presence of a protein denaturing agent to form a multiple complex.
  • A3-1) A step of detecting a multiple complex.
  • each step will be described in order.
  • the step (A1-1) is not particularly limited as long as it is a method capable of contacting the target substance in the sample with the first antibody to form a complex of the target substance and the first antibody.
  • the step (A1-1) may be carried out in an aqueous solvent or by developing in an insoluble membrane, but is preferably carried out in an aqueous solvent.
  • contacting the target substance with the first antibody means a state in which the target substance and the first antibody coexist in one reaction vessel so that they can come into contact with each other.
  • the aqueous solvent is not particularly limited as long as it is an aqueous solvent that enables the immunoassay method of the present invention, and examples thereof include deionized water, distilled water, and a buffer solution, and a buffer solution is preferable.
  • the aqueous solvent may contain salts, metal ions, sugars, preservatives, proteins, surfactants, protein stabilizers and the like.
  • salts include lithium chloride, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, ammonium chloride, lithium bromide, sodium bromide, potassium bromide, calcium bromide, magnesium bromide, ammonium bromide and the like. ..
  • Examples of the metal ion include magnesium ion, manganese ion, zinc ion and the like.
  • saccharides include mannitol and sorbitol.
  • Examples of preservatives include sodium azide, antibiotics (streptomycin, penicillin, gentamicin, etc.), bioace, Proclin 300, Proxel GXL, and the like.
  • Examples of the protein include bovine serum albumin (hereinafter referred to as BSA) and the like.
  • Examples of the surfactant include nonionic surfactants and the like.
  • Examples of the protein stabilizer include a peroxidase stabilizing buffer (Peroxidase Stabilizing Buffer, manufactured by DakoCytomation) and the like.
  • the temperature of the reaction for forming the complex is not particularly limited as long as it is a temperature that enables the immunoassay method of the present invention, and is usually 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C., 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, 40 °C , 41 ° C., 42
  • the reaction time is not particularly limited as long as it enables the immunoassay method of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes. Minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes, 160 minutes, 170 minutes, 180 minutes, 190 minutes , 200 minutes, 210 minutes, 220 minutes, 230 minutes, 240 minutes and the like.
  • the concentration of the first antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. 9,1,2,3,4,5,6,7,8,9,10,20,30,40,50,60,70,80,90,100 ⁇ g / mL, etc., 0.1 to 20 ⁇ g The / mL range is preferred.
  • the first antibody is preferably immobilized on an insoluble carrier.
  • the insoluble carrier include slide glass, ELISA plate (microtiter plate), beads, latex particles, magnetic particles, filters, films, membranes and the like.
  • the material of the insoluble carrier glass, glass fiber, silicon, ceramic, cellulose, nitrocellulose, nylon, polycarbonate, polyethylene, polypurpyrene, polystyrene, polyethylene terephthalate, polyurethane and the like can be used.
  • Methods for immobilizing an antibody on an insoluble carrier are well known in the art, and for example, a method such as a chemical bond method (a method for immobilizing by covalent bond) or a method for physically adsorbing an antibody can be applied.
  • the antibody may be immobilized on an insoluble carrier using a very strong binding reaction such as the avidin-biotin reaction.
  • the biotinylated antibody in which biotin is bound to the antibody may be immobilized on a streptavidin plate coated with streptavidin.
  • the antibody may be immobilized on an insoluble carrier via a linker.
  • the linker is a molecule capable of covalently binding the functional group on the surface of the insoluble carrier and the functional group of the antibody, for example, a first reactive active group capable of reacting with the functional group of the antibody, and an insoluble carrier.
  • a molecule having a second reactive active group capable of reacting with a functional group on the surface and having a different first reactive active group and a second reactive active group is preferably used.
  • the functional group contained in the antibody and the functional group retained on the surface of the insoluble carrier include a carboxyl group, an amino group, a glycidyl group, a sulfhydryl group, a hydroxyl group, an amide group, an imino group, an N-hydroxysuccinyl group and a maleimide group. And so on.
  • Examples of the reactive group in the linker include allyl azide, carbodiimide, hydrazide, aldehyde, hydroxymethylphosphine, imide ester, isocyanate, maleimide, N-hydroxysuccinimide ester, pentafluorophenyl (PFP) ester, solarene, pyridyl disulfide, vinyl sulfone and the like. The basis of.
  • a cleaning step may be included between the steps (A1-1) and the step (A2-1). However, as will be described later, the cleaning step may be omitted.
  • the cleaning solution for example, a PBS solution, a PBS solution containing a surfactant, and the like have been widely used.
  • the surfactant include nonionic surfactants such as Tween 20 and the like.
  • step (A2-1) corresponds to a denaturant treatment step.
  • step (A2-1) the complex and the complex-recognizing antibody can be brought into contact with each other in the presence of a protein denaturing agent to form a multiple complex of the target substance, the first antibody, and the complex-recognizing antibody.
  • a protein denaturing agent there is no particular limitation as long as it is a method.
  • the step (A2-1) may be carried out in an aqueous solvent or by developing in an insoluble membrane, but it is preferably carried out in an aqueous solvent.
  • contacting the complex with the complex recognition type antibody means that the complex and the complex recognition type antibody coexist in one reaction vessel so that they can come into contact with each other. Means.
  • the temperature of the reaction for forming the multiple complex is not particularly limited as long as it is a temperature that enables the immunoassay method of the present invention, and is usually 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C.
  • the reaction time is not particularly limited as long as it enables the immunoassay method of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes.
  • the concentration of the complex recognition type antibody in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method of the present invention, and is 0.01, 0.02, 0.03, 0.04, 0.
  • the protein denaturant may be added directly into the reaction solution.
  • the protein denaturant may be added to the complex recognition antibody solution, in which case the complex recognition antibody solution containing the protein denaturant may be added to the reaction solution.
  • the concentration of the protein denaturant in the solution in which the complex and the complex recognition antibody are reacted is as described above.
  • a cleaning step may be included between the steps (A2-1) and the step (A3-1). However, as will be described later, the cleaning step may be omitted.
  • the cleaning step may be omitted.
  • impurities and unreacted antibodies in the sample are removed from the surface of the insoluble carrier by washing, and only the multiplex complex formed on the surface of the carrier is used. Can be separated.
  • the step (A3-1) can be carried out, for example, by detecting the multiplex complex using a labeling substance.
  • the labeling substance may be bound to either the first antibody or the complex recognition type antibody, but when the first antibody is immobilized on an insoluble carrier, the labeling substance binds to the complex recognition type antibody. It is preferable that it is.
  • Labeling substances include enzymes, radioactive isotopes, fluorescent substances, luminescent substances, DNA, RNA, coenzymes, or substances that specifically bind to coenzymes (biotin, avidin) used in ordinary immunoassay methods. , Tags, substances that absorb in the ultraviolet to infrared regions, color-developing fine particles, fluorescent fine particles, metallic fine particles, magnetic substances, substances having properties as spin labeling agents, and the like.
  • the enzyme include alkaline phosphatase, peroxidase, galactosidase, glucuronidase, luciferase and the like.
  • Radioisotopes include, for example, 3 H, 14 C, 35 S, 32 P, 125 P and 131 I.
  • Examples of the fluorescent substance include FITC (fluorescein isothiocyanate) and RITC (rhodamine B-isothiocyanate).
  • Examples of the luminescent substance include aclidinium and its derivatives, ruthenium complex compounds, loffin and the like.
  • Methods for binding a labeling substance to an antibody are known in the art of immunoassay.
  • the labeling substance can be attached to the antibody via one or several amino acids, or via a linker with one or several amino acids.
  • various kits for binding the labeling substance to the antibody are commercially available.
  • the multiplex complex can be detected by detecting the signal generated from the labeling substance of the first antibody and / or the complex recognition type antibody forming the multiplex complex.
  • the method for measuring the signal generated from the labeling substance may be appropriately selected depending on the labeling substance to be used.
  • the labeling substance is a coloring substance, that is, a substance that absorbs light of a certain wavelength
  • the labeling substance can be measured by measuring the absorbance using a spectrophotometer, a multi-well plate reader, or the like.
  • the labeling substance is a fluorescent substance
  • the labeling substance can be measured by measuring the fluorescence intensity using a fluorometer, a fluorescent multi-well plate reader, or the like.
  • the labeling substance When the labeling substance is a luminescent substance, the labeling substance can be measured by measuring the luminescence intensity using a luminescent photometer, a luminescent multi-well plate reader, or the like.
  • the labeling substance When the labeling substance is a radioisotope, the labeling substance can be measured by measuring the radioactivity with a scintillation counter, a ⁇ -well counter, or the like.
  • the labeling substance is an enzyme
  • the labeling substance can be measured by measuring the enzyme activity. For example, the labeling substance can be measured by reacting the substrate of the enzyme with the enzyme and measuring the produced substance.
  • the first antibody that forms the non-specific complex is generated.
  • a signal is also generated from the labeling substance of the complex recognition antibody and / or the complex recognition type antibody.
  • the signal from the labeling substance of the non-specific complex becomes noise with respect to the signal generated from the labeling substance of the multiple complex, and becomes a factor of lowering the S / N ratio.
  • the first antibody and the complex recognition type antibody are combined with the first antibody by holding them in a solvent containing a protein denaturant.
  • Non-specific binding with body-recognizing antibody can be suppressed. Therefore, it is possible to suppress the detection of noise from the labeling substance of the first antibody and / or the complex recognition type antibody forming the non-specific complex, and improve the S / N ratio. Therefore, especially when the cleaning step between the step (A1-1) and the step (A2-1) and / or between the step (A2-1) and the step (A3-1) is omitted. Also, a high S / N ratio can be achieved.
  • the step (A3-1) includes the following steps, so that the concentration of the target substance in the sample can be determined. Performing up to the detection of multiple complexes using a target substance with a known concentration, and creating a calibration curve showing the relationship between the target substance concentration and the measured value (hereinafter referred to as the "calibration curve creation step"); and creating a calibration curve. Determine the concentration of the target substance in the sample from the calibration curve created in the process and the measured value of the sample.
  • This embodiment can be suitably implemented as a sandwich ELISA including, for example, the following steps.
  • [A1-1] Contacting the target substance in the sample with the first antibody to form a complex;
  • [A2-1] Contacting a complex with a complex-recognizing antibody in the presence of a protein denaturing agent to form a multiple complex;
  • [A3-1] Measuring multiple complexes;
  • this embodiment can also be preferably implemented as a latex agglutination method including, for example, the following steps.
  • [A1-1] The target substance in the sample and the first antibody to which the insoluble carrier particles are bound are brought into contact with each other in an aqueous solvent to form a complex of the target substance and the first antibody to which the insoluble carrier particles are bound.
  • [A2-1] A complex recognition antibody to which insoluble carrier particles are bound is added to the solution after the step [A1-1], and the complex and the complex recognition antibody to which the insoluble carrier particles are bound are combined with a protein.
  • the insoluble carrier particles used in the latex agglutination method are not particularly limited, and examples thereof include latex particles and magnetic particles, and latex particles are preferable.
  • examples of the material of the latex particles include polystyrene, polyvinyl chloride, polypropylene and the like.
  • the particle size of the insoluble carrier particles is not particularly limited, but the average particle size is, for example, 30 to 800 nm, preferably 100 to 500 nm, and more preferably 150 to 450 nm.
  • the insoluble carrier particles to which the first antibody binds and the insoluble carrier particles to which the complex recognition type antibody binds may be the same or different, but are preferably the same.
  • the concentration of the insoluble carrier particles in the reaction solution is not particularly limited as long as it is a concentration that enables the immunoassay method of the present invention, and is 0.00005, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0. 008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% by weight, etc. Can be mentioned.
  • Aggregation can be measured by, for example, a method of measuring absorbance, a method of measuring scattered light, or the like.
  • Embodiment 1-2 The method according to this embodiment includes the following steps.
  • A1-2 A step of contacting a target substance in a sample with a first antibody in the presence of a protein denaturing agent to form a complex.
  • A2-2 A step of contacting a complex and a complex-recognizing antibody in the presence of a protein denaturing agent to form a multiple complex.
  • A3-2 A step of detecting a multiple complex.
  • the step (A1-2) is particularly limited as long as it is a method capable of contacting the target substance and the first antibody in the sample in the presence of a protein denaturant to form a complex of the target substance and the first antibody. There is no.
  • the step (A1-2) can be carried out in the same manner as the above-mentioned step (A1-1) except that the step (A1-2) is carried out in the presence of a protein denaturing agent.
  • the protein denaturant may be added directly into the reaction solution.
  • the protein denaturant may be added to the first antibody solution and / or the solvent for diluting the sample, or the first antibody solution and / or the sample solution containing the protein denaturant may be added to the reaction solution. good.
  • the concentration of the protein denaturant in the solution in which the target substance and the first antibody are reacted may be the same as the concentration of the protein denaturant in the solution in which the complex and the complex recognition type antibody are reacted as described above.
  • a cleaning step may be included between the steps (A1-2) and the step (A2-2). However, as will be described later, the cleaning step may be omitted.
  • the step (A2-2) corresponds to a denaturant treatment step.
  • the step (A2-2) can be performed in the same manner as the step (A2-1).
  • the protein denaturant is newly added to the reaction solution. If no wash is performed between step (A1-2) and step (A2-2), the protein denaturant may have been added to the reaction solution in step (A1-2). It may be newly added to the reaction solution in this step.
  • a cleaning step may be included between the steps (A2-2) and the steps (A3-2). However, as will be described later, the cleaning step may be omitted.
  • Process (A3-2) The step (A3-2) can be performed in the same manner as the step (A3-1).
  • the first antibody and the complex recognition type antibody are retained in a solvent containing a protein denaturant to obtain the first antibody.
  • Non-specific binding with complex recognition type antibody can be suppressed. Therefore, it is possible to suppress the detection of noise from the labeling substance of the first antibody and / or the complex recognition type antibody forming the non-specific complex, and improve the S / N ratio. Therefore, especially when the cleaning step between the step (A1-2) and the step (A2-2) and / or between the step (A2-2) and the step (A3-2) is omitted. Also, a high S / N ratio can be achieved.
  • This embodiment can be suitably implemented as an immunochromatographic method including, for example, the following steps. [1] Supplying a sample to the sample supply section of the test strip; [A1-2] The target substance in the sample is brought into contact with the first antibody held in the labeled antibody holding portion in the presence of a protein denaturing agent to form a complex of the target substance and the first antibody. ; [A2-2] In the detection unit, the complex is brought into contact with the complex in the presence of a protein denaturant to form a multiple complex of the target substance, the first antibody, and the complex recognition antibody; [A3-2] To measure a multiplex complex.
  • the concentration of the target substance in the sample can be determined by including the following steps. [4] Using a target substance having a known concentration as a sample, the steps [A1-2]-[A3-2] are performed to prepare a calibration curve showing the relationship between the target substance concentration and the measured value of the multiplex complex. And [5] Determine the concentration of the target substance in the sample from the calibration curve prepared in step [4] and the measured value obtained by the measurement in step [A3-2].
  • the test strip is provided with a sample supply unit, and with this as the uppermost stream, a labeled antibody holding unit, a developing unit, a detecting unit, and an absorbing unit are further provided in order according to the direction in which the liquid sample develops.
  • the sample supply unit is a place where a liquid sample is supplied, and may be any substance and form that can absorb the sample and allow the liquid and the target substance to pass through.
  • the material constituting the sample supply unit is not particularly limited as long as it has the property of absorbing the liquid sample and developing the liquid sample.
  • cellulose, nitrocellulose, glass fiber, polyester, etc. examples thereof include polyurethane, polyacetate, cellulose acetate, nylon and cotton cloth.
  • the labeled antibody holding section is a place where the first antibody to which the labeling substance is bound is held so that it can be eluted, and is located downstream of the sample supply section.
  • the material constituting the labeled antibody holding portion is not particularly limited as long as it is a material that retains the first antibody to which the labeling substance is bound so that it can be eluted.
  • Examples of the labeling substance include the labeling substance described in the above step (3A-1).
  • Examples of the method for binding the labeling substance to the first antibody include the binding method described in the above step (3A-1).
  • the unfolding part is a place where the sample containing the target substance is unfolded by the capillary phenomenon.
  • a complex recognition type antibody is immobilized on the detection unit.
  • the developing part and the detecting part are made of a membrane made of a porous body.
  • the material of the membrane is not particularly limited as long as it can absorb and move a liquid sample by capillarity.
  • polyurethane and the like can be mentioned.
  • the absorption unit is a place where a liquid sample is absorbed.
  • the material of the absorbing portion is not particularly limited as long as it has an ability to absorb a sample, and examples thereof include cellulose, nitrocellulose, glass fiber, cotton cloth, and a composite material containing a water-absorbing polymer.
  • the protein denaturant is contained in at least one site of the sample supply part, the labeled antibody holding part and the developing part, so that the target substance and the first antibody are brought into contact with each other in the presence of the protein denaturing part in the developing part. Subsequently, in the detection unit, the complex and the complex recognition type antibody immobilized on the detection unit come into contact with each other in the presence of a protein denaturing agent to form a multiple complex.
  • the protein denaturant may be added to a solvent for diluting the sample, and by supplying the sample diluted with the solvent to the sample supply section, the target substance and the first antibody become the protein denaturant in the developing section. Are contacted in the presence of. Subsequently, in the detection unit, the complex and the complex recognition type antibody immobilized on the detection unit come into contact with each other in the presence of a protein denaturing agent to form a multiple complex.
  • Embodiment 1-3 A step of contacting a target substance in a sample, a first antibody, and a complex recognition type antibody in the presence of a protein denaturing agent to form a multiple complex.
  • A3-3 A step of detecting a multiple complex.
  • each step will be described in order. Items that may be the same as those in the first embodiment in each step will not be described.
  • step (A1-3) corresponds to the denaturant treatment step.
  • step (A1-3) the target substance, the first antibody, and the complex recognition type antibody in the sample are brought into contact with each other in the presence of a protein modifier, and the target substance, the first antibody, and the complex recognition type antibody are duplicated.
  • a protein modifier There is no particular limitation as long as it is a method capable of forming a coalescence.
  • the step (A1-3) may be carried out in an aqueous solvent or by developing in an insoluble membrane, but it is preferably carried out in an aqueous solvent.
  • contacting the target substance with the first antibody and the complex recognition type antibody means that the target substance, the first antibody, and the complex recognition type antibody coexist in one reaction vessel, and these are used. It means a state in which they can come into contact with each other.
  • the target substance, the first antibody, and the complex recognition type antibody may be added in any order and brought into contact with each other.
  • the temperature of the reaction for forming the multiple complex is not particularly limited as long as it is a temperature that enables the immunoassay method of the present invention, and is usually 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C.
  • the reaction time is not particularly limited as long as it enables the immunoassay method of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes.
  • concentrations of the first antibody and the complex recognition type antibody in the reaction solution are not particularly limited as long as they are concentrations that enable the immunoassay method of the present invention, and are 0.01, 0.02, 0.03, 0.
  • the first antibody or complex recognition type antibody is preferably immobilized on an insoluble carrier.
  • the protein denaturant may be added directly into the reaction solution.
  • the protein denaturant may be added to the first antibody solution, the complex recognition antibody solution and / or the solvent for diluting the sample, in which case the first antibody solution, the complex containing the protein denaturant.
  • the recognition antibody solution and / or the sample solution may be added to the reaction solution.
  • the concentration of the protein denaturant in the solution in which the target substance, the first antibody and the complex recognition antibody are reacted is the same as the concentration of the protein denaturant in the solution in which the complex and the complex recognition antibody are reacted as described above. It can be the same.
  • a cleaning step may be included between the steps (A1-3) and the steps (A3-3). However, as will be described later, the cleaning step may be omitted.
  • Process (A3-3) The step (A3-3) can be performed in the same manner as the step (A3-1).
  • the first antibody and the complex recognition type antibody are retained in a solvent containing a protein denaturant to obtain the first antibody.
  • Non-specific binding with complex recognition type antibody can be suppressed. Therefore, it is possible to suppress the detection of noise from the labeling substance of the first antibody and / or the complex recognition type antibody forming the non-specific complex, and improve the S / N ratio. Therefore, a high S / N ratio can be realized even when the cleaning step between the steps (A1-3) and the step (A3-3) is omitted.
  • This embodiment can be suitably implemented as a sandwich ELISA including, for example, the following steps.
  • [A1-3] The target substance in the sample, the first antibody, and the complex recognition type antibody are brought into contact with each other in the presence of a protein denaturing agent to form a multiple complex;
  • [A3-3] To measure the multiplex complex.
  • [4] Using a target substance with a known concentration as a sample, perform steps [A1-3] and [A3-3] to create a calibration curve showing the relationship between the target substance concentration and the measured value of the multiple complex. That; and [5] Determine the concentration of the target substance in the sample from the calibration curve created in the calibration curve preparation step and the measured value obtained by the measurement in step [A3-3].
  • Latex agglutination method 2 Latex agglutination method 2
  • this embodiment can also be preferably implemented as a latex agglutination method including, for example, the following steps.
  • A1-3 In an aqueous solvent, the target substance in the sample, the first antibody to which the insoluble carrier particles are bound, and the complex recognition type antibody to which the insoluble carrier particles are bound are contacted in the presence of a protein modifier.
  • the concentration of the target substance in the sample can be determined by including the following steps. [4] Using a target substance having a known concentration as a sample, the steps [A1-3] and [A3-3] are performed to prepare a calibration curve showing the relationship between the target substance concentration and the measured value of the multiplex complex. And [5] Determine the concentration of the target substance in the sample from the calibration curve prepared in step [4] and the measured value obtained by the measurement in step [A3-3].
  • the structure of the test strip is used in the above-mentioned immunochromatography method 1 except that the labeled antibody holding portion retains the complex recognition type antibody to which the labeling substance is bound so that it can be eluted and the first antibody is immobilized on the detecting portion. Similar to the test strip.
  • the protein denaturant is contained in at least one site of the sample supply section, the labeled antibody holding section, the developing section or the detection section, and in the detection section, the target substance, the first antibody and the complex recognition type antibody are proteins. Contact in the presence of a denaturant to form a multiplex complex.
  • the protein denaturant may be added to a solvent for diluting the sample, and by supplying the sample diluted with the solvent to the sample supply unit, the detection unit recognizes the target substance, the first antibody, and the complex.
  • the type antibody comes into contact with the protein denaturant in the presence of a protein denaturant to form a multiplex complex.
  • the second embodiment of the immunoassay method (a form in which the denaturant treatment step is performed in the absence of the target substance) 3-1.
  • Embodiment 2-1 The method according to this embodiment includes the following steps.
  • (B1a) A step of dissolving a first antibody and a complex recognition type antibody in a solvent containing a protein denaturing agent to prepare an antibody solution.
  • (B1b) A step of replacing the solvent of the antibody solution with a solvent containing no protein denaturant.
  • B2 A step of mixing the antibody solution after solvent replacement and the sample, and contacting the target substance, the first antibody, and the complex recognition type antibody to form a multiple complex.
  • (B3) A step of detecting a multiple complex.
  • step (B1a) corresponds to a denaturant treatment step.
  • step (B1a) the first antibody and the complex recognition type antibody are brought into contact with each other by dissolving them in a solvent containing a protein denaturing agent.
  • "contacting the complex with the complex recognition type antibody” means that the complex and the complex recognition type antibody coexist in one reaction vessel so that they can come into contact with each other. Means.
  • the protein denaturant may be added directly into the solvent.
  • the protein denaturant may be added to the first antibody solution and / or the complex recognition antibody solution, in which case the first antibody solution and / or the complex recognition antibody solution containing the protein denaturant is used as a solvent. It may be added to.
  • the concentration of the protein denaturant in the solution containing the first antibody, the complex recognition type antibody and the protein denaturant is as described above.
  • Process (B1b) The solvent substitution in the step (B1b) can be performed by, for example, any of the following methods. (1) Utilizing the difference in molecular weight between the first antibody and the complex recognition type antibody and the protein denaturing agent, the solvent is replaced by dialysis or gel filtration chromatography. (2) The solvent is replaced by ion exchange chromatography by utilizing the difference in electrical properties between the first antibody and the complex recognition type antibody and the protein denaturing agent. (3) Utilizing the difference in hydrophobicity between the first antibody and the complex recognition type antibody and the protein denaturing agent, the solvent is replaced by hydrophobic chromatography. (4) The first antibody and the complex recognition type antibody are immobilized on an insoluble carrier, and the solvent is replaced by performing B / F separation. (5) In the immunochromatography method, the solvent is replaced by utilizing the difference in the developing rate between the first antibody and the complex recognition type antibody and the protein denaturing agent.
  • the step (B2) is particularly limited as long as it is a method capable of contacting the target substance, the first antibody, and the complex recognition type antibody to form a multiple complex of the target substance, the first antibody, and the complex recognition type antibody.
  • contacting the target substance with the first antibody and the complex recognition type antibody means that the target substance, the first antibody, and the complex recognition type antibody coexist in one reaction vessel, and these are used. It means a state in which they can come into contact with each other.
  • the temperature of the reaction for forming the multiple complex is not particularly limited as long as it is a temperature that enables the immunoassay method of the present invention, and is usually 1 ° C., 2 ° C., 3 ° C., 4 ° C., 5 ° C., 6 ° C.
  • the reaction time is not particularly limited as long as it enables the immunoassay method of the present invention, and is 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes.
  • concentrations of the first antibody and the complex recognition type antibody in the reaction solution are not particularly limited as long as they are concentrations that enable the immunoassay method of the present invention, and are 0.01, 0.02, 0.03, 0.
  • the first antibody or complex recognition type antibody is preferably immobilized on an insoluble carrier.
  • a cleaning step may be included between the steps (B2) and the step (B3).
  • Process (B3) The step (B3) can be performed in the same manner as the step (A3-1).
  • the first antibody that forms the non-specific complex is generated.
  • a signal is also generated from the labeling substance of the complex recognition antibody and / or the complex recognition type antibody.
  • the signal from the labeling substance of the non-specific complex becomes noise with respect to the signal generated from the labeling substance of the multiple complex, and becomes a factor of lowering the S / N ratio.
  • a non-specific complex may be formed during the storage period to generate noise. There is.
  • the first antibody and the complex recognition type antibody are retained in a solvent containing a protein denaturant to recognize the first antibody and the complex.
  • Non-specific binding to type antibody can be suppressed. Therefore, for example, even when an antibody solution in which the first antibody and the complex recognition type antibody are mixed is used for measurement after long-term storage, the detection of noise from the labeling substance of the non-specific complex is suppressed. , The S / N ratio can be improved.
  • Sandwich ELISA3 This embodiment can be suitably implemented as a sandwich ELISA including, for example, the following steps. [B1a] To prepare an antibody solution by dissolving the first antibody and the complex recognition type antibody in a solvent containing a protein denaturant; [B1b] Replacing the solvent of the antibody solution with a solvent containing no protein denaturant; [B2] The antibody solution after solvent replacement and the sample are mixed, and the target substance, the first antibody, and the complex recognition type antibody are brought into contact with each other to form a multiple complex; [B3] To measure the multiplex complex.
  • step [4] Using a target substance of a known concentration as a sample, perform step [B1a] -step [B2] to prepare a calibration curve showing the relationship between the target substance concentration and the measured value of the multiple complex; and [ 5] Determine the concentration of the target substance in the sample from the calibration curve created in the calibration curve preparation step and the measured value obtained by the measurement in step [B3].
  • this embodiment can also be preferably implemented as a latex agglutination method including, for example, the following steps.
  • [B1a] To prepare an antibody solution by dissolving the first antibody to which the insoluble carrier particles are bound and the complex recognition type antibody to which the insoluble carrier particles are bound in a solvent containing a protein denaturing agent; [B1b] Replacing the solvent of the antibody solution with a solvent containing no protein denaturant; [B2] The antibody solution after solvent replacement and the sample are mixed, and the target substance, the first antibody to which the insoluble carrier particles are bound, and the complex recognition type antibody to which the insoluble carrier particles are bound are brought into contact with each other to cause aggregation.
  • the immunoassay kit includes the following (1)-(3).
  • the protein denaturant may be provided alone in the form of a solid or liquid in a suitable container.
  • one or more of a first antibody solution, a complex recognition type antibody solution, a solvent for redissolving the first antibody and / or a complex recognition type antibody, or a solvent for diluting a sample containing a target substance It may be provided by being dissolved in the liquid of the above and contained in a suitable container.
  • the protein denaturant is one or more of a sample supply part, a labeled antibody holding part, a developing part and a detection part, and / or a solvent for diluting the sample. It suffices if it is included in.
  • the sample and target substance to which the immunoassay kit according to the present invention is applied, and the first antibody and complex recognition type antibody included in the kit are as described above.
  • the first antibody and the complex recognition type antibody may be provided in a suitable container as a dried product or a lysate. In the case of a dried product, the first antibody and the complex recognition type antibody are redissolved in a suitable solvent and used.
  • the first antibody and the complex recognition type antibody can be provided alone or in combination.
  • the solvent of the first antibody and / or the complex recognition type antibody and the sample is not particularly limited, but an aqueous solvent such as deionized water, distilled water, or a buffer solution is preferable.
  • the medium may contain the above-mentioned salts, metal ions, sugars, preservatives, proteins, protein stabilizers and the like.
  • the concentration of the first antibody when used is not particularly limited, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09. , 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 ⁇ g / mL and the like, preferably in the range of 0.1 to 20 ⁇ g / mL.
  • the concentration of the complex recognition antibody when used is not particularly limited, and is 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.
  • the first antibody and the complex recognition type antibody may be provided by being immobilized on an insoluble carrier.
  • the protein denaturant may be the chaotropic denaturant, surfactant and / or reducing agent described above.
  • concentration of the protein denaturant in the solution in which the first antibody and the complex recognition antibody are in contact is, for example, 0.0005% (w / v) or more and 48% (w / v) or less, preferably 0.001%. (W / v) or more and 36% (w / v) or less, more preferably 0.0025% (w / v) or more and 24% (w / v) or less, which is included in the kit. ..
  • the concentration of the protein denaturant in the solution is, for example, 0.1M, 0.2M, 0.3M, 0.4M, 0.5M, 0.6M, 0.7M, 0.8M. , 0.9M, 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M, for example, 0.1M or more and 8M or less, preferably 0.5M or more and 6M or less, more preferably 2M or more and 4M or less. ..
  • the concentration of the protein modifier in the solution is, for example, 0.0005%, 0.001%, 0.0025%, 0.005%, 0.01%.
  • Examples thereof include 4%, 4.5%, 5%, 6%, 7%, 8%, 9% and 10%, for example, 0.0005% or more and 10% or less, preferably 0.001% or more and 5% or less. It can be more preferably 0.0025% or more and 2.5% or less, and particularly preferably 0.25% or more and 0.5% or less.
  • the immunoassay kit may contain a cleaning solution, but the immunoassay method according to the present invention can preferably omit the cleaning step, and thus can be free of the cleaning solution.
  • the immunoassay kit comprises, for example, the following components.
  • the immunoassay kit according to the present invention comprises, for example, the following components.
  • the immunoassay kit comprises, for example, the following components.
  • a test strip with a sample supply section, a labeled antibody holding section, a developing section, a detection section and an absorption section.
  • the first antibody that is eluted and retained in the labeled antibody retainer.
  • a complex recognition type antibody immobilized on the detection part.
  • the immunoassay kit may include, for example, the following components.
  • a test strip comprising a sample supply section, a labeled antibody holding section containing a protein denaturant, a developing section, a detecting section and an absorbing section.
  • the first antibody that is eluted and retained in the labeled antibody retainer.
  • a complex recognition type antibody immobilized on the detection part.
  • the immunoassay kit comprises, for example, the following components.
  • the immunoassay kit according to the present invention comprises, for example, the following components.
  • the immunoassay kit comprises, for example, the following components.
  • a test strip with a sample supply section, a labeled antibody holding section, a developing section, a detection section and an absorption section.
  • a complex-recognizing antibody that is eluted and retained in the labeled antibody retainer.
  • the first antibody immobilized on the detection part.
  • the immunoassay kit may include, for example, the following components.
  • a test strip comprising a sample supply section, a labeled antibody holding section containing a protein denaturant, a developing section, a detecting section and an absorbing section.
  • a complex-recognizing antibody that is eluted and retained in the labeled antibody retainer.
  • the first antibody immobilized on the detection part.
  • the immunoassay kit according to the present invention comprises, for example, the following components.
  • First antibody Complex recognition type antibody.
  • the immunoassay kit according to the present invention comprises, for example, the following components.
  • the reagents commonly used in this example are as follows. Aldosterone (manufactured by Sigma-Aldrich) Disodium hydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Inc.) Sodium dihydrogen phosphate (phosphate buffer; manufactured by Kanto Chemical Co., Inc.) Bovine serum albumin (BSA: manufactured by Oriental Yeast Co., Ltd.) Tris hydroxymethylaminomethane (Tris: manufactured by Kanto Chemical Co., Inc.) Sodium chloride (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Urea (manufactured by Kanto Chemical Co., Inc.) Saccharose (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Sodium cholic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) Casein (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.)
  • Hybridoma cells were suspended in RPMI1640 medium (manufactured by Thermo Fisher Scientific) containing 10% FCS and HAT, and then seeded and cultured on a microwell plate. The culture supernatant of the grown hybridoma cells was obtained.
  • Hybridoma cells KTM-2012 were cultured in HyClone SFM4MAb-Utility medium (manufactured by Global Life Science Technologies Japan). The cell suspension was collected and centrifuged at 3000 rpm and 4 ° C. for 20 minutes, and the culture supernatant was collected. Harlow et. al. , Antibodies: A Laboratory Manual, Cold Spring Harbor, NY: The anti-aldosterone monoclonal antibody KTM-2012 was purified from the culture supernatant using Protein A Sepharose (manufactured by Global Life Science Technologies Japan) according to the method described in 1988.
  • Protein A Sepharose manufactured by Global Life Science Technologies Japan
  • F (ab') 2 and aldosterone were mixed in a PBS solution at a molar ratio of 1: 540 and left at 25 ° C. for 1 hour to prepare an antigen solution.
  • An emulsion was prepared by mixing an equal amount of the antigen solution and the adjuvant.
  • the emulsion was subcutaneously injected into Balb / c mice (manufactured by Nippon SLC Co., Ltd.) three times in total.
  • Freund's Complete Adjuvant manufactured by Sigma-Aldrich was used as an adjuvant.
  • Freund's Incomplete Adjuvant manufactured by Sigma-Aldrich
  • Blood was collected after immunization a total of 3 times at 2-week intervals, and the antibody titer in serum was evaluated by ELISA according to the procedure (2) described later. After confirming that the antibody titer in serum was elevated, an equal amount of PBS solution and antigen solution were injected subcutaneously into the mouse, and the spleen was removed from the mouse 3 days later.
  • the reaction was carried out to form a complex of KTM-2012 antibody and aldosterone.
  • a well to which only the sample diluent was added was prepared. After washing the wells three times (B / F separation) with a washing solution (PBS (pH 7.4) containing 0.05% tween 20), serum collected from immunized mice is diluted 10000 times with a sample diluent. The serum diluent was added to the wells (50 ⁇ L) and reacted at 25 ° C. for 1 hour.
  • hybridoma by cell fusion
  • the spleen was excised from the mouse in which an increase in antibody titer was confirmed, and spleen cells were prepared according to a conventional method.
  • a hybridoma was prepared by fusing spleen cells with mouse myeloma cells (P3-U1) by an electrofusion method using a super cell fusion device EGFG21 (manufactured by Neppagene).
  • Hybridoma cells were suspended in 10% BM Conditioned H1 Hybridoma Cloning Supplement (10 ⁇ ) (manufactured by Roche Diagnostics) and HAT-containing GIT medium (manufactured by Kojin Bio), and then seeded and cultured on a microwell plate. .. The culture supernatant of the grown hybridoma cells was obtained.
  • the culture supernatant obtained in (3) was diluted 2-fold with a sample diluent and added to the wells (50 ⁇ L), and the mixture was added to the wells at 25 ° C. for 1 hour. It was reacted.
  • a peroxidase-labeled anti-mouse IgG antibody manufactured by Millipore
  • diluted 10000 times with a sample diluent was added to the wells (50 ⁇ L), and the temperature was 25 ° C. for 1 hour. It was reacted.
  • hybridoma cell line KTM-611 producing a monoclonal antibody against the aldosterone-anti-aldosterone antibody complex was established.
  • Hybridoma KTM-611 is a depositary organization based on the provisions of Article 27-2 and 3 of the Patent Law Enforcement Regulations, and an international depositary authority based on the Budapest Treaty on International Approval of Deposits of Microorganisms. It has been internationally deposited at the Japan Biotechnology Center, Patented Microbial Deposit Center (NPMD) (Room 122, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture).
  • NPMD Patented Microbial Deposit Center
  • KTM-611 were cultured in Hybridoma-SFM medium (manufactured by Thermo Fisher Scientific). The cell suspension was collected and centrifuged at 3000 rpm and 4 ° C. for 20 minutes, and the culture supernatant was collected. Harlow et. al. , Antibodies: A Laboratory Manual, Cold Spring Harbor, NY: Monoclonal antibody against aldosterone-anti-aldosterone antibody complex from culture supernatant using protein A Sepharose (manufactured by Global Life Science Technologies Japan) according to the method described in 1988. KTM-611 was purified.
  • Example 1 Preparation of aldosterone measuring reagent 1 An aldosterone measuring reagent 1 containing the following KTM-611 antibody-immobilized membrane and a QuartzDot (QD) -labeled KTM-2012 antibody solution was prepared.
  • KTM-611 antibody-immobilized membrane As the membrane, a Hi-Flow Plus HF135 (Millipore company system) cut into a width of 2.5 cm and a length of 25 cm was used. A detection region is formed by linearly applying KTM-611 antibody at Rate 1.0 ⁇ L / cm using a BioJet Quonti dispensing module (manufactured by BioDot) at a position 11.5 mm away from one end in the minor axis direction of the membrane. bottom. After application, it was dried at 25 ° C. for 2 hours.
  • a BioJet Quonti dispensing module manufactured by BioDot
  • aldosterone measuring reagent 2 Preparation of aldosterone measuring reagent 2 An aldosterone measuring reagent 2 containing the following KTM-2012 antibody-immobilized membrane and QD-labeled KTM-611 antibody solution was prepared.
  • KTM-2012 antibody-immobilized membrane As the membrane, a Hi-Flow Plus HF135 cut into a width of 2.5 cm and a length of 25 cm was used. A detection region was formed by linearly applying the KTM-2012 antibody at a rate of 1.0 ⁇ L / cm using a BioJet Quonti dispensing module at a position 11.5 mm away from one end in the minor axis direction of the membrane. After application, it was dried at 25 ° C. for 2 hours. After drying, it was immersed in the blocking buffer for 30 minutes and then in the washing solution for 30 minutes. The washed membrane was dried in a desiccator overnight or longer.
  • a membrane and CELLULOSE FIBER SAMPLE PADS were immobilized on a precut backing sheet, and the sheet was cut to a width of 5 mm to prepare a KTM-2012 antibody-immobilized membrane.
  • Table 1 shows the results (fluorescence intensity and S / N ratio) of measuring the sample mixture and the blank mixture using the aldosterone measuring reagents 1 to 4.
  • the S / N ratio was calculated by the following formula.
  • S / N ratio Fluorescence intensity of sample mixture / Fluorescence intensity of blank mixture
  • the S / N ratio was 1.0 or more in the measurement using the aldosterone measuring reagents 1 and 2 (the labeled antibody diluent contained urea).
  • the fluorescence intensity of the blank mixture is higher than that of the sample mixture, and the S / N ratio is 1.0.
  • the measurement noise caused by the binding between the KTM611 antibody and the KTM2012 antibody was considered to be the cause of the decrease in the S / N ratio. From these results, it is clear that the use of urea suppresses non-specific binding between the KTM611 antibody and the KTM2012 antibody and improves the measurement accuracy.
  • aldosterone measuring reagent 1B good measurement was possible in the range of aldosterone concentration of 31 to 2000 pg / mL.
  • the aldosterone measuring reagent 3 Comparative Example 1
  • strong fluorescence intensity was measured even in the blank mixed solution, and quantitative measurement of aldosterone was impossible.
  • Aldosterone was diluted with the sample diluent described in Test Example 1 (1) to prepare 62, 125, 250, 500, 1000, 2000 and 4000 pg / mL aldosterone samples.
  • the sample diluent was used as a blank sample (aldosterone concentration 0 pg / mL).

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Abstract

En tant que technologie permettant de mesurer avec précision la concentration de substances telles que des hormones peptidiques et des haptènes contenus dans un échantillon, l'invention concerne un procédé d'immunoessai étant destiné à une substance dans un échantillon et étant basé sur la détection d'un complexe multiple comprenant la substance cible, un premier anticorps reconnaissant la substance cible, et un anticorps de type à reconnaissance de complexe reconnaissant spécifiquement un complexe entre la substance cible et le premier anticorps. Le procédé comprend une étape de mise en contact du premier anticorps avec l'anticorps de type à reconnaissance de complexe en présence d'un agent de dénaturation de protéine.
PCT/JP2021/009722 2020-03-12 2021-03-11 Procédé et kit pour immunoessai de substance cible dans un échantillon WO2021182558A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63112599A (ja) * 1986-10-09 1988-05-17 ベーリングヴェルケ・アクチエンゲゼルシャフト 免疫複合体レセプター
JPH09507910A (ja) * 1993-12-10 1997-08-12 チバ−ガイギー アクチェンゲゼルシャフト 同時でのサンプル抽出及び免疫原性反応を含む抗体又は抗原の検査のための迅速イムノアッセイ
WO2019098314A1 (fr) * 2017-11-17 2019-05-23 富士レビオ株式会社 Liquide de traitement destiné à être utilisé dans la désorption d'un stéroïde inclus dans la cyclodextrine

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63112599A (ja) * 1986-10-09 1988-05-17 ベーリングヴェルケ・アクチエンゲゼルシャフト 免疫複合体レセプター
JPH09507910A (ja) * 1993-12-10 1997-08-12 チバ−ガイギー アクチェンゲゼルシャフト 同時でのサンプル抽出及び免疫原性反応を含む抗体又は抗原の検査のための迅速イムノアッセイ
WO2019098314A1 (fr) * 2017-11-17 2019-05-23 富士レビオ株式会社 Liquide de traitement destiné à être utilisé dans la désorption d'un stéroïde inclus dans la cyclodextrine

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