WO2023163155A1 - 検出用デバイス - Google Patents

検出用デバイス Download PDF

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Publication number
WO2023163155A1
WO2023163155A1 PCT/JP2023/006958 JP2023006958W WO2023163155A1 WO 2023163155 A1 WO2023163155 A1 WO 2023163155A1 JP 2023006958 W JP2023006958 W JP 2023006958W WO 2023163155 A1 WO2023163155 A1 WO 2023163155A1
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Prior art keywords
antibody
detection device
sample
target substance
control
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English (en)
French (fr)
Japanese (ja)
Inventor
亮吾 東
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Kaneka Corp
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Kaneka Corp
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Priority to JP2024503282A priority Critical patent/JPWO2023163155A1/ja
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a detection device for detecting target substances in samples.
  • a substance (antibody/antigen) that specifically binds to the target substance is used to induce an antigen-antibody reaction to detect the target substance.
  • Immunoassays for detection are widely available.
  • a capture substance (antibody/antigen) that specifically binds to the target substance is immobilized at a predetermined position on a flaky carrier, and the sample is placed thereon with the labeled antibody.
  • An immunochromatographic method is known in which a target substance is developed in a mixed state, captured at the predetermined position, and visually detected (for example, Patent Document 1). This method does not require special equipment, etc., and can obtain detection results in about 5 to 30 minutes. Therefore, it is widely used mainly in clinical practice to aid diagnosis of infectious diseases.
  • a detection device used for immunochromatography is usually provided with a control section at a predetermined position on the carrier for confirming that the sample/reagent has been normally developed on the carrier.
  • a control section There are various configurations of the control section, but most of the currently used ones have a configuration in which an antibody (control antibody) that captures unreacted labeled antibody is immobilized.
  • Immunochromatography is usually performed by using a long thin strip-shaped thin piece as a solid phase carrier, and a capture portion is provided by immobilizing a capture substance in a line perpendicular to the longitudinal direction of the solid phase carrier, and the control is provided in parallel with the capture portion.
  • a portion is provided on the downstream side in the deployment direction (for example, Patent Document 1).
  • a detection device for detecting a target substance in a sample a first portion which is a solid-phase carrier comprising a target substance capturing portion and a control portion; a second portion containing a labeled antibody that specifically binds to the target substance; Capture molecules that specifically bind to the target substance are immobilized on the target substance capture portion, A control antibody that specifically binds to the labeled antibody is immobilized on the control portion, A detection device, wherein the capture molecules and the labeled antibodies are derived from different organisms, and the immobilized amount of the control antibody on the first portion is less than the immobilized amount of the capture molecules.
  • a detection device for detecting two or more target substances in a sample a first portion that is a solid-phase carrier comprising two or more target substance-capturing portions and one or more control portions; a second portion comprising two or more labeled antibodies; The two or more labeled antibodies specifically bind to different target substances, Capturing molecules that specifically bind to different target substances are immobilized on the two or more target substance capturing portions, A control antibody that specifically binds to one or more of the two or more labeled antibodies is immobilized on the one or more control regions, At least one of the capture molecules and the labeled antibody are derived from different organisms, and the immobilized amount of the control antibody in each control portion on the first portion is equal to the immobilized amount of the capture molecule in each target substance capturing portion.
  • a detection device for detection comprising detecting a target substance in a sample using the detection device according to any one of [1] to [10].
  • a kit for detecting a target substance in a sample comprising the detection device according to any one of [1] to [10] and a sample pretreatment reagent.
  • FIG. 1A and 1B are a top view (A) and an aa' cross-sectional view (B) of a detection device according to a first embodiment of the present invention
  • FIG. FIG. 2A is a top view (A) and a cross-sectional view (B) taken along the line aa' of a detection device according to a second embodiment of the present invention
  • 4 is a photograph showing the colored state of the solid phase carriers of Example 2 and Comparative Example 3.
  • FIG. 10 is a graph showing the relationship between the color intensity of the control line of the negative samples produced in Examples 4 and 5 and Comparative Examples 5 and 6 and the elapsed time.
  • 2 is a photograph showing the colored state of solid phase carriers of Examples 4 and 5 and Comparative Examples 5 and 6.
  • FIG. 2 is a photograph showing the colored state of the solid phase carriers of Examples 6 and 7 and Comparative Example 7.
  • molar concentration refers to the molar amount per unit volume of solution (mol/dm 3 ) unless otherwise specified.
  • % concentration refers to weight concentration (wt %) unless otherwise specified.
  • antibody refers not only to intact antibodies, but also to antigen-specific binding fragments such as Fab, Fab' and F(ab') 2 .
  • the detection device of the first embodiment of the present invention is a detection device for detecting a target substance in a sample and having the following characteristics: target
  • the first part is a solid phase carrier comprising a substance capturing part and a control part
  • the second part contains a labeled antibody that specifically binds to the target substance.
  • a capture molecule that specifically binds is immobilized
  • a control antibody that specifically binds to the labeled antibody is immobilized on the control portion
  • the organisms derived from the capture molecule and the labeled antibody are different, and
  • the immobilized amount of the control antibody on the first portion is less than the immobilized amount of the capture molecule.
  • the detection device of this embodiment is capable of more accurate detection due to the features described above. More specifically, it is possible to prevent defective coloring of the control portion and suppress coloring of the background.
  • sample is not particularly limited as long as it may contain the target substance, but it can be a sample taken from a living subject, that is, a biological sample.
  • subject refers to mammals and birds. Birds refer to animals belonging to the subphylum Avians of the phylum Chordata, subphylum Vertebrates, and include, for example, chickens, ducks, quails, geese, ducks, turkeys, budgerigars, parrots, mandarin ducks, and swans. Mammals refer to animals belonging to the subphylum Mammalia, phylum Chordata, subphylum Mammalia, including humans and non-humans.
  • Examples include livestock animals such as sheep and goats, rodents such as mice and rats, and mammals kept in zoos.
  • the subject herein is preferably human.
  • biological samples include, but are not limited to, nasal swabs, pharyngeal swabs, nasal discharge, saliva, sputum, gargle, blood (e.g., whole blood, serum, plasma), urine, feces, milk, tissue or cells. It can be an extract or a mixture thereof.
  • the sample may be subjected to pretreatment such as dissolution, dilution, filtration, centrifugation, solvent extraction, etc., if necessary.
  • test sample a sample that can be directly loaded onto a detection device is referred to as a "test sample", regardless of the presence or absence of pretreatment.
  • the "target substance” is not particularly limited as long as it can bind to any of the capture molecules described below, and may be any of polypeptides, polysaccharides, low-molecular compounds, and the like. Polypeptides are preferred.
  • biomarkers that exist in a biological sample and can assist in diagnosing a subject's health condition, disease, or the like based on the presence or absence thereof are preferred.
  • target substances include polypeptides derived from infectious disease pathogens such as influenza virus, SARS-CoV-2, Zika virus, tuberculosis, and pneumococcus, disease markers such as tumor markers, sex hormones, antibodies (IgG, IgM etc.) are known, but are not limited to these.
  • the detection device of the present embodiment is particularly suitable for detection of polypeptides derived from pathogens of infectious diseases and detection of acute disease markers, for which rapid treatment policy determination is required in clinical practice.
  • FIG. 1 shows the configuration of an example of the detection device of this embodiment.
  • FIG. 1A is a top view of an example of a chromatographic apparatus
  • FIG. 1B is a cross-sectional view taken along the line aa' of FIG. 1A.
  • the detection device shown in FIG. 1 has at least a labeled antibody holding portion 12 and a solid phase carrier 13.
  • the solid phase carrier 13 has a target substance capturing portion 13a and a control portion 13b.
  • the detection device of this embodiment may include a sample receiving section.
  • the sample receiving portion 11 is, for example, a sample pad, and is a portion onto which a test sample is loaded (eg dropped).
  • a sample pad for example, a glass fiber sample pad or a cellulose fiber sample pad can be used.
  • the test sample loaded in the sample receiving portion 11 is deployed rightward.
  • the solid phase carrier 13 of the detection device 10 corresponds to the first part of this embodiment.
  • the solid-phase carrier 13 is, for example, a thin piece such as a membrane, and is a member on which a mobile phase containing labeled antibodies capable of binding to a test sample and a target substance is developed. When the target substance is present in the test sample, the mobile phase contains a complex of the target substance and the labeled antibody on the solid phase carrier 13 .
  • the shape of the solid-phase carrier 13 is not particularly limited, but it is particularly preferable to have a long and narrow strip-like shape as shown in the figure. Examples of membranes that can be used include nitrocellulose membranes, cellulose membranes, cellulose acetate membranes, polyethersulfone membranes, nylon membranes, polyester membranes, and glass fiber membranes. .
  • the target substance-capturing portion 13a on the solid-phase carrier 13 is a region where capture molecules capable of binding to the target substance are immobilized. It is a line-shaped part that has been When the test sample contains a target substance, in the target substance capturing section 13a, a complex of the target substance and a labeled antibody described later binds to the capture molecule to form a three-component complex of labeled antibody-target substance-capture molecule. A sandwich-type complex is formed.
  • any capture molecule may be used as long as it can bind to the target substance.
  • the target substance is an antigen such as a polypeptide
  • an antibody against the antigen can be used as the capture molecule.
  • the target molecule may be a monoclonal antibody or a polyclonal antibody.
  • the target molecule is an antibody (IgG and/or IgM)
  • the antigen for that antibody can be used as the capture molecule.
  • a receptor, lectin, or the like that can bind to the target substance can be used.
  • These capturing molecules can be appropriately selected according to the type of target substance.
  • the capture molecule is an antibody, more preferably an IgG antibody, whose antigen is the target substance. That is, a preferred aspect of this embodiment is an immunoassay device, and further an immunochromatography device using the principle of chromatography.
  • the organism from which the capture molecule is derived must be different from the labeled antibody described below. It is not particularly limited as long as it is derived from a different organism from the labeled antibody. Examples include mice, rats, rabbits, goats, cows, pigs, sheep, dogs, cats, monkeys, camels, alpacas, birds, fish, and plants (lentils, etc.). may be either.
  • the capture molecule is derived from rabbits.
  • the capture molecule may contain multiple species of derived organisms, but at least one or more of the multiple species should be different from the labeled antibody.
  • the capture molecule is preferably other than a mouse antibody.
  • antibody drugs are used for immunosuppression and for the treatment and prevention of recurrence of malignant tumors.
  • Antibody drugs are mainly classified into four types: mouse antibodies, chimeric antibodies, humanized antibodies, and human antibodies. be.
  • HAMA Human Anti-Mouse Antibody
  • HAMA Human Anti-Mouse Antibody
  • this HAMA may bind to mouse molecules and cause false positives and the like. Therefore, it is preferable to use a capturing molecule other than a mouse antibody to avoid problems such as false positives.
  • the solid phase carrier 13 has a control section 13b.
  • the control section 13b is a site that captures labeled antibodies, particularly free labeled antibodies that have not formed a complex between the target substance and the labeled antibodies, and is usually referred to as a control line.
  • the control section 13b is normally located downstream of the target substance capturing section 13a, as shown in the figure.
  • the control section 13b located downstream of the target substance capturing section 13a may capture surplus complexes that could not be captured by the target substance capturing section 13a.
  • the control portion 13b is a portion containing an antibody that specifically binds to the labeled antibody, and particularly a portion containing an antibody capable of capturing the antibody portion of the labeled antibody.
  • An antibody that specifically binds to the labeled antibody corresponds to the control antibody in the detection device of this embodiment.
  • the control antibody may be a monoclonal antibody or a polyclonal antibody as long as it specifically binds to the labeled antibody.
  • a control antibody can be, for example, an anti-IgG antibody.
  • the origin of the control antibody is not particularly limited, and may be, for example, mouse, rat, rabbit, goat, cow, pig, sheep, dog, cat, monkey, camel, alpaca, bird, fish, or the like.
  • the organism of origin of the control antibody is preferably different from the organism of origin of the capture molecule.
  • the organism from which the control antibody is derived is preferably different from the organism from which the labeled antibody described below is derived.
  • the organisms of origin of the capture molecule, control antibody and labeled antibody are all different.
  • the control antibody is derived from goats.
  • the control antibody may contain more than one species of derived organism, but preferably at least one or more of the multiple species is different from the capture molecule. Alternatively, it is preferable that at least one or more derived organisms out of a plurality of species are different from the labeled antibody described below.
  • the immobilized amount of the control antibody on the control portion 13b of the solid phase carrier 13 needs to be less than the immobilized amount of the captured molecules on the target substance capturing portion 13a.
  • the "immobilized amount” is preferably compared using the total number of moles of molecules (capture molecule/control antibody) immobilized on the solid phase carrier as an indicator. However, when one capture molecule binds to a plurality of target molecules, it is necessary to compare the total number of moles of theoretically bindable target molecules. In the detection device 10 of the illustrated example, the areas of the target substance capturing portion 13a and the control portion 13b are the same.
  • the molar concentration of the solution coated on the solid-phase carrier can be used as an indicator for comparison. It is possible. Alternatively, if the capture molecules and the control antibody have approximately the same molecular weight, for example, both are intact antibodies, the total weight of the molecules immobilized on the solid-phase carrier can be used as an indicator for comparison. Furthermore, if the area of the coating region is the same, it is also possible to compare using the weight concentration (wt %) as an index.
  • the immobilized amount of the control antibody on the solid-phase carrier is 0.1 to 0.9 times, particularly 0.15 to 0.85 times, further 0.2 to 0.8 times the immobilized amount of the capture molecule. It is preferable to
  • the amount of capture molecules immobilized on the solid-phase carrier is not particularly limited, and can be changed as appropriate depending on the size and shape of the device, the type of target substance, and the type and condition of the sample.
  • the capture molecule is an intact antibody, it may be 0.001-10.0 ⁇ g/mm 2 , especially 0.01-1.0 ⁇ g/mm 2 , even 0.1-0.5 ⁇ g/mm 2 . can.
  • a small amount of capture molecules immobilized on the target substance capturing portion may be released and move downstream.
  • the capturing molecule and the labeled antibody are derived from the same organism, especially when the capturing molecule is an antibody, the captured molecule released and migrated downstream may have a structure similar to that of the labeled antibody.
  • a problem may arise that the binding site of the control antibody is masked by binding to the control antibody that has been detected. As a result, binding of the labeled antibody to the control portion is inhibited, resulting in insufficient coloration of the control portion, making it difficult to determine whether detection was performed normally.
  • the detection device of the present embodiment since the capture molecules and the labeled antibodies are derived from different organisms, even if the capture molecules are released from the target substance capture portion, the risk of masking the control antibody is extremely low. As described above, the detection device of the present embodiment has a configuration in which defective coloring of the control portion is less likely to occur.
  • control section is arranged further downstream in order to prevent the labeled antibody from stagnation upstream of the control section, background coloration is certainly less likely to occur.
  • placing the control section on the downstream side slows down the development speed of the test sample in the control section, further increasing the risk that the capture molecules released and moved downstream will mask the binding sites of the control antibody. Become.
  • the binding of the labeled antibody to the control portion is inhibited, resulting in insufficient coloration of the control portion, making it difficult to determine whether the detection was performed normally.
  • the amount of solid phase in the control portion by making the amount of solid phase in the control portion smaller than that in the target substance capturing portion, excessive coloring of the control portion is suppressed, while coloring of the target substance capturing portion is increased. can be made certain.
  • the first part (the solid phase carrier 13 in the detection device 10) of this embodiment preferably contains sugars and/or sugar alcohols.
  • sugars include, but are not limited to, sucrose, glucose, galactose, lactose, palatinose, maltose, isomaltose, trehalose, lactosucrose, maltotriose, maltotetraose, cyclodextrin, cyclodextran, and dextran.
  • Sugar alcohols include, but are not limited to, erythritol, sorbitol, maltitol, isomaltitol, lactitol, palatinit and the like. At least one of these sugars and sugar alcohols is preferably contained.
  • Sugars and sugar alcohols have the effect of stabilizing polypeptides such as antibodies in a dry state.
  • the sugar and/or sugar alcohol is preferably contained in at least one of the first portion and the second portion described below, particularly both.
  • the labeled antibody holding portion 12 of the detection device 10 corresponds to the second portion of this embodiment.
  • the labeled antibody holding part 12 is, for example, a conjugate pad, and is a member containing a labeled antibody that specifically binds to the target substance.
  • a labeled antibody refers to an antibody bound to any known labeling substance such as colored particles, enzymes, biotin, digoxigenin and the like.
  • the labeling substance it is preferable to use colored particles because they are particularly strongly colored and the results can be obtained quickly.
  • Colored particles include, for example, metal particles, colored latex particles, colored polystyrene particles, colored cellulose particles, fluorescent cellulose particles, and pigment-containing silica nanoparticles.
  • metal particles examples include colloidal metal particles such as colloidal gold particles, colloidal silver particles, and colloidal platinum particles.
  • conjugate pad for example, a glass fiber conjugate pad, a cellulose fiber conjugate pad, a polyester fiber conjugate pad, a polyethylene fiber conjugate pad, a polypropylene fiber conjugate pad, or the like is used. be able to.
  • the labeled antibody may be either a polyclonal antibody or a monoclonal antibody as long as it can bind to the target substance.
  • the biological origin of the antibody used for the above-mentioned labeled antibody must be different from that of the capture molecule. If the antibody is derived from a different organism from the capturing molecule, the antibody is usually derived from an organism used in immunoassays, such as mice, rats, rabbits, goats, cows, pigs, sheep, dogs, cats, monkeys, and camels. , alpaca, birds, fish, and the like. In addition, the organism from which the labeled antibody is derived is preferably different from that of the control antibody. Preferably, the organism of origin of the labeled antibody is mouse. In addition, although the labeled antibody may contain a plurality of derived organisms, it is required that at least one of the plurality of derived organisms is different from the capture molecule.
  • the second portion (the labeled antibody holding portion 12 in the detection device 10) of the present embodiment preferably contains saccharides and/or sugar alcohols.
  • sugars include, but are not limited to, sucrose, glucose, galactose, lactose, palatinose, maltose, isomaltose, trehalose, lactosucrose, maltotriose, maltotetraose, cyclodextrin, cyclodextran, and dextran.
  • Sugar alcohols include, but are not limited to, erythritol, sorbitol, maltitol, isomaltitol, lactitol, palatinit and the like.
  • sugars and sugar alcohols are preferably contained.
  • Sugars and sugar alcohols have the effect of stabilizing polypeptides such as antibodies in a dry state.
  • colloidal metal particles are used as a labeling substance for labeled antibodies, they have the effect of suppressing aggregation of particles.
  • Sugars and/or sugar alcohols are preferably contained in at least one, especially both, of the second portion and the first portion.
  • the detection device 10 preferably has an absorbent pad 14.
  • the absorbent pad 14 holds the mobile phase so that it does not flow back after the mobile phase developed upstream, that is, from the sample receiving portion 11 on the left side of the detection device 10 toward the downstream side, that is, toward the right side, passes through the solid phase carrier 13 .
  • the absorbent pad for example, an absorbent pad made of cellulose fiber, an absorbent pad made of glass fiber, or an absorbent pad made of polystyrene fiber can be used.
  • the detection device 10 preferably has a backing sheet 15.
  • the backing sheet 15 may have an adhesive layer on a part of the upper surface or the front surface, and the solid phase carrier 3 may be fixed on the upper surface.
  • one or more of the sample receiving portion 11 , the labeled antibody holding portion 12 and the absorbent pad 14 may be fixed to the backing sheet 15 .
  • all of the sample receiving portion 11, the labeled antibody holding portion 12 and the absorbent pad 14 may be fixed to the backing sheet 15 as shown in the illustrated example.
  • the backing sheet for example, a backing sheet made of polypropylene, a backing sheet made of polystyrene, a backing sheet made of polyester, and a backing sheet made of vinyl chloride can be used.
  • the detection device 10 is not particularly limited in terms of shape and size. can be The shape and size can be changed as appropriate.
  • the detection device 10 has a labeled antibody holding portion 12, a solid phase carrier 13, and preferably a housing case (see Fig. not shown).
  • the housing case is made of a water-impermeable moldable material, such as polyethylene, polypropylene, polystyrene, polyethylene terephthalate, polyvinyl chloride, etc., and covers the entire test strip and encloses the sample receiver 11 and the target substance. It has a shape in which an opening or window is provided at a position corresponding to the capturing portion 13a, preferably further at a position corresponding to the control portion 13b. By providing the housing, the developed mobile phase can be prevented from leaking.
  • the detection device 10 of the illustrated example is a lateral flow type device in which the test sample develops in a substantially horizontal direction, but the detection device of the present embodiment is not limited to this format. It may be a strip-type device in which the end of the test piece is directly immersed and the test sample is spread using capillary action.
  • the first portion and the second portion are formed of separate members, but they may be formed of one member, and furthermore, if necessary, the sample receiving portion and the absorbent pad may be formed. One or more of them may be combined with the first portion and the second portion to form one member.
  • a second embodiment of the present invention is a detection device for detecting two or more target substances in a sample, which has the following characteristics: A detection device for detecting more than one type of target substance, comprising: a first portion that is a solid-phase carrier having two or more target substance-capturing portions and one or more control portions; and two or more types of labeled antibodies. and a second portion, wherein the two or more labeled antibodies specifically bind to different target substances, and the two or more target substance-capturing portions contain capture molecules that specifically bind to different target substances.
  • a control antibody that specifically binds to one or more of the two or more labeled antibodies is immobilized on the one or more control parts, and the captured molecule and the organism derived from the labeled antibody are immobilized. It is different, and the immobilized amount of control antibody in each control portion on the first portion is smaller than the immobilized amount of capturing molecules in each target substance capturing portion.
  • the detection device of this embodiment has the above-described features, so that it has the advantage of the first embodiment, that is, it can prevent coloring defects in the control section and suppress background coloring. In addition, it has the advantage of being able to detect two or more types of target substances in a sample with one detection device.
  • the two or more target substances referred to here are not particularly limited, and the above-described capture molecules (e.g., influenza virus, SARS-CoV-2, Zika virus, tuberculosis, pneumococcus, and other infectious disease pathogens-derived polypeptides, Any combination of disease markers such as tumor markers, sex hormones, antibodies (IgG, IgM, etc.) may be used, for example, by combining multiple disease markers with similar symptoms, these diseases It can be used to assist differential diagnosis.
  • disease markers such as tumor markers, sex hormones, antibodies (IgG, IgM, etc.
  • Such marker combinations include, for example, SARS-CoV-2 antigen and influenza virus antigen (type A and/or type B).
  • FIG. 2 shows the configuration of an example of the detection device of this embodiment.
  • FIG. 2A is a top view of an example of a chromatographic apparatus
  • FIG. 2B is a cross-sectional view taken along the line aa' of FIG. 1A.
  • the detection device 20 shown in FIG. 2 has a labeled antibody holding portion 22 and a solid phase carrier 23.
  • the solid phase carrier 23 has two target substance capturing portions 23a and 23a' and a control portion 23b. .
  • the configuration of the sample receiving section 21 of the detection device 20 is common to the configuration of the sample receiving section in the detection device of the first embodiment.
  • the solid phase carrier 23 of the detection device 20 corresponds to the first part of this embodiment.
  • the two target substance-capturing portions 23a and 23a' on the solid-phase carrier 23 are sites on which capture molecules capable of binding to different target substances are immobilized. According to the detection device 20, it is possible to capture two different target molecules in the sample with different target substance capture portions.
  • the detection device 20 in the illustrated example is provided with two target substance capturing portions, three or more target substance capturing portions may be provided in the present embodiment.
  • the preferred number of target substance capturing portions is 2-6, especially 2-4.
  • the solid phase carrier 23 has a control section 23b.
  • the control section 23b is normally located downstream of the target substance capturing sections 23a and 23a' as shown in the illustrated example.
  • the control unit 23b captures a free labeled antibody that does not particularly form a complex with the target substance in at least one of the two or more labeled antibodies described below.
  • the control section 23b may capture two or more types of labeled antibodies.
  • one control section is provided in the detection device 20 in the illustrated example, two or more control sections may be provided in the present embodiment.
  • the number of controls is one.
  • the source organism of the capture molecule must be different from any of the labeled antibodies described below.
  • Two or more capture molecules may be derived from the same organism as long as they are derived from different organisms than the labeled antibody. Preferably, all of the capture molecules are derived from rabbits.
  • two or more capture molecules may each contain a plurality of derived organisms, provided that at least one or more of the multiple derived organisms is different from any of the labeled antibodies.
  • the organism from which the control antibody is derived is not particularly limited, it is preferably different from any of the capture molecules.
  • the organism from which the control antibody is derived is preferably different from any of the labeled antibodies described below. If more than one control antibody is used, these control antibodies may be derived from the same organism.
  • control antibody is derived from goats.
  • control antibody (group) may contain more than one species of origin, but it is preferred that at least one of the plurality of species of origin is different from any of the two or more capture molecules.
  • at least one of the plurality of species is derived from a different organism from any of the two or more labeled antibodies.
  • the organisms of origin of the capture molecule group, the control antibody(groups) and the labeled antibody group are different from each other, but the organisms of origin within the same group are the same.
  • At least one of the capture molecules is preferably other than a mouse antibody in order to avoid the effects of HAMA. Also, it is more preferable that all of the capture molecules are other than mouse antibodies.
  • the immobilized amount of the control antibody on the control portion 23b of the solid phase carrier 23 is required to be less than the immobilized amount of any captured molecules on the target substance capturing portions 23a and 23a'.
  • the immobilized amount of the control antibody on the solid-phase carrier is 0.1 to 0.9 times, particularly 0.15 to 0.85 times, more preferably 0.15 to 0.85 times the average immobilized amount of the capturing molecules. It is preferably 2 to 0.8 times.
  • the control section As in the first embodiment, it is possible to prevent poor coloring of the control section by using different organisms for the capture molecules of the target substance capture section and the labeled antibody. Further, by making the immobilized amount of the control antibody smaller than the immobilized amount of the capturing molecule, the coloration of the target substance-capturing portion can be ensured as in the first embodiment. be.
  • the labeled antibody holding portion 22 of the detection device 20 corresponds to the second portion of this embodiment.
  • the labeled antibody holding part 22 is a member containing labeled antibodies that specifically bind to each of two or more target substances, that is, two or more labeled antibodies.
  • the organism from which the labeled antibody is derived must be a different organism from any of the two or more capture molecules.
  • two or more labeled antibodies may be derived from the same organism.
  • both of the two or more labeled antibodies are derived from mice.
  • two or more types of labeled antibodies may each contain multiple types of derived organisms, but at least one or more of the multiple types of derived organisms should be different from any of the two or more types of capture molecules.
  • the detection device 20 preferably has an absorbent pad 24.
  • the detection device 20 also preferably has a backing sheet 25 .
  • a housing case (not shown) may be provided.
  • Other configurations of the detection device of this embodiment are the same as those of the first embodiment unless there is a particular contradiction.
  • the detection method of the third embodiment of the present invention is characterized by detecting a target substance in a sample using the detection device described in the section "1. Detection device".
  • the target substance in the sample can be detected more accurately with a low risk of detection failure due to poor control coloring and false negatives due to background coloring. is.
  • the detection method of this embodiment is not particularly limited, it preferably includes the following steps.
  • Step 1) Step of preparing a test sample;
  • Step 2) A step of loading the test sample onto a predetermined position of the detection device; and
  • Step 3) A step of determining whether or not the target substance capturing portion and the control portion on the detection device are colored. Each step will be described below.
  • Step 1 Step of Preparing Test Sample
  • the detection method of the present embodiment preferably uses a sample removed from a subject.
  • subject refers to mammals and birds.
  • the “sample” referred to here is preferably a sample taken from a living subject, that is, a biological sample. Since biological samples are not necessarily suitable for development on a detection device, various methods such as dissolution, dilution, filtration, centrifugation, solvent extraction, etc., are required depending on the type and state of the biological sample and the type of target substance to be detected.
  • Pretreatment may be performed. This step includes this pretreatment step.
  • the pretreatment step is preferably a step using a sample pretreatment reagent.
  • the sample pretreatment reagent is not particularly limited, but may be a cell lysate, a sample diluent, an organic solvent, or the like.
  • a cell lysate containing SDS or the like is used to prepare the cells.
  • a step of removing the solid matter by filtration or centrifugation after diluting the sample with a sample diluent may be provided.
  • a step of extracting with an organic solvent may be provided.
  • a swab containing a nasopharyngeal swab or the like is used as a sample
  • a step of immersing the swab in a sample diluent may be provided.
  • a liquid sample such as urine or blood
  • a step of diluting the sample with a sample diluent to make the sample have a viscosity, pH, etc. suitable for development may be performed.
  • the term "test sample” refers to a sample ready for development on a detection device, with or without pretreatment.
  • Step 2 Step of Loading Test Sample into Predetermined Position of Detection Device
  • This embodiment includes a step of loading a sample into a predetermined position of the detection device.
  • a method of dropping the test sample onto the sample receiving portion 11 with a dropper, pipette, or the like can be adopted.
  • a method of immersing a portion corresponding to the sample receiving portion in a test sample such as a beaker or cup can be adopted.
  • Step 3 Step of Determining Whether or Not the Target Substance-Capturing Part and Control Part on the Detection Device Are Colored expand.
  • a developing solution can be additionally added.
  • the test sample develops from left to right.
  • the time required for deployment varies depending on the size of the device, the type and condition of the specimen, etc., but is usually about 5 to 30 minutes.
  • colored particles are used as the labeling substance, it is possible to directly confirm the coloring of the target substance capturing portion (test line) and the control portion (control line) after development.
  • a coloring component such as an enzyme substrate may be added to the additional developing solution.
  • the enzyme substrate or the like may be arranged in advance on the detection device, and the enzyme substrate or the like may be developed by loading the sample or developing solution.
  • test line and control line on the solid phase carrier are colored.
  • the determination of coloring may be performed visually, but it is also possible to determine the presence or absence of coloring using image processing software, or to quantify the strength of coloring, for example. For example, if both the test line and the control line are colored, it can be determined that the target substance has been detected. If no coloration is observed in the test line, it can be determined that there was no detectable amount of the target substance in the sample if there was coloration in the control line. If no coloration can be confirmed in the control line, it can be determined that correct detection was not possible (defective detection) due to poor development, deterioration of the reagent, or the state of the sample.
  • the detection method of the present embodiment can detect a target substance in a biological sample in a very simple manner, and thus can rapidly obtain detection results particularly in clinical practice. It is suitable for detection to assist diagnosis of desired infectious diseases, acute diseases, and the like.
  • a detection kit according to the fourth embodiment of the present invention is for detecting a target substance in a sample, comprising the detection device and the sample pretreatment reagent described in the section "1. Detection Device”. It's a kit. Further, the detection kit of the present embodiment is a kit used for the method described in the section "2. Detection method”.
  • sample pretreatment reagent is not particularly limited as long as it is a reagent used to make the sample developable on the device. It can be used as a solvent or the like.
  • sample pretreatment reagent is a sample diluent.
  • the labeled antibody used in the detection device contains colored particles such as metal particles, colored latex particles, colored polystyrene particles, colored cellulose particles, fluorescent cellulose particles, silica nanoparticles containing pigments, etc. as labeling substances. It is preferable to use a labeled antibody containing
  • the kit of this embodiment may include a filter for filtration, a tube for centrifugation, a cotton swab for sample collection, a cup/dropper, a squeeze tube, and the like. Furthermore, a color gauge or an instruction manual may be provided for assisting in determining the line after development.
  • Example 1 (1) Preparation of anti-SARS-CoV-2 NP antibody-bound colloidal gold A colloidal gold solution adjusted to pH 7.5 with 10 mM Tris-HCl buffer (particle size: 40 nm, concentration: 9.0 ⁇ 10 10 [number of particles/mL ], manufactured by BBI Solutions) was mixed with 1 mL of a 100 ⁇ g/mL mouse anti-SARS-CoV-2 NP monoclonal antibody aqueous solution and incubated at room temperature for 10 minutes.
  • the antibody-bound colloidal gold suspension prepared in (1) above contains 5 wt% trehalose, 0.05 wt% polyethylene glycol (average molecular weight: 20,000), and bovine
  • An antibody-bound colloidal gold coating solution was prepared by adding serum albumin at 1 wt %.
  • An antibody-bound colloidal gold coating solution was uniformly applied to a glass fiber pad cut into a shape of 7 mm in height and 300 mm in length at 0.5 ⁇ L/mm 2 . Thereafter, the glass fiber pad coated with the antibody-bound colloidal gold coating solution was dried in a vacuum dryer to obtain a conjugate pad.
  • Control line coloring evaluation A cotton swab (FLOQ (registered trademark) swab 534100CS01-E, manufactured by Copan) from which a SARS-CoV-2 negative nasopharyngeal swab was collected was placed in 425 ⁇ L of the sample dilution solution, and the nasopharyngeal swab was used as the sample. A negative sample was prepared by suspending it in a diluent. 75 ⁇ L of the negative sample was dropped onto the sample pad, and the colored state of the control line was visually confirmed after 10 minutes.
  • FLOQ registered trademark
  • swab 534100CS01-E manufactured by Copan
  • Example 2 The procedure of Example 1 was repeated except that the 1.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody in (3) of Example 1 was changed to 1 mg/mL goat anti-mouse immunoglobulin polyclonal antibody.
  • Example 3 The procedure was carried out in the same manner as in Example 1, except that the 1.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody in (3) of Example 1 was changed to 0.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody. .
  • Example 1 The procedure of Example 1 was repeated except that the 1.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody in (3) of Example 1 was changed to 2 mg/mL goat anti-mouse immunoglobulin polyclonal antibody.
  • Example 3 The 2 mg/mL rabbit anti-SARS-CoV-2 NP monoclonal antibody in (3) of Example 1 was changed to 2 mg/mL mouse anti-SARS-CoV-2 NP monoclonal antibody, and 1.5 mg/mL goat anti- The procedure was carried out in the same manner as in Example 1, except that the mouse immunoglobulin polyclonal antibody was changed to 1 mg/mL goat anti-mouse immunoglobulin polyclonal antibody.
  • Example 4 The 2 mg/mL rabbit anti-SARS-CoV-2 NP monoclonal antibody in (3) of Example 1 was changed to 2 mg/mL mouse anti-SARS-CoV-2 NP monoclonal antibody, and 1.5 mg/mL goat anti- The procedure was carried out in the same manner as in Example 1, except that the mouse immunoglobulin polyclonal antibody was changed to 0.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody.
  • Table 1 shows the evaluation results of the control lines of the devices of Examples 1-3 and Comparative Examples 1-4.
  • photographs of Example 2 and Comparative Example 3 are shown in FIG.
  • Comparative Example 1 in which the same amount of antibody was applied to the test line and control line, the control line was the darkest, but the background was colored.
  • Comparative Examples 2 to 4 in which the same mouse-derived anti-SARS-CoV-2 antibody as the mouse antibody used for the conjugate pad was used for the test line, the control line was thin.
  • Example 4 The 2 mg / mL rabbit anti-SARS-CoV-2 NP monoclonal antibody in (3) of Example 1 was changed to 1 mg / mL rabbit anti-SARS-CoV-2 NP monoclonal antibody, and 1.5 mg / mL goat anti- An immunochromatographic device and a sample diluent were prepared in the same manner as in Example 1, except that the mouse immunoglobulin polyclonal antibody was changed to 0.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody, and the amount of the application liquid was doubled. .
  • a sample diluent to which no antigen was added was used as a negative sample.
  • 75 ⁇ L of the negative sample was dropped onto the sample pad, and the coloring intensity of the control line and the test line was measured every 30 seconds until 15 minutes had passed using an immunochromatographic reader (C10066, manufactured by Hamamatsu Photonics).
  • C10066 manufactured by Hamamatsu Photonics
  • Example 5 The mouse anti-SARS-CoV-2 NP monoclonal antibody aqueous solution in (1) of Example 1 was changed to the rabbit anti-SARS-CoV-2 NP monoclonal antibody aqueous solution, and 2 mg / mL rabbit anti-SARS in (3) of Example 1 - CoV-2 NP monoclonal antibody changed to 1 mg/mL mouse anti-SARS-CoV-2 NP monoclonal antibody and 1.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody to 0.5 mg/mL goat anti- An immunochromatographic device was prepared in the same manner as in Example 1, except that the rabbit immunoglobulin polyclonal antibody was used and the amount of the coating solution was doubled. Negative samples were developed as in Example 4.
  • Example 5 The procedure was carried out in the same manner as in Example 4, except that the 1 mg/mL rabbit anti-SARS-CoV-2 NP monoclonal antibody in Example 4 was changed to 1 mg/mL mouse anti-SARS-CoV-2 NP monoclonal antibody.
  • Example 6 The mouse anti-SARS-CoV-2 NP monoclonal antibody aqueous solution in (1) of Example 1 was changed to the rabbit anti-SARS-CoV-2 NP monoclonal antibody aqueous solution, and 2 mg / mL rabbit anti-SARS in (3) of Example 1 - CoV-2 NP monoclonal antibody changed to 1 mg/mL rabbit anti-SARS-CoV-2 NP monoclonal antibody and 1.5 mg/mL goat anti-mouse immunoglobulin polyclonal antibody to 0.5 mg/mL goat anti- An immunochromatographic device was prepared in the same manner as in Example 1, except that the rabbit immunoglobulin polyclonal antibody was used and the amount of the coating liquid was doubled. Negative samples were developed as in Example 4.
  • Table 2 shows the evaluation results of control lines and test lines of the devices of Examples 4 and 5 and Comparative Examples 5 and 6. Further, FIG. 4 shows the relationship between the coloring intensity of the control line of the negative samples prepared in Examples 4 and 5 and Comparative Examples 5 and 6 and the elapsed time. In addition, none of the test lines had a coloring intensity detected. FIG. 5 shows a photograph of each device after the negative sample was developed. In Comparative Examples 5 and 6, although the derived antibody used for the conjugate pad and the derived antibody used for the test line were the same type, it was confirmed that the coloration of the control line was low.
  • Example 6 An immunochromatographic device was prepared in the same manner as in Example 4. A sample dilution solution to which HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche) was added as an interfering factor to a 20-fold dilution was used as a negative sample. 75 ⁇ L of the negative sample was dropped onto the sample pad, and the colored state of the control line and the test line after 15 minutes was visually confirmed.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche
  • Example 7 An immunochromatographic device was prepared in the same manner as in Example 5. A sample dilution solution to which HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche) was added as an interfering factor to a 20-fold dilution was used as a negative sample. 75 ⁇ L of the negative sample was dropped onto the sample pad, and the colored state of the control line and the test line after 15 minutes was visually confirmed.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche
  • Comparative Example 7 An immunochromatographic device was prepared in the same manner as in Comparative Example 5. A sample dilution solution to which HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche) was added as an interfering factor to a 20-fold dilution was used as a negative sample. 75 ⁇ L of the negative sample was dropped onto the sample pad, and the colored state of the control line and the test line after 15 minutes was visually confirmed.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche
  • Table 3 shows the control line and test line evaluation results of the devices of Examples 6 and 7 and Comparative Example 7.
  • 6 shows photographs of the negative samples of the devices of Examples 6 and 7 and Comparative Example 7 after development.
  • Comparative Example 7 the antibody-derived organism used for the conjugate pad and the antibody-derived organism used for the test line were both mice, but the control line was faint and the test line showed non-specific HAMA-derived coloration was confirmed.
  • the antibody-derived organism used for the conjugate pad and the antibody-derived organism used for the test line were different, but in both cases, the control line was darkly colored, and the test line No non-specific staining was seen. In the examples, it was shown that more accurate detection is possible without being affected by HAMA interference.
  • Example 8 Preparation of antibody-bound colloidal gold In the same manner as in Example 1 (1), a suspension of anti-SARS-CoV-2 NP antibody-bound colloidal gold (antibody-bound colloidal gold suspension) was obtained.
  • Example 1 (1) In the same manner as in Example 1 (1), except that the mouse anti-SARS-CoV-2 NP monoclonal antibody in Example 1 (1) was changed to a mouse anti-influenza A virus NP monoclonal antibody, anti-A A suspension of type influenza virus NP antibody-bound colloidal gold (antibody-bound colloidal gold suspension) was obtained.
  • Example 8 In the same manner as in Example 1 (1), except that the mouse anti-SARS-CoV-2 NP monoclonal antibody in Example 1 (1) was changed to a mouse anti-influenza B virus NP monoclonal antibody, anti-B A suspension of type influenza virus NP antibody-bound colloidal gold (antibody-bound colloidal gold suspension) was obtained. That is, in Example 8, three types of antibody-bound colloidal gold suspensions were prepared.
  • a control line was prepared by applying 1 ⁇ L/cm in a line shape with a width of 1 mm perpendicular to the developing direction.
  • the nitrocellulose membrane on which the test line and control line were prepared by coating was dried at 50°C for 30 minutes to obtain an antibody-coated membrane.
  • Example 9 1 mg/mL mouse anti-influenza A NP monoclonal antibody in (3) of Example 8 was changed to 1 mg/mL rabbit anti-influenza A NP polyclonal antibody, and 1 mg/mL mouse anti-influenza B NP monoclonal antibody was changed to 1 mg/mL rabbit anti-influenza B NP polyclonal antibody, but the procedure was performed in the same manner as in Example 8.
  • Example 8 In the same manner as in Example 8, except that the 1 mg / mL rabbit anti-SARS-CoV-2 NP monoclonal antibody in (3) of Example 8 was changed to 1 mg / mL mouse anti-SARS-CoV-2 NP monoclonal antibody. went.
  • Table 4 shows the control line and test line evaluation results of the devices of Examples 8 and 9 and Comparative Example 8.
  • the antibody-derived organism used for the conjugate pad and the antibody-derived organism used for all the test lines were mice, but it was confirmed that the coloration of the control line was low.
  • the control line was strongly colored, and no conspicuous coloration was observed in the background.
  • Example 9 Although the organism of origin of the antibody used in the conjugate pad was different from the organism of origin of the antibody used in all the test lines, the control line showed the strongest staining, and no conspicuous staining was observed in the background. I could't.
  • Example 10 An immunochromatographic device was prepared in the same manner as in Example 8. As an interfering factor, HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche) was added to the sample diluent at a 20-fold dilution to obtain a negative sample. Thereafter, development was carried out in the same manner as in Example 8.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche
  • Example 11 An immunochromatographic device was prepared in the same manner as in Example 8. Mouse IgG was added to 100 ⁇ g/mL, and HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterile distilled water) manufactured by Roche) as an interfering factor was added to the sample dilution solution at a 20-fold dilution, It was taken as a negative sample. Thereafter, development was carried out in the same manner as in Example 8.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterile distilled water) manufactured by Roche
  • Example 12 An immunochromatographic device was prepared in the same manner as in Example 9. As an interfering factor, HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche) was added to the sample diluent at a 20-fold dilution to obtain a negative sample. Thereafter, development was carried out in the same manner as in Example 9.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche
  • Comparative Example 9 An immunochromatographic device was prepared in the same manner as in Comparative Example 8. As an interfering factor, HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche) was added to the sample diluent at a 20-fold dilution to obtain a negative sample. Thereafter, development was carried out in the same manner as in Comparative Example 8.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterilized distilled water) manufactured by Roche
  • Comparative Example 10 An immunochromatographic device was prepared in the same manner as in Comparative Example 8. Mouse IgG was added to 100 ⁇ g/mL, and HAMA serum (HAMA Serum Type II (dissolved in 1 mL of sterile distilled water) manufactured by Roche) as an interfering factor was added to the sample dilution solution at a 20-fold dilution, It was taken as a negative sample. Thereafter, development was carried out in the same manner as in Comparative Example 8.
  • HAMA serum HAMA Serum Type II (dissolved in 1 mL of sterile distilled water) manufactured by Roche
  • Table 5 shows the evaluation results of the control line and test line of the devices of Examples 10-12 and Comparative Examples 9 and 10.
  • Comparative Example 9 the antibody used in the conjugate pad and the antibody used in all test lines were both derived from mice, but non-specific coloring was confirmed in all test lines.
  • Comparative Example 10 is an example in which mouse IgG was added to the negative sample of Comparative Example 9. Since non-specific coloring of the test line was suppressed, the coloring of the test line in Comparative Example 9 was due to the influence of HAMA. It was speculated that there was In Comparative Example 10, the coloration of the added control line was significantly reduced.
  • Example 10 the antibody used for the conjugate pad was a mouse-derived antibody, whereas the antibody used for the A line and B line was derived from a mouse, and the antibody used for the S line was derived from a mouse.
  • Example 11 is an example in which mouse IgG was added to the negative sample of Example 10, but non-specific coloring of the test line was not detected. In addition, although a decrease in coloring of the control line was observed, it was at a sufficiently visible level.
  • Example 12 the antibody used for the conjugate pad was derived from a mouse, and the antibody used for the A line, B line and S line was derived from a rabbit. No non-specific staining was observed in the test line of either.

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