Multimerized polypeptide, immunochromatographic strip, immunochromatographic device, immunochromatographic kit, and analyte detection method

Abstract

This multimerized polypeptide for an immunochromatographic strip comprises at least one type of hydrophilic polymer carrier selected from the group consisting of dextran, polyethylene glycol, and derivatives of these, and a plurality of polypeptides carried on the hydrophilic polymer carrier. The polypeptide is of 200 amino acid residues or less and capable of binding to an analyte.

Description

Multimerized polypeptide, immunochromatographic strip, immunochromatographic device, immunochromatographic kit, and method for detecting analyte

[0001] The present invention relates to a multimerized polypeptide, an immunochromatographic strip, an immunochromatographic device, an immunochromatographic kit, and a method for detecting a test substance, and more particularly to a multimerized polypeptide, an immunochromatographic strip, an immunochromatographic device, an immunochromatographic kit comprising the same, and a method for detecting a test substance using these.

[0002] In order to determine whether an infectious disease is positive or negative and to identify its cause, it is necessary to detect pathogens such as bacteria and viruses and antibodies induced by these pathogens in samples collected from a subject. It is also known that, in order to determine whether a specific disease is positive or negative, substances that serve as markers for that disease can be detected in samples such as blood. Immunoassays that utilize antigen-antibody reactions are widely used as methods for detecting such test substances as pathogens, antibodies, and markers.

[0003] Among immunoassays, immunochromatography (immunochromatography) is a simple and rapid detection method. In immunochromatography, a probe molecule capable of binding to an analyte in a sample is immobilized as a capturer on a solid-phase matrix, thereby capturing and detecting the analyte. For example, a sample is developed from one end of an immunochromatography strip (test piece) consisting of a porous membrane (matrix) to which the probe molecule is immobilized. During the development process, a complex is formed between the analyte and a label capable of specifically binding to the analyte (a labeled substance (e.g., a labeled antibody) capable of specifically binding to the analyte). The complex is then indirectly or directly captured by the probe molecule (capturer), thereby detecting the analyte. This method is simple to operate, as the analyte in the sample can be detected simply by applying or dropping the sample onto the immunochromatography strip and allowing the sample or the complex to develop on the porous membrane by capillary action. Furthermore, since it is possible to detect test substances in a short time and visual judgment is also possible, it is widely used in research tests and clinical examinations.

[0004] In such immunochromatography techniques, the probe molecules include antigens and antibodies against the analyte. Physical adsorption is a common method for immobilizing these molecules on the matrix, as it is simple and does not impair the activity of the antigens or antibodies. However, when physical adsorption is used to immobilize small polypeptides such as antigen peptides, the immobilization amount or immobilization strength is insufficient, resulting in insufficient detection sensitivity. To address this issue, for example, Japanese Patent Laid-Open Publication No. 2004-340722 (Patent Document 1) describes a polyamino acid carrier for peptide binding, which comprises a polyamino acid consisting of amino acid residues having a reactive group selected from an amino group, a carboxy group, a sulfhydryl group, or a hydroxyl group in the side chain, and a diol or aminoalcohol structure bound to all or part of the reactive group. The carrier is then immobilized on a nitrocellulose membrane, and antibodies against the antigen peptide are detected.

[0005] Japanese Patent Application Laid-Open No. 2004-340722

[0006] The present inventors further investigated methods for detecting analytes in samples by immunochromatography and found that, for example, when a polypeptide is immobilized on a matrix using the carrier described in Patent Document 1, sufficient detection sensitivity can be achieved and nonspecific reactions in which a portion of the immunoglobulins contained in the sample reacts with the carrier can also be suppressed. However, depending on the sample, such nonspecific reactions may not be sufficiently suppressed. Therefore, the present inventors found that a method for detecting analytes in samples by immunochromatography requires even higher detection sensitivity and suppression of nonspecific reactions.

[0007] The present invention has been made in view of the above-mentioned problems, and aims to provide a polymerized polypeptide that, when immobilized on the matrix of an immunochromatographic strip and used as a capture body for capturing a test substance, enables highly sensitive detection of the test substance and suppression of non-specific reactions; an immunochromatographic strip, an immunochromatographic device, an immunochromatographic kit that enable highly sensitive detection of the test substance and suppression of non-specific reactions; and a test substance detection method that uses these to enable highly sensitive detection of the test substance in immunochromatography and suppression of non-specific reactions.

[0008] The present inventors have conducted extensive research to solve the above-mentioned problems, and have found that by using, as a carrier for a polypeptide to be immobilized on a matrix of a strip used for immunochromatographic detection of a test substance in a sample, at least one hydrophilic polymer carrier selected from among many materials, particularly from the group consisting of dextran, polyethylene glycol, and derivatives thereof, to polymerize the polypeptide, the test substance can be detected with high sensitivity even if the total amount of polypeptide immobilized on the matrix is ​​reduced, and furthermore, non-specific reactions that could sometimes be detected with the technology described in Patent Document 1 can also be highly suppressed, thereby completing the present invention.

[0009] That is, the present invention relates to a multimerizing polypeptide, an immunochromatographic strip, an immunochromatographic device, an immunochromatographic kit comprising the same, and a method for detecting an analyte using the same. More specifically, the present invention provides the following: [1] A multimerizing polypeptide for an immunochromatographic strip, the multimerizing polypeptide comprising at least one hydrophilic polymer carrier selected from the group consisting of dextran, polyethylene glycol, and derivatives thereof, and a plurality of polypeptides supported on the hydrophilic polymer carrier, the polypeptides being polypeptides of 200 amino acid residues or less capable of binding to an analyte. [2] The multimerizing polypeptide according to [1], wherein the amount of the polypeptide is 2 to 30 moles per mole of the hydrophilic polymer carrier. [3] The multimerizing polypeptide according to [1] or [2], wherein the content of the polypeptide is 10 to 70% by mass. [4] The multimerizing polypeptide according to any one of [1] to [3], wherein the polypeptide is an antigen peptide of the analyte. [5] An immunochromatographic strip used for detecting a test substance in a sample by immunochromatography, comprising a matrix and a polypeptide-immobilizing section in which the multimerized polypeptide according to any one of [1] to [4] is immobilized on the matrix. [6] The immunochromatographic strip according to [5], wherein the matrix is ​​a nitrocellulose membrane. [7] The immunochromatographic strip according to [5] or [6], wherein the amount of the multimerized polypeptide immobilized on the polypeptide-immobilizing section is 0.001 to 2.0 μg in terms of polypeptide. [8] The immunochromatographic strip according to any one of [5] to [7], comprising, upstream of the polypeptide-immobilizing section on the matrix, a label-containing section that elutably holds a label comprising a probe molecule capable of binding to the test substance and a labeling substance. [9] The immunochromatographic strip described in [8], wherein the labeling substance is an enzyme, and the matrix is ​​provided with a substrate-containing section that holds a substrate of the enzyme in an elutable manner or a substrate-adding section that adds a substrate of the enzyme, upstream of the polypeptide-immobilized section.

[10] An immunochromatographic device used for detecting a test substance in a sample by immunochromatography, the immunochromatographic device comprising the immunochromatographic strip according to any one of [5] to [9].

[11] An immunochromatographic kit used for detecting a test substance in a sample by immunochromatography, the immunochromatographic kit comprising the immunochromatographic strip according to any one of [5] to [9] or the immunochromatographic device according to

[10] .

[12] A method for detecting a test substance in a sample by immunochromatography, the method comprising: a capture step of capturing the test substance in the sample at the polypeptide immobilization part; and a detection step of detecting the captured test substance, in an immunochromatography strip comprising a matrix and a polypeptide immobilization part where a multimerized polypeptide is immobilized on the matrix, the multimerized polypeptide comprising at least one hydrophilic polymer carrier selected from the group consisting of dextran, polyethylene glycol, and derivatives thereof, and a plurality of polypeptides supported on the hydrophilic polymer carrier, and the polypeptides being polypeptides of 200 amino acid residues or less that can bind to the test substance.

[13] The method for detecting a test substance according to

[12] , wherein the immunochromatography strip comprises a label-containing section upstream of the polypeptide-immobilized section on the matrix, which holds a label in an elutable manner, the label containing a probe molecule capable of binding to the test substance and a labeling substance; the method further comprises a labeling step of forming a complex between the test substance in the sample and the label; the capture step is a step of capturing the complex at the polypeptide-immobilized section; and the detection step is a step of detecting the captured complex.

[0010] According to the present invention, it is possible to provide a polymerized polypeptide that, when immobilized on the matrix of an immunochromatographic strip and used as a capture body for capturing a test substance, enables highly sensitive detection of the test substance and suppression of non-specific reactions, as well as an immunochromatographic strip, an immunochromatographic device, an immunochromatographic kit that enable highly sensitive detection of the test substance and suppression of non-specific reactions, and a test substance detection method that uses these to enable highly sensitive detection of the test substance in immunochromatography and suppression of non-specific reactions.

[0011] 1 is a schematic plan view showing one embodiment of the immunochromatographic strip of the present invention. 2 is a schematic cross-sectional view of FIG. 1 showing one embodiment of the immunochromatographic strip of the present invention. 3 is a schematic cross-sectional view of FIG. 2 showing one embodiment of the main part of the immunochromatographic device of the present invention. 4 is a schematic plan view showing one embodiment of the immunochromatographic device of the present invention. 5 is a schematic cross-sectional view of FIG. 3 showing one embodiment of the immunochromatographic device of the present invention. 6 is a diagram showing the appearance and evaluation results of the test window of the immunochromatographic device when a polypeptide (HIV2 peptide) or a complex (Dextran-HIV2, PEG-HIV2) is used in immunochromatographic assay evaluation 1. 7 is a diagram showing the appearance and evaluation results of the test window of the immunochromatographic device when Poly Lys-HIV2 or Dextran-HIV2 is used for sample 1 and sample 2 in immunochromatographic assay evaluation 2. FIG. 10 shows the appearance of the test window of an immunochromatographic device and the evaluation results when Poly Lys-HIV2 or PEG-HIV2 was used for Sample 1 and Sample 2 in immunochromatographic assay evaluation 2.

[0012] The present invention will be described in detail below based on preferred embodiments thereof.

[0013] <Multimerized Polypeptide> The multimerized polypeptide of the present invention is intended for use in an immunochromatographic strip, more specifically, for immobilization on the matrix of the immunochromatographic strip, and functions as a capture body that captures a test substance in the immunochromatographic strip. However, this does not exclude its use in applications other than immunochromatography, such as a sandwich method or a competitive method, using a capture body in which the multimerized polypeptide of the present invention is immobilized on an insoluble carrier.

[0014] The multimerized polypeptide of the present invention comprises at least one hydrophilic polymer carrier selected from the group consisting of dextran, polyethylene glycol, and derivatives thereof, and a plurality of polypeptides supported on the hydrophilic polymer carrier, wherein the polypeptides are polypeptides of 200 amino acid residues or less that can bind to a test substance.

[0015] (Hydrophilic Polymer Carrier) The hydrophilic polymer carrier contained in the multimerized polypeptide of the present invention functions as a carrier for supporting the polypeptide and also mediates the bond between the polypeptide and the matrix of the immunochromatography strip. The hydrophilic polymer carrier of the present invention is at least one selected from the group consisting of dextran, polyethylene glycol, and derivatives thereof, and may be, for example, a mixture of dextran and / or a derivative thereof with polyethylene glycol and / or a derivative thereof. In this specification, "hydrophilic" refers to a solubility in water at room temperature and normal pressure of more than 0.01 g / mL, preferably 0.05 g / mL or more, and more preferably 0.1 g / mL or more.

[0016] The branched structure of dextran is primarily a structure in which glucose is linked via an α-1,6-glycosidic bond, but the dextran according to the present invention may have one or more of the following structures: a structure in which glucose is linked via an α-1,4-glycosidic bond and an α-1,6-glycosidic bond; a structure in which glucose is linked via an α-1,3-glycosidic bond and an α-1,6-glycosidic bond; or a structure in which glucose is linked via an α-1,2-glycosidic bond and an α-1,6-glycosidic bond. Furthermore, the dextran derivative according to the present invention is not particularly limited as long as it is hydrophilic, and examples thereof include aminodextran, diethylaminoethyldextran (DEAE-dextran), dextran sulfate, and carboxymethyldextran. Of these, dextran is preferred as the dextran and its derivative according to the present invention.

[0017] The dextran and derivatives thereof according to the present invention preferably have a mass (mass determined by calibration with markers by gel filtration chromatography (GFC); the same applies hereinafter) of 5,000 to 650,000 Da, more preferably 15,000 to 250,000 Da, and even more preferably 25,000 to 100,000 Da.

[0018] The polyethylene glycol according to the present invention may be linear, cyclic, branched, or star-shaped (having three or more (divalent) branched chains extending radially from a common center, preferably divalent to octavalent). The derivative of polyethylene glycol according to the present invention is also not particularly limited as long as it is hydrophilic, and examples thereof include modified PEG in which at least one (preferably all) of the terminal hydroxy groups of polyethylene glycol have been converted to a group such as a carboxy group, an amino group, a thiol group, an acryloyl group (acrylic group), a succinimidyl carboxymethyl ester group (SCM group), a maleimide group, or a silane group, or which has been biotinylated; polyethylene glycol; or a crosslinked product of the modified PEG. Among these, the polyethylene glycol and derivatives thereof according to the present invention are preferably modified PEGs in which the terminal hydroxy groups (preferably all hydroxy groups) of branched or star-shaped polyethylene glycol have been converted to SCM groups (e.g., 8-ArmPEG-SCM (manufactured by Biopharma PEG Scientific Inc.) and 4-ArmPEG-SCM (manufactured by Biopharma PEG Scientific Inc.)).

[0019] The polyethylene glycol and derivatives thereof according to the present invention preferably have a mass of 1,000 to 40,000 Da, more preferably 10,000 to 40,000 Da.

[0020] (Polypeptide) The polypeptide contained in the multimerized polypeptide of the present invention is capable of binding to a analyte to be detected by immunochromatography (preferably capable of specifically binding to the analyte), and directly captures the analyte on an immunochromatographic strip. The polypeptide according to the present invention is a low-molecular-weight polypeptide having 200 amino acid residues or less. In this specification, the term "polypeptide" refers to two or more amino acid residues bonded by amide bonds. The polypeptide according to the present invention preferably has 3 to 200 amino acid residues, more preferably 3 to 120 amino acid residues, and even more preferably 3 to 30 amino acid residues.

[0021] The polypeptide of the present invention is not particularly limited as long as it is capable of binding to a test substance. For example, it is preferable that the test substance is an antibody and the polypeptide is an antigenic peptide for the antibody. Such an antigenic peptide may contain at least one epitope for the antibody, and may be a complete peptide, a fragment containing only the epitope, or one or more of the epitopes. Such polypeptides are not particularly limited, and include, for example, naturally occurring polypeptides, polypeptides expressed from recombinant DNA by genetic engineering techniques, chemically synthesized polypeptides, and polypeptides produced by enzymatic treatment of proteins. Furthermore, these polypeptides may be appropriately modified or have an active group introduced therein. For example, in the following examples, a synthetic HIV-2 gp36 polypeptide is used, which has an amino acid sequence derived from the HIV-2 envelope protein gp36, is composed of 18 amino acid residues, and has a hydrazino group introduced therein.

[0022] (Configuration of Multimerized Polypeptide) The multimerized polypeptide of the present invention comprises the hydrophilic polymer carrier and a plurality of polypeptides supported on the hydrophilic polymer carrier. Here, "a plurality" means that there are two or more molecules of the polypeptide per molecule of the hydrophilic polymer carrier, but the amount of the polypeptide (if there are two or more types of polypeptides, the total amount; the same applies hereinafter) per mole of the hydrophilic polymer carrier (if there are two or more types of hydrophilic polymer carriers, the total amount; the same applies hereinafter) is preferably 2 to 35 moles, more preferably 2 to 30 moles, even more preferably 4 to 20 moles, and even more preferably 8 to 20 moles.

[0023] Furthermore, the content of the polypeptide in the multimerized polypeptide of the present invention is preferably 10 to 70% by mass, more preferably 20 to 60% by mass, and even more preferably 30 to 50% by mass.

[0024] (Method for Producing Multimeric Polypeptide) The multimeric polypeptide of the present invention can be produced by directly or indirectly binding the polypeptide to the hydrophilic polymer carrier. As such a production method, a conventionally known method or a method similar thereto can be appropriately adopted.

[0025] Examples of methods for directly binding the polypeptide to the hydrophilic polymer carrier include a method of imparting an active group such as a carboxyl group, an epoxy group, a tosyl group, an amino group, a hydroxyl group, an isothiocyanate group, an isocyanate group, an azide group, an aldehyde group, a carbonate group, an allyl group, an aminooxy group, a maleimide group, a thiol group, an SCM group, or a hydrazino group (HBz) to the hydrophilic polymer carrier and / or the polypeptide, or a method of covalently binding the hydrophilic polymer carrier and / or the polypeptide via the active group. The hydrophilic polymer carrier and the polypeptide to which the active group has been imparted may be commercially available products, and may be prepared by introducing the active group into the hydrophilic polymer carrier and / or the polypeptide under appropriate reaction conditions.

[0026] Furthermore, examples of methods for indirectly binding the polypeptide to the hydrophilic polymer carrier include methods of binding via a linker such as polyhistidine, oligopeptide, or a linker molecule having the active group. The selection and size of the linker can be appropriately determined taking into consideration the strength of the bond between the hydrophilic polymer carrier and the polypeptide, steric hindrance, etc. When the linker contains amino acid residues, the number of residues is not included in the number of residues of the polypeptide. However, the number of amino acid residues in the polyhistidine or oligopeptide is usually 35 or less, and the linker molecule is preferably used as the linker.

[0027] The ratio of the hydrophilic polymer carrier and the polypeptide used in these production methods can be appropriately selected so as to achieve the preferred ranges of the contents of each component in the multimeric polypeptide. Furthermore, the obtained multimeric polypeptide may be purified and concentrated, preferably by chromatography or the like, as necessary.

[0028] <Immunochromatography Strip and Immunochromatography Device> The immunochromatography strip of the present invention is a strip used for detecting a test substance in a sample by immunochromatography, and comprises a matrix and a polypeptide immobilization section in which the multimerized polypeptide of the present invention is immobilized on the matrix. Furthermore, the immunochromatography device of the present invention comprises the immunochromatography strip of the present invention.

[0029] Preferred embodiments of the immunochromatographic strip and immunochromatographic device of the present invention will be described in detail below with reference to the drawings, but the present invention is not limited thereto. In the following description and drawings, the same or corresponding elements are designated by the same reference numerals, and redundant description will be omitted.

[0030] FIG. 1 is a schematic plan view of one embodiment of the immunochromatographic strip of the present invention (immunochromatographic strip 101), and FIG. 2 is a cross-sectional view thereof. FIG. 3 is a schematic cross-sectional view of one embodiment of the main part of the immunochromatographic device of the present invention (immunochromatographic device 102). FIG. 4 is a schematic plan view of one embodiment of the immunochromatographic device of the present invention, and FIG. 5 is a cross-sectional view thereof. In FIGS. 1 to 5, the immunochromatographic strip is a lateral flow immunochromatographic strip, but is not limited thereto. In the following description, the absorption zone 5 side of the matrix 2 is referred to as downstream (downstream in the sample development direction 14), and the opposite side is referred to as upstream (upstream in the sample development direction 14).

[0031] (Test Substance) In the present invention, examples of the "test substance" to be detected include antibodies; pathogens such as bacteria, protozoa, fungi, and viruses, and substances such as antigen peptides, sugars, and glycoproteins derived therefrom; substances such as peptides and albumin that serve as markers for specific diseases; and low molecular weight compounds such as vitamins, hormones, coenzymes, toxins, and antibiotics. Among these, the test substance according to the present invention is preferably an antibody against an antigen peptide, since antigen peptides are suitable as polypeptides contained in the multimerized polypeptide. In this specification, the "antibody" may be a polyclonal antibody or a monoclonal antibody, and may be a complete antibody or an antibody fragment (e.g., Fab, Fab', F(ab')). ２ , Fv, single-chain antibodies, diabodies, etc.), and minibodies in which the variable regions of antibodies are bound.

[0032] (Sample) In the present invention, the "sample" used for detecting a test substance is not particularly limited as long as it is a sample in which the test substance may be present. Examples of the sample include, but are not limited to, specimens such as whole blood, serum, plasma, urine, feces, mucosal secretions (sputum, throat swab, nasal swab, saliva, and otorrhea) collected from a subject (e.g., a mammal, preferably a human), and test antibody solutions. The sample is preferably an aqueous sample, more preferably an aqueous solution or aqueous dispersion. If necessary, the sample may be diluted with a diluent, treated with various pretreatment solutions, or filtered through a filtration filter. Examples of the diluent include water, physiological saline, and buffer solutions. Examples of the buffer solution include buffer solutions that can be adjusted to a pH suitable for the target immunochromatographic reaction, more specifically, phosphate buffer, Tris buffer, Good's buffer, borate buffer, etc. The diluent may also contain a stabilizing protein such as BSA (bovine serum albumin), serum, etc. The pretreatment liquid may include, for example, a surfactant.

[0033] The matrix according to the present invention is an insoluble carrier that functions as an immunochromatographic medium (stationary phase). The matrix also serves as an antigen / antibody-containing membrane that serves as the main body of an immunochromatographic strip and has a polypeptide-immobilized portion and, if necessary, a label-containing portion and a development-confirming portion (e.g., matrix 2 shown in Figures 1 to 3).

[0034] The matrix according to the present invention is preferably a porous membrane, and examples thereof include nitrocellulose membrane, nitrocellulose mixed ester membrane, cellulose membrane, acetylcellulose membrane, polysulfone membrane, polyethersulfone membrane, nylon membrane, glass fiber, nonwoven fabric, cloth, and filter paper. Among these, nitrocellulose membrane is preferred as the matrix according to the present invention, since it tends to further improve detection sensitivity when the multimerized polypeptide of the present invention is immobilized thereon. The migration speed of the sample developed in such a matrix can be changed by the material, size, pore size, and distribution of the porous membrane, and can be appropriately adjusted depending on the type and concentration of the analyte, the purpose of detection, and the like.

[0035] The size of the matrix according to the present invention is not particularly limited, but may be, for example, a strip (long, thin piece) having a width of about 3 to 10 mm and a length of about 30 to 100 mm. The thickness of the matrix is ​​also not particularly limited, but may be, for example, in the range of 100 μm to 1 mm.

[0036] (Polypeptide immobilization site) The polypeptide immobilization site of the present invention is a site where the above-mentioned multimerized polypeptide of the present invention is immobilized on the matrix. Since the multimerized polypeptide functions as a capture agent, it also functions as a capture agent immobilization site or a detection site for confirming the presence or absence of a test substance (for example, polypeptide immobilization site 6 shown in Figures 1 and 2).

[0037] 1 and other figures, the polypeptide-fixing portion 6 is formed in a line shape as a test line, but the shape of the polypeptide-fixing portion according to the present invention is not limited to this and can be any shape, such as a circle, a polygon, etc. Among these shapes, the shape of the polypeptide-fixing portion according to the present invention is preferably a line, and more preferably a line with a width of, for example, 0.5 to 3.0 mm.

[0038] Furthermore, as shown in Figures 3 and 4, the polypeptide-immobilizing portion according to the present invention may be multiple (two polypeptide-immobilizing portions 6a and 6b in Figures 3 and 4) depending on the type of analyte and the detection method. For example, when label A containing antibody a capable of binding to analyte a and label B containing antibody b capable of binding to analyte b are used as labels described below, the polypeptide-immobilizing portion may correspondingly include polypeptide-immobilizing portion A to which a multimerized polypeptide containing polypeptide a capable of binding to analyte a is immobilized and polypeptide-immobilizing portion B to which a multimerized polypeptide containing polypeptide b capable of binding to analyte b is immobilized.

[0039] The multimerized polypeptide of the present invention can be easily immobilized, for example, even when the matrix is ​​a nitrocellulose membrane. Therefore, the method for immobilizing the multimerized polypeptide to the polypeptide immobilization site of the present invention is not particularly limited. For example, a multimerized polypeptide solution containing the multimerized polypeptide can be spotted onto the matrix and dried using a conventionally known method such as physical adsorption, or a method similar thereto, to bind the multimerized polypeptide. Furthermore, after binding, blocking may be performed as necessary. The solvent for the multimerized polypeptide solution is not particularly limited as long as it has a pH (5.0 to 9.0) that does not inhibit adsorption, and examples thereof include buffers such as phosphate buffer, Tris buffer, Good's buffer, and borate buffer.

[0040] The amount of the multimerized polypeptide immobilized on the polypeptide immobilization moiety according to the present invention can be adjusted appropriately depending on the form of the sample, the type of antibody contained in the label, the purpose of detection, etc., and can be, for example, in the range of 0.001 to 2.0 μg in polypeptide equivalent. Note that, in the present invention, by using the multimerized polypeptide, highly sensitive detection is possible even with a small amount of polypeptide, for example, at least 0.03 to 0.4 μg or 1 to 40 ng in polypeptide equivalent.

[0041] In the immunochromatographic strip of the present invention, an analyte in a sample that has migrated through the matrix is ​​captured by a multimerized polypeptide immobilized on a polypeptide-immobilized portion via the polypeptide. As a result, the analyte accumulates at the polypeptide-immobilized portion, and can be detected by labeling it with, for example, the following label.

[0042] (Label-Containing Portion) The immunochromatographic strip of the present invention preferably includes a label-containing portion located upstream of the polypeptide-immobilized portion on the matrix, which elutably holds a label containing a probe molecule capable of binding to an analyte and a label. The label-containing portion functions as a label-holding portion that elutably holds the label. The label-containing portion may be integral with the matrix 2, as in the label-containing portion 4 shown in FIGS. 1 and 2, or may be a label pad 4a, as shown in FIG. 3, in which the label is elutably held in a separate water-absorbent material, which is layered on the label zone 4b of the matrix 2. The label-containing portion preferably also functions as a sample application portion 8 that receives a dropped or applied sample, as in the label pad 4a. The water-absorbent material is preferably a porous material, such as the porous membrane.

[0043] The size of the labeled substance-containing portion is not particularly limited, but examples include a width of 1 to 10 mm and a length of 3 to 30 mm, and the thickness of the labeled substance-containing pad is, for example, in the range of 0.3 to 2 mm.

[0044] [Probe molecule] The probe molecule contained in the label is a probe molecule capable of binding to the analyte. Examples of such probe molecules include antibodies, antigens, antigenic peptides, lectins, Fc-binding proteins, vitamins, hormones, coenzymes, toxins, antibiotics, etc., depending on the analyte. Furthermore, for example, when the polypeptide contained in the multimerized polypeptide is an antigen or antigenic peptide, the probe molecule may be the same antigen or antigenic peptide as the polypeptide, as long as it does not inhibit the effects of the present invention. Such probe molecules can be produced by appropriately adopting and improving conventionally known production methods, and commonly available probe molecules may also be used as appropriate.

[0045] [Labeling substance] The labeling substance according to the present invention functions as a label for the test substance in detecting the test substance, and any labeling substance used in known immunoassays can be used without any particular limitation.

[0046] Examples of labeling substances of the present invention include enzymes; radioisotopes (isotopes such as iodine, tritium, and carbon); luminescent substances such as acridinium derivatives; fluorescent substances such as europium; fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE); low-molecular-weight labeling substances such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); gold particles; latex; dinitrophenyl phosphate (DNP); and digoxigenin (DIG). These may be used alone or in combination of two or more. Among these, enzymes are preferred as labeling substances of the present invention in terms of the ease of label development and visual detection. Examples of the enzyme include various enzymes conventionally used in enzyme immunoassays, such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), glucose oxidase, and luciferase. When an enzyme is used as the labeling substance, various detection methods can be performed depending on the substrate by using a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like as the substrate.

[0047] [Constitution of Labeled Body and Production Method] In the labeled body according to the present invention, the molar ratio of the labeling substance to the probe molecule is not particularly limited and can be appropriately adjusted depending on the combination of these types, ease of binding to the test substance, etc. For example, the ratio of the probe molecule to 1 mole of labeled substance is preferably 1 to 30 moles, more preferably 1 to 20 moles.

[0048] The labeled product according to the present invention can be produced by binding the labeling substance and the probe molecule. As the production method, a conventionally known method or a method based thereon can be appropriately adopted depending on the types of the labeling substance and the probe molecule, and the labeling substance and the probe molecule may be directly or indirectly bound to each other.

[0049] Examples of methods for directly binding the labeling substance and the probe molecule include a method of adding an active group (e.g., one of the active groups listed in the method for producing a multimerized polypeptide) to the labeling substance and / or the probe molecule, or a method of using a labeling substance and / or a probe molecule having such an active group and covalently binding the labeling substance and / or the probe molecule via the active group. The labeling substance and the probe molecule to which the active group has been added may be commercially available products, or may be prepared by introducing the active group onto the surface of the labeling substance and / or the probe molecule under appropriate reaction conditions. Examples of methods for indirectly binding the labeling substance to the probe molecule include a method of binding via polyhistidine, polyethylene glycol, oligopeptide, linker molecule, etc. Alternatively, one may be modified in some way and the other may be attached with a substance that captures the modified portion, and then the two may be bound via these. For example, one may be biotinylated and the other may be avidinized, and a binding method using avidin-biotin bonding may be employed. The ratio of labeling substance to probe molecule used in such a production method can be appropriately selected so as to achieve the preferred range for the above-mentioned label. Furthermore, as such a label, for example, commercially available products such as enzyme-labeled antibodies may be used appropriately.

[0050] The method for retaining the label in the label-containing portion is not particularly limited as long as it allows the label to be eluted into a sample (preferably an aqueous solution or aqueous dispersion) or the developing solution described below when developed, and examples thereof include a method in which a labeled body fluid containing the label is applied to or impregnated into the material of the matrix or label pad described above, and then dried. Examples of solvents for the labeled body fluid include buffer solutions, such as phosphate buffer, Tris buffer, Good's buffer, and borate buffer. The amount of the label retained in the label-containing portion according to the present invention can be adjusted as appropriate depending on the form of the sample, the sensitivity of the probe molecule contained in the label to the analyte, the purpose of detection, etc.

[0051] When the immunochromatographic strip of the present invention comprises the label-containing portion, upon contact of the sample with the label-containing portion, the label is eluted, and if the analyte is present in the sample, a complex (e.g., an immune complex) is formed in which the analyte binds to the label through a reaction between the analyte and the probe molecule contained in the label. Furthermore, in this case, when the label that has bound to the analyte to form a complex migrates to the polypeptide-immobilized portion, the complex is captured by the multimerized polypeptide immobilized on the polypeptide-immobilized portion via the bond between the polypeptide and the analyte, and the labeled substance that constitutes the complex accumulates on the polypeptide-immobilized portion. As a result, the polypeptide-immobilized portion exhibits a signal due to the accumulation of the labeled substance, and by detecting this signal, the analyte in the sample can be detected.

[0052] (Development Confirmation Section) Furthermore, in the immunochromatography strip of the present invention, the matrix may further include a development confirmation section (e.g., development confirmation section 10 shown in Figures 3 and 4) downstream of the label-containing section, to which a substance capable of capturing the label is fixed.

[0053] The development confirmation section functions as a reference section for confirming whether the development of the developing solution in the immunochromatographic strip of the present invention has been successful and for determining whether the test is successful or not. Such a development confirmation section is preferably located downstream of the polypeptide-immobilized section.

[0054] In the present invention, a "substance capable of capturing a label (hereinafter sometimes referred to as a "control substance")" is a substance capable of binding to the label and capturing the label. Such a control substance is preferably a substance that does not bind to the test substance, and is typically an antibody capable of binding to the label or probe molecule contained in the label. As such a control substance, for example, an anti-enzyme antibody can be used when the label contained in the label is an enzyme, or an anti-rabbit antibody can be used when the probe molecule contained in the label is a rabbit polyclonal antibody specific to the test substance.

[0055] 4, the development confirmation section 10 is formed in a line shape as a reference line, but the shape of the development confirmation section in the present invention is not limited to this and can be any shape such as a circle, a polygon, etc. Among these shapes, the shape of the development confirmation section in the present invention is preferably a line shape, and more preferably a line shape with a width of, for example, 0.5 to 3.0 mm.

[0056] The method for immobilizing the control substance on the development confirmation section according to the present invention is not particularly limited, and any conventionally known method or a method based thereon can be appropriately adopted, including the same methods as those exemplified as the method for immobilizing the multimerized polypeptide on the polypeptide immobilization section. The amount of the control substance immobilized on the development confirmation section according to the present invention can be appropriately adjusted depending on the form of the sample, the type of labeling substance or probe molecule contained in the label, the purpose of detection, etc.

[0057] In the immunochromatographic strip of the present invention, when the labeled entity (or the labeled entity not captured by the polypeptide-immobilizing section, if the development confirmation section is located downstream of the polypeptide-immobilizing section) moves to the development confirmation section, the labeled entity is captured by the control entity immobilized in the development confirmation section, and the labeled substance constituting the labeled entity accumulates in the development confirmation section. As a result, the development confirmation section shows a signal due to the accumulation of the labeled substance, and by detecting this signal, it can be confirmed that the labeled entity has developed to the development confirmation section.

[0058] (Absorption Zone) In the immunochromatography strip of the present invention, the matrix may further include an absorption zone (e.g., absorption zone 5 shown in Figures 1 to 3 and 5) as a member having the function of absorbing a sample or developing solution that has migrated through the matrix, which is a chromatographic medium, and a labeled substance that has not been captured in the polypeptide fixing section or development confirmation section. The absorption zone according to the present invention is preferably located at the most downstream position of the matrix.

[0059] The absorption zone may be integral with the matrix 2, but is preferably a separate member (absorbent pad) made of a water-absorbent material, like the absorption zone 5. The material of the absorption zone is not particularly limited, and examples thereof include water-absorbent materials such as cellulose filter paper, nonwoven fabric, cloth, and cellulose acetate membrane.

[0060] The size of the absorbent zone is not particularly limited, but may range, for example, from 1 to 10 mm in width and from 4 to 20 mm in length, and the thickness of the absorbent pad may range, for example, from 0.5 to 2 mm.

[0061] (Substrate-containing section, substrate addition section) In the immunochromatographic strip of the present invention, when the labeling substance contained in the label is an enzyme, the substrate may be pre-contained in the developing solution, but it is preferable that the matrix further comprises a substrate-containing section (e.g., substrate-containing section 7 shown in Figure 3) that retains the enzyme substrate in an elutable manner, or an addition section (substrate addition section) to which the enzyme substrate is added during or after development of the developing solution. The substrate-containing section or substrate addition section according to the present invention is located upstream of the polypeptide immobilization section. Furthermore, the substrate-containing section or substrate addition section according to the present invention is preferably located upstream of the label-containing section.

[0062] The method for retaining the substrate in the substrate-containing region is not particularly limited as long as it allows the substrate to be eluted into the sample or the developing solution when the sample or the developing solution is developed, and examples thereof include the same methods as those exemplified as the methods for retaining the label in the label-containing region. The amount of the substrate retained in the substrate-containing region can be appropriately adjusted depending on the type of enzyme or substrate, etc.

[0063] Furthermore, the immunochromatography strip of the present invention may further include a developer pad (developer pad 3 in Figures 3 and 5) for supplying developer to the matrix, as needed, as shown in Figures 3 and 5, and may further include a sheet (not shown, for example, an adhesive sheet made of plastic, metal, paper, etc.) for supporting the matrix and each pad.

[0064] The immunochromatographic device of the present invention may include the immunochromatographic strip of the present invention, and may further include, for example, a developer tank (developer tank 11 in FIGS. 3 and 5 ) for storing a developer, upstream of the matrix of the immunochromatographic strip, a protrusion (protrusion 13 in FIG. 5 ) for supplying the developer in the developer tank to the matrix through the developer pad, and a pusher (pusher 12 in FIGS. 3 and 5 ) for applying pressure to move the protrusion. The device may also include a case that houses the immunochromatographic strip of the present invention and has a determination window for checking the polypeptide-immobilized portion (e.g., test line) and the development confirmation portion (e.g., reference line).

[0065] <Analyte detection method> The analyte detection method of the present invention is a method for detecting a analyte in a sample by immunochromatography, and includes, in the immunochromatography strip of the present invention, a capture step of capturing the analyte in the sample at the polypeptide immobilization portion, and a detection step of detecting the captured analyte.

[0066] In the present invention, "detection of a test substance" includes detection to confirm the presence or absence of the test substance, and quantification or semi-quantification of the amount of the test substance. In the present invention, detection of a test substance is carried out by detecting the test substance accumulated at the polypeptide-immobilized region, preferably by labeling the test substance with a labeling substance, detecting a signal derived from the labeling substance, and quantifying its intensity as needed. Furthermore, if necessary, the success or failure of the detection is determined by detecting the signal at the development confirmation region. In this specification, "signal" includes color development (color development), reflected light, luminescence, fluorescence, radiation from radioisotopes, etc., and includes signals that can be confirmed with the naked eye as well as signals that can be confirmed using detection methods and devices appropriate for the type of signal.

[0067] In the analyte detection method of the present invention, the analyte may be captured by the polypeptide-immobilized portion (capture step), and then, for example, the labeled body fluid may be added thereto to detect the captured analyte (detection step). However, it is preferable to perform a labeling step in which a complex between the analyte in the sample and the label is formed, and then capture the complex at the polypeptide-immobilized portion (capture step), and then detect the complex captured at the polypeptide-immobilized portion (detection step). Hereinafter, as one embodiment of the analyte detection method of the present invention, an analyte detection method (immunochromatography method) using the immunochromatographic strip of Figures 1 and 2 and the immunochromatographic device including the same of Figures 3 to 5 will be described as an example.

[0068] First, a sample is dropped or applied to the matrix 2 and accepted therein (Step 1). The sample is preferably accepted so as to come into direct contact with the label, and may be mixed with the label before being accepted in the matrix 2. However, for example, as shown in Figure 3, it is preferable to drop or apply the sample 9 to the sample application section 8 on the label-containing section 4 (label pad 4a).

[0069] Furthermore, a developing solution may be dropped or applied to the matrix 2, simultaneously with or separately from the sample, at the same position as or upstream of the label-containing portion 4. Examples of the developing solution include buffer solutions that can be adjusted to a pH suitable for the target immunochromatographic reaction, more specifically, phosphate buffer solutions, Tris buffer solutions, Good's buffer solutions, borate buffer solutions, 2-amino-2-methyl-1-propanol (AMP) buffer solutions, etc., and the buffer solutions may further contain salts, sugars, proteins, surfactants, etc.

[0070] The sample received in the matrix 2 migrates to the label-containing region 4 by capillary action. This brings the sample into contact with the label contained in the label-containing region 4, eluting the label. Furthermore, if the analyte is present in the sample, a complex (e.g., an immune complex) between the analyte and the label is formed by reaction between the analyte and the probe molecule constituting the label (Step 2, labeling step). Alternatively, in the immunochromatographic strip of the present invention, for example, as shown in FIGS. 3 to 5 , in which the label-containing region 4 is the sample application region 8, sample 9 dropped into the sample application region 8 elutes the label from the label pad 4 a and is received by the matrix 2 in the label zone 4 b. If the analyte is present in the sample, a complex between the analyte and the label is formed (Step 2, labeling step).

[0071] When the immunochromatographic strip of the present invention is, for example, one of the embodiments shown in Figures 3 to 5, the sample and label (and the formed complex) received in the matrix 2 are developed in the matrix 2 by capillary action. Furthermore, by applying pressure to the pusher 12 to move the protrusion 13, the developer pad 3 can be inserted into the developer tank 11, and the developer can be supplied to the matrix 2 through the developer pad 3. If the labeling substance contained in the label is an enzyme, the substrate is eluted into the developer as the developer passes through the substrate-containing section 7, causing the developer containing the substrate to flow. The substrate may be added to the matrix 2 by dropping or applying a substrate solution to the substrate application section simultaneously with or separately from the sample or the developer solution. Examples of the solvent for the substrate solution include the buffer solutions listed as examples of the solvent for the labeled body fluid. As a result, the developer solution containing the sample, label (and the complex), and substrate flows through the developer solution in the matrix 2.

[0072] The complex then migrates downstream by capillary action and reaches the polypeptide-immobilizing section 6 (test line). The complex is then captured by the multimerized polypeptide immobilized on the polypeptide-immobilizing section 6 via the bond between the polypeptide and the test substance (step 3, capture step). As a result of the complex being captured on the polypeptide-immobilizing section 6, the polypeptide-immobilizing section 6 exhibits coloration or the like due to the labeling substance that constitutes the label, which can be detected as a signal (detection step). If necessary, the amount can be quantified based on the signal intensity.

[0073] In the test substance detection method of the present invention, the capture step and the detection step may be performed simultaneously, for example, by visual inspection, or the detection step may be performed after the capture step. For example, after capturing the complex in the capture step, a detection step may be performed in which a treatment appropriate for the labeled substance contained in the complex (addition of a substrate, observation with a fluorescent microscope, detection with a fluorometer, detection with a scintillation counter, etc.) is performed.

[0074] If the sample does not contain the analyte, the label will not be immobilized on the polypeptide-immobilized section 6 and will migrate further downstream, resulting in no signal being detected at the polypeptide-immobilized section 6. Furthermore, the label eluted from the label-containing section 4 (if the development confirmation section 10 is located downstream of the polypeptide-immobilized section 6, the label not completely captured by the polypeptide-immobilized section 6) migrates downstream by capillary action and reaches the development confirmation section 10. As a result, the label is captured and detected by the control immobilized on the development confirmation section 10 in the development confirmation section 10 (reference line) (step 4). In the immunochromatographic strip 101 of FIG. 1 , step 4 is performed after step 3; however, steps 3 and 4 may be performed independently or simultaneously at the same position. Furthermore, step 3 may be performed after step 4. As a result of the capture of the label in the development confirmation section 10, the development confirmation section 10 exhibits coloration or the like due to the labeling substance that constitutes the label, which can be detected as a signal, and by quantifying the signal intensity as necessary, it can be determined whether the sample has migrated normally on the immunochromatography strip. The criteria for this determination can be set appropriately depending on the purpose of detection, the type of test substance, etc. The developing solution is finally absorbed in the absorption zone 5 downstream thereof.

[0075] <Immunochromatography Kit> The immunochromatography kit of the present invention includes at least the immunochromatography strip of the present invention. The immunochromatography strip may be included in the immunochromatography device of the present invention.

[0076] Furthermore, the immunochromatography kit of the present invention may further include at least one selected from the group consisting of a sample diluent, a pretreatment solution, and the developing solution, as needed. Furthermore, the labeled substance, the control substance, and their solvents, etc., may be combined. Furthermore, when the labeled substance is an enzyme, for example, a substrate necessary for detecting the label, its solvent, a reaction stop solution, etc. may also be included. Furthermore, the immunochromatography kit of the present invention may further include instructions for use of the kit.

[0077] The multimerized polypeptide, immunochromatographic strip, immunochromatographic device, test substance detection method, and immunochromatographic kit of the present invention can be used not only for research purposes, but also, for example, as in vitro diagnostic pharmaceuticals for detecting the test substance that forms the basis for diagnosing a disease related to the test substance.

[0078] The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. In particular, the type of each buffer, reaction conditions, purification conditions, etc. can be appropriately adjusted depending on the type and amount of antigen or antibody.

[0079] (Preparation Example 1) Preparation of HIV-2 gp36 synthetic polypeptide having a hydrazino group As an antigen peptide for detecting antibodies induced by HIV-2 envelope protein gp36 (HIV-2 gp36), a HIV-2 gp36 synthetic polypeptide having a hydrazino group (hydrazino peptide: comparative example) was prepared based on the amino acid sequence of HIV-2 gp36 in the same manner as in Example 3 of Japanese Patent Laid-Open No. 2004-340722 (Patent Document 1). That is, 17 residues (SEQ ID NO: 1) were selected from the amino acid sequence of HIV-2 gp36, and these 17 amino acid residues were linked together using a peptide synthesizer PSSM-8 (Shimadzu Corporation) according to the known Fmoc method. Finally, Nα-(9-fluorenylmethoxycarbonyl)-Nε-(4-(t-butoxycarbonylhydrazino)benzoyl)-L-lysine was added to prepare a synthetic HIV-2 gp36 polypeptide (hydrazino peptide) consisting of 18 amino acid residues and bearing a hydrazino group (HBz) at the N-terminal lysine. This dried powder was used in the following test examples. The sequence of the hydrazinopeptide is: Lys(HBz)-Gln-Asp-Gln-Ala-Arg-Leu-Asn-Ser-Trp-Gly-Cys-Ala-Phe-Arg-Gln-Val-Cys (the 17 residues following Lys(HBz) are shown in SEQ ID NO: 1).

[0080] (Preparation Example 2) Preparation of Dextran Carrier-Polypeptide Complex 30 mg of dextran (Dextran 70K, mass: 70 KDa) was dissolved in 0.6 mL of 0.1 M phosphate buffer (pH 7.0), and 0.4 mL of 150 mM sodium periodate aqueous solution was added. The mixture was stirred at room temperature in a dark place for 30 minutes. The reaction solution after stirring was applied to a PD-10 column (manufactured by Cytiva) equilibrated with 0.1 M phosphate buffer (pH 6.0) and eluted with the same buffer. The first 2.5 mL of the eluate was not collected, and the subsequent 2.5 mL was collected to exchange the buffer for 0.1 M phosphate buffer (pH 6.0). Next, the hydrazinopeptide prepared in Preparation Example 1 was added to the collected solution so that the molar ratio relative to dextran was 10, 20, or 35, and the mixture was stirred at room temperature in a dark place for 120 minutes. Next, 1 mg of dimethylamine borane (DMAB) was added thereto, and the mixture was stirred at room temperature in the dark for 60 minutes to obtain a conjugate of dextran and hydrazinopeptide (Dextran carrier-polypeptide conjugate: Example). The mixture was then applied to a PD-10 column (manufactured by Cytiva) equilibrated with 0.1 M phosphate buffer (pH 6.0) and eluted with the same buffer. The first 2.5 mL of the eluate was not collected, and the subsequent 1.5 mL was recovered to remove unreacted polypeptide. The recovered solution was used in the following test examples as a Dextran carrier-polypeptide conjugate solution. Note that in the following test examples, a solution with a 20-fold molar ratio of hydrazinopeptide to dextran and a polypeptide content of 30 to 40% by mass was used.

[0081] Preparation Example 3: Preparation of PEG Carrier-Polypeptide Conjugate 2 mg of a polyethylene glycol derivative (8-ArmPEG-SCM, an octavalent polyethylene glycol in which the terminal hydroxyl group has been converted to a succinimidyl carboxymethyl ester group (SCM group), mass: 20 kDa, manufactured by BioPharma PEG Scientific) was dissolved in 1 mL of 50 mM phosphate buffer, and the hydrazinopeptide prepared in Preparation Example 1 was added thereto so that the molar ratio relative to the polyethylene glycol derivative was 8 and the polypeptide content was 40 to 50% by mass. The mixture was stirred at room temperature in a dark place for 16 to 24 hours. After stirring, 1 / 10 the volume of 1 M Tris-HCl buffer (pH 8.0) was added to the reaction solution, and the mixture was stirred at room temperature for 30 minutes to mask the unreacted SCM groups, thereby obtaining a conjugate of the polyethylene glycol derivative and the hydrazinopeptide (PEG carrier-polypeptide conjugate: Example). This was then diluted with Superdex ＴＭ The product was purified by gel filtration chromatography using 200 Increase 10 / 300 GL (Cytiva), and the purified solution was used as a PEG carrier-peptide complex solution in the following test examples.

[0082] (Preparation Example 4) Preparation of Polylysine Carrier-Polypeptide Complex A polylysine carrier-polypeptide complex liquid containing a conjugate of serine-modified polylysine and hydrazinopeptide (Poly Lys-HIV2: Comparative Example), obtained in the same manner as in Example 4 of JP-A No. 2004-340722 (Patent Document 1), was used in the following test examples.

[0083] <Test Example 1> Detection of HIV-2 antibodies 1 Using the polypeptides or complexes prepared in Preparation Examples 1 to 3, an immunochromatographic device was prepared with the following configuration, and a simple immunochromatographic assay was performed using, as an example, the detection of antibodies induced by HIV-2 gp36 (HIV-2 antibodies).

[0084] (1) Materials (Capturer) Hydrazinopeptide solution containing hydrazinopeptide powder prepared in Preparation Example 1 (HIV2 peptide) Dextran carrier-polypeptide complex solution (Dextran-HIV2) prepared in Preparation Example 2 PEG carrier-polypeptide complex solution (PEG-HIV2) prepared in Preparation Example 3 (Labeled substance) ALP-labeled hydrazinopeptide (hydrazinopeptide prepared in Preparation Example 1 bound to alkaline phosphatase) (Control) Anti-ALP antibody (Developing solution) 2-amino-2-methyl-1-propanol (AMP) buffer solution (containing metal ions, NaCl, sugar, protein, and surfactant) (Substrate) 5-bromo-4-chloro-3-indolyl-phosphate (BCIP).

[0085] (2) Preparation of Immunochromatography Device 1: One short side of a 3.65 mm wide, 50 mm long strip of nitrocellulose membrane was positioned at the right end. 0.5 to 1.5 μL of the above-mentioned hydrazinopeptide solution (HIV2 peptide), Dextran carrier-peptide conjugate solution (Dextran-HIV2), or PEG carrier-peptide conjugate solution (PEG-HIV2) was spotted and dried on the nitrocellulose membrane at a position 15 mm from the right end to form a polypeptide immobilization zone (test line). The amounts of polypeptide or conjugate immobilized on the polypeptide immobilization zone were 1.20 μg for HIV2 peptide, 0.34 μg for Dextran-HIV2, and 0.11 μg for PEG-HIV2, respectively, calculated as hydrazinopeptide.

[0086] A substrate solution containing the substrate was spotted in a line at a position 47.5 mm from the right end of each nitrocellulose membrane so that the substrate amount was 80 μg, and then dried to form a substrate-containing area.Furthermore, a control solution containing the anti-ALP antibody was spotted in a line at a position 11 mm from the right end of each nitrocellulose membrane so that the antibody amount was 11 ng, and then dried to form a development confirmation area (reference line).

[0087] Furthermore, a labeled body solution containing ALP-labeled hydrazinopeptide was spotted and dried on a glass fiber membrane 3.65 mm wide and 15 mm long, with one short side positioned at the right end, to form a sample application area and a label-containing area. This produced a labeled pad equipped with a sample application area and a label-containing area. The right end of the resulting labeled pad was attached to each of the nitrocellulose membranes so that its right end was positioned 29 mm from the right end. Immunochromatography strips equipped with a substrate-containing area, a sample application area and label-containing area (label pad), a polypeptide-immobilized area (test line), and a development confirmation area (reference line) were produced on the nitrocellulose matrix. These immunochromatographic strips were set in a liquid storage container and a developer (developer tank) described in JP-A-10-104236, as well as in a test tool equipped with protrusions and deformable elements (protrusions and push-in portions), to prepare an immunochromatographic device to be used in the immunochromatographic assay evaluation 1 described below.

[0088] (3) Immunochromatographic Assay Evaluation 1: A positive specimen prepared by diluting anti-HIV-2 antibody-positive serum with anti-HIV-2 antibody-negative serum was used as a sample. The sample was dropped into the sample application section of each immunochromatographic device prepared in (2) above, and immediately after dropping, the protrusion of the immunochromatographic device was pressed to develop the developing solution. The color development in the test window was visually confirmed 15 minutes and 30 minutes after the start of development. Since a positive sample was used as the specimen in this test, if detection was correct, blue lines would be observed in both the polypeptide fixing section (test line) and the development confirmation section (reference line) of the test window.

[0089] The appearance of the test window of the immunochromatography device when each polypeptide or complex was used is shown in Figure 6. In Figure 6, the upper line in each test window is the reference line and the lower line is the test line. The appearance of the test window was visually evaluated according to the following criteria: - (negative): only the reference line is visible; + (positive): both the reference line and the test line are visible; Indeterminate: the reference line cannot be confirmed. The evaluation results are also shown in Figure 6.

[0090] As shown in Figure 6, when Dextran-HIV2 or PEG-HIV2, which are complexes of a hydrophilic polymer carrier and a polypeptide according to the present invention, were immobilized on a nitrocellulose membrane, a significant positive reaction was observed in either case. On the other hand, when only the polypeptide (HIV2 peptide) was immobilized on the nitrocellulose membrane, no positive reaction was observed, despite the large amount of immobilized polypeptide, confirming that the detection sensitivity was insufficient.

[0091] Test Example 2: Detection of HIV-2 antibodies 2 An immunochromatographic device was fabricated using the complexes of the hydrophilic polymer carrier and the polypeptide prepared in Preparation Examples 2 and 3, and the complex of the polylysine and the polypeptide prepared in Preparation Example 4, and a simple immunochromatographic assay was performed.

[0092] (1) Preparation of Immunochromatographic Device 2 Using the Dextran carrier-polypeptide complex solution prepared in Preparation Example 2 or the PEG carrier-polypeptide complex solution prepared in Preparation Example 3, immunochromatographic devices to be used in the immunochromatographic assay evaluation 2 described below were prepared in the same manner as in (2) of <Test Example 1>, except that the amount of the complex immobilized on the polypeptide immobilization site was set to 0.11 μg for Dextran-HIV2 and 0.04 μg for PEG-HIV2, calculated as hydrazinopeptide. An immunochromatographic device to be used in the following immunochromatographic assay evaluation 2 was prepared in the same manner as in (2) of <Test Example 1>, except that the polylysine carrier-polypeptide complex liquid (Poly Lys-HIV2) prepared in Preparation Example 4 was used instead of the hydrazinopeptide liquid (HIV2 peptide) containing the hydrazinopeptide powder prepared in Preparation Example 1, and the amount of polypeptide immobilized on the polypeptide immobilization part was set to 0.38 μg.

[0093] (2) Immunochromatographic Assay Evaluation 2 An HIV-2 antibody-negative specimen (Trina Inc., Switzerland) that shows a false positive for Poly Lys-HIV2, as described in JP 2004-340722 A (Patent Document 1), was used as Sample 1. The same positive specimen (anti-HIV-2 antibody-positive serum diluted with anti-HIV-2 antibody-negative serum) as in (3) of Test Example 1 above was used as Sample 2.

[0094] Each sample was dropped into the application section of each immunochromatographic device prepared in (1) above, and a simple immunochromatographic assay was performed in the same manner as in (3) of <Test Example 1>. The color development in the test window was visually confirmed 15 minutes and 30 minutes after the development of the developer. In this test, when a negative sample (sample 1) was correctly detected, a blue line was observed only in the development confirmation section (reference line) of the test window, and when a positive sample (sample 2) was correctly detected, a blue line was observed in both the polypeptide fixing section (test line) and the development confirmation section (reference line) of the test window.

[0095] The appearance of the test window of the immunochromatography device when each conjugate was used for each sample is shown in Figures 7 and 8. In Figures 7 and 8, the upper line in each test window is the reference line, and the lower line is the test line. The appearance of the test window was visually evaluated according to the following criteria: - (negative): For a negative sample (sample 1), only the reference line is visible. +W (false positive): For a negative sample (sample 1), the reference line is visible, and a test line is also visible, although it is unclear. +' (false positive): For a negative sample (sample 1), both the reference line and the test line are visible. + (positive): For a positive sample (sample 2), both the reference line and the test line are visible. Indeterminate: Other than the above. The evaluation results are also shown in Figures 7 and 8.

[0096] 7 and 8, when Dextran-HIV2 or PEG-HIV2, which are complexes of a hydrophilic polymer carrier and a polypeptide according to the present invention, were immobilized on a nitrocellulose membrane, both showed a markedly positive reaction to the positive specimen (sample 2) and a correct negative reaction to the negative specimen (sample 1). On the other hand, when Poly Lys-HIV2, which is a complex of polylysine and a polypeptide, was immobilized on a nitrocellulose membrane, it showed a positive reaction to the positive specimen (sample 2), but also a positive reaction (false positive) to the negative specimen (sample 1), preventing correct detection.

[0097] As described above, according to the present invention, it is possible to provide a polymerized polypeptide that, when immobilized on the matrix of an immunochromatographic strip and used as a capture body for capturing a test substance, enables highly sensitive detection of the test substance and suppression of non-specific reactions, as well as an immunochromatographic strip, an immunochromatographic device, an immunochromatographic kit that enable highly sensitive detection of the test substance and suppression of non-specific reactions, and a test substance detection method that uses these to enable highly sensitive detection of the test substance in immunochromatography and suppression of non-specific reactions.

[0098] 101... immunochromatography strip, 102... immunochromatography device, 2... matrix, 3... developer pad, 4, 4b, 4a... label-containing section (4a... label pad, 4b... label zone), 5... absorption zone, absorption pad, 6, 6a, 6b... polypeptide immobilization section (test line), 7... substrate-containing section, 8... sample addition section, 9... sample, 10... development confirmation section (reference line), 11... developer tank, 12... push-in section, 13... protrusion, 14... development direction

Claims

1. A polymerized polypeptide for immunochromatographic strips, comprising at least one hydrophilic polymer carrier selected from the group consisting of dextran, polyethylene glycol, and derivatives thereof, and multiple polypeptides supported on the hydrophilic polymer carrier, wherein the polypeptides are polypeptides of 200 amino acid residues or less that can bind to a test substance.

2. The multimerized polypeptide according to claim 1, wherein the amount of the polypeptide is 2 to 30 moles per mole of the hydrophilic polymer carrier.

3. The multimeric polypeptide according to claim 1, wherein the content of the polypeptide is 10 to 70% by mass.

4. The multimerized polypeptide of claim 1, wherein the polypeptide is an antigen peptide of the test substance.

5. A strip for immunochromatography used to detect a test substance in a sample by immunochromatography, comprising a matrix and a polypeptide immobilization section on which a multimerized polypeptide described in any one of claims 1 to 4 is immobilized on the matrix.

6. The immunochromatographic strip according to claim 5, wherein the matrix is a nitrocellulose membrane.

7. The immunochromatographic strip according to claim 5, wherein the amount of the multimerized polypeptide immobilized on the polypeptide immobilization portion is 0.001 to 2.0 μg in terms of polypeptide.

8. An immunochromatographic strip as described in claim 5, which has a label-containing portion upstream of the polypeptide-immobilized portion on the matrix, which holds a label containing a probe molecule capable of binding to a test substance and a labeling substance in an elutable manner.

9. An immunochromatographic strip as described in claim 8, wherein the labeled substance is an enzyme, and the matrix is provided with a substrate-containing section that holds a substrate for the enzyme in an elutable manner or a substrate-addition section that adds a substrate for the enzyme, upstream of the polypeptide-immobilized section.

10. An immunochromatographic device used for detecting a test substance in a sample by immunochromatography, comprising the immunochromatographic strip according to claim 5.

11. An immunochromatography kit used for detecting a test substance in a sample by immunochromatography, comprising the immunochromatography strip according to claim 5.

12. A method for detecting a test substance in a sample by immunochromatography, comprising: a capture step of capturing the test substance in the sample at the polypeptide immobilization part; and a detection step of detecting the captured test substance, in an immunochromatography strip comprising a matrix and a polypeptide immobilization part where a multimerized polypeptide is immobilized on the matrix, the multimerized polypeptide comprising at least one hydrophilic polymer carrier selected from the group consisting of dextran, polyethylene glycol, and derivatives thereof, and a plurality of polypeptides supported on the hydrophilic polymer carrier, and the polypeptides being polypeptides of 200 amino acid residues or less that can bind to the test substance.

13. The method for detecting a test substance according to claim 12, wherein the immunochromatographic strip comprises a label-containing section upstream of the polypeptide-immobilized section on the matrix, which holds a label containing a probe molecule capable of binding to the test substance and a labeling substance in an elutable manner, and further comprises a labeling step of forming a complex between the test substance in the sample and the label, wherein the capture step is a step of capturing the complex at the polypeptide-immobilized section, and the detection step is a step of detecting the captured complex.

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