Immunochromatographic test strip

Abstract

An immunochromatographic test strip (100) includes: a sample pad (101) into which a sample is introduced; and a detection unit (103) for detecting a substance to be detected in the sample. The detection unit (103) of the immunochromatographic test strip (100) carries: a proteinaceous or peptidic capture substance that specifically binds to the substance to be detected; and at least one sugar selected from a group consisting of sugars having three or more saccharide units and polysaccharides.

Description

Immunochromatographic test strip

[0001] The present invention relates to an immunochromatographic test strip.

[0002] In recent years, various test reagents and kits for detecting the presence or absence of pathogen infections such as viruses and bacteria, pregnancy, etc., utilizing antigen-antibody reactions or binding reactions between substances having interactions, have been continuously developed. Among them, many simple test reagents have the characteristics of not requiring special equipment, being easy to operate, and inexpensive, and are widely used not only in large hospitals and medical examination centers but also in general hospitals and clinics, and are often used by users other than inspection experts. Therefore, it is very important that the test accuracy of the reagent is high. Examples of simple test reagents currently on the market include simple test reagents for detecting pathogen infections and simple test reagents for pregnancy diagnosis. These test reagents are often implemented in medical institutions where patients first visit, and it is possible to determine the presence or absence of infection or pregnancy on the spot for the specimens collected from patients, and treatment measures can be taken at an early stage. Therefore, the importance of simple test reagents in medicine is increasing. With the increasing use of simple test reagents, users are demanding higher reproducibility of test results and test accuracy as the performance of the reagents.

[0003] Currently, as a representative reagent for simple test methods, immunoassay methods utilizing antigen-antibody reactions, especially the immunochromatographic method, are generally known. The immunochromatographic method forms a complex of a capture agent (capturing substance) that specifically binds to the detected substance and a labeled agent that specifically binds to the detected substance on a membrane, and detects or quantifies the label to detect (measure or quantify) the detected substance. The immunochromatographic method has a simple measuring device and is also excellent in terms of cost, so it is widely used for detecting a variety of detected substances.

[0004] In one form of immunochromatography, a test device is equipped with a detection unit on which an antibody that specifically binds to the substance to be detected is immobilized as a capture substance on a membrane strip such as nitrocellulose, and a labeling unit containing a label that specifically binds to the substance to be detected. A sample containing the substance to be detected is dropped onto the device, and a complex of the substance to be detected and the label is formed and spread, and this complex is captured by the detection unit to detect or quantify the label. Furthermore, there are technologies related to immunochromatography described in Patent Documents 1 to 3.

[0005] Patent Document 1 (International Publication No. 2020 / 085289) describes an immunochromatographic test piece having at least a sample supply section, a development section, and a detection section on which an antibody is immobilized, wherein the detection section contains an antibody together with one or more sugars selected from the group consisting of reducing monosaccharides and reducing disaccharides (Claim 1), and it is stated that this can reduce the color development due to the blank reaction (blank color development) while maintaining the detection sensitivity of the target substance (paragraph 0008). The reason why reducing sugars of three saccharides or more are undesirable in the detection section is that they have low solubility and are difficult to handle, and also that the proportion of reducing ends per molecular weight is low, so it is not possible to effectively prevent denaturation of the antibody by oxidation (paragraph 0017).

[0006] Patent Document 2 (International Publication No. 2002 / 040999) describes an in vitro diagnostic reagent for measuring a test substance in a sample, comprising a reagent that specifically reacts with the test substance and a compound comprising at least one hydroxyl group and at least one aldehyde group or ketone group, wherein these are supported on a support (claims 1 and 12). Sucrose and sorbitol are given as preferred examples of the compound (page 13, lines 1-2). This allows for highly accurate diagnosis and detection even after long-term storage, including but not limited to one month, two months, three months, six months, and one year (page 20, bottom line 2 to page 21, line 1).

[0007] Furthermore, Patent Document 3 (Japanese Patent Application Publication No. 2000-310639) describes an immunochromatographic apparatus that provides an immunochromatographic apparatus in which latex particles do not clog during development (paragraph 0006), comprising latex particles carrying a substance that immunologically binds to the substance to be measured, and a porous strip provided with a site for determining the substance to be measured, wherein at least one type of carbohydrate is carried on the development path of the test solution in an amount of 50 to 4,000 parts by weight per 1 part by weight of the latex particles (Claim 1).

[0008] International Publication No. 2020 / 085289, International Publication No. 2002 / 040999, Japanese Patent Publication No. 2000-310639

[0009] Regarding clinical diagnostic agents and research reagents, including immunochromatography, there is a demand from clinical settings for more reliable diagnostic results, and further improvement in reagent reliability is a challenge. A highly reliable diagnostic reagent is one that has high sensitivity and specificity and is less likely to cause misdiagnosis. Regarding specificity, there are concerns that it may decrease due to factors such as long-term storage of the reagent, heat generated during manufacturing, the influence of ambient temperature during reagent transport or storage in summer, and storage or transport in geographically hot countries. Countermeasures against these are still being explored, and more effective technologies for improving specificity are an extremely important issue for all simple diagnostic methods. In this regard, even with the technologies described in the aforementioned Patent Documents 1 to 3, the situation was still not entirely satisfactory.

[0010] Furthermore, commercially available test kits for immunochromatography generally have a shelf life of 1 to 2 years. Longer storage periods can lead to deterioration of biological raw materials within the test kit, potentially causing nonspecific reactions and reducing detection specificity. Specific examples of factors that degrade reagent performance include heat generated during manufacturing, the effects of ambient temperatures during kit transport and storage in summer, and storage and use in geographically high-temperature countries. Therefore, there is a need to suppress changes in test kit performance due to storage period and environment. However, even with the technologies described in the aforementioned Patent Documents 1 to 3, there was still room for improvement.

[0011] This invention provides a technology to improve the storage stability of immunochromatographic reagents and suppress the decrease in specificity.

[0012] The present invention provides the following immunochromatographic test pieces, immunochromatographic devices, test kits, and detection methods: [1] An immunochromatographic test piece having a sample supply unit into which a sample is introduced, and a detection unit for detecting a substance to be detected in the sample, wherein the detection unit supports on the immunochromatographic test piece a proteinaceous or peptide capture substance that specifically binds to the substance to be detected, and at least one sugar selected from the group consisting of 3 or more saccharides and polysaccharides. [2] The immunochromatographic test piece according to [1], wherein the detection unit is provided on a membrane-like support, and the capture substance and the sugar are contained in the support in the detection unit. [3] The immunochromatographic test piece according to [1] or [2], wherein the sugar comprises one or more selected from the group consisting of isomaltoligosaccharides, galactooligosaccharides, fructooligosaccharides, raffinose, and dextran. [4] The mass of the sugar per unit area in the detection unit is 2 μg / cm² 2The immunochromatographic test piece according to any one of [1] to [3]. [5] The immunochromatographic test piece according to any one of [1] to [4], comprising: a sample pad having the sample supply unit; a conjugate pad provided in contact with the sample pad and having a labeling unit portion on which a labeling substance that specifically binds to the substance to be detected is movably provided; a membrane-like support provided in contact with the conjugate pad and having the detection unit; and an absorption unit provided downstream of the detection unit and in contact with the support, which absorbs the sample. [6] An immunochromatographic device comprising the immunochromatographic test piece according to any one of [1] to [5] on a substrate. [7] A test kit having the immunochromatographic device according to [6]. [8] A method for detecting a substance to be detected in a sample by immunochromatography, comprising the steps of: introducing the sample into an immunochromatographic test piece; and contacting the sample with a detection unit provided on the immunochromatographic test piece, which includes a proteinaceous or peptide capture substance that specifically binds to the substance to be detected, and at least one sugar selected from the group consisting of three or more sugars and polysaccharides. [9] The detection method according to [8], wherein the detection unit is provided on a membrane-like support, and in the detection unit, the capture substance and the sugar are contained in the support.

[10] The detection method according to [8] or [9], wherein the sugar comprises one or more selected from the group consisting of isomaltoligosaccharides, galactooligosaccharides, fructooligosaccharides, raffinose and dextran.

[0013] Furthermore, according to the present invention, for example, it is also possible to provide the use of the immunochromatographic test piece described above for suppressing a decrease in the detection accuracy of the sample to be detected.

[0014] According to the present invention, the storage stability of immunochromatographic reagents can be improved and the decrease in specificity can be suppressed.

[0015] This is a perspective view showing an example of the configuration of an immunochromatographic test specimen in an embodiment. This is a cross-sectional view showing an example of the configuration of an immunochromatographic test specimen in an embodiment.

[0016] Embodiments of the present invention will be described below with reference to the drawings. The drawings are schematic diagrams and do not correspond to actual dimensional ratios. In this embodiment, the numerical range indicated by "~" represents "greater than or equal to" and "less than or equal to," and both values ​​at both ends are included. Furthermore, each component may be included individually or in combination of two or more components.

[0017] (Immunochromatographic Test Specimen) Figures 1 and 2 are a perspective view and a cross-sectional view, respectively, showing an example of the configuration of an immunochromatographic test specimen in this embodiment. The immunochromatographic test specimen 100 shown in Figures 1 and 2 includes a sample supply unit (sample pad 101) into which the sample is introduced, and a detection unit 103 for detecting the substance to be detected in the sample. In the detection unit 103, the immunochromatographic test specimen 100 is supported with a proteinaceous or peptide capture substance that specifically binds to the substance to be detected, and at least one sugar selected from the group consisting of three or more sugars and polysaccharides.

[0018] In this embodiment, since the detection unit 103 of the immunochromatographic test piece 100 is supported with a specific capture substance and at least one of three-saccharide or more oligosaccharides and polysaccharides, it has excellent storage stability, and even if the immunochromatographic test piece 100 is exposed to harsh conditions, for example, the decrease in specificity in the detection unit 103 can be effectively suppressed. Harsh conditions include, for example, long-term storage of the immunochromatographic test piece 100, during manufacturing or transportation / storage in summer, or transportation / storage in countries with high geographical temperatures. In this embodiment, even if the immunochromatographic test piece 100 is exposed to such harsh conditions as described above, it is possible to detect the target substance with excellent detection accuracy (precision). More specifically, according to this embodiment, false positives can be reduced and specificity can be improved even after storage of the immunochromatographic test piece 100. Furthermore, according to this embodiment, it is also possible to suitably suppress, for example, the denaturation of the capture substance due to storage and the resulting nonspecific adsorption in the detection unit 103.

[0019] First, we will specifically describe the sample introduced into the sample pad 101 and the capture substance and sugar supported on the detection unit 103.

[0020] (Sample) The sample should be of a nature that allows it to be introduced into the immunochromatographic test piece 100. From the viewpoint of improving fluidity in the flow path of the immunochromatographic test piece 100, the sample is preferably a liquid or semi-solid. The sample may also be a biological sample, for example. Specific examples of biological samples include at least one liquid or semi-solid sample selected from the group consisting of nasal swabs, nasopharyngeal swabs, nasal aspirates, pharyngeal swabs, saliva, nasal secretions, urine, feces, vomit, blood, serum, plasma, skin exudate, and surface scrapings. The sample may also be a liquid or semi-solid sample containing components derived from the above-mentioned biological sample. Here, a semi-solid sample is, for example, feces or vomit.

[0021] (Capture substance) The capture substance is a proteinaceous or peptide-like substance that specifically binds to the substance to be detected. The capture substance may also contain sugar chains. Specific examples of the capture substance include antigen-binding substances such as antibodies, and at least one selected from the group consisting of avidins such as avidin and streptavidin. For example, the capture substance may be an antibody against the substance to be detected or an antigen-binding fragment thereof, and is preferably an antibody. The antibody may be a polyclonal antibody or a monoclonal antibody. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies.

[0022] When the substance to be detected is a virus-derived antigen, the capture substance may be an antibody against the viral antigen or an antigen-binding fragment thereof, and may be selected from the group consisting of, for example, anti-hMPV antibody, anti-RSV antibody, anti-FluA antibody, anti-FluB antibody, anti-SARS-CoV-2 antibody and antigen-binding fragments thereof.

[0023] (Sugar) The sugar supported on the detection unit 103 includes at least one sugar selected from the group consisting of three or more saccharides (oligosaccharides) and polysaccharides. Preferably, the sugar functions as a durability enhancer to support the durability of the capture substance. Specifically, durability of the capture substance includes durability against heat and durability for long-term storage. Hereinafter, the at least one sugar selected from the group consisting of three or more saccharides (oligosaccharides) and polysaccharides supported on the detection unit 103 will also be referred to as the "durability enhancer" as appropriate. The durability enhancer is, for example, a durability improver for the capture substance. The durability enhancer may also be a denaturation inhibitor for the capture substance. By using a configuration in which the above sugar is supported as a durability enhancer, for example, denaturation of the capture substance due to exposure to harsh conditions as described above and the occurrence of non-specific adsorption to the detection unit 103 associated with this can be suppressed more stably.

[0024] The number of constituent sugars in the oligosaccharide is three or more, preferably 10 or less, and more preferably 5 or less. This allows for more stable suppression of the decrease in specificity due to exposure to harsh conditions such as those described above. The oligosaccharide may consist of one constituent sugar or may have two or more constituent sugars. Preferably, the oligosaccharide contains one or more selected from the group consisting of isomaltoligosaccharide, galactooligosaccharide, fructooligosaccharide, and raffinose, and more preferably at least one of galactooligosaccharide and isomaltoligosaccharide. This allows for a more stable improvement in the specificity and detection accuracy of the immunochromatographic test piece 100.

[0025] Galactooligosaccharides are oligosaccharides whose main component is 4'-galactosyl lactose, with 2 to 6 galactose molecules. The glucosidic bond is basically a β-1,4 bond, but it may also contain a β-1,3 bond. Isomaltooligosaccharides are oligosaccharides whose constituent sugar is glucose and which contain an α-1,6 glucosidic bond. In addition to the α-1,6 glucosidic bond, they may also contain an α-1,4 glucosidic bond, etc. Isomaltooligosaccharides include one or more oligosaccharides selected from the group consisting of, for example, panose (trisaccharide, α-1,6 and α-1,4 bonds), isomalttriose (trisaccharide, α-1,6 bond), and isomalttetratriose (tetrasaccharide, α-1,6 bond), which are oligosaccharides having three or more constituent sugars.

[0026] Furthermore, specific examples of polysaccharides include one or more selected from the group consisting of dextran, dextrin, and amylose. The constituent sugars of the polysaccharide include, for example, glucose, and preferably glucose. The average molecular weight of the polysaccharide is preferably 1000 or more, preferably 2000 or more, more preferably 3000 or more, and may also be 10000 or more, and preferably 100000 or less, more preferably 50000 or less, for example, 10000 or less or 5000 or less. This allows for more efficient suppression of the decrease in specificity.

[0027] The sugar used as a durability enhancer preferably comprises one or more selected from the group consisting of isomaltoligosaccharides, galactooligosaccharides, fructooligosaccharides, raffinose, and dextran, and more preferably galactooligosaccharides or isomaltoligosaccharides. This further improves the storage stability and specificity of the immunochromatographic test specimen 100.

[0028] Furthermore, the durability enhancer may further include at least one selected from the group consisting of monosaccharides and disaccharides. Examples of monosaccharides and disaccharides include isomaltoligosaccharides and other monosaccharides and disaccharides that are included in the above-mentioned oligosaccharides or polysaccharides due to the manufacturing process. Other examples of monosaccharides and disaccharides include glucose, ribose, sucrose, maltose, galactose, lactose, trehalose, and the like.

[0029] Returning to Figures 1 and 2, the configuration of the immunochromatographic test piece 100 having a durability enhancer in the detection unit 103 will be described in more detail.

[0030] The immunochromatographic test specimen 100 includes a sample pad 101 having a sample supply section, a conjugate pad 107 provided in contact with the sample pad 101 and having a labeling section on which a labeling substance that specifically binds to the substance to be detected is movably provided, a film-like support 105 provided in contact with the conjugate pad 107 and having a detection section 103, and an absorption section 109 provided downstream of the detection section 103 and in contact with the support 105 for absorbing the sample. In the example shown in Figures 1 and 2, the support 105 is provided on a substrate 111, the conjugate pad 107 and the absorption section 109 are provided on the support 105 at both ends of the support 105, and the sample pad 101 is provided on the conjugate pad 107 at the end of the conjugate pad 107 opposite to the support 105, resulting in a laminated structure. The sample introduced into the sample pad 101 is transported in the following order from the sample introduction side to the sample discharge side (along the direction of travel d in Figure 2): conjugate pad 107, support 105, and absorbent section 109.

[0031] Specifically, the substrate 111 functions as a holding member for the support 105. In the immunochromatographic test specimen 100, the substrate 111 is a member to which the sample pad 101, conjugate pad 107, support 105, and absorbent portion 109 are attached and fixed with partial overlap. By providing the substrate 111, the manufacturing stability and stability during use of the immunochromatographic test specimen 100 can be further improved. Specifically, the material of the substrate 111 can be one or more selected from the group consisting of glass such as quartz glass, soda-lime glass, and borosilicate glass; and resin materials such as poly(meth)acrylate such as polymethyl (meth)acrylate, polyester, polyolefin, polystyrene, polycarbonate, fluororesin, polyvinyl chloride, polyamide, and polyimide. The thickness of the substrate 111 is, for example, 0.2 mm or more, preferably 0.5 mm or more, in terms of improving the strength of the immunochromatographic test specimen 100. Furthermore, in terms of thinning the immunochromatographic test specimen 100, the thickness of the substrate 111 is, for example, 5 mm or less, preferably 4 mm or less.

[0032] The sample pad 101 functions as a sample supply unit into which the sample is introduced. Examples of materials for the sample pad 101 include filter paper, glass fiber, and nonwoven fabric. The thickness of the sample pad 101 can be, for example, about 0.3 to 1 mm.

[0033] The conjugate pad 107 is positioned between the sample pad 101 and the detection unit 103 and functions as a labeling unit on which a labeling substance that specifically binds to the substance to be detected is movably provided. In the example of the immunochromatographic test piece 100, the labeling substance is provided over the entire conjugate pad 107, and the substance to be detected in the sample that reaches the conjugate pad 107 specifically binds to the labeling substance in the conjugate pad 107 and moves to the support 105. The conjugate pad 107 can be made of, for example, a porous substrate containing the labeling substance. For example, glass fibers, nonwoven fabrics, etc., can be used as the porous substrate. The thickness of the conjugate pad 107 is preferably about 0.3 to 0.6 mm in order to sufficiently hold the labeling substance while making the entire immunochromatographic test piece 100 thinner.

[0034] Specific examples of labeling substances that specifically bind to the substance to be detected include labeled antibodies against the substance to be detected. For example, labeled antibodies used in immunoassays such as enzyme-linked immunosorbent assay (ELISA) and fluorescence immunoassay can be used.

[0035] Examples of labels for labeled antibodies include enzymes such as alkaline phosphatase and horseradish peroxidase; metal colloids such as gold colloid; and particles such as silica particles, cellulose particles, magnetic particles, fluorescent particles, and colored particles (colored polystyrene particles, colored latex particles, etc.). When metal colloid particles or colored particles are used as labels, the labeled antibody is immobilized into particles, and the coloration caused by the aggregation of the labeled particles can be detected or measured.

[0036] The concentration of the labeling substance in the conjugate pad 107 can be, for example, 0.005% by mass to 4.00% by mass. This ensures a sufficient amount to label the substance to be detected while reducing the detection background.

[0037] The support 105 is specifically a sheet-like member that supports the detection unit 103 and has a channel for transporting the sample. Examples of materials for the support 105 include cellulose, nitrocellulose, cellulose acetate, polyvinylidene difluoride (PVDF), glass fiber, nylon, and polyketone. The support 105 is preferably a nitrocellulose membrane. This allows the detection unit 103 to stably support the capture substance and durability enhancer.

[0038] The thickness of the support 105 is, for example, 50 μm or more, preferably 100 μm or more, and more preferably 150 μm or more, in terms of improving the strength of the immunochromatographic test specimen 100. Also, in terms of making the entire immunochromatographic test specimen 100 thinner, the thickness of the support 105 is, for example, 2000 μm or less, and preferably 1000 μm or less.

[0039] The detection unit 103 is a region of the support 105 on which the capture substance and durability enhancer are supported. In the immunochromatographic test specimen 100, the detection unit 13 is provided on the film-like support 105, and the capture substance and durability enhancer are contained in the support 105 within the detection unit 103. There may be one or more detection units 103. In the example of the immunochromatographic test specimen 100, there are two detection units 103, which function as the test line TL and the control line CL.

[0040] The width of one detection unit 103, that is, the length in the direction perpendicular to the direction of travel d, is preferably 0.5 mm or more, more preferably 0.8 mm or more, and preferably 3 mm or less, and more preferably 1.5 mm or less. This improves detection stability while reducing the required amount of sample. The spacing between multiple detection units 103 is preferably 1 mm or more, more preferably 2 mm or more, and preferably 10 mm or less, and more preferably 5 mm or less. This allows for miniaturization of the support unit 105 while suppressing the contamination of adjacent detection units 103 with components.

[0041] Here, the durability aid is preferably provided in direct contact with the detection unit 103, and more preferably adheres to the support 105 in the detection unit 103. The durability aid may be immobilized on the surface of the detection unit 103, for example, or may exist reversibly without being immobilized. More specifically, the durability aid is either directly adsorbed or bound to the surface of the detection unit 103, or is in contact without adsorption or binding. The durability aid may be physically adsorbed or chemically adsorbed or bound to the detection unit 103. The capture substance only needs to be contained in the detection unit 103. For example, it may adhere to the durability aid, may be provided on the surface of the detection unit 103 via the durability aid, or may adhere to the durability aid and the support 105. The capture substance is preferably immobilized on the detection unit 103. Thereby, the substance to be detected can be surely captured by the detection unit 103.

[0042] The mass per unit area of the durability aid in the detection unit 103 is preferably 2 μg / cm 2 or more, more preferably 3 μg / cm 2 or more, and even more preferably 4 μg / cm 2 or more. Thereby, a decrease in specificity in the detection unit 103 can be more surely suppressed. Also, from the point of more efficiently suppressing a decrease in specificity in the detection unit 103, the mass per unit area of the durability aid in the detection unit 103 is preferably 150 μg / cm 2 or less, more preferably 100 μg / cm 2 or less, and even more preferably 50 μg / cm 2 or less.

[0043] Further, the support 105 further has a developing portion 113 (FIG. 2) that develops the sample introduced into the sample pad 101 on the upstream side of the detection unit 103. In the immunochromatographic test strip 100, the durability aid is selectively arranged on the support 105 in the detection unit 103.

[0044] The absorption part 109 is provided in contact with the support 105 on the downstream side of the support 105, and absorbs the components that have flowed through the support 105. For example, the components that are supplied to the support 105 and are not involved in the detection in the detection part 103 are absorbed by the absorption part 109. Examples of the material of the absorption part 109 include filter paper and porous bodies made of, for example, paper or polymer compounds. From the viewpoint of smoothly advancing the development of the sample, it is preferable to use a material with high water absorption for the absorption part 109.

[0045] Next, a modified example of the immunochromatographic test piece will be described. Hereinafter, the description will focus on the points different from the immunochromatographic test piece 100 shown in FIGS. 1 and 2.

[0046] In the immunochromatographic test piece 100, an example where the durability assisting agent is selectively arranged in the detection part 103 on the support 105 has been shown. However, the durability assisting agent may also be provided in the developing part 113 of the support 105, or may be provided, for example, throughout the support 105.

[0047] Further, in the immunochromatographic test piece 100, an example where the durability assisting agent is selectively provided in the detection part 103 and the conjugate pad 107 that functions as the labeling body part does not have the durability assisting agent has been illustrated. However, the durability assisting agent may be further provided in the conjugate pad 107.

[0048] Next, a method for manufacturing the immunochromatographic test piece 100 will be described. The method for manufacturing the immunochromatographic test piece 100 includes, for example, a step of applying a capture substance and a durability assisting agent to a predetermined region of the film-like support 105 to obtain the detection part 103 having the capture substance and the durability assisting agent. At this time, the capture substance and the durability assisting agent may be applied together or sequentially. For example, after applying the durability assisting agent to a designated region on the surface of the support 105, the capture substance may be applied. Also, in the example of the immunochromatographic test piece 100, the application regions of the capture substance and the durability assisting agent are the same region, but these may be different regions. For example, the durability assisting agent may be applied to the entire support 105, and the capture substance may be applied to the detection part 103.

[0049] Furthermore, the method for manufacturing the immunochromatographic test specimen 100 may include, for example, the steps of: arranging a support 105 on a substrate 111; laminating a conjugate pad 107 and an absorption portion 109 in contact with the end of the support 105 on the substrate 111; and laminating a sample pad 101 in contact with the end of the conjugate pad 107 opposite to the support 105. The immunochromatographic test specimen 100 obtained in this embodiment may be used as is, or may be applied to, for example, the following immunochromatographic device.

[0050] The immunochromatographic test strip 100 can be used, for example, to detect SARS-CoV-2, influenza A virus, influenza B virus, human metapneumovirus, RSV, adenovirus, rhinovirus, group A streptococcus, mycoplasma pneumoniae, norovirus, rotavirus, Campylobacter, Helicobacter pylori, pathogenic Escherichia coli, Staphylococcus aureus, MRSA, Streptococcus pneumoniae, Legionella, Bordetella pertussis, procalcitonin, presepsin, brain natriuretic peptide, cardiac troponin T, D-dimer, H-FABP, luteinizing hormone, and human chorionic gonadotropin.

[0051] (Immunochromatographic Device) In this embodiment, the immunochromatographic device comprises the immunochromatographic test specimen of this embodiment on a substrate. The substrate may be, for example, a housing that holds the immunochromatographic test specimen 100. This improves the mechanical strength of the immunochromatographic device having the immunochromatographic test specimen 100, and reduces or prevents damage to the test specimen due to transportation or external forces. It also improves usability by providing functions such as improved operability or reduction or prevention of secondary infection.

[0052] The housing may have, for example, a bottom portion for supporting the immunochromatographic specimen 100, side portions for holding the outer circumference of the side of the immunochromatographic specimen 100, and a lid portion provided on the top of the immunochromatographic specimen 100, with an opening provided at a predetermined position on the lid portion. The opening may include, for example, a sample inlet located on the top of the sample pad 101. In addition, an opening or window portion may be provided on the lid portion above the control lines CL and TL.

[0053] (Packaging) In this embodiment, the packaging contains the immunochromatographic device of this embodiment housed in a packaging container. For example, by using a material with excellent moisture resistance and gas barrier properties as the packaging material, the storage stability of the immunochromatographic test piece 100 can be further improved. Examples of packaging container materials include one or more resin materials selected from the group consisting of polyethylene, polypropylene, polyethylene terephthalate, polyvinyl chloride, and polyvinylidene chloride; and metal materials such as aluminum packaging materials. The packaging container may be, for example, a single-layer film or a multi-layer film of these materials.

[0054] (Test Kit) In this embodiment, the test kit includes the immunochromatographic device of this embodiment. The immunochromatographic device may be included in the test kit as the packaging described above. The test kit may also be a test reagent containing the immunochromatographic device and other reagents. For example, the test kit may include, in addition to the immunochromatographic device, at least one selected from the group consisting of a sample extraction solution for suspending the sample and a reagent applied to the immunochromatographic device. The test kit may also appropriately include sample collection equipment such as cotton swabs.

[0055] (Detection Method) The detection method in this embodiment is a method for detecting the substance to be detected in a sample by immunochromatography, and includes the following steps: (Step 10) Introducing the sample into an immunochromatographic test piece 100 (Step 20) Contacting the sample with a detection unit 103 provided on the immunochromatographic test piece 100, which includes a proteinaceous or peptide capture substance that specifically binds to the substance to be detected, and at least one sugar (durability enhancer) selected from the group consisting of 3 or more sugars and polysaccharides.

[0056] In step 10, the sample is introduced, for example, by dropping it onto the sample pad 101. The following steps may also be included between steps 10 and 20: (Step 12) A step to move the sample introduced into the immunochromatographic test piece 100 to the conjugate pad 107 to specifically bind the labeled substance provided on the conjugate pad 107 to the substance to be detected. (Step 14) A step to move the substance to be detected, which has been specifically bound to the labeled substance, to the development section 113 and then the detection section 103 of the support 105, and guide it to the detection section 103.

[0057] In step 20, the substance to be detected comes into contact with at least the capture substance in the detection unit 103, and the substance to be detected is captured in the detection unit 103 by a specific interaction between the capture substance and the substance to be detected. The substance to be detected may or may not come into contact with the durability enhancer in the detection unit 103. In this embodiment, since the capture substance and durability enhancer are provided in the detection unit 103, the storage stability of the capture substance in the detection unit 103 is excellent, and the decrease in specificity in the detection unit 103 can be effectively suppressed even after the immunochromatographic test piece 100 has been stored. For this reason, the decrease in the measurement accuracy of the substance to be detected can be suitably suppressed even after the immunochromatographic test piece 100 has been stored.

[0058] Furthermore, the following steps may be added after step 20: (Step 30) A step to detect or quantify the substance to be detected in the sample by detecting the substance to be detected that has been captured in the detection unit 103. In step 30, a detection method can be used that corresponds to the type of labeling substance used in step 12. For example, if a fluorescently labeled antibody is used in step 12, the substance to be detected can be detected or quantified in step 30 by measuring the presence or absence of fluorescence or the fluorescence intensity in the detection unit 103. Alternatively, if an enzyme-labeled antibody is used in step 12, in step 30, a substrate for the enzyme immobilized on the labeled antibody is introduced into the detection unit 103, and the substance to be detected can be detected or quantified by measuring the presence or absence of color development based on the substrate, absorbance, presence or absence of fluorescence, fluorescence intensity, presence or absence of chemiluminescence, chemiluminescence intensity, etc.

[0059] The embodiments of the present invention have been described above with reference to the drawings, but these are merely examples of the present invention, and various other configurations can also be adopted.

[0060] The embodiment will be described in detail below with reference to examples, but this embodiment is not limited to these examples.

[0061] (Examples 1-4) Immunochromatographic specimens were prepared and evaluated using the following methods.

[0062] 1.1. Preparation of Labeled Substrates One antibody each was used from the following: anti-influenza A virus (FluA), anti-influenza B virus (FluB), anti-RSV virus (RSV), anti-human metapneumovirus (hMPV), and anti-SARS-CoV-2. One of the above antibodies was covalently bound to colored latex particles, suspended in buffer solution, and thoroughly dispersed to prepare an antibody-conjugated colored latex suspension. The antibody-conjugated colored latex suspension was applied to a 20 cm x 1 cm nonwoven glass fiber fabric, thoroughly dried under warm air, and a labeled substrate pad was prepared by forming a dried mixture.

[0063] 2. Preparation of the detection membrane A nitrocellulose membrane with a thickness of 0.3 mm was cut to a size of 2 cm x 20 cm. An amount of anti-FluA antibody, anti-FluB antibody, anti-RSV antibody, or anti-hMPV antibody (the same type of antibody used for the labeling pad) in a line about 1.0 cm from the bottom edge was applied in a line about 1 mm wide. The membrane was then immersed in a solution containing 50 mM Tris (pH 7.0) and 0.25-10% sugars, and thoroughly dried under warm air to solidify the antibody and prepare the detection membrane.

[0064] 3. Preparation of Immunochromatographic Specimens The immunochromatographic specimen used had the same configuration as shown in Figure 1. A filter paper measuring 3 cm x 20 cm was overlapped by 5 mm on the upper end of the detection membrane to create a developing solution absorption section. Furthermore, a label section was overlapped by 2 mm on the lower end of the detection membrane to create a label section, and a sample supply section was overlapped at a position 7 mm away from the upper end of the label section to create a sample addition section. Next, the specimen was cut into 5 mm wide strips with a cutter to create an integrated immunochromatographic specimen.

[0065] In addition, the following oligosaccharides and dextrans were used: Isomaltooligosaccharide: Isomaltooligosaccharide, manufactured by Fujifilm Wako Pure Chemical Industries; Galactooligosaccharide: Galactooligosaccharide, manufactured by Fujifilm Wako Pure Chemical Industries; Fructooligosaccharide: Fructooligosaccharide, manufactured by Fujifilm Wako Pure Chemical Industries; Raffinose: D(+)-raffinose pentahydrate, manufactured by Fujifilm Wako Pure Chemical Industries; Dextran: Average molecular weight 40,000, dextran 40,000, manufactured by Fujifilm Wako Pure Chemical Industries.

[0066] (Example 1) In this example, the effect of oligosaccharides on improving specificity was evaluated. First, anti-hMPV antibody, anti-RSV antibody, anti-FluA antibody, or anti-FluB antibody was applied in a line pattern to the detection membrane, and then immersed in an aqueous solution containing 10% disaccharide (trehalose) or 10% oligosaccharide {isomaltooligosaccharide (IMO)}. Immunochromatographic test pieces were prepared using the above detection membrane as described above. Next, the prepared immunochromatographic test pieces were heated at 60°C for more than 3 days. After that, a predetermined amount of nasal aspirate specimens (hereinafter referred to as "specimens") that are negative for the target virus and are expected to be used in clinical settings were added, and the test was performed, and the color development of the false-positive lines on the test pieces was observed. False-positive lines were compared with a predetermined color chart (Denka Co., Ltd.'s in-house prepared product, the same applies hereinafter) and scored. The scores for each specimen are shown in Table 1 (Tables 1-1 to 1-4). The color sample scores were assigned as follows: "0" represents no color development (false positive), "±" represents the limit of visible color development, and scores range from "1+" to "8+" as the color development becomes stronger. When observed with the naked eye, the degree of color development for each score can be expressed as follows: "0": no color development, "±": suspected very slight color development, "1+": slight coloring, "2+": weak color development, "3+, 4+, 5+": clear color development, and "6+, 7+, 8+": prominent color development.

[0067]

[0068] Tables 1-1 to 1-4 show that test pieces using trehalose exhibited strong false-positive results in all antibody samples after being stored under harsh conditions. On the other hand, test pieces containing IMO showed significantly improved false-positive results compared to the trehalose-containing test pieces in all samples. From these results, it was found that test pieces using isomaltoligosaccharide can significantly improve specificity for multiple different antibodies.

[0069] (Example 2) In this example, the effect of oligosaccharides on improving storage stability was evaluated.

[0070] (Test Example 2-1) First, an anti-FluA antibody was applied in a line to the detection membrane, and then it was immersed in an aqueous solution containing either 2% disaccharide (trehalose) or 10% oligosaccharide (isomaltooligosaccharide). Immunochromatographic test pieces were prepared using the above detection membranes. Next, of the prepared immunochromatographic test pieces, those immersed in trehalose were stored at 4°C for 23 months, at 33°C for 23 months, or at 60°C for 3 days. The test pieces immersed in isomalttooligosaccharide were stored at 60°C for 28 days. After storage, a predetermined amount of buffer solution without the detection antigen was added to the test pieces, and the test was performed, and the color development of the false-positive lines on the test pieces was observed. False-positive lines were compared with a predetermined color chart and scored. The scores for each sample are shown in Table 2. The color sample scores were assigned as follows: "0" represents no color development (false positive), "±" represents the limit of visible color development, and scores range from "1+" to "8+" as the color development becomes stronger. When observed with the naked eye, the degree of color development for each score can be expressed as follows: "0": no color development, "±": suspected very slight color development, "1+": slight coloring, "2+": weak color development, "3+, 4+, 5+": clear color development, and "6+, 7+, 8+": prominent color development.

[0071]

[0072] Table 2 shows that test specimens immersed in trehalose did not show false positive reactions after 23 months of storage at 4°C, but a weak false positive reaction was observed in the FluA detection line after 23 months of storage at 33°C. Furthermore, when the same test specimens were stored at 60°C for 3 days and the same test was performed, a moderate false positive reaction was observed. From this, it was found that storing the test specimens at 60°C for 3 days is a harsher storage condition than storing them at 33°C for 23 months. On the other hand, no false positives were observed in test specimens immersed in IMO even after 28 days of storage at 60°C, suggesting that oligosaccharides have the effect of improving the storage stability of test specimens.

[0073] (Test Example 2-2) First, anti-SARS-CoV-2 antibody was applied in a line to the detection membrane, and then immersed in an aqueous solution containing 2% disaccharide (trehalose) or 10% oligosaccharide (isomaltooligosaccharide). Immunochromatographic test pieces were prepared using the above detection membrane as described above. Next, of the prepared immunochromatographic test pieces, the test piece immersed in trehalose was stored at 4°C for 23 months, at 33°C for 23 months, or at 60°C for 3 days. The test piece immersed in isomalttooligosaccharide was stored at 60°C for 28 days. After storage, a predetermined amount of buffer solution without the detection antigen was added to the test piece, and the test was performed, and the color development of the false-positive line on the test piece was observed. False-positive lines were compared with a predetermined color chart and scored. The results are shown in Table 3. The color sample scores were assigned as follows: "0" represents no color development (false positive), "±" represents the limit of visible color development, and scores range from "1+" to "8+" as the color development becomes stronger. When observed with the naked eye, the degree of color development for each score can be expressed as follows: "0": no color development, "±": suspected very slight color development, "1+": slight coloring, "2+": weak color development, "3+, 4+, 5+": clear color development, and "6+, 7+, 8+": prominent color development.

[0074]

[0075] Table 3 shows that while the test specimens immersed in trehalose showed no false positive reactions after 23 months of storage at 4°C, a weak false positive reaction was observed in the SARS-CoV-2 detection line after 23 months of storage at 33°C. Furthermore, a weak false positive reaction was observed when the same test specimens were stored at 60°C for 3 days and the same test was performed. This indicates that the storage conditions of 23 months at 33°C and 3 days at 60°C are of similar severity. On the other hand, since no false positives were observed in the test specimens immersed in IMO even after 28 days of storage at 60°C, it was inferred that oligosaccharides similarly improve storage stability in this test specimen as well.

[0076] (Example 3) In this example, the effect of improving the specificity of various oligosaccharides and polysaccharides was evaluated.

[0077] (Test Example 3-1) First, anti-FluA antibody was applied in a line to the detection membrane, and then it was immersed in an aqueous solution containing 6% disaccharide (trehalose or maltose), 6% oligosaccharide (isomaltooligosaccharide, galactooligosaccharide, fructooligosaccharide or raffinose), or polysaccharide (dextran molecular weight 40,000, indicated as "Dextran40000" in Table 4). Immunochromatographic test pieces were prepared using the above detection membrane as described above. Next, the prepared immunochromatographic test pieces were heated at 60°C for 3 days or more. After that, a predetermined amount of buffer solution without the detection antigen or a nasal aspirate sample negative for the target virus, which is expected to be used in a clinical setting, was added, and the test was performed, and the color development of the false-positive lines on the test pieces was observed. False-positive lines were compared with a predetermined color chart and scored. The results are shown in Table 4. The color sample scores were assigned as follows: "0" represents no color development (false positive), "±" represents the limit of visible color development, and scores range from "1+" to "8+" as the color development becomes stronger. When observed with the naked eye, the degree of color development for each score can be expressed as follows: "0": no color development, "±": suspected very slight color development, "1+": slight coloring, "2+": weak color development, "3+, 4+, 5+": clear color development, and "6+, 7+, 8+": prominent color development.

[0078]

[0079] Table 4 shows that trehalose, a disaccharide, produced strong false positives in both the buffer and the sample. For other oligosaccharides and polysaccharides, the false positive signal intensity was reduced or completely eliminated in both the buffer and the sample compared to when trehalose was used. In a comparison of different oligosaccharides and polysaccharides, isomaltoligosaccharides and galactooligosaccharides completely suppressed the false positive reaction, demonstrating a significantly stronger false positive suppression effect than fructooligosaccharides and raffinose. While its false positive suppression effect was inferior to isomaltoligosaccharides and galactooligosaccharides, Dextran 40000 also showed a stronger false positive suppression effect than fructooligosaccharides and raffinose.

[0080] (Test Example 3-2) First, anti-FluA antibody and anti-FluB antibody were applied in lines to the detection membrane, and then immersed in an aqueous solution containing 6% disaccharide (trehalose or maltose) or 6% oligosaccharide (isomaltooligosaccharide). Immunochromatographic test pieces were prepared using the above detection membrane as described above. Next, the prepared immunochromatographic test pieces were heated at 60°C for 3 days or more. After that, a predetermined amount of buffer without the detection antigen or a nasal aspirate sample negative for the target virus, which is expected to be used in a clinical setting, was added, and the test was performed, and the color development of the false-positive lines on the test pieces was observed. False-positive lines were compared with a predetermined color chart and scored. The results are shown in Table 5. The color sample scores were assigned as follows: "0" represents no color development (false positive), "±" represents the limit of visible color development, and scores range from "1+" to "8+" as the color development becomes stronger. When observed with the naked eye, the degree of color development for each score can be expressed as follows: "0": no color development, "±": suspected very slight color development, "1+": slight coloring, "2+": weak color development, "3+, 4+, 5+": clear color development, and "6+, 7+, 8+": prominent color development.

[0081]

[0082] Table 5 shows that isomaltoligosaccharide can suppress false positive reactions that could not be suppressed by trehalose or maltose.

[0083] (Example 4) In this example, the effective concentration of oligosaccharides and polysaccharides was evaluated. First, anti-FluA antibody was applied in a line to the detection membrane, and then each was immersed in a solution containing either 0.5% trehalose or one of the following concentrations of isomaltoligosaccharide (IMO 0.25%, 0.5%, 1.0%, 8.0%). Immunochromatographic test pieces were prepared using the above detection membrane as described above. Next, the prepared immunochromatographic test pieces were heated at 60°C for 3 days or more. After that, a predetermined amount of buffer without the detection antigen was added dropwise, and the test was performed, and the degree of color development of the false positive lines on the test pieces was observed. False positive lines were compared with a predetermined color chart and scored. The results are shown in Table 6. The color chart scores were as follows: "0" was considered as no color development (false positive), "±" as the limit of visible color development, and then scores from "1+" to "8+" as the color development became stronger. The degree of color development for each score can be expressed as follows when observed with the naked eye: "0": no color development, "±": suspected very slight color development, "1+": slight color development, "2+": weak color development, "3+, 4+, 5+": clear color development, "6+, 7+, 8+": prominent color development.

[0084]

[0085] Table 6 shows that test specimens immersed in 0.25% isomaltoligosaccharide showed weaker false positives than test specimens immersed in 0.5% trehalose. Similarly, test specimens immersed in IMO at concentrations of 0.25% or higher also showed a significant reduction in false positives.

[0086] This application claims priority based on Japanese Patent Application No. 2024-208790, filed on 29 November 2024, and incorporates all of its disclosures herein.

[0087] 100 Immunochromatographic specimen 101 Sample pad 103 Detection unit 105 Support 107 Conjugate pad 109 Absorption unit 111 Substrate 113 Deployment unit CL Control line d Direction of travel TL Test line

Claims

1. An immunochromatographic test piece comprising: a sample supply unit into which a sample is introduced; and a detection unit for detecting a substance to be detected in the sample, wherein the detection unit supports the immunochromatographic test piece with a proteinaceous or peptide capture substance that specifically binds to the substance to be detected, and at least one sugar selected from the group consisting of 3 or more sugars and polysaccharides.

2. The immunochromatographic test piece according to claim 1, wherein the detection unit is provided on a membrane-like support, and the capture substance and the sugar are contained in the support within the detection unit.

3. The immunochromatographic test piece according to claim 1 or 2, wherein the sugar comprises one or more selected from the group consisting of isomaltoligosaccharides, galactooligosaccharides, fructooligosaccharides, raffinose, and dextran.

4. The mass of the sugar per unit area in the detection unit is 2 μg / cm². 2 The immunochromatographic specimen described in claim 2 is as described above.

5. An immunochromatographic test piece according to claim 1 or 2, comprising: a sample pad having the sample supply section; a conjugate pad provided in contact with the sample pad and having a labeling section on which a labeling substance that specifically binds to the substance to be detected is movably provided; a membrane-like support provided in contact with the conjugate pad and having the detection section; and an absorption section provided downstream of the detection section and in contact with the support, which absorbs the sample.

6. An immunochromatographic device comprising an immunochromatographic test specimen according to claim 1 or 2 on a substrate.

7. A test kit having the immunochromatographic device according to claim 6.

8. A method for detecting a substance to be detected in a sample by immunochromatography, comprising the steps of: introducing the sample into an immunochromatographic test piece; and contacting the sample with a detection unit provided on the immunochromatographic test piece, which includes a proteinaceous or peptide capture substance that specifically binds to the substance to be detected, and at least one sugar selected from the group consisting of three or more sugars and polysaccharides.

9. The detection method according to claim 8, wherein the detection unit is provided on a membrane-like support, and the capture substance and the sugar are contained in the support.

10. The detection method according to claim 8 or 9, wherein the sugar comprises one or more selected from the group consisting of isomaltoligosaccharides, galactooligosaccharides, fructooligosaccharides, raffinose, and dextran.

Images