JP2017134027A - Quantitative chromatography analysis strip - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a chromatography analysis strip which can highly accurately determine whether or not a target substance exists in a sample, or which can quantify a target substance in a sample.SOLUTION: A chromatography analysis strip includes, in a sequential arrangement, a sample pad for adding a sample on a base material, a solid-phase carrier including a plurality of capture regions where a capture substance for capturing a capture object in the sample is arranged and fixed in different amounts, and an absorption pad.SELECTED DRAWING: None

Description

  The present invention relates to a chromatographic strip capable of determining the presence or absence and amount of a target substance in a sample, and a method for quantifying a target substance in a sample using the same.

  In recent years, immunochromatography has attracted attention as a technique for detecting a target substance in a sample. For example, a sample is dropped onto a solid phase carrier such as a nitrocellulose membrane in which a capturing means capable of specifically binding to a target substance is placed and fixed in a predetermined region, and the sample becomes a capillary phenomenon. When the solid phase carrier is moved by this, the target substance in the sample comes into contact with the capture means, and the target substance is bound to the capture means. Subsequently, the presence or absence of the target substance in the sample can be determined by detecting the target substance bound to the capturing means. This method is excellent in speed and simplicity, and today, using the principle of this method, a chromatographic strip for detecting viruses, bacteria, hormones and various disease-related biomarkers has been developed. It is used in various fields.

  On the other hand, detection of a target substance in a chromatographic analysis strip depends on, for example, the intensity of the detection signal based on the target substance bound to the capturing means arranged and fixed in a line at one place. When the value is low, there is a risk of erroneous determination regarding the presence or absence of the target substance in the sample.

  In addition, there is a need to analyze the amount of the target substance in the sample with a detection system using a chromatographic analysis strip because of the speed and simplicity of the operation, but the target substance can be quantified. The detection sensitivity was not sufficient and it was difficult to realize.

  Therefore, in this field, a strip for chromatographic analysis that can determine the presence or absence of a target substance in a sample with high accuracy and / or enables quantification of the target substance in a sample has been desired.

  An object of the present invention is to provide a chromatographic strip capable of determining the presence or absence of a target substance in a sample with high accuracy.

  It is another object of the present invention to provide a chromatographic analysis strip that enables quantification of a target substance in a sample.

  The present inventors prepare a solid phase carrier in which a capturing substance for capturing a capturing target such as a target substance in a sample is arranged and fixed in a plurality of regions in different amounts, and using this, a sample is prepared. When the target substance in the sample was detected, based on the amount of target substance in the sample and the capture efficiency of the capture target in each area, different detection signal intensities were obtained in each of the plurality of areas. It was found that a pattern of detection signal intensity corresponding to the amount of target substance in the sample (hereinafter sometimes referred to as “detection pattern”) is obtained. It has been found that by using the detection pattern, the presence or absence of the target substance in the sample can be determined with high accuracy, and the amount of the target substance in the sample can be determined.

The present invention has been made based on these findings, and includes the following inventions.
[1] A solid phase carrier including a plurality of capture regions on which a sample pad for adding a sample and a capture substance for capturing a capture target in the sample are arranged and fixed in different amounts on a substrate. And a strip for chromatographic analysis, in which absorption pads are sequentially arranged.
[2] The strip for chromatographic analysis according to [1], wherein the amount of the capture substance arranged and fixed in the capture region is larger in the upstream capture region.
[3] The strip for chromatographic analysis according to [1], wherein the amount of the capture substance arranged and fixed in the capture region is larger in the downstream capture region.
[4] A method for quantifying a target substance contained in a sample,
The sample is brought into contact with a solid phase carrier including a plurality of capture regions in which capture substances for capturing the capture target in the sample are arranged and fixed in different amounts, and the capture target in the sample is used as the capture substance. Capture process,
Detecting a target substance by detecting a capture target captured by the capture substance;
Obtaining a detection pattern including detection results of the target substance in the plurality of capture regions; and
Determining the amount of the target substance contained in the sample based on the obtained detection pattern,
Including the above method.
[5] The amount of the capture substance arranged and fixed in the capture region is carried out using a solid phase carrier that is larger in the upstream capture region, and the step of detecting the target substance is performed by the sandwich method. [4] the method of.
[6] The amount of the capture substance arranged and fixed in the capture region is carried out using a solid phase carrier that is larger in the downstream capture region, and the step of detecting the target substance is performed by a competitive method. [4] the method of.

  According to the present invention, since detection of a target substance in a sample can be determined based on detection signals in a plurality of regions, the presence or absence of the target substance in the sample can be determined with high accuracy.

  Further, according to the present invention, the detection signal intensity pattern (detection pattern) in a plurality of regions obtained by detecting the target substance in the sample has a correlation with the amount of the target substance in the sample. Therefore, the amount of the target substance in the sample can be determined based on the detection pattern.

FIG. 1 is a schematic diagram showing an example of a lateral flow type chromatographic analysis strip. (1) Substrate, (2) Sample pad, (3) Solid support, (4) Absorption pad, (5) Capture region (line 1), (6) Capture region (line 2), (7) Capture region (Line 3). FIG. 2 is a photograph showing the detection result of the target substance (cortisol) in the sample by the competitive method using each chromatographic analysis strip prepared in Example 1. FIG. 3 shows the relationship between the coloring amount of each line obtained by the detection of the target substance (cortisol) in the sample by the competitive method using each chromatographic strip produced in Example 1 and the amount of cortisol in the sample. FIG. The coloring amount (vertical axis) is indicated by a relative value where the uncolored value is “1”. FIG. 4 is a photographic diagram showing the detection result of the target substance (influenza A antigen) in the sample by the sandwich method using each chromatographic analysis strip prepared in Example 2.

1. The strip for chromatographic analysis The strip for chromatographic analysis according to the present invention has a sample pad for adding a sample and a capture substance for capturing a capture target in the sample in different amounts on a substrate. A solid support including a plurality of capture regions and an absorption pad are sequentially arranged.

  Hereinafter, the chromatographic analysis strip of the present invention may be described using an embodiment described in a schematic diagram, but the chromatographic analysis strip of the present invention is not limited to this embodiment. In addition, the code | symbol attached | subjected to each member respond | corresponds to the code | symbol shown in a figure.

  The strip for chromatographic analysis in FIG. 1 comprises a sample pad (2) for adding a sample, a solid phase carrier (3), and an absorption pad (4) sequentially arranged on a substrate (1). The solid phase carrier (3) is provided with a capture region (line 1) (5), a capture region (line 2) (6), and a capture region (line 3) (7) for capturing the capture target. It has a structure. “Upstream” means an upstream region, that is, a region on the sample pad side with respect to a direction in which a sample dropped on the sample pad is sequentially developed into a solid phase carrier and an absorption pad by capillary action. In addition, “downstream” means a region downstream of the sample dripped onto the sample pad, ie, the region on the absorption pad side in the direction in which the sample is sequentially developed to the solid phase carrier and the absorption pad by capillary action.

  The base material only needs to be able to support various members disposed thereon, for example, resin (plastic, polyester resin, polyethylene resin, polystyrene resin, polypropylene resin, ABS resin, polycarbonate resin, polyurethane resin, vinyl chloride resin, etc.) A material made of a metal or an inorganic material (glass, silicon, ceramic, etc.) can be used (but is not limited thereto). The substrate is not particularly limited as long as it facilitates the operation of the strip for chromatographic analysis, and includes, for example, those having a flat shape such as a flat plate, a flat membrane, and a film, and other members. It may have a three-dimensional shape (for example, a housing case).

  The sample pad, the solid phase carrier, and the absorption pad may be any material as long as the sample dropped on the sample pad can be developed in and between these members by capillary action. For example, resin (plastic, polyester resin, polyethylene resin, polystyrene) Resin, polypropylene resin, ABS resin, polycarbonate resin, polyurethane resin, vinyl chloride resin, etc.), metals, inorganic materials (glass, silicon, ceramics, etc.), natural polymers (chitin, chitosan, cellulose, etc.) are used. (But not limited to). These members preferably have a porous structure. For example, a nitrocellulose membrane, a polyether sulfone membrane, a nylon membrane, filter paper or various dried gels (gelatin gel, agarose gel, dextran gel, silica gel), etc. should be used. Can do.

  The sample pad, the solid phase carrier, and the absorption pad may be made of different materials, or may be made of the same material. As the size and form of the sample pad, solid phase carrier and absorption pad, those suitable for the operation of the strip for chromatographic analysis and detection of the target substance can be appropriately selected.

  The sample pad, the solid phase carrier, and the absorption pad may be arranged so that the dropped sample can be sequentially developed from the sample pad to the solid phase carrier and then from the solid phase carrier to the absorption pad. Are arranged to contact directly or indirectly. For example, as shown in FIG. 1, the sample pad (2), the solid phase carrier (3), and the absorption pad (4) can be sequentially stacked.

  The capture substance for capturing the capture target in the sample may be any substance that can specifically bind to the capture target, and includes a substance that specifically binds by interaction with the capture target. The interaction between the capture target and the capture substance includes, for example, interactions such as antibody-antigen, nucleic acid-nucleic acid having a complementary sequence thereof, aptamer-its target molecule, avidin-biotin, or lectin-sugar chain. However, an appropriate substance can be selected as the capture substance depending on the capture target. For example, as a capture substance, an antibody (IgG, IgM, IgE, IgD, IgA, etc.) that can specifically bind to the capture target or a fragment thereof, or when the capture target is an antibody or a fragment thereof, Antigens and fragments thereof can be used.

  When the “detection and quantification method of a target substance in a sample” performed using the strip for chromatographic analysis of the present invention described in detail below is based on the sandwich method, the “capture target” means the target substance in the sample. can do. “Target substance” means a substance whose presence and content in a sample is desired to be determined, and includes proteins, polypeptides, peptides, DNA, RNA, compounds, etc. There is no limitation. Examples of target substances include various viruses such as influenza virus, HIV and hepatitis virus and antigens and antibodies thereof, various bacteria such as Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Streptococcus, and Streptococcus pneumoniae and antigens and antibodies thereof Metabolites, various hormones such as human chorionic gonadotropin, growth hormone, thyroid stimulating hormone, DiAcSpm, CEA, CA19-9, PSA, various tumor markers such as fecal occult blood, IFN-α, IFN-β, IL-2, Various cytokines including chemokines such as IL-6, MCP-1 and MCP-2, various cytokeratins such as CK-8 and CK-18, various growth factors such as HGF, EGF, FGF and VEGF, IgG and IgM Various serum protein components such as immunoglobulin and albumin are not limited thereto.

  On the other hand, when the “method for detecting and quantifying a target substance in a sample” performed using the strip for chromatographic analysis of the present invention, which is described in detail below, is based on a competitive method, the “capture target” The substance is not limited to the target substance, and may be a substance that competes with the target substance and binds to the capture substance, or a substance that competes and binds to the target substance and the capture substance. The “substance that competes with the target substance and binds to the capture substance” only needs to be able to compete with the target substance and bind to the capture substance, and includes at least the portion of the target substance that is required to bind to the capture substance. Any substance may be used, and preferably the same substance as the target substance. The “substance to which the target substance and the capture substance compete and bind” may be any substance that can bind to both the target substance and the capture substance. Examples of such substances include the target substance and the capture substance, respectively. And a substance that specifically binds by interaction. Such interactions include, but are not limited to, interactions such as antibody-antigen, nucleic acid-nucleic acid having its complementary sequence, aptamer-target molecule, avidin-biotin, or lectin-sugar chain. ), An appropriate substance can be selected according to the target substance and the capture substance. For example, an antibody (IgG, IgM, IgE, IgD, IgA, etc.) or a fragment thereof that can specifically bind to the target substance and the capture substance as “substance that the target substance and the capture substance compete and bind” Alternatively, when the target substance and the capture substance are antibodies or fragments thereof, the antigens or fragments thereof can be used. Hereinafter, the “substance that competes with the target substance and binds to the capture substance” and “the substance that competes and binds the target substance and the capture substance” in this specification may be referred to as “competitive substance”. .

  The arrangement and immobilization of the capture substance on the solid phase carrier can be appropriately selected depending on the capture substance and the solid phase carrier to be used, and suitable peptides, proteins, nucleic acids, functional groups (amino group, carboxyl group, hydroxyl group, sulfide) Group, mercapto group, dithiol group, etc.), or may be performed using electrostatic interaction, hydrophobic interaction, hydrogen bond, covalent bond, or the like.

  The arrangement / fixation of the capture substance on the solid phase carrier is preferably performed limited to a specific region using an ink jet method, a printing method, a spray coating method, or the like. By arranging and fixing the trapping substance in a specific region, the trapped capture target is limited to a predetermined region, so that it is possible to easily determine whether or not the capture target is bound. In the present specification, a region where a capture substance is locally arranged and fixed to a solid phase carrier may be referred to as a “capture region” or “line”.

  Capture substances can be immobilized on the plurality of capture regions in different amounts. By immobilizing different amounts of the capture substance for each capture region, it is possible to vary the intensity of the detection signal based on the binding target to be captured for each capture region. The detection signal obtained in each capture region as a whole can show a detection pattern corresponding to the amount of the target substance in the sample. In addition, this invention does not exclude that the capture area | region where the quantity of the capture | acquisition substance fixed is the same is included in part.

  The number of capture areas and the amount of capture substance immobilized on the capture areas are described in the “Method for detecting and quantifying target substance in sample” performed using the strip for chromatographic analysis of the present invention described in detail below. Based on this, it is possible to adjust the target substance in the sample so that the target substance can be detected and quantified satisfactorily. For example, the number of capture regions can be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more (particularly These are not limited). Further, the amount of the capture substance immobilized on the capture region can be determined based on the amount of the capture target that can be included in the sample. For example, more capture substances may be fixed in the upstream capture area, or more capture substances may be fixed in the downstream capture area. For example, when tested using a sample containing a known amount of capture target, capture that can capture and detect the capture target in multiple capture regions and produce a detection pattern depending on the amount of capture target in the sample The number of regions and / or the amount of capture substance to be immobilized can be appropriately selected and determined. At this time, a detection pattern obtained by using a sample containing a known amount of a capture target can be used as a reference detection pattern, which is performed using the chromatographic analysis strip of the present invention described in detail below. It can be used in the "detection and quantification method of target substance".

  When the “detection and quantification method of target substance in a sample” performed using the strip for chromatographic analysis of the present invention is based on the sandwich method, more capture substances can be immobilized in the upstream capture region. The amount of trapped material can be reduced stepwise from the upstream capture region. For example, when the amount of the capture substance immobilized on the plurality of capture regions is 1, the amount of the capture substance immobilized on the other capture regions is 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0 to decrease stepwise from the upstream capture region. The amount to be a ratio of 1 can be appropriately selected (not particularly limited thereto). Here, “decreasing in a stepwise manner from the upstream capture region” does not exclude that a capture region having the same amount of the capture substance to be immobilized is included in part.

  According to the sandwich method, the detection signal intensity is generally higher in the upstream capture region and lower in the downstream capture region by gradually reducing the amount of the capture substance immobilized on the upstream capture region. A detection pattern with a low detection signal intensity can be obtained. The detection signal intensity of each capture region can vary based on the amount of capture target in the sample, and the detection pattern corresponds to the amount of capture target in the sample.

  In addition, when the “method for detecting and quantifying a target substance in a sample” performed using the strip for chromatographic analysis of the present invention is based on a competitive method, more capture substances can be immobilized in the downstream capture region. The amount of the capture substance to be immobilized can be increased stepwise from the upstream capture region. For example, when the amount of the capture substance fixed to the plurality of capture regions is 1, the amount of the capture material fixed to the other capture regions is 1 , A ratio of 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 so as to decrease stepwise from the downstream side Can be selected (particularly without limitation). Here, “increase stepwise from the upstream capture region” does not exclude that a capture region having the same amount of the capture substance to be immobilized is included in part.

  According to the competition method, the sample contains a certain amount of the competitor substance together with the target substance. The competitive substance can bind to the capture substance present in the capture region at a certain ratio depending on the amount of the target substance contained in the sample. That is, when there is no target substance present in the sample, the competitive substance can bind to the capture substance existing in the capture region without being affected by the target substance, but the target substance exists in the sample. In some cases, the binding of the competitive substance to the capture substance present in the capture region is affected by the target substance, and the amount of the binding decreases depending on the amount of the target substance. The competing substance affected by the target substance moves to the next (downstream) capture region with the development of the sample, and can bind to the capture substance present there and according to the amount of the target substance. Therefore, by increasing the amount of the capture substance to be immobilized in a stepwise manner from the upstream capture area, it is possible to easily capture and detect the competitive substance in the sample over a plurality of capture areas. The detection signal intensity based on the competitor in each capture region can vary based on the amount of target substance in the sample, and a detection pattern corresponding to the amount of target substance in the sample can be obtained.

The chromatographic analysis strip of the present invention may further comprise a labeling site.
When the “detection and quantification method of a target substance in a sample” performed using the strip for chromatographic analysis of the present invention is based on the sandwich method, the labeling site is labeled with a target to be captured (that is, a target substance) in the sample. In addition, a substance that can be specifically bound to the target to be captured (hereinafter, referred to as “labeling substance”) labeled with a labeling substance can be included. Examples of the “substance that can specifically bind to the capture target” used herein include a substance that specifically binds by interaction with the capture target. The interaction between the capture target and the substance includes, for example, an antibody-antigen, nucleic acid-nucleic acid having a complementary sequence thereof, aptamer-target molecule, avidin-biotin, or lectin-sugar chain. However, it is possible to select an appropriate substance depending on the capture target. For example, as a “substance that can specifically bind to the capture target”, an antibody (IgG, IgM, IgE, IgD, IgA, etc.) that can specifically bind to the capture target, or a fragment thereof, or capture When the subject is an antibody or fragment thereof, the antigen or fragment thereof can be used. Preferably, one that does not hinder the binding between the capture target and the capture substance is selected.

  Examples of “substances to be labeled” include metal colloid (eg, gold, silver, copper, platinum, etc.) particles, colored latex particles, silica nanoparticles including dyes, dyes, enzymes (peroxidase, alkaline phosphatase, luciferase, etc.) ), Low molecular compounds (for example, biotin, digoxigenin, FITC, etc.) and the like can be used. The substance to be labeled is not particularly limited as long as it does not hinder the movement of the capturing target bound to the labeling substance or the binding with the capturing substance, and can be clearly detected at the time of detection. Preferably, metal colloids, colored latex particles, silica nanoparticles including pigments, pigments, and the like that can be visually checked for detection without further processing are required. As such a labeling substance, for example, a substance having a particle size of about 500 nm or less, preferably 10 nm or more and 100 nm or less can be used.

  When the “method for detecting and quantifying a target substance in a sample” performed using the strip for chromatographic analysis of the present invention is based on a competition method, the above-mentioned competitor substance can be included in the labeling site. The competitive substance can be labeled with a substance that becomes a label. Here, as the “substance to be labeled”, those described above can be used, and preferably those that do not interfere with the binding to the target substance or the capture substance are selected.

  Any labeling site can be included at any position upstream from the most upstream capture region. For example, in the chromatographic analysis strip of FIG. 1, the labeling site may be included in the sample pad (2) or in the solid phase carrier (3). Alternatively, a solid phase carrier different from the sample pad (2) and the solid phase carrier (3) is prepared, and a labeling substance is included in the solid phase carrier to form a labeling site, and the sample pad (2) and the solid phase carrier (3) You may arrange | position in any position in between.

  When the labeling part contains a labeling substance, the target to be captured in the sample binds to the labeling substance by contact and interaction when passing through the labeling part. Thereby, the capture target is labeled with the labeling substance. In addition, when the labeled site contains a competitive substance, the competitive substance can be included in the sample when the sample passes through the labeled site.

  The labeling substance or the competing substance can be contained in the sample pad or the solid phase carrier using an ink jet method, a printing method, a spray coating method, an impregnation method or the like. The labeling substance or the competing substance may be arranged / fixed in a specific region or may not be restricted.

2. Method for detecting and quantifying target substance in sample The method for detecting and quantifying a target substance in a sample according to the present invention comprises the following steps:
The sample is brought into contact with a solid phase carrier including a plurality of capture regions in which capture substances for capturing the capture target in the sample are arranged and fixed in different amounts, and the capture target in the sample is used as the capture substance. Capture process,
Next, a step of detecting a target substance by detecting a capture target captured by the capture substance,
Next, obtaining a detection pattern including the detection result of the target substance in the plurality of capture regions, and
Next, a step of determining the amount of the target substance contained in the sample based on the obtained detection pattern,
including.
This method can be carried out using the above-described chromatographic analysis strip according to the present invention.

  As the “sample”, any substance that can contain a target substance can be used. For example, a subject's tissue, cell, body fluid (blood, serum, plasma, lymph, spinal fluid, urine, semen, saliva, sweat, Tears, ascites, amniotic fluid, etc.), mucous membranes, mucus, stool, etc., but are not limited thereto. The sample may be subjected to a treatment that allows the target substance to be extracted, purified, or roughly purified.

  The sample may be used as it is, or may be diluted or suspended in an appropriate buffer (or developing solution). As the buffer (or developing solution), purified water, physiological saline, phosphate buffer, Tris buffer, Good buffer, SSC buffer, or the like can be used. Buffers (or developing solutions) include surfactants (nonionic surfactants, amphoteric surfactants, anionic surfactants, etc.), protein components (bovine serum albumin (BSA), human, as necessary. Serum albumin (HSA), casein, etc.), denaturing agents (urea, guanidine hydrochloride, thiocyanate, etc.) and the like can be included.

  When the strip for chromatographic analysis does not include the labeling site, the labeling substance or the above-described substance in the buffer solution (or developing solution) is detected according to the target substance detection / quantification method (sandwich method or competitive method) in the sample. Competing substances can be added. As a result, the target substance in the sample can be bonded to the labeling substance by contact and interaction and labeled with the labeling substance, or a competing substance can be included in the sample.

  The sample dropped on the sample pad is developed by capillary action from the sample pad to the solid phase carrier and then from the solid phase carrier to the absorption pad.

  In the case where the chromatographic strip is provided with the labeling site, the target substance binds to the labeling substance by contact and interaction when the sample passes through the labeling site and is labeled with the labeling substance, or in the sample. Competing substances can be included.

  The capture target contained in the sample reaches a plurality of capture regions and binds to the immobilized capture substance by contact and interaction.

  In this method, the target substance in the sample can be detected based on the sandwich method or the competitive method.

  According to the sandwich method, the detection pattern obtained generally has a positive result in the upstream capture region, and a high detection signal intensity can be obtained. Therefore, when the target substance is not present in the sample, detection signals are not obtained in all the capture areas including the most upstream capture area, while the more target substance is present in the sample, Detection signals can be obtained in a large number of capture regions up to the downstream side, and a higher detection signal intensity can be obtained in the upstream capture region.

  The detection of the target substance captured by the capture substance in each capture region can be performed by detecting the label substance by a technique according to the substance to be used as the label. If the used labeling substance can be visually confirmed, the capture region is colored due to the labeling substance. Thereby, it is possible to obtain a detection pattern including the presence / absence and degree (for example, color intensity) of the target substance in each capture region.

  On the other hand, according to the competitive method, the sample contains a certain amount of competing substances together with the target substance. Thereby, the competitive substance can be bound to the capture substance existing in the capture region at a certain ratio according to the amount of the target substance contained in the sample. The detection pattern obtained based on the competitive substance can change depending on the amount of the target substance. For example, when the target substance is not present in the sample, the competitive substance binds to the capture substance in the most upstream capture region. Therefore, a high detection signal intensity can be obtained in the most upstream capture region. On the other hand, when the target substance is contained in the sample, the binding of the competitive substance to the capture substance in the capture region is limited depending on the amount of the target substance. In the process of the development of the sample, the competitive substance is transported to the capture region on the downstream side, and is captured there depending on the amount of the target substance contained in the sample. For this reason, a detection signal can be obtained even in the downstream capture region, and the binding of the competitor to the upstream capture region is limited, so that the detection signal intensity decreases in the upstream capture region, or Disappearance can occur. When the amount of the target substance in the sample is larger, the binding between the competitor substance and the capture substance in the capture region is reduced, so that the detection signal intensity in the capture region may be reduced or eliminated.

  The detection of the competitive substance captured by the capture substance in each capture region can be performed by detecting the competitive substance by a technique according to the substance to be used as the label. If the used labeling substance can be visually confirmed, the capture region is colored due to the labeling substance. Thereby, it is possible to obtain a detection pattern including the presence / absence and degree (for example, intensity of coloration) of the competitive substance in each capture region.

  As described above, the detection pattern based on the detection signal intensity in the plurality of capture regions is determined according to the amount of the target substance. Therefore, the obtained detection pattern has a correlation with the amount of the target substance in the sample.

  The amount of the target substance in the sample can be determined by comparing the obtained detection pattern with a “reference detection pattern” obtained in advance using a sample containing a known amount of the target substance.

  Hereinafter, the present invention will be described specifically by way of examples. However, the present invention is not limited to these examples.

(Example 1) Detection / quantification of a target substance (cortisol) in a sample by a competitive method 1-1. Preparation of chromatographic strip A nitrocellulose membrane (Merck Millipore) was used as a solid support, and a cortisol antigen (Fitzgerald) was applied in three lines as a capture substance for capturing an anti-cortisol antibody. (Hereinafter, each line is described as “line 1”, “line 2”, “line 3” from the upstream side). Cortisol antigen was applied to and fixed on lines 1, 2 and 3 in amounts of 0.2 mg / cm, 0.5 mg / cm and 0.75 mg / cm, respectively.

  Next, the obtained solid phase carrier was dried at 50 ° C. for 30 minutes to obtain a solid phase carrier having lines 1, 2 and 3 as capture regions. A glass fiber sample pad (Merck Millipore) and a cellulose absorbent pad (Merck Millipore) were overlapped and bonded together as shown in FIG. 1 to produce a lateral flow type chromatographic analysis strip.

1-2. Detection and quantification of target substance (cortisol) in sample Anticortisol antibody (rat monoclonal antibody) labeled with gold colloid on PBS 0.1 mg / mL and cortisol antigen (SIGMA-ALDRICH Hydrocortisone) 0 μg / dL, 5 μg / Samples were added in amounts of dL, 10 μg / dL, 15 μg / dL, 20 μg / dL, 25 μg / dL, 30 μg / dL, or 40 μg / dL.

1-1. The sample (100 μL) was added to the sample pad of the chromatographic analysis strip prepared in step 1 and developed at room temperature for 10 minutes.
Next, the presence / absence / degree (detection pattern) of coloration on line 1, line 2, and line 3 on the solid support was confirmed. Moreover, the coloring amount of each line was measured using the immunoleader (Trust Medical TOR-500).

FIG. 2 shows the coloring of each line on the solid support. Moreover, the relationship between the coloring amount of each line and the amount of cortisol in the sample is shown in FIG.
It was confirmed that the coloring amount of each line changes according to the amount of cortisol in the sample.

  The amount of cortisol in the sample with the highest coloring amount is different for each of line 1, line 2, and line 3 (FIG. 3). Therefore, depending on the amount of cortisol in the sample, the amounts of line 1, line 2, and line 3 are different. A difference was observed in the amount of coloring (FIG. 2).

  That is, when the amount of cortisol in the sample was 0 μg / dL, a detection pattern in which only line 1 was strongly colored was shown.

  When the amount of cortisol in the sample was 5 μg / dL, a detection pattern in which only line 1 and line 2 were colored and line 1 was colored more strongly than line 2 was shown.

  When the amount of cortisol in the sample is 10 μg / dL, a detection pattern in which coloring is observed in line 1, line 2, and line 3 and intense coloring in the order of line 1, line 2, and line 3 is shown. It was.

  When the amount of cortisol in the sample was 15 μg / dL, a detection pattern in which lines 1, 2 and 3 were colored to the same extent was shown.

  When the amount of cortisol in the sample is 20 μg / dL or 25 μg / dL, color is detected in line 1, line 2, and line 3, and detection is strong in the order of line 3, line 2, and line 1 A pattern was shown.

  When the amount of cortisol in the sample was 30 μg / dL, coloring was observed in line 2 and line 3, and a detection pattern in which coloring of line 1 was hardly recognized was shown.

  When the amount of cortisol in the sample was 40 μg / dL, a detection pattern in which coloring was hardly observed in any of the lines 1, 2, and 3 was shown.

  When no cortisol was present in the sample (0 μg / dL), only line 1 was colored. This indicates that the anti-cortisol antibody present in the sample was mostly captured by the cortisol on the solid phase carrier, which is a capture substance, particularly in line 1 where the coating amount is large.

  When cortisol was present in the sample (5 μg / dL to 15 μg / dL), coloring was observed not only in the line 1 but also in the downstream line. The cortisol affects the binding of the anti-cortisol antibody present in the sample to the line 1 and shows that the anti-cortisol antibody that could not bind to the line 1 caused the binding in the downstream line.

  When a large amount of cortisol was present in the sample (20 μg / dL to 40 μg / dL), a decrease in coloring was observed from line 1. This indicates that the amount of anti-cortisol antibody bound to the line decreased with an increase in the cortisol content in the sample. If it is necessary to obtain a clear detection pattern even in the presence of a large amount of cortisol, the number of lines can be increased and / or the amount of cortisol immobilized as a capture means in each line can be increased.

  From the above results, since the detection patterns of line 1, line 2, and line 3 differ according to the amount of cortisol in the sample, it is confirmed that the amount of cortisol in the sample can be determined based on the detection pattern. It was.

(Example 2) Detection and quantification of target substance (influenza A antigen) in a sample by sandwich method 2-1. Production of Strip for Chromatographic Analysis An anti-influenza A mouse monoclonal antibody (Fitzgerald Influenza A) as a capture substance for capturing a target substance (influenza A antigen) on a nitrocellulose membrane (Merck Millipore) as a solid support. ) In three lines (hereinafter, each line is described as “line 1”, “line 2”, “line 3” from the upstream side). The antibody was applied and fixed on lines 1, 2 and 3 in amounts of 0.75 mg / cm, 0.5 mg / cm and 0.2 mg / cm, respectively.

  Next, the obtained solid phase carrier was dried at 50 ° C. for 30 minutes to obtain a solid phase carrier having lines 1, 2 and 3 as capture regions. A glass fiber sample pad (Merck Millipore) and a cellulose absorbent pad (Merck Millipore) were overlapped and bonded together as shown in FIG. 1 to produce a lateral flow type chromatographic analysis strip.

  Further, as a comparative control, a lateral flow type chromatographic analysis strip was prepared in which the amount of antibody immobilized on line 1, line 2 and line 3 was all changed to 0.7.

2-2. Detection and quantification of target substance (influenza A antigen) in a sample 100 ngP / mL, 200 ngP / mL, 400 ngP / mL, 800 ngP / mL of influenza A antigen (A-California 07/2009) using PBS as a specimen extract Alternatively, the sample was added in an amount of 1200 ng P / mL.

  2-1. The sample (100 μL) was added to the sample pad of the chromatographic analysis strip prepared in Step 1, and developed at room temperature for 15 minutes.

  Next, the presence / absence / degree (detection pattern) of coloration on line 1, line 2, and line 3 on the solid support was confirmed. Each line was detected by colloidal gold colloid-labeled anti-influenza virus A antibody mouse monoclonal antibody (binding to influenza A antigen at a site different from that of anti-influenza A antibody mouse monoclonal antibody immobilized on a solid phase carrier ( Fitzgerald Influenza A).

The results are shown in FIG.
It was confirmed that the coloring amount of each line changes according to the amount of influenza A antigen in the sample.

  As the amount of influenza A antigen in the sample increases, the line colored in the order of line 1, line 2, and line 3 changes, and the color of each line increases the amount of influenza A antigen in the sample. It was confirmed that it became stronger with

  That is, when the amount of influenza A antigen in the sample was 100 ngP / mL, a detection pattern in which only line 1 was colored was shown.

  When the amount of influenza A antigen in the sample was 200 ngP / mL, a detection pattern in which line 1 was strongly colored and line 2 was slightly colored was shown.

  When the amount of influenza A antigen in the sample was 400 ngP / mL, a detection pattern in which line 1 was strongly colored, then line 2 was observed, and line 3 was slightly colored was shown.

  When the amount of influenza A antigen in the sample is 800 ngP / mL, a detection pattern in which coloring is observed in lines 1, 2, and 3 and is strongly colored in the order of lines 1, 2, and 3 Indicated.

  When the amount of influenza A antigen in the sample was 1200 ngP / mL, coloring was observed in line 1, line 2, and line 3, and in particular, a detection pattern in which lines 1 and 2 were intensely colored was shown.

  From the above results, the detection patterns of line 1, line 2, and line 3 differ corresponding to the amount of influenza A antigen in the sample, so the amount of influenza A antigen in the sample is determined based on the detection pattern. It was confirmed that it was possible.

  On the other hand, in the comparative control in which the same amount of antibody was immobilized on line 1, line 2, and line 3, regardless of the amount of influenza A antigen in the sample, on line 1, line 2, and line 3 A degree of coloring was observed. Since a different detection pattern corresponding to the amount of influenza A antigen in the sample was not obtained, the amount of the antigen in the sample could not be determined in the comparative chromatographic analysis strip.

Claims (6)

  A sample pad for adding a sample on a substrate, a solid phase carrier including a plurality of capture regions in which capture substances for capturing a capture target in the sample are arranged and fixed in different amounts, and absorption A strip for chromatographic analysis in which pads are sequentially arranged.   The strip for chromatographic analysis according to claim 1, wherein the amount of the capture substance arranged and fixed in the capture region is larger in the upstream capture region.   The strip for chromatographic analysis according to claim 1, wherein the amount of the capture substance arranged and fixed in the capture region is larger in the downstream capture region. A method for quantifying a target substance contained in a sample, comprising:
The sample is brought into contact with a solid phase carrier including a plurality of capture regions in which capture substances for capturing the capture target in the sample are arranged and fixed in different amounts, and the capture target in the sample is used as the capture substance. Capture process,
Detecting a target substance by detecting a capture target captured by the capture substance;
Obtaining a detection pattern including detection results of the target substance in the plurality of capture regions; and
Determining the amount of the target substance contained in the sample based on the obtained detection pattern,
Including the above method.   The amount of the capture substance arranged and fixed in the capture region is carried out using a solid phase carrier that is larger in the upstream capture region, and the step of detecting the target substance is performed by a sandwich method. Method.   The amount of the capture substance arranged / fixed in the capture region is carried out using a solid phase carrier that is larger in the downstream capture region, and the step of detecting the target substance is performed by a competitive method. Method.

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