JP6677284B2 - Analyte detection method and lateral flow test strip - Google Patents

Description

  The present invention relates to a method for detecting an analyte and a test strip for lateral flow. In particular, the present invention relates to a technique suitable for a method for quantitatively and quickly measuring the concentration of extracellular granules in a sample using the extracellular granules as an analysis target.

  Although excellent therapeutics and advanced medical technologies have been developed due to the remarkable progress in basic medicine, various factors such as cancer, autoimmune diseases, infectious diseases, circulatory diseases, etc., depend on various factors such as dietary habits and living environment. Anxiety due to various diseases is extremely large. Early detection of the disease is important for resolving such anxiety. In recent years, extracellular granules secreted from normal cells and cancer cells have attracted attention as biomarkers indicating a disease state.

  The secreted extracellular granules are generally called microvesicles or exosomes. This exosome is present in all samples in the body such as blood, saliva, sweat, urine, semen, and lymph. In 2007, it was discovered that microRNA was present in this exosome, suggesting a new signal transmission by intercellular transport (Non-Patent Document 1). It has also been suggested that microRNA indicates a pathological condition of cancer (Non-Patent Document 2). In other words, microRNAs contained in exosomes have a disease-specific role, and it is suggested that detection of exosomes and microRNAs in exosomes is useful for diagnosis, and secretory cells such as normal cell-specific exosomes and cancer-specific exosomes It is thought that early diagnosis is possible by quickly and simply quantifying another exosome.

  However, since exosomes have a diameter of about 40 to 100 nm, it is necessary to use very complicated techniques such as ultracentrifugation, ultrafiltration, chromatography, and immunocapture to isolate them from a sample. . In general, when these exosomes are isolated and collected by these methods to measure the recovered exosomes, an apparatus and a complicated technique are required.

  As a method for specifically and quantitatively detecting exosomes to solve the above problem, there is a method of preparing two kinds of beads labeled with an antibody that recognizes an exosome surface antigen and detecting the beads (Patent Document 1). In this method, two types of acceptor beads and donor beads are prepared, and each of them is labeled with an antibody that recognizes a different exosome surface antigen. If exosomes are present, the exosomes are bound to the two beads by an antigen-antibody reaction. Thereafter, by exciting the donor beads, singlet oxygen reaches the acceptor and emits fluorescence. In this method, the sample and the beads are mixed, reacted, and detected by an apparatus. Therefore, the operation is simple and quantitative, and the exosome in the sample can be detected.

International Publication 2013/094307

Nat Cell Biol (2007) 9: 654-659. Cancer Sci (2010) 101: 2087-2092.

However, in Patent Document 1, exosomes can be measured simply and quantitatively simply by mixing and reacting a sample and beads. However, since excitation by fluorescence and fluorescence detection are required, a dedicated device for an optical system is indispensable. is there. Therefore, when a dedicated device cannot be introduced, measurement is impossible.
When measuring the concentration of an analyte contained in a sample by the above method, it is necessary to wait for a sufficient signal to be detected, such as a time limit in actual operation, which can be a major obstacle to rapid detection. is there. For this reason, there is a need for a simple and quick method capable of analyzing an analyte.

  Therefore, the present invention has been made in view of the above problems, and compared with the conventional art, a detection method capable of more quickly measuring an analyte contained in a biological sample and a lateral method using the method. It is intended to provide a test strip for flow.

To solve the problem, the detection method of an analyte which is one embodiment of the present invention, the reaction of the reaction of the analyte and the first substance contained in a biological sample, and the analyte and a second substance A signal derived from the complex formed by the method is measured, and the analyte is an exosome .
Further, lateral flow test strip which is one embodiment of the present invention, a composite forming unit for forming a composite by reaction of the analyte and the first substance contained in a biological sample, said compound and the second material A signal generating portion capable of generating a detectable signal by reacting , the first substance is fixed to a first support, the first support is dried and solidified in the complex forming portion, the The analyte is an exosome .

According to one embodiment of the present invention, a detection signal is measured at a predetermined time after the start of the reaction of the analyte contained in the sample.
For example, by using a lateral flow assay, exosomes for each secretory cell can be easily detected, and a disease diagnosis targeting exosomes becomes possible.

FIG. 1 is a schematic diagram showing a basic structure of a lateral flow test strip according to an embodiment of the present invention.

First, the sample used in the present embodiment and the analyte to be measured in the present embodiment will be described with reference to specific examples.
(About sample)
The sample used in the present embodiment is a biological sample and is preferably a liquid sample. Biological samples include, for example, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostate fluid, Cowper's fluid or pre-ejaculatory fluid, sweat, feces, hair, tears, cystic fluid, pleural or ascites fluid, pericardial fluid, lymph, chyme, chylous, bile, interstitial fluid, menstrual discharge, pus, sebum, vomit , Vaginal secretions, mucosal secretions, stool, pancreatic juice, nasal washings, broncho-pulmonary aspirates, blastocyst fluid, umbilical cord blood, and the like. An undiluted sample may be used as a measurement sample, or a sample diluted with a solvent may be used as a measurement sample.

(About the analyte)
Analyte in the present embodiment, for example, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid, sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, Semen, prostate fluid, Cowper's fluid or pre-ejaculatory fluid, sweat, feces, hair, tears, cystic fluid, pleural effusion or ascites, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menstrual discharge, pus Exosomes present in any one of sebum, vomiting, vaginal secretions, secretions from mucous membranes, faeces, pancreatic juice, nasal washings, broncho-pulmonary aspirate, blastocyst fluid, and umbilical cord blood . Hereinafter, specific examples of these analytes will be described.

(About exosomes)
Exosomes are formed in a phospholipid bilayer with a diameter of 40 to 100 nm, and have tetraspanins (CD9, CD63, CD81, etc.), antigen presenting proteins (MHCI, MHCII), EGFR, ICAM-1, TGF-β For example, nucleic acids (microRNA) and heat shock proteins are present in the membrane. In the present invention, a substance having a specific affinity for a molecule existing in a membrane is detected and isolated. For example, a protein on the surface of a membrane can be detected or isolated as an antigen using an antibody of a substance having specific affinity for the protein.

(Test strip for lateral flow)
Hereinafter, a test strip for lateral flow used in the method for measuring the concentration of an analyte according to the present embodiment will be described.
FIG. 1 shows the basic structure of a test strip for lateral flow in the present embodiment.
The lateral flow test strip 1 according to the present embodiment detects an analyte contained in a sample and quantifies the concentration. The sample pad 11, which is a sample dropping member, and the analyte flows in a direction in which the sample flows. Binding pad 12 in which particles labeled with a first detection target capture substance (first substance) having specific affinity for are dried and solidified, and a second detection target having specific affinity for the analyte A developing membrane (membrane) having a part where the capture substance (second substance) is immobilized (test line 13a) and a part where the capture substance for recognizing the capture substance to be detected on the particle surface is immobilized (control line 13b). 13) Four types of members of an absorption pad 14 for absorbing a sample infiltrated further downstream by a capillary phenomenon are connected in series via a backing sheet 10.

The binding pad 12 constitutes a complex forming section that forms a complex by a reaction between the analyte contained in the biological sample and the first substance. The membrane 13 constitutes a signal generating unit capable of generating a detectable signal by reacting the complex with the second substance.
The particles labeled with the first substance having a specific affinity for the analyte are, for example, gold colloid particles having an average particle diameter of 20 to 100 nm. In addition, the second substance for detecting the analyte is, for example, linearly immobilized on the membrane 13. The width of the test strip 1 for lateral flow is, for example, 1.5 mm to 5 mm, and the volume of the sample containing the analyte to be dropped on the sample pad 11 is, for example, 25 μl to 100 μl.

  In the test strip 1 for lateral flow according to the present embodiment, the detection intensity is increased by forming a complex of the mixture of the analyte and the first substance and the second substance in the test line 13a. The control line 13b detects the first substance labeling particles flowing by capillary action. More specifically, from the time when the sample is dropped onto the sample pad 11, the signal at the site where the second substance is immobilized is detected over time using an optical method.

Hereinafter, materials and configurations of members constituting the lateral flow test strip 1 according to the present embodiment will be described in detail.
(About the material of the member)
Each of the first substance and the second substance is, for example, any one of an amino acid, a nucleic acid, a polypeptide, a polynucleotide, a lipid, a phospholipid, a saccharide, a polysaccharide, a low molecular compound, an inorganic substance, and a fusion thereof. One.

  In addition, each of the above-described amino acids and polypeptides is, for example, a protein, a protein fragment, a binding domain, a target binding domain, a binding protein, a binding protein fragment, an antibody, an antibody fragment, an antibody heavy chain, an antibody light chain, or a single-chain antibody. Single domain antibody, Fab antibody fragment, Fc antibody fragment, Fv antibody fragment, F (ab ') 2 antibody fragment, Fab' antibody fragment, single chain Fv (scFv) antibody fragment, antibody binding domain, antigen, antigen determination Group, epitope, hapten, immunogen, immunogen fragment, biotin, biotin derivative, avidin, streptavidin, substrate, enzyme, antibody enzyme, cofactor, receptor, receptor fragment, receptor subunit, receptor subunit fragment , A hormone, a lectin, or a polyhistidine.

The above-described nucleic acid is, for example, any one of mRNA, total RNA, genomic DNA, plasmid DNA, and plant DNA.
The particles labeled with the first substance (first support) are, for example, metal colloid particles including gold colloid, silver colloid, iron colloid, aluminum colloid, or platinum colloid, or a polymer compound. Particles. More specifically, the particles made of a polymer compound are fluorescent dye-containing particles or visible dye-containing particles.

  The fluorescent dye included in the fluorescent dye-containing particles may be, for example, divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or tetravalent. Titanium, copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound, tin compound, thallium Compound, bismuth compound, tungstate compound, molybdate compound, sulfur, vanadium compound, lanthanum compound, praseodymium compound, neodymium compound, promethium compound, gadolinium compound, thulium compound, ytterbium compound, lutetium compound, organic fluorescent dye compound, organic visible dye At least one substance selected from the group of compounds It is an inclusive dye.

  Examples of the organic fluorescent dye compound and the organic visible dye compound include DY630, DY631, DY633, DY635, DY636, DY650, and DY651 (manufactured by Dyomics GmbH), Oyster643, and Oyster656 (manufactured by Denovo Biolabels Inle-Carboxy). , 6-carboxy-fluorescein, 5,6-dicarboxy-fluorescein, 6-carboxy-2 ', 4,4', 5 ', 7,7'-hexachlorofluorescein, 6-carboxy-2', 4,7, 7'-tetrachlorofluorescein, 6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein, naphthofluorescein, 5-carboxy-rhodamine, 6-carboxy-rhodamine, 5,6-dicarboxy C-rhodamine, rhodamine 6G, tetramethylrhodamine, X-rhodamine, and Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500, Alexa Fluor 514, Alexa Fluor 514, Alexa Fluor 514 555; Alexa Fluor 568; Alexa Fluor 594; Alexa Fluor 610; Alexa Fluor 633; PY FL, BODIPY TMR, BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650, 66 Methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like.

The membrane 13 (second support) on which the second substance is immobilized includes, for example, polymethyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane, nitrocellulose, silicone resin, copolymer or composite containing their high molecular compounds, quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate It is composed of any one of an acid glass, a ceramic, and a composite thereof.
The above-mentioned first substance is fixed to the first support by a chemical bond or a physical bond. Further, the above-mentioned second substance is immobilized on the second support by a chemical bond or a physical bond.

(About structure)
The shape of the test strip 1 for lateral flow is not particularly limited as long as the sample to be dropped can move in the test strip by capillary action. For example, a typical example is a structure in which four members of a sample pad 11, a bonding pad 12, a membrane 13, and a water absorbing pad are serially connected in one direction in this order on a backing sheet serving as a substrate and fixed. After each member is fixed to the backing sheet, it is preferable that the backing sheet is cut into a width of 2 mm to 5 mm by a cutting machine to form a test strip.

Hereinafter, the details of the four members constituting the test strip 1 for lateral flow will be described.
(1) Sample Pad The sample pad 11 is a member that holds a dropped liquid sample (biological sample) containing an analyte for a certain period of time. The sample pad 11 is preferably made of cellulose, and more specifically, for example, Cellulose Absorbent Pad (manufactured by Pall) or the like. In consideration of the fact that the pH differs depending on the sample dropped, it is preferable to use the sample pad 11 after infiltrating the sample pad 11 with a buffer solution and drying the buffer.

(2) Binding Pad The binding pad 12 is a member in which the labeled particles for the analyte are dried and solidified, and the analyte contained in the sample that has moved from the sample pad 11 due to the capillary action is a specific substance such as an antigen-antibody reaction. This is a portion that is bound to the above-mentioned labeled particles in a recognition reaction. The material of the bonding pad 12 is preferably non-woven glass fiber, and more specifically, for example, Glass Fiber Pad (manufactured by Millipore) or the like. The above-described reaction between the analyte and the antibody (first substance) provided on the labeled particles is performed using one of physical affinity, chemical affinity, chemical bonding, and immunological methods. It is something to be done.

The content of the labeled particles per unit area (cm ² ) in the binding pad 12 is not particularly limited, but is preferably 1 μg or more and 4 μg or less. As a method of infiltrating the binding particles into the bonding pad 12, for example, a method in which a liquid in which the label particles are suspended and dispersed is applied to the bonding pad 12, dropped or sprayed, and then the bonding pad 12 is dried. Is preferred.
Further, as the labeling particles, among the above-mentioned materials, metal colloid particles, fluorescent dye particles, colored latex particles, up-conversion phosphor particles, quantum dot particles, and the like are preferable. Further, the particle size of the labeling particles is preferably 20 nm or more and 1000 nm or less.
In addition, among the above-mentioned antibodies, mouse IgG and goat IgG, as well as rabbit IgG, chicken IgY, mouse IgE, human IgG, human IgE, and the like are preferable as the antibodies provided in the labeled particles.

(3) Membrane The membrane 13 is a member in which the complex of the labeled particles and the analyte, the labeled particles and the sample are moved by capillary action, and the immune reaction composed of the immobilized antibody, the analyte, and the labeled particles is carried out. It has an antibody immobilization section (test line 13a) to be performed and an antibody immobilization section (control line 13b) for performing an immune reaction composed of immobilized antibody-labeled particles. The above-described reaction between the analyte and the immobilized antibody (second substance) is performed using any one of physical affinity, chemical affinity, chemical bonding, and immunological methods. .

The membrane 13 is preferably composed of a material capable of physically adsorbing proteins, particularly preferably composed of nitrocellulose. More specifically, examples include a Hi-Flow Plus 120 membrane (manufactured by Millipore).
The shape of the antibody immobilization section (determination section) in the membrane 13 is not particularly limited as long as the capture antibody is locally immobilized, and examples thereof include a line shape, a circle shape, and a band shape. Among these shapes, it is preferable that the shape is a line shape having a width of 0.5 to 2.0 mm, and the amount of antibody per cm is preferably 0.1 to 2 μg.

  As the antibody provided in the membrane 13, among the above-mentioned antibodies, in addition to mouse IgG and goat IgG, for example, rabbit IgG, chicken IgY, mouse IgE, human IgG, human IgE and the like are preferable. For example, when mouse IgG is surface-modified on the labeled particles, an antibody-immobilized portion on which a goat anti-mouse IgG antibody is immobilized can be used as the control line 13b. After the antibody is immobilized on the membrane 13, it is preferable to dry the membrane 13 after infiltrating the membrane 13 with a solution of a blocking agent such as albumin, casein, or polyvinyl alcohol in order to prevent nonspecific adsorption of proteins. .

  In the test line 13a, the formed complex (analyte-labeled particles) is captured by an immune reaction consisting of the immobilized antibody-analyte-labeled particles. Then, in the test line 13a, the presence or absence of the analyte is determined based on the intensity (signal) of color development or fluorescence or the like (specifically, fluorescence, light emission, coloration, coloring, or the like) derived from the captured labeled particles. Judgment, that is, judgment of positive / negative. On the other hand, in the control line 13b, the movement of the sample can be confirmed by the immune reaction composed of the immobilized antibody and the labeled particles. Thus, the labeled particles are concentrated in the antibody-immobilized portion, and the signal can be detected and determined visually or using a detection device.

(4) Water-absorbing pad The water-absorbing pad is a member for absorbing the sample and the labeled particles that have moved through the membrane 13 due to the capillary phenomenon, and constantly generating a constant flow. The material of the water-absorbing pad is preferably a fibrous material of a cellulose material, and more specifically, for example, Cellulose Fiber Sample Pad (Millipore) or the like.

(5) Backing sheet The backing sheet is an adhesive sheet for fixing the four members of the sample pad 11, the bonding pad 12, the membrane 13, and the absorption pad 14. Examples of the backing sheet include a backing sheet AR9020 (manufactured by Adhesives Research).

Hereinafter, a method for detecting a signal derived from the labeled particles captured on the test line 13a will be described.
In the test strip, a signal (detection signal) generated by a reaction between the analyte and the particle labeled with the analyte-specific substance is preferably detected by an optical method. Optical methods include, for example, reflected light intensity or fluorescence detection, luminescence detection, or electrochemiluminescence. More specifically, examples of an apparatus capable of detecting a red / blue color over time include an immunochromatograph reader C10066-10 (manufactured by Hamamatsu Photonics).

Next, a measuring method will be described. As described above, the analyte and the analyte-specific substance are detected on a test strip using an immunological reaction. A sample with a known concentration in the analyte is dropped on the test strip to start measurement. Preferably, the test strip is developed for about 30 minutes to obtain a constant signal.
Here, the immunological methods include enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescent immunoassay (FIA), cell immunostaining, tissue immunostaining, immunoprecipitation, and flow cytometry. Method, fluorescence-activated cell sorting (FACS), immunochromatography, quartz crystal microbalance (QCM), surface plasmon resonance (SPR), dual-polarization interferometry (DPI), ellipsometry, ELISPOT And Western blotting.

The method for measuring the exosome of the analyte according to the present embodiment uses a test strip, and measures a detectable signal generated by reacting the complex formed by the reaction of the exosome with the first substance and the second substance. Then, the exosome concentration is determined by comparing with a preset reference value of the signal.
In the above embodiment, the case where the reaction between the analyte and the first substance is started at the bonding pad 12 has been described, but the present invention is not limited to this. In the above embodiment, the case where the reaction between the composite and the second substance is started by the membrane 13 has been described, but the present invention is not limited to this.

Hereinafter, the present invention will be described in more detail based on examples. The present invention is not limited to these examples.
(1) Antibody preparation In order to detect exosomes using an exosome surface antigen (CD9, CD63, CD81) as an analyte, an antibody immobilized on the membrane 13 is an anti-CD9 mouse monoclonal antibody: Clone M-L13, an anti-CD63 mouse monoclonal antibody. : Clone H5C6, anti-CD81 mouse monoclonal antibody: Clone JS81 (above, BD Pharmingen) was used. The control line 13b used anti-mouse immunoglobulin G (Dako). The two antibodies were dialyzed against 50 mM phosphate buffer (pH 7.2) at 4 ° C. with a dialysis cartridge Slide-A-Lyzer 10,000 MWCO (trade name, Pierce). The antibody concentration after dialysis was measured, and the anti-exosome surface antigen antibody was adjusted to 1 mg / ml, and the anti-mouse immunoglobulin was adjusted to 0.5 mg / ml.

(2) Preparation of Colloidal Gold Solution The above three anti-exosome surface antigen antibodies were dialyzed, adjusted to 60 μg / ml with a phosphate buffer (pH 7.4), and adjusted to 1 OD (particle size). (40 nm, Tanaka Kikinzoku Kogyo Co., Ltd.) solution and a mixture of 9 ml of potassium dihydrogen phosphate solution (pH 8.0), add this antibody solution, pipette, allow to stand at room temperature for 15 minutes, A 10% BSA solution was added and mixed. The mixed solution was centrifuged at 8000 × g for 15 minutes, and the supernatant was discarded. The coating solution for the binding pad was added thereto, followed by stirring and ultrasonic treatment. After centrifugation, the supernatant was discarded. This operation was repeated twice in total to obtain a colloidal gold solution.

(3) Preparation of Sample Pad A sample pad made of cellulose (Cellulose Absorbent Pad # 111, Pall) was cut into 1.8 mm × 150 mm, and 1 ml of 50 mM Tris-HCl (pH 7.4) was added dropwise to the sample pad. Dried for 1 hour at ° C.

(4) Preparation of bonding pad A coating solution and a gold colloid solution were added to a 1.5 ml Eppendorf tube, and after sonication at 28 kHz, a total of 900 μl of the gold colloid coating solution was added to a glass fiber pad (glass fwiber pad, 10 mm × 150 mm). (Millipore) and dried for 18 hours under reduced pressure with a vacuum reduced pressure pump.

(5) Preparation of Membrane The immobilized antibody (1) was applied at 1 μl / cm to HiFlow Plus HF120 (Milipore), which is a membrane, using an antibody coating machine XYZ3060 (trade name, BioDot). After the application, it was blocked with a 0.5% milk casein solution, washed and stabilized with a washing solution containing sucrose and a surfactant, and dried overnight at room temperature.

(6) Preparation of test strip A dried membrane, an absorption pad (CFSP203000, manufactured by Millipore), a bonding pad, and a sample pad were attached to a backing sheet (AdhesiveResearch), and 5 mm was cut with a cutting machine CM4000 (trade name, BioDot). After being cut by the width, it was housed in a housing case to obtain a test strip kit.

(7) Preparation of antigen solution Serum was used as a sample containing exosomes as detection targets. This antigen solution was diluted to 50% with 1 × PBS, 5% BSA solution to obtain an antigen solution for measurement. Serum was prepared for a total of 8 individuals.

(8) Measurement of object on test strip The test strip prepared in (6) was placed horizontally, and the antigen solution of (7) was dropped at 100 μl per test strip. After the start of the dropping, the sample was placed at a measurement position in an immunochromatographer C10066 (trade name, Hamamatsu Photonics), and data was acquired 20 minutes after the start of the reaction. The measurement data was scanned in the developing direction of the test strip, and the intensity at the peak center (Height: peak top) in the test line and control line was used as the measurement data.

(9) Grasping the amount of the target substance in the sample From the result of (8) above, measure the intensity at 20 minutes after the start of the reaction, and confirm the intensity ratio at the test line and the intensity other than the test line (S / N ratio). did. The result of the S / N ratio of each sample was measured. When the S / N ratio was compared, it was 5 or more, and it was confirmed that the achievement of this S / N ratio indicates the presence of exosomes.

Reference Signs List 1 Test strip for lateral flow 10 Backing sheet 11 Sample pad 12 Bonding pad 13 Membrane 13a Test line 13b Control line 14 Absorption pad

Claims (13)

The reaction between the analyte and the first substance contained in the biological sample and the presence or absence of the analyte by measuring a signal derived from a complex formed by the reaction between the analyte and the second substance Judge,
The analyte is a method for detecting an analyte that is an exosome ,
The second substance is fixed to a second substance fixing portion on the second support,
The analyte that has reacted with the first substance moves on the second support from upstream to downstream from the second substance immobilization section, and the second substance in the second substance immobilization section. Reacts with
The signal is generated from the second substance immobilization unit,
The first substance that has not reacted with the analyte does not form the complex in the second substance immobilization section, and moves downstream from the second substance immobilization section. Of the analyte to be analyzed.   The method for detecting an analyte according to claim 1, wherein the concentration of the analyte is determined by comparing the signal with a signal reference value measured in advance.   The method for detecting an analyte according to claim 1 or 2, wherein the reaction between the analyte and the first substance is performed by an immunological method. The analyte of claim 1, wherein the first substance is fixed to a first support, and the signal is derived from the first support . Detection method.   The first support is a metal colloid particle containing a gold colloid, a silver colloid, an iron colloid, an aluminum colloid, or a platinum colloid, or a particle made of any one or more of a group consisting of a polymer compound. The method for detecting an analyte according to claim 4, wherein:   The method for detecting an analyte according to claim 4, wherein the first support is a particle made of a polymer compound, and is a fluorescent dye-containing particle or a visible dye-containing particle.   The first support is a particle made of a polymer compound, divalent manganese, lead, antimony, cerium, trivalent cerium, trivalent chromium, divalent or trivalent iron, trivalent or Tetravalent titanium, copper, silver, divalent samarium, divalent or trivalent europium, trivalent terbium, trivalent dysprosium, trivalent holmium, trivalent erbium, uranyl compound, ruthenium compound, tin compound , Thallium compounds, bismuth compounds, tungstate compounds, molybdate compounds, sulfur, vanadium compounds, lanthanum compounds, praseodymium compounds, neodymium compounds, promethium compounds, gadolinium compounds, thulium compounds, ytterbium compounds, lutetium compounds, organic fluorescent dye compounds, organic Select from the group of visible dye compounds and metal colloid particles Method of detecting an analyte according to claim 4, wherein the enclosing at least one substance. The method for detecting an analyte according to any one of claims 4 to 7 , wherein the size of the first support is 20 nm or more and 1000 nm or less. The first support has been dried and solidified in a bonding region that is fixed continuously with the second support and that is upstream of the second substance fixing portion,
The method for detecting an analyte according to any one of claims 4 to 8, wherein the analyte and the first substance react in the binding region. The method for detecting an analyte according to claim 9, wherein the analyte is moved from an upstream side of the binding region. The method for detecting an analyte according to any one of claims 1 to 10, wherein the analyte is concentrated in the second substance immobilizing section. A complex forming unit that forms a complex by a reaction between the analyte contained in the biological sample and the first substance, and a signal generating unit that can generate a detectable signal by reacting the complex with the second substance With
The first substance is fixed to a first support,
The first support is dried and solidified in the composite forming section,
A test strip for lateral flow, wherein the analyte is an exosome. The reaction between the analyte contained in the biological sample and the first substance immobilized on the first support , and the second substance immobilized on the second substance immobilized portion on the analyte and the second support Determine the presence or absence of the analyte by measuring the signal derived from the complex formed by the reaction with the substance,
The analyte is an exosome,
Method for detecting the signal, a method of the second material to detect the signal in an optical method in the second material immobilized portion above immobilized,
The analyte that has reacted with the first substance moves on the second support from upstream to downstream from the second substance immobilization section, and the second substance in the second substance immobilization section. Reacts with
The first substance that did not react with the analyte does not form the complex in the second substance immobilization section, and moves downstream from the second substance immobilization section,
An analysis object, wherein a ratio (S / N ratio) of the signal in the second substance immobilization section to the signal on the second support other than the second substance immobilization section is 5 or more. Object detection method.

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