JP2019007984A - Detection method of analysis object and test strip for lateral flow - Google Patents

Abstract

To enable measure of an analysis object included in a biological specimen more quickly.SOLUTION: Detection of an analysis object is configured to: measure a signal which can be detected, and which is generated by reaction of a composite formed by reaction of the analysis object included in a biological specimen and first material, and second material; and compare the measured signal with a signal reference value which is determined in advance, thereby for determining density of the analysis object.SELECTED DRAWING: None

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 rapidly measuring the concentration of extracellular granules in a sample using extracellular granules as an analysis target.

  Although excellent therapeutic drugs and advanced medical technologies have been developed due to remarkable advances in basic medicine, various factors such as cancer, autoimmune diseases, infectious diseases, cardiovascular diseases, etc., due to various factors such as eating habits and living environment Anxiety due to various diseases is extremely large. Early detection of diseases is important for relieving such anxiety. In recent years, extracellular granules secreted from normal cells and cancer cells have attracted attention as biomarkers indicating disease states.

  Secreted extracellular granules are generally called microvesicles or exosomes. This exosome is present in every sample in the living body such as blood, saliva, sweat, urine, semen and lymph. In 2007, it was discovered that microRNAs exist in this exosome, and information transmission by new cell-to-cell transport was suggested (Non-patent Document 1). It has also been suggested that microRNA indicates cancer pathology (Non-patent Document 2). In other words, microRNAs contained in exosomes have a disease-specific role, and detection of exosomes and microRNAs in exosomes is suggested to be useful for diagnosis. Secretory cells such as normal cell-specific exosomes and cancer-specific exosomes It is considered that early diagnosis is possible by quickly and simply quantitatively measuring another exosome.

  However, since the exosome has a diameter of about 40 to 100 nm, it is necessary to use a very complicated method such as ultracentrifugation, ultrafiltration, chromatography, or immunocapture to isolate it from the sample. . In general, when measuring exosomes isolated and recovered by these methods, an apparatus or a complicated technique is required.

  In order to solve the above-mentioned problem, there is a method for preparing and detecting two types of beads labeled with antibodies recognizing exosome surface antigens as a method for specifically and quantitatively detecting exosomes (Patent Document 1). In this method, two types of acceptor beads and donor beads are prepared and labeled with antibodies that recognize different exosome surface antigens. If exosomes are present, exosomes are bound to two kinds of beads by antigen-antibody reaction. Thereafter, singlet oxygen reaches the acceptor by exciting the donor bead and emits fluorescence. In this method, the sample and the beads are mixed, reacted, and detected only with an apparatus, so that the operation can easily and quantitatively detect exosomes in the sample.

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 by simply mixing and reacting the sample and beads. However, since excitation and detection of fluorescence are necessary, a dedicated device for the optical system is essential. is there. Therefore, if a dedicated device cannot be introduced, measurement is impossible.
When measuring the concentration of the analyte contained in the sample by the above method, it takes time to actually work, such as waiting for sufficient signal detection, which can be a major obstacle to rapid detection. is there. For this reason, there is a need for a simple and quick method that can analyze an analysis object.

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

In order to solve the problem, a method for detecting an analyte that is one embodiment of the present invention includes a reaction between a complex formed by a reaction between an analyte contained in a biological sample and the first substance, and the second substance. Detecting a detectable signal generated by measuring the concentration of the analyte by comparing the measured signal with a signal reference value measured in advance.
Moreover, the test strip for lateral flow according to one aspect of the present invention includes a composite forming unit that forms a composite by a reaction between an analyte contained in a biological sample and a first substance, the composite and the second substance. And a signal generating part capable of generating a detectable signal by reacting with the above.

If it is one aspect | mode of this invention, the detection signal in predetermined time will be measured after reaction start of the analyte contained in a sample.
For example, by using a lateral flow assay, exosomes classified by secretory cells can be easily detected, and disease diagnosis targeting exosomes becomes possible.

It is a schematic diagram which shows the basic structure of the test strip for lateral flows which concerns on embodiment based on this invention.

First, the sample used in this embodiment and the analyte to be measured in this embodiment will be described with specific examples.
(About the sample)
The sample used in this 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 fluid or pre-ejaculate fluid, sweat, feces, hair, tears, cyst fluid, pleural effusion or ascites, pericardial fluid, lymph fluid, sputum, milk fistula, bile, interstitial fluid, menstrual discharge, pus, sebum, vomiting Vaginal secretions, mucous secretions, water stool, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, blastocyst fluid, umbilical cord blood and the like. Further, an undiluted sample may be used as a measurement sample, or a sample diluted with a solvent may be used as a measurement sample.

(Analytical objects)
Analytes in the present embodiment 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-ejaculate fluid, sweat, feces, hair, tears, cyst fluid, pleural effusion or ascites, pericardial fluid, lymph fluid, sputum, milk, bile, interstitial fluid, menstrual discharge, pus , An exosome present in any one of sebum, vomiting, vaginal secretion, mucous secretion, stool, pancreatic juice, nasal wash, bronchopulmonary aspirate, blastocyst fluid, umbilical cord blood . Hereinafter, specific examples of these analysis objects will be described.

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

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

The binding pad 12 constitutes a composite formation unit that forms a composite by the reaction between the analyte contained in the biological sample and the first substance. The membrane 13 constitutes a signal generating part capable of generating a detectable signal by reacting the composite with the second substance.
In addition, the particle | grains by which the 1st substance with specific affinity to the above-mentioned to-be-analyzed object was labeled are gold colloid particles with an average particle diameter of 20-100 nm, for example. In addition, the second substance for detecting the analysis target is immobilized in a linear shape on the membrane 13, for example. The width of the lateral flow test strip 1 is, for example, 1.5 mm to 5 mm, and the volume of the sample including the analyte to be dropped on the sample pad 11 is, for example, 25 μl to 100 μl.

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

Hereinafter, materials and configurations of members constituting the lateral flow test strip 1 according to the present embodiment will be described in detail.
(About material of parts)
Each of the first substance and the second substance is, for example, any one of amino acids, nucleic acids, polypeptides, polynucleotides, lipids, phospholipids, carbohydrates, polysaccharides, low molecular compounds, inorganic substances, and fusions thereof. One.

  Each of the above-mentioned amino acids and polypeptides includes, 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, and 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, immunogenic fragment, biotin, biotin derivative, avidin, streptavidin, substrate, enzyme, antibody enzyme, cofactor, receptor, receptor fragment, receptor subunit, receptor subunit fragment , Any one of hormone, lectin, and polyhistidine.

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

  The fluorescent dyes encapsulated in the fluorescent dye-encapsulated particles are, 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, tungstic acid 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, DY651 (above, manufactured by Dynamics GmbH), Oyster643, Oyster656 (above, made by Denovo Biolabels, Inc.) 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-dicarboxyl Cyrhodamine, 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 532, Alexa Fluor 532, Alexa Fluor 532 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 635, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 50 680, Alexa Fluor 680, Alexa Fluor 680, Alexa Fluor 680 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 650/666, BODIPY 650/666 Manufactured), methoxycoumarin, eosin, NBD, pyrene, Cy5, Cy5.5, Cy7 and the like.

The membrane 13 (second support) on which the second substance is immobilized is, for example, polymethyl methyl methacrylate (PMMA), polycarbonate, polypropylene, polyethylene, polymethylpentene, polystyrene, polytetrafluoroethylene, ABS resin, polydimethylsiloxane, nitrocellulose, silicone resin, copolymer or composite containing these polymer compounds, quartz glass, Pyrex (registered trademark) glass, soda glass, borate glass, silicate glass, borosilicate It is comprised by any one of acid glass, ceramics, and its composite_body | complex.
The first substance described above is immobilized on the first support by a chemical bond or a physical bond. Moreover, the above-mentioned second substance is immobilized on the second support by chemical bonding or physical bonding.

(About structure)
The shape of the lateral flow test strip 1 is not particularly limited as long as the dropped sample can move in the test strip by capillary action. For example, a structure in which four members of a sample pad 11, a bonding pad 12, a membrane 13, and a water absorbing pad are connected in series in one order in this order on a backing sheet serving as a substrate and fixed. After each member is fixed to the backing sheet, the backing sheet is preferably 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 lateral flow test strip 1 will be described.
(1) Sample pad The sample pad 11 is a member that holds a dropped liquid sample (biological sample) containing an analysis object for a certain period of time. As the sample pad 11, it is preferable to use a material made of cellulose. More specifically, for example, Cellulose Absorbent Pad (manufactured by Pall) or the like can be used. In view of the difference in pH depending on the dropped sample, the sample pad 11 is preferably used after the sample pad 11 is infiltrated with a buffer solution and dried.

(2) Binding pad The binding pad 12 is a member obtained by drying and solidifying the labeled particles for the analyte, and the analyte contained in the sample that has moved from the sample pad 11 by capillary action is specific to an antigen-antibody reaction or the like. This is the part that is bound to the labeling particle in the recognition reaction. The material of the bonding pad 12 is preferably a non-woven glass fiber, and more specifically, for example, Glass Fiber Pad (manufactured by Millipore). The reaction between the analyte and the antibody (first substance) provided on the labeled particle is performed using any one of physical affinity, chemical affinity, chemical binding, and immunological methods. It is what is said.

The content of the labeling particles per unit area (cm ² ) in the bonding pad 12 is not particularly limited, but is preferably 1 μg or more and 4 μg or less. In addition, as a method for infiltrating the labeled particles into the bonding pad 12, for example, a method of drying the bonding pad 12 after applying, dripping or spraying a liquid in which the labeled particles are suspended and dispersed to the bonding pad 12. Is preferred.
The labeling particles are preferably metal colloid particles, fluorescent dye particles, colored latex particles, up-conversion phosphor particles, quantum dot particles, etc. among the above materials. The particle diameter of the labeling particles is preferably 20 nm or more and 1000 nm or less.
As the antibody provided in the labeled particles, 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.

(3) Membrane The membrane 13 is a member in which the complex of the labeled particle and the analyte, the labeled particle, and the sample move by capillary action, and an immune reaction consisting of immobilized antibody-analyte-labeled particle is performed. It has an antibody immobilization section (test line 13a) to be performed and an antibody immobilization section (control line 13b) to perform an immune reaction composed of immobilized antibody-labeled particles. The reaction between the analyte and the immobilized antibody (second substance) is performed using any one of physical affinity, chemical affinity, chemical binding, and immunological method. .

The membrane 13 is preferably composed of a material capable of physically adsorbing protein, particularly composed of nitrocellulose, and more specifically, exemplified Hi-Flow Plus 120 membrane (manufactured by Millipore).
The shape of the antibody immobilization portion (determination portion) 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 circular shape, and a belt shape. Among these shapes, a line shape having a width of 0.5 to 2.0 mm is preferable, and an antibody amount per 1 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 immobilization part on which a goat anti-mouse IgG antibody is immobilized can be used as the control line 13b. Further, after immobilizing the antibody on the membrane 13, in order to prevent non-specific adsorption of the protein, for example, the membrane 13 is preferably infiltrated with a solution of a blocking agent such as albumin, casein, or polyvinyl alcohol and then dried. .

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

(4) Water-absorbing pad The water-absorbing pad is a member that absorbs the sample and marker particles that have moved through the membrane 13 by capillary action and always generates a constant flow. The material of the water absorption pad is preferably a fibrous material of cellulose material, and more specifically, for example, Cellulose Fiber Sample Pad (manufactured by Millipore).

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

Hereinafter, a method for detecting a signal derived from the captured labeled particles in the test line 13a will be described.
In the test strip, it is preferable to detect a signal (detection signal) generated by the reaction between the analyte and the particles labeled with the object-specific substance by an optical method. Examples of the optical method include reflected light intensity or fluorescence detection, luminescence detection, or electrochemiluminescence. More specifically, as an apparatus capable of detecting red / blue system over time, an immunochromatography reader C10066-10 (manufactured by Hamamatsu Photonics) and the like can be mentioned.

Next, a measurement method will be described. As described above, the analyte and the object-specific substance are detected by the test strip using an immunological reaction. A sample having a known concentration in the analysis object is dropped on the test strip and measurement is started. It is preferable to develop the test strip for about 30 minutes in order to obtain a constant signal.
Here, immunological methods include enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), cell immunostaining, tissue immunostaining, immunoprecipitation, flow cytometry. Method, fluorescence-labeled cell sorting method (FACS), immunochromatography method, quartz crystal microbalance method (QCM), surface plasmon resonance method (SPR), two-plane polarization interference method (DPI), ellipsometry method, ELISPOT Method and Western blotting.

The method for measuring an exosome of an analyte according to the present embodiment uses a test strip to measure a detectable signal generated by reacting a complex formed by the reaction of the exosome and the first substance with the second substance. Then, the concentration of exosome is judged by comparing with a preset reference value of the signal.
In the above-described embodiment, the case where the reaction between the analyte and the first substance is started by the bonding pad 12 has been described, but the present invention is not limited to this. In the above-described embodiment, the case where the reaction between the composite and the second substance is initiated 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 an exosome using an exosome surface antigen (CD9, CD63, CD81) as an analysis target, the membrane 13 solid-phase antibody 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 was anti-mouse immunoglobulin G (Dako). These two antibodies were dialyzed at 4 ° C. with a dialysis cartridge Slide-A-Lyzer 10,000 MWCO (trade name, Pierce) with 50 mM phosphate buffer (pH 7.2). The antibody concentration after dialysis was measured, and anti-exosome surface antigen antibody was prepared at 1 mg / ml and anti-mouse immunoglobulin was prepared at 0.5 mg / ml.

(2) Preparation of colloidal gold solution The above-mentioned three kinds of anti-exosome surface antigen antibodies were dialyzed, prepared to 60 μg / ml with phosphate buffer (pH 7.4), and colloidal gold (particle size prepared to 1 OD) 40 nm, Tanaka Kikinzoku Kogyo Co., Ltd.) The antibody solution was added to a mixed solution of 9 ml of a solution and 1 ml of potassium dihydrogen phosphate solution (pH 8.0). After pipetting, the mixture was allowed to stand at room temperature for 15 minutes, and 1% PEG 20,000 solution, 10% BSA solution was added and mixed. The mixed solution was centrifuged at 8000 × g for 15 minutes and the supernatant was discarded. The binding pad coating solution was added, stirred and sonicated, and centrifuged again, and the supernatant was discarded. This operation was repeated twice in total to obtain a gold colloid 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 dropped. Dry at 1 ° C. for 1 hour.

(4) Preparation of binding pad Add the coating solution and colloidal gold solution to a 1.5 ml Eppendorf tube, and after ultrasonic treatment at 28 kHz, add a total of 900 μl of colloidal gold coating solution to a 10 mm × 150 mm glass fiber pad (glass fwiber pad, millipore) and dried under reduced pressure with a vacuum vacuum pump for 18 hours.

(5) Production of membrane The above-mentioned solid-phased 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% dairy 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 Dried membrane, absorbent pad (CFSP203000, Milipore), bonding pad, sample pad are attached to a backing sheet (Adhesive Research), and 5 mm with cutting device CM4000 (trade name, BioDot). After cutting by width, it was housed in a housing case to make 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 and 5% BSA solution to obtain an antigen solution for measurement. Serum was prepared for a total of 8 individuals.

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

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

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

Claims (13)

Determine the presence or absence of the analyte by measuring the signal generated by reacting the second substance with the complex formed by the reaction of the analyte contained in the biological sample and the first substance,
The method for detecting an analyte, wherein the analyte is an exosome.   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 method for detecting an analyte according to any one of claims 1 to 3, wherein the first substance is fixed to a first support.   The first support is a metal colloidal particle containing gold colloid, silver colloid, iron colloid, aluminum colloid, or platinum colloid, or a particle made of one or more of the group consisting of polymer compounds. The method for detecting an analysis object according to claim 4, wherein:   The method for detecting an analyte according to claim 4, wherein the first support is particles made of a polymer compound, and is a fluorescent dye-containing particle or a visible dye-containing particle.   The method for detecting an analyte according to any one of claims 4 to 6, wherein the size of the first support is 20 nm or more and 1000 nm or less.   The method for detecting an analyte according to any one of claims 1 to 7, wherein the second substance is fixed to a second support. The reaction between the analyte and the first substance forms a complex, and the reaction between the complex and the second substance is performed on one test strip,
The method for detecting an analyte according to any one of claims 1 to 8, wherein these reactions are performed at different positions on the test strip.   The method according to claim 9, wherein the complex reacts with the second substance after moving on the test strip. A complex formation part that forms a complex by the reaction between the analyte contained in the biological sample and the first substance, and a signal generation part that can generate a detectable signal by reacting the complex with the second substance. And
The first substance is fixed to a first support;
The first support is dried and solidified in the composite forming part,
A test strip for lateral flow, wherein the analyte is an exosome.   The lateral flow test strip according to claim 11, wherein the signal generation unit is present on a member in which the composite is moved by capillary action. The second substance is an antibody;
The lateral flow test strip according to claim 11 or 12, wherein the second substance is immobilized on the signal generating portion.

Images