Testing method and test kit for detecting target substance

Abstract

To provide an inspection method for a target substance, having high reliability and a low cost and capable of easily performing inspection.SOLUTION: An analysis method for detecting a target substance in an analyte, includes the steps of: preparing a composite composed of a binding partner and a labeling substance bound to the binding partner, the labeling substance having a dissociation rate from the binding partner faster than that of the target substance; sensitizing the analyte likely to contain the target substance to the composite; acquiring image data in which the target substance may be replaced with the labeling substance to cause light intensity to change; and determining the presence or absence of the target substance on the basis of the image data.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a test method and a test kit for detecting a target substance.

Background Art

[0002] Currently, the novel coronavirus (SARS-CoV-2), which has caused an unprecedented disaster worldwide, is one of the coronaviruses and the seventh virus among the coronaviruses that infect humans. Coronaviruses are a type of RNA virus (single-stranded RNA virus) that has RNA as genetic information and has a double membrane made of lipids called an "envelope" on the outermost side of the particles. It cannot multiply on its own, but can attach to cells such as mucous membranes, enter them, and multiply. Regarding the novel coronavirus infection, knowledge about the pathogen and the disease is gradually accumulating (see, for example, Non-Patent Document 1). For the determination of the presence or absence of infection with the novel coronavirus, mainly PCR tests are used. The PCR test has high sensitivity, which indicates the proportion of people who are determined to be positive among those who are actually infected, and also high specificity, which indicates the proportion of people who are determined to be negative among those who are actually not infected. Therefore, it is considered effective to perform a PCR test as a medical diagnosis for subjects with symptoms. However, it has also been pointed out that the PCR test has a high cost per test and a heavy burden on medical staff because it is necessary to perform the test at a designated medical institution. Therefore, improvement of the test method has been demanded.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

[0004] One proposed solution to the aforementioned problems is to first perform a rapid test, and then only perform a PCR test on those suspected of being positive, rather than performing a PCR test on everyone. However, rapid test kits require time-consuming steps such as mixing solutions, so there was a need for a more convenient testing method. It was also pointed out that the cost was high, though not as high as PCR tests. Therefore, there was a need for a test strip and a testing method using it that could maintain the same level of reliability as PCR tests while being cheaper and easier to use than conventional rapid test methods. The virus multiplies in the throat and respiratory tract, and is said to be shed 2-3 days before the onset of symptoms, infecting others. In people with mild symptoms, it is believed that viral shedding stops after 7-8 days. Since the virus multiplies transiently in the early stages of infection, it is hoped that detecting the virus released in the early stages of infection can effectively prevent infection. Furthermore, recent technical literature has reported that a large amount of the novel coronavirus is released in exhaled breath. Therefore, there has been a need for a simple test method that can detect the virus with high sensitivity even when the sample is exhaled breath. Furthermore, there was a need for a method that could be used not only for countermeasures against COVID-19, but also for detecting various viruses and pathogens.

[0005] The object of the present invention is to provide a test specimen, a test kit, and a testing method that are not limited to the object to be tested, and that allow for reliable, inexpensive, and easy testing. [Means for solving the problem]

[0006] This invention relates to the following: <1> A test specimen for detecting a target substance in a sample, comprising a carrier and a composite arranged in a test area on the carrier, wherein the composite consists of a binding partner and a labeling substance bound to the binding partner, the labeling substance dissociates from the binding partner faster than the target substance, and the test specimen detects the target substance from the change in color of the test area when the target substance is replaced by the labeling substance. <2> The test specimen described in (1), wherein the target substance is a protein. <3> The test specimen according to (2), wherein the labeling substance has been modified to alter the amino acid sequence of the target protein. <4> The test specimen according to (2), wherein the labeling substance is obtained by chemically modifying the target protein. <5> The test piece according to (2), wherein the binding partner is an antibody and the labeling substance is an antigen labeled with a marker. <6> The test specimen described in <5> is an antibody, Anti-RBD antibody CR3022, and the antigen is the receptor binding domain of the novel coronavirus (SARS-COVID-19). <7> The test specimen according to (1), wherein the labeled substance is labeled with a marker selected from the group consisting of gold colloid particles, colored latex particles, metal complexes, fluorescent substances, agglomerating luminescent materials, chemiluminescent substances, and electrochemiluminescent substances. <8> The test specimen according to (1), wherein the inspection areas are arranged in multiple locations on the support. <9> The test specimen described in (1) is capable of detecting multiple types of target substances, with multiple test areas arranged for each type of target substance. <10> The test specimen described in (1), wherein the target substance is a microorganism. <11> The test specimen described in (1), wherein the target substance is a virus. <12> The test specimen according to (1), wherein the target substance is SARS-COVID-19 or a variant thereof. <13> The test specimen described in (1) is the exhaled breath of the subject. <14> The test specimen described in (1) is a sample that is a body fluid of a subject. <15> A mask comprising the test specimen described in (1). <16> A test kit comprising the test specimen described in (1). <17> An analytical method for detecting a target substance in a sample, comprising: a step of preparing a complex comprising a binding partner and a labeling substance bound to the binding partner, wherein the labeling substance dissociates from the binding partner faster than the target substance; a step of sensitizing a sample that may contain the target substance to the complex; a step of acquiring image data in which the target substance may have replaced the labeling substance and a change in light intensity may have occurred; and a determination step of determining whether or not the target substance is present based on the image data. <18> An analytical system for detecting a target substance in a sample, comprising the steps of: receiving image data in which a change in light intensity may have occurred due to the replacement of the target substance by the labeling substance of a complex comprising a binding partner and a labeling substance bound to the binding partner with a weaker binding force than the target substance; retrieving known image data stored in association with positive or negative results from a database, comparing the image data with the known image data to determine whether or not the target substance is present; storing the image data in the database as new known image data in association with positive or negative results based on the determination result; and transmitting the determination result to a user terminal. [Effects of the Invention]

[0007] According to the present invention, there are provided a test piece, a test kit, and a test method that are highly reliable, inexpensive, and easy to test. According to the present invention, even when the sample is exhaled breath, it can be easily tested with high sensitivity. Further, according to the present invention, the test target is not limited to the novel coronavirus, and the presence of various target substances can be detected.

Brief Description of the Drawings

[0008] [Figure 1] FIG. 1A, FIG. 1B, and FIG. 1C are diagrams showing the principle of the competitive displacement method. [Figure 2] FIG. 2 is a conceptual diagram showing the color development state of the labeling substance of the complex arranged on the membrane (carrier). [Figure 3] FIG. 3 is a diagram showing the amino acid sequence of the target substance and the position where it is mutated. [Figure 4] FIG. 4A (the antibody is Anti-RBD antibody CR3022), FIG. 4B (the antibody is ARG66740 [anti-SARS-CoV-2 Spike protein (RBD) antibody]) are diagrams showing the change in light intensity when the labeling substance is replaced by the target substance, respectively. [Figure 5] FIG. 5A, FIG. 5B, and FIG. 5C are diagrams showing the change in light intensity when the labeling substance is replaced by the target substance, respectively. [Figure 6] FIG. 6A and FIG. 6B are diagrams showing the change in light intensity when the chemically modified labeling substance is replaced by the target substance, respectively. [Figure 7] FIG. 7A, FIG. 7B, and FIG. 7C are diagrams showing specific examples of the test area of the test piece, respectively. [Figure 8] FIG. 8 is a diagram showing a specific example in which a test area and a control area are arranged on the test piece. [Figure 9] FIG. 9A is a positive simulation image, and FIG. 9B is a negative simulation image. FIGS. 9C and 9D are diagrams showing the change in light intensity when the labeling substance is replaced by the target substance, respectively, and compare the performance of the machine learning engine. FIG. 9E is a diagram showing the test result. [Figure 10]Figure 10A is an image of the actual testing area, and Figure 10B is a positive simulation image created from the actual testing area. Figures 10C and 10D show the change in light intensity when the labeling substance is replaced with the target substance, comparing the performance of the machine learning engines. Figure 10E shows the test results. [Figure 11] Figure 11A is an image of the actual test area, and Figure 11B is an image of the actual positive result after the target substance was introduced into the actual test area. Figures 11C and 11D show the change in light intensity when the labeling substance is replaced with the target substance, and compare the performance of the machine learning engine. Figure 10E shows the test results. [Figure 12] Figures 12A, 12B, and 12C show one method of using the test specimen (mask). [Figure 13] Figures 13A and 13B show images when the test area is divided into 5 spots per row. Figure 13A is a positive simulation image, and Figure 13B is a negative simulation image. Figures 13C and 13D show the change in light intensity when the labeling substance is replaced with the target substance, comparing the performance of the machine learning engine. Figure 13E shows the test results. [Figure 14] Figures 14A and 14B show images when the test area is divided into one row and two spots. Figure 14A is a positive simulation image, and Figure 14B is a negative simulation image. Figures 14C and 14D show the change in light intensity when the labeling substance is replaced with the target substance, comparing the performance of the machine learning engine. Figure 14E shows the test results. [Figure 15] Figure 15 shows one way in which the test specimen is used (a test kit). [Figure 16A] Figure 16A is a flowchart of the inspection method. [Figure 16B] Figure 16B is a flowchart of the inspection method. [Figure 17] Figure 17 is a flowchart of the inspection system. [Modes for carrying out the invention]

[0009] The present invention will be described below with reference to embodiments, but the present invention is not limited to the embodiments described below.

[0010] [Test specimen for detecting the target substance] The present invention relates to a test piece for detecting a target substance in a sample, comprising a carrier and a complex disposed on a test area on the carrier. The complex consists of a binding partner and a labeling substance bound to the binding partner. The labeled substance dissociates from its binding partner faster than the target substance. In other words, the binding force between the labeled substance and its binding partner is weaker than the binding force between the target substance and its binding partner. With this configuration, the target substance can be detected by the color change in the inspection area when the target substance replaces the labeled substance and binds to its binding partner.

[0011] [Competitive Substitution Law] Figures 1A to 1C illustrate the principle of the competitive substitution method, which is the solution principle of the present invention. The competitive substitution method will be explained using Figures 1A to 1C. Here, a membrane will be used as an example of the carrier. As shown in Figure 1A, a complex consisting of a binding partner 1 and a labeled substance 2, in which substance 2a is labeled with labeling substance 2b, is immobilized on the membrane 5 of the test specimen 10 in a predetermined test area. The labeling substance 2 dissociates from the binding partner 1 faster than the target substance 4. Therefore, as shown in Figure 1B, when a sample containing the target substance 4 is introduced into the test specimen 10, substitution of the labeling substance 2 with the target substance 4 occurs, as shown in Figure 1C. In other words, if the target substance is present in the sample, the light intensity decreases and the color of the test area becomes lighter; if it is not present, there is no change in light intensity and the color of the test area remains dark. The target substance 4 can be detected from the change in color of the test area when the substitution of the labeling substance 2 with the target substance 4 occurs. Here, the change in color of the test area includes changes in the shade of color, and changes in the brightness and saturation of the color.

[0012] [complex] As described above, the complex consists of a binding partner and a labeling substance. As a binding partner, a variety of substances can be used without particular limitations, as long as they can be immobilized on the carrier and can bind to the labeled substance and / or the target substance. Antibodies of the labeled substance and / or the target substance when those substances are used as antigens can be used as binding partners. The labeled substance is labeled with a marker. A variety of markers can be used without particular limitations, as long as they do not inhibit the binding of the binding partner to the labeled substance and / or the target substance. Existing materials used in the analysis of biological samples can be used as markers, such as gold colloid particles, colored latex particles, metal complexes, fluorescent substances, agglomerative luminescent materials, chemiluminescent substances, electrochemiluminescent substances, etc. This is because it makes it easier to observe the color change when the labeled substance and the target substance are replaced, for example, changes in color intensity, brightness, or saturation. It is preferable to use a labeled substance with high light intensity. Preferably, the labeling substance has its binding affinity to the binding substance weakened by various methods so that it can be substituted for the target substance. One method for reducing the binding affinity is to mutate a predetermined position in the amino acid sequence of the target substance. Figure 3 shows the amino acid sequence of the Receptor Binding Domain (RBD) of the novel coronavirus (SARS-COVID-19) and the positions that are thought to affect the reduction in binding ability, assuming the target substance is the RBD. By identifying the positions from the amino acid sequence where a decrease in the binding ability of the RBD to an antibody (e.g., Anti-RBD antibody CR3022 (commercial product)) is expected, and preparing RBD variants (K378N, V382E, R346S), it is possible to prepare substances in which the binding ability is expected to decrease and the dissociation rate is expected to increase. One method for reducing the binding strength is to chemically modify the target substance. Examples of chemical modifications include reductive methylation of lysine, acetylation of tyrosine with N-acetylimidazole, acetylation with sulfo-NHS acetate, and succinylation. Among these, acetylation and succinylation of tyrosine with N-acetylimidazole are preferred. We have discussed amino acid mutations and chemical modifications as methods to reduce binding strength, but these can also be used in combination.

[0013] [Specimen] There are no particular restrictions on the target substance to be detected in the test specimen; various substances can be used as the target substance. Examples of target substances include proteins, viruses, and pathogens. The target substance includes substances contained within microorganisms and viruses. Examples of target substances include the novel coronavirus (SARS-COVID-19) or its variants, influenza viruses, and SARS viruses. Sample forms include the subject's bodily fluids and breath. Using breath as a sample allows the subject to more easily determine the presence or absence of the target substance than before. Body fluids include not only fluids in the narrow sense, such as blood, lymph, tissue fluid, and body cavity fluid, but also fluids in the broader sense, such as digestive fluids (saliva, gastric juice, bile, pancreatic juice, intestinal juice), sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, and breast milk. Furthermore, solutions containing cell fragments detached from the cell surface during nasal or oral swab examinations are also included in the definition of body fluids.

[0014] Various studies are progressing on the novel coronavirus. For example, there are reports that the novel coronavirus is present in human exhaled breath (Reference 1: High infectiousness immediately before COVID-19 symptom onset highlights the importance of continued contact tracing, eLife 2021;10:e65534. DOI: https: / / doi.org / 10.7554 / eLife.65534). Reference 2: Evolution of SARS-CoV-2 Shedding in Exhaled Breath Aerosols (https: / / doi.org / 10.1101 / 2022.07.27.22278121). There are also reports of cases where the novel coronavirus has been detected in exhaled breath (Reference 3: Wearable materials with embedded synthetic biology sensors for biomolecule detection, Nature Biotechnology 39, pages 1366-1374 (2021) https: / / doi.org / 10.1038 / s41587-021-00950-3). As research into transmission routes progressed, it is now believed that the main route of infection is the inhalation of virus-containing droplets and aerosols (particles containing water, even smaller than droplets) expelled from infected individuals (including asymptomatic carriers) during coughing, sneezing, and talking (Reference 4: "Guidelines for the Treatment of Novel Coronavirus Infection (COVID-19), Version 8.1," Ministry of Health, Labour and Welfare, Pathogens and Immunology, p. 6). Given that exhaled breath is an important route of infection and that it is easy to test subjects, it is preferable to use exhaled breath as the sample.

[0015] The binding partner can be an antibody, and the labeling substance can be an antigen labeled with the labeling substance. The antigen can be the receptor binding domain (RBD) of the novel coronavirus (SARS-COVID-19), and the antibody can be Anti-RBD antibody CR3022 (commercial product).

[0016] [Carrier] There are no particular restrictions on the type of carrier material that can be used, but a membrane is one example. The membrane can be any existing material used in the analysis of biological samples, such as nitrocellulose or PVDF membranes, without any particular restrictions.

[0017] [Examination Area] On the surface of the carrier, an inspection area is formed where the complex is fixed via a binding partner. Figure 2 is a conceptual diagram showing the color development state of the labeling substance of the complex placed on a membrane as a carrier. As detailed in the Examples section, the binding partner (antibody) is immobilized in a spot-like manner on the surface of the membrane, and then the labeling substance (labeled antigen) is spread onto the membrane to form a spot-like testing area on which the complex is immobilized. There are no particular restrictions on the number of testing areas, but it is preferable to arrange multiple testing areas on the membrane. This is because arranging multiple testing areas is expected to increase the opportunities for contact with the target substance, thereby improving accuracy. Furthermore, if there are analytical deficiencies in one testing area, the other testing areas can compensate for them. There are no particular restrictions on the arrangement pattern of the inspection areas, and various arrangement patterns can be used. For example, one arrangement is to place multiple inspection areas at equal intervals in both the vertical and horizontal directions of the membrane. Figures 7A, 7B, and 7C show specific examples of inspection areas for test specimens. Examples include a 5x5 layout in Figure 7A, a 5x3 layout in Figure 7B, and a 6x6 layout in Figure 7C. As shown in Figure 7A, the effect of achieving 100% accuracy is obtained. According to Figure 7B, the same effect as in Figure 7A can be obtained even when the number (area) of inspection areas is reduced. As shown in Figure 7C, the same effect as in Figure 7A can be obtained even when an inspection area without gaps is set. Multiple test areas may be arranged for each type of target substance to enable detection of multiple types of target substances. This is because the presence of multiple types of substances can be determined in a single test. Figure 8 shows a specific example of a test specimen with an inspection area and a control area. In addition to the inspection area, a control area with a labeled substance fixed adjacent to the inspection area may be placed on the membrane of the test specimen. For example, as shown in Figure 8, the inspection area (changing spot) and the control area (unchanging spot) may be placed in parallel. This has the effect of reducing the influence of the image acquisition (shooting) environment and improving the accuracy of the judgment by making a relative comparison between the inspection area and the control area.

[0018] [Examples of test specimen applications (other embodiments)] As described above, the present invention has been described by embodiments, but the descriptions and drawings that constitute part of this disclosure should not be understood as limiting the invention. Various alternative embodiments, examples, and operational techniques will become apparent to those skilled in the art from this disclosure. Figures 12A and 12B show one way of using the test specimen (mask). The above-mentioned test specimen may be used as the test specimen itself, but for example, as shown in Figure 12A, test specimens 85a and 85b may be fixed to a holder 83, and the holder may be attached to the back side (the side that touches the skin) of the mask 81 in a removable manner, as shown in Figure 12B. Alternatively, as shown in Figure 12B, test specimens 85a and 85b may be directly fixed to the mask 81. This is because if the target substance (e.g., novel coronavirus) is present in the subject's exhaled breath, the presence of the target substance can be easily detected from the change in light intensity of the test area 85a2 shown in the enlarged view of test specimen 85a in Figure 12C. Specifically, by comparing the color intensity (light intensity) of the control area 85a1 and the test area 85a2, a positive result can be determined if the color of the test area 85a2 is lighter than the color of the control area 85a1, and a negative result can be determined if there is no difference in color intensity.

[0019] Figure 15 shows one embodiment of the use of the test specimen (test kit). In addition to the above, as shown in Figure 15A, for example, the test kit 9 ​​may be used in which test areas 91, 92, 93, 94, and 95 are formed at the ends of a strip-shaped test specimen. This is because the presence of the target substance can be easily determined by applying the subject's saliva to the test areas 91-95 of the test kit 9 ​​or by blowing their breath onto them. In this case, the presence or absence of multiple target substances can be determined at once by arranging the SARS-COVID-19 test area 91, the SARS test area 92, the Soviet influenza test area 93, the Hong Kong influenza test area 94, and the other virus test areas 95.

[0020] Thus, the present invention naturally includes various embodiments and the like that are not described herein. Therefore, the technical scope of the present invention is determined solely by the inventive features relating to the claims that are appropriate from the above description.

[0021] [Analysis method A] Next, we will explain the analytical method using the test specimens described above. Figure 16A is a flowchart of the inspection method. (i) First, a test specimen is prepared comprising a composite consisting of a binding partner and a labeled substance bound to the binding partner (S101). For example, a test kit is prepared comprising a test specimen as shown in Figure 15A. (b) Next, a subject's sample that may contain the target substance is sensitized. For example, saliva is applied to the test piece or breath is blown onto it (S103). (h) As shown in Figure 15C, the color intensity (light intensity) of the control area 91a1 and the test area 91a2 are compared. If the color of the test area 91a2 is lighter than the color of the control area 91a1, the result is positive; if there is no difference in color intensity, the result is negative (S110). Therefore, the target substance can be detected from the change in light intensity.

[0022] In step S110 described above, if it is difficult to determine the intensity of the color, an AI trained in machine learning to determine the presence or absence of the target substance may be used. The following describes an analysis method (system) using AI. For convenience, the explanation will be divided into the user side and the server side. [Analysis method B] Figure 16B is an overall flowchart of the inspection method (user side). (i) Perform steps S101 and S103 in Figure 16A. (b) Take a photograph of the test specimen and obtain image data (S105). (h) The application installed on the user terminal is activated and the obtained image data is transferred to the server (S108). (ii) Receive the results of the inspection system's judgment. Therefore, subjects can determine whether they are positive or negative.

[0023] [Analysis System] Figure 17 is a flowchart of the testing system (server side). The testing system consists of a server (not shown) equipped with a transmitting / receiving unit, a storage unit (database), an output unit, and an arithmetic control unit. These components can utilize hardware such as the CPU, GPU, memory, and programs loaded into memory of any computer. The storage unit pre-stores known image data associated with positive or negative results. The server and user terminals are connected via communication methods such as the internet or an intranet. Information can be exchanged between the user terminal and the server by running applications installed on the user terminal. (i) The server receives the image data (S201). (b) Retrieve known image data that is close to the image data from the server's storage (S203). (h) The image data is compared with known image data, and the positive or negative result is determined from the difference in the intensity of the color in the test area (S205). (ii) The judgment result and image data are associated and saved as known image data in the server's storage (S208). (e) Send the decision result to the user terminal (S210). [Examples]

[0024] The present invention will be described in more detail below based on test examples. However, the scope of the present invention is not limited by these manufacturing and test examples.

[0025] [Test Example 1] Preparation of antibody-loaded membrane The target substance was the receptor binding domain (RBD) of the novel coronavirus (SARS-COVID-19), and the label was a gold colloid (manufactured by Jackson ImmunoResearch, product name "40 nm Colloidal Gold Streptavidin"). The gold colloid label was then attached to the RBD to obtain a labeled substance (gold colloid-labeled RBD). FF170HP Plus Thick (Cytiva) was prepared as the carrier, and CR3022 (Abcam, CR3022) (anti-RBD antibody) was prepared as the antibody. The antibody was then conjugated in a spot pattern onto nitrocellulose to obtain an antibody-supported membrane. As shown in Figure 2, the labeling substance was poured over the entire surface of the antibody-loaded membrane and then incubated. During incubation, it was observed that the color intensity of the antibody-supported spots on the antibody-supported membrane (hereinafter also referred to as "spot areas") increased. This is thought to be because the labeling substance accumulated on the antibody-supported spots during incubation, causing the spots to become more intense. After incubation, the samples were washed with PBST (PBS containing 0.05% Tween-20). From the above, it was confirmed that the labeling substance binds to the antibody, forming a complex consisting of the antibody and the labeling substance (anti-RBD antibody-gold colloid-labeled RBD complex).

[0026] [Test Example 2] Preparation of an antigen with a fast dissociation rate Figure 3 shows the amino acid sequence of the target substance RBD and the locations of mutations in that amino acid sequence. The mutation locations were determined based on technical literature (see Yi et al. Genome Medicine (2021) 13:164, Fig. 4). Then, RBD mutants (K378N, V382E, R346S) were prepared, which were expected to have reduced binding ability to the antibody (Anti-RBD antibody CR3022 (commercial product)). Protein preparation was performed using cell-free protein synthesis, and the increase in dissociation rate due to the reduced binding affinity to the antibody was investigated.

[0027] [Test Example 3] Confirmation of antigens with a fast dissociation rate Figures 4A and 4B show the changes in light intensity when the RBD variant (labeling substance) is replaced by RBD (target substance), respectively. In Figure 4A, Anti-RBD antibody CR3022 (commercially available) was used as the antibody, and in Figure 4B, ARG66740 (commercially available, manufactured by Arigo Biolaboratories) was used as the antibody. Figure 4A shows a significant decrease in binding affinity to the antibody (anti-RBD antibody CR3022) for RBD(K378N) and RBD(V382E), and approximately a 35% decrease for RBD(R346R).

[0028] [Test Example 4] Confirmation of substitution by the antigen to be detected Figures 5A, 5B, and 5C show the changes in light intensity when the labeled substance is replaced with the target substance, respectively. By reacting the antibody-antigen complex with a trimer of spike protein (Trimeric Spike Protein), we obtained results showing that RBD(K378N) and RBD(V382E) were replaced by RBD(WT) by approximately 10-20% within 2 hours.

[0029] [Test Example 4] Confirmation of increased dissociation rate due to chemical modification of antigen Figures 6A and 6B show the changes in light intensity when the chemically modified labeling substance is replaced by the target substance, respectively. Multiple RBD (WT) antigens were prepared, and each was chemically modified by (1) reductive methylation of lysine, (2) acetylation of tyrosine with N-acetylimidazole, (3) acetylation with sulfo-NHS acetate, and (4) succinylation. The effect of chemically modified antigens on reducing their binding affinity to antibodies (anti-RBD antibody ARG66740) was investigated. As a result, it was confirmed that the dissociation rate of the light source from the antibody increased due to the decreased binding affinity of the chemically modified antigens in (2) and (4) to the antibody.

[0030] Figure 9A is a positive simulation image, and Figure 9B is a negative simulation image. Figures 9C and 9D show the change in light intensity when the labeling substance is replaced with the target substance. Figure 9E shows the test results.

[0031] Figure 10A is an actual spot image, and Figure 10B is a positive simulation image created from the actual spot image. Figures 10C and 10D show the change in light intensity when the labeling substance is replaced with the target substance. Figure 10E shows the test results.

[0032] Figure 11A is an image of the actual test area, and Figure 11B is an image of the actual positive result after the target substance was introduced into the actual test area. Figures 11C and 11D show the change in light intensity when the labeling substance is replaced by the target substance. Figure 11E shows the test result. [Industrial applicability]

[0033] The present invention provides a highly reliable, inexpensive, and easy-to-use testing method. According to the present invention, even if the sample is exhaled breath, testing can be performed easily and with high sensitivity. Furthermore, according to the present invention, the presence of various target substances can be detected without being limited to the novel coronavirus. By applying the principles of the present invention, it can be used in test specimens, test kits, masks, and testing methods, such as AI-based testing methods.

Claims

1. A test piece for detecting a target substance in a sample, comprising a carrier and a composite arranged on the carrier in a test area, The aforementioned complex consists of a binding partner and a labeled substance bound to the binding partner. The labeled substance dissociates from its binding partner at a faster rate than the target substance. A test specimen that detects the target substance based on the change in color of the inspection area when the target substance replaces the labeling substance.

2. The test specimen according to claim 1, wherein the target substance is a protein.

3. The test specimen according to claim 2, wherein the labeling substance is a substance that has mutated the amino acid sequence of the target protein.

4. The test specimen according to claim 2, wherein the labeling substance is a target protein that has been chemically modified.

5. The test piece according to claim 2, wherein the binding partner is an antibody and the labeling substance is an antigen labeled with a marker.

6. The test specimen according to claim 5, wherein the antibody is Anti-RBD antibody CR3022 and the antigen is the receptor binding domain of the novel coronavirus (SARS-COVID-19).

7. The test specimen according to claim 1, wherein the labeled substance is labeled with a marker selected from the group consisting of gold colloid particles, colored latex particles, metal complexes, fluorescent substances, cohesive light-emitting materials, chemiluminescent substances, and electrochemiluminescent substances.

8. The test specimen according to claim 1, wherein a plurality of the inspection areas are arranged on the carrier.

9. The test piece according to claim 1, wherein multiple inspection areas are arranged for each type of target substance, enabling detection of multiple types of target substances.

10. The test specimen according to claim 1, wherein the target substance is a microorganism.

11. The test specimen according to claim 1, wherein the target substance is a virus.

12. The test specimen according to claim 1, wherein the target substance is SARS-COVID-19 or a variant thereof.

13. The test specimen according to claim 1, wherein the sample is the exhaled breath of a subject.

14. The test specimen according to claim 1, wherein the sample is the bodily fluid of a subject.

15. A mask comprising the test piece described in claim 1.

16. An inspection kit comprising the test piece described in claim 1.

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