Testing method and test kit for detecting target substance

Abstract

To provide highly reliable, inexpensive, and easy-to-use testing methods for target substances. [Solution] The solution consists of a binding partner and a labeled substance bound to the binding partner. The labeling substance prepares a complex in which the dissociation rate from its binding partner is faster than that of the target substance. The process involves a step of sensitizing a sample that may contain the target substance to the composite, and the target substance However, the process of acquiring image data in which the labeling substance may have been replaced and a change in light intensity may have occurred. A specimen having a determination step that determines whether or not a target substance is present based on image data. An analytical method for detecting the target substance inside.

Description

Technical Field

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

Background Art

[0002] Currently, the novel coronavirus (SARS-CoV-2) that is causing an unprecedented disaster worldwide is one of the coronaviruses and is 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 does not increase on its own, but can attach to cells such as mucous membranes, enter, and increase. Regarding coronavirus disease 2019 (COVID-19), 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 large burden on medical staff because it needs to be performed at a designated medical institution. Therefore, an improvement in the inspection method has been demanded.

Prior Art Documents

Non-Patent Documents

[0003] [Non-Patent Document 1] Ministry of Health, Labour and Welfare: Q&A regarding the novel coronavirus: https: / / www.mhlw.go.jp / stf / seisakunitsuite / bunya / kenkou_iryou / dengue_fever_qa_00001.html [Overview of the project] [Problems that the invention aims to solve]

[0004] One way to address the aforementioned challenges is, for example, not to conduct PCR tests on everyone, but first It has been proposed to conduct rapid tests and then perform PCR tests only on those suspected of being positive. However, simple test kits require the effort of mixing solutions, so they are not ideal for easy testing. A viable method was needed. It was also pointed out that the cost was high, though not as high as PCR testing. Therefore, it was possible to maintain a level of reliability comparable to PCR testing while being cheaper than conventional rapid testing methods. There was a need for a test specimen that could be easily and inexpensively tested, as well as a testing method using it. The infected virus multiplies in the throat and respiratory tract, and is shed 2-3 days before the onset of symptoms, thus infecting others. It is said that it can infect people. For those with mild symptoms, it is believed that the virus stops being shed within 7-8 days. Because viruses multiply transiently in the early stages of infection, the viruses released in the early stages of infection... It is hoped that if the virus can be detected, infection can be effectively prevented. Also, recent technical literature suggests that According to reports, a large amount of the novel coronavirus is released through exhalation. Therefore, there was a need for a simple testing method that could perform highly sensitive tests even when the sample was exhaled. Furthermore, this is not limited to measures against COVID-19, but also applies to testing for various viruses and pathogens. A method that could also be applied to knowledge was needed.

[0005] The object of this invention is to provide a reliable, inexpensive, and easy-to-use test that has no limitations on the object to be tested. The objective is to provide test specimens, test kits, and testing methods. [Means for solving the problem]

[0006] This invention relates to the following: <1> A device comprising a carrier and a composite arranged in a testing area on the carrier, for the target substance in a sample A test specimen for detecting quality, wherein the composite comprises a binding partner and the binding partner A bound labeled substance and a labeling substance, wherein the labeling substance has a dissociation rate from its binding partner However, the inspection when the target substance is replaced by the labeled substance faster than the target substance A test specimen for detecting the target substance based on the color change of a region. <2> The test specimen described in (1), wherein the target substance is a protein. <3> The labeling substance is one in which the amino acid sequence of the target protein has been mutated, as described in <2>. The test specimen. <4> The labeling substance is obtained by chemically modifying the target protein as described in (2). Test specimen. <5> The binding partner is an antibody, and the labeling substance is an antigen labeled with a marker. The test specimen described in <2>. <6> The antibody is Anti-RBD antibody CR3022, and the antigen is the novel coronavirus (SARS-CoV-19). The test specimen described in <5> is the receptor binding domain of ). <7> The labeling substance may be gold colloid particles, colored latex particles, metal complexes, fluorescent substances, or agglomerating substances. A test piece according to <1>, labeled with a marker selected from the group consisting of a luminescent material, a chemiluminescent substance, and an electrochemiluminescent substance. The test piece according to <1>, which is labeled as described above. <8> The test piece according to <1>, wherein a plurality of the inspection regions are arranged on the carrier. <9> A test piece according to <1>, wherein a plurality of inspection regions are arranged for each type of target substance so as to be able to detect a plurality of types of target substances. The test piece according to <1>, which is arranged as described above. <10> The test piece according to <1>, wherein the target substance is a microorganism. <11> The test piece according to <1>, wherein the target substance is a virus. <12> The test piece according to <1>, wherein the target substance is SARS-COVID-19 or a variant thereof. <13> The test piece according to <1>, wherein the sample is the exhaled breath of a subject. <14> The test piece according to <1>, wherein the sample is the body fluid of a subject. <15> A mask comprising the test piece according to <1>. <16> An inspection kit comprising the test piece according to <1>. <17> A complex comprising a binding partner and a labeling substance bound to the binding partner, wherein the dissociation rate of the labeling substance from the binding partner is faster than that of the target substance; a step of sensitizing a sample that may contain the target substance to the complex; a step of obtaining an image data in which the target substance may have replaced the labeling substance and a change in light intensity has occurred; and a determination step of determining the presence or absence of the target substance based on the image data. An analysis method for detecting a target substance in a sample. The above steps are used to prepare a complex with a faster dissociation rate of the labeling substance from the binding partner than that of the target substance; sensitize a sample that may contain the target substance to the complex; obtain image data where the target substance may have replaced the labeling substance and a change in light intensity has occurred; and determine the presence or absence of the target substance based on the image data. The above steps are used to prepare a complex with a faster dissociation rate of the labeling substance from the binding partner than that of the target substance; sensitize a sample that may contain the target substance to the complex; obtain image data where the target substance may have replaced the labeling substance and a change in light intensity has occurred; and determine the presence or absence of the target substance based on the image data. The above steps are used to prepare a complex with a faster dissociation rate of the labeling substance from the binding partner than that of the target substance; sensitize a sample that may contain the target substance to the complex; obtain image data where the target substance may have replaced the labeling substance and a change in light intensity has occurred; and determine the presence or absence of the target substance based on the image data. The above steps are used to prepare a complex with a faster dissociation rate of the labeling substance from the binding partner than that of the target substance; sensitize a sample that may contain the target substance to the complex; obtain image data where the target substance may have replaced the labeling substance and a change in light intensity has occurred; and determine the presence or absence of the target substance based on the image data. An analysis method for detecting a target substance in a sample, comprising the above steps. <18> A binding partner and a label bonded to the binding partner with a weaker bonding force than that of the target substance. When the labeling substance of a composite consisting of a substance is replaced by the target substance, the light intensity changes. The process of receiving potentially generated image data; and associating it with whether it is positive or negative. The stored known image data is retrieved from the database, and the image data and the known image A step of comparing the data to determine whether or not the target substance is present; and based on the determination result, The aforementioned image data is associated with whether it is positive or negative and added to the database as new known The process of saving the result as image data; and the process of transmitting the determination result to the user terminal; An analytical system for detecting target substances in a sample. [Effects of the Invention]

[0007] According to the present invention, a test specimen, a test kit, and a test that can be easily performed with high reliability, low cost, and ease of use are available. A testing method is provided. According to the present invention, even if the sample is breath, it can be tested easily and with high sensitivity. Furthermore, according to the present invention, the test subject is not limited to the novel coronavirus, but can be various It can detect the presence of the target substance. [Brief explanation of the drawing]

[0008] [Figure 1] Figures 1A, 1B, and 1C illustrate the principle of competitive substitution. [Figure 2] Figure 2 is a conceptual diagram showing the color development state of the labeling substance of the complex placed on the membrane (support). [Figure 3] Figure 3 shows the amino acid sequence of the target substance and the location of the mutation. [Figure 4]Figures 4A (antibody is Anti-RBD antibody CR3022) and 4B (antibody is ARG66740 [anti-SARS-CoV-2 Spike protein (RBD) antibody]) show the change in light intensity when the labeling substance is replaced with the target substance, respectively. [Figure 5] Figures 5A, 5B, and 5C show the changes in light intensity when the labeled substance is replaced with the target substance, respectively. [Figure 6] Figures 6A and 6B show the changes in light intensity when the chemically modified labeling substance is replaced by the target substance, respectively. [Figure 7] Figures 7A, 7B, and 7C show specific examples of inspection areas for test specimens. [Figure 8] Figure 8 shows a specific example in which an inspection area and a control area are arranged on a test specimen. [Figure 9] 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 labeled substance is replaced with the target substance, respectively, and compare the performance of the machine learning engines. Figure 9E shows the test results. [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 following embodiments. It is not something that can be done.

[0010] [Test specimen for detecting the target substance] The present invention comprises a carrier and a composite arranged in a test area on the carrier, in a sample Regarding test specimens for detecting target substances. The complex consists of a binding partner and a binding partner. It consists of a combined labeled substance and The labeled substance dissociates from its binding partner faster than the target substance. The bonding force between a substance and its bonding partner is greater than the bonding force between the target substance and its bonding partner. weak. Because of this configuration, the target substance replaces the labeled substance, and the binding partner The target substance can be detected by observing the color change in the test area when it binds to the target substance.

[0011] [Competitive Substitution Law] Figures 1A and 1C illustrate the principle of the competitive substitution method, which is the solution principle of the present invention. Figure 1A The competitive substitution method will be explained using Figure 1C. Here, a membrane will be used as an example of the carrier. I will list and explain. As shown in Figure 1A, the membrane 5 of the test specimen 10 has bonded parts in a predetermined inspection area. A composite consisting of toner 1 and labeled substance 2, in which substance 2a is labeled with labeled substance 2b, is fixed. The labeling substance 2 dissociates from its 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 piece 10, As shown in 1C, substitution occurs between the labeled substance 2 and the target substance 4. In other words, the target substance is in the sample. If present, the light intensity decreases and the color of the inspection area becomes lighter; if not present, the light intensity changes. Since there is no chemical reaction, the color of the inspection area remains dark. When substitution occurs between labeling substance 2 and target substance 4. The target substance 4 can be detected by the change in color of the inspection area. Here, the color of the inspection area These changes include variations in color intensity, brightness, and saturation.

[0012] [complex] As described above, the complex consists of a binding partner and a labeling substance. The binding partner is a labeling substance and / or target substance that can be fixed onto the carrier. Various materials can be used without particular restrictions, as long as they are compatible with the quality. Combination partners For example, antibodies can be used when the labeled substance and the target substance are used as antigens. The labeled substance is labeled with a marker. The marker is labeled with its binding partner. Various substances are available without particular limitation, as long as they do not inhibit the binding of the substance and / or the target substance. Existing materials used in the analysis of biological samples can be used as markers. For example, gold colloid particles, colored latex particles, metal complexes, fluorescent substances, and aggregated luminescent materials. Chemiluminescent substances, electrochemiluminescent substances, etc., can be used. To make the color changes that occur when a color is changed, such as changes in shade, brightness, or saturation, easier to see. This is because it is possible to do so. It is preferable to use a labeling substance with high light intensity. The labeling substance is bonded to the binding substance by various methods so that it can be replaced by the target substance. It is preferable that the bonding force is weakened. Methods for reducing the bonding force include, for example, the target substance One method involves mutating specific positions in the amino acid sequence. Figure 3 shows the target substance as the receptor-binding region of the novel coronavirus (SARS-COVID-19). The amino acid sequence of the Binding Domain (hereinafter also referred to as "RBD") and its binding ability. This diagram shows the positions that are thought to affect the decrease. From the amino acid sequence, antibodies (e.g., Anti - Identify the site where the binding ability of RBD to the RBD antibody CR3022 (commercial product) is expected to decrease. By preparing RBD mutants (K378N, V382E, R346S), the binding ability is reduced and the dissociation rate is It is possible to prepare a substance that is expected to increase in quantity. One method to reduce the bonding strength is to chemically modify the target substance. Examples of chemical modifications include reductive methylation of lysine and methylation by N-acetylimidazole. Examples include acetylation of rosine, acetylation by sulfo-NHS acetate, and succinylation. This can be achieved. In particular, acetylation of tyrosine by N-acetylimidazole, succinyl Modification is preferable. We have explained amino acid mutations and chemical modifications as methods to reduce binding strength, but You can also combine them.

[0013] [Specimen] There are no particular restrictions on the target substance to be detected in the test specimen; various substances can be detected. It is possible to do this. Target substances include proteins, viruses, pathogens, etc. Target substances include substances found in microorganisms and viruses. Examples of target substances include: For example, the novel coronavirus (SARS-COVID-19) or its variants, and the influenza virus. Examples include the SARS virus. The form of the sample can include the subject's bodily fluids or breath. Using breath as a sample... Therefore, subjects can more easily determine the presence or absence of the target substance than before. Body fluids include not only blood, lymph, tissue fluid, and coelomic fluid in the narrow sense, but also digestive fluids ( A broad definition including saliva, gastric juice, bile, pancreatic juice, intestinal juice, sweat, tears, nasal mucus, urine, semen, vaginal fluid, amniotic fluid, and breast milk. It also includes bodily fluids. In addition, it includes fluids that have been detached from the cell surface in conjunction with swab tests taken from the nasal cavity or oral cavity. Solutions containing cell fragments are also included in body fluids.

[0014] Various studies are progressing on the novel coronavirus. For example, studies are underway to determine if the novel coronavirus is present in human exhaled breath. There are reports of the presence of coronavirus (Reference 1: High infectiousness immedi ately before COVID-19 symptom onset highlights the importance of continued conta ct 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). Also, there have been cases of coronavirus detection through exhaled breath. There are reports such as (Reference 3: Wearable materials with embedded synthetic biology) sensors for biomolecule detection, Nature Biotechnology 39, pages 1366-1374 (20 21) https: / / doi.org / 10.1038 / s41587-021-00950-3). Furthermore, research into transmission routes has progressed, and currently, it is understood that the virus is transmitted from infected individuals (including asymptomatic carriers). Virus-containing droplets and aerosols (from droplets) are expelled when coughing, sneezing, talking, etc. Furthermore, inhalation of even smaller, moisture-containing particles is now considered to be the primary route of infection. (Reference 4: "Guidelines for the Treatment of Novel Coronavirus Infection (COVID-19)" Section 8.1) (See "Ministry of Health, Labour and Welfare, Pathogens and Immunology, p. 6"). Thus, since exhaled breath is an important route of infection and subjects are easy to test, From a physical standpoint, it is preferable to use exhaled air.

[0015] The binding partner can be an antibody, and the labeling substance can be an antigen labeled with the labeling substance. The antigen is the receptor binding region of the novel coronavirus (SARS-COVID-19). Domain) (hereinafter also referred to as "RBD"), and the antibody shall be Anti-RBD antibody CR3022 (commercial product). It is possible.

[0016] [Carrier] There are no particular restrictions on the carrier material, and various materials can be used, but as an example, A membrane can be mentioned. There are no particular restrictions on the membrane, and it can be used for the analysis of biological samples. Existing materials such as nitrocellulose and PVDF membranes can be used. .

[0017] [Examination Area] On the surface of the carrier, an inspection area is formed where the complex is fixed via a binding partner. It is. Figure 2 shows the color development state of the labeling substance of the complex placed on the membrane as a carrier. This is a diagram. As detailed in the Examples section, a binding partner (antibody) is attached to the surface of the membrane. The process involves fixing the substance in a spot-like manner, and then spreading the labeled substance (labeled antigen) onto the membrane. Then, a spot-like inspection area is formed where the complex is fixed. There is no particular limit to the number of test areas, but multiple test areas can be arranged on the membrane. This is preferable. By arranging multiple inspection areas, the opportunity for contact with the target substance is increased. This is because it is expected to improve accuracy. Also, there may be analytical deficiencies in one of the testing areas. Even if such a condition exists, other areas of testing can compensate for it. There are no particular restrictions on the arrangement pattern of the inspection area; various arrangement patterns can be used. It is possible to have multiple test areas at equal intervals in both the vertical and horizontal directions of the membrane. Examples include those that define a zone. Figures 7A, 7B, and 7C show specific examples of the inspection area of ​​the test specimen. Examples include A with 5 rows and 5 columns, Figure 7B with 5 rows and 3 columns, and Figure 7C with 6 rows and 6 columns. Thus, the effect of being able to make a 100% judgment is obtained. According to Figure 7B, the number of test areas ( Even when the area is reduced, the same effect as in Figure 7A can be obtained. As shown in Figure 7C, Even when setting an inspection area without gaps, the same effects as in Figure 7A can be obtained. Multiple types of target substances can be detected, with multiple testing areas arranged for each type of target substance. This is acceptable because it allows for the detection of the presence of multiple substances in a single test. Figure 8 shows a specific example of a test specimen with an inspection area and a control area. On the plane, in addition to the test area, there is a control area adjacent to the test area where a labeled substance is fixed. They may be arranged as shown in Figure 8, the inspection area (changing spot) and The control area (unchanging spot) may be arranged in parallel. Image acquisition By reducing the influence of the (photography) environment and other factors, and by making a relative comparison between the inspection area and the control area, This is because it has the effect of improving the accuracy of the judgment.

[0018] [Examples of test specimen applications (other embodiments)] As described above, the present invention has been described by embodiments, but the discussion and which form part of this disclosure The drawings should not be understood as limiting this invention. From this disclosure, those skilled in the art will understand... Various alternative embodiments, examples, and operational techniques will become apparent. Figures 12A and 12B show one aspect of the use of the test specimen (mask). The above-mentioned test The piece may be used as the test specimen itself, but for example, as shown in Figure 12A, 85a and 85b are fixed to the holder 83, and the holder is removable as shown in Figure 12B. It may also be used by attaching it to the back side of the mask 81 (the side that touches the skin). As shown in Figure 12B, The test pieces 85a and 85b may be directly fixed to the mask 81. The target substance (e.g.) is present in the subject's exhaled breath. For example, if it contains the novel coronavirus, it is shown in the enlarged view of test specimen 85a in Figure 12C. Is it possible to easily detect the presence of the target substance from the change in light intensity in the inspection area 85a2? Specifically, the color intensity (light intensity) of the control area 85a1 and the test area 85a2 are compared. If the color of the test area 85a2 is lighter than the color of the control area 85a1, the result is positive. If the test is not performed, the result can be considered negative.

[0019] Figure 15 shows one way of using the test specimen (test kit). In addition to the above, for example... As shown in Figure 15A, the ends of the strip-shaped test piece are the test areas 91, 92, 93, 94, 9 5 may be used as the formed test kit 9. Test areas 91-95 of test kit 9 The presence of the target substance can be easily determined by applying the subject's saliva or blowing their breath onto it. This is because it is possible. Here, SARS-COVID-19 testing area 91, SARS testing area 9 2. Influenza Soviet type testing area 93, Influenza Hong Kong type testing area 94, Others The presence of the virus testing area 95 allows for the simultaneous determination of the presence or absence of multiple target substances. It can be determined.

[0020] Thus, the present invention naturally includes various embodiments not described herein. Yes. Therefore, the technical scope of the present invention is reasonable from the above description and relates to the claims. It is determined solely by clearly specified matters.

[0021] [Analysis method A] Next, we will explain the analytical method using the test specimens described above. Figure 16A shows the inspection method. This is a drawing. (i) First, a complex consisting of a binding partner and a labeled substance bound to the binding partner. Prepare a test specimen equipped with (S101). For example, an inspection kit equipped with a test specimen like the one in Figure 15A Prepare the box. (b) Next, a subject's sample that may contain the target substance in the test specimen is sensitized. Alternatively, apply saliva to the test specimen or blow exhaled air onto it (S103). (h) As shown in Figure 15C, the contrast in color intensity (light intensity) between the control area 91a1 and the test area 91a2. The degree of color is compared, and 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 intensity, it is judged as negative (S110). Therefore, the target substance can be detected from the change in light intensity.

[0022] In S110 described above, if it is difficult to determine the shade of color, the presence or absence of the target substance is determined. You may also use an AI that has been trained through machine learning to make a decision. Below is an analysis method using AI (system This section will explain (M). For convenience, the explanation will be divided into user-side and server-side sections. [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) Run the application installed on the user terminal and obtain the image data. Transfer 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 (server side) of the inspection system. The inspection system is a transmission and reception system. It consists of a server equipped with a signal unit, a storage unit (database), an output unit, and an arithmetic control unit. (Not shown in the diagram). These configurations include, as hardware, any computer It is possible to use the CPU, GPU, memory, and programs loaded into memory. It is possible. The memory unit has pre-stored known image data associated with whether the result is positive or negative. The server and user terminals are connected by communication methods such as the internet or intranet. It is done by running the application installed on the user terminal. It is possible to exchange information between the server and the system. (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) Compare the image data with known image data and determine whether the result is positive or negative based on the difference in color intensity of the test area. Determine the sex (S205). (ii) Associate the judgment result with the image data and save it 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 limited to the following. This is not limited to the manufacturing and testing examples provided.

[0025] [Test Example 1] Preparation of antibody-loaded membrane The target substance is the receptor binding region of the novel coronavirus (SARS-COVID-19). (R&D Domain) (hereinafter also referred to as "RBD"), with gold colloid as a label (Jackson ImmunoResearch) We prepared a product from ch company, product name "40 nm Colloidal Gold Streptavidin". Then, we applied it to RBD. A gold colloid label was attached to obtain a labeled substance (gold colloid-labeled RBD). The carrier is FF170HP Plus Thick (manufactured by Cytiva), and the antibody is CR3022 (manufactured by Abcam, CR Antibody 3022 (anti-RBD antibody) was prepared. The antibody was then conjugated in a spot-like manner onto nitrocellulose. This process was used to obtain an antibody-supported membrane. As shown in Figure 2, the labeling substance is poured so that it spreads across the entire surface of the antibody-supported membrane. They were mixed and incubated. During incubation, the spots on the antibody-carrying membrane where the antibody is loaded (hereinafter referred to as "spots") It was confirmed that the color of the "pot portion" (also called the "pot part") increased during incubation. As a result, the labeling substance accumulates in the antibody-supported spot area, increasing the density of the spot area. It is thought that the amount has increased. After incubation, the samples were washed with PBST (PBS containing 0.05% Tween-20). Therefore, when a labeling substance binds to an antibody, a complex consisting of the antibody and the labeling substance (anti-RBD antibody) is formed. It was confirmed that a gold colloid-labeled RBD complex was formed.

[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 positions where the amino acid sequence has been mutated. The location to be mutated is described in the technical literature (Yi et al. Genome Medicine (2021) 13:164, Fig.4). (Reference) was used as a reference. And, RB against antibody (Anti-RBD antibody CR3022 (commercial product)) RBD mutants (K378N, V382E, R346S) with reduced D binding ability were prepared, and cell-free protein Proteins are prepared by a synthesis method, and the dissociation rate is increased due to a decrease in binding affinity to antibodies. I considered promoting him.

[0027] [Test Example 3] Confirmation of antigens with a fast dissociation rate Figures 4A and 4B show the results when the RBD mutant (labeling substance) is replaced with RBD (target substance), respectively. This figure shows the change in light intensity at the time. Figure 4A shows Anti-RBD antibody CR3022 (market price). (Commercial product), Figure 4B shows the use of ARG66740 (commercial product, manufactured by Arigo Biolaboratories) as the antibody. . Figure 4A shows that the difference is significant for RBD(K378N) and RBD(V382E), and 35% for RBD(R346R). A decrease in binding affinity to the antibody (anti-RBD antibody CR3022) was observed to some extent.

[0028] [Test Example 4] Confirmation of substitution by the antigen to be detected Figures 5A, 5B, and 5C show the light intensity when the labeled substance is replaced with the target substance, respectively. This is a diagram showing the changes. The trimer of the spike protein is reacted to the antibody-antigen complex. By responding accordingly, RBD(K378N) and RBD(V382E) will be replaced by RBD(WT) in 2 hours. The results showed a percentage of approximately 10-20%.

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

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

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

[0032] Figure 11A is an image of the actual inspection area, and Figure 11B shows the actual inspection area with the target substance introduced. These are images of the positive result at that time. Figures 11C and 11D show the result when the labeled substance is replaced with the target substance. This figure shows the change in light intensity. Figure 11E shows the inspection results. [Industrial applicability]

[0033] According to the present invention, a highly reliable, inexpensive, and easy-to-use testing method is provided. According to the present invention, even if the sample is breath, it can be tested easily and with high sensitivity. Furthermore, according to the present invention, The test can detect the presence of various target substances without being limited to the novel coronavirus. The present invention provides a test specimen, an inspection kit and a mask, and a test kit, by using the principle of the present invention. This can be used in inspection methods, such as those using AI.

Claims

1. A device comprising a carrier and a composite arranged in a testing area on the carrier, for the target substance in a sample A test specimen for detecting quality, The complex comprises a binding partner and a labeled substance bound to the binding partner. It became, The labeled substance dissociates from its binding partner at a faster rate than the target substance. From the change in color of the inspection area when the target substance is replaced by the labeling substance, the target A test specimen used to detect a target substance.

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

3. Claim 2, wherein the labeling substance is a substance that has altered the amino acid sequence of the target protein. The test specimen described.

4. Claim 2 states that the labeling substance is obtained by chemically modifying the target protein. The test specimen.

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

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

7. The labeling substance may be gold colloid particles, colored latex particles, metal complexes, fluorescent substances, or agglomerating substances. Labeled with a marker selected from the group consisting of luminescent materials, chemiluminescent materials, and electrochemiluminescent materials. The test specimen described in claim 1.

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

9. Multiple types of target substances can be detected, with multiple testing areas arranged for each type of target substance. The test specimen according to claim 1.

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.

17. It consists of a binding partner and a labeled substance bound to the binding partner, and the label The substance prepares a composite in which the dissociation rate from the binding partner is faster than that of the target substance. The process and A step of sensitizing the composite with a sample that may contain the target substance, Image data where the target substance may have replaced the labeling substance, potentially causing a change in light intensity. The process of obtaining data, The process includes a determination step of determining whether or not the target substance is present based on the image data. An analytical method for detecting a target substance in a sample.

18. A binding partner and a label bonded to the binding partner with a weaker bonding force than that of the target substance. When the labeling substance of a composite consisting of a substance is replaced by the target substance, the light intensity changes. The process of receiving potentially generated image data, Retrieve known image data stored in association with positive or negative results from the database. Then, by comparing the image data with the known image data, the presence or absence of the target substance can be determined. The decision-making process, Based on the judgment result, the image data is associated with whether it is positive or negative. The process involves saving the data as new known image data in the database, The process of transmitting the aforementioned determination result to the user terminal, An analytical system for detecting a target substance in a sample containing the following characteristics.

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