Method for analyzing molecule-molecule interaction and device for detecting interaction-interfering substance using same

Abstract

The present invention relates to a method for analyzing a molecule-molecule interaction and a device for detecting substances that interfere with such interactions. For example, the present invention relates to a method for analyzing protein–protein interactions, more specifically the interaction between the spike protein of SARS-CoV-2 and its receptor, human ACE2 protein, and a device capable of detecting neutralizing antibodies generated by vaccination on the basis of the analysis.

Description

Method for analyzing molecule-molecule interactions and device for detecting substances interfering with the interactions using the same

[0001] The present invention relates to a method for analyzing molecule-molecule interactions and a device for detecting substances that interfere with such interactions using the same, and for example, to a method for analyzing protein-protein interactions, more specifically, the interaction between the spike protein of SARS-CoV-2 and its receptor, human ACE2 protein, and a device capable of detecting neutralizing antibodies induced by vaccination based on the same.

[0002] The degree of immunity acquired through the formation of neutralizing antibodies following vaccination varies from individual to individual, and the duration of immunity also varies significantly. Breakthrough infections occur due to viral mutations and a decrease in the amount of neutralizing antibodies formed in the body over time, which requires management. Furthermore, when utilizing novel vaccine platforms developed in a short period of time, such as for COVID-19, concerns about side effects may arise, leading to hesitancy toward additional vaccinations. Therefore, the development of methods to specifically detect neutralizing antibodies present in the body is essential for assessing vaccine efficacy, determining whether immunity has been formed after vaccination, and determining the timing of additional vaccinations through analysis of the duration of immunity.

[0003] Looking at existing neutralizing antibody detection technologies, first, there is a cell-based in vitro assay method that analyzes the degree to which the virus infects the host cell by treating the host cell with the target serum (neutralizing antibody) and the actual virus. This method allows for accurate neutralizing antibody detection and neutralizing activity analysis, but it requires an analysis time of more than 24 hours and requires specialized facilities (biosafety level 3) and specialized equipment because it uses a virus (due to infection risk issues).

[0004] Next, there is a competitive ELISA method that analyzes the degree to which the binding between the virus's outer membrane protein (e.g., spike) and the host cell's receptor (e.g., ACE2), which is the mechanism of action of neutralizing antibodies, is inhibited. It can analyze the presence of actual neutralizing antibodies among the antibodies formed after vaccination on a large scale, so it has been approved for emergency use by the FDA, and it has the advantage of being safer than cell-based experiments and requiring a short analysis time (within 3 hours), but it has the problem of requiring large-scale automated equipment and skilled professional personnel.

[0005] In addition, we can consider the method of the COVID-19 rapid diagnostic kits (RDTs) currently approved and sold, but in these cases, they are only used to check for immune responses or infection after vaccination by detecting IgG / IgM antibodies, and it is impossible to determine and detect actual neutralizing antibodies.

[0006] Recently, research has been reported on the development of a rapid neutralizing antibody diagnostic kit that applies the competitive ELISA principle to a rapid diagnostic platform. A typical method produces a positive signal in the absence of neutralizing antibodies and a weakening signal depending on the amount of neutralizing antibodies present. However, this method, in stark contrast to the detection method of existing rapid diagnostic kits, only provides accurate results when neutralizing antibody titers are high, and quantitative analysis of neutralizing antibodies is also impossible. These critical shortcomings have prevented commercialization after development or limited practical use.

[0007] Therefore, the inventors of the present invention sought to develop a new technology that can overcome the problems of the existing technology as described above, and can analyze interactions between molecules and detect neutralizing antibodies quickly, accurately, safely, and easily.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Korean Patent Publication No. 10-2023-0005288

[0011] The main purpose of the present invention is to provide a method and device capable of analyzing interactions between molecules.

[0012] In addition, the present invention provides a method and device that can detect neutralizing antibodies quickly, accurately, safely, and easily.

[0013] According to one aspect of the present invention, the present invention is a method for analyzing the interaction between a first molecule and a second molecule,

[0014] A molecule-molecule contact step of bringing a first molecule labeled with a first labeling substance into contact with a second molecule labeled with a second labeling substance;

[0015] A first detection unit contact step of bringing the resultant product that has gone through the above molecule-molecule contact step into contact with a first detection unit, wherein a first capturing body that captures the first molecule is fixed to the first detection unit;

[0016] A second detection unit contact step in which the resultant product that has gone through the above molecule-molecule contact step is brought into contact with a second detection unit, wherein a second capturing body that captures the second molecule is fixed to the second detection unit; and

[0017] A third detection unit contact step of bringing the resultant product that has gone through the above molecule-molecule contact step into contact with a third detection unit, wherein the first capture body and the second capture body are fixed to the third detection unit;

[0018] A method is provided wherein the first label material and the second label material are materials exhibiting different colors.

[0019] In the method of the present invention, the interaction between the first molecule and the second molecule may be a protein-protein interaction.

[0020] In the method of the present invention, a sample contact step may be further included prior to the molecule-molecule contact step, wherein the sample is brought into contact with at least one selected from the first molecule and the second molecule.

[0021] The method of the present invention may be for detecting a substance that interferes with the interaction between the first molecule and the second molecule in the sample by analyzing the interaction between the first molecule and the second molecule.

[0022] In the method of the present invention, the substance that interferes with the interaction between the first molecule and the second molecule may be a compound, protein, peptide or aptamer that inhibits protein-protein binding.

[0023] In the method of the present invention, the first molecule and the second molecule may include a capture mediator that mediates the capture.

[0024] In the method of the present invention, the first labeling material and the second labeling material may be colored beads or fluorescent molecules.

[0025] In the method of the present invention, the first capture body and the second capture body may be antibodies or aptamers.

[0026] In the method of the present invention, the molar ratio of the first capture body and the second capture body of the third detection unit may be 1:3 to 3:1.

[0027] The method of the present invention may be a lateral flow-based method.

[0028] According to another aspect of the present invention, the present invention is a lateral flow-based device for detecting a substance that interferes with the interaction between a first molecule and a second molecule,

[0029] A sample introduction section for receiving a sample;

[0030] A sample expansion means for expanding a sample from the sample introduction section;

[0031] A first contact portion located in a path through which a sample is developed from the sample introduction portion, and containing a first molecule labeled with a first labeling material, so that the sample comes into contact with the first molecule;

[0032] A second contact portion located in a path following the first contact portion in the path through which the sample is developed from the sample introduction portion, and containing a second molecule labeled with a second labeling material, such that the first molecule in contact with the sample contacts the second molecule;

[0033] A first detection unit located in a path following the second contact unit in the path through which the sample is developed from the sample introduction unit, and having a first capturing body for capturing the first molecule fixed thereon;

[0034] A second detection unit, located at a location spaced apart from the first detection unit in the path following the second contact unit in the path through which the sample is developed from the sample introduction unit, and having a second capturing body for capturing the second molecule fixed thereto; and

[0035] A third detection unit is located at a distance from the first detection unit and the second detection unit in a path following the second contact unit in which a sample is developed from the sample introduction unit, and the first capture body and the second capture body are fixed thereto;

[0036] A device is provided wherein the first label material and the second label material are materials exhibiting different colors.

[0037] In the device of the present invention, the interaction between the first molecule and the second molecule may be a protein-protein interaction.

[0038] In the device of the present invention, the substance that interferes with the interaction between the first molecule and the second molecule may be a compound, protein, peptide or aptamer that inhibits protein-protein binding.

[0039] In the device of the present invention, the first molecule and the second molecule may include a capture mediator that mediates the capture.

[0040] In the device of the present invention, the first labeling material and the second labeling material may be colored beads or fluorescent molecules.

[0041] In the device of the present invention, the first capture body and the second capture body may be antibodies or aptamers.

[0042] In the device of the present invention, the molar ratio of the first capture body and the second capture body of the third detection unit may be 1:3 to 3:1.

[0043] According to the present invention, molecule-molecule interactions, for example, protein-protein interactions, can be analyzed quickly, accurately, safely, and easily.

[0044] As a more specific example, neutralizing antibodies that interfere with protein-protein interactions can also be detected rapidly, accurately, safely, and easily.

[0045] And through these effects, it is possible to quickly, accurately, safely, and easily determine whether an individual has developed immunity after vaccination and whether an individual needs additional vaccination, thereby minimizing vaccine side effects and reducing costs by preventing unnecessary additional vaccinations.

[0046] In addition, the present invention has the advantage of being usable as a simple platform for diagnosing neutralizing antibodies for various infectious diseases, including COVID-19, influenza, and viral hepatitis, as well as for analyzing various other protein-protein interactions, as a universal platform.

[0047] Figure 1 shows exemplary results of a conventional method based on the clarity of a positive signal (left) and a method of the present invention based on color change (right).

[0048] Figure 2 illustrates a device (lateral flow immunoassay strip sensor) according to one embodiment of the present invention that can implement the method of the present invention.

[0049] Figure 3 illustrates a device according to another embodiment capable of implementing the method of the present invention and its application method.

[0050] Figure 4 illustrates the operation of a device (lateral flow immunoassay strip sensor) according to one embodiment of the present invention capable of implementing the method of the present invention. Upper part, when the first molecule and the second molecule interact (bind); Lower part, when the first molecule and the second molecule do not interact (bind) due to a PPI (protein-protein interaction) inhibitor.

[0051] Figure 5 illustrates the operation of a device (lateral flow immunoassay strip sensor) according to another embodiment of the present invention capable of implementing the method of the present invention. Upper section, when the first molecule and the second molecule interact (bind); Lower section, when the first molecule and the second molecule do not interact (bind) due to a neutralizing antibody.

[0052] Figure 6 illustrates an implementation example according to another embodiment of the present invention, showing results when the first and second labeling materials are green fluorescent substances and red fluorescent substances, respectively. Upper part, when the first and second molecules interact (bind); lower part, when the first and second molecules do not interact (bind) due to a neutralizing antibody.

[0053] Figures 7 and 8 illustrate the results of experiments to determine whether the method and device of the present invention are operable. Spike, SARS-CoV-2 Spike-RBD-mFc-blue CNB conjugate (first molecule labeled with a first label); ACE2, ACE2-rFC-red CNB conjugate (second molecule labeled with a second label); Unit, ng / ml.

[0054] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0055] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0056] Terms such as first, second, etc. may be used to describe various components, but these components should not be limited by these terms. These terms are used solely to distinguish one component from another.

[0057] The present invention relates to a method for analyzing an interaction between a first molecule and a second molecule, comprising: a molecule-molecule contact step of contacting a first molecule labeled with a first labeling substance with a second molecule labeled with a second labeling substance; a first detection unit contact step of contacting a resultant of the molecule-molecule contact step with a first detection unit, wherein a first capture agent for capturing the first molecule is immobilized on the first detection unit; a second detection unit contact step of contacting a resultant of the molecule-molecule contact step with a second detection unit, wherein a second capture agent for capturing the second molecule is immobilized on the second detection unit; And a third detection unit contact step of contacting the resultant product that has gone through the molecule-molecule contact step with a third detection unit, wherein the first capture body and the second capture body are fixed to the third detection unit; The method relates to a method in which the first labeling material and the second labeling material are materials that exhibit different colors when themselves or a molecule labeled therewith is captured by the relevant capture body.

[0058] In addition, the present invention is a lateral flow-based device for detecting a substance that interferes with the interaction between a first molecule and a second molecule, comprising: a sample introduction unit for receiving a sample; a sample development means for developing a sample from the sample introduction unit; a first contact unit located in a path along which a sample is developed from the sample introduction unit, the first contact unit including a first molecule labeled with a first labeling substance, such that the sample comes into contact with the first molecule; a second contact unit located in a path following the first contact unit in a path along which a sample is developed from the sample introduction unit, the second contact unit including a second molecule labeled with a second labeling substance, such that the first molecule that has come into contact with the sample comes into contact with the second molecule; a first detection unit located in a path following the second contact unit in a path along which a sample is developed from the sample introduction unit, the first capture body for capturing the first molecule being fixed thereto; The present invention relates to a device comprising: a second detection unit, which is located at a location spaced from the first detection unit in a path following the second contact unit in a path through which a sample is developed from the sample introduction unit, and to which a second capture body for capturing the second molecule is fixed; and a third detection unit, which is located at a location spaced from the first detection unit and the second detection unit in a path following the second contact unit in a path through which a sample is developed from the sample introduction unit, and to which the first capture body and the second capture body are fixed; wherein the first labeling material and the second labeling material are materials exhibiting different colors.

[0059] The present invention is characterized in that the first labeling material and the second labeling material exhibit different colors, and thus can analyze the interaction between molecules based on the color change. Compared to existing technologies based on the clarity of positive signals (e.g., technologies for confirming the presence or absence of neutralizing antibodies that interfere with binding between two proteins through weakening of the intensity of positive signals), more clear analysis is possible (see Fig. 1).

[0060] In the present invention, the first molecule and the second molecule may each be a compound, and may be a biological molecule, such as a bacterium, a virus, a protein, or a nucleic acid. In one embodiment, the first molecule and the second molecule are proteins that bind to each other.

[0061] In the present invention, the first molecule and the second molecule may include a substance for mediating capture by the first capture body or the second capture body, such as a capture mediator protein. Such labeling substances may include, but are not limited to, tagging proteins such as mouse or rabbit-derived antibody fragments (e.g., mFc, rFc, etc.), hemagglutinin (HA), MYC protein (e.g., c-Myc), Flag, 3xFlag, 6xHis, Strep, etc.

[0062] In the present invention, the interaction between the first molecule and the second molecule may be a protein-protein interaction (PPI), i.e., a phenomenon in which one protein binds to another protein, for example, an interaction between a viral protein and a host cell receptor protein. In one embodiment, the interaction between the first molecule and the second molecule is an interaction between the spike receptor binding domain (RBD) of SARS-CoV-2 and human ACE2 (angiotensin-converting enzyme-2).

[0063] In the present invention, the labeling substances that label the first molecule or the second molecule, i.e., the first labeling substance and the second labeling substance, are characterized in that they are substances that exhibit different colors. The first labeling substance and the second labeling substance of the present invention may be, for example, labeling substances commonly used in the field of biosensors, and may be, for example, color beads or fluorophores that exhibit different colors. The first labeling substance and the second labeling substance of the present invention may exhibit a unique color by themselves or when a molecule labeled with them is captured by a related capture body. The first labeling substance and the second labeling substance of the present invention may exhibit a unique color always or temporarily. In addition, when the first labeling substance and the second labeling substance exist together, they may exhibit a third different color that is different from their respective unique colors.

[0064] A molecule labeled with a labeling agent may have the labeling agent directly or indirectly linked to the molecule. The linkage between the labeling agent and the molecule may be, for example, using conventional techniques used to link labeling agents to proteins. Furthermore, the indirect linkage may be via a linker, for example.

[0065] In the present invention, the first detection unit is characterized in that the first capture body is fixed thereto. The first capture body can capture the first molecule, and may be, for example, an antibody or an antigen-binding fragment thereof for the first molecule, or may be an aptamer for the first molecule. For example, when the first molecule comes into contact with the first detection unit and is captured by the first capture body, the first detection unit exhibits a unique color of the first labeling substance labeled with the first molecule, and thus, whether the first molecule has been captured by the first detection unit can be determined through this color. If the first molecule and the second molecule are bound in the molecule-molecule contact step, the first detection unit captures not only the first molecule but also the second molecule bound thereto, and in this case, the first detection unit exhibits not only the color of the first labeling substance labeled with the first molecule but also the color of the second labeling substance labeled with the second molecule.

[0066] In the present invention, the second detection unit is characterized in that the second capture body is fixed thereto. The second capture body can capture the second molecule, and may be, for example, an antibody or an antigen-binding fragment thereof for the second molecule, or may be an aptamer for the second molecule. For example, when the second molecule comes into contact with the second detection unit and is captured by the second capture body, the second detection unit exhibits a unique color of the second labeling substance labeled with the second molecule, and thus, whether the second molecule has been captured by the second detection unit can be determined through this color. If the first molecule and the second molecule are bound in the molecule-molecule contact step, the second detection unit captures not only the second molecule but also the first molecule bound thereto, and in this case, the second detection unit exhibits not only the color of the second labeling substance labeled with the second molecule but also the color of the first labeling substance labeled with the first molecule.

[0067] In the present invention, the third detection unit is characterized in that the first capture body and the second capture body are fixed thereto. At this time, the first capture body and the second capture body are as described above. Since both the first capture body and the second capture body are fixed to the third detection unit, both the first molecule and the second molecule can be captured regardless of whether the first molecule and the second molecule are bound. In one embodiment, the mole ratio of the first capture body and the second capture body of the third detection unit is 1:10 to 10:1, 1:5 to 5:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.

[0068] The present invention may further include a sample contact step prior to the molecule-molecule contact step, wherein the sample is brought into contact with at least one selected from among a first molecule and a second molecule. This step may be, for example, an additional step for detecting a substance in the sample that interferes with the first molecule-second molecule interaction or a substance that induces or assists the interaction.

[0069] In the present invention, the sample may be a sample for detecting a substance that interferes with the first molecule-second molecule interaction, or a substance that induces or assists the interaction, or a sample for confirming the presence or concentration of the substance. Furthermore, the sample of the present invention may be a biological sample, such as a tissue, cell, body fluid, or blood sample isolated from a subject, or may be a non-biological sample.

[0070] In the present invention, the substance that interferes with the interaction between the first molecule and the second molecule may be a substance that inhibits the binding of proteins to proteins, and such substances may include, but are not limited to, compounds, proteins (e.g., antibodies), peptides, aptamers, etc. In one embodiment, the substance that interferes with the interaction between the first molecule and the second molecule is a neutralizing antibody, for example, a neutralizing antibody that interferes with the binding between an outer membrane protein (e.g., spike protein) of a virus (e.g., SARS-CoV-2) and a receptor (e.g., ACE2) of a host cell (e.g., human).

[0071] The present invention will be described in more detail based on the lateral flow method, which is an exemplary implementation method of the present invention. This can also serve as an explanation of the operating method of the present invention's device.

[0072] FIG. 2 illustrates a device according to one embodiment of the present invention, comprising: a sample introduction unit (11) for receiving a sample; a sample development means (17) for developing a sample from the sample introduction unit (11); a first contact unit (12) located in a path along which a sample is developed from the sample introduction unit (11), the first contact unit including a first molecule labeled with a first labeling substance, such that the sample comes into contact with the first molecule; a second contact unit (13) located in a path following the first contact unit (12) in a path along which a sample is developed from the sample introduction unit (11), the second contact unit including a second molecule labeled with a second labeling substance, such that the first molecule that has come into contact with the sample comes into contact with the second molecule; a first detection unit (14) located in a path following the second contact unit (13) in a path along which a sample is developed from the sample introduction unit (11), the first capture body for capturing the first molecule being fixed thereto; It comprises a second detection unit (15) located at a location spaced from the first detection unit (14) in a path following the second contact unit (13) in a path along which a sample is developed from the sample introduction unit (11), and to which a second capture body for capturing the second molecule is fixed; and a third detection unit (16) located at a location spaced from the first detection unit (14) and the second detection unit (15) in a path following the second contact unit (13) in a path along which a sample is developed from the sample introduction unit (11), and to which the first capture body and the second capture body are fixed.

[0073] When a sample is introduced into the sample introduction unit (11), the sample passes through the first contact unit (12) by the sample spreading means (17) and reaches the second contact unit (13) in a state containing the first molecule, thereby allowing contact between the first molecule and the second molecule (molecule-molecule contact step). Meanwhile, the molecule-molecule contact step of the present invention may not be performed on the above-described device, but may be performed in a separate location, for example, in a separate container (for example, a test tube) (see FIG. 3).

[0074] Next, by allowing the sample (containing the first molecule and the second molecule) to reach the first detection unit (14) by the sample spreading means (17), the resultant product that has gone through the molecule-molecule contact step can come into contact with the first detection unit (14) (first detection unit contact step). If the first molecule and the second molecule are combined in the preceding process (molecule-molecule contact step), the first molecule is captured by the first capture body at the same time as the second molecule is also captured in a form combined with the first molecule, and accordingly, the colors of both the first labeling substance and the second labeling substance are expressed (see the upper part of FIGS. 4 and 5). Conversely, if the first molecule and the second molecule are not combined in the preceding process (e.g., by a PPI inhibitor or a neutralizing antibody), only the first molecule is captured by the first capture body in the first detection unit (14), and thus only the color of the first labeling substance is expressed (see the lower part of FIGS. 4 and 5).

[0075] Next, by the sample spreading means (17), the sample passes through the first detection unit (14) and reaches the second detection unit (15), so that the resultant product that has gone through the molecule-molecule contact step can come into contact with the second detection unit (15) (second detection unit contact step). If the first molecule and the second molecule are combined in the preceding process (molecule-molecule contact step), the second molecule is captured by the second capture body at the second detection unit (15) and at the same time, the first molecule is also captured in a form combined with the second molecule, whereby the colors of both the first labeling substance and the second labeling substance are expressed (see the upper part of FIGS. 4 and 5). Conversely, if the first molecule and the second molecule are not combined in the preceding process (e.g., by a PPI inhibitor or a neutralizing antibody), only the second molecule is captured by the second capture body in the second detection unit (15), and thus only the color of the second labeling substance is expressed (see the lower part of FIGS. 4 and 5).

[0076] Next, the sample can pass through the second detection unit (15) by the sample spreading means (17) and reach the third detection unit (16), thereby bringing the resultant product that has gone through the molecule-molecule contact step into contact with the third detection unit (16) (third detection unit contact step). Since both the first capture body and the second capture body are present in the third detection unit (16), both the first molecule and the second molecule are captured regardless of whether the first molecule and the second molecule are bound, and accordingly, the colors of both the first labeling substance and the second labeling substance are expressed (see FIGS. 4 and 5).

[0077] In this way, by using the method and / or device of the present invention, it is possible to analyze the interaction between the first molecule and the second molecule and to detect a substance that interferes with the interaction between the first molecule and the second molecule, depending on the colors of the first detection unit, the second detection unit, and the third detection unit.

[0078] Meanwhile, when fluorescent materials are used as the first and second labeling materials, for example, when cy3, a green fluorescent material, is used as the first labeling material and cy5, a red fluorescent material, is used as the second labeling material, when the first molecule and the second molecule are combined, the first detection unit, the second detection unit, and the third detection unit all exhibit yellow fluorescence (see the upper part of Fig. 6), and conversely, when the first molecule and the second molecule are not combined, the first detection unit exhibits green fluorescence, the second detection unit exhibits red fluorescence, and the third detection unit exhibits yellow fluorescence (see the lower part of Fig. 6).

[0079] Hereinafter, the present invention will be described in more detail through examples. These examples are intended merely to illustrate the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

[0080] Example

[0081] As shown in Fig. 2, a lateral flow immunoassay (LFIA) strip sensor was manufactured by fixing a sample input pad (11), a first contact pad (12), a second contact pad (13), and an absorption pad (17) to a nitrocellulose (NC) membrane equipped with a first detection line (14), a second detection line (15), and a third detection line (16), and the strip was uniformly cut to 38 mm.

[0082] The first contact pad (12) contained SARS-CoV-2 spike-RBD-mFc (in which SARS-CoV-2 spike-RBD is linked to the Fc portion of a mouse) (first molecule) conjugated with blue cellulose nanobeads (CNB, Nano ActTM, Asahi Kasei, Japan) (first labeling material), and the second contact pad (13) contained ACE2-rFc (in which human ACE2 is linked to the Fc portion of a rabbit) (second molecule) conjugated with red cellulose nanobeads (CNB, Nano ActTM) (second labeling material).

[0083] Conjugation of each colored cellulose nanobead was performed as follows: Protein (0.5 mg / mL in 120 μL conjugation buffer) was mixed with colored CNB (0.5% CNB in ​​120 μL conjugation buffer) and incubated at 37°C for 2 h. Then, 7.2 mL of blocking buffer was added to block the CNB surface. After blocking at 37°C for 2 h, unconjugated CNB was removed, and the protein-CNB conjugate was washed with 7.5 mL of wash buffer by centrifugation at 14,400 g for 20 min at 4°C. Finally, the pellet was gently suspended in 300 μL of wash buffer. The concentration of the protein-CNB conjugate was measured using UV-vis spectrophotometry.

[0084] The first contact pad (12) and the second contact pad (13) were prepared in the following manner: Before fixation, each contact pad was immersed in 0.1% triton X-100 and dried at 37°C for 1 hour. The SARS-CoV-2 spike-RBD-mFc-blue CNB conjugate (the first molecule labeled with the first labeling substance) was sprayed onto the first contact pad (12), and the ACE2-rFC-red CNB conjugate (the second molecule labeled with the second labeling substance) was sprayed onto the second contact pad (13), and then each contact pad was dried at 37°C for 1 hour in a vacuum drying oven.

[0085] Nitrocellulose (NC) membranes were prepared as follows: Each detection line was dispensed onto the NC membrane using a line dispenser (BioDot Inc., Irvine, CA, USA) at a dispensing speed of 50 mm / s and 1 μl / cm. Anti-mFc antibody (50 μg / ml in sample dilution buffer) (first capture agent) was dispensed into the first detection line (14), and anti-rFc antibody (50 μg / ml in sample dilution buffer) (second capture agent) was dispensed into the second detection line (15). A mixture of anti-mFc antibody (first capture agent) and anti-rFc antibody (second capture agent) (1:1 mixture of 50 μg / ml in sample dilution buffer) was dispensed into the third detection line (16). The membrane was dried at 37°C for 1 hour, blocked with a blocking solution at 37°C for 1 hour, and then dried again in a vacuum drying oven at 37°C for 1 hour.

[0086] Experimental example

[0087] The operation of the LFIA strip sensor assembled in the above example was tested.

[0088] First, a LFIA strip sensor was manufactured by applying different concentrations of SARS-CoV-2 spike-RBD-mFc-blue CNB conjugate (first molecule labeled with a first labeling substance) and ACE2-rFC-red CNB conjugate (second molecule labeled with a second labeling substance) to the first contact pad (12) and the second contact pad (13), respectively, and then adding PBS buffer to the sample input pad (11) and checking the color of the detection line after 20 minutes.

[0089] As a result, as shown in FIG. 7, when only the SARS-CoV-2 spike-RBD-mFc-blue CNB conjugate (the first molecule labeled with the first labeling substance) was applied, blue was implemented in the second detection line (15) and the third detection line (16), when only the ACE2-rFC-red CNB conjugate (the second molecule labeled with the second labeling substance) was applied, red was implemented in the first detection line (14) and the third detection line (16), and when both the SARS-CoV-2 spike-RBD-mFc-blue CNB conjugate (the first molecule labeled with the first labeling substance) and the ACE2-rFC-red CNB conjugate (the second molecule labeled with the second labeling substance) were applied, purple was implemented in all of the first detection line (14), the second detection line (15), and the third detection line (16), confirming that it worked as expected.

[0090] In addition, a LFIA strip sensor was manufactured by applying 10 ng / mL of SARS-CoV-2 spike-RBD-mFc-blue CNB conjugate (first molecule labeled with a first labeling substance) and ACE2-rFC-red CNB conjugate (second molecule labeled with a second labeling substance) to the first contact pad (12) and the second contact pad (13), respectively, and 100 μL of neutralizing antibody was applied to the sample input pad (11) at a concentration of 0, 1, 10, or 25 ng / mL, and then the color of the detection line was confirmed after 20 minutes.

[0091] As a result, as shown in Fig. 8, when a neutralizing antibody (hNab) was not applied, purple was implemented in all of the first detection line (14), the second detection line (15), and the third detection line (16), and as the concentration of the applied neutralizing antibody increased, blue was implemented in the first detection line (14), red in the second detection line (15), and purple in the third detection line (16), confirming that it worked as expected.

[0092] Although the present invention has been illustrated and described above with respect to specific preferred embodiments, it will be apparent to those skilled in the art that the present invention may be variously modified and changed without departing from the technical features or scope of the present invention as defined by the following claims.

[0093] [Explanation of symbols]

[0094] 11: Sample introduction section (sample introduction pad)

[0095] 12: First contact (first contact pad)

[0096] 13: Second contact (second contact pad)

[0097] 14: 1st detection unit (1st detection line)

[0098] 15: Second detection unit (second detection line)

[0099] 16: Third detection unit (third detection line)

[0100] 17: Sample distribution means (absorbent pad)

Claims

1. A method for analyzing the interaction between a first molecule and a second molecule, A molecule-molecule contact step of bringing a first molecule labeled with a first labeling substance into contact with a second molecule labeled with a second labeling substance; A first detection unit contact step of bringing the resultant product that has gone through the above molecule-molecule contact step into contact with a first detection unit, wherein a first capturing body that captures the first molecule is fixed to the first detection unit; A second detection unit contact step in which the resultant product that has gone through the above molecule-molecule contact step is brought into contact with a second detection unit, wherein a second capturing body that captures the second molecule is fixed to the second detection unit; and A third detection unit contact step of bringing the resultant product that has gone through the above molecule-molecule contact step into contact with a third detection unit, wherein the first capture body and the second capture body are fixed to the third detection unit; A method wherein the first labeling material and the second labeling material are materials that exhibit different colors.

2. A method according to claim 1, wherein the interaction between the first molecule and the second molecule is a protein-protein interaction.

3. In the first paragraph, before the molecule-molecule contact step A method further comprising a sample contacting step of bringing the sample into contact with at least one selected from the first molecule and the second molecule.

4. In the third paragraph, the method is for detecting a substance that interferes with the interaction between the first molecule and the second molecule in the sample by analyzing the interaction between the first molecule and the second molecule.

5. In the fourth paragraph, the substance that interferes with the interaction between the first molecule and the second molecule is a compound, protein, peptide or aptamer that inhibits protein-protein binding.

6. A method according to claim 1, wherein the first molecule and the second molecule comprise a capture mediator that mediates the capture.

7. A method according to claim 1, wherein the first labeling material and the second labeling material are colored beads or fluorescent molecules.

8. A method according to claim 1, wherein the first capture body and the second capture body are antibodies or aptamers.

9. A method in the first paragraph, wherein the molar ratio of the first capture body and the second capture body of the third detection unit is 1:3 to 3:

1.

10. In the first paragraph, the method is a lateral flow-based method.

11. A lateral flow-based device for detecting a substance that interferes with the interaction between a first molecule and a second molecule, A sample introduction section for receiving a sample; A sample expansion means for expanding a sample from the sample introduction section; A first contact portion located in a path through which a sample is developed from the sample introduction portion, and containing a first molecule labeled with a first labeling material, so that the sample comes into contact with the first molecule; A second contact portion located in a path following the first contact portion in the path through which the sample is developed from the sample introduction portion, and containing a second molecule labeled with a second labeling material, such that the first molecule in contact with the sample contacts the second molecule; A first detection unit located in a path following the second contact unit in the path through which the sample is developed from the sample introduction unit, and having a first capturing body for capturing the first molecule fixed thereon; A second detection unit, located at a location spaced apart from the first detection unit in the path following the second contact unit in the path through which the sample is developed from the sample introduction unit, and having a second capturing body for capturing the second molecule fixed thereto; and A third detection unit is located at a distance from the first detection unit and the second detection unit in a path following the second contact unit in which a sample is developed from the sample introduction unit, and the first capture body and the second capture body are fixed thereto; A device wherein the first label material and the second label material are materials that exhibit different colors.

12. A device according to claim 11, wherein the interaction between the first molecule and the second molecule is a protein-protein interaction.

13. In the 11th paragraph, the device, wherein the substance that interferes with the interaction between the first molecule and the second molecule is a compound, protein, peptide or aptamer that inhibits protein-protein binding.

14. A device according to claim 11, wherein the first molecule and the second molecule include a capture mediator that mediates the capture.

15. A device according to claim 11, wherein the first labeling material and the second labeling material are colored beads or fluorescent molecules.

16. A device according to claim 11, wherein the first capture body and the second capture body are antibodies or aptamers.

17. In the 11th paragraph, the device has a molar ratio of the first capture body and the second capture body of the third detection unit of 1:3 to 3:1.

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