KR-102963355-B1 - Method for analyzing molecule-molecule interaction, and device for detecting inhibitor of the interaction using the same

KR102963355B1KR 102963355 B1KR102963355 B1KR 102963355B1KR-102963355-B1

Abstract

The present invention relates to a method for analyzing the interaction between a first molecule and a second molecule, comprising: a molecule-molecular 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 detector contact step of contacting the result obtained from the molecule-molecular contact step with a first detector, wherein a first capture body for capturing the first molecule is fixed to the first detector; and a second detector contact step of contacting the result obtained from the molecule-molecular contact step with a second detector, wherein a second capture body for capturing the second molecule is fixed to the second detector. The present invention relates to a method and a lateral flow-based device for detecting a substance that interferes with the interaction between a first molecule and a second molecule using the same, comprising: a third detector contact step in which the result obtained from the molecule-molecular contact step is brought into contact with a third detector, wherein the first capture agent and the second capture agent are fixed to the third detector; wherein the first labeling substance and the second labeling substance are substances that exhibit different colors.

Inventors

박창균
김승일
김우영

Assignees

한국기초과학지원연구원

Dates

Publication Date: 20260511
Application Date: 20240322

Claims (17)

As a method for analyzing the interaction between a first molecule and a second molecule, A molecule-molecular 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 the result obtained from the above molecule-molecular contact step with a first detection unit, wherein a first capture body for capturing the first molecule is fixed to the first detection unit; A second detector contact step of contacting the result obtained from the above molecule-molecular contact step with a second detector, wherein a second capture body for capturing the second molecule is fixed to the second detector; A third detector contact step of contacting the result obtained from the above molecule-molecular contact step with a third detector, wherein the first capture body and the second capture body are fixed to the third detector; and The method includes the step of analyzing the interaction between the first molecule and the second molecule; The above-mentioned first label material and second label material are materials that exhibit different colors, and Analyzing the interaction between the first molecule and the second molecule is, When the colors of the first label substance and the second label substance are expressed together in all of the first, second, and third detectors, it is determined that the first molecule and the second molecule have combined, and A method characterized by determining that the first molecule and the second molecule are not bound when only the color of the first labeled substance labeled on the first molecule is expressed in the first detection unit, only the color of the second labeled substance labeled on the second molecule is expressed in the second detection unit, and the colors of the first labeled substance and the second labeled substance are expressed together in the third detection unit.
A method according to claim 1, wherein the interaction between the first molecule and the second molecule is a protein-protein interaction.
In claim 1, prior to the molecule-molecular contact step A method further comprising a sample contact step of contacting a sample with one or more selected from the first molecule and the second molecule.
In paragraph 3, the method is for detecting a substance that interferes with the interaction between a first molecule and a second molecule in a sample by analyzing the interaction between a first molecule and a second molecule.
In claim 4, the substance interfering with the interaction between the first molecule and the second molecule is a compound, protein, peptide, or aptamer that inhibits protein-protein binding.
In paragraph 1, The fact that the colors of the first labeling material and the second labeling material are expressed together is, A method in which the colors of the first labeling material and the second labeling material are expressed together, and a third color different from the colors of the first labeling material and the second labeling material is expressed.
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A method according to claim 1, wherein the molar ratio of the first capture body to the second capture body of the third detection unit is 1:3 to 3:1.
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As 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 development means for developing a sample from the above sample introduction section; A first contact portion located in the path where the sample is developed from the sample introduction portion, comprising a first molecule labeled with a first labeling substance, wherein the sample comes into contact with the first molecule; A second contact portion located in the path following the first contact portion in the path where the sample is developed from the sample introduction portion, and containing a second molecule labeled with a second labeling substance, wherein the first molecule in contact with the sample comes into contact with the second molecule; A first detector located in the path following the second contact part in the path where the sample unfolds from the sample introduction part, and having a first capture body fixed thereon that captures the first molecule; A second detector located at a location spaced apart from the first detector in the path following the second contact part in the path where the sample is developed from the sample introduction part, and having a second capture body fixed thereon that captures the second molecule; and It includes a third detector located at a location spaced apart from the first detector and the second detector in the path following the second contact part in the path where the sample is developed from the sample introduction part, and on which the first capture body and the second capture body are fixed. A device in which the first labeling material and the second labeling material are materials that exhibit different colors.
A device according to claim 11, wherein the interaction between the first molecule and the second molecule is a protein-protein interaction.
In claim 11, the substance interfering with the interaction between the first molecule and the second molecule is a compound, protein, peptide, or aptamer that inhibits protein-protein binding.
In Paragraph 11, If the sample does not contain a substance that interferes with the interaction between the first molecule and the second molecule, the colors of the first label substance and the second label substance are all expressed together in the first detector, the second detector, and the third detector. A device in which, when a substance interfering with the interaction between the first molecule and the second molecule is included in the sample, only the color of the first labeled substance labeled on the first molecule is expressed in the first detector, only the color of the second labeled substance labeled on the second molecule is expressed in the second detector, and the colors of the first labeled substance and the second labeled substance are expressed together in the third detector.
In Paragraph 14, The fact that the colors of the first labeling material and the second labeling material are expressed together is, A device in which the colors of the first labeling material and the second labeling material are expressed together, and a third color different from the colors of the first labeling material and the second labeling material is expressed.
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An apparatus according to claim 11, wherein the molar ratio of the first capture body to the second capture body of the third detection unit is 1:3 to 3:1.

Description

Method for analyzing molecule-molecule interaction, and device for detecting inhibitor of the interaction using the same The present invention relates to a method for analyzing molecular-molecular interactions and a device for detecting substances that interfere with said interactions using the same, for example, a method for analyzing protein-protein interactions, more specifically, the interaction between the spike protein of SARS-CoV-2 and its receptor, the human ACE2 protein, and a device capable of detecting neutralizing antibodies resulting from vaccination based on the same. There is a problem in that the degree of immunity acquired through the formation of neutralizing antibodies following vaccination varies by individual, and the duration of such immunity also differs significantly. Since breakthrough infections occur due to viral mutations and the gradual decrease in the amount of neutralizing antibodies formed in the body over time, it is necessary to manage this situation. Furthermore, when novel vaccine platforms developed in a short period, such as for COVID-19, are utilized, concerns regarding associated side effects may lead to resistance to booster shots. Therefore, it is essential to develop a method that specifically detects only the neutralizing antibodies present in the body to evaluate vaccine efficacy, determine whether immunity has been formed after vaccination, and decide on the timing for booster shots based on the analysis of immunity duration. Looking at existing neutralizing antibody detection technologies, there is a cell-based in vitro assay method that analyzes the extent to which the virus infects the host cell by treating the host cell with the serum (neutralizing antibody) and the actual virus. While this method enables accurate detection of neutralizing antibodies and analysis of neutralization ability, it requires more than 24 hours of analysis time and, due to the use of viruses (due to the risk of infection), requires specialized facilities (Biosafety Level 3) and specialized equipment. Next, there is the competitive ELISA method, which analyzes the degree to which neutralizing antibodies inhibit 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. It has been approved for emergency use by the FDA because it can analyze the presence of actual neutralizing antibodies among antibodies formed after vaccination on a large scale. It has the advantages of being safer and requiring less time for analysis (around 3 hours) compared to cell-based experiments, but it has the problem of requiring large automated equipment and skilled professionals. In addition, one could consider the approach of currently approved and sold COVID-19 rapid diagnostic kits (RDTs); however, these kits detect IgG/IgM antibodies and are intended only to confirm the immune response or infection status after vaccination, making it impossible to actually identify or detect neutralizing antibodies. Recently, research has been reported on the development of rapid neutralizing antibody diagnostic kits that apply the principles of competitive ELISA to rapid diagnostic platforms. A representative method involves displaying a positive signal when neutralizing antibodies are absent and showing a weakening signal depending on the amount of neutralizing antibodies. However, this approach has limitations as it can only provide accurate test results when the titer of neutralizing antibodies is high—contrary to the detection methods of existing rapid diagnostic kits—and it is also impossible to perform quantitative analysis of neutralizing antibodies. Due to these critical drawbacks, commercialization has not progressed since development, or actual use remains restricted. Therefore, the inventors intended to develop a new technology capable of overcoming the problems of the existing technology described above, which can analyze interactions between molecules and detect neutralizing antibodies quickly, accurately, safely, and easily. 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). FIG. 2 shows a device (lateral flow immunoassay strip sensor) according to one embodiment of the present invention capable of implementing the method of the present invention. FIG. 3 shows an apparatus according to another embodiment capable of implementing the method of the present invention and a method of applying the same. FIG. 4 illustrates the operation of a device (lateral flow immunoassay strip sensor) according to an embodiment of the present invention capable of implementing the method of the present invention. An upper portion, when a first molecule and a second molecule interact (bind); a lower portion, when the first molecule and the second molecule cannot interact (bind) due to a PPI (