Method for detecting phosphorylated protein

Abstract

This method for detecting a phosphorylated protein having two or more phosphorylation sites in a sample comprises: a step for introducing, into a well, the sample, a first specific binding substance for a first phosphorylation site in a target protein labeled with a first single-stranded nucleic acid fragment, and a second specific binding substance for a second phosphorylation site in the target protein labeled with a second single-stranded nucleic acid fragment, and consequently, forming a complex containing the target protein, the first specific binding substance, and the second specific binding substance when the target protein is present in the sample, and forming a double-stranded nucleic acid by hybridizing at least a part of the first single-stranded nucleic acid fragment and at least a part of the second single-stranded nucleic acid fragment; and a step for detecting the formation of the double-stranded nucleic acid. The detection of the formation of the double-stranded nucleic acid indicates the presence of the target protein.

Description

Method for detecting phosphorylated proteins

[0001] The present invention relates to a method for detecting phosphorylated proteins. This application claims priority to Japanese Patent Application No. 2025-010700, filed in Japan on January 24, 2025, the contents of which are incorporated herein by reference.

[0002] Quantitative detection of target substances in biological samples is used for early disease detection and prediction of drug efficacy. Conventionally, protein quantification has been performed using enzyme-linked immunosorbent assay (ELISA), and nucleic acid quantification has been performed using real-time PCR. Furthermore, Patent Document 1, Non-Patent Documents 1 and 2 report on Proxy Ligation Assay (PLA) and Proxy Extension Assay (PEA), which are methods for detecting proteins using antibodies modified with oligonucleotides. These methods utilize PCR for detection.

[0003] On the other hand, in recent years, there has been a growing need to detect target substances with greater accuracy, for purposes such as earlier disease detection. Techniques that perform enzymatic reactions within numerous micro-compartments are being investigated as methods for accurately detecting target substances. These methods are called digital measurement. Examples of digital measurement include digital ELISA and digital PCR.

[0004] In digital measurement, the sample solution is divided into a very large number of minute solutions. Then, the signal from each minute solution is binarized to determine only whether or not the target substance is present, and the number of molecules of the target substance is measured. Compared to conventional methods such as ELISA and real-time PCR, digital measurement can significantly improve detection sensitivity and quantitative accuracy.

[0005] For example, Non-Patent Document 3 describes a microwell array having channels for supplying microwells and reagents, and describes that digital ELISA was performed using this microwell array.

[0006] Furthermore, living organisms possess mechanisms for transmitting external stimuli into cells. In this process, various proteins temporarily bind to each other to transmit information. This information transmission system is called a signal transduction system, and various protein molecules that mediate stimuli play a role in this system. In many signal transduction systems, signals are transmitted downstream by controlling protein phosphorylation. Non-patent document 4 describes a microwell array having microwells and channels for supplying reagents, and describes the use of cells within this microwell array to represent signal transduction and detect phosphorylated proteins. The signal transduction process is monitored by detecting phosphorylated proteins.

[0007] In digital measurement devices, the target substance or antibody is encapsulated within the well or droplet, making it difficult to perform a washing step. Therefore, Patent Document 2 discloses a method for detecting a target substance without performing a washing step, which uses two types of antibodies labeled with single-stranded nucleic acids that can hybridize with each other to detect the target substance. In this method, the target substance is detected by detecting the double-stranded nucleic acid formed by the two antibodies bound to the target substance, thus enabling detection of the target substance without removing unreacted antibodies through a washing step.

[0008] Furthermore, Patent Document 3 discloses a method for investigating the activity of a BRAF mutant having a gene mutation of unknown clinical significance (VUS) by detecting the phosphorylated protein of MEK using digital measurement. BRAF is a kinase involved in mitogen-activated protein kinase (MAPK) signaling and uses MEK as a substrate. In the method described in the document, the BRAF mutant to be evaluated is reacted with MEK, and then the phosphorylated MEK is complexed with anti-MEK antibodies and anti-pMEK antibodies labeled with mutually hybridizable single-stranded nucleic acids, respectively, and the double-stranded nucleic acid formed on the complex is detected. If the BRAF mutant is a normal mutant (a mutant that does not affect activity), MEK will not be phosphorylated unless there is an upstream stimulus, and the double-stranded nucleic acid will not be detected. If the BRAF mutant is a constitutively activated mutant, MEK will be phosphorylated even without an upstream stimulus, and the double-stranded nucleic acid will be detected.

[0009] International Publication No. 2012 / 160083, International Publication No. 2023 / 080137, International Publication No. 2024 / 029511

[0010] Mohammed H., et al., Approaches for Assessing and Discovering Protein Interactions in Cancer., Mol Cancer Res, 11 (11), 1295-1302, 2013.Assarsson E., et al., Homogenous 96-Plex PEA Immunoassay Exhibiting High Sensitivity, Specificity, and Excellent Scalability., PLoS One, 9 (4), e95192, 2014.Kan CW, et al., Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies., Lab on a Chip, 12 (5), 977-985, 2012.Blazek M., et al., Proximity Ligation Assay for High-content Profiling of Cell Signaling Pathways on a Microfluidic Chip., Molecular & Cellular Proteomics 12: 10.1074 / mcp.M113.032821, 3898-3907, 2013.

[0011] The method described in Patent Document 3 allows for the detection of phosphorylated MEK using digital measurement without requiring a complicated cleaning process. However, further improvement in detection sensitivity is desired.

[0012] The present invention aims to provide a method for detecting phosphorylated proteins with high sensitivity.

[0013] The present invention includes the following aspects: [1] A method for detecting a target protein in a sample, comprising: introducing the sample, a first specific binding substance for the target protein labeled with a first single-stranded nucleic acid fragment, and a second specific binding substance for the target protein labeled with a second single-stranded nucleic acid fragment into a well, wherein if the target protein is present in the sample, a complex comprising the target protein, the first specific binding substance, and the second specific binding substance is formed, and at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment hybridize to form a double-stranded nucleic acid; and detecting the formation of the double-stranded nucleic acid by Invasive Clevage Assay, wherein the target protein is a phosphorylated protein having two or more phosphorylation sites, and the first specific binding substance is a substance that specifically recognizes a first phosphorylation site in the target protein. A method wherein the second specific binding substance is a substance that specifically recognizes a second phosphorylation site in the target protein that is different from the first phosphorylation site, and the detection of the formation of the double-stranded nucleic acid indicates the presence of the target protein. [2] The method of [1], wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.05 to 2 nM. [3] The method of [1], wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 0.1 to 4 nM, and the concentration of the other single-stranded nucleic acid fragment is 1 to 4 nM. [4] The method of [1], wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.1 to 1 nM. [5] The method of [1], wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.05 to 2 nM. [6] The method of [1], wherein the concentration of either the first or second specific binding substance in the solution in the well is 0.1 to 4 nM, and the concentration of the other specific binding substance is 1 to 4 nM.[7] The method of [1], wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.1 to 1 nM. [8] The method of any one of [2] to [7], wherein the target protein is phosphorylated MEK. [9] The method of any one of [2] to [7], wherein t is the time from the start of measurement of the Invasive Clevage Assay. The measurement time T at which the St / Nt ratio takes its maximum value, where St is the fluorescence intensity value detected at measurement time t in the well in which the complex is formed, and Nt is the fluorescence intensity value detected at measurement time t in the well in which the complex is not formed. ｍａｘ In which, any of the methods [1] to [8] above, the St / Nt ratio is greater than 4.0.

[10] The measurement time T ｍａｘ The method of [9], wherein the duration is 40 minutes or less.

[11] The method of [9] or

[10] , wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.5 to 2 nM.

[12] The method of any of [9] to

[11] , wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 4 nM, and the concentration of the other single-stranded nucleic acid fragment is 1 to 4 nM.

[13] The method of any of [9] to

[12] , wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.8 to 1.2 nM.

[14] The method of [9] or

[10] , wherein the concentration of one of the first and second specific binding substances in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.5 to 2 nM.

[15] The method of any of [9],

[10] and

[14] , wherein the concentration of one of the first and second specific binding substances in the solution in the well is 1 to 4 nM, and the concentration of the other specific binding substance is 1 to 4 nM.

[16] The method of any of [9],

[10] ,

[14] and

[15] , wherein the concentration of one of the first and second specific binding substances in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.8 to 1.2 nM.

[17] The measurement time Tｍａｘ The method of any of [9] to

[16] , wherein St is greater than 200 and Nt is less than 400.

[18] The method of any of [1] to

[17] , wherein the volume of the well is 10 fL to 100 pL.

[19] The method of any of [1] to

[18] , wherein the distance between the first phosphorylation site and the second phosphorylation site is such that at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment can hybridize.

[20] The method of any of [1] to

[19] , wherein the base lengths of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment are 10 to 200 bases each.

[21] The method of any of [1] to

[20] , further comprising the step of introducing the sample, the first specific binding agent, and the second specific binding agent into the well, and then sealing the opening of the well.

[22] The method of

[21] , wherein the wells, whose openings are sealed, include a first specific binding agent not bound to the target protein, and / or a second specific binding agent not bound to the target protein.

[23] A kit for detecting a target protein, comprising: a well array having a plurality of wells; a first specific binding agent for the target protein labeled with a first single-stranded nucleic acid fragment; and a second specific binding agent for the target protein labeled with a second single-stranded nucleic acid fragment, wherein the target protein is a phosphorylated protein having two or more phosphorylation sites; the first specific binding agent is a substance that specifically recognizes a first phosphorylation site in the target protein; and the second specific binding agent is a substance that specifically recognizes a second phosphorylation site in the target protein that is different from the first phosphorylation site.

[24] The kit of

[23] , further comprising a sealing solution for sealing the openings of the wells.

[25] The kit of

[23] or

[24] , further comprising a reagent for detecting a double-stranded nucleic acid formed by the hybridization of at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment.

[0014] According to the present invention, a technique for detecting phosphorylated proteins with higher sensitivity can be provided.

[0015] Figure 1 is a schematic diagram illustrating the detection method for the target protein in this embodiment. Figure 2 is a schematic cross-sectional view showing an example of a fluid device. Figure 3 is a schematic cross-sectional view showing an example of a fluid device. Figure 4 is a schematic cross-sectional view showing an example of a fluid device. Figure 5 is a schematic cross-sectional view showing an example of a fluid device. Figure 6 is a schematic cross-sectional view showing an example of a fluid device. Figure 7 is a schematic cross-sectional view showing an example of a fluid device. Figure 8 is a schematic diagram illustrating an example of the Invasive Clevage Assay (ICA) method. Figure 9 shows the results of measuring the fluorescence intensity of each reaction solution over time when an immuno-ICA reaction was performed on a phosphorylation reaction solution (S) containing wild-type BRAF and active MEK, and a phosphorylation reaction solution (N) containing wild-type BRAF and inactive MEK, respectively, under PC conditions, condition 1, or condition 2 reaction conditions. Figure 10 shows a histogram of the brightness values ​​of each well detected by a microwell device for the immunoICA reaction performed under each reaction condition in Example 2. Figure 11 shows the measurement results of the brightness value of the positive reaction (S) (Figure 11(A)) and the background brightness value (N) (Figure 11(B)) at a reaction time of 60 minutes for the ICA reaction performed in Example 3 with the concentration of DNA1-modified antibody shown on the horizontal axis and the concentration of DNA2-modified antibody shown on the vertical axis. Figure 12 shows the maximum value of the St / Nt ratio (Figure 12(A)) and the measurement time T at which the St / Nt ratio reaches its maximum value for the ICA reaction performed in Example 3 with the concentration of DNA1-modified antibody shown on the horizontal axis and the concentration of DNA2-modified antibody shown on the vertical axis. ｍａｘ Figure 12(B) shows the measurement results. Figure 13 shows the maximum value of the St / Nt ratio (Figure 13(A)) and the measurement time T at which the St / Nt ratio reaches its maximum value in the ICA reaction performed in Example 4, with the concentration of DNA1-modified antibody shown on the horizontal axis and the concentration of DNA2-modified antibody shown on the vertical axis. ｍａｘ This figure shows the measurement results (Figure 13(B)).

[0016] The embodiments of the present invention will be described in detail below, with reference to the drawings as appropriate. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant explanations are omitted. Note that the dimensional ratios in each figure are exaggerated for illustrative purposes and do not necessarily correspond to the actual dimensional ratios.

[0017] [Method for detecting target protein] In one embodiment, the present invention provides a method for detecting a target protein in a sample, comprising introducing the sample, a first specific binding agent for the target protein labeled with a first single-stranded nucleic acid fragment, and a second specific binding agent for the target protein labeled with a second single-stranded nucleic acid fragment into a well, wherein if the target protein is present in the sample, a complex is formed comprising the target protein, the first specific binding agent, and the second specific binding agent, and at least a portion of the first single-stranded nucleic acid fragment and at least of the second single-stranded nucleic acid fragment The present invention provides a method comprising the steps of: a portion hybridizing to form a double-stranded nucleic acid; and detecting the formation of the double-stranded nucleic acid by ICA, wherein the target protein is a phosphorylated protein having two or more phosphorylation sites, the first specific binding substance is a substance that specifically recognizes a first phosphorylation site in the target protein, and the second specific binding substance is a substance that specifically recognizes a second phosphorylation site in the target protein that is different from the first phosphorylation site, and the detection of the formation of the double-stranded nucleic acid indicates the presence of the target protein.

[0018] Figure 1 is a schematic diagram illustrating the method of this embodiment. As shown in Figure 1, in the method of this embodiment, first, the sample, a first specific binding substance 120 labeled with a first single-stranded nucleic acid fragment 121, and a second specific binding substance 130 labeled with a second single-stranded nucleic acid fragment 131 are introduced into the wells. As a result, if the target protein 110 is present in the sample, a complex 100 is formed containing the target protein 110, the first specific binding substance 120, and the second specific binding substance 130. At least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 hybridize to form a double-stranded nucleic acid (also called a double-stranded nucleic acid region) 140.

[0019] Next, the formation of double-stranded nucleic acid 140 is detected. If the formation of double-stranded nucleic acid 140 is detected, it indicates the presence of the target protein 110. The method of this embodiment may also be applied to a sample that does not contain the target protein 110, in which case the formation of double-stranded nucleic acid 140, i.e., the presence of the target protein 110, will not be detected. The detection of the formation of double-stranded nucleic acid 140 will be described later.

[0020] As shown in Figure 1, the first specific binding substance 120 recognizes the first phosphorylation site 111 of the target protein 110. The second specific binding substance 130 recognizes the second phosphorylation site 112 of the target protein 110. Thus, the first phosphorylation site 111 and the second phosphorylation site 112 are different binding sites. For example, if the target protein is a protein, the first phosphorylation site 111 and the second phosphorylation site 112 can also be considered the first epitope 111 and the second epitope 112, respectively.

[0021] Examples of samples include biological samples and environmental samples. Biological samples are not particularly limited and include serum, plasma, urine, and cell culture media. Examples of environmental samples include river water and factory wastewater. The target protein 110 may be any phosphorylated protein having two or more phosphorylation sites, and may be a complex substance with one or more selected from the group consisting of other proteins, glycans, nucleic acids, lipid membrane structures, bacteria, viruses, and cells. In other words, it may be a complex substance containing a phosphorylated protein having two or more phosphorylation sites and one or more selected from the group consisting of other proteins, glycans, nucleic acids, lipid membrane structures, bacteria, viruses, and cells. Examples of target proteins 110 include phosphorylated MEK, phosphorylated SHC, and phosphorylated ERK.

[0022] The target protein may be a protein phosphorylated by a kinase. Examples of kinases include serine / threonine kinases such as BRAF and MEK; tyrosine kinases such as RET (Rearranged During Transfer) and FGFR; and lipid kinases.

[0023] The target protein to be detected is not particularly limited as long as it is a phosphorylated protein having two or more phosphorylation sites, and may be a glycoprotein or a membrane protein. Examples of complex substances include glycoproteins in which sugar chains and proteins are bound, and fusion proteins in which multiple proteins are fused. Examples of lipid membrane structures include vesicles composed of artificial or natural lipids, such as liposomes, exosomes, and intracellular organelles. In the detection of lipid membrane structures, bacteria, viruses, and cells, phosphorylated proteins having two or more phosphorylation sites present in these target molecules may be detected.

[0024] The method of this embodiment is characterized by using two types of specific binding substances that recognize and specifically bind to the phosphorylation site of the target protein when detecting the target protein with ICA. In the ICA reaction, in order to detect the target protein with sufficient sensitivity, it is preferable that the intensity ratio (S / N ratio) between the intensity of the fluorescent signal (S) derived from the double-stranded nucleic acid formed by a complex containing the target protein, the first specific binding substance and the second specific binding substance (hereinafter sometimes referred to as "positive reaction fluorescence intensity" or "S value") and the intensity of the fluorescent signal (noise: N) detected in the absence of the target protein (hereinafter sometimes referred to as "background fluorescence intensity" or "N value") is sufficiently large. On the other hand, in the ICA reaction, the background increases with time elapsed from the start of the reaction, but if the increase is too large, the S / N ratio will decrease. In the method of this embodiment, by making both the first specific binding substance and the second specific binding substance substances that specifically bind to different phosphorylation sites in the target protein, the background of the ICA reaction can be reduced, the signal-to-noise ratio can be increased, and the detection sensitivity of the target protein can be improved.

[0025] In the target protein 110, the distance between the first phosphorylation site 111 and the second phosphorylation site 112 is preferably such that at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 can hybridize. Here, the distance at which hybridization is possible is the distance at which the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 are close enough to hybridize.

[0026] For example, assuming that the target protein 110 is a sphere with a diameter of 10 nm, the distance between the first phosphorylation site 111 and the second phosphorylation site 112 is preferably 3 to 10 nm, and more preferably 3.5 to 9 nm. As an example, assuming that the target protein 110 is a sphere with a diameter of 10 nm, and the angle between the first phosphorylation site 111 and the second phosphorylation site 112 with respect to the center of the target protein 110 is 30 to 120°, the distance between the first phosphorylation site 111 and the second phosphorylation site 112 is 4.14 to 8.66 nm. Here, the distance between the first phosphorylation site 111 and the second phosphorylation site 112 is defined as the shortest distance between the first phosphorylation site 111 and the second phosphorylation site 112 along the surface of the target protein 110.

[0027] Specifically, for example, the base length of the first single-stranded nucleic acid fragment 121 may be 10 to 200 bases. Similarly, the base length of the second single-stranded nucleic acid fragment 131 may also be 10 to 200 bases. Furthermore, the length of the double-stranded nucleic acid 140 formed by the hybridization of at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 may be approximately 7 to 30 bases, or 7 to 20 bases. The hybridized region of the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 may include the ends of each single-stranded nucleic acid fragment. The hybridized region of the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 does not have to include the ends of the single-stranded nucleic acid fragments; for example, it may be located 7 to 13 bases from the ends of each single-stranded nucleic acid fragment. From the viewpoint of improving detection sensitivity, it is preferable that the hybridized region of the first single-stranded nucleic acid fragment 121 and the second single-stranded nucleic acid fragment 131 includes the ends of each single-stranded nucleic acid fragment.

[0028] In the method of this embodiment, the magnitude of the background reduction effect of the ICA reaction, which is obtained by using both the first specific binding substance and the second specific binding substance as substances that specifically bind to different phosphorylation sites in the target protein, is affected by the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment in the reaction solution. Since the fluorescence intensity of the positive reaction is sufficiently large and it is easy to sufficiently reduce the fluorescence intensity of the background, the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment in the ICA reaction solution are such that the concentration of either one of the first and second single-stranded nucleic acid fragments is 1 to 12 nM and the concentration of the other single-stranded nucleic acid fragment is preferably 0.05 to 2 nM, and more preferably the concentration of either one of the first and second single-stranded nucleic acid fragments is 1 to 12 nM and the concentration of the other single-stranded nucleic acid fragment is 0.1 to 1 nM. Also, it is preferable that the concentration of either one of the first and second single-stranded nucleic acid fragments is 1 to 4 nM and the concentration of the other single-stranded nucleic acid fragment is 0.1 to 4 nM. When the concentrations of the first and second single-stranded nucleic acid fragments are satisfied, the target protein is preferably phosphorylated MEK.

[0029] In the method of this embodiment, in order to further increase the detection sensitivity of the target protein, the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment in the ICA reaction solution are preferably such that the concentration of one of the first and second single-stranded nucleic acid fragments is 1 to 12 nM and the concentration of the other single-stranded nucleic acid fragment is 0.5 to 2 nM, and more preferably such that the concentration of one of the first and second single-stranded nucleic acid fragments is 1 to 12 nM and the concentration of the other single-stranded nucleic acid fragment is 0.8 to 1.2 nM. It is also preferable that the concentration of one of the first and second single-stranded nucleic acid fragments is 1 to 4 nM and the concentration of the other single-stranded nucleic acid fragment is 1 to 4 nM. Furthermore, it is also preferable that the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment in the ICA reaction solution be 0.1 nM for one and 1 to 12 nM for the other; 1 nM for one and 0.1 to 12 nM for the other; 4 nM for one and 0.1 to 4 nM for the other; 8 nM for one and 0.1 to 1 nM for the other; or 12 nM for one and 0.1 to 1 nM for the other. When the concentrations of the first and second single-stranded nucleic acid fragments are met, the target protein is preferably phosphorylated MEK. It is preferable to adjust the concentrations of the first and second single-stranded nucleic acid fragments so that they fall within these concentration ranges. Furthermore, the concentration of the first single-stranded nucleic acid fragment in the ICA reaction solution can be adjusted to a desired range by adjusting the amount of the first specific binding substance contained in the ICA reaction solution, and the concentration of the second single-stranded nucleic acid fragment in the ICA reaction solution can be adjusted to a desired range by adjusting the amount of the second specific binding substance contained in the ICA reaction solution.

[0030] Another aspect of the method of this embodiment is that, in order to further increase the detection sensitivity of the target protein, the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment in the ICA reaction solution are preferably such that the concentration of one of the first and second single-stranded nucleic acid fragments is 0.05 to 4 nM and the concentration of the other single-stranded nucleic acid fragment is 0.005 to 1 nM, and more preferably such that the concentration of one of the first and second single-stranded nucleic acid fragments is 0.1 to 4 nM and the concentration of the other single-stranded nucleic acid fragment is 0.01 to 1 nM. It is also preferable that the concentration of one of the first and second single-stranded nucleic acid fragments is 0.5 to 4 nM and the concentration of the other single-stranded nucleic acid fragment is 0.01 to 1 nM. Furthermore, it is also preferable that the concentrations of the first and second single-stranded nucleic acid fragments in the ICA reaction solution are such that one of them is 0.1 to 4 nM and the concentration of the other single-stranded nucleic acid fragment is 0.05 to 1 nM. Furthermore, it is also preferable that the concentration of one of the first and second single-stranded nucleic acid fragments is 0.005 nM and the concentration of the other single-stranded nucleic acid fragment is 0.5 to 4 nM; the concentration of one of the first and second single-stranded nucleic acid fragments is 0.01 nM and the concentration of the other single-stranded nucleic acid fragment is 0.1 to 4 nM; the concentration of one of the first and second single-stranded nucleic acid fragments is 0.05 nM and the concentration of the other single-stranded nucleic acid fragment is 0.05 to 4 nM; the concentration of one of the first and second single-stranded nucleic acid fragments is 0.1 nM and the concentration of the other single-stranded nucleic acid fragment is 0.05 to 4 nM; the concentration of one of the first and second single-stranded nucleic acid fragments is 0.5 nM and the concentration of the other single-stranded nucleic acid fragment is 0.05 to 4 nM; or the concentration of one of the first and second single-stranded nucleic acid fragments is 1 nM and the concentration of the other single-stranded nucleic acid fragment is 0.5 to 4 nM. Furthermore, when the concentrations of the first and second single-stranded nucleic acid fragments described above are met, the target protein is preferably phosphorylated SHC.

[0031] In FIG. 1, one first single-stranded nucleic acid fragment is labeled with one first specific binding substance, and one second single-stranded nucleic acid fragment is labeled with one second specific binding substance. The first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment described in FIG. 1 are single-stranded nucleic acid fragments that hybridize with each other to form a double-stranded nucleic acid. The concentrations of the above-mentioned first and second single-stranded nucleic acid fragments also mean the concentrations of the single-stranded nucleic acid fragments involved in the formation of the double-stranded nucleic acid.

[0032] However, two or more first single-stranded nucleic acid fragments may be labeled with one first specific binding substance. In this case, one of the two or more first single-stranded nucleic acid fragments hybridizes with the second single-stranded nucleic acid fragment to form a double-stranded nucleic acid region. Also, two or more second single-stranded nucleic acid fragments may be labeled with one second specific binding substance. One of the two or more second single-stranded nucleic acid fragments hybridizes with the first single-stranded nucleic acid fragment to form a double-stranded nucleic acid region.

[0033] Since the fluorescence intensity of the positive reaction is sufficiently large and it is easy to sufficiently reduce the fluorescence intensity of the background, the concentrations of the first specific binding substance and the second specific binding substance in the ICA reaction solution are preferably such that the concentration of either one of the first and second specific binding substances is 1 to 12 nM and the concentration of the other specific binding substance is 0.05 to 2 nM, more preferably the concentration of either one of the first and second specific binding substances is 1 to 12 nM and the concentration of the other specific binding substance is 0.1 to 1 nM. Also, it is preferable that the concentration of either one of the first and second specific binding substances is 1 to 4 nM and the concentration of the other specific binding substance is 0.1 to 4 nM. When the concentrations of the first and second specific binding substances are satisfied, the target protein is preferably phosphorylated MEK.

[0034] In the method of this embodiment, in order to further increase the detection sensitivity of the target protein, it is preferable that the concentration of the first specific binding substance and the second specific binding substance in the ICA reaction solution be such that the concentration of one of the first and second specific binding substances is 1 to 12 nM and the concentration of the other specific binding substance is 0.5 to 2 nM, and more preferably that the concentration of one of the first and second specific binding substances is 1 to 12 nM and the concentration of the other specific binding substance is 0.8 to 1.2 nM. It is also preferable that the concentration of one of the first and second specific binding substances is 1 to 4 nM and the concentration of the other specific binding substance is 1 to 4 nM. Furthermore, it is also preferable that the concentrations of the first specific binding substance and the second specific binding substance in the ICA reaction solution be within these ranges: one is 0.1 nM and the other is 1 to 12 nM; one is 1 nM and the other is 0.1 to 12 nM; one is 4 nM and the other is 0.1 to 4 nM; one is 8 nM and the other is 0.1 to 1 nM; or one is 12 nM and the other is 0.1 to 1 nM. It is preferable to adjust the concentrations of the first specific binding substance and the second specific binding substance so that they fall within these concentration ranges. When the concentrations of the first and second specific binding substances are met, the target protein is preferably phosphorylated MEK.

[0035] Another aspect of the method of this embodiment is that, in order to further increase the detection sensitivity of the target protein, the concentrations of the first specific binding substance and the second specific binding substance in the ICA reaction solution are preferably such that the concentration of one of the first and second specific binding substances is 0.05 to 4 nM and the concentration of the other specific binding substance is 0.005 to 1 nM, and more preferably such that the concentration of one of the first and second specific binding substances is 0.1 to 4 nM and the concentration of the other specific binding substance is 0.01 to 1 nM. It is also preferable that the concentration of one of the first and second specific binding substances is 0.5 to 4 nM and the concentration of the other specific binding substance is 0.01 to 1 nM. Furthermore, it is also preferable that the concentrations of the first and second specific binding substances in the ICA reaction solution are such that one of them is 0.1 to 4 nM and the concentration of the other specific binding substance is 0.05 to 1 nM. Furthermore, it is also preferable that the concentration of either the first or second specific binding substance is 0.005 nM and the concentration of the other specific binding substance is 0.5 to 4 nM; either the first or second specific binding substance is 0.01 nM and the concentration of the other specific binding substance is 0.1 to 4 nM; either the first or second specific binding substance is 0.05 nM and the other is 0.05 to 4 nM; either the first or second specific binding substance is 0.1 nM and the concentration of the other specific binding substance is 0.05 to 4 nM; either the first or second specific binding substance is 0.5 nM and the concentration of the other specific binding substance is 0.05 to 4 nM; or either the first or second specific binding substance is 1 nM and the concentration of the other specific binding substance is 0.5 to 4 nM. It is preferable to adjust the concentrations of the first and second specific binding substances so that they fall within these concentration ranges. Furthermore, when the concentrations of the first and second single-stranded nucleic acid fragments are met, the target protein is preferably phosphorylated SHC.

[0036] In the ICA reaction, both the fluorescence intensity of the positive reaction and the fluorescence intensity of the background increase with time from the start of the reaction, and the S / N ratio at each measurement time also changes with time. Let the time from the start of measurement of the ICA reaction be t, the fluorescence intensity of the positive reaction detected at the measurement time t (i.e., the fluorescence intensity of the reaction solution in which the complex is formed) be St, and the fluorescence intensity of the background detected at the measurement time t (i.e., the fluorescence intensity of the reaction solution in which the complex is not formed) be Nt. Then, the measurement time T at which the St / Nt ratio takes the maximum value ｍａｘ Then, the measurement sensitivity is highest at the measurement time T ｍａｘ point

[0037] In the method of this embodiment, T ｍａｘ is preferably 96 minutes or less, more preferably 60 minutes or less, still more preferably 40 minutes or less, and particularly preferably 30 minutes or less. When the ICA reaction is carried out using a real-time PCR apparatus, the reaction time of the ICA reaction preferably exceeds T ｍａｘ When the ICA reaction is carried out using a measuring apparatus utilizing the endpoint method, the reaction time of the ICA reaction is preferably T ｍａｘ The shorter T ｍａｘ is, the shorter the measurement time of the ICA reaction can be made.

[0038] In the method of this embodiment, when the ICA reaction is carried out using a real-time PCR apparatus, the detection sensitivity can be further increased. Therefore, the St / Nt ratio at the measurement time T ｍａｘ may be 2.05 or more, may be 2.86 or more, preferably exceeds 4.0, more preferably 4.5 or more, and still more preferably 5.0 or more. The upper limit of the St / Nt ratio at the measurement time T ｍａｘ is not particularly limited, and the St / Nt ratio is generally 10.0 or less.

[0039] In the method of this embodiment, since the detection sensitivity can be further increased, the measurement time T ｍａｘIn this case, St preferably has a sufficient signal intensity. For example, when performing the ICA reaction using a 96-well real-time PCR instrument, "Light Cycler® 480" (manufactured by Roche Diagnostics), the measurement time T is preferable. ｍａｘ St in this case is preferably greater than 200, more preferably 250 or greater, and even more preferably 300 or greater. Measurement time T ｍａｘ The upper limit of St in this case is not particularly limited and can be, for example, 1000 or less.

[0040] In the method of this embodiment, detection sensitivity can be increased, thus reducing the measurement time T ｍａｘ In this case, Nt is preferably relatively small. For example, when the ICA reaction is performed using the "Light Cycler® 480" (manufactured by Roche Diagnostics), a 96-well real-time PCR instrument, the measurement time T is preferable. ｍａｘ The Nt in this case is preferably less than 400, more preferably 350 or less, and even more preferably 300 or less. Measurement time T ｍａｘ The lower limit of Nt in this case is not particularly limited and can be, for example, 10 or more.

[0041] In the method of this embodiment, the measurement time T ｍａｘ In this case, it is preferable that the St / Nt ratio is greater than 4.0, St is greater than 200, and Nt is less than 400. To achieve this, it is preferable to appropriately adjust the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment in the reaction solution. For example, in the method of this embodiment, it is preferable that the concentration of either the first single-stranded nucleic acid fragment or the second single-stranded nucleic acid fragment in the ICA reaction solution is 0.5 to 2 nM and the other single-stranded nucleic acid fragment is 1 to 12 nM; one is 1 to 4 nM and the other is 1 to 4 nM; or one is 0.8 to 1.2 nM and the other is 1 to 12 nM. It is preferable to adjust the concentrations of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment so that they fall within these concentration ranges. When the concentrations of the first and second single-stranded nucleic acid fragments are met, the target protein is preferably phosphorylated MEK.

[0042] In the method of this embodiment, the measurement time T ｍａｘ In this case, it is preferable that the St / Nt ratio is greater than 4.0, St is greater than 200, and Nt is less than 400. To achieve this, it is preferable to appropriately adjust the concentrations of the first specific binding substance and the second specific binding substance in the reaction solution. For example, in the method of this embodiment, it is preferable that the concentration of either the first specific binding substance or the second specific binding substance in the ICA reaction solution is 0.5 to 2 nM and the other specific binding substance is 1 to 12 nM; one is 1 to 4 nM and the other is 1 to 4 nM; or one is 0.8 to 1.2 nM and the other is 1 to 12 nM. It is preferable to adjust the concentrations of the first specific binding substance and the second specific binding substance so that they fall within these concentration ranges. When the concentrations of the first and second specific binding substances are met, the target protein is preferably phosphorylated MEK.

[0043] (Wells) The method of this embodiment is suitable for detecting target proteins by digital measurement. When the method of this embodiment is performed by digital measurement, it is preferable that the wells constitute a well array in which multiple wells are arranged. Furthermore, it is preferable that the well array is located within the flow path of a fluid device.

[0044] (Fluid Device) Figure 2 is a schematic cross-sectional view showing an example of a fluid device. As shown in Figure 2, the fluid device 200 comprises a substrate 210 and a lid member 220 positioned opposite the substrate 210. The lid member 220 has a protrusion 221, the tip of which is in contact with the substrate 210. In the fluid device 200, the well array 240 is integrally molded with the substrate 210 on one surface of the substrate 210. The surface of the substrate 210 on which the well array 240 is molded faces the lid member 220. The well array 240 has a plurality of wells 241. The lid member 220 may be welded or bonded to the substrate 210.

[0045] The wells 241 open to the surface of the substrate 210. The shape, dimensions, and arrangement of the wells 241 are not particularly limited, but it is preferable that one target protein is introduced into each well 241. The wells 241 are preferably small wells with a small volume. For example, the volume of one well 241 may be about 10 fL to 100 pL. In the fluid device 200, multiple wells 241 of the same shape and size constitute a well array 240. By "same shape and size," they should be identical in shape and volume to the extent required for digital measurement, and variations within the range of manufacturing tolerances are acceptable.

[0046] The diameter of the well 241 may be, for example, about 1 to 10 μm. The depth of the well 241 may also be, for example, about 1 to 10 μm. Furthermore, the arrangement of the wells 241 is not particularly limited; for example, they may be arranged in a triangular lattice, a square lattice, or randomly.

[0047] In the fluid device 200, the presence of the protrusion 221 creates a space between the well array 240 and the lid member 220. This space constitutes a flow channel 230. The flow channel 230 functions as a path for delivering a liquid in which the target protein, the first specific binding substance, the second specific binding substance, etc., are dispersed, and a sealing liquid described later. The shape, structure, and volume of the flow channel 230 are not particularly limited, but the height of the flow channel 230 (i.e., the distance between the surface of the substrate 210 facing the lid member 220 and the portion where the well array is not formed, and the surface of the lid member 220 facing the substrate 210) may be, for example, 500 μm or less, for example, 300 μm or less, for example, 200 μm or less, or for example, 100 μm or less.

[0048] The protrusion 221 may be molded integrally with the lid member 220. The lid member 220 can be formed into a plate shape having the protrusion 221 by, for example, molding a fluid of thermoplastic resin using a mold. The lid member 220 may also have a reagent introduction port 222 and an outlet port 223 formed therein.

[0049] If the lid member 220 has a protrusion 221, the lid member 220 and the substrate 210 are stacked such that the protrusion 221 contacts the surface of the substrate 210 where the well 241 opens. As a result, the space between the lid member 220 and the substrate 210 becomes a flow path 230. The lid member 220 and the substrate 210 may be welded together by laser welding or the like.

[0050] (Modified Fluid Device 1) The fluid device used in the method of this embodiment is not limited to the fluid device 200 described above. Figure 5 is a schematic cross-sectional view showing an example of a fluid device. As shown in Figure 5, the fluid device 500 comprises a substrate 210 and a wall member 510. In the fluid device 500, the well array 240 is integrally molded with the substrate 210 on one side of the substrate 210. The well array 240 has a plurality of wells 241.

[0051] The fluid device 500 differs from the fluid device 200 described above mainly in that it does not have a lid member 220. Therefore, the fluid device 500 does not have a flow path.

[0052] (Modification 2 of the fluid device) In the fluid device 200 described above, the lid member 220 and the protrusion 221 are integrally molded. However, the lid member 220 and the protrusion 221 may be molded as separate parts.

[0053] Furthermore, in the fluid devices 200 and 500 described above, the well array 240 is integrally molded with the substrate 210 on one surface of the substrate 210. However, the well array does not have to be integrally molded with the substrate 210. For example, the well array 240, which is molded separately from the fluid device, may be placed on the substrate 210 of the fluid device. Alternatively, a resin layer may be laminated on the surface of the substrate 210, and the well array may be formed in the resin layer by etching or the like.

[0054] (Material of the fluid device) The substrate 210 is formed using, for example, a resin. The type of resin is not particularly limited, but it is preferable to use a resin that is resistant to reagents and sealing solutions. Furthermore, if the signal to be detected is fluorescence, it is preferable to use a resin with low autofluorescence. Examples of resins include, but are not limited to, cycloolefin polymers, cycloolefin copolymers, silicon, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins.

[0055] Multiple wells 241 may be formed on one side of the substrate 210 in the thickness direction. Methods for forming wells using resin include injection molding, thermal imprinting, and optical imprinting.

[0056] Alternatively, for example, a fluororesin may be laminated on the substrate 210, and the fluororesin may be processed by etching or the like to form a well array. As the fluororesin, for example, CYTOP® (Asahi Glass Co., Ltd.) can be used.

[0057] Furthermore, if the fluid device has a lid member 220, the material of the lid member 220 is preferably a resin with low autofluorescence, and may be a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0058] Furthermore, the lid member 220 may be made of a material that does not transmit light of wavelengths near the wavelength detected when observing the signal by fluorescence, or it may be made of a material that does not transmit light at all. For example, the lid member 220 may be made of a thermoplastic resin to which carbon or metal particles have been added.

[0059] (Method of this Embodiment) Next, the method of this embodiment will be described, with reference to Figures 2 to 4 as an example, using a fluid device 200. The method of this embodiment is a method for detecting a target protein in a sample, and includes the steps of: introducing a sample, a first specific binding substance 120 labeled with a first single-stranded nucleic acid fragment 121 for the target protein 110, and a second specific binding substance 130 labeled with a second single-stranded nucleic acid fragment 131 for the target protein 110 into a well 241, and if the target protein 110 is present in the sample, a complex 100 including the target protein 110, the first specific binding substance 120, and the second specific binding substance 130 is formed, and at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 hybridize to form a double-stranded nucleic acid 140; and detecting the formation of the double-stranded nucleic acid 140, where detection of the formation of the double-stranded nucleic acid 140 indicates the presence of the target protein 110. According to the method of this embodiment, target proteins can be detected without performing a washing step.

[0060] <Introduction Process> First, as shown in Figure 2, reagent solution L210 is introduced from the introduction port 222 of the fluid device 200 and delivered to the flow path 230. Reagent solution L210 is a liquid in which a first specific binding substance 120, a second specific binding substance 130, and, if present, a target protein 110 are dispersed, and also includes a reagent for detecting the formation of double-stranded nucleic acid 140.

[0061] The reagent solution L210 delivered to the flow path 230 comes into contact with the well array 240. The reagent solution L210 is then contained within the well 241. As a result, the first specific binding substance 120, the second specific binding substance 130, and, if present, the target protein 110, as well as reagents for detecting the formation of double-stranded nucleic acid 140, are introduced into the well 241.

[0062] The number of target proteins 110 introduced into a single well 241 is not particularly limited, but preferably, one or fewer, i.e., zero or one target protein 110, is introduced into each well 241. This allows for detection of the target protein 110 on a single-unit basis, i.e., digital measurement becomes possible. Furthermore, it is not necessary to introduce the target protein 110 into all wells of the well array.

[0063] The means for introducing the target protein 110 into the wells are not particularly limited, and an appropriate means can be selected according to the chosen target protein 110. For example, one method is to allow the target protein 110 to settle in the fluid device (flow channel) by its own gravity and then distribute it into the wells. Alternatively, a substance that captures the target protein 110 (capturer) may be used, and the capturer may be attached to the target protein 110, which is difficult to settle by its own gravity, and then delivered. The efficiency of introducing the target protein 110 into the wells can also be improved by pre-immobilizing the capturer in the wells and capturing the delivered target protein 110.

[0064] The step of binding the captured substance to the target protein 110 can be performed at any point in the method of this embodiment. For example, this step may be performed by bringing the target protein 110 and the captured substance into contact in the sample tube before introducing the target protein 110 into the well 141. Alternatively, the target protein 110 may be introduced into the well after the captured substance has been introduced into the well 141, and the captured substance and the target protein 110 may be brought into contact in the well.

[0065] The capture substance is a substance capable of capturing the target protein 110. The capture substance may be, for example, a conjugate of a solid phase and a substance that specifically binds to the target protein 110.

[0066] Examples of solid phases include particles, films, and substrates. Furthermore, the specific binding substance to the target protein 110 may be one type or two or more types. For example, it may be three types, four types, or five or more types.

[0067] The particles are not particularly limited and include polymer particles, magnetic particles, and glass particles. Particles that have undergone surface treatment to avoid nonspecific adsorption are preferred. Furthermore, particles having functional groups such as carboxyl groups on their surface are preferred for immobilizing specific binding substances. More specifically, JSR Corporation's product name "Magnosphere LC300" can be used.

[0068] Examples of the first specific binding substance 120, the second specific binding substance 130, and the specific binding substance in the capture include antibodies, antibody fragments, and aptamers. Examples of antibody fragments include Fab, F(ab'). ２ Examples include Fab', single-chain antibodies (scFv), disulfide-stabilized antibodies (dsFv), dimerized V-region fragments (Diabody), and peptides containing CDRs. The antibody may be a monoclonal antibody or a polyclonal antibody. Commercially available antibodies may also be used.

[0069] In the method of this embodiment, the target protein 110 is a phosphorylated protein having two or more phosphorylation sites, and the first specific binding substance 120 and the second specific binding substance 130 are phosphorylation site binding substances that recognize different phosphorylation sites of the target protein. Preferably, the first specific binding substance 120 and the second specific binding substance 130 are antibodies, antibody fragments, or aptamers that have different phosphorylation sites of the target protein as epitopes.

[0070] One method for labeling a single-stranded nucleic acid fragment with a specific binding substance is to use a crosslinking agent. The single-stranded nucleic acid fragment may be labeled with the specific binding substance via a linker molecule. The linker is not particularly limited and includes, for example, polyethylene chains, hydrocarbon chains, and peptides. The single-stranded nucleic acid fragment may be DNA or RNA. It may also contain artificial nucleic acids such as BNA and LNA.

[0071] The method for immobilizing a specific binding substance on the particle surface is not particularly limited and includes methods by physical adsorption, chemical bonding, avidin-biotin bonding, and bonding between protein G or protein A and an antibody. Methods by physical adsorption include immobilizing the specific binding substance on the particle surface through hydrophobic and electrostatic interactions. Methods by chemical bonding include using a crosslinking agent. For example, if the particle surface has hydroxyl groups, the specific binding substance can be immobilized on the particle surface by reacting a crosslinking agent with the carboxyl groups of the specific binding substance to activate esterification, and then reacting the hydroxyl groups with these ester groups. Furthermore, it is preferable to provide a spacer between the specific binding substance and the particle surface so as not to inhibit the target molecule recognition ability of the specific binding substance.

[0072] As described above, when introducing the target protein 110 into the well 241 using a capture agent, it is preferable to form a conjugate between the capture agent and the target protein 110 under conditions where zero or one target protein 110 is captured by one capture agent. Furthermore, it is preferable that zero or one capture agent is introduced into each well 241. This enables digital measurement.

[0073] When the target protein 110, the first specific binding substance 120, and the second specific binding substance 130 are brought into contact, a complex 100 containing these is formed, and at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 hybridize to form a double-stranded nucleic acid 140. The formation of the complex 100 may be carried out in the sample tube or in the well 241.

[0074] <Sealing Process> After introducing the sample, the first specific binding substance 120, and the second specific binding substance 130 into the well 241, a process of sealing the opening of the well 241 may be performed.

[0075] The method for sealing the openings of the wells 241 is not particularly limited, as long as it can prevent the liquid contained in one well 241 from mixing with the liquid contained in another well 241. For example, the openings of the wells 241 may be sealed by covering them with a sealing liquid. Alternatively, the openings of the wells 241 may be sealed by laminating a plate-like member, such as a glass plate, over them.

[0076] For example, as shown in Figure 3, sealing liquid L220 is delivered from the introduction port 222 of the lid member 220 to the flow channel 230 between the substrate 210 and the lid member 220. The sealing liquid L220 delivered to the flow channel 230 comes into contact with the well array 240. The sealing liquid L220 then displaces the reagent liquid L210 delivered to the flow channel 230 that is not contained in the wells 241. As a result, the sealing liquid L220 individually seals each of the multiple wells 241 containing reagent liquid L210 containing the target protein 110, and the wells 241 become independent reaction spaces (micro-compartments 242). When the flow channel 230 is filled with sealing liquid L220, the excess sealing liquid L220 is discharged from the discharge port 223. Figure 4 shows the state in which all the wells 241 of the well array 240 are sealed with sealing liquid L220, and sealed wells (micro-compartments) 242 are formed.

[0077] Alternatively, by dissolving lipids in reagent solution L210, sending sealing solution L220 into the flow path 230, and then sending another lipid-containing liquid, a lipid bilayer can be formed at the opening of well 241. Multiple wells 241 can then be individually sealed with this lipid bilayer to form sealed wells 242. Examples of lipids used to form the lipid bilayer include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), and mixtures thereof.

[0078] The well 242 sealed by any of the sealing methods described above may contain a first specific binding substance 120 that is not bound to the target protein 110, and / or a second specific binding substance 130 that is not bound to the target protein 110.

[0079] The sealing liquid is a liquid that can individually seal the liquids introduced into multiple wells 241 so that they do not mix with each other, thereby forming droplets (microdroplets). Preferably, it is an oily solution, and more preferably, it is an oil. As the oil, a fluorinated oil, a silicone oil, a hydrocarbon oil, or a mixture thereof can be used. More specifically, a product such as "FC-40" manufactured by Sigma Corporation can be used. FC-40 (CAS number: 86508-42-1) is a fluorinated aliphatic compound with a specific gravity of 1.85 g / mL at 25°C.

[0080] <Detection Step> Next, the formation of double-stranded nucleic acid 140 is detected. The detection of the formation of double-stranded nucleic acid 140 is preferably carried out using a signal amplification reaction. An example of a signal amplification reaction is ICA.

[0081] The ICA reaction is related to the principle that signal amplification proceeds through a cycle of two reactions: (1) complementary binding between nucleic acids, and (2) recognition and cleavage of the triple-strand structure by an enzyme.

[0082] The ICA reaction is less susceptible to reaction cycle inhibition by contaminants. Therefore, the target molecule 110 can be detected with high accuracy by using the ICA reaction. When the ICA reaction is used for the signal amplification reaction, the reagent solution L210 (a liquid containing the target molecule 110, the first specific binding substance 120, and the second specific binding substance 130) contains the reaction reagents necessary for the ICA reaction.

[0083] Reaction reagents required for the ICA reaction include flap probes, flap endonucleases (FEN), and fluorescent substrates. The flap probe is a nucleic acid fragment designed to hybridize with a first single-stranded nucleic acid fragment 121 or a second single-stranded nucleic acid fragment 131 to form a flap structure with a double-stranded nucleic acid 140.

[0084] Figure 8 is a schematic diagram illustrating an example of the ICA method. In the example shown in Figure 8, the ICA method detects a double-stranded nucleic acid 140 formed by the hybridization of at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131.

[0085] First, the flap probe is hybridized to either the first single-stranded nucleic acid fragment 121 or the second single-stranded nucleic acid fragment 131. In the example shown in Figure 8, the flap probe 810 hybridizes to the first single-stranded nucleic acid 121. As a result, the first flap region 811 is formed.

[0086] Next, when FEN is reacted with the first flap region 811, the first flap region 811 is cleaved, and a nucleic acid fragment 811 is generated. Subsequently, the nucleic acid fragment 811 hybridizes with a fluorescent substrate (nucleic acid fragment 820) to form a second flap region 821.

[0087] In the example shown in Figure 8, a fluorescent substance F is bound to the 5' end of nucleic acid fragment 820, and a quenching substance Q is bound to the 3' end of the nucleic acid fragment 820, a few bases from the 5' end. Subsequently, when FEN is reacted with the second flap site 821, the second flap site 821 is cleaved, and nucleic acid fragment 821 is generated. As a result, the fluorescent substance F detaches from the quenching substance Q, generating a fluorescence signal. By detecting this fluorescence signal, the formation of double-stranded nucleic acid 140 can be detected.

[0088] The reagent solution L210 can be a general liquid used in biochemical analysis performed using a fluid device, and is preferably an aqueous solution. Furthermore, the reagent solution L210 may contain a surfactant or the like to facilitate the sealing of the liquid within the well.

[0089] When the ICA reaction is used to detect the formation of double-stranded nucleic acid 140, if double-stranded nucleic acid 140 is present, the fluorescent substance F is released from the quenching substance Q by an isothermal enzymatic reaction, and emits a predetermined fluorescence signal in response to excitation light.

[0090] The detection of double-stranded nucleic acid 140 formation can be performed by selecting an appropriate known method depending on the type of signal to be detected. For example, when observing a fluorescent signal, excitation light corresponding to the fluorescent substance is irradiated into well 142, and the fluorescence emitted by the fluorescent substance is observed. For example, as shown in Figure 4, a predetermined reaction is performed in a sealed well 242, and the resulting signal is observed. Well 242R is the well in which a signal was detected, and well 242 is the well in which no signal was detected.

[0091] Next, with reference to Figures 5 to 7, the method of this embodiment will be explained using the case where the fluid device 500 is used as an example.

[0092] First, as shown in Figure 5, the reagent solution L210 is introduced into the fluid device 500. The reagent solution L210 is a liquid in which the target protein 110, the first specific binding substance 120, and the second specific binding substance 130 are dispersed, and also includes a reagent for detecting the formation of double-stranded nucleic acid 140. In the reagent solution L210, it is preferable that the concentration of the target protein 110 is adjusted so that one molecule or less of the target protein is present in each well 241.

[0093] Next, as shown in Figure 6, the sealing solution L220 is introduced into the fluid device 500. The specific gravity of the sealing solution L220 is greater than that of the reagent solution L210. Therefore, the sealing solution L220 sinks below the portion of the reagent solution L210 that is not contained in the wells 241, and comes into contact with the well array 240. The sealing solution L220 then individually seals each of the multiple wells 241 containing the reagent solution L210 containing the target protein, creating independent reaction spaces (microcompartments 142).

[0094] Next, as shown in Figure 7, a predetermined reaction is carried out in well 242, and the generated signal is observed. Well 242R is the well in which a signal was detected, and well 242 is the well in which no signal was detected.

[0095] [Complex] In one embodiment, the present invention provides a complex 110 comprising a target protein 110, a first specific binding substance 120 labeled with a first single-stranded nucleic acid fragment 121 for the target protein 110, and a second specific binding substance 130 labeled with a second single-stranded nucleic acid fragment 131 for the target protein 110, wherein at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 hybridize to form a double-stranded nucleic acid 140.

[0096] In the complex 110, the flap probe may further hybridize to the first single-stranded nucleic acid fragment 121 or the second single-stranded nucleic acid fragment 131.

[0097] As described above, by using the complex of this embodiment, target proteins can be detected without performing a washing step, even when using digital measurement.

[0098] [Kit] In one embodiment, the present invention provides a kit for detecting a target protein 110, comprising a well array 240 having a plurality of wells 241, a first specific binding substance 120 for the target protein 110 labeled with a first single-stranded nucleic acid fragment 121, and a second specific binding substance 130 for the target protein 110 labeled with a second single-stranded nucleic acid fragment 131.

[0099] The kit of this embodiment allows for the detection of the target protein described above to be performed suitably. Therefore, the kit of this embodiment includes the steps of introducing the target protein 110, a first specific binding agent 120 labeled with a first single-stranded nucleic acid fragment 121 for the target protein 110, and a second specific binding agent 130 labeled with a second single-stranded nucleic acid fragment 131 for the target protein 110 into well 241, thereby forming a complex 100 containing the target protein 110, the first specific binding agent 120, and the second specific binding agent 130, and at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131 hybridizing to form a double-stranded nucleic acid 140, and detecting the formation of the double-stranded nucleic acid 140, and the detection of the formation of the double-stranded nucleic acid 140 can be said to be used as a method to indicate the presence of the target protein 110.

[0100] The well array may be placed inside the fluid device described above. In the kit of this embodiment, the target protein, the first single-stranded nucleic acid fragment, the first specific binding agent, the second single-stranded nucleic acid fragment, and the second specific binding agent are the same as those described above.

[0101] The kit of this embodiment may further include a sealing liquid L220 for sealing the opening of the well 241. The sealing liquid L220 is the same as described above.

[0102] The kit of this embodiment may further include a reagent for detecting a double-stranded nucleic acid 140 formed by the hybridization of at least a portion of the first single-stranded nucleic acid fragment 121 and at least a portion of the second single-stranded nucleic acid fragment 131.

[0103] Examples of such reagents include the reagents for the ICA reaction mentioned above, specifically flap probes, flap endonucleases (FEN), and fluorescent substrates.

[0104] [Materials and Methods] As anti-MEK antibodies, we used anti-MEK1 / 2 (47E6) rabbit monoclonal antibody (clone #9126, manufactured by Cell Signaling Technologies, Inc.) (hereinafter sometimes referred to as "anti-MEK antibody"), anti-MEK1 / 2 (Ser217 / 221) (41G9) rabbit monoclonal antibody (manufactured by Cell Signaling Technologies, Inc.) (hereinafter sometimes referred to as "anti-pMEK (pS) antibody"), and anti-MEK1 (phospho T286) antibody (EPR7226-8) rabbit monoclonal antibody (manufactured by Cell Signaling Technologies, Inc.) (hereinafter sometimes referred to as "anti-pMEK (pT) antibody").

[0105] As anti-SHC antibodies, we used anti-SHC (phosphho Y427) antibody (EPR778(2)Y, manufactured by abcam Inc.) (hereinafter sometimes referred to as "anti-pSHC (Y427) antibody") and Phosphho-SHC (Tyr239 / 240) antibody (manufactured by abcam Inc.) (hereinafter sometimes referred to as "anti-pSHC (Tyr239 / 240) antibody").

[0106] DNA1 (5'-TTTGTCACTGTTCCTCCTTTTGTTTTCCTTTCTGTGAGCAATTTCACCCAA-3', SEQ ID NO: 1) and DNA2 (5'-GCATGGTTCCAATTTGGGTGAT-3', SEQ ID NO: 2) (both manufactured by Integrated DNA Technologies) were used as oligonucleotides to modify the antibody.

[0107] [Oligonilonucleotide Modification of Antibodies] Using an antibody-oligonucleotide conjugation tool (product name "oYo-Link Antibody Labeling Reagent", Funakoshi Co., Ltd.), DNA1 or DNA2 was conjugated to each antibody. Hereafter, an antibody in which DNA1 is conjugated to an anti-MEK antibody will be referred to as a DNA1-modified anti-MEK antibody, an antibody in which DNA2 is conjugated to an anti-MEK antibody will be referred to as a DNA2-modified anti-MEK antibody, an antibody in which DNA1 is conjugated to an anti-pMEK(pS) antibody will be referred to as a DNA1-modified anti-pMEK(pS) antibody, an antibody in which DNA2 is conjugated to an anti-pMEK(pS) antibody will be referred to as a DNA2-modified anti-pMEK(pS) antibody, an antibody in which DNA1 is conjugated to an anti-pMEK(pT) antibody will be referred to as a DNA1-modified anti-pMEK(pT) antibody, and an antibody in which DNA2 is conjugated to an anti-pMEK(pT) antibody will be referred to as a DNA2-modified anti-pMEK(pT) antibody. Furthermore, an antibody in which DNA1 is conjugated to an anti-pSHC (Y427) antibody is called a DNA1-modified anti-pSHC (Y427) antibody, and an antibody in which DNA2 is conjugated to an anti-pSHC (Tyr239 / 240) antibody is called a DNA2-modified anti-pSHC (Tyr239 / 240) antibody.

[0108] [Example 1] Phosphorylated MEK was detected using wild-type BRAF, active MEK (MEK phosphorylated by BRAF), and inactive MEK (mutant MEK not phosphorylated by BRAF).

[0109] (Preparation of a mixture of antigen-antibody reaction reagent and ICA reaction reagent) Two types of oligonucleotide-modified antibodies, MgCl ２A mixture was prepared by mixing the two oligonucleotide-modified antibodies with the ICA reaction reagent for detection. The volume of each mixture was 20 μL. The two oligonucleotide-modified antibodies were prepared to have final concentrations of 1 nM or 4 nM, respectively. Can Get Signal buffer (TOYOBO) solution A was used as the buffer. The ICA reaction reagent consists of 2 μM allele probe (5'-AGGCGCACGGAGGAATTGCTCACAGAAAGGA-3') (Fasmac, SEQ ID NO: 3), 4 μM FRET cassette 1 (5'-XTTCTYAGCCGGTTTTCCGGCTGAGACCTCCGTGCGCT-3': a nucleotide sequence represented by SEQ ID NO: 4 with fluorescent substrate X (Alexa 488 + Amino C6) added to the 5' end, and quenching agent Y (Black Hole Quencher (BHQ) 1-dT) inserted between the 4th and 5th bases of the nucleotide sequence; manufactured by Nippon Bioservices Co., Ltd.), 0.108 μM flap endonuclease (FEN)-1, 50 mM Tris-HCl (pH 8.5), and 20 mM MgCl ２ It also contained 0.05% Tween 20. Note that the concentrations of each component in these reaction reagents are the final concentrations in the reaction solution.

[0110] (Phosphorylation reaction) 2.8 nM wild-type BRAF, 7 nM active MEK, 100 μM ATP, 5 mM MgCl ２ A phosphorylation reaction solution (S) was prepared by mixing with blocking buffer to measure the fluorescence intensity of the positive reaction. Also, wild-type BRAF, inactive MEK, ATP, and MgCl were used. ２ A phosphorylation reaction solution (N) was prepared by mixing the BRAF with a blocking buffer to measure the background fluorescence intensity. The volume of each reaction solution was 7.5 μL. Wild-type BRAF was prepared to produce either active or inactive MEK. These are the final concentrations in the reaction solution. Each phosphorylation reaction solution was reacted at 30°C for 60 minutes to carry out the phosphorylation reaction.

[0111] (Immuno-ICA reaction) A mixture of antigen-antibody reaction reagent and ICA reaction reagent was added to the phosphorylation reaction solution (S) after the phosphorylation reaction to prepare ICA reaction solution (S) for measuring the fluorescence intensity of the positive reaction. Similarly, a mixture of antigen-antibody reaction reagent and ICA reaction reagent was added to the phosphorylation reaction solution (N) after the phosphorylation reaction to prepare ICA reaction solution (N) for measuring the background fluorescence intensity. The combination conditions of the two oligonucleotide-modified antibodies contained in the antigen-antibody reaction reagent used are shown in Table 1. The composition of the mixture of antigen-antibody reaction reagent and ICA reaction reagent is shown in Table 2.

[0112]

[0113]

[0114] Each ICA reaction solution was reacted using a T-Gradient (Biometler) at 37°C for 60 minutes, followed by a Light Cycler 480 (Roche Diagnostics) at 66°C for 120 minutes, and the fluorescence of Alexa 488 was detected.

[0115] Figure 9 shows the results of measuring the fluorescence intensity of phosphorylation reaction solution (S) and phosphorylation reaction solution (N) over time, after performing immuno-ICA reactions under the three reaction conditions shown in Table 1. In Figure 9, the vertical axis represents fluorescence intensity, and the horizontal axis represents time. As shown in Figure 9, the fluorescence intensity of ICA reaction solution (S) and ICA reaction solution (N) increased over time under all reaction conditions. The increase in fluorescence intensity of ICA reaction solution (N) was suppressed more significantly in reaction solutions under conditions 1 or 2 than in reaction solutions under PC conditions. These results indicate that using two oligonucleotide-modifying antibodies against phosphorylated MEK in the immuno-ICA reaction reduces background noise more effectively than using one antibody against phosphorylated MEK and one against unphosphorylated MEK.

[0116] [Example 2] The immuno-ICA reaction was performed using digital measurement with the fluid device shown in Figure 2, and the effect of the combination of oligonucleotide-modified antibodies used on background fluorescence intensity was investigated. Specifically, the phosphorylation reaction solution was prepared in the same manner as in Example 1, reacted at 30°C for 60 minutes, and then a mixture of antigen-antibody reaction reagent and ICA reaction reagent was added, and the mixture was incubated at 37°C for 60 minutes using a T-Gradient (biometler). Subsequently, the mixture was transferred to a microwell and reacted at 66°C for 90 minutes, and the fluorescence of Alexa 488 was detected using a fluorescence microscope. The antibody combination conditions for the antigen-antibody reaction solution used at this time were the same as in Example 1.

[0117] Figure 10 shows the results of detecting the immuno-ICA reaction performed under each reaction condition using a microwell device. Brightness data was extracted from images taken with a microscope (product name "All-in-One Fluorescence Microscope", model "BZ-X810", Keyence Corporation) and a histogram was created. In Figure 10, the vertical axis represents frequency and the horizontal axis represents brightness. The upper part of Figure 10 shows the results for ICA reaction solution (S), and the lower part shows the results for ICA reaction solution (N).

[0118] As shown in Figure 10, under PC conditions, a large number of wells with a brightness of 36,000 or higher were detected in ICA reaction solution (N), similar to ICA reaction solution (S). In contrast, under conditions 1 and 2, the number of wells with a brightness of 36,000 or higher in ICA reaction solution (N) was very small. These results clearly demonstrate that background noise can be reduced even in digital measurements using fluid devices by using two types of phosphorylated antibodies.

[0119] [Example 3] The effect of the concentrations of two types of oligonucleotide-modified antibodies in the ICA reaction solution on the background was investigated. Specifically, after the phosphorylation reaction, the antibody set of Condition 1 in Table 1 was used at concentrations of 0.01 to 12 nM as shown in Figure 11, and the fluorescence detection time of Alexa 488 was set to 120 minutes, except that the procedure was the same as in Example 1.

[0120] Figure 11(A) shows the fluorescence intensity (S) of the positive reaction (fluorescence intensity of ICA reaction solution (S)) at a reaction time of 60 minutes, and Figure 11(B) shows the background fluorescence intensity (S) (fluorescence intensity of ICA reaction solution (N)) at a reaction time of 60 minutes. In the matrix, the horizontal axis represents the concentration of the DNA1-modified antibody, and the vertical axis represents the concentration of the DNA2-modified antibody.

[0121] As shown in Figure 11, when the concentration ratio of the two oligonucleotide-modified antibodies in the ICA reaction solution was 0.1 nM for DNA1-modified antibody and 1-12 nM for DNA2-modified antibody; 1 nM for DNA1-modified antibody and 0.1-12 nM for DNA2-modified antibody; 4 nM for DNA1-modified antibody and 0.1-4 nM for DNA2-modified antibody; 8 nM for DNA1-modified antibody and 0.1-1 nM for DNA2-modified antibody; and 12 nM for DNA1-modified antibody and 0.1-1 nM for DNA2-modified antibody, the brightness value (S) for the positive reaction was 200 or higher, and the background brightness value (S) was less than 400, indicating that sufficient detection sensitivity was achieved.

[0122] Figure 12 shows the maximum value of the St / Nt ratio (the ratio of the fluorescence intensity value of the ICA reaction solution (S) (St) to the fluorescence intensity value of the ICA reaction solution (N) (Nt) at time t from the start of measurement) when the ICA is measured over time (Figure 12(A)), and the measurement time T at which the St / Nt ratio reaches its maximum value. ｍａｘ This figure shows the measurement results (Figure 12(B)).

[0123] As shown in Figures 12(A) and (B), when the concentration ratio of the two oligonucleotide-modified antibodies in the ICA reaction solution is 1 nM for DNA1-modified antibody and 1 to 12 nM for DNA2-modified antibody; 4 nM for DNA1-modified antibody and 1 to 4 nM for DNA2-modified antibody; 8 nM for DNA1-modified antibody and 1 nM for DNA2-modified antibody; and 12 nM for DNA1-modified antibody and 1 nM for DNA2-modified antibody, the St / Nt ratio is sufficiently large at 4.0 or higher, and T ｍａｘ The detection time was sufficiently short, less than 30 minutes. The brightness value (S) for positive responses was 200 or higher, and the background brightness value (S) was less than 400, indicating that sufficient detection sensitivity was achieved.

[0124] [Example 4] Phosphorylated SHC was detected using RET (Rearranged During Transaction) and SHC (phosphorylated by RET).

[0125] (Preparation of a mixture of antigen-antibody reaction reagent and ICA reaction reagent) Anti-pSHC (Y427) antibody, anti-pSHC (Tyr239 / 240) antibody, MgCl ２ A mixture was prepared by mixing the ICA reaction reagent for detection. The volume of each mixture was 20 μL. The final concentrations of the anti-pSHC (Y427) antibody were adjusted to 0.001 nM, 0.005 nM, 0.01 nM, 0.05 nM, 0.1 nM, or 0.5 nM. The final concentrations of the anti-pSHC (Tyr239 / 240) antibody were adjusted to 0.05 nM, 0.1 nM, 0.5 nM, 1 nM, 2 nM, or 4 nM. Can Get Signal buffer (TOYOBO) solution A was used as the buffer. The ICA reaction reagent consists of 2 μM allele probe (5'-AGGCGCACGGAGGAATTGCTCACAGAAAGGA-3') (Fasmac, SEQ ID NO: 3), 4 μM FRET cassette 1 (5'-XTTCTYAGCCGGTTTTCCGGCTGAGACCTCCGTGCGCT-3': a nucleotide sequence represented by SEQ ID NO: 4 with fluorescent substrate X (Alexa 488 + Amino C6) added to the 5' end, and quenching agent Y (Black Hole Quencher (BHQ) 1-dT) inserted between the 4th and 5th bases of the nucleotide sequence; manufactured by Nippon Bioservices Co., Ltd.), 0.108 μM flap endonuclease (FEN)-1, 50 mM Tris-HCl (pH 8.5), and 5 mM MgCl ２ It also contained 0.05% Tween 20. Note that the concentrations of each component in these reaction reagents are the final concentrations in the reaction solution.

[0126] (Phosphorylation Reaction) A phosphorylation reaction solution (S) was prepared by mixing 0 nM or 250 pM RET, 7 nM SHC, 100 μM ATP, and solution A to measure the background fluorescence intensity or the fluorescence intensity of the positive reaction. The volume of each reaction solution was 7.5 μL. The above concentrations are the final concentrations in the reaction solution. Each phosphorylation reaction solution was reacted at 30°C for 60 minutes to carry out the phosphorylation reaction.

[0127] (Immuno-ICA reaction) A mixture of antigen-antibody reaction reagent and ICA reaction reagent was added to the phosphorylation reaction solution (S) after the phosphorylation reaction to prepare ICA reaction solution (S) for measuring the fluorescence intensity of the positive reaction. Similarly, a mixture of antigen-antibody reaction reagent and ICA reaction reagent was added to the phosphorylation reaction solution (N) after the phosphorylation reaction to prepare ICA reaction solution (N) for measuring the background fluorescence intensity. The composition of the mixture of antigen-antibody reaction reagent and ICA reaction reagent is shown in Table 3.

[0128]

[0129] Each ICA reaction solution was reacted using a T-Gradient (Biometler) at 37°C for 60 minutes, followed by a Light Cycler 480 (Roche Diagnostics) at 66°C for 120 minutes, and the fluorescence of Alexa 488 was detected.

[0130] Figure 13 shows the maximum value of the St / Nt ratio (the ratio of the fluorescence intensity value of the ICA reaction solution (S) (St) and the fluorescence intensity value of the ICA reaction solution (N) (Nt) at time t from the start of measurement) when the ICA is measured over time (Figure 13(A)), and the measurement time T at which the St / Nt ratio reaches its maximum value. ｍａｘ This figure shows the measurement results (Figure 13(B)). In the matrix, the horizontal axis represents the concentration of DNA1 modification anti-pSHC (Y427) antibody, and the vertical axis represents the concentration of DNA2 modification anti-pSHC (Tyr239 / 240) antibody.

[0131] As shown in Figures 13(A) and (B), the maximum St / Nt ratio was 2.05 or higher when the concentration ratio of the two oligonucleotide-modified antibodies in the ICA reaction solution was 0.005 nM for DNA1-modified antibody and 0.5-4 nM for DNA2-modified antibody; 0.01 nM for DNA1-modified antibody and 0.1-4 nM for DNA2-modified antibody; 0.05 nM for DNA1-modified antibody and 0.05-4 nM for DNA2-modified antibody; 0.1 nM for DNA1-modified antibody and 0.05-4 nM for DNA2-modified antibody; 0.5 nM for DNA1-modified antibody and 0.05-4 nM for DNA2-modified antibody; and 1 nM for DNA1-modified antibody and 0.5-4 nM for DNA2-modified antibody.

[0132] Furthermore, when the concentration ratio of the two oligonucleotide-modified antibodies in the ICA reaction solution is as follows: 0.05 nM for DNA1-modified antibody and 2-4 nM for DNA2-modified antibody; 0.1 nM for DNA1-modified antibody and 1-4 nM for DNA2-modified antibody; 0.5 nM for DNA1-modified antibody and 0.5-4 nM for DNA2-modified antibody; or 1 nM for DNA1-modified antibody and 0.1-4 nM for DNA2-modified antibody, T ｍａｘ But it was sufficiently short, under 30 minutes.

[0133] The maximum value of St / Nt was high, above 2.86, when the concentration ratio of the two oligonucleotide-modified antibodies in the ICA reaction solution was as follows: when the DNA1-modified antibody was 0.005 nM and the DNA2-modified antibody was 1-4 nM; when the DNA1-modified antibody was 0.01 nM and the DNA2-modified antibody was 0.5-4 nM; when the DNA1-modified antibody was 0.05 nM and the DNA2-modified antibody was 0.1-4 nM; when the DNA1-modified antibody was 0.1 nM and the DNA2-modified antibody was 0.1-4 nM; when the DNA1-modified antibody was 0.5 nM and the DNA2-modified antibody was 0.1 nM; and when the DNA1-modified antibody was 1 nM and the DNA2-modified antibody was 2-4 nM.

[0134] According to the present invention, it is possible to provide a technique for detecting phosphorylated proteins with higher sensitivity by reducing background noise when detecting them using the ICA reaction.

[0135] 100...complex, 110...target protein, 111...first phosphorylation site, 112...second phosphorylation site, 120...first specific binding substance, 121...first single-stranded nucleic acid fragment, 130...second specific binding substance, 131...second single-stranded nucleic acid fragment, 140...double-stranded nucleic acid, 200, 500...fluid device, 210...substrate, 220...lid member, 221...protrusion, 222...inlet port, 223...outlet port L210...well, 230...channel, 240...well array, 241...well, 242...sealed well (microcompartment), L210...reagent solution, L220...sealing solution, 242R...well where signal was detected, 510...wall member, 810...flap probe, 811...first flap site (nucleic acid fragment), 821...second flap site (nucleic acid fragment), 820, 820'...nucleic acid fragment, F...fluorescent substance, Q...quenching substance.

Claims

1. A method for detecting a target protein in a sample, comprising: introducing the sample, a first specific binding substance for the target protein labeled with a first single-stranded nucleic acid fragment, and a second specific binding substance for the target protein labeled with a second single-stranded nucleic acid fragment into a well, and if the target protein is present in the sample, a complex comprising the target protein, the first specific binding substance, and the second specific binding substance is formed, and at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment hybridize to form a double-stranded nucleic acid; and detecting the formation of the double-stranded nucleic acid by Invasive Clevage Assay, wherein the target protein is a phosphorylated protein having two or more phosphorylation sites, and the first specific binding substance is a substance that specifically recognizes a first phosphorylation site in the target protein. A method wherein the second specific binding substance is a substance that specifically recognizes a second phosphorylation site in the target protein that is different from the first phosphorylation site, and the detection of the formation of the double-stranded nucleic acid indicates the presence of the target protein.

2. The method according to claim 1, wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.05 to 2 nM.

3. The method according to claim 1, wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 0.1 to 4 nM, and the concentration of the other single-stranded nucleic acid fragment is 1 to 4 nM.

4. The method according to claim 1, wherein the concentration of either the first or second single-stranded nucleic acid fragment in the solution in the well is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.1 to 1 nM.

5. The method of claim 1, wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.05 to 2 nM.

6. The method of claim 1, wherein the concentration of either the first or second specific binding substance in the solution in the well is 0.1 to 4 nM, and the concentration of the other specific binding substance is 1 to 4 nM.

7. The method of claim 1, wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.1 to 1 nM.

8. The method according to any one of claims 2 to 7, wherein the target protein is phosphorylated MEK.

9. Let t be the time from the start of measurement of the Invasive Clevage Assay, St be the fluorescence intensity value detected at measurement time t in the well where the complex is formed, and Nt be the fluorescence intensity value detected at measurement time t in the well where the complex is not formed, then the measurement time T at which the St / Nt ratio is maximum is... ｍａｘ The method according to claim 1, wherein the St / Nt ratio is greater than 4.

0.

10. The measurement time T ｍａｘ The method according to claim 9, wherein the duration is 40 minutes or less.

11. The method according to claim 9, wherein the concentrations of the first and second single-stranded nucleic acid fragments in the solution in the well are such that when the concentration of one of the first and second single-stranded nucleic acid fragments is 1 to 12 nM, the concentration of the other is 0.5 to 2 nM.

12. The method according to claim 9, wherein the concentrations of the first and second single-stranded nucleic acid fragments in the solution in the well are such that the concentration of one of the first and second single-stranded nucleic acid fragments is 1 to 4 nM, and the concentration of the other single-stranded nucleic acid fragment is 1 to 4 nM.

13. The method according to claim 9, wherein the concentrations of the first and second single-stranded nucleic acid fragments in the solution in the well are such that the concentration of one of the first and second single-stranded nucleic acid fragments is 1 to 12 nM, and the concentration of the other single-stranded nucleic acid fragment is 0.8 to 1.2 nM.

14. The method according to claim 9, wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.5 to 2 nM.

15. The method according to claim 9, wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 4 nM, and the concentration of the other specific binding substance is 1 to 4 nM.

16. The method according to claim 9, wherein the concentration of either the first or second specific binding substance in the solution in the well is 1 to 12 nM, and the concentration of the other specific binding substance is 0.8 to 1.2 nM.

17. The measurement time T ｍａｘ The method according to claim 9, wherein St is greater than 200 and Nt is less than 400.

18. The method according to claim 1, wherein the volume of the well is 10 fL to 100 pL.

19. The method according to claim 1, wherein the distance between the first phosphorylation site and the second phosphorylation site is such that at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment can hybridize.

20. The method according to claim 1, wherein the base lengths of the first single-stranded nucleic acid fragment and the second single-stranded nucleic acid fragment are each 10 to 200 bases.

21. The method according to claim 1, further comprising the step of introducing the sample, the first specific binding substance, and the second specific binding substance into the well, and then sealing the opening of the well.

22. The method according to claim 21, wherein the well with a sealed opening contains a first specific binding agent that is not bound to the target protein, and / or a second specific binding agent that is not bound to the target protein.

23. A kit for detecting a target protein, comprising: a well array having a plurality of wells; a first specific binding substance for the target protein labeled with a first single-stranded nucleic acid fragment; and a second specific binding substance for the target protein labeled with a second single-stranded nucleic acid fragment, wherein the target protein is a phosphorylated protein having two or more phosphorylation sites; the first specific binding substance is a substance that specifically recognizes a first phosphorylation site in the target protein; and the second specific binding substance is a substance that specifically recognizes a second phosphorylation site in the target protein that is different from the first phosphorylation site.

24. The kit according to claim 23, further comprising a sealing liquid for sealing the opening of the well.

25. The kit according to claim 23 or 24, further comprising a reagent for detecting a double-stranded nucleic acid formed by the hybridization of at least a portion of the first single-stranded nucleic acid fragment and at least a portion of the second single-stranded nucleic acid fragment.

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