Highly sensitive method for detecting target antigen using isothermal nucleic acid amplification

Abstract

The present invention relates to an antibody-nucleic acid probe complex in which an antibody specifically binding to a target antigen and a nucleic acid probe for isothermal nucleic acid amplification are bound directly or through a mediator such as a nanoparticle, etc. and to a method for detecting a target antigen with higher sensitivity using same. When the target antigen is present, an amplified detection signal can be generated by driving an isothermal nucleic acid amplification reaction through the nucleic acid probe immobilized by antigen-antibody binding. Accordingly, the method is an assay having higher sensitivity than conventional antigen detection methods, and since the method is performed at an isothermal temperature and does not require a device for temperature control, the method can also be applied to point-of-care diagnosis of various diseases.

Description

High-sensitivity target antigen detection method using isothermal nucleic acid amplification

[0001] The present invention relates to a method for detecting a target antigen with high sensitivity using an isothermal amplification method, and more specifically, to an antibody-nucleic acid probe complex in which an antibody that specifically binds to a target antigen and a nucleic acid probe for isothermal amplification are directly bound or bound through a medium such as nanoparticles, and a method for detecting a target antigen by using said complex to drive an isothermal amplification reaction through a nucleic acid probe immobilized by the specific binding of the antigen and antibody when the target antigen is present, and measuring the amplified signal.

[0002]

[0003] Target antigen detection technology plays a pivotal role in the fields of modern medicine and life sciences. Its importance in clinical applications is further emphasized as it provides essential information not only for disease diagnosis but also for treatment, such as vaccine development. Currently, representative antigen detection technologies primarily utilized are LFA (Lateral Flow Assay)-based rapid antigen detection technology and ELISA (Enzyme-Linked Immunosorbent Assay). LFA-based antigen detection technology detects target antigens by utilizing gold nanoparticles and paper strips conjugated with antibodies capable of specifically binding to the target antigen; while this enables very rapid detection, it has limitations in detecting low concentrations of target antigens due to low sensitivity (Banerjee Ruptanu and Amit Jaiswal, Analyst 143.9 (2018): 1970-1996). ELISA is an antigen detection method that offers relatively high sensitivity; generally, it binds to the target antigen by immobilizing antibodies that specifically bind to the target antigen on the surface of a test plate where the reaction takes place. Subsequently, a detection antibody capable of binding to the conjugated target antigen in a sandwich form is added. Peroxidases, which can generate colorimetric signals through reactions with specific substrates, are immobilized on the detection antibody to detect the target antigen through the generation of a final signal (Falzone, Luca, et al., International journal of molecular medicine 47.6 (2021): 1-23). ​​Although ELISA offers higher sensitivity compared to LFA-based antigen analysis methods, it exhibits limitations in situations requiring point-of-care diagnosis or rapid response due to disadvantages such as complex procedures, long testing times, and high costs; furthermore, the issue of sensitivity to low concentrations of target antigens has not yet been completely overcome (Peng, Ping, et al., TraAC Trends in Analytical Chemistry 152 (2022): 116605).

[0004]

[0005] The information described above in the background section is intended solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to those skilled in the art to which the present invention belongs.

[0006]

[0007] Summary of the Invention

[0008] The objective of the present invention is to provide an antibody-nucleic acid probe complex comprising an antibody and a nucleic acid probe, and a method for more sensitively detecting or quantifying a target antigen through an isothermal amplification reaction using the same.

[0009]

[0010] To achieve the above objective, the present invention provides an antibody-nucleic acid probe complex comprising an antibody that specifically binds to a target antigen and a nucleic acid probe for isothermal amplification.

[0011] The present invention also provides a kit for detecting a target antigen comprising the antibody-nucleic acid probe complex.

[0012] The present invention also provides a method for detecting a target antigen comprising the following steps: (a) contacting a sample containing a target antigen with the antibody-nucleic acid probe complex, then adding a composition for isothermal nucleic acid amplification, and performing an isothermal amplification reaction to produce a nucleic acid product; and (b) adding a signal-generating substance to the nucleic acid product produced in step (a), and measuring the generated signal to detect the target antigen.

[0013]

[0014] FIG. 1 is a schematic diagram of a complex of a target antigen-specific antibody and a signal amplification nucleic acid probe according to the present invention. The antibody and the nucleic acid probe may bind directly or may bind through a medium such as nanoparticles.

[0015] FIG. 2 schematically illustrates a method for detecting a target antigen using isothermal nucleic acid amplification technology according to the present invention. When a target antigen is present in a sample, the target antigen binds in a sandwich form with an antibody immobilized on a detection plate or LFA strip and an antibody-nucleic acid probe complex, and through this process, an isothermal nucleic acid amplification reaction is driven through the immobilized nucleic acid probe, and the target antigen can be detected through the reaction product.

[0016] FIG. 3 is a graph showing the results of an efficacy experiment of the target antigen detection technology according to the present invention (respectively, 1: a sample containing the N protein of the SARS-CoV-2 virus which is the target antigen; 2: a sample containing the S protein of the SARS-CoV-2 virus; 3: real-time fluorescence signal values ​​generated from a sample not containing the antigen).

[0017] FIG. 4 is a graph showing the results of a sensitivity experiment of the target antigen detection method according to the present invention (real-time fluorescence signal values ​​generated from samples of N protein 1: 10 ng / mL; 2: 5 ng / mL; 3: 2.5 ng / mL; 4: 1.25 ng / mL; 5: 0.625 ng / mL; 6: 0.3125 ng / mL; 7: 0 ng / mL, respectively).

[0018] FIG. 5 is a graph showing the results of a specificity experiment of the target antigen detection method according to the present invention (the concentration of SARS-CoV-2 N protein is 1 ng / mL; the concentration of N protein of Influenza A, Influenza B, MERS-CoV, and HCoV-229E is 10 ng / mL).

[0019]

[0020] Detailed Description of the Invention and Preferred Embodiments

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a skilled expert in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0022]

[0023] The inventors of the present invention aimed to develop a new target antigen detection technology that can overcome the limitations of existing technologies and simultaneously satisfy rapid result generation and high sensitivity. By utilizing isothermal nucleic acid amplification technology, which does not require temperature control and can produce a specific nucleic acid sequence to amplify the final signal, the inventors developed a technology with higher sensitivity than existing target antigen detection technologies, thereby completing the present invention.

[0024]

[0025] The inventors have developed a high-sensitivity target antigen detection technology by utilizing isothermal nucleic acid amplification technology to improve the relatively low sensitivity of existing target antigen detection technologies. In one embodiment of the present invention, an antibody-nucleic acid probe complex (Fig. 1) is utilized in which an antibody that specifically binds to a target antigen and a nucleic acid probe for an isothermal amplification reaction are directly bound or bound through a medium such as a nanoparticle. When a target antigen is present, the antibody-nucleic acid probe complex is immobilized on a plate or LFA by antigen-antibody binding, and an isothermal amplification composition including a promoter, RNA polymerase, etc. is administered thereto to drive an isothermal nucleic acid amplification reaction by the nucleic acid probe conjugated to the antibody-nucleic acid probe complex (Fig. 2). Through this, a method is implemented to detect the target antigen more sensitively by detecting a signal from a fluorescent substance that can generate a signal by binding to the nucleic acid amplification product, a colorimetric signal generated through the characteristics of the nucleic acid amplification product, or an electrochemical signal generated through the nucleic acid amplification product.

[0026]

[0027] Accordingly, in one aspect, the present invention relates to an antibody-nucleic acid probe complex comprising an antibody that specifically binds to a target antigen and a nucleic acid probe for isothermal amplification.

[0028]

[0029] In this specification, the term "isothermal amplification" refers to a method of amplifying a target nucleic acid under constant reaction temperature conditions. While the conventional Polymerase Chain Reaction (PCR) method requires temperature changes during the steps of denaturation, annealing, and extension, the isothermal amplification method has the advantage of enabling annealing and extension at a constant temperature.

[0030] In the present invention, the isothermal amplification may be characterized by being selected from the group consisting of TMA (Transcription-Mediated Amplification), LAMP (Loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification), HDA (Helicase-Dependent Amplification), NEAR (Nicking Enzyme Amplification Reaction), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid-Sequence-Based Amplification), RCA (Rolling Circle Amplification), SMART (Signal Mediated Amplification of RNA Technology), IMDA (Isothermal Multiple Displacement Amplification), and SPIA (Single Primer Isothermal Amplification), but is not limited thereto.

[0031]

[0032] In this specification, the term “antibody” is a substance that specifically binds to an epitope of an antigen to induce an antigen-antibody reaction, and said antibody may include variants thereof containing an antigen / epitope-binding moiety, antibody fragments or derivatives, antibody analogs, engineered antibodies, or substances that bind to an antigen in a manner similar to that of an antibody. Typically, an antibody comprises variable domains of a heavy chain and a light chain. Both the heavy chain and the light chain are divided into regions of structural and functional homology. Generally, the N-terminal portion of the antibody chain is the variable portion, the C-terminal portion is the constant region, and the CH3 and CL domains typically comprise the C-terminals of the heavy chain and the light chain, respectively. Generally, the variable region enables the antibody to selectively recognize and specifically bind to an epitope on an antigen. Thus, the VL domain and VH domain of the antibody, or a subset of complementarity determining regions (CDRs) within these variable domains, combine to form a variable region that forms an antigen-binding domain. The antigen-binding domain is typically defined by three CDRs on each VL and VH domain. The six complementation determining regions, or CDRs, typically present on each antigen-binding domain are short, discontinuous sequences of amino acids specifically positioned to form the antigen-binding domain. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the antigen. This complementary surface facilitates the non-covalent binding of the antibody to that cognate epitope.

[0033] In the present invention, the antibody or its antigen-binding fragment comprises an invariant region including an IgA, IgD, IgE, IgG, or IgM domain. The antibody or its antigen-binding fragment may be derived from a mouse, rat, goat, rabbit, chicken, guinea pig, hamster, horse, or sheep.

[0034]

[0035] In the present invention, the nucleic acid probe may be characterized by including the nucleotide sequence of SEQ ID NO. 1, but is not limited thereto.

[0036] In this specification, the term “probe” is a nucleic acid capable of binding to a target nucleic acid of a complementary sequence through one or more types of chemical bonds, generally through the formation of complementary base pairs, usually through the formation of hydrogen bonds, and thus forming a duplex structure. To facilitate the detection of the probe when the probe hybridizes to the probe’s complementary target, the probe may be labeled with a detectable label, or the probe may not be labeled but may be detected directly or indirectly by specific binding with a labeled ligand.

[0037] In this specification, the term “nucleic acid” means a polymer of nucleotides. The nucleic acid may be DNA, RNA, or an analog thereof (e.g., a phosphorothioate analog). The nucleic acid may also include modified bases and / or a backbone (e.g., a modified phosphate linkage or a modified sugar moiety). Non-limiting examples of synthetic backbones that impart stability and / or other advantages to the nucleic acid may include a phosphorothioate linkage, a peptide nucleic acid, a lock nucleic acid, a xylose nucleic acid, or an analog thereof.

[0038]

[0039] In the present invention, the antibody and the nucleic acid probe may be characterized by being directly bound or bound via a nanoparticle.

[0040] In the present invention, the antibody and the nucleic acid probe may be characterized by independently binding to the surface of the nanoparticles to form an antibody-nucleic acid probe complex.

[0041] In the present invention, the nanoparticles may be metal nanoparticles, and the metal nanoparticles may be gold, platinum, silver, copper, or palladium nanoparticles, and more specifically, may be gold nanoparticles, but are not limited thereto.

[0042]

[0043] In another aspect, the present invention relates to a kit for detecting a target antigen comprising the antibody-nucleic acid probe complex.

[0044] In the present invention, the kit may further comprise a composition for isothermal nucleic acid amplification for an isothermal amplification reaction or a signal generating material for detecting nucleic acid products, but is not limited thereto.

[0045]

[0046] In one embodiment of the present invention, an isothermal amplification reaction using T7 polymerase and Cas13a was performed as one example among various isothermal nucleic acid amplification reactions. According to the present invention, an antibody capable of specifically binding to a target antigen is immobilized on a detection plate or LFA and a complex thereon. Only when the target antigen is present in the sample is an antibody-nucleic acid probe complex formed via sandwich binding and immobilized on the plate or LFA. Subsequently, after a washing process, when an isothermal nucleic acid amplification composition (a composition comprising a T7 promoter, T7 RNA polymerase, and rNTPs) is added to the plate or LFA, an in-vitro transcription reaction is driven by the composition and the nucleic acid probe conjugated to the antibody-nucleic acid probe complex, and a specific nucleic acid sequence is amplified. The amplified RNA nucleic acid sequence binds to a crRNA sequence to induce non-specific cleavage activity of Cas13a, and during this process, a fluorescent signal is generated by the cleavage of the FAM-UUUUUU-BHQ1 reporter. It was confirmed that by measuring the above signal value, the presence or absence of the target antigen (SARS-CoV-2 N protein) could be verified and it could be distinguished from the non-target antigen (SARS-CoV-2 S protein) (Fig. 3).

[0047]

[0048] Accordingly, in another aspect, the present invention relates to a method for detecting a target antigen comprising the following steps:

[0049] (a) a step of contacting a sample containing a target antigen with the antibody-nucleic acid probe complex, then adding a composition for isothermal nucleic acid amplification, and performing an isothermal amplification reaction to produce a nucleic acid product; and

[0050] (b) A step of adding a signal-generating substance to the nucleic acid product generated in step (a) above, and detecting a target antigen by measuring the generated signal.

[0051] In the present invention, the antibody-nucleic acid probe complex may be characterized by contacting the target antigen-containing sample with the antibody-nucleic acid probe complex to fix the antibody-nucleic acid probe complex through antigen-antibody binding, but is not limited thereto.

[0052] In the present invention, the sample may include a target antigen or a non-target antigen, and if a target antigen is present in the sample, the complex may be characterized by being immobilized through antigen-antibody binding with the antibody included in the antibody-nucleic acid probe complex.

[0053] In the present invention, the isothermal amplification reaction may be characterized as being selected from the group consisting of TMA (Transcription-Mediated Amplification), LAMP (Loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification), HDA (Helicase-Dependent Amplification), NEAR (Nicking Enzyme Amplification Reaction), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid-Sequence-Based Amplification), RCA (Rolling Circle Amplification), SMART (Signal Mediated Amplification of RNA Technology), IMDA (Isothermal Multiple Displacement Amplification), and SPIA (Single Primer Isothermal Amplification), but is not limited thereto.

[0054] In the present invention, the isothermal nucleic acid amplification composition may be characterized by comprising a T7 promoter, a T7 RNA polymerase, and rNTPs, but is not limited thereto.

[0055] In the present invention, the signal generating substance is a substance that generates a detection signal through interaction with a nucleic acid product generated by an isothermal amplification reaction, and the detection signal may be characterized as a fluorescent signal, a luminescent signal, a chemiluminescent signal, a colorimetric signal, an immunofluorescence signal, or an electrochemical signal, but is not limited thereto.

[0056] In the present invention, the signal generating substance can be bound to the complementary nucleic acid in various ways, including direct or indirect attachment of a detectable moiety.

[0057] In one embodiment of the present invention, crRNA, Cas13a, and FAM-UUUUUU-BHQ1 were used as signal-generating substances, and the nucleic acid product generated by the isothermal amplification reaction bound to the crRNA sequence to induce non-specific cleavage activity of Cas13a, and the fluorescent signal generated by the cleavage of the FAM-UUUUUU-BHQ1 reporter was detected.

[0058] Examples of fluorescent signal-generating substances include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, monochloride, phycocyanin, phycoerythrin, etc.

[0059] In additional, non-limiting examples, the fluorescent label may include any combination of the fluorescent groups ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594 and ALEXA FLUOR™ 647.

[0060] Commercially available fluorescent nucleotide analogs that are easily introduced into nucleic acid sequences include Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences: Pitzkataway, NJ, USA), Fluorescein-12-dUTP, Tetramethylrhodamine-6-dUTP, Texas Red™-5-dUTP, Cascade Blue™-7-dUTP, Bodipy™FL-14-dUTP, Bodipy™TMR-14-dUTP, Bodipy™TR-14-dUTP, Rhodamine Green™-5-dUTP, Oregon Green™ 488-5-dUTP, Texas Red™-12-dUTP, and Bodipy™ 630 / 650-14-dUTP, BODIPY™ 650 / 665-14-dUTP, AlexaFluor™ 488-5-dUTP, AlexaFluor™ 532-5-dUTP, AlexaFluor™ 568-5-dUTP, AlexaFluor™ 594-5-dUTP, AlexaFluor™ 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, Texas Red™-5-UTP, mCherry, Cascade Blue™-7-UTP, BODIPY™ FL-14-UTP, BODIPY TMR-14-UTP, BODIPY™ TR-14-UTP, Rhodamine Green™-5-UTP, AlexaFluor™ 488-5-UTP, Lexa LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc.: Eugene, Oregon, USA) is included, but not limited to. Nucleic acids may also be stained a priori with intercalating dyes, such as DAPI, YOYO-1, ethidium bromide, cyanine dyes (e.g., SYBR® Green), etc.

[0061]

[0062] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited to these examples.

[0063]

[0064] Example 1: Establishment of reaction conditions for target antigen detection and quantification

[0065] In order to detect and quantify a target antigen in a sample using the target antigen detection technology according to the present invention, an analysis was performed using the nucleocapsid (N) protein of the SARS-CoV-2 virus as the target antigen.

[0066] The method for manufacturing the antibody-nucleic acid probe complex used in this technology is as follows:

[0067] Nucleic acid probes with the activated sulfhydryl (SH) form are prepared using dithiothreitol (DTT) treatment and an NAP-5 purification column (Cytiva).

[0068] Adjust 1 mL of gold nanoparticles (AuNP) to pH 7.4. Mix 10 μl of N protein detection antibody (SARS-CoV-2 Nucleocapsid Antibody (Sekbio Co., Ltd. Cat#: ZLA911-37R)) and 100 μL of activated SH-type nucleic acid probe with the prepared 1 mL of AuNP in a protein low-binding tube and react at -80°C for about 15 minutes.

[0069] After thawing the reaction mixture at room temperature, centrifuge it using a centrifuge (9000 rpm, 10℃), and resuspend it in PBS (pH 7.4) to adjust the final volume to 1 mL. Then, mix the obtained antibody-nucleic acid probe complex with 1% BSA and react at room temperature for 60 minutes.

[0070] After the reaction is finished, the complex is centrifuged again with a centrifuge (9000 rpm, 10℃), resuspended in 100 μL of PBS, and stored in a refrigerator at 4℃.

[0071] The process for preparing the reaction solution of this technology is as follows:

[0072] 50 μL of 2 μg / ml of N protein capture antibody (SARS-CoV-2 Nucleocapsid Antibody (Sekbio Co., Ltd. Cat#: ZLA911-23M)) was added to polystyrene plates (384 wells), incubated at 37°C for 120 minutes, and washed with PBS-Tween buffer. Afterward, 100 μL of w / v 1% BSA (Bovine serum albumin) was added to each plate, incubated at 37°C for 60 minutes, and then washed with PBS-Tween buffer to prepare plates with immobilized antibodies.

[0073] 50 μL of a sample containing a target antigen or non-target substance was added to the plate, incubated at 37°C for 60 minutes, and washed with PBS-Tween buffer. Subsequently, 25 μL of a solution containing an antibody-nucleic acid probe complex was added, incubated at 37°C for 15 minutes, and washed with PBS-Tween buffer. Afterward, the isothermal amplification reaction solution was added to the plate, and the reaction was carried out. The reaction solution (final 20 μL) contained 8.5 μL of DEPC-distilled water, 1 μL of 10X transcription buffer, 1 μL of 10X Cas13a buffer, 1 μL of 200 mM MgCl2, 1 μL of 25 mM rNTP mix, 2 μL of 1 μM T7 promoter, 2 μL of 1 μM crRNA, 0.5 μL of 20 μM FAM-UUUUUU-BHQ1 probe, 1 μL of 50 U / μL T7 RNA polymerase, and 2 μL of 1 μM Cas13a. The prepared reaction solution was placed in a fluorescence analyzer equipped with a temperature control device, and an isothermal nucleic acid amplification reaction was performed at 37°C for 30 minutes, while fluorescence signal values ​​were measured in real time.

[0074] The nucleic acid sequences used in this example are as described in Table 1 below.

[0075]

[0076] Example 2: Verification of the Validity of Target Antigen Detection Technology

[0077] Validation experiments for the present invention were conducted using the reaction conditions described in Example 1.

[0078] As a result of analyzing an analysis sample containing the target antigen SARS-CoV-2 N protein and the non-target antigen S protein using the target antigen detection technology, it was confirmed that only the analysis sample containing the N protein showed significantly high fluorescence signal values ​​(Fig. 3).

[0079]

[0080] Example 3: Verification of sensitivity of target antigen detection technology

[0081] A detection sensitivity verification experiment of the present invention was conducted using the reaction conditions described in Example 1. After preparing analysis samples containing N protein at various concentrations (0, 0.3125, 0.625, 1.25, 2.5, 5, and 10 ng / mL), the samples were analyzed using the target antigen detection technology. As a result, it was confirmed that 5 ng / mL of N protein could be detected within 10 minutes and 0.3125 ng / mL of N protein could be detected within 20 minutes (Fig. 4).

[0082]

[0083] Example 4: Verification of the specificity of target antigen detection technology

[0084] A specificity verification experiment of the detection technology of the present invention was conducted using the reaction conditions described in Example 1.

[0085] As a result of analyzing an analysis sample containing the target antigen SARS-CoV-2 N protein (1 ng / mL) and non-target antigens N protein of Influenza A, influenza B, MERS-CoV, and HCoV-229E (10 ng / mL) using the target antigen detection technology, it was confirmed that only the analysis sample containing the target antigen SARS-CoV-2 N protein showed significantly high fluorescence signal values ​​(Fig. 5). Consequently, it was confirmed that the target antigen of the antibody constituting the antibody-nucleic acid probe complex according to the present invention can be detected with high specificity.

[0086] Therefore, since the above complex can be modified to use any antibody specific to any specific antigen to be detected, it suggests its usefulness as a general-purpose antigen detection method.

[0087]

[0088] The target antigen detection method according to the present invention has high specificity based on antigen-antibody binding using an antibody that specifically binds to a target antigen, and can provide high sensitivity compared to existing target antigen detection technologies by amplifying the signal through isothermal amplification technology.

[0089]

[0090] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0091]

[0092]

[0093] I have attached the electronic file.

Claims

1. An antibody-nucleic acid probe complex comprising an antibody that specifically binds to a target antigen and a nucleic acid probe for isothermal amplification.

2. An antibody-nucleic acid probe complex according to claim 1, characterized in that the nucleic acid probe comprises the nucleotide sequence of SEQ ID NO.

1.

3. An antibody-nucleic acid probe complex according to claim 1, characterized in that the antibody and the nucleic acid probe bind directly or bind via a nanoparticle.

4. A kit for detecting a target antigen comprising the antibody-nucleic acid probe complex of claim 1.

5. A method for detecting a target antigen comprising the following steps: (a) a step of contacting a sample containing a target antigen with the antibody-nucleic acid probe complex of claim 1, then adding a composition for isothermal nucleic acid amplification, and performing an isothermal amplification reaction to produce a nucleic acid product; and (b) A step of adding a signal-generating substance to the nucleic acid product generated in step (a) above, and detecting a target antigen by measuring the generated signal.

6. A method for detecting a target antigen according to claim 5, wherein the isothermal amplification reaction is selected from the group consisting of TMA (Transcription-Mediated Amplification), LAMP (Loop-mediated isothermal amplification), RPA (Recombinase Polymerase Amplification), HDA (Helicase-Dependent Amplification), NEAR (Nicking Enzyme Amplification Reaction), SDA (Strand Displacement Amplification), NASBA (Nucleic Acid-Sequence-Based Amplification), RCA (Rolling Circle Amplification), SMART (Signal Mediated Amplification of RNA Technology), IMDA (Isothermal Multiple Displacement Amplification), and SPIA (Single Primer Isothermal Amplification).

7. A method for detecting a target antigen according to claim 5, characterized in that the isothermal nucleic acid amplification composition comprises a T7 promoter, a T7 RNA polymerase, and rNTPs.

8. A method for detecting a target antigen according to claim 5, wherein the signal generating substance is a substance that generates a detection signal through interaction with a nucleic acid product generated by an isothermal amplification reaction, and the detection signal is a fluorescent signal, a luminescence signal, a chemiluminescence signal, a colorimetric signal, an immunofluorescence signal, or an electrochemical signal.

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