KR20210133846A - Diagonstic kits for SARS coronavirus 2 comprising the receptor and the antibody binding to SARS coronavirus 2 spike protein - Google Patents

Abstract

The present invention relates to a composition for detecting SARS coronavirus 2 (SARS-CoV-2) using a receptor and antibody binding to SARS coronavirus 2 spike protein for detecting SARS-CoV-2 or diagnosing SARS-CoV-2 infection (COVID-19), to a composition for diagnosing the SARS-CoV-2 infection, to a method for detecting the SARS-CoV-2, and to a kit for diagnosing the SARS-CoV-2 Infection.

Description

SARS coronavirus 2 diagnostic kit comprising the receptor and antibody binding to the SARS coronavirus 2 spike protein {Diagonstic kits for SARS coronavirus 2 comprising the receptor and the antibody binding to SARS coronavirus 2 spike protein}

The present invention relates to a diagnostic kit using a receptor and an antibody that binds to the SARS coronavirus 2 spike protein for effective detection or diagnosis of the SARS coronaviru 2 (SARS-CoV-2) spike protein.

As the gene sequence of SARS-CoV-2, which first started in Wuhan, China and spread as an epidemic, was released, the WHO released the molecular genetic diagnosis method for SARS-CoV-2 on its website ( https://www.who.int/emergencies/diseases/ novel-coronavirus-2019/technical-guidance ).

SARS-CoV-2 is an RNA virus belonging to Coronaviridae, classified as a class 1 infectious disease novel infectious disease syndrome. Until now, it is spread through droplets (saliva) and contact. it is known It is usually known with an incubation period of 1 to 14 days, and has an average incubation period of about 4 to 7 days. The main symptoms include fever, malaise, cough, shortness of breath, and pneumonia, and various other respiratory infections, including sputum, sore throat, headache, hemoptysis, nausea, and diarrhea. Although symptomatic treatment and general-purpose antiviral agents are being administered in clinical practice, there is no specific anti-barrier agent. In order to diagnose the SARS-CoV-2 infection, the virus is isolated from an upper or lower respiratory tract sample and a specific gene is used to diagnose infection through real-time gene amplification.

The name of the coronavirus, first discovered in the 1960s, is derived from the Latin word corona, meaning crown, because of its unique shape of the spike protein. When infected with humans, it causes common cold symptoms, but the mutant viruses SARS (2003) and MERS (2012) caused pneumonia and severe acute respiratory syndrome, leading to death with a high fatality rate. SARS coronavirus 2 is the most recently identified strain of coronavirus, whose genetic structure is 79.5% identical to the SARS virus and 96% identical to the bat coronavirus.

SARS coronavirus 2, like the existing SARS coronavirus, mainly infects the human body through the oral mucosa and lungs. The reason is that a receptor called angiotensin-converting enzyme 2 (ACE2) and the spike protein of SARS coronavirus 2 must bind to penetrate into human host cells. ACE2 is an enzyme that acts on heart function and blood pressure regulation. It is found in the heart, kidneys, gastrointestinal mucosa or lungs, and among them, it is known that the rate of infection through the respiratory tract is higher than that of the internal organs that have to go through the bloodstream. As a result of studying how SARS-CoV-2 penetrates human cells, a joint research team at the West Lake Advanced Research Institute in China and Tsinghua University succeeded in identifying the binding mechanism between the ACE2 receptor and the SARS-CoV-2 Spite protein. In addition, the joint research team at the American Institute of Allergy and Infectious Diseases Vaccine Research Center at the University of Texas in the U.S., using Cryo-EM technology, similarly to the Chinese joint research team, found that the SARS-CoV-2 spike protein has a much higher binding affinity to the ACE2 receptor than the existing SARS-CoV-2 spike protein. reported the results. This difference in binding force may be a clue to explain the high infectivity of SARS-CoV-2 because it binds better with ACE2, a receptor on human host cells.

Existing virus detection uses molecular biological methods such as PCR (gene amplification), but since the PCR technique is amplified exponentially in proportion to time, the sensitivity is high, but it requires specialized pre-processing, complex and specialized experiments, and equipment. There are difficulties in field application. Viruses through real-time RT-PCR using primers and/or probes that specifically bind to cDNA obtained by extracting viral RNA from a sample from a patient currently infected with SARS-CoV-2 in the laboratory and reverse transcribed it Diagnosing infection. However, such real-time gene amplification has a disadvantage in that it is difficult to utilize it for rapid pint-of care.

Currently, a diagnostic method for detecting antibodies through a serological test used for the SARS coronavirus 2 diagnostic test has been developed. This has a difficult drawback.

Although the enzyme immunoassay method was developed for the diagnosis of the SARS coronavirus 2 antigen, it is difficult to make a simple and rapid diagnosis because it requires several processes, takes a lot of time, and requires malfunctioning equipment. The disadvantage is that it is expensive. On the other hand, the immunoassay using the lateral flow assay is a method of reading nanoparticles or fluorescence that can visually identify the presence or absence of antigen-antibody reaction, enabling rapid diagnosis and lower cost compared to enzyme immunoassay there is a thief However, the rapid test method using the existing lateral flow assay method has disadvantages in that it takes a lot of time and money to develop an antibody, such as requiring two types of antibody pairs that bind to an antigen.

Therefore, in the present invention, it was attempted to develop a rapid diagnostic technology using a lateral flow assay method using a pair between a receptor and an antibody instead of two types of antibody pairs that bind to an existing antigen.

Korean Patent No. 1593641 Korean Patent No. 2100813

The present invention was derived from the above needs, and the present inventors completed the present invention by confirming that the receptor and antibody binding to the SARS coronavirus 2 spike protein are effective for detection or diagnosis of the SARS coronavirus 2 spike protein. .

In order to solve the above problems, the present invention is a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody provides a composition for detecting SARS-CoV-2 comprising a secondary antibody to which a visibly identifiable nanostructure is bound, or a visibly identifiable nanostructure is bound to the antibody.

In addition, the present invention is a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody provides a composition for diagnosing SARS-CoV-2 infection comprising a secondary antibody to which a visibly identifiable nanostructure is bound, or a visibly identifiable nanostructure is bound to the antibody.

In another example, the present invention comprises the steps of reacting the separated sample with the composition for detecting SARS coronavirus 2 of claim 1; reacting a sample containing no antigen as a control; reacting for 20 minutes; and analyzing the signal coming from the detection point; provides a method for detecting SARS coronavirus 2 comprising a.

The present invention relates to a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody is a SARS-CoV-2 containing a composition for detecting SARS-CoV-2 containing a secondary antibody to which the eye-identifiable nanostructure is bound, or a secondary antibody to which the eye-identifiable nanostructure is bound to recognize the antibody, and instructions for use. A kit for diagnosing infectious diseases is provided.

The present invention relates to a diagnostic kit comprising a receptor and an antibody binding to the SARS coronavirus 2 spike protein, developed by replacing the immunodiagnostic method using a capture antibody and a detection antibody as a representative method for detecting an antigenic protein, and an antibody. It can be usefully used for detecting virus 2 or diagnosing SARS-CoV-2 infection.

1 shows an intracellular receptor (ACE2)-based LFIA according to an embodiment of the present invention. a) Schematic diagram of ACE2 receptor recognition by SARS coronavirus 2. ACE2 is a cellular receptor for SARS coronavirus 2 as a type 1 membrane protein expressed in lung, heart, kidney and intestine. b) Schematic diagram of E2-based LFIA consisting of sample pad, bonding pad, nitrocellulose membrane and absorbent pad. The detection line located on the nitrocellulose membrane contains ACE2 for detection of the SARS coronavirus 2 spike antigen. Anti-IgG antibodies are used in control lines. The proposed LFIA can achieve sensitive and selective detection for the SARS coronavirus 2 spike antigen within 20 minutes.
2 shows the indirect ELISA results for the spike antigen of three different coronaviruses (SARS coronavirus S1, SARS coronavirus 2 S1 and MERS S1) in one embodiment of the present invention. a) The interaction between the S1 antigen and ACE2 was tested in serially diluted samples (concentrations ranged from 200 to 0.05 ng/mL). In addition, three other antibodies, CR3022 (black) (b); F26G19 (red) (c); and S1-mAb (orange) (d) were used at the same concentration to examine the interaction with the spike antigen.
3 shows the biolayer interference (BLI) results of ACE2, CR3022, F26G19 and S1-mAb against the SARS coronavirus 2 S1 antigen according to an embodiment of the present invention. The dotted line represents the response curve of the BLI measurement, and the solid line represents the titration curve based on the 1:1 binding model. Binding kinetics was measured for four different concentrations of the S1 antigen.
4 shows the results of identification of a sandwich pair for detection of the SARS coronavirus 2 spike antigen according to an embodiment of the present invention. a) Schematic diagram of LFIA using ACE2 as capture probe and sandwich assay results from paired antibodies (CR3022, F26G19 and S1mAb). SARS coronavirus 2 S1 antigen (50 ng) was used as a positive control, and a buffer without S1 antigen was used as a negative control. After 20 minutes, the strips were taken with a smartphone and the peak density was analyzed. b) LFIA schematic diagram and results of sandwich analysis when antibodies were used as detection probes. c) detecting probe (capture probe, P _C) - Diagnostic probe (detection probe, P _D) of the peak density in pairs. A total of 12 pairs of positive controls (50 ng S1 antigen) were tested and densities analyzed. The peak density was calculated by subtracting the background density of the strip from the average density of dots.
5 shows the sensitivity and specificity of ACE2-based LFIA according to an embodiment of the present invention. a) Results of ACE2-based LFIA for detection sensitivity of SARS coronavirus 2 S1 antigen. ACE2-based LFIAs were tested at serially diluted antigen concentrations (concentrations ranged from 500 to 5 ng/mL). After 20 minutes, the LFIA strip was filmed with a smartphone. The density of the detection and control lines was transformed into a peak histogram in the image analyzer. b) Comparative assay of detection selectivity: positive control - SARS coronavirus S1, negative control - SARS coronavirus S1, MERS S1 and buffer solution. The detection efficiency of ACE2-based LFIA was demonstrated using antigen samples at three different concentrations (1 μg/mL, 200 ng/mL, and 50 ng/mL). c) Histogram of peak density for the detection line. After 20 minutes for sample flow, the density of the detection line was measured with a hand-held line analyzer. Inset) detection density for 5 ng antigen per reaction of each control. The limit of detection (LOD) is determined as the mean value of the negative control plus the standard deviation of three trials. P-values: ns > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
6 shows laboratory confirmation results of ACE2-based LFIA using clinical samples according to an embodiment of the present invention. a) Schematic diagram of the COVID-19 test using ACE2-based LFIA. A nasopharyngeal smear from a COVID-19 patient is placed in a UTM. 50 µL of UTM containing SARS-CoV-2 is mixed in the running buffer at a 1:1 (v/v) ratio, and 100 µL of the mixed solution is injected into the LFIA device. After 20 minutes, the line density of the LFIA strips is semi-quantified on a handheld analyzer. b) Results of ACE2-based LFIA on detection sensitivity of cultured SARS coronavirus 2. Serially diluted virus concentrations (concentration ranges from 1.07 x 10 ⁸ copies/mL to 5.35 x 10 ⁶ copies/mL) were tested. After 20 minutes, the LFIA strips were taken with a smartphone and scanned with an image analyzer. The line densities of the detection and control lines were converted into peak histograms. In addition, the density of the _{detection line was measured with a portable line analyzer (I L} : line density of the detection line). Additionally, human coronavirus (OC43) was used as a negative control. c) Histogram of density versus detection line measured on handheld analyzer. The limit of detection (LOD) is determined as the mean value of the negative control (0 copies/mL SARS coronavirus 2) plus the standard deviation of three trials. d) Laboratory validation of ACE2-based LFIA compared to RT-qPCR using clinical samples. i) Nasopharyngeal smear samples from COVID-19 patients (n=4) and healthy individuals (n=4) were measured in both ACE2-based LFIA and RT-qPCR. Sensitivity was determined by dividing the number of actual positive samples by the number of positive samples tested. Specificity was determined by dividing the number of actual negative samples by the number of negative samples tested. ii) RT-qPCR results for detection of SARS coronavirus 2 specific gene (Env gene). Ct values and relative viral load in clinical samples were evaluated. e) ACE2-based LFIA results for laboratory validation using clinical samples of COVID-19 patients. Twenty minutes after sample loading, the detection line density of the LFIA strip was measured with a hand-held line analyzer. The limit of detection (LOD) is determined as the mean value of the negative control (health control) plus the standard deviation of 3 trials.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, in the following description, many specific details such as specific components are shown, which are provided to help a more general understanding of the present invention, and it is common in the art that the present invention can be practiced without these specific details. It will be self-evident to those who have the knowledge of And, in describing the present invention, if it is determined that a detailed description of a related known function or configuration may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted.

In order to achieve the object of the present invention, the present invention provides a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody provides a composition for detecting SARS-CoV-2 comprising a secondary antibody to which a visibly identifiable nanostructure is bound, or a visibly identifiable nanostructure is bound to the antibody.

The present invention uses the binding between the receptor expressed on the surface of a host cell and the SARS coronavirus 2 spike protein, and can detect or diagnose the SARS coronavirus 2 spike protein using a pair between the receptor and the antibody binding to the spike protein. means the skills

The receptor binding to the SARS coronavirus 2 spike protein according to an embodiment of the present invention includes angiotensin converting enzyme 2 (ACE2), a lysate of a cell line expressing or overexpressing ACE2, and preferably an ACE2 protein, but is not limited thereto. does not

The antibody to the SARS coronavirus 2 spike protein according to an embodiment of the present invention is a SARS coronavirus 2 spike protein antibody capable of pairing with ACE2, and is a complete antibody, an antigen-binding fragment of an antibody molecule, a synthetic antibody, a recombinant antibody, or an antibody It may include an antibody hybrid. Preferably, it comprises a monoclonal antibody or a polyclonal antibody, and more preferably, the antibody binding to the SARS coronavirus 2 spike protein recognizes any one of S1, RBD (Receptor binding domain) and RBM (Receptor binding motif) as an antigen. It includes, but is not limited to, any one selected from the group consisting of antibodies.

The nanostructure according to an embodiment of the present invention is a nanostructure that can be identified by the eye, and is a nanostructure to which a dye that can be identified by the eye is bound, preferably cellulose nanoparticles or gold nanoparticles to which a dye that can be identified by the eye is bound. includes, but is not limited to.

The visually identifiable dye has a label that generates a detectable signal as a detection antibody or a secondary antibody. The label can include chemicals (eg biotin), enzymes (alkaline phosphatase, β-galactosidase, horse radish peroxidase and cytochrome P450), radioactive substances (eg C14, I125). , P32 and S35), a fluorescent material (eg, fluorescein), a light emitting material, a chemiluminescent material, and fluorescence resonance energy transfer (FRET).

The sample may be a biological material derived from a subject. The subject may be a vertebrate. The vertebrate may be a mammal. The mammal may be a primate including humans and non-human primates, a camel, or a rodent including mice and rats. The sample may be stored frozen or left in a natural state. The sample may be a water sample, a soil sample, a food sample, an air sample, a nasal swab sample, a nasopharyngeal wash, a branchioalveolar lavage, or a pleural fluid. The biological material may include nasal swap, nasal aspirate, nasopharyngeal swab, nasopharyngeal aspirate, blood or blood constituent, body fluid. fluid), saliva, sputum, or a combination thereof.

The present invention relates to a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody provides a composition for diagnosing SARS-CoV-2 infection comprising a secondary antibody to which a visibly identifiable nanostructure is bound, or a visibly identifiable nanostructure is bound to the antibody.

The present invention provides a composition for diagnosing SARS-CoV-2 infection (COVID-19) in various individuals that can be infected with SARS-CoV-2. The SARS-CoV-2 infection may include flu, cold, sore throat, bronchitis, or pneumonia, but is not limited thereto as long as it is a disease caused by SARS-CoV-2 infection.

In addition, the present invention comprises the steps of reacting the separated sample with the composition for detecting SARS coronavirus 2 of claim 1; reacting a sample containing no antigen as a control; reacting for 20 minutes; and analyzing the signal coming from the detection point; provides a method for detecting SARS coronavirus 2 comprising a.

The present invention relates to a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody is a SARS-CoV-2 containing a composition for detecting SARS-CoV-2 containing a secondary antibody to which the eye-identifiable nanostructure is bound, or a secondary antibody to which the eye-identifiable nanostructure is bound to recognize the antibody, and instructions for use. A kit for diagnosing infectious diseases is provided.

In the diagnostic kit of the present invention, the receptor binding to the SARS coronavirus 2 spike protein may be used in a state immobilized on a solid support. Various materials may be used as the solid support, and examples thereof include, but are not limited to, cellulose, nitrocellulose, polyvinyl chloride, silica gel, polystyrene, nylon, activated beads, and the like.

Such a diagnostic kit may further include tools, reagents, and the like generally used for immunological analysis in the field of the present invention. Such tools or reagents include, but are not limited to, suitable carriers, labels capable of generating a detectable signal, solubilizing agents, detergents, buffers, stabilizers, and the like.

In one embodiment of the present invention, a "kit" is a sample pad, which is a site to which a liquid sample containing an analyte is applied, is fluidly connected to the sample pad, and a dye that can be identified by the eye is bound. As a conjugate pad on which the nanostructure is movably supported, the nanostructure to which the eye identifiable dye is bound is an antibody or fragment thereof that binds to the SARS coronavirus 2 spike protein and the eye identifiable dye is bound. a chromatographic membrane material fluidly connected to the binding pad and to which the liquid sample moves by capillary movement, and a receptor that binds to the SARS coronavirus 2 spike protein downstream of the binding pad or its A chromatography membrane material comprising a detection region to which a fragment is non-diffusively immobilized, an absorbent pad fluidly connected to the chromatography membrane material, and the sample pad, a binding pad, a chromatography membrane material, and It may be a diagnostic kit for detecting SARS coronavirus 2 comprising a solid support supporting a moisture-absorbing pad.

1B is a schematic diagram illustrating an example of a diagnostic kit for detecting or diagnosing SARS coronavirus 2 or a spike protein of SARS coronavirus 2 of the present invention.

In the kit, a chromatographic material membrane having a detection region (T) and a control region (C) on which a receptor capable of binding to the SARS coronavirus 2 spike protein is immobilized is supported on a solid support, and a sample pad is supported therein. , A binding pad and a moisture absorption pad on which a nanostructure in which an antibody binding to the SARS coronavirus 2 spike protein and a visually identifiable dye are supported are superimposed and connected. When the sample is added to the sample pad on the strip, it moves to the binding pad due to capillary action, and the antigen in the sample binds to the nanostructure in which the antibody binding to the SARS coronavirus 2 spike protein in the binding pad and the eye identifiable dye are bound. There is capillary migration in the direction of the absorbent pad through the membrane of the chromatography material. When the antigen and the nanostructure conjugate reach the detection region (T), color is developed, and when the conjugate is not present, color is not developed. In addition, when the conjugate of the antibody and the nanostructure together with the sample passes through the control region (C), it is combined with an antibody specific for the conjugate and develops color, thereby confirming whether the capillary movement is correct.

The kit may be an immunoassay kit using a porous material as a solid carrier of an immunochemical component such as an antigen or antibody. The sample pad, binding pad, chromatography membrane material and moisture absorption pad used in one embodiment of the present invention are materials having sufficient porosity and volume to contain and contain a liquid sample to be analyzed, for example, a microporous membrane. It may be a substance. Examples of the microporous membrane material include nylon, cellulosic material, polysulfone, polyvinylidene difluoride, polyester, and glass fiber. A preferred example of the microporous membrane material is a nitrocellulose membrane. The analysis device may have the form of a strip.

The nanostructure in which the antibody used in the kit of one embodiment of the present invention is coupled with an eye-identifiable dye may be prepared by a method well known in the art.

Advantages and features of the present invention, and methods for achieving them, will become apparent with reference to the embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only these embodiments allow the disclosure of the present invention to be complete, and common knowledge in the technical field to which the present invention belongs It is provided to fully inform the possessor of the scope of the invention, and the present invention is only defined by the scope of the claims.

<Example 1> Enzyme-linked immunosorbent assay (ELISA)

Maxisorp immunoplates (ThermoFisher SCIENTIFIC, MA, USA) were applied to various concentrations (200, 100, 50, 25, 12.5, 6.25, 3.12, 0 ng/mL) per well of SARS-CoV-2 S1 protein (S1 subunit, Cat. No 40591-V08H; Sino Biological, Beijing, China) was aliquoted in 100 μL and coated overnight at 4 °C. Immunoplates were blocked with blocking buffer (Cat. No. DS98200; Invitrogen, CA, USA) for 1 hour, and then hACE2, CR3022, F26G19 or S1-mAb was dissolved in 100 μL blocking buffer and added to each well for 1 hour. reacted while After washing with washing buffer (Cat. No. WB01; Invitrogen), the bound receptor and antibody were horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:2000; Cat. No. 31437; Invitrogen), anti-human IgG (1:2000; Cat. No. 31413; Invitrogen) or anti-rabbit IgG (1:2000; Cat. No. 31463; Invitrogen) detection antibody was reacted for 1 hour. Continue the washing process again, and after dispensing 100 μL of TMB solution (TMB solution, Cat. No. SB01; Invitrogen) to watch the process of color change in the immunoplate, STOP solution (1M HCl) is dispensed to initiate the reaction. stop Then, the absorbance of the sample was measured at 450 nm using a microplate reader (BioTek, VT, USA).

<Example 2> Biolayer interferometry (BLI, biolayer interferometry)

Fc-tag-bound human ACE2 (ACE2, Cat. No. 10108-H05H) and SARS coronavirus 2 spike monoclonal antibody (S1-mAb, Cat. No. 40150-R007), SARS coronavirus 2 spike protein (S1 subunit) , Cat. No. 40591-V08H) and SARS coronavirus 2 RBD (Cat. No. 40592-V08B) were purchased from Sino Biological. Plasmids encoding the heavy and light chains of each antibody were mixed at a ratio of 1:6 and transiently co-infected into 293-F cells using PEI solution (PolyScience, PA, USA). The supernatant was collected 6 days after infection, and CR3022 and F26G19 were purified using a protein A column (GE Healthcare, IL, USA). BLI was assayed on a BLItz instrument (ForteBio, CA, USA) with binding affinity to the SARS coronavirus 2 spike antigen (S1 and RBD) and four different antibodies (ACE2, CR3022, F26G19, and S1-mAb). After the biosensor (ForteBio, USA) was hydrated in tertiary distilled water for 10 minutes, the binding affinity measurement using the biolayer interferometry was performed in five steps [1. Initial baseline setting (30 sec), 2. Antibody immobilization (300 sec), 3. Second baseline setting (120 sec), 4. Antibody-antigen binding (300 sec), 5. Antibody-antigen dissociation (300 sec)] . All procedures are performed using sample dilution buffer (0.02% Tween 20, 150 mM NaCl, and 1 mg/mL BSA in 10 mM PBS with 0.05% sodium azide, pH 7.4). In the sample loading step, each antibody (100 μg/mL) is immobilized through binding of protein A coated on the biosensor surface to the Fc domain of the antibody (or receptor). After setting the initial baseline, four different concentrations of antigen were reacted with the antibody-binding biosensor to confirm antibody-antigen binding. Four types of binding curves were analyzed based on a 1:1 binding model, and binding constants were obtained based on this.

<Example 3> Antibody-binding CNB preparation

Red cellulose nanobeads (CNBs) 1% stock solution was purchased from Asahi Kasei Fibers Corporation (NanoAct, Cat. No. RE2AA; Miyazaki, Japan), and the average diameter of the beads was 345 nm. The CNB binding kit includes a binding buffer, a blocking buffer, and a washing buffer, and was purchased from DCN Diagnostics (CA, USA). SARS coronavirus 2 antigen-specific antibodies (CR3022, F26G19, S1mAb) and binding protein (ACE2) were bound to the surface of CNB and the test was performed according to the manufacturer's instructions. Briefly, 0.12 mL of 0.5 mg/mL antibody was mixed with 0.60 mL of 1% CNB stock solution and 0.12 mL of binding buffer and reacted at 37 °C for 2 h. Then, 7.2 mL of blocking buffer was mixed and reacted at 37 °C for 1 hour. The solution was centrifuged (14,400 x g , 20 minutes, 4 °C), and the pellet was resuspended in 7.2 mL of washing buffer. After centrifugation (14,400 x g , 20 minutes, 4 °C), the washed pellet was resuspended in 0.5 mL of washing buffer. The final concentration of antibody-bound CNB was approximately 0.1%. The exact concentration of CNB was calculated by measuring the absorbance at 554 nm with UV-vis spectrophotometry (Synergy H1; BioTek).

<Example 4> LFIA strip preparation

As shown in FIG. 1b, the LFIA strips were prepared from a sample pad (Ahlstrom, Helsinki, Finland), a binding pad (Ahlstrom, Helsinki, Finland), a nitrocellulose membrane (NC membrane), and an absorbent pad (Ahlstrom, Helsinki, Finland). Binding pads were treated with 0.1% Triton X-100 (Cat. No. T8787; Sigma-Aldrich, MO, USA) prior to CNB fixation. After complete drying, stabilization buffer containing 0.05% antibody-binding CNB solution (10 mM 2-amino-2-methyl-1-propanol (pH 9.0), 0.5% BSA, 0.5% β-Lactose, 0.05% Triton X -100, and 0.05% sodium azide) was sprayed onto the bonding pad, and reacted in a vacuum dryer (FDU-1200, EYELA, Tokyo, Japan; JSVO-30T, JSR, Gongju, Korea) at 37°C for 1 hour. A test line and a control line containing a capture probe were divided into the following conditions on a nitrocellulose membrane using a line dispenser (BTM Inc., Uiwang, Korea). speed, 50 mm/s; dispensing rate, 1 uL/cm). A mixture of ACE2 (1 mg/mL) and 0.5 mg/mL anti-immunoglobulin G antibody [1:1:1 (v/v/v)] in sample dilution buffer (Cat. No. ab154873; Abcam, Cambridge, UK) anti-human IgG (Cat. No. I2136, Sigma-Aldrich)/anti-rabbit IgG antibody (Cat. No. R5506, Sigma-Aldrich)/anti-mouse IgG antibody (Cat. No. A4416, Sigma-Aldrich)] was used for the formation of test lines and control lines, respectively. After line separation, the nitrocellulose membrane was dried at 3 °C for 1 h. To reduce non-specific reactions between the diagnostic probe and the capture probe at the detection line, a blocking solution (10 mM 2-amino-2-methyl-1-propanol (pH) 9.0), 0.5% BSA, 0.5% β-Lactose, 0.05% Triton X-100, 0.05% sodium azide) in a vacuum dryer (37°C) for 1 hour. Each component of the LFIA strip was correctly assembled and cut to 38 mm wide and stored for single LFIA testing.

<Example 5> Dot-blot assay for the discovery of sandwich pairs

Twelve detection probe-diagnostic probe pairs were tested to find a suitable pair for detecting the SARS coronavirus 2 spike antigen. The affinities of four different SARS coronavirus 2 S1 and RBD antibodies were confirmed by ELISA, Western blot and BLI. Anti-human IgG/anti-rabbit IgG antibody/anti-mouse IgG antibody in a mixture of detection probe (1 mg/mL) and 0.5 mg/mL anti-immunoglobulin G antibody [1:1:1 (v/v/v) ] was used for the formation of test dots and control dots, respectively. The dots were immobilized by dropping 0.5 uL of detection or control solution onto nitrocellulose membranes (Advanced Microdevices). The nitrocellulose membrane was dried in a vacuum with a blocking solution (10 mM 2-amino-2-methyl-1-propanol (pH 9.0), 0.5% BSA, 0.5 % β-lactose, 0.05% Triton X-100, 0.05% sodium azide). (1 h, 37 °C). LFIA strips for dot-blot analysis were prepared by the method presented above. For comparative analysis of 12 pairs, 50 ng of the target antigen (SARS coronavirus 2 spike S1) was added to each detection probe in running buffer [10 mM adenosine monophosphate (AMP, pH 9.0), 5 mM EDTA, 200 mM Urea) , 1% Triton X-100, 0.5% Tween 20, 500 mM NaCl, 1% PEG (MW 200)] for 10 minutes at room temperature, and then the mixed sample was loaded onto the LFIA strip. After 20 minutes, a red signal indicating antigen detection was confirmed through direct observation (naked eye) and a smartphone camera. Additionally, all dots were quantitatively analyzed using an image analyzer (Sapphire Biomolecular Imager, Azure Biosystems, CA, USA), and the relative density of each dot was calculated based on the average density of dots excluding the background.

<Example 6> Sensitivity and specificity of LFIA assay

For the development of LFIA with high sensitivity and specificity for the detection of SARS-CoV-2, the concentration of CNB, the concentration of immobilized ACE2, and the composition of the development buffer were optimized. Finally, 0.05% CNB for the detection probe, 1 mg/mL ACE2 for the immobilized capture probe, and development buffer [10 mM AMP, (pH 9.0), 5 mM EDTA, 200 mM urea, 1% Triton X-100, 0.5% Tween 20, 500 mM NaCl, 1% PEG (MW 200)] was selected. SARS-CoV-2 S1 and SARS-CoV-2 RBD antigens were diluted in a sample dilution buffer (Cat. No. ab154873; Abcam) by serial dilution, and the diluted sample was added to the developing buffer at a ratio of 1:9 (v/v). mixed. The final concentrations of the diluted samples ranged from 500 to 5 ng/mL. 100 uL of the development buffer containing each antigen concentration was dropped into the inside of the LFIA device. In this system, the sample flows with capillary force along the LFIA strip and first encounters the antibody (S1-mAb)-bound CNB. When the antigen of the sample binds to the CNB bound to the S1-mAb and an antigen-probe complex is formed, the detection probe (ACE2) pre-immobilized to the nitrocellulose membrane is diagnosed. At 20 minutes after loading the sample, the detection and control lines appear red and analyzed on the Sapphire Biomolecular Imager.

For the specificity test, two different corona-associated spike antigens (eg, SARS coronavirus S1 and MERS S1 antigen) were prepared at three concentrations (100, 20 and 50 ng/mL). Three concentrations of each antigen were loaded into the LIFA apparatus, and the line density was quantified with a portable line analyzer (Light-G; WellsBio, Seoul, Korea). The positive density in the detection line was 50 or higher according to the manufacturer's instructions for the portable analyzer. All experiments were performed in triplicate, and the detection limit in FIG. 5c was calculated as the mean value of the negative control plus the standard deviation of 3 times.

<Example 7> Isolation and copy number quantification of human coronavirus-OC43 (HCoV-OC43) and SARS coronavirus 2

Virus of HCoV-OC43, RNA isolation of gamma-irradiated SARS coronavirus 2 (Cat. No. NR-52287, BEI Resources, VA, USA) was performed using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. was performed with Nucleocapsid (N) gene of HCoV-OC43 (1.16 x 10 ¹² -10 ⁰ copies/μL) or envelope of SARS coronavirus 2 (7.7 x 10 ⁶ -10 ⁰ copies/μL) by serial dilution of viral RNA (Env ) gene was targeted as standard, isolated viral RNA was synthesized with complementary DNA, and continuous RT-qPCR was performed with the Luna® Universal Probe One-Step RT-qPCR Kit (New England BioLabs, MA, USA) according to the manufacturer's instructions. was performed according to

Primers of the HCoV-OC43 gene:

Forward 5'-AGC AAC CAG GCT GAT GTC AAT ACC

Reverse 5'-AGC AGA CCT TCC TGA GCC TTC AAT

Primers of the SARS coronavirus 2 Env gene:

Forward 5'-ACA GGT ACG TTA ATA GTT AAT AGC GT

Reverse 5'-ATA TTG CAG CAG TAC GCA CAC A

FAM (6-carboxyfluorescein) and BHQ-1 (Back Hole Quencher-1) labeled probes:

ACA CTA GCC ATC CTT ACT GCG CTT CG

Single-step RT-qPCR was set for reverse transcription at 55 °C for 10 min, 45 cycles of amplification at 95 °C for 10 sec, and at 60 °C for 30 sec. The reaction was analyzed by Bio-Rad CFX 96 Touch Real-Time PCR System (CA, USA).

<Example 8> Evaluation of clinical use of ACE2-based LFIA

Serial dilutions were made in the development buffer of SARS coronavirus 2 treated with gamma radiation, and a cultured SARS coronavirus 2 sample of ^{1.07 X 10 8} copies/mL to 5.35 X 10 ^{6 copies/mL was prepared.} 100 µL of development buffer containing each virus concentration was dropped into the LFIA device. After 20 minutes, it was confirmed that the detection line appeared red, and it was quantified with a _{LightG portable analyzer (I L: line intensity).} Additionally, the densities of detection and control lines were converted to peak histograms using a Sapphire Biomolecular Imager. Human coronavirus-OC43 (HCoV-OC43) was tested as a negative control in RT-qPCR. Two different concentrations (5 X 10 ⁷ copies/mL, and 5 X 10 ⁶ copies/mL) were loaded into the LFIA device. Twenty minutes after sample loading, non-specific reactions in the detection line were evaluated in the same manner. To evaluate the clinical potential of ACE2-based LFIA, nasopharyngeal smear samples from COVID-19 patients (n=4) and healthy subjects (n=4) were applied to ACE2-based LFIA. Nasopharyngeal smear samples in universal transport media (UTM) collected from COVID-19 patients were provided by Chonbuk National University Hospital (Korea). Nasopharyngeal smear samples from healthy subjects were purchased from LEE Biosolutions (Cat. No. 991-31-NC, MO, USA). A healthy nasopharyngeal smear sample was suspended in UTM (Cat. No. UTNFS-3B-1, Noble Bio, Korea) and used for the LFIA test. 50 uL of UTM obtained from nasopharyngeal smears taken from COVID-19 patients and healthy subjects was mixed in the development buffer at a ratio of 1:1 (v/v) and loaded into the LFIA device. After 20 minutes, the density of the detection line was analyzed with a LightG portable analyzer. The limit of detection was determined as the mean value of the healthy control group plus three standard deviations. RT-qPCR was also used to compare test efficiencies using clinical samples. A primer-probe set was used to detect the SARS coronavirus 2 specific envelope gene (Env gene). The primer is as follows.

Primers of the SARS coronavirus 2 Env gene:

Forward 5'-ACA GGT ACG TTA ATA GTT AAT AGC GT

Reverse 5'-ATA TTG CAG CAG TAC GCA CAC A

FAM (6-carboxyfluorescein) and BHQ-1 (Back Hole Quencher-1) labeled probes:

ACA CTA GCC ATC CTT ACT GCG CTT CG

Single-step RT-qPCR was set for reverse transcription at 55 °C for 10 min, 45 cycles of amplification at 95 °C for 10 sec, and at 60 °C for 30 sec. The reaction was analyzed by Bio-Rad CFX 96 Touch Real-Time PCR System (CA, USA).

<Test Example 1> The interaction of SARS coronavirus 2 S1 antigen and human cell receptor (ACE2)

The surface spike (S) glycoprotein of SARS coronavirus is recognized by the ACE2 receptor, resulting in endocytosis. The S protein of SARS coronavirus 2 and SARS coronavirus are very similar (amino acid sequence homology: -77%). In some studies, the S protein of SARS-CoV-2 outperforms SARS-CoV in ACE2 and reaction affinity. The strong affinity between the target antigen and the antibody (or receptor) is essential for the development of a highly sensitive and accurate diagnostic platform for antigen detection as well as the development of therapeutic agents and vaccines. The interaction of SARS coronavirus 2 S1 protein with ACE2 was determined by detection of SARS coronavirus 2 S protein by Western blot analysis, indirect ELISA and BLI.

The interaction of the receptor (or antibody) with the SARS coronavirus 2 S1 protein was measured by Western blot. Anti-SARS coronavirus 2 antibodies (CR3022, F26G19, S1-mAb) and human FC labeled ACE2 receptor (ACE2-Fc) were shown to be able to detect SARS coronavirus 2 S1 protein. This interaction was confirmed by ELISA, and the result was that the ACE2 receptor binds more strongly to the SARS coronavirus 2 S1 protein compared to the SARS coronavirus S1 protein, but not the MERS coronavirus S1 protein. In contrast, the commercial anti-SARS coronavirus 2 antibody showed similar affinity to the SARS coronavirus S1 protein and the SARS coronavirus 2 S1 protein. The detection limits of the ACE2 receptors, CR3022, F26G19 and S1-mAb for the SARS coronavirus 2 S1 protein in the ELISA were approximately 125, 3.13, 3.13 and 0.78 ng/mL. In addition, the detection limits of the ACE2 receptors, CR3022, F26G19 and S1-mAb for SARS coronavirus 2 RBD were 3.13, 125, 0.05 and 0.05 ng/mL. As a result of using the BLI method to confirm the detailed binding kinetics of ACE2 and the SARS coronavirus 2 S1 protein, the affinity of ACE2 for two mutations of the SARS coronavirus 2 spike (S1 and RBD) protein was measured. Three different commercial antibodies (CR3022, F26G19 and S1-mAb) were used to confirm binding to SARS coronavirus 2 S1 in the BLI assay. BLI is a label-exclusion technique for measuring biomolecular interactions related to changes in interference patterns after binding. The end of the BLI biosensor was coded for protein A, which was used to detect an effective target antibody. Three different commercial antibodies (CR3022, F26G19 and S1-mAb) and ACE2-Fc were immobilized on the surface of the BLI biosensor through specific interactions with protein A. The interaction with these antibodies was tested with SARS coronavirus 2 S1 and RBD. Representative real-time binding sensegrams (dotted lines) and calibration curves (solid lines) for receptor (or antibody)-antigens are shown in FIG. 3 . Binding kinetic analysis was performed at 4 different antigen concentrations, and kinetic constants were calculated from 4 curves based on a 1:1 binding model. _{The K D} values of ACE for S1 and RBD were 319.7 and 13.18 nM. The RBD of S1 plays a major role in the reaction with host cell receptor ACE2. Previous studies have suggested that each monolith of the trimeric S protein of SARS coronavirus binds to ACE. It is known that the proteolytically activated S protein of SARS coronavirus 2 by furin, a proproteinase, has a higher binding affinity to ACE2 through RBD. Therefore, in the present invention, since RBD can bind to ACE2 more effectively compared to S1, the K _D value of ACE2 is expected to be lower in RBD compared to S1 binding. This also indicates that targeting the monomer of SARS coronavirus 2 S1 may be more effective compared to targeting the S1 protein by using ACE2 and antibody pairing to detect the antigen, and why the detection of RBD itself was not performed. It is also

_{In contrast, the K D} values of CR3022, F26G19 and S1-mAb were 185.1, 242.3 and 73.35 nM for S1 and 21.52, 17.99 and 12.42 nM for RBD, respectively. Among the antibodies, S1-mAb showed the highest affinity for SARS coronavirus 2 S1, which corresponds to the result of ELISA. CR3022 and F26G19 are antibodies that target and neutralize the RBD of the SARS coronavirus. Although recent studies have shown that these antibodies are capable of responding to the RBD of SARS-Coronavirus 2, the affinity of these antibodies to SARS-CoV-2 S1 and RBD uses SARS-CoV-2 S1 as the immunizing antigen. was lower than that of the S1-mAb prepared by Even considering the binding of S1 of SARS coronavirus 2, ACE2 had the highest K _D value. However, the affinity of ACE2 for the SARS coronavirus 2 RBD was similar to that of other commercial antibodies, which raises an important possibility of using ACE as a replacement for commercial antibodies in diagnostic platforms for antigen detection. In the present invention, the K _D value shows a similar tendency despite the different _{K D} values used in each experiment.

<Test Example 2> Sandwich pair for detection of SARS coronavirus 2 S1 antigen

For successful detection of a target antigen, two types of probes, a capture probe and a diagnostic probe, are required, which recognize different sites of specific antigens. However, in an unpredictable environment such as the current COVID-19 epidemic, the development of two different antibody pairs can be very time consuming and costly, so a rapid diagnostic field that needs the purpose of preventing the epidemic of the disease. It is difficult to apply to In order to overcome this disadvantage of antibody production, human ACE2, an intracellular receptor, was used in the present invention, which indicates that the binding affinity for the target antigen is similar to that of the antibody instead of the antibody.

In the present invention, dot-blot analysis was used to find a mate for sandwich antigen detection. ACE2 and three commercially available antibodies (CR3022, F26G19 and S1-mAb) were immobilized as detection probes on a nitrocellulose membrane (NC membrane). After that, other proteins (antibodies or receptors) were not used for immobilization, but were bound to cellulose nanoparticles (CNB) for signal generation as diagnostic probes, and the detection efficiency was evaluated. As shown in FIGS. 4A and 4B , a total of 12 pairs were used for the detection of the SARS coronavirus 2 S1 antigen, and non-specific interactions between the detection probe and the diagnostic probe were evaluated. Additionally, the density of each dot was analyzed with an image scanner. ACE2 was used as a capture probe, and sandwich detection of the S1 antigen was successfully achieved by three different detection probes. This indicates that the binding site for the SARS coronavirus 2 S1 antigen of ACE does not overlap with the epitope of the antibody. Additionally, in the present invention, no false-positive signal was observed in the negative control (eg, no antigen).

On the other hand, when an antibody was used as a detection probe, a false-positive signal was found due to the non-specific interaction between the detection and diagnostic antibodies. As is known, non-specific interactions occurred between S1-mAb and CR3022 and S1-mAb and F26G19 (red circles in the negative control group in Fig. 4b). Additionally, since the epitope of CR3022 overlapped with the epitope of F26G19, the signal at the detection point decreased. The results of the present invention indicate that finding suitable antibody pairs for sandwich assays is complicated by some factors. The use of ACE2 to replace antibodies accelerates the development of antigen diagnostic kits and helps to effectively manage disease outbreaks. Moreover, as a total signal, both the detection point and the control point showed a decrease in the case of CNB to which ACE2 was bound. Evaluation of 12 detection-diagnostic probe pairs showed that the most suitable pair capable of sensitively detecting the SARS coronavirus-2 S1 antigen was ACE2 as detection probe and S1-mAb as diagnostic probe.

<Test Example 3> Sensitivity and specificity of ACE2-based LFIA for SARS coronavirus 2 S1

The LFIA sensor strip consists of a sample pad, a bonding pad, a nitrocellulose membrane and an absorbent pad. A sample comprising the target antigen is injected into the sample pad, continuously contacting the dried CNB on the binding pad. CNBs are coated with a detection antibody (S1-mAb) through hydrophobic and/or electrostatic interactions. The diagnostic antibody-coated CNBs bind to the target SARS coronavirus 2 S1 antigen and migrate to the nitrocellulose membrane. The detection line comprising the detection probe (ACE2) detects the SARS coronavirus 2 S1 antigen previously bound by the diagnostic probe (S1-mAb binding CNB), enabling sandwich detection of the SARS coronavirus 2 S1 antigen. On the other hand, the control line serves to ensure that the biological molecules on the binding pad are active and that the sample is migrated. For this purpose, anti-IgG antibodies are used to identify all antibodies already bound to CNB. A detection line and a control line are formed on the nitrocellulose membrane using a line dispenser. Since the target antigen is detected at the detection line of LFIA, the red signal of CNB allows visual confirmation of whether the sample contains the target antigen. In order to avoid non-specific interactions between the detection probe and the diagnostic probe in the detection line, the nitrocellulose membrane was appropriately treated with a blocking solution. The unbound diagnostic probe passes through the nitrocellulose membrane and reaches an absorbent pad located at the end of the strip, which is held by capillary forces.

In order to prove the detection ability of ACE-2-based LFIA (ACE2-LFIA), in the present invention, using serially diluted samples of SARS coronavirus 2-specific antigens (S1 and RBD, concentration range is 5-500 ng/mL), Sensitivity analysis was performed 20 minutes after loading the sample, the window of the LFIA device was photographed using a smartphone, and the densities of the detection and control lines were analyzed using an image scanner and analyzer that converts the color density of the lines into signal peaks. In the case of S1 detection, it was found that the detection signal was gradually decreased according to the dilution factor, and it was confirmed that the signal was present even in the case of 5 ng antigen. Additionally, in the present invention, it was confirmed that the detection signal was higher in the case of RBD compared to S1 in all the diluted samples, and it was confirmed that the detection signal was detected in ACE2-LFIA even at 1 ng of RBD. Here, no false-positive signals were observed in negative controls (eg, absence of target antigen). Cellulose nanoparticles (CNB) are used for color labeling in LFIA and are known to have higher sensitivity than colloidal gold. Thus, in the present invention, the detection sensitivities of colloidal gold and CNB were compared. Colloidal gold-based LFIA detected the RBD antigen at a concentration of 20 ng, which showed lower sensitivity compared to CNB-based LFIA. Because CNB has a higher color density and surface area compared to colloidal gold at the same dose, CNB-based LFIA exhibits about 10-fold higher sensitivity compared to colloidal gold-based LFIA, consistent with recent studies. it will pass

Next, the present invention confirmed the detection sensitivity of ACE2-LFIA using the S1 antigen of other coronaviruses (SARS coronavirus and MERS coronavirus). Three different concentrations (100, 20 and 5 ng/reaction) of each S1 antigen were injected into the LFIA device, and the density of the detection line was measured after 20 minutes using a portable line analyzer. The line density of each LFIA device was measured within 10 seconds, in order to confirm that rapid and accurate diagnosis is possible in a point-of-care or laboratory setting. SARS coronavirus 2 S1 (< 5 ng antigen) and RBD (< 1 ng antigen) were successfully detected in the LFIA device, even at low concentrations. The sensitivity difference between SARS coronavirus 2 S1 and RBD shown using ACE2-based LFIA was _{related to the K D} value of ACE2, and S1-mAb bound less to RBD compared to S1. On the other hand, MERS coronavirus S1 antigen was not detected even at a relatively high concentration (100 ng antigen), and SARS coronavirus S1 100 ng antigen was weakly detected. This means that 100 ng SARS coronavirus S1 antigen was not differentiated using ACE2-LFIA. Density increased slightly at high concentrations of SARS-CoV S1, indicating that the proposed ACE2-LFIA can distinguish SARS-CoV-2 from other coronaviruses due to significant differences in density. As shown in Figure 5c, the detection density for the S1 antigen was 5 ng antigen concentration in three different coronaviruses. SARS coronavirus 2 S1 detection signal was higher than the limit of detection (LOD, mean value of negative controls + 3 Х standard deviation), and SARS coronavirus S1 and MERS coronavirus S1 were similar to negative controls. Thus, it was confirmed that the proposed ACE2-LFIA exhibited high specificity to the SARS coronavirus 2 antigen without significant cross-reactivity to other coronaviruses.

<Test Example 4> Laboratory confirmation using clinical samples

The efficacy of ACE2-LFIA was confirmed in the laboratory using cultured virus and clinical samples. The virus amounts of the cultured SARS coronavirus 2 and human coronavirus OC43 were measured by quantitative RT-PCR, and the E and N genes were used for standard curves, respectively. The detection limit of ACE2-LFIA was 5.35 x 10 ⁶ copies/mL in the cultured SARS coronavirus 2 sample, but there was no positive signal (>50) in the ACE2-LFIA test in the human coronavirus OC43 culture sample.

RT-qPCR analysis was performed with nasopharyngeal and nasal smear samples taken from COVID-19 patients (n=4) and healthy individuals (n=4) to determine the amount of virus (copy/mL) in clinical samples. The amount of virus in the nasopharyngeal smear from COVID-19 patients was investigated as a standard curve for the E gene of SARS coronavirus 2, patient 1 2.49x10 ⁷ copies/mL Patient 2 1.15x10 ⁶ copies/mL, Patient 4 1.86x10 ⁵ copies/mL. No SARS coronavirus 2 RNA was detected in patient 3. This result was also confirmed by other independent RT-qPCR assays and is expected to be associated with the degradation of viral RNA in UTM. Laboratory confirmation of ACE2-LFIA with clinical samples from COVID-19 patients showed positive results in only three clinical samples, as in RT-qPCR analysis. The detection limit of ACE2-LFIA was 1.86x10 ⁵ copies/mL (patient 4), but no positive signal was observed when the ACE2-LFIA test was performed with nasal smears from healthy subjects (n=4). Therefore, the ACE2-based LFIA test could be helpful in detecting the S1 antigen of SARS coronavirus 2 from COVID-19 patients. The development of additional ACE2-based LFIAs using more specific antibodies, aptamers, apimers or nanobodies will enable more sensitive and specific SARS coronavirus 2 S1 antigen detection.

Claims (12)

a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody is a composition for detecting SARS coronavirus 2 comprising a secondary antibody to which a visibly identifiable nanostructure is bound, or a visibly identifiable nanostructure is bound to the antibody The method according to claim 1, wherein the receptor binding to the SARS coronavirus 2 spike protein comprises at least one selected from the group consisting of angiotensin converting enzyme 2 (ACE2) and a lysate of a cell line expressing or overexpressing ACE2. The composition for detecting SARS coronavirus 2 According to claim 1, wherein the antibody binding to the SARS coronavirus 2 spike protein is any one selected from the group consisting of antibodies that recognize any one of S1, RBD (Receptor binding domain) and RBM (Receptor binding motif) as an antigen. Which, comprising, a composition for detecting SARS coronavirus 2 The composition of claim 1, wherein the nanostructure comprises any one selected from the group consisting of cellulose nanoparticles and gold nanoparticles. According to claim 1, wherein the sample is soil, water, air, food, nasal swabs, nasopharyngeal lavage solution, bronchoalveolar lavage fluid, pleural fluid, nasal smear, nasal aspirate, nasopharyngeal smear, nasopharyngeal aspirate, blood, blood composition Components, body fluids, saliva, sputum, and a composition for detecting SARS coronavirus 2 comprising at least one selected from the group consisting of combinations thereof a receptor that binds to the SARS coronavirus 2 spike protein; an antibody capable of pairing with the receptor and binding to the SARS coronavirus 2 spike protein; The antibody is a composition for diagnosing SARS coronavirus 2 infection comprising a secondary antibody to which a visibly identifiable nanostructure is bound, or a visibly identifiable nanostructure is bound to the antibody The method of claim 6, wherein the receptor binding to the SARS coronavirus 2 spike protein comprises at least one selected from the group consisting of angiotensin converting enzyme 2 (ACE2) and a lysate of a cell line expressing or overexpressing ACE2. The composition for diagnosing SARS coronavirus 2 infection The method of claim 6, wherein the antibody binding to the SARS coronavirus 2 spike protein comprises any one selected from the group consisting of antibodies that recognize any one of S1, RBD (Receptor binding domain) and RBM (Receptor binding motif) as an antigen. Composition for diagnosing SARS coronavirus 2 infection The composition for diagnosing SARS coronavirus 2 infection according to claim 6, wherein the nanostructure comprises any one selected from the group consisting of cellulose nanoparticles and gold nanoparticles. According to claim 6, wherein the sample is nasal swab, nasopharyngeal lavage fluid, bronchoalveolar lavage fluid, pleural fluid, nasal smear, nasal aspirate, nasopharyngeal smear, nasopharyngeal aspirate, blood, blood components, body fluid, saliva, sputum and A composition for diagnosing SARS coronavirus 2 infection, which includes at least one selected from the group consisting of combinations thereof reacting the composition for detecting SARS-CoV-2 of claim 1 and the separated sample;
reacting a sample containing no antigen as a control;
reacting for 20 minutes; and
SARS coronavirus 2 detection method comprising; analyzing the signal from the detection point A kit for diagnosing SARS coronavirus 2 infection comprising the composition of claim 1 and instructions for use

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