A novel coronavirus neutralizing antibody detection kit

Abstract

The application provides a kit for detecting neutralizing antibodies of a novel coronavirus, comprising an immunochromatography test paper, the immunochromatography test paper comprising a backing plate and a sample pad, a conjugate pad, a detection pad and a water absorption pad arranged on the backing plate in sequence; the detection pad is provided with a detection line and a quality control line; the sample pad is coated with a recombinant antigen, the recombinant antigen being a fusion protein SARS-CoV-2S-RBD-Fc formed by fusing SARS-CoV-2S-RBD with the Fc fragment of IgG antibody; the conjugate pad is coated with SARS-CoV-2ACE2 labeled with nanoparticles; and the detection line is coated with a polyantibody of anti-IgG antibody. The kit for detecting the application utilizes the principle of competition and sandwich detection, can quickly and simply realize effective detection of neutralizing antibodies of the novel coronavirus, has low cost, high sensitivity and high specificity, and has a good popularization and application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection, and particularly relates to a novel coronavirus neutralizing antibody detection kit. BACKGROUND

[0002] The novel coronavirus (SARS-CoV-2) has the characteristics of high infectivity and high concealment, and has caused a large number of people to be infected and led to the novel coronavirus pneumonia (Corona Virus Disease 2019, COVID-19). The novel coronavirus pneumonia has fever, dry cough, fatigue and other symptoms as the main manifestations, and a small number of patients have nasal congestion, runny nose, diarrhea and other upper respiratory and digestive symptoms. Severe cases often develop respiratory distress after 1 week, and severe cases rapidly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, coagulation dysfunction and multiple organ failure, etc., which seriously endanger human health.

[0003] In order to control the spread of the novel coronavirus, much progress has been made in vaccine research and development, and as the number of people vaccinated increases, the demand for detection of whether protective antibodies are produced after vaccination is also increasing.

[0004] The spike protein (S) of the novel coronavirus (SARS-CoV-2) contains a receptor-binding domain (RBD) responsible for recognizing the cell surface receptor angiotensin-converting enzyme-2 (ACE2). It is found that the RBD of the SARS-CoV-2 S protein strongly interacts with the human ACE2 receptor, leading to endocytosis into host cells deep in the lungs and viral replication. Infection with SARS-CoV-2 initiates an immune response, which includes the production of antibodies or binding antibodies in the blood. Not all binding antibodies can block cell infiltration and replication of the SARS-CoV-2 virus. The subpopulation of binding antibodies that can block viral cell infiltration and replication is called neutralizing antibodies. Neutralizing antibodies of the SARS-CoV-2 S1 protein RBD fragment can bind to the RBD region of SARS-CoV-2, block the binding of RBD to the ACE2 receptor, and thus block the invasion of the virus into human cells. Detection of neutralizing antibodies of the novel coronavirus helps to assess the risk of reinfection of recovered patients or asymptomatic infected persons, and can also be used as an evaluation index of the effect of the vaccine, which has very important significance.

[0005] Currently, there are reports on detecting neutralizing antibodies of the new coronavirus by enzyme-linked immunoassay, chemiluminescence and other detection methods. However, the above methods are complex and time-consuming. Among many detection technologies, lateral flow immunoassay (LFIA) has gained more and more attention due to its simple and fast characteristics. The basic principle of LFIA is that specific antibodies (antigens) are pre-coated in a certain zone of a nitrocellulose (NC) membrane. During the test, the sample moves forward along the NC membrane by capillary action. When it reaches the zone coated with antibodies (antigens), the corresponding antigens (antibodies) in the sample specifically bind to the antibodies (antigens) on the NC membrane to form an immune complex. After the reaction is complete, the results are read by visual observation or instrument detection according to different markers.

[0006] Currently, research on products and methods for detecting neutralizing antibodies of the new coronavirus based on LFIA has made many progress. For example, patent application CN112485436A discloses a new coronavirus neutralizing antibody test strip, which uses the principle of neutralizing antibodies competing with ACE2 to bind gold-labeled RBD antigens to achieve the detection of neutralizing antibodies by competition method. Patent CN112730851B discloses a neutralizing antibody test kit, which adds a capture line to capture non-neutralizing antibodies targeting RBD in advance, improves the detection sensitivity, and ensures the specificity of subsequent detection of neutralizing antibodies by sandwich method. However, the above prior art does not disclose the sensitivity and specificity of the detection. Li et al. developed a LFIA for rapid detection, which can detect IgM and IgG of SARS-CoV-2 in human blood within 15 min (Li Z, Yi Y, Luo X, et al. Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis [J]. Journal of Medical Virology, 2020, 92(9): 1518-1524.). However, its sensitivity and specificity are only 88.66% and 90.63%, respectively. In addition, the concentration of antibodies / antigens coated in some neutralizing antibody test products is high, and the amount used is large, which to some extent increases the production cost and is not conducive to industrialization and application.

[0007] It is the goal of researchers to develop antibody detection reagents with high sensitivity and high specificity, and it is also the difficulty of developing detection reagents. Moreover, the market has increasingly high requirements for detection efficiency and the cost of detection reagents. Therefore, it is of great significance to provide a novel coronavirus neutralizing antibody detection reagent that can be quickly and simply realized, and has low cost, excellent sensitivity and specificity. SUMMARY

[0008] The purpose of the present application is to provide a novel coronavirus neutralizing antibody detection kit with low cost and excellent sensitivity and specificity.

[0009] The present application provides a kit for detecting novel coronavirus neutralizing antibodies, which comprises an immunochromatography test paper, the immunochromatography test paper comprising a backing plate and a sample pad, a conjugate pad, a detection pad and a water absorption pad arranged on the backing plate in sequence; the detection pad is provided with a detection line and a quality control line;

[0010] The sample pad is coated with a recombinant antigen, which is a fusion protein SARS-CoV-2S-RBD-Fc formed by fusing SARS-CoV-2S-RBD with the Fc fragment of IgG antibody;

[0011] The conjugate pad is coated with a nanoparticle-labeled SARS-CoV-2ACE2;

[0012] The detection line is coated with a polyantibody against IgG antibody.

[0013] Further, the coating concentration of the above-mentioned SARS-CoV-2S-RBD-Fc is 0.1-100 μg / mL, for example, it can be 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 8 μg / mL, 10 μg / mL, 15 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL, etc.

[0014] Further, the above-mentioned nanoparticle-labeled SARS-CoV-2ACE2 is a colloidal gold-labeled SARS-CoV-2ACE2, and the coating concentration is O.D. value = 1-30; that is, the coating concentration of the SARS-CoV-2ACE2 is 9.75-12.75 μg / mL, preferably 11.25 μg / mL.

[0015] Further, the coating concentration of the anti-IgG antibody polyclonal antibody is 0.1-2 mg / mL, for example, it can be 0.1 mg / mL, 0.3 mg / mL, 0.5 mg / mL, 0.8 mg / mL, 1 mg / mL, 1.2 mg / mL, 1.5 mg / mL, 1.8 mg / mL, or 2 mg / mL, etc.

[0016] Further, the IgG antibody is a human IgG antibody, and the anti-IgG antibody polyclonal antibody is a mouse anti-human IgG polyclonal antibody (i.e., a mouse anti-human IgG polyclonal antibody).

[0017] Or, the IgG antibody is a mouse IgG antibody, and the anti-IgG antibody polyclonal antibody is a goat anti-mouse IgG polyclonal antibody (i.e., a goat anti-mouse IgG polyclonal antibody).

[0018] Further, the IgG antibody is a human IgG antibody, and the anti-IgG antibody polyclonal antibody is a mouse anti-human IgG polyclonal antibody (i.e., a mouse anti-human IgG polyclonal antibody).

[0019] Preferably, the IgG antibody is a colloidal gold-labeled IgG antibody.

[0020] More preferably, the colloidal gold-labeled IgG antibody is a colloidal gold-labeled rabbit IgG antibody, and the anti-IgG antibody polyclonal antibody is a goat anti-rabbit IgG polyclonal antibody (i.e., a goat anti-rabbit IgG polyclonal antibody).

[0021] Further, the kit further comprises a sample diluent, which is physiological saline or a solution containing 0.85% NaCl.

[0022] The present application also provides a preparation method of the above-mentioned kit, which comprises the following steps of preparing an immunochromatography test paper:

[0023] (1) Sample pad pretreatment: the sample pad is soaked in a treatment solution containing a mouse anti-human red blood cell monoclonal antibody, incubated, and then dried for standby use; the mouse anti-human red blood cell monoclonal antibody serves to remove red blood cells in blood, and when the sample itself does not contain red blood cells (e.g., is not whole blood), the mouse anti-human red blood cell monoclonal antibody can not be used.

[0024] (2) Antigen, antibody coating: SARS-CoV-2 S-RBD-Fc is fixed on the pretreated sample pad, nanoparticle-labeled SARS-CoV-2 ACE2 and nanoparticle-labeled IgG antibody are fixed on the binding pad, and anti-IgG antibody multivalent is fixed on the detection pad to form a detection line, and the multivalent antibody specifically combined with the nanoparticle-labeled IgG antibody is fixed on the detection pad to form a quality control line; dry for standby;

[0025] (3) Immune layer test paper assembly: sequentially connect the sample pad, the binding pad, the detection pad, and the water absorption pad on the backing plate to obtain the immune layer test paper;

[0026] Preferably, the sample pad and the binding pad are glass cellulose membranes, the detection pad is a nitrocellulose membrane, and the water absorption pad is a filter paper fiber.

[0027] The application further provides a detection method of a novel coronavirus neutralizing antibody, comprising the following steps:

[0028] (1) First, the sample to be tested is added to the sample pad of the immune chromatography test paper of the above-mentioned kit, and then the sample to be tested is added to the sample pad of the immune chromatography test paper of the above-mentioned kit;

[0029] (2) Place at room temperature for 15-30 min, and observe the color development result of the immune chromatography test paper.

[0030] Further, the sample to be tested is serum, plasma or whole blood; the plasma or whole blood is plasma or whole blood treated with an anticoagulant.

[0031] The application has the following beneficial effects: the detection kit of the application uses an improved competition method, and can quickly and simply realize effective detection of the novel coronavirus neutralizing antibody by using the detection principle of the combination of the competition method and the sandwich method. In the case of a lower amount of SARS-CoV-2 S-RBD-Fc recombinant antigen, high detection sensitivity and specificity can be achieved, and the detection kit is low in cost and excellent in sensitivity and specificity.

[0032] The detection principle is as follows:

[0033] If the sample to be tested contains neutralizing antibodies against the novel coronavirus, the SARS-CoV-2 S-RBD on the sample pad will bind to the recombinant antigen SARS-CoV-2 S-RBD-Fc fused with the Fc fragment of IgG antibody to form an immune complex, which will move to the conjugate pad under the effect of lateral flow chromatography. The nanoparticle (colloidal gold) labeled SARS-CoV-2 ACE2 protein cannot bind to SARS-CoV-2 S-RBD-Fc. Then, the complex continues to move to the detection pad and reacts with the anti-IgG polyclonal antibody coated on the detection line to form a sandwich structure complex of "neutralizing antibodies" - "SARS-CoV-2 S-RBD-Fc" - "anti-IgG polyclonal antibody". Since the complex is not labeled with nanoparticles (colloidal gold), the detection line does not develop color or shows a light red band. At the same time, the nanoparticle labeled IgG antibody (such as rabbit IgG antibody) on the conjugate pad also moves to the control line under the effect of lateral flow chromatography and reacts with the polyclonal antibody (such as goat anti-rabbit IgG polyclonal antibody) coated on the control line, which can specifically bind to the nanoparticle labeled IgG antibody. The control line shows a neat red band.

[0034] If the sample to be tested does not contain neutralizing antibodies against the novel coronavirus, the SARS-CoV-2 S-RBD on the sample pad will bind to the recombinant antigen SARS-CoV-2 S-RBD-Fc fused with the Fc fragment of IgG antibody to form an immune complex, which will move to the conjugate pad under the effect of lateral flow chromatography. The nanoparticle (colloidal gold) labeled SARS-CoV-2 ACE2 protein cannot bind to SARS-CoV-2 S-RBD-Fc. Then, the complex continues to move to the detection pad and reacts with the anti-IgG polyclonal antibody coated on the detection line to form a sandwich structure complex of "nanoparticle labeled SARS-CoV-2 ACE2" - "SARS-CoV-2 S-RBD-Fc" - "anti-IgG polyclonal antibody". Since the complex is labeled with nanoparticles (colloidal gold), the detection line shows a red band. At the same time, the nanoparticle labeled IgG antibody (such as rabbit IgG antibody) on the conjugate pad also moves to the control line under the effect of lateral flow chromatography and reacts with the polyclonal antibody (such as goat anti-rabbit IgG polyclonal antibody) coated on the control line, which can specifically bind to the nanoparticle labeled IgG antibody. The control line shows a neat red band.

[0035] Wherein, when the IgG antibody is human IgG antibody, the anti-IgG polyclonal antibody is murine anti-human IgG polyclonal antibody; when the IgG antibody is murine IgG antibody, the anti-IgG polyclonal antibody is goat anti-murine IgG polyclonal antibody; and so on. That is, the Fc fragment of the IgG antibody fused with S-RBD has a specific binding relationship with the polyclonal antibody on the detection line.

[0036] If the control line does not show a red band, the test paper is invalid.

[0037] In the present application, the "fusion protein SARS-CoV-2 S-RBD-Fc" refers to "RBD recombinant protein with Fc tag". The Fc tag can be human or murine. An exemplary method for preparing RBD recombinant protein with murine Fc tag includes: cloning the gene sequence of the RBD region in the S protein of the novel coronavirus COVID-19 (its encoding nucleic acid sequence has been disclosed on NCBI); inserting the synthesized gene sequence into a pcDNA3.1 expression vector with a mouse Fc fragment, transfecting HEK293 cells with the expression vector, and then purifying the protein through a Protein G affinity purification column.

[0038] In the present application, the coating concentration is O.D. value = 1-30, which means that the optical density value of the solution in the specific concentration range is within the range of 1-30.

[0039] In the present application, the polyclonal antibodies of ACE2 and anti-IgG antibodies are respectively combined with different sites of the RBD recombinant protein to form a sandwich structure, which has stronger affinity, so that the band of the negative sample on the detection line is more obvious, further enhancing the brightness difference between the positive sample and the negative sample, and improving the accuracy of test paper interpretation.

[0040] Obviously, according to the above content of the present application, according to the ordinary technical knowledge and conventional means in the art, other various forms of modifications, substitutions or changes can be made without departing from the above basic technical idea of the present application.

[0041] The above content of the present application will be further described in detail through the specific embodiments below. However, it should not be understood that the scope of the above subject matter of the present application is limited to the following examples. Any technology realized based on the above content of the present application belongs to the scope of the present application. DETAILED DESCRIPTION

[0042] The raw materials and equipment used in the present application are known products, which are obtained by purchasing commercially available products.

[0043] For example, the fusion protein SARS-CoV-2 S-RBD-Fc (mFc) is purchased from Sichuan Ankoresi New Material Technology Co., Ltd. (item number: XCL2103), the SARS-CoV-2 ACE2 protein is purchased from Sichuan Ankoresi New Material Technology Co., Ltd. (item number: XCL2104), the goat anti-mouse IgG polyclonal antibody is purchased from Sichuan Ankoresi New Material Technology Co., Ltd. (item number: XCL2105), the rabbit IgG antibody is purchased from Sichuan Ankoresi New Material Technology Co., Ltd. (item number: XCL1048), and the goat anti-rabbit IgG polyclonal antibody is purchased from Sichuan Ankoresi New Material Technology Co., Ltd. (item number: XCL1205).

[0044] Example 1, preparation method of the detection kit of the present application

[0045] (1) Sample pad pretreatment: the sample pad is soaked in a treatment solution containing a mouse anti-human red blood cell monoclonal antibody (used to remove red blood cells in the blood, so that the test paper of the present application is suitable for detecting whole blood samples) and incubated, and then dried for standby use; the treatment solution used is common technical knowledge in the art, and a person skilled in the art can select and adjust it according to the actual situation. An exemplary treatment solution can also contain Tris, sodium chloride, BSA, casein, PVP-40, surfactant, preservative, etc.

[0046] (2) Antigen and antibody coating: a solution of SARS-CoV-2 S-RBD with a concentration of 4 μg / mL and a recombinant antigen S-RBD-hFc fused with the Fc fragment of human IgG antibody is sprayed or line-fixed on the pretreated sample pad, the conjugate pad is soaked in a mixed solution of colloidal gold-labeled SARS-CoV-2 ACE2 and colloidal gold-labeled rabbit IgG antibody to fix, and a mouse anti-human IgG polyclonal antibody with a concentration of 2 mg / mL is sprayed or line-fixed on the detection pad to form a detection line, and a goat anti-rabbit IgG polyclonal antibody solution is sprayed or line-fixed on the detection pad to form a quality control line; dry for standby use; the amount of antigen / antibody fixed on the test paper can be adjusted by a person skilled in the art according to common technical knowledge in the art. In this embodiment, the amount of S-RBD-hFc fixed is: 90 μL of solution for 65 people of test paper, and the amount of mouse anti-human IgG polyclonal antibody fixed is: 33 μL of solution for 65 people of test paper.

[0047] (3) Immune layer system test paper assembly: the sample pad, the conjugate pad, the detection pad, and the water absorption pad are connected in sequence on a backing plate to obtain an immune layer system test paper.

[0048] The sample pad and the conjugate pad are glass cellulose membranes, the detection pad is a nitrocellulose membrane, and the water absorption pad is filter paper.

[0049] (4) The immune layer system test paper and the sample diluent to be tested are combined to obtain the detection kit of the present application. The sample diluent to be tested is a physiological saline or a solution containing 0.85% NaCl.

[0050] Example 2, preparation method of the detection kit of the present application

[0051] (1) Sample pad pretreatment: same as example 1;

[0052] (2) Antigen, antibody coating: 4 pg / mL of SARS-CoV-2 S-RBD was sprayed or scribed on the pre-processed sample pad to fix the recombinant antigen S-RBD-mFc fused with the Fc fragment of mouse IgG antibody, and the mixed solution of colloidal gold-labeled SARS-CoV-2 ACE2 and colloidal gold-labeled rabbit IgG antibody was soaked in the conjugate pad, and 2 mg / mL of goat anti-mouse IgG polyclonal antibody was fixed on the detection pad to form a detection line, and goat anti-rabbit IgG polyclonal antibody was fixed on the detection pad to form a quality control line; dry for standby; the amount of antigen / antibody fixed on the test paper can be adjusted by those skilled in the art according to common knowledge in the art. In this embodiment, the amount of S-RBD-mFc fixed is: 90 pL of solution is used for 65 test papers, and the amount of mouse anti-human IgG polyclonal antibody fixed is: 33 pL of solution is used for 65 test papers.

[0053] (3) Immune layer system test paper assembly: same as example 1.

[0054] (4) The immune layer system test paper, the sample to be tested is combined to obtain the test kit of the application. The sample to be tested is physiological saline or a solution containing 0.85% NaCl.

[0055] Example 3, detection method of the kit of the application

[0056] 1. The sample to be tested is serum or plasma treated with an anticoagulant (EDTA)

[0057] 1) Take out the immune chromatography test paper;

[0058] 2) Take 3 drops (about 50 pL) of 10 pL of the sample to be tested serum / plasma sample to the sample addition hole of the test paper card;

[0059] 3) Add 1 drop of the sample to be tested vertically to the sample addition hole of the test paper card, and place it at room temperature;

[0060] 4) Start timing after adding the sample, and observe the results within 15-30 minutes, and judge invalid after 30 minutes.

[0061] 2. The sample to be tested is venous whole blood or fingertip whole blood treated with an anticoagulant (EDTA)

[0062] 1) Take out the immune chromatography test paper;

[0063] 2) Take 3 drops (about 50 pL) of EDTA venous whole blood / fingertip whole blood to the sample addition hole of the test paper card;

[0064] 3) Add 3 drops of the sample to be tested vertically to the sample addition hole of the test paper card, and place it at room temperature;

[0065] 4) After adding sample, start timing, observe results within 15-30 minutes, and judge invalid after 30 minutes.

[0066] Comparative Example 1: Test kit prepared by using traditional competition method

[0067] Compared with Example 2, no SARS-CoV-2 S-RBD-Fc is coated on the sample pad; "colloidal gold-labeled SARS-CoV-2 S-RBD-Fc" and "colloidal gold-labeled rabbit IgG antibody" are coated on the binding pad; "SARS-CoV-2 ACE2" is coated on the T line of the chromatography membrane, and "goat anti-rabbit IgG polyclonal antibody" is coated on the C line.

[0068] The beneficial effects of the present application are demonstrated by the following experimental examples.

[0069] Experimental Example 1, detection performance of the kit of the present application

[0070] The performance of the test kit prepared in Example 2 was investigated, and the detection results were as follows:

[0071]

[0072]

[0073] The performance of the test kit prepared in Comparative Example 1 was investigated, and the detection results were as follows:

[0074]

[0075] As can be seen, the test kit prepared by using the traditional competition method has poor "minimum detection limit" performance, i.e. poor sensitivity, and needs to be further improved.

[0076] Experimental Example 2, detection of clinical samples by the test kit

[0077] 1. Experimental method

[0078] The kit of Example 2 was used to detect the samples of injected vaccine (positive sample) and non-injected vaccine (negative sample) according to the detection method of Example 3, and the same clinical samples were detected according to the instructions of the commercially available ELISA detection kit (product name: cPassTM SARS-CoV-2 Neutralization Antibody Detection Kit; manufacturer: GenScript USA, Inc. The neutralizing antibody detection kit is authorized by the US FDA EUA (For In Vitro Diagnostic Use Under FDA Emergency Use Authorization)) of GenScript, and the detection results were compared.

[0079] 2. Experimental results

[0080] The coincidence rate data of the detection kit of the present application and the GenScript detection product are as follows:

[0081]

[0082] The specific detection data are as follows:

[0083] Among them, the detection results of the kit of the present application show that G6 and below are positive, G7 and above are negative, G6-G7 (i.e., G6 or G7 cannot be distinguished) is recommended to be retested after 24 hours; G*: the larger the value, the stronger the line color.

[0084] The judgment standard of GenScript results is that the inhibition rate ≥ 30% is positive, and the inhibition rate < 30% is negative.

[0085] In the table, "+" represents a positive result, and "-" represents a negative result.

[0086]

[0087]

[0088]

[0089]

[0090] As can be seen from the above table, the color development of the positive and negative samples of the kit is greatly different, the interpretation is accurate, the detection results are consistent with the internationally recognized GenScript Elisa detection kit, and the color development brightness is basically consistent with the inhibition rate of the GenScript Elisa kit, which reflects that the kit has high detection accuracy, excellent specificity and sensitivity. The detection kit of the present application is rapid and convenient, and has significant advantages.

[0091] In conclusion, the application provides a kit for rapidly and simply detecting neutralizing antibodies of the novel coronavirus, which has high sensitivity and specificity, low cost and good popularization and application prospect under the condition of using a lower amount of SARS-CoV-2 S-RBD-Fc recombinant antigen.

Claims

1. A kit for detecting neutralizing antibodies against novel coronavirus, characterized in that: The invention comprises an immunochromatographic test paper, which comprises a lining plate and a sample pad, a binding pad, a detection pad and a water-absorbing pad which are arranged on the lining plate and connected in sequence; a detection line and a quality control line are arranged on the detection pad; The sample pad is coated with a recombinant antigen, which is a fusion protein SARS-CoV-2S-RBD-Fc formed by the fusion of SARS-CoV-2S-RBD and the Fc fragment of IgG antibody; The conjugate pad is coated with nanoparticle-labeled SARS-CoV-2 ACE2; The detection line is coated with a polyclonal antibody with anti-IgG antibodies; The coating concentration of the SARS-CoV-2S-RBD-Fc is 0.1 to 100 μg / mL; The nanoparticle-labeled SARS-CoV-2 ACE2 is colloidal gold-labeled SARS-CoV-2 ACE2, and the coating concentration is OD value = 1 to 30; The coating concentration of the anti-IgG antibody polyclonal antibody is 0.1-2 mg / mL; The IgG antibody is a human IgG antibody, and the anti-IgG antibody polyclonal antibody is a mouse anti-human IgG polyclonal antibody; or, the IgG antibody is a mouse IgG antibody, and the anti-IgG antibody polyclonal antibody is a goat anti-mouse IgG polyclonal antibody; The conjugate pad is also coated with a nanoparticle-labeled IgG antibody, and the quality control line is coated with a polyclonal antibody that specifically binds to the nanoparticle-labeled IgG antibody; the nanoparticle-labeled IgG antibody does not bind to SARS-CoV-2S-RBD-Fc; The nanoparticle-labeled IgG antibody is a colloidal gold-labeled IgG antibody; The nanoparticle-labeled IgG antibody is a nanoparticle-labeled rabbit IgG antibody, and the polyclonal antibody specifically binding to the nanoparticle-labeled IgG antibody is a goat anti-rabbit IgG polyclonal antibody.

2. The method for preparing the kit according to claim 1, wherein The method comprises the following steps of preparing an immunochromatographic test paper: (1) Sample pad pretreatment: soak the sample pad in a treatment solution containing mouse anti-human erythrocyte monoclonal antibody, incubate, and then dry for later use; (2) Antigen and antibody coating: SARS-CoV-2S-RBD-Fc is fixed on the pretreated sample pad, SARS-CoV-2 ACE2 labeled with nanoparticles and IgG antibodies labeled with nanoparticles are fixed on the binding pad, polyclonal antibodies against IgG antibodies are fixed on the detection pad to form a detection line, and polyclonal antibodies that specifically bind to the IgG antibodies labeled with nanoparticles are fixed on the detection pad to form a quality control line; and the mixture is dried for later use; (3) Assembly of the immune layer test paper: Connect the sample pad, conjugate pad, detection pad, and absorbent pad on the backing plate in sequence to obtain the immune layer test paper.

3. The preparation method according to claim 2, wherein The sample pad and the conjugation pad are glass cellulose membranes, the detection pad is nitrocellulose membrane, and the absorbent pad is filter paper.

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