CN111303254A - Novel coronavirus (SARS-CoV-2) antigen detection kit - Google Patents

Abstract

The invention relates to the field of biotechnology, and particularly provides a novel coronavirus (SARS-CoV-2) antigen detection kit. The SARS-CoV-2 antigen detecting kit provided by the invention utilizes a colloidal gold double-antibody sandwich method to detect the SARS-CoV-2 antigen, adopts two monoclonal antibodies and colloidal gold mixed markers, not only has accurate detection result and high sensitivity for the SARS-CoV-2 antigen, but also can obviously improve the detection rate. The detection kit fills the blank of immunological detection of SARS-CoV-2, can realize on-site detection without a detection instrument, and has the advantages of time saving, labor saving and flexible operation.

Description

Novel coronavirus (SARS-CoV-2) antigen detection kit

Technical Field

The invention relates to the field of biotechnology, in particular to a novel coronavirus (SARS-CoV-2) antigen detection kit.

Background

Since 2019 new coronavirus (SARS-CoV-2) outbreak, both confirmed cases and suspected cases rapidly grow in one time, but the currently adopted mode for the confirmation and detection of the suspected cases is mainly a dual fluorescence PCR method for detection, the sample can be confirmed to be positive only if the specific real-time fluorescence RT-PCR detection results of 2 targets (ORF1ab, N) of the new coronavirus in the same sample are positive, the detection method is time-consuming and labor-consuming, meanwhile, the sample needs to be sent in a centralized manner and detected by a specific instrument, in addition, the sample preparation process is complicated and time-consuming, and the sample is very easy to be polluted in the process, so that the method has great limitation. However, in the prior art, the product called SARS-CoV-2 antigen detection is mostly detected by using the antibody in the previous research of other coronaviruses, for example, the homology between new coronaviruses and SARS is very high, and the antibody of SARS can be used for temporary emergency, but the specificity and the sensitivity are difficult to ensure, so that the detection accuracy of the infection in China is reduced.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a SARS-CoV-2 antigen.

The second objective of the invention is to provide an antibody against SARS-CoV-2.

The third object of the present invention is to provide a method for producing the above antibody.

The fourth object of the present invention is to provide the use of the above antigen or antibody.

The fifth purpose of the invention is to provide a product for detecting SARS-CoV-2 antigen by using double antibody sandwich.

The sixth purpose of the invention is to provide a test paper for detecting SARS-CoV-2 antigen.

The seventh object of the present invention is to provide a method for preparing the above test paper.

The eighth object of the present invention is to provide a SARS-CoV-2 antigen detection kit.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

SARS-CoV-2 antigen, the antigen is protein composed of amino acid sequence shown in any one of SEQ ID NO.2-SEQ ID NO. 11.

An antibody against SARS-CoV-2, which antibody specifically binds to the above-mentioned antigen.

Further, the antibody is a monoclonal antibody.

The preparation method of the antibody adopts protein consisting of amino acid sequences shown in any one of SEQ ID NO.2-SEQ ID NO.11 as antigen, prepares immunogen with adjuvant, and immunizes animals to obtain the antibody.

Preferably, a protein consisting of an amino acid sequence shown in any one of SEQ ID NO.2-SEQ ID NO.11 is used as an antigen, an immunogen is prepared by an adjuvant, after an animal is immunized, the titer of the antibody to be detected meets 10-fold dilution activity above L6, the animal is immunized again, spleen cells of the immunized animal are taken to be subjected to cell fusion with myeloma cells, and the antibody is obtained by screening positive clones, amplification and purification.

Further, the animal is a mouse, preferably a BALB/c mouse;

preferably, the adjuvant comprises freund's complete adjuvant or freund's incomplete adjuvant;

preferably, the step of immunizing the animal comprises: performing primary immunization by using the antigen and the immunogen prepared by Freund's complete adjuvant by back multi-point injection, and performing secondary, tertiary and quaternary immunization by using the antigen and the immunogen prepared by Freund's incomplete adjuvant every three weeks;

preferably, the amount of antigen for both the first and second immunizations is independently 40-60 μ g;

preferably, the amount of antigen for each of the second, third and fourth immunizations is independently 20-30 μ g;

preferably, the ratio of the number of spleen cells to the number of myeloma cells is 1X 10⁷-1×10⁹：1×10⁷；

Preferably, cell fusion uses peritoneal macrophages as feeder cells;

preferably, amplification of positive clones is made using ascites fluid.

The application of the antigen or the antibody in preparing SARS-CoV-2 antigen detection products;

preferably, the product comprises a test strip or kit.

A product for detecting SARS-CoV-2 antigen by double antibody sandwich method, the antibody includes the above-mentioned antibody.

The SARS-CoV-2 antigen detecting test paper includes base plate, sample pad, combining pad, coating film and absorbing pad, and the sample pad, the combining pad, the coating film and the absorbing pad are fixed successively on the base plate along the flow direction of the liquid sample to be detected; the binding pad is loaded with gold-labeled protein, and the gold-labeled protein comprises the antibody; the coating film is provided with a detection area and a quality control area, the detection area is loaded with a specific antibody, and the quality control area is loaded with a goat anti-mouse IgG antibody; the specific antibody is an anti-N protein specific antibody, an anti-S protein specific antibody, an anti-M protein specific antibody or an anti-E protein specific antibody; the amino acid sequence of the N protein is shown as SEQ ID NO.1, the amino acid sequence of the S protein is shown as SEQ ID NO.12, the amino acid sequence of the M protein is shown as SEQ ID NO.13, and the amino acid sequence of the E protein is shown as SEQ ID NO. 14;

preferably, the gold-labeled protein comprises monoclonal antibodies respectively prepared by SEQ ID NO.2 and SEQ ID NO. 3;

preferably, the specific polyclonal antibody is an anti-N protein specific antibody;

preferably, the liquid sample to be tested comprises whole blood, serum or plasma;

preferably, the particle size of the colloidal gold is 15-25 nm;

preferably, each centimeter of the bonding pad is loaded with 0.8-1.5 mug of gold-labeled protein;

preferably, the coating film is loaded with 1.5-2.5 μ g of specific antibody per cm of detection zone;

preferably, the coating film is loaded with 1.0-1.5 mu g of goat anti-rabbit polyclonal antibody per centimeter of quality control area;

preferably, the sample pad is soaked with a solution containing 0.5-1.5 v/v% Tween20, 0.4-0.6 w/v% PVA, and 0.1-0.2mg/ml anti-RBC antibody.

A preparation method of test paper comprises sequentially fixing a sample pad, a bonding pad, a coating film and a water absorption pad on a bottom plate along the flowing direction of a liquid sample to be detected; the binding pad is loaded with gold-labeled protein, and the gold-labeled protein comprises the antibody; the coating film is provided with a detection area and a quality control area, the detection area is loaded with a specific antibody, and the quality control area is loaded with a goat anti-mouse IgG antibody; the specific antibody is an anti-N protein specific antibody, an anti-S protein specific antibody, an anti-M protein specific antibody or an anti-E protein specific antibody; the amino acid sequence of the N protein is shown as SEQ ID NO.1, the amino acid sequence of the S protein is shown as SEQ ID NO.12, the amino acid sequence of the M protein is shown as SEQ ID NO.13, and the amino acid sequence of the E protein is shown as SEQ ID NO. 14;

preferably, the preparation method of the gold-labeled protein comprises the following steps: uniformly mixing the colloidal gold solution with the antibody prepared by SEQ ID NO.2 for reaction to obtain a labeled antibody 1; uniformly mixing the colloidal gold solution with the antibody prepared by SEQ ID NO.3 for reaction to obtain a labeled antibody 2; mixing the labeled antibody 1 and the labeled antibody 2 according to the volume ratio of 2-4:7 to obtain gold labeled protein;

preferably, the specific polyclonal antibody is an anti-N protein specific antibody;

preferably, the particle size of the colloidal gold is 15-25 nm;

preferably, each centimeter of the bonding pad is loaded with 0.8-1.5 mug of gold-labeled protein;

preferably, the coating film is loaded with 1.5-2.5 μ g of specific antibody per cm of detection zone;

preferably, the coating film is loaded with 1.0-1.5 mu g of goat anti-rabbit polyclonal antibody per centimeter of quality control area;

preferably, the coating buffer solution on the coating film, the detection area and the quality control area is PBS independently;

preferably, the sample pad is soaked with a solution containing 0.5-1.5 v/v% Tween20, 0.4-0.6 w/v% PVA, and 0.1-0.2mg/ml anti-RBC antibody.

A detection kit for SARS-CoV-2 antigen, comprising the above-mentioned detection test paper.

Compared with the prior art, the invention has the beneficial effects that:

the SARS-CoV-2 antigen provided by the invention is a protein composed of any one of the amino acid sequences shown in SEQ ID NO.2-SEQ ID NO. 11. The ten antigens can obtain various specific binding antibodies, and the various antibodies can be used for detecting SARS-CoV-2 antigen, wherein, the inventor obtains the antibody obtained by the immunization of two antigens of SEQ ID NO.2 and SEQ ID NO.3 through multiple selection and repeated verification, the antibody is not only used for the detection result of the SARS-CoV-2 antigen with accurate result and high sensitivity, but also the two monoclonal antibodies can be mixed and marked for the detection of the SARS-CoV-2 antigen, and the detection rate can be obviously improved. The antibody provided by the invention can be applied to various immunological detection means, such as colloidal gold double-antibody sandwich detection and the like, fills the blank of the immunological detection of SARS-CoV-2, realizes field detection, and has the advantages of time saving, labor saving and flexible operation.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a technical route flow diagram of an embodiment of the present invention;

FIG. 2 is a schematic diagram showing the evaluation of color development standard in the titer detection in example 1 of the present invention;

fig. 3 is a schematic structural view of a test paper in embodiment 4 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention protects a SARS-CoV-2 antigen, which is a protein composed of any amino acid sequence shown in SEQ ID NO.2-SEQ ID NO. 11.

The ten antigens can obtain various specific binding antibodies, and the various antibodies can be used for detecting SARS-CoV-2 antigen, wherein, the inventor obtains the antibody obtained by the immunization of two antigens of SEQ ID NO.2 and SEQ ID NO.3 through multiple selection and repeated verification, the antibody is not only used for the detection result of the SARS-CoV-2 antigen with accurate result and high sensitivity, but also the two monoclonal antibodies can be mixed and marked for the detection of the SARS-CoV-2 antigen, and the detection rate can be obviously improved.

The invention also provides an antibody against SARS-CoV-2, which antibody specifically binds to the antigen of the invention.

The antibody has high immunological activity, and can specifically bind SARS-CoV-2 efficiently, rapidly and sensitively, and the antibody is preferably a monoclonal antibody. The antigen provided by the invention is ten kinds of SEQ ID NO.2-SEQ ID NO.11, and various antibodies can be obtained by respectively immunizing the antigen, and can be used for detecting SARS-CoV-2, and the detection effect is good.

The invention also provides a preparation method of the antibody, which adopts protein consisting of any amino acid sequence shown in SEQ ID NO.2-SEQ ID NO.11 as an antigen, prepares immunogen with adjuvant, and immunizes animals to obtain the antibody. Further, for example, a protein consisting of an amino acid sequence shown in any one of SEQ ID NO.2 to SEQ ID NO.11 is used as an antigen, an immunogen is prepared by an adjuvant, after an animal is immunized, the antibody titer is detected to meet 10-fold dilution activity and be more than L6, the spleen cell is immunized again, and cell fusion is carried out on myeloma cells, and the antibody is obtained by screening positive clone, amplification and purification. The inventors have determined the preparation scheme provided by the present invention by exploring conditions for animal immunization, cell fusion, screening of specific hybridoma cells, and mass production of monoclonal antibodies, and the antigen of the present invention can obtain a large amount of target antibodies by this method.

In a preferred embodiment, the animal is a mouse, preferably a BALB/c mouse; the adjuvant comprises Freund's complete adjuvant or Freund's incomplete adjuvant; the step of immunizing the animal further comprises: using back multi-point injection, performing primary immunization by using an immunogen prepared by an antigen and Freund's complete adjuvant, and performing secondary, tertiary and quaternary immunization by using an immunogen prepared by an antigen and Freund's incomplete adjuvant every three weeks, wherein the antigen amount of the primary immunization and the secondary immunization is independently preferably 40-60 mu g for the mice, and can be, but not limited to, 40 mu g, 45 mu g, 50 mu g, 55 mu g or 60 mu g; the amount of antigen for the second, third and fourth immunizations is each independently preferably 20-30. mu.g, and may be, for example, but not limited to, 20. mu.g, 25. mu.g or 30. mu.g.

In a preferred embodiment, the cell ratio of splenocytes to myeloma cells is 1X 10⁷-1×10⁹：1×10⁷Further, cell fusion preferably employs peritoneal macrophages as feeder cells; furthermore, the amplification of positive clones is preferably prepared using ascites.

The invention also protects the application of the antigen or the antibody in the preparation of the SARS-CoV-2 antigen detection product. Wherein, the product can be test paper or a kit and the like.

The invention also protects a product for detecting SARS-CoV-2 antigen by using double antibody sandwich method, the antibody used in the product is the antibody provided by the invention.

The invention also provides SARS-CoV-2 antigen test paper, which comprises a bottom plate, a sample pad, a combination pad, a coating film and an absorption pad, wherein the sample pad, the combination pad, the coating film and the absorption pad are fixed on the bottom plate in sequence along the flowing direction of the liquid sample to be tested; the binding pad is loaded with gold-labeled protein, and the gold-labeled protein is the antibody provided by the invention; the coating film is provided with a detection area and a quality control area, the detection area is loaded with a specific antibody, and the quality control area is loaded with a goat anti-mouse IgG antibody; the specific antibody is an anti-N protein specific antibody, an anti-S protein specific antibody, an anti-M protein specific antibody or an anti-E protein specific antibody; the amino acid sequence of the N protein is shown as SEQ ID NO.1, the amino acid sequence of the S protein is shown as SEQ ID NO.12, the amino acid sequence of the M protein is shown as SEQ ID NO.13, and the amino acid sequence of the E protein is shown as SEQ ID NO. 14. The inventor searches through a large amount of test conditions, utilizes 4 proteins (N, S, M, E) expressed by SARS-CoV-2 recombination to carry out immunization, and selects coating raw materials and selects the optimal coating conditions according to the detection results through debugging and detection of different coating buffer solutions, sample pad treatment, colloidal gold labeling process, monoclonal antibody pairing screening and the like. The polyclonal antibody prepared by using N, S, M or E protein as immunogen is used as detection zone protein, the antibody provided by the invention is gold-labeled protein, and the colloidal gold double-antibody sandwich principle is utilized to detect SARS-CoV-2 antigen, thus filling the blank of immunological detection of SARS-CoV-2, realizing on-site detection, avoiding using detection instrument to save time and labor and having flexible operation.

In a preferred embodiment, the gold-labeled protein is a mixture of monoclonal antibodies respectively prepared from SEQ ID No.2 and SEQ ID No.3, and the two antibodies are mixed and labeled, so that the detection rate can be remarkably improved. The inventor determines that the monoclonal antibody resisting a plurality of proteins is the most suitable gold-labeled antibody through a large amount of condition exploration and screening. And detecting the coating antibody and the labeled antibody through cross combination, and selecting the combination condition of the two antibodies for marking as the optimal mixed label. The liquid sample to be detected of the detection test paper can be whole blood, serum or plasma;

in a preferred embodiment, the particle size of the colloidal gold is 15 to 25 nm; on the bonding pad, each centimeter of the bonding pad is preferably loaded with 0.8-1.5 mug of gold-labeled protein; on the coating film, 1.5-2.5 mu g of specific polyclonal antibody is preferably loaded on each centimeter detection area; on the coating film, each centimeter of quality control area is preferably loaded with 1.0-1.5 mu g goat anti-rabbit polyclonal antibody; the sample pad is loaded with anti-RBC antibody and is used for removing red blood cells in the liquid sample to be detected; the sample pad is soaked by a solution containing 0.5-1.5 v/v% Tween20 and 0.4-0.6 w/v% PVA, which is beneficial to the release of the colloidal gold.

The invention also provides a preparation method of the detection test paper, wherein the sample pad, the combination pad, the coating film and the absorbent pad are sequentially fixed on the bottom plate along the flowing direction of the liquid sample to be detected; the conjugate pad is loaded with gold-labeled protein, and the gold-labeled protein comprises the antibody of the invention; the coating film is provided with a detection area and a quality control area, the detection area is loaded with a specific antibody, and the quality control area is loaded with a goat anti-mouse IgG antibody; the specific antibody is an anti-N protein specific antibody, an anti-S protein specific antibody, an anti-M protein specific antibody or an anti-E protein specific antibody; the amino acid sequence of the N protein is shown as SEQ ID NO.1, the amino acid sequence of the S protein is shown as SEQ ID NO.12, the amino acid sequence of the M protein is shown as SEQ ID NO.13, and the amino acid sequence of the E protein is shown as SEQ ID NO. 14.

In a preferred embodiment, the method for preparing the gold-labeled protein comprises: uniformly mixing the colloidal gold solution with the antibody prepared by SEQ ID NO.2 for reaction to obtain a labeled antibody 1; uniformly mixing the colloidal gold solution with the antibody prepared by SEQ ID NO.3 for reaction to obtain a labeled antibody 2; and mixing the labeled antibody 1 and the labeled antibody 2 according to the volume ratio of 2-4:7 to obtain the gold-labeled protein.

In a preferred embodiment, the particle size of the colloidal gold is 15 to 25 nm; on the bonding pad, each centimeter of the bonding pad is preferably loaded with 0.8-1.5 mug of gold-labeled protein; on the coating film, 1.5-2.5 mu g of specific polyclonal antibody is preferably loaded on each centimeter detection area; on the coating film, each centimeter of quality control area is preferably loaded with 1.0-1.5 mu g goat anti-rabbit polyclonal antibody; the sample pad is loaded with anti-RBC antibody and is used for removing red blood cells in the liquid sample to be detected; the sample pad is soaked by a solution containing 0.5-1.5 v/v% Tween20 and 0.4-0.6 w/v% PVA, which is beneficial to the release of the colloidal gold.

In a preferred embodiment, the coating buffer in the detection zone and the quality control zone is PBS independently. The inventor finds that the PBS can obviously improve the detection accuracy and sensitivity through experimental research.

The invention finally provides a detection kit for SARS-CoV-2 antigen, which comprises the detection test paper of the invention.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

The technical route of the invention is shown in figure 1, and the following embodiment scheme is further provided.

EXAMPLE 1 preparation of monoclonal antibodies

Animal immunization

① antigen preparation.

For the preparation of immunizing antigens for monoclonal antibodies, the selected antigens of this example were as follows 10 types: n1 antigen (SEQ ID NO.2), N2 antigen (SEQ ID NO.4), N3 antigen (SEQ ID NO.3), N4 antigen (SEQ ID NO.5), S1 antigen (SEQ ID NO.6), S2 antigen (SEQ ID NO.7), S3 antigen (SEQ ID NO.8), M1 antigen (SEQ ID NO.9), M2 antigen (SEQ ID NO.10), and E antigen (SEQ ID NO. 11).

② selection of immunized animals.

Mice can be selected as the immunized animal depending on the myeloma cells used. Because, the mouse myeloma cell lines for hybridoma technology are all derived from BALB/c mice. The mice are preferably 8-12 weeks old in the primary immunization, and female mice are convenient to operate.

③ determination of immunization protocol.

The appropriate immunization protocol is selected based on the specificity of the antigen. For soluble antigens, which are poorly immunogenic, adjuvants are commonly used, including Freund's complete adjuvant and Freund's incomplete adjuvant. The antigen and adjuvant are required to be mixed together in equal volumes to prepare a water-in-oil state.

④ immunization.

The antigen is immunized 50 mug/mouse for the first time, the antigen is mixed with Freund's complete adjuvant in equal volume, and prepared into water-in-oil state, and the back of the mouse is injected with multiple points, each mouse is injected with 0.3ml, and the interval is three weeks.

Immunizing 25 μ g antigen per mouse for the second time, mixing antigen with Freund's adjuvant in equal volume, and making into water-in-oil state, injecting the antigen into the back of the mouse at multiple points, injecting 0.3ml antigen per mouse, and separating three weeks.

Immunizing 25 μ g antigen/mouse for the third time, mixing antigen with Freund's adjuvant in equal volume, and making into water-in-oil state, injecting the mouse back with multiple injection points, each mouse injecting 0.3ml, and separating three weeks.

Immunizing 25 μ g antigen per mouse for the fourth time, mixing antigen with Freund's non-adjuvant in equal volume, and making into water-in-oil state, injecting mouse back with multiple injection points, and injecting 0.3ml per mouse. One week later, blood was taken to test activity.

⑤ detecting the result.

The activity of 10-fold dilution was above L6 (see FIG. 2 for reference standard), the booster was raised with 50. mu.g of antigen, i.e., i.p., and three days later, the spleen was fused.

Cell fusion

Preparation of mouse peritoneal macrophages

① BALB/c mice 6-10 weeks old were used.

② neck-pulled to kill, soaking in 75% ethanol, sterilizing for 3min, cutting skin with sterile scissors, exposing peritoneum, injecting 6ml 1640 culture solution containing no calf serum with a suction tube, repeatedly washing, and sucking out the washing solution.

③ were placed in a 10ml centrifuge tube and centrifuged at 1200rpm for 5 min.

④ the suspension was suspended in 20% calf serum 1640 medium, and the number of cells was adjusted to 1X 10⁵And/ml. Add 96 well plates, 100. mu.l/well. Adding 5% CO at 37 deg.C₂An incubator.

Preparation of myeloma cells

① myeloma cells are expanded and cultured 48 to 36 hours before fusion.

② on the day of fusion, the cells were gently blown down from the vial wall using an elbow dropper and collected in a 50ml centrifuge or fusion tube.

③ 1000r/min for 5-10 minutes, and discarding the supernatant.

④ adding 30ml of 1640 culture solution without calf serum, centrifuging and washing once, then resuspending the cells in 10ml of 1640 culture solution without calf serum, and mixing well.

⑤ taking myeloma cell suspension, adding 0.4% of Taiwan phenol blue staining solution to count the living cells for standby.

Preparation of splenocytes

① immunized BALB/c mouse is taken, the eyeball is removed to collect blood, and serum is separated to be used as positive control serum in antibody detection, meanwhile, the mouse is killed by cervical dislocation, the mouse is soaked in 75% alcohol for 5 minutes, after being fixed on a culture dish, the left abdominal skin is opened, the spleen can be seen, the forceps are cut for ophthalmology, the peritoneum is cut by aseptic operation in a super clean bench, the spleen is taken out and placed in a flat dish which is already filled with 10ml of 1640 culture solution without calf serum, the flat dish is lightly washed, and the surrounding connective tissue is carefully stripped.

② placing on stainless steel screen in a plate, grinding with syringe needle core to obtain cell suspension, counting, allowing spleen cells to enter into 1640 culture solution containing no calf serum in the plate, and blowing with suction tube for several times to obtain single cell suspension, wherein each mouse is usually 1 × 10⁸-2.5×10⁸And (4) spleen cells.

Cell fusion

① mixing the raw materials together by 1 × 10⁸Spleen cells and 1X 10⁷Myeloma cells SP2/0 were mixed in 1 fusion tube of 50ml, supplemented with incomplete medium to 30ml, and mixed well.

② 1000 centrifugal for 5-10 minutes at 1000r/min, and the supernatant is sucked up as clean as possible.

③ the bottom of the fusion tube is tapped on the palm to loosen and homogenize the precipitated cells.

④ Pre-heated 50% PEG 1ml was added over 30s with a 1ml pipette with gentle stirring.

⑤ sucking the suction tube and standing for 1 min.

⑥ adding preheated incomplete culture solution to stop PEG action, and adding 1ml, 2ml, 3ml, 4ml, 5ml and 10ml every 2min continuously.

⑦ 800rpm, 5 minutes, and discarding the supernatant.

⑧ adding 5ml of 20% calf serum 1640HAT culture medium, gently sucking the precipitated cells, suspending and mixing, supplementing 80-90ml of 20% calf serum 1640HAT culture medium, subpackaging 96-well cell culture plates with each well of 100 μ l, placing the culture plates at 37 deg.C, and adding 5% CO₂Culturing in an incubator.

After ⑨ 6 days, half-exchange the solution with HAT medium 1640 containing 20% calf serum.

⑩ the growth of the hybridoma cells is often observed and when they grow to a depth above the bottom area 1/10 of the well, the supernatant is aspirated for antibody detection.

Selection of specific hybridoma cells

① sucking 100 μ l cell supernatant to 96-well plate, inserting antibody gold label strip for detection, subcloning positive polyclonal antibody, cloning by limiting dilution method, and preparing mouse peritoneal macrophage as feeder cell.

② hybridoma cell suspensions to be cloned are prepared and diluted with HAT medium containing 20% serum to 3 different dilutions of 5, 10 and 20 cells per ml.

③ adding 5X 10 per ml⁴-1×10⁵The ratio of cells, abdominal cavity macrophage is added into the hybridoma cell suspension respectively.

④ Each hybridoma cell was dispensed into a 96-well plate at 100. mu.l per well.

⑤37℃、5％CO₂After culturing for 6 days, observing under an inverted microscope, marking out the hole with only single clone growing, and sucking out the supernatant for antibody gold mark detection when the hole grows to a bottom area of 1/10.

⑥ cells were cryopreserved.

⑦ when the cell coverage reaches 80%, it can be transferred into bottles for amplification, the culture bottles are taken out from the incubator with 37 deg.C and 5% CO2, the culture bottles are placed in a clean bench, the cells are lightly blown by aseptic bent tubes twenty times, and the blown cells are transferred to 110cm at the ratio of 1 bottle to 6 bottles²In culture flasks (about 5ml per flask). Each flask was then supplemented with 25ml of 10% calf serum in 1640 medium. Sealing (the culture bottle cap is not too tight and is provided with an upper buckle, thus ensuring the CO in the culture bottle and the culture box₂Open circuit), put in 5% CO at 37 ℃₂And (5) standing and culturing in an incubator.

⑧ when the cells in 6 culture bottles after amplification grow to 80% of the bottom area of the culture bottles, gently blowing the cells with a sterile elbow tube twenty times, collecting the cells cultured in the culture bottles, transferring the cell suspension to a 50ml centrifuge tube, centrifuging at 1500 rpm for 5min, discarding the supernatant, adding 1640 culture solution without calf serum to count the cells, adjusting the cell concentration to 1.2 × 10⁶/ml。

⑨ only one cell can be collected at a time, and the collected monoclonal antibody cells must be injected into the mice within 3 hours.

Large scale preparation of monoclonal antibodies (ascites preparation):

① mouse sensitization, the paraffin oil is sterilized by high pressure before use, F1 mouse is sensitized by liquid paraffin, the weight of the mouse reaches 22-24g after 6-8 weeks, and the injection volume of each mouse is 0.2ml per mouse in abdominal cavity, and the mouse can be used for cell inoculation for 7-15 days.

② cell injection, wherein the collected monoclonal cell is injected into 1 ml/mouse abdominal cavity, the injection device is sterile, 7-15 days show that the mouse is inactive and the mouse abdominal cavity is enlarged, only one mouse abdominal cavity cell injection can be carried out at the same time, ascites production of different types of mice can be carried out in different rooms, and cross contamination caused by cage crossing of mice is avoided.

③ ascites collection, periodically observing growth condition of inoculated mouse, making abdominal cavity round and round, timely extracting ascites when state is not good, combining low speed centrifugation for 1500 r/min, 10min, collecting 500ml, bottling, mixing, taking 1ml as ascites detection sample, placing at-20 deg.C for cryopreservation, collecting only one kind of ascites at the same time, centrifuging, bottling, and cryopreserving, and strictly prohibiting collection of two or more kinds of ascites at the same time, collecting ascites within 15 days after cell injection, and purifying.

EXAMPLE 2 preparation of polyclonal antibodies

Immunization

The antigen is selected from N protein (SEQ ID NO.1), S protein (SEQ ID NO.12), M protein (SEQ ID NO.13) and E protein (SEQ ID NO.14), and multiple antibodies are respectively prepared.

The first immunization is carried out, the antigen is taken to be mixed with Freund's complete adjuvant according to the volume of 1:1, the mixture is stirred by a vortex stirrer to prepare the water-in-oil adjuvant, and each guinea pig is injected with 2ml of adjuvant by subcutaneous immunization at multiple points. Each guinea pig was 0.5mg antigen.

And (3) carrying out second immunization, namely mixing the antigen and Freund's incomplete adjuvant in a volume ratio of 1:1 after 3 weeks of the first immunization, stirring by using a vortex stirrer, and preparing the water-in-oil adjuvant, wherein 2ml of the antigen is injected into each guinea pig subcutaneously in a multipoint way, and 0.25mg of the antigen is injected into each guinea pig subcutaneously.

And 3 weeks after the third immunization, mixing the antigen with Freund's incomplete adjuvant at a volume of 1:1, stirring with a vortex stirrer, and preparing into water-in-oil adjuvant, wherein each guinea pig is injected with 2ml of antigen at multiple points by subcutaneous immunization, and each guinea pig is 0.25mg of antigen.

Serum collection

And (3) after three times of immunization for one week, taking blood from veins, detecting titer by using a gold label, wherein the titer reaches L6-L7, taking blood from veins, putting the blood in a refrigerator with 4 ℃ for overnight, centrifuging the blood the next day, collecting serum, centrifuging the blood at 9500 rpm, centrifuging the blood for 20 minutes, and storing the serum in a refrigerator with 20 ℃ below zero for purification.

Example 3 antibody purification

(1) Ammonium sulfate precipitation

Collected ascites or serum is slowly dripped with equal volume of saturated (NH)₄)₂SO₄. And after all the materials are completely stirred, continuously stirring the materials at room temperature for 2 hours, and standing the materials at the temperature of 2-8 ℃ for 12-16 hours.

The next day, the sample was centrifuged at 15,000g for 20min at 4 ℃. Taking out the centrifugal cup, unscrewing the cup cover, observing, approaching the centrifugal sedimentation position to the palm center (the centrifugal object is upward when pouring), slowly and continuously pouring the centrifugal supernatant into the waste liquid barrel, continuously inverting the centrifugal cup for 3-5 s after the supernatant is completely poured out, and draining the liquid.

Measuring PBS by using 50ml PBS per liter of culture supernatant, dividing into centrifuge cups, sucking with a suction tube for 1min, and dissolving the centrifuged precipitate sufficiently.

Centrifuge again at 15,000g, 4 ℃ for 20 min. Taking out the centrifuge cup, unscrewing the cup cover, observing, approaching the centrifugal sedimentation position to the palm center (the centrifugal substance is upward when pouring), slowly and continuously pouring the centrifugal supernatant into a clean container, and obtaining the ProteinA column chromatography sample.

(2) Protein A affinity chromatography purification

Column assembling:

an already assembled chromatography column can be used, wherein the volume of Protein A chromatography packing used should not be less than 1/20 of the volume of the sample loaded, and the height of the column is 10 cm-12 cm. The filler should be used for fixing the antibody, and the repeated use cannot exceed 10 times.

And (4) balancing.

The packed Protein A column was fully equilibrated with PBS buffer. And (4) communicating and starting an online ultraviolet detector, and selecting a wavelength of 280nm and a 0.2A gear. A linear flow rate of 60cm/h was used. And after the detector value is stable, resetting the baseline to zero and injecting the sample.

Loading:

after the equilibration was complete, the sample was injected at a linear flow rate of 60cm/h, and the breakthrough fluid was collected when the UV detector reading rose to 15, and the peak of the maximum absorption of the breakthrough fluid was recorded. And after the sample introduction is finished, washing residual samples in the pipeline and the chromatographic column by PBS (phosphate buffer solution), and stopping collecting the penetration liquid when the reading of the ultraviolet detector is reduced to 15 until the baseline of the detector is stable.

And (3) elution:

the bound antibody was eluted with 0.1mol/L Gly buffer (pH 2.4). The linear flow rate was 60cm/h, the elution peak was collected when the UV detector reading rose to 15, the change in value was observed, the highest peak was recorded, and collection was stopped when the UV detector reading fell to 15.

Neutralizing:

and dropwise adding 1mol/L Tris into the elution sample to adjust the pH value to 6.0-6.5.

And (3) filtering:

filtering the eluted sample with 0.45 μm water system filter membrane, measuring the volume of the filtered sample with a graduated cylinder, and mixing. Protein concentration was calculated by diluting 1 volume sample with 24 volumes of PBS using PBS as a blank and measuring a 280. Protein concentration was calculated as C ═ a280 × sample dilution factor/1.36. The eluted sample was diluted exactly to 5mg/ml with PBS. Adding Proclin-300 with two ten-thousandth concentration for preservation. And marking the date outside the container, and sealing and temporarily storing the container at 2-8 ℃.

EXAMPLE 4 screening of antibodies

Method for screening raw material activity

The above antibodies were coated with each other by diluting the specific antibody to 10-20. mu.g/ml with a coating buffer (10mM PBS, pH 7.2). Labeling specific antibodies on colloidal gold with the diameter of 15-25nm, adjusting the pH value of a gold solution to 6-7, adjusting the concentration of the antibodies to 10-20 mu g/ml, sealing with 20% BSA, suspending with TB9 solution to 1/10 of the original volume after centrifugation, redissolving the antibodies with DOA redissolution, and uniformly coating the labeled colloidal gold solution on a glass cellulose membrane (1200 glass fibers) and drying for later use. The coated specific antibody and the labeled specific antibody are subjected to a cross method to detect the activity of the raw materials. The results are shown in the following table:

as can be seen from the above table: the activity of the raw material screening N1+ N, N3+ N is high without background, the activity of N1+ S/M/E is high with background, and the activity of N3+ S/M/E is also high with background.

Method for screening coating buffer

A comparative experiment was conducted on the coating buffer of the coating film, and the above-mentioned antibody specific to the anti-N protein having high coating activity was coated at 1.5mg/ml to 2.0mg/ml using three buffers, PBS (10mM PBS, pH 7.2), CB (50mM PBS, pH 9.6) and TBS (20mM PBS, pH 8.0), respectively, and the results of the activity detection were as shown in the following table:

as can be seen from the above table: the buffer screening was low in CB (50mM Ph9.6 PBS), TBS (20mM pH8.0 PBS) and PBS (10mM pH7.2 PBS) activity similar, but PBS (10mM pH7.2 PBS) better.

Method for determining mixed mark proportion

Respectively and independently labeling N1 and N3, then mixing the labeled antibody N1 and the labeled antibody N3 according to different proportions, redissolving the antibody by using DOA redissolving solution, and uniformly coating the labeled colloidal gold solution on a glass cellulose membrane (1200 glass fibers) and drying for later use. The results of the activity measurements are shown in the following table:

as can be seen from the above table, the optimum ratio of N1/N3 is between 1/3 and 1/2.

EXAMPLE 5 preparation of the kit

The main components of the kit

The nitrocellulose membrane and the coating solution are purchased from Hipposhu scientific Co., Ltd. The novel coronavirus specific antibodies (monoclonal antibody and polyclonal antibody) and goat anti-mouse IgG are all produced by Beijing Biotechnology Ltd.

Preparation method of nitrocellulose membrane

The goat anti-mouse IgG antibody was coated on the control line of NC membrane at 1.5mg/ml-2.0mg/ml with coating buffer (10mM PBS, pH7.2), the anti-N protein specific polyclonal antibody was coated on the detection line of NC membrane at 1.5mg/ml-2.0mg/ml, and the NC membrane was dried (temperature 24 + -4 deg.C, humidity < 25%) (1-2 h). And (3) storage: and filling the dried NC film into an aluminum foil bag for sealing, adding a drying agent, vacuumizing, packaging and storing at 4-30 ℃.

Preparation method of sample pad

The treatment for removing red blood cells in whole blood is carried out on the sample pad: adding anti-RBC; promoting the release of the colloidal gold: 1% Tween20+ 0.5% PVA was added.

Preparation method of colloidal gold labeled anti-N protein specific monoclonal antibody N1 and N3 mixed label

1) Labeling antibody N1 on colloidal gold with diameter of 15-25nm, adjusting pH value of gold solution to 6-7, antibody concentration of 15-20 μ g/ml, blocking with 20% BSA, centrifuging, and resuspending with TB9 solution to 1/10 of the original volume;

2) labeling antibody N3 on colloidal gold with diameter of 15-25nm, adjusting pH value of gold solution to 7-8, antibody concentration to 10-20 μ g/ml, blocking with 20% BSA, centrifuging, and resuspending with TB9 solution to 1/10 of the original volume;

3) mixing the labeled antibody N1 with the labeled antibody N3 according to the ratio of 3: 7;

4) the antibody was then reconstituted with DOA in original volume 9/10.

5) The labeled colloidal gold solution was uniformly coated on a glass cellulose film (1200 glass fiber) at 42 ml/sheet.

6) And (4) freeze-drying or drying the gold pad by using a freeze dryer.

7) And (3) putting the dried gold pad into an aluminum foil bag for sealing, adding a drying agent, vacuumizing, packaging and storing at 4-30 ℃.

Assembly of colloidal gold test strip

And sequentially fixing the sample pad, the combination pad, the nitrocellulose membrane and the absorption pad on the bottom plate along the flow direction of the liquid sample to be detected to obtain the detection test paper, wherein the schematic structural diagram is shown in fig. 3.

Detection method of kit

1) Numbering: and taking out the test paper, sticking the test paper to a corresponding position on the adhesive sticker recording paper, horizontally placing the test paper and making a mark.

2) Sample adding: 60 μ l of fresh whole blood (or serum, plasma) is aspirated by a sample applicator or a disposable blood collection tube, and slowly dropped onto a pad or a sample application well of a test card below the arrow of the test paper (the volume of the disposable blood collection tube is 80 ul).

3) Interpretation: to prevent the missed detection of the samples of Wen Yang, please observe and record the results 10 minutes after the sample is added.

Interpretation of test results

1) Positive: the test paper has two purple red lines at the detection line and the comparison line.

2) Negative: the test paper only shows a purple red line at the position of the contrast line.

3) And (3) failure: the test paper has no purple red line or only has a purple red line on the detection line.

Example 6 kit Performance testing

The following tests were carried out according to the test method of the kit in example 5:

(1) specific experiments:

detection of negative samples: the test results of 1000 cases of common clinical blood are negative.

Detection of positive samples: 453 cases of novel coronavirus infection serum confirmed by the Hubei province disease control center are detected by the kit, namely the detection rate is 90.6%.

(2) Stability test:

dividing the randomly-extracted kit to be detected produced in the same batch into 2 parts, placing 1 part in a refrigerator at 4 ℃, placing the other 1 part in a thermostat at 37 ℃ for 6 days, taking out, placing in the refrigerator at 4 ℃ for balancing overnight, and performing stability detection by using positive key quality control products.

The interpretation method comprises the following steps: the detection line shows the color strength, and the interpretation is from L0 to L10, see FIG. 2 in particular.

And (4) judging a result: the sensitivity of the kit placed at 37 ℃ for 6 days is judged to be consistent with that of the kit placed at 4 ℃ for initial detection, and the more consistent the color development, the better the stability.

Test result of stability of kit

Storage conditions of the kit	Preliminary examination 4 ℃ placing kit	Standing at 37 deg.C for 6 days	Residual rate of activity at 37 ℃
				Positive key quality control product	L4	L4	100％

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

<110> Beijing Biotechnology Ltd

<120> novel coronavirus (SARS-CoV-2) antigen detection kit

<160>14

<170>PatentIn version 3.5

<210>1

<211>419

<212>PRT

<213> Artificial sequence

<400>1

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 510 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr

100 105 110

Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp

115 120 125

Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp

130 135 140

His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln

145 150 155 160

Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser

165 170175

Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn

180 185 190

Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala

195 200 205

Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu

210 215 220

Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln

225 230 235 240

Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys

245 250 255

Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln

260 265 270

Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp

275 280 285

Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile

290 295 300

Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile

305 310 315 320

Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Ala Ala

325 330335

Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu

340 345 350

Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro

355 360 365

Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln

370 375 380

Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu

385 390 395 400

Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser

405 410 415

Thr Gln Ala

<210>2

<211>100

<212>PRT

<213> Artificial sequence

<400>2

Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr

1 5 10 15

Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg

20 25 30

Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn

35 40 45

Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu

50 55 60

Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro

65 70 75 80

Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly

85 90 95

Gly Asp Gly Lys

100

<210>3

<211>112

<212>PRT

<213> Artificial sequence

<400>3

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

1 5 10 15

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

20 25 30

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

35 40 45

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

50 55 60

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

65 70 75 80

Glu Leu Ile Arg Gln Gly ThrAsp Tyr Lys His Trp Pro Gln Ile Ala

85 90 95

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

100 105 110

<210>4

<211>109

<212>PRT

<213> Artificial sequence

<400>4

Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly

1 5 10 15

Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp

20 25 30

Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr

35 40 45

Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly

50 55 60

Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly Ser

65 70 75 80

Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn

85 90 95

Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg

100 105

<210>5

<211>112

<212>PRT

<213> Artificial sequence

<400>5

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

1 5 10 15

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

20 25 30

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

35 40 45

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

50 55 60

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

65 70 75 80

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

85 90 95

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

100 105 110

<210>6

<211>112

<212>PRT

<213> Artificial sequence

<400>6

Met AlaGly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

1 5 10 15

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

20 25 30

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

35 40 45

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

50 55 60

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

65 70 75 80

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

85 90 95

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

100 105 110

<210>7

<211>112

<212>PRT

<213> Artificial sequence

<400>7

Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp

1 5 10 15

Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln Gln

2025 30

Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys

35 40 45

Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala

50 55 60

Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln

65 70 75 80

Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala

85 90 95

Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly

100 105 110

<210>8

<211>88

<212>PRT

<213> Artificial sequence

<400>8

Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly

1 5 10 15

Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val

20 25 30

Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr

35 40 45

Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu

50 55 60

Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn

65 70 75 80

Phe Tyr Glu Pro Gln Ile Ile Thr

85

<210>9

<211>128

<212>PRT

<213> Artificial sequence

<400>9

Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu

1 5 10 15

Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile

20 25 30

Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile

35 40 45

Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys

50 55 60

Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile

65 70 75 80

Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe

85 90 95

Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr ArgSer Met Trp Ser Phe

100 105 110

Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile

115 120 125

<210>10

<211>103

<212>PRT

<213> Artificial sequence

<400>10

Met Asn Val Pro Leu His Gly Thr Ile Leu Thr Arg Pro Leu Leu Glu

1 5 10 15

Ser Glu Leu Val Ile Gly Ala Val Ile Leu Arg Gly His Leu Arg Ile

20 25 30

Ala Gly His His Leu Gly Arg Cys Asp Ile Lys Asp Leu Pro Lys Glu

35 40 45

Ile Thr Val Ala Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Leu Gly Ala

50 55 60

Ser Gln Arg Val Ala Gly Asp Ser Gly Phe Ala Ala Tyr Ser Arg Tyr

65 70 75 80

Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp His Ser Ser Ser Ser Asp

85 90 95

Asn Ile Ala Leu Leu Val Gln

100

<210>11

<211>75

<212>PRT

<213> Artificial sequence

<400>11

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 25 30

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val

65 70 75

<210>12

<211>1301

<212>PRT

<213> Artificial sequence

<400>12

Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro

1 5 10 15

Arg Gly Ser His Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser

20 25 30

Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr

35 40 45

Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg

50 55 60

Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser

65 70 75 80

Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr

85 90 95

Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe

100 105 110

Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr

115 120 125

Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr

130 135 140

Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe

145 150 155 160

Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu

165 170 175

Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser

180 185 190

Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn

195200 205

Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr

210 215 220

Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe

225 230 235 240

Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr

245 250 255

Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly

260 265 270

Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly

275 280 285

Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr

290 295 300

Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys

305 310 315 320

Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser

325 330 335

Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile

340 345 350

Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala

355360 365

Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp

370 375 380

Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr

385 390 395 400

Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr

405 410 415

Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro

420 425 430

Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp

435 440 445

Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys

450 455 460

Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn

465 470 475 480

Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly

485 490 495

Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu

500 505 510

Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr

515 520 525

Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val

530 535 540

Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn

545 550 555 560

Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn

565 570 575

Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr

580 585 590

Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr

595 600 605

Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr

610 615 620

Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val

625 630 635 640

Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr

645 650 655

Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly

660 665 670

Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala

675 680685

Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala

690 695 700

Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly

705 710 715 720

Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr

725 730 735

Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr

740 745 750

Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu

755 760 765

Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn

770 775 780

Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu

785 790 795 800

Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp

805 810 815

Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro

820 825 830

Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu

835 840845

Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile

850 855 860

Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val

865 870 875 880

Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala

885 890 895

Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala

900 905 910

Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly

915 920 925

Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala

930 935 940

Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser

945 950 955 960

Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala

965 970 975

Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala

980 985 990

Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu

995 1000 1005

Ala Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser

1010 1015 1020

Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile

1025 1030 1035

Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val

1040 1045 1050

Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His

1055 1060 1065

Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu

1070 1075 1080

His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala

1085 1090 1095

Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly

1100 1105 1110

Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn

1115 1120 1125

Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser

1130 1135 1140

Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr

1145 1150 1155

Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp

1160 1165 1170

Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp

1175 1180 1185

Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile

1190 1195 1200

Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile

1205 1210 1215

Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro

1220 1225 1230

Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val

1235 1240 1245

Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys

1250 1255 1260

Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu

1265 1270 1275

Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr

1280 1285 1290

Leu Glu His His His His His His

1295 1300

<210>13

<211>222

<212>PRT

<213> Artificial sequence

<400>13

Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu

1 5 10 15

Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile

20 25 30

Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile

35 40 45

Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys

50 55 60

Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile

65 70 75 80

Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe

85 90 95

Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe

100 105 110

Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile

115 120 125

Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile

130 135 140

Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp

145 150 155 160

Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu

165 170 175

Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly

180 185 190

Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr

195 200 205

Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln

210 215 220

<210>14

<211>247

<212>PRT

<213> Artificial sequence

<400>14

Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser

1 5 10 15

Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala

20 25 30

Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn

35 40 45

Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn

50 55 60

Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val Gly Gly Gly Gly Ser

65 70 75 80

Ala Ser Gly Gly Gly Ser Met Tyr Ser Phe Val Ser Glu Glu Thr Gly

85 90 95

Thr Leu Ile Val Asn Ser Val Leu Leu Phe Leu Ala Phe Val Val Phe

100 105 110

Leu Leu Val Thr Leu Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr

115 120 125

Cys Cys Asn Ile Val Asn Val Ser Leu Val Lys Pro Ser Phe Tyr Val

130 135 140

Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser Arg Val Pro Asp Leu Leu

145 150 155 160

Val Gly Gly Gly Gly Ser Ala Ser Gly Gly Gly Ser Met Tyr Ser Phe

165 170 175

Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser Val Leu Leu Phe

180 185 190

Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala Ile Leu Thr Ala

195 200 205

Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn Val Ser Leu Val

210 215 220

Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser

225230 235 240

Arg Val Pro Asp Leu Leu Val

245

Claims (10)

1. SARS-CoV-2 antigen, wherein the antigen is a protein composed of an amino acid sequence shown in any one of SEQ ID NO.2-SEQ ID NO. 11.

2. An antibody against SARS-CoV-2 that specifically binds to the antigen of claim 1.

3. The antibody of claim 2, wherein the antibody is a monoclonal antibody.

4. A method for preparing the antibody of claim 2 or 3, wherein the antibody is obtained by immunizing an animal with an immunogen prepared by using a protein consisting of an amino acid sequence shown in any one of SEQ ID NO.2 to SEQ ID NO.11 as an antigen and an adjuvant.

5. The method for preparing the antibody according to claim 4, wherein the immunogen is prepared by using a protein consisting of an amino acid sequence shown in any one of SEQ ID No.2 to SEQ ID No.11 as an antigen and an adjuvant, after an animal is immunized, the titer of the antibody to be detected meets the requirement that the dilution activity is more than 10 times that of L6, the animal is immunized again, then spleen cells of the immunized animal are taken to be subjected to cell fusion with myeloma cells, and the antibody is obtained by screening positive clones, amplification and purification;

preferably, the animal is a mouse, preferably a BALB/c mouse;

preferably, the adjuvant comprises freund's complete adjuvant or freund's incomplete adjuvant;

preferably, the step of immunizing the animal comprises: performing primary immunization by using the antigen and the immunogen prepared by Freund's complete adjuvant by back multi-point injection, and performing secondary, tertiary and quaternary immunization by using the antigen and the immunogen prepared by Freund's incomplete adjuvant every three weeks;

preferably, the amount of antigen for both the first and second immunizations is independently 40-60 μ g;

preferably, the amount of antigen for each of the second, third and fourth immunizations is independently 20-30 μ g;

preferably, the ratio of the number of spleen cells to the number of myeloma cells is 1X 10⁷-1×10⁹：1×10⁷；

Preferably, cell fusion uses peritoneal macrophages as feeder cells;

preferably, amplification of positive clones is made using ascites fluid.

6. Use of the antigen of claim 1 or the antibody of claim 2 or 3 for the preparation of a detection product for detecting the SARS-CoV-2 antigen;

preferably, the product comprises a test strip or kit.

7. A product for detecting SARS-CoV-2 antigen using a double antibody sandwich method, wherein the antibody used comprises the antibody of claim 2 or 3.

8. The SARS-CoV-2 antigen detecting test paper includes one base plate, one sample pad, one combining pad, one coating film and one absorbing pad fixed successively on the base plate; the conjugate pad is loaded with a gold-labeled protein comprising the antibody of claim 2 or 3; the coating film is provided with a detection area and a quality control area, the detection area is loaded with a specific antibody, and the quality control area is loaded with a goat anti-mouse IgG antibody; the specific antibody is an anti-N protein specific antibody, an anti-S protein specific antibody, an anti-M protein specific antibody or an anti-E protein specific antibody; the amino acid sequence of the N protein is shown as SEQ ID NO.1, the amino acid sequence of the S protein is shown as SEQ ID NO.12, the amino acid sequence of the M protein is shown as SEQ ID NO.13, and the amino acid sequence of the E protein is shown as SEQ ID NO. 14;

preferably, the gold-labeled protein comprises monoclonal antibodies respectively prepared by SEQ ID NO.2 and SEQ ID NO. 3;

preferably, the specific polyclonal antibody is an anti-N protein specific antibody;

preferably, the liquid sample to be tested comprises whole blood, serum or plasma;

preferably, the particle size of the colloidal gold is 15-25 nm;

preferably, the binding pad is loaded with 0.8-1.5 μ g of gold-labeled protein per square centimeter of the binding pad;

preferably, the coating film is loaded with 1.5-2.5 μ g of specific antibody per cm of detection zone;

preferably, the coating film is loaded with 1.0-1.5 mu g of goat anti-rabbit polyclonal antibody per centimeter of quality control area;

preferably, the sample pad is soaked with a solution containing 0.5-1.5 v/v% Tween20, 0.4-0.6 w/v% PVA, and 0.1-0.2mg/ml anti-RBC antibody.

9. The method for preparing a test strip according to claim 8, wherein the sample pad, the conjugate pad, the envelope and the absorbent pad are sequentially fixed on the base plate along the flow direction of the liquid sample to be tested; the conjugate pad is loaded with a gold-labeled protein comprising the antibody of claim 2 or 3; the coating film is provided with a detection area and a quality control area, the detection area is loaded with a specific antibody, and the quality control area is loaded with a goat anti-mouse IgG antibody; the specific antibody is an anti-N protein specific antibody, an anti-S protein specific antibody, an anti-M protein specific antibody or an anti-E protein specific antibody; the amino acid sequence of the N protein is shown as SEQ ID NO.1, the amino acid sequence of the S protein is shown as SEQ ID NO.12, the amino acid sequence of the M protein is shown as SEQ ID NO.13, and the amino acid sequence of the E protein is shown as SEQ ID NO. 14;

preferably, the preparation method of the gold-labeled protein comprises the following steps: uniformly mixing the colloidal gold solution with the antibody prepared by SEQ ID NO.2 for reaction to obtain a labeled antibody 1; uniformly mixing the colloidal gold solution with the antibody prepared by SEQ ID NO.3 for reaction to obtain a labeled antibody 2; mixing the labeled antibody 1 and the labeled antibody 2 according to the volume ratio of 2-4:7 to obtain gold labeled protein;

preferably, the specific polyclonal antibody is an anti-N protein specific antibody;

preferably, the particle size of the colloidal gold is 15-25 nm;

preferably, each centimeter of the bonding pad is loaded with 0.8-1.5 mug of gold-labeled protein;

preferably, the coating film is loaded with 1.5-2.5 μ g of specific antibody per cm of detection zone;

preferably, the coating film is loaded with 1.0-1.5 mu g of goat anti-rabbit polyclonal antibody per centimeter of quality control area;

preferably, the coating buffer solution on the coating film, the detection area and the quality control area is PBS independently;

preferably, the sample pad is soaked with a solution containing 0.5-1.5 v/v% Tween20, 0.4-0.6 w/v% PVA, and 0.1-0.2mg/ml anti-RBC antibody.

10. A kit for detecting SARS-CoV-2 antigen, comprising the test paper according to claim 8.

Images