Novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip and preparation method

Abstract

The present invention belongs to the field of immunological detection, and relates to a preparation method and a detection reagent strip of a novel coronavirus monoclonal antibody visualization signal enhancement antigen detection reagent strip, wherein the preparation method includes the preparation of a nitrocellulose membrane detection layer, the preparation of a conjugation pad, and the preparation of a water-absorbing pad; the end of the nitrocellulose membrane detection layer close to the conjugation pad is coated with an anti-novel coronavirus N protein double epitope mixed antibody and an anti-novel coronavirus S protein monoclonal antibody; the end of the nitrocellulose membrane detection layer close to the water-absorbing pad is coated with a goat anti-mouse IgG antibody; the conjugation pad is sprayed with a colloidal gold-labeled anti-novel coronavirus N protein double epitope mixed antibody and a colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody. The present invention provides a preparation method and a detection reagent strip of a novel coronavirus monoclonal antibody visualization signal enhancement antigen detection reagent strip, which has high sensitivity of the kit, low missed detection rate of mutants, is quick, simple, highly sensitive, and highly specific.

Description

Technical Field

[0001] The present invention belongs to the field of immunological detection, and relates to a virus detection reagent strip and a preparation method, and in particular to a novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip and a preparation method. Background Art

[0002] Coronavirus belongs to the Coronavirus family and the Coronavirus genus. It is a type of single-stranded positive-sense RNA virus. Since the first virus was discovered in 1937, a number of coronaviruses have been identified, belonging to the α, β, γ and δ genera. Among them, 7 coronaviruses can infect humans, namely HCoV-229E and HCoV-NL63 of the α genus and HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV of the β genus, and the new coronavirus SARS-CoV-2 identified at the end of 2019. The coronavirus genome encodes the spike protein, envelope protein, membrane protein and nucleocapsid protein in sequence. Among them, the spike protein is the most important surface membrane protein of the coronavirus, containing two subunits S1 and S2. Among them, S1 mainly contains the receptor binding domain (RBD), which is responsible for recognizing the cell's receptors. S2 contains the basic elements required for the membrane fusion process. The Spike protein is responsible for the binding and membrane fusion of the virus and host cell membrane receptors. It is an important site for host neutralizing antibodies and a key target for vaccine design. The Spike protein of SARS-CoV-2 interacts with human ACE2 to infect human respiratory epithelial cells. The nucleocapsid protein is the most abundant protein in coronaviruses. During the assembly of the virion, the N protein binds to the viral RNA and leads to the formation of a helical nucleocapsid.

[0003] There are colloidal gold immunoassay kits targeting viral N or S proteins on the market, but the monoclonal antibodies used are a pair that only target two epitopes of viral antigens. Although they have high specificity, their sensitivity and anti-mutation ability are relatively weak, which is far behind the nucleic acid detection method and greatly limits their application. Summary of the invention

[0004] In order to solve the above-mentioned technical problems existing in the background technology, the present invention provides a novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip and a preparation method thereof, which has high sensitivity, low missed detection rate of mutants, and is quick, simple, highly sensitive and highly specific.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip, the novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip comprising a sample pad, a first conjugation pad, a second conjugation pad, a nitrocellulose membrane detection layer, a water absorbent pad and a rigid polyvinyl chloride backing plate; the nitrocellulose membrane detection layer is laid on the upper surface of the rigid polyvinyl chloride backing plate; the end of the nitrocellulose membrane detection layer close to the second conjugation pad is coated with an anti-novel coronavirus N protein double-epitope mixed antibody and an anti-novel coronavirus S protein monoclonal antibody; the end of the nitrocellulose membrane detection layer close to the water absorbent pad is coated with a goat anti-mouse IgG antibody; the first conjugation pad is sprayed with a colloidal gold-labeled anti-novel coronavirus N protein double-epitope mixed antibody; the second conjugation pad is sprayed with a colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody; the sample pad, the first conjugation pad and the second conjugation pad are sequentially connected from front to back and are jointly arranged at one end of the nitrocellulose membrane detection layer; the water absorbent pad is arranged at the other end of the nitrocellulose membrane detection layer.

[0007] The novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip provided by the present invention is used to detect the 2019-nCoV (officially renamed COVID-19 on February 11-12, 2020) virus, and the "novel coronavirus" in this application document refers to this virus. A method for preparing the novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip as described above, the method comprising the preparation of a nitrocellulose membrane detection layer, the preparation of a first conjugation pad, the preparation of a second conjugation pad, and the preparation of an absorbent pad; the end of the nitrocellulose membrane detection layer close to the conjugation pad is coated with an anti-novel coronavirus N protein dual-epitope mixed antibody and an anti-novel coronavirus S protein monoclonal antibody; the end of the nitrocellulose membrane detection layer close to the absorbent pad is coated with a goat anti-mouse IgG antibody; the conjugation pad is sprayed with a colloidal gold-labeled anti-novel coronavirus N protein dual-epitope mixed antibody and a colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody.

[0008] Preferably, the preparation method of the nitrocellulose membrane detection layer adopted in the present invention is: two monoclonal antibodies targeting different epitopes of the new coronavirus N protein are mixed in equal proportions (referring to equimolar mixing) to prepare a dual-epitope mixed antibody against the new coronavirus N protein, and at the same time, a monoclonal antibody against the new coronavirus S protein is prepared, and the two antibodies are coated on the membrane surface using a film drawing instrument to form T1 and T2 detection lines, respectively, and sheep anti-mouse IgG is coated on the membrane surface using a film drawing instrument to form a quality control C line.

[0009] Preferably, the anti-novel coronavirus N protein dual-epitope mixed monoclonal antibody coated on the nitrocellulose membrane detection layer of the detection reagent strip, one of which is characterized in that it comprises three heavy chain complementary determining regions (A-CDR-H1, A-CDR-H2 and A-CDR-H3) and three light chain complementary determining regions (A-CDR-L1, A-CDR-L2 and A-CDR-L3), wherein the amino acid sequence of A-CDR-H1 is GYTFTDYS, the amino acid sequence of A-CDR-H2 is INTETGEA, the amino acid sequence of A-CDR-H3 is TRSTATDD, the amino acid sequence of A-CDR-L1 is QSVDYDGDSY, the amino acid sequence of A-CDR-L2 is TAS, and the amino acid sequence of A-CDR-L3 is QQSNEDPLT;

[0010] Further, the anti-novel coronavirus N protein dual-epitope mixed monoclonal antibody coated on the nitrocellulose membrane detection layer of the detection reagent strip, wherein another monoclonal antibody is characterized in that it comprises three heavy chain complementary determining regions (B-CDR-H1, B-CDR-H2 and B-CDR-H3) and three light chain complementary determining regions (B-CDR-L1, B-CDR-L2 and B-CDR-L3), wherein the amino acid sequence of B-CDR-H1 is GYTFTNYL, the amino acid sequence of B-CDR-H2 is INLYNDVT, the amino acid sequence of B-CDR-H3 is ARSYHGFYWYIDV, the amino acid sequence of B-CDR-L1 is EEINNYGISF, the amino acid sequence of B-CDR-L2 is AAS, and the amino acid sequence of B-CDR-L3 is QQSKEVPWT;

[0011] Preferably, the anti-novel coronavirus S protein monoclonal antibody coated on the nitrocellulose membrane detection layer of the detection reagent strip is characterized in that it comprises three heavy chain complementary determining regions (S-CDR-H1, S-CDR-H2 and S-CDR-H3) and three light chain complementary determining regions (S-CDR-L1, S-CDR-L2 and S-CDR-L3), wherein the amino acid sequence of S-CDR-H1 is AYSITSDYA, the amino acid sequence of S-CDR-H2 is ISYSGRT, the amino acid sequence of S-CDR-H3 is AREAFYFDY, the amino acid sequence of S-CDR-L1 is SSVSY, the amino acid sequence of S-CDR-L2 is RTS, and the amino acid sequence of S-CDR-L3 is HQYHSYPWT;

[0012] Preferably, the concentrations of the anti-novel coronavirus N protein dual-epitope mixed antibody and the anti-novel coronavirus S protein monoclonal antibody used in the present invention are 0.1-3.0 mg / ml, and the streaking coating amount is 0.2-2.0 ul / cm.

[0013] Preferably, the concentration of goat anti-mouse IgG coated on the nitrocellulose membrane detection layer used in the present invention is 0.1-3.0 mg / ml, and the streaking coating amount is 0.2-2.0 ul / cm.

[0014] Preferably, the specific preparation method of the first conjugate pad used in the present invention is: prepare a conjugate pad, prepare a colloidal gold solution by a trisodium citrate reduction method, prepare a colloidal gold-labeled anti-novel coronavirus N protein double-epitope mixed antibody based on the prepared colloidal gold solution, and spray the prepared colloidal gold-labeled anti-novel coronavirus N protein double-epitope mixed antibody onto the conjugate pad surface by a biofilm gold spraying instrument to form a first conjugate pad;

[0015] Preferably, the anti-novel coronavirus N protein dual-epitope mixed antibody on the first conjugate pad of the present invention, one of the monoclonal antibodies, is characterized in that it comprises three heavy chain complementary determining regions (A-CDR-H1, A-CDR-H2 and A-CDR-H3) and three light chain complementary determining regions (A-CDR-L1, A-CDR-L2 and A-CDR-L3), wherein the amino acid sequence of A-CDR-H1 is GYSFTGYN, the amino acid sequence of A-CDR-H2 is IDPYYGDT, the amino acid sequence of A-CDR-H3 is ARDGSTDGYYLDY, the amino acid sequence of A-CDR-L1 is QSVSTSSYNY, the amino acid sequence of A-CDR-L2 is YAS, and the amino acid sequence of A-CDR-L3 is QNSWEIPLT;

[0016] Further, the anti-novel coronavirus N protein dual-epitope mixed antibody on the first conjugate pad of the present invention, wherein another monoclonal antibody is characterized in that it comprises three heavy chain complementary determining regions (B-CDR-H1, B-CDR-H2 and B-CDR-H3) and three light chain complementary determining regions (B-CDR-L1, B-CDR-L2 and B-CDR-L3), wherein the amino acid sequence of B-CDR-H1 is GYTFTGYT, the amino acid sequence of B-CDR-H2 is IGYINPSSEFA, the amino acid sequence of B-CDR-H3 is ARESLDY, the amino acid sequence of B-CDR-L1 is QSLLDSGNQKNY, the amino acid sequence of B-CDR-L2 is GAS, and the amino acid sequence of B-CDR-L3 is QNDHSYPLT;

[0017] The specific preparation method of the second conjugate pad is: take another conjugate pad, prepare a colloidal gold solution by a trisodium citrate reduction method, prepare a colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody based on the prepared colloidal gold solution, and spray the prepared colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody onto the surface of the conjugate pad by a bio-scratch gold sprayer to form a second conjugate pad.

[0018] Preferably, the anti-novel coronavirus S protein monoclonal antibody on the second conjugate pad of the present invention is characterized in that it comprises three heavy chain complementary determining regions (S-CDR-H1, S-CDR-H2 and S-CDR-H3) and three light chain complementary determining regions (S-CDR-L1, S-CDR-L2 and S-CDR-L3), wherein the amino acid sequence of S-CDR-H1 is GYALTNYL, the amino acid sequence of S-CDR-H2 is INPGSGAP, the amino acid sequence of S-CDR-H3 is ALDSSGYGYALDY, the amino acid sequence of S-CDR-L1 is QSVLTN, the amino acid sequence of S-CDR-L2 is SAF, and the amino acid sequence of S-CDR-L3 is HQYNSYPLT;

[0019] Preferably, the preparation method of the first binding pad and the preparation method of the second binding pad used in the present invention further include: transferring the prepared first binding pad and the second binding pad to a space with a relative humidity lower than 30%, and placing them for 1-2 hours for standby use.

[0020] Preferably, the specific implementation method of step 1) adopted in the present invention is:

[0021] 1.1) preparing a binding pad treatment solution: the content and components of the binding pad treatment solution are 0.01M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone; the pH of the binding pad treatment solution is 8.7;

[0022] 1.2) Soaking treatment: Soak the two conjugate pads in the conjugate pad treatment solution for 1-2 hours and then dry them in a drying room for 12-16 hours;

[0023] 1.3) Collect the dried bonding pads, cut them and store them in a dry and sealed environment for future use.

[0024] Preferably, the method for preparing the novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip adopted by the present invention further comprises preparing a sample pad, and the specific preparation method of the sample pad is:

[0025] a) preparing a sample pad treatment solution: the components and contents of the sample pad treatment solution are 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone, and the pH of the sample pad treatment solution is 8.7;

[0026] b) Soaking treatment: Soak a single conjugate pad in the conjugate pad treatment solution for 1-2 hours and then dry it in a drying room for 12-16 hours;

[0027] c) Collect the dried bonding pads, cut them and store them in a dry and sealed environment for future use.

[0028] Preferably, the binding pad used in the present invention is a glass fiber pad; and the sample pad is a glass fiber pad or a polyester pad.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] First, the present invention uses three pairs of monoclonal antibodies to recognize six different epitopes of N and S antigens. The antibodies have a synergistic effect and can efficiently capture related antigens on colloidal gold test paper, making the test paper highly sensitive and specific. The lower limit of N antigen detection in the test paper is 2×10 2 CFU / ml, which basically reaches the sensitivity of the nucleic acid fluorescence quantification kit and can realize rapid detection of asymptomatic carriers with low virus load.

[0031] Secondly, since the S protein content of the virus itself is lower than that of the N protein, the co-testing of N and S can provide relative quantitative data of the patient's viral load. If both N and S antigens are positive, it proves that the patient's viral load is high, he is in a diseased state, and is highly contagious; if the N antigen is positive and the S antigen is negative, it means that the viral load is low. Therefore, the co-testing of N and S antigens can quickly determine the patient's infection status and infectivity, which is conducive to the design of treatment plans.

[0032] Third, the present invention uses two conjugate pads in series, which can prevent the dead gold effect and gold removal effect caused by antibody aggregation caused by spraying excessive gold-labeled antibodies on one conjugate pad, thereby reducing the sensitivity of the test paper and generating false positives. At the same time, the loading capacity of the gold-labeled antibodies is greatly increased, so that the visualization signal of the colloidal gold precipitation is enhanced, and the sensitivity is greatly improved.

[0033] Finally, the monoclonal antibody of the present invention has high specificity and no cross-reaction with other common respiratory pathogens including SARS. The kit of the present invention has good specificity and stability, which is significantly higher than the traditional microbial detection method, and has the advantages of rapidity and high efficiency.

[0034] The novel coronavirus monoclonal antibody visualized signal enhanced antigen detection reagent strip provided by the present invention is a double antibody sandwich method principle, combined with immunochromatography and colloidal gold surface antibody directional labeling technology to achieve a joint qualitative detection of the novel coronavirus N and S antigens in the sample, overcoming the defects of the prior art novel coronavirus detection kits with relatively poor sensitivity, and the existence of many missed diagnoses and false positives. By using three pairs of monoclonal antibodies, different epitopes of the N and S antigens can be detected simultaneously in one test, solving the defects of the current detection kits with low sensitivity and missed detection of mutants, and having the advantages of being quick, simple, highly sensitive and highly specific. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings:

[0036] Figure 1 It is a schematic diagram of the structure of the test strip provided by the present invention;

[0037] in:

[0038] 1-sample pad; 2-first conjugation pad; 3-second conjugation pad; 4-nitrocellulose membrane detection layer; 5-quality control line; 6-N antigen detection line; 7-S antigen detection line; 8-rigid polyvinyl chloride backing plate; 9-water absorbent pad. DETAILED DESCRIPTION

[0039] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0040] Example 1 Preparation of monoclonal antibodies against novel coronavirus N protein and S protein

[0041] 1) Preparation of monoclonal antibodies against the novel coronavirus N protein

[0042] 1.1) Animal immunization

[0043] The recombinant protein of the N protein of the new coronavirus (SARS-CoV-2 (2019-nCoV)) with the catalog number 40588-V08B of Beijing Yiqiao Shenzhou Technology Co., Ltd. was used as an antigen to immunize 5 8-week-old BALB / c mice, and 2 mice were not immunized as negative controls. After the initial immunization antigen was fully emulsified with an equal amount of Freund's complete adjuvant, the mice were immunized by multiple subcutaneous injections on the back, 100μg / mouse. After an interval of three weeks, the same dose of antigen was fully emulsified with Freund's incomplete adjuvant and then intraperitoneally injected for the second immunization. After an interval of 2 weeks, the same dose of antigen was fully emulsified with Freund's incomplete adjuvant and then intraperitoneally injected for the third immunization. Blood was collected from the tail vein 15 days after the third immunization to detect the titer of the antiserum.

[0044] 1.2) Antiserum titer detection

[0045] Antiserum titer was determined by indirect ELISA method: dilute the antigen concentration to 3 μg / mL with PBS (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, pH 7.4), add to 96-well plate, 100 μL / well, incubate at 37°C for 2 h or 4°C overnight. Wash the plate 3 times with PBST (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, 0.5% (v / v) Tween-20, pH 7.4) and tap the plate. 1% bovine serum albumin was dissolved in PBS solution, 250 μL / well, blocked at 37°C for 1 h or 4°C overnight. Wash the plate 3 times with PBST and tap the plate. The sera of 4 immune mice were diluted with PBS and added to the corresponding wells, 100 μL / well, the blank control was PBS solution, and the negative control was the mouse serum before immunization. The plate was washed after coating at 37℃ for 1 hour. HRP-labeled goat anti-mouse IgG was added at a dilution of 1:3000, 100 μL / well, and the plate was washed after coating at 37℃ for 1 hour. 100 μL of freshly prepared TMB color substrate solution was added to each well. After 10 minutes at 37℃, 100 μL of 1M hydrochloric acid was added to each well to terminate the reaction. The OD450nm value was measured with an enzyme-linked detector, and the results were read and observed. The highest titer (titer 51200) was selected, and the third immunization was strengthened one month later. After 5 days, the mouse spleen cells were taken for cell fusion.

[0046] 1.3) Recovery and culture of SP2 / 0 myeloma cells

[0047] The frozen myeloma cells (SP2 / 0) were revived in advance. The myeloma cells frozen in liquid nitrogen were quickly taken out and placed in a 37°C water bath with slight shaking to melt them quickly. Note that the freezing tube mouth cannot touch water to avoid contamination. Then the cells were transferred to a 24-well plate containing 2 ml of RPMI-1640 complete medium (RPMI-1640 medium containing 20% ​​fetal bovine serum, and the fetal bovine serum was purchased from Shanxi Runsheng Daye Biomaterials Co., Ltd.) and placed in a 37°C, 5% CO2 incubator for half an hour. When all the cells grew attached to the wall, the medium was changed in time. After that, the cells were passaged every 3 days, and the cells were adjusted to the optimal growth density. When the cells reached a certain activity, they were counted and prepared for fusion. Two days before cell fusion, the cells were passaged 1 to 4, and the cell concentration in each bottle was adjusted to 1×10 5 / ml.

[0048] 1.4) Preparation of feeder cells

[0049] 1.4.1) After blood was collected from the eyeballs of BALB / c mice, the mice were killed by pulling their necks apart, and the mice were completely immersed in 75% alcohol for 5 min. The mice were then transferred to a flat dish on a clean bench with their abdomen facing upward.

[0050] 1.4.2) Use forceps to lift the mouse chest and abdominal skin, cut a small hole with scissors, use two forceps to tear a larger hole in the skin, lift the mouse peritoneum, cut it open, find the mouse spleen, use forceps and small scissors to carefully remove the spleen, put it in a disposable dish, carefully peel off the fat, connective tissue, etc. attached to the spleen, add 5 ml of RPMI-1640 culture medium (purchased from Hyclone, product number SH30809.01), puncture the spleen with the needle of a syringe filled with 5 ml of RPMI-1640 culture medium, carefully wash out the spleen cells, and then sieve, add the spleen cell suspension to a 10 ml centrifuge tube, centrifuge at 1100 rpm for 5 minutes, discard the supernatant, and wash twice with RPMI-1640 culture medium.

[0051] 1.4.3) Gently resuspend the cells in 5 ml of HAT medium and mix well, count, and add HAT medium until the cell concentration is 1×10 5 / ml.

[0052] 1.4.4) Drop the cell suspension into a 96-well cell culture plate, 130 μl / well, and culture in a 37°C, 5% CO2 incubator.

[0053] 1.5) Preparation of immune spleen cell suspension

[0054] 1.5.1) Five days after booster immunization, select BALB / c mice with the highest serum titer, remove their eyeballs, bleed, and collect and separate serum as a positive control for antibody detection.

[0055] 1.5.2) The mice were killed by cervical dislocation and then immersed in 75% alcohol for 5 min. The mice were taken out and placed in a plate on a sterile clean bench with their abdomen facing upwards.

[0056] 1.5.3) Use forceps to lift the mouse chest and abdominal skin, cut a small hole with scissors, and then use two forceps to tear a larger hole in the skin. Then use new forceps to lift the mouse peritoneum, cut it open, find the mouse spleen, carefully remove the spleen, place it in a disposable dish, and carefully remove the fat and connective tissue.

[0057] 1.5.4) After rinsing with RPMI-1640 solution, add new RPMI-1640 solution, puncture the spleen with the needle of a syringe filled with 5 ml of RPMI-1640 medium to carefully wash out the splenocytes, then sieve to squeeze as many splenocytes as possible through the mesh into the solution, transfer the splenocyte suspension into a centrifuge tube, centrifuge at 1100 rpm for 5 min, discard the supernatant, and centrifuge and wash twice.

[0058] 1.5.5) Gently resuspend the spleen cells in RPMI-1640 culture medium, count and set aside.

[0059] 1.6) Preparation of SP2 / 0 myeloma cell suspension

[0060] 1.6.1) Take 2 bottles of myeloma cells cultured in T75 culture flasks (change the medium one day before fusion, and the cells should be in the logarithmic growth phase during fusion) and collect them in 50 ml centrifuge tubes.

[0061] 1.6.2) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0062] 1.6.3) Add 30 ml of RPMI-1640 washing solution to the precipitate, gently resuspend, mix, and centrifuge and wash again in the same way.

[0063] 1.6.4) Gently resuspend the spleen cells in 10 ml RPMI-1640 culture medium and mix evenly, count and set aside.

[0064] 1.7) Cell fusion

[0065] 1.7.1) will contain 1×10 8 A suspension of spleen cells containing 1×10 7 Mix the suspension of 100 SP2 / 0 myeloma cells in a 50ml centrifuge tube, add culture medium to 40ml, and mix thoroughly.

[0066] 1.7.2) Centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

[0067] 1.7.3) Gently tap the bottom of the centrifuge tube to loosen and evenly distribute the cell clumps into a paste.

[0068] 1.7.4) Take out the prepared 50% PEG (MW1450) and RPMI-1640 washing solution from the 4°C refrigerator and place it in a 37°C water bath to pre-warm it for use.

[0069] 1.7.5) Use a 1 ml pipette to draw 0.8 ml of 50% PEG (MW1450) and slowly add it into the centrifuge tube while stirring for 60 seconds.

[0070] 1.7.6) Then gradually add 40 ml of preheated RPMI-1640 washing solution over 60 seconds to dilute the PEG and make it lose its fusogenic effect.

[0071] 1.7.7) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0072] 1.7.8) Add 400 mL of HAT culture medium (purchased from Sigma, catalog number H0262) and gently pipette to resuspend the precipitated cells.

[0073] 1.7.9) Add the fused cell suspension into a 96-well culture plate containing feeder cells, 50 μl / well, and spread 20 culture plates in total. Then, place the culture plates in an incubator at 37°C and 5% CO2 for culture.

[0074] 1.8) Screening and cloning of positive clones

[0075] Starting from the third day after fusion, observe the cell growth in each well every day. If there is contamination, treat it with sodium azide immediately. On the 7th day after fusion, replace the medium with HT culture medium (HT, 50×, purchased from Sigma, item number H0137). The next day after the medium change, aspirate the supernatant of the well where the clone appears for specific detection. The recombinant antigen of the new coronavirus N protein was used as the coating antigen and detected by indirect ELISA. OD450 was measured with an enzyme-linked detector, and the P / N value was >2.1 for positive. The positive wells were replaced with HT culture medium again, and the ELISA test was positive again the next day. The clone wells with continuous positive results were selected for 2-3 subcloning to screen out monoclonal hybridoma cells. Specific steps of subcloning: ① Blow and mix the positive fusion well hybridoma cells and measure the cell concentration; ② Prepare feeder cells in advance and suspend them in HT culture medium, 130ul / well, and spread them into 96-well plates, and then place the culture plates in an incubator at 37°C and 5% CO2 for use; ③ Take the positive fusion well hybridoma cells and evenly distribute them into the 96-well plates in step ②; ④ Place the culture plates in an incubator at 37°C and 5% CO2 for 7 days; ⑤ Use ELISA to screen the positive single colony wells and subclone again; ⑥ After 2-3 consecutive subclones, the subclone cell colony wells can be made positive and the values ​​are similar, that is, monoclonal hybridoma cells are obtained; expand the culture of monoclonal hybridoma cells to obtain the cell culture supernatant containing monoclonal antibodies. Human Mycoplasma pneumoniae (ATCC15531, 1×10 8 Pseudomonas aeruginosa (ATCC27853, 1×10 8 bacteria / well), Moraxella catarrhalis (ATCC25240, 1×10 8 baumannii (ATCC19606, 1×10 8 bacteria / well), Haemophilus parainfluenzae (ATCC7901, 1×10 8 bacteria / well), Legionella pneumophila (ATCC33152, 1×10 8 bacteria / well), Streptococcus pyogenes (ATCC19615, 1×10 8 bacteria / well), Staphylococcus aureus (ATCC25923, 1×10 8 bacteria / well), Haemophilus influenzae (ATCC49247, 1×10 8 bacteria / well), Klebsiella pneumoniae (ATCC 700603, 1×10 8 bacteria / well), Enterobacter cloacae (ATCC13047, 1×10 8 Escherichia coli (ATCC25922, 1×10 8 cells / well), Candida (ATCC10231, 1×10 8The ELISA plates were coated with respiratory pathogens such as influenza virus (ATCC VR-1743) and the specificity of the screened antibodies was tested by ELISA to eliminate the monoclonal antibodies that reacted positively with these pathogens. In this step, 4 qualified cell lines were screened.

[0076] 1.9) Ascites preparation and cell preservation

[0077] The four positive monoclonal hybridoma cells screened out were expanded and cultured. Monoclonal antibody ascites was prepared by conventional ascites in vivo induction method.

[0078] (1) Western blot detection of mAb: The specificity of ascites at a dilution of 1:2000 was determined using conventional Western blot assays. The results showed that the four antibodies only bound to the SARS-CoV-2 N protein.

[0079] (2) Antibody purification and titer determination: Ascites was purified by Protein A affinity chromatography as follows:

[0080] Use GE-HiTrap Protein A HP prepacked column to purify the antibody according to the instructions. The specific steps are as follows:

[0081] a. Take 5 mL of hybridoma supernatant, add 0.5 mL of 1 M Tris (pH 8.0) to adjust to pH 8.0, and centrifuge at 20,000 g for 20 min to remove the precipitate.

[0082] b. After loading the column, wash with 10 column volumes of buffer A (100 mM Tris-Cl, pH 8.0), and then wash with 10 column volumes of buffer B (10 mM Tris-Cl, pH 8.0).

[0083] c. Elute IgG with approximately three column volumes of IgG elution buffer (100 mM glycine, pH 3.0). (Pre-load 0.1 mL of IgG-neutralization buffer (1 M Tris-Cl, pH 8.0) in the collection tube, and load 0.9 mL of elution buffer in each tube)

[0084] d. Dialyze with 50 volumes of PBS (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, pH 7.4).

[0085] e. After ultrafiltration and concentration, adjust the concentration to 1 mg / ml with PBS and store at -70°C for future use.

[0086] The purified 4 antibodies were diluted in multiples and then the titers were determined by indirect ELISA. The purity of the antibodies was above 95% by SDS-PAGE analysis, and the ELISA titers were above 1:1000000. The purified antibodies were adjusted to a concentration of 1 mg / ml and stored at -70°C for future use. The four monoclonal antibodies were named AbN1, AbN2, AbN3 and AbN4.

[0087] 1.10) Analysis of antibody recognition epitopes

[0088] Different concentrations (0, 30, 100, 300 and 1 000 nmol / L) of AbN1, AbN2, AbN3 and AbN4 monoclonal antibodies were reacted with the same concentration of N antigen. The specific steps were as follows: the AMC sensor was immersed in PBS for 60 seconds, then immersed in the antibody to be tested for solidification for 180 seconds, then immersed in PBS for 60 seconds, then immersed in antigen dilution for reaction for 120 seconds, then immersed in PBS for dissociation for 120 seconds, and finally immersed in regeneration solution (10 mmol / L glycine, pH 1.7) for regeneration for 30 seconds. The above process was detected in real time using the Octet Red96e molecular interaction instrument. At the same time, a competition experiment was used to test the antibody recognition of the antigen site. The N antigen was immobilized on the Ni-NTA sensor, and then interacted with AbN1, AbN2, AbN3 and AbN4 monoclonal antibodies in turn, and the binding signals of each monoclonal antibody were detected to determine whether the four antibodies recognized the same epitope. A monoclonal antibody binding signal ratio of >60% indicated that there was no competition between the antibodies, and <20% indicated complete competition. The above process was detected in real time using the Octet Red96e molecular interaction instrument. The results of the binding kinetic constant analysis showed that the affinity of the four antibodies to the antigen could reach 10 9 The above results indicate that the interaction between antigen and antibody is strong. The results of the binding and dissociation diagram analysis show that the binding rates of the four monoclonal antibodies are fast, the binding signal values ​​are good, and the dissociation rates are slow, indicating that the four selected monoclonal antibodies have strong binding force with the N antigen. The results of the competition experiment show that the N antigen is immobilized on the Ni-NTA sensor and interacts with AbN1, AbN2, AbN3 and AbN4 monoclonal antibodies in turn. According to the sensor technical manual, the binding signal ratios of AbN2, AbN3 and AbN4 are all greater than 60%, indicating that AbN1, AbN2, AbN3 and AbN4 monoclonal antibodies recognize different antigenic epitopes of the same antigen.

[0089] The hybridoma cells were sequenced and analyzed according to conventional methods to determine the gene sequences of monoclonal antibodies AbN1, AbN2, AbN3 and AbN4, wherein AbN1 comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein the amino acid sequence of CDR-H1 is GYTFTDYS, the amino acid sequence of CDR-H2 is INTETGEA, the amino acid sequence of CDR-H3 is TRSTATDD, the amino acid sequence of CDR-L1 is QSVDYDGDSY, the amino acid sequence of CDR-L2 is TAS, and the amino acid sequence of CDR-L3 is QQSNEDPLT;

[0090] AbN2 comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein the amino acid sequence of CDR-H1 is GYTFTNYL, the amino acid sequence of CDR-H2 is INLYNDVT, the amino acid sequence of CDR-H3 is ARSYHGFYWYIDV, the amino acid sequence of CDR-L1 is EEINNYGISF, the amino acid sequence of CDR-L2 is AAS, and the amino acid sequence of CDR-L3 is QQSKEVPWT;

[0091] AbN3 comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein the amino acid sequence of CDR-H1 is GYSFTGYN, the amino acid sequence of CDR-H2 is IDPYYGDT, the amino acid sequence of CDR-H3 is ARDGSTDGYYLDY, the amino acid sequence of CDR-L1 is QSVSTSSYNY, the amino acid sequence of CDR-L2 is YAS, and the amino acid sequence of CDR-L3 is QNSWEIPLT;

[0092] AbN4 comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein the amino acid sequence of CDR-H1 is GYTFTGYT, the amino acid sequence of CDR-H2 is IGYINPSSEFA, the amino acid sequence of CDR-H3 is ARESLDY, the amino acid sequence of CDR-L1 is QSLLDSGNQKNY, the amino acid sequence of CDR-L2 is GAS, and the amino acid sequence of CDR-L3 is QNDHSYPLT.

[0093] 2) Preparation of monoclonal antibodies against the novel coronavirus S protein

[0094] 2.1) Animal immunization

[0095] The recombinant protein of the S protein of the new coronavirus (SARS-CoV-2 (2019-nCoV)) with the catalog number 40592-V08H80 of Beijing Yiqiao Shenzhou Science and Technology Co., Ltd. was used as an antigen to immunize 5 8-week-old BALB / c mice, and 2 mice were not immunized as negative controls. After the initial immunization antigen was fully emulsified with an equal amount of Freund's complete adjuvant, the mice were immunized by multiple subcutaneous injections on the back, 100μg / mouse. After an interval of three weeks, the same dose of antigen was fully emulsified with Freund's incomplete adjuvant and then intraperitoneally injected for the second immunization. After an interval of 2 weeks, the same dose of antigen was fully emulsified with Freund's incomplete adjuvant and then intraperitoneally injected for the third immunization. Blood was collected from the tail vein 15 days after the third immunization to detect the titer of the antiserum.

[0096] 2.2) Antiserum titer detection

[0097] The antiserum titer was determined by indirect ELISA method: dilute the S antigen concentration to 3 μg / mL with PBS (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, pH 7.4), add 100 μL / well to a 96-well plate, and incubate at 37°C for 2 h or at 4°C overnight. Wash the plate three times with PBST (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, 0.5% (v / v) Tween-20, pH 7.4) and tap the plate. 1% bovine serum albumin was dissolved in PBS solution, 250 μL / well, and blocked at 37°C for 1 h or at 4°C overnight. Wash the plate three times with PBST and tap the plate. The sera of 4 immune mice were diluted with PBS and added to the corresponding wells, 100 μL / well, the blank control was PBS solution, and the negative control was the mouse serum before immunization. The plate was washed after coating at 37℃ for 1 hour. HRP-labeled goat anti-mouse IgG was added at a dilution of 1:3000, 100 μL / well, and the plate was washed after coating at 37℃ for 1 hour. 100 μL of freshly prepared TMB color substrate solution was added to each well. After 10 minutes at 37℃, 100 μL of 1M hydrochloric acid was added to each well to terminate the reaction. The OD450nm value was measured with an enzyme-linked detector, and the results were read and observed. The highest titer (titer 51200) was selected, and the third immunization was strengthened one month later. After 5 days, the mouse spleen cells were taken for cell fusion.

[0098] 2.3) Recovery and culture of SP2 / 0 myeloma cells

[0099] The frozen myeloma cells (SP2 / 0) were revived in advance. The myeloma cells frozen in liquid nitrogen were quickly taken out and placed in a 37°C water bath with slight shaking to melt them quickly. Note that the freezing tube mouth cannot touch water to avoid contamination. Then the cells were transferred to a 24-well plate containing 2 ml of RPMI-1640 complete medium (RPMI-1640 medium containing 20% ​​fetal bovine serum, and the fetal bovine serum was purchased from Shanxi Runsheng Daye Biomaterials Co., Ltd.) and placed in a 37°C, 5% CO2 incubator for half an hour. When all the cells grew attached to the wall, the medium was changed in time. After that, the cells were passaged every 3 days, and the cells were adjusted to the optimal growth density. When the cells reached a certain activity, they were counted and prepared for fusion. Two days before cell fusion, the cells were passaged 1 to 4, and the cell concentration in each bottle was adjusted to 1×10 5 / ml.

[0100] 2.4) Preparation of feeder cells

[0101] 2.4.1) After blood was collected from the eyeballs of BALB / c mice, the mice were killed by pulling their necks apart, and the mice were completely immersed in 75% alcohol for 5 min. The mice were then transferred to a flat dish on a clean bench with their abdomen facing upward.

[0102] 2.4.2) Use forceps to lift the mouse chest and abdominal skin, cut a small hole with scissors, use two forceps to tear a larger hole in the skin, lift the mouse peritoneum, cut it open, find the mouse spleen, use forceps and small scissors to carefully remove the spleen, place it in a disposable dish, carefully peel off the fat, connective tissue, etc. attached to the spleen, add 5 ml of RPMI-1640 culture medium (purchased from Hyclone, product number SH30809.01), puncture the spleen with the needle of a syringe filled with 5 ml of RPMI-1640 culture medium, carefully wash out the spleen cells, and then sieve, add the spleen cell suspension into a 10 ml centrifuge tube, centrifuge at 1100 rpm for 5 min, discard the supernatant, and wash twice with RPMI-1640 culture medium.

[0103] 2.4.3) Gently resuspend the cells in 5 ml of HAT medium and mix evenly, count, and add HAT medium until the cell concentration is 1×10 5 / ml.

[0104] 2.4.4) Drop the cell suspension into a 96-well cell culture plate, 130 μl / well, and culture in a 37°C, 5% CO2 incubator.

[0105] 2.5) Preparation of immune spleen cell suspension

[0106] 2.5.1) Five days after booster immunization, select BALB / c mice with the highest serum titer, remove their eyeballs, bleed, and collect and separate serum as a positive control for antibody detection.

[0107] 2.5.2) The mice were killed by cervical dislocation and then immersed in 75% alcohol for 5 min. The mice were taken out and placed in a plate on a sterile clean bench with their abdomen facing upwards.

[0108] 2.5.3) Use forceps to lift the mouse chest and abdominal skin, cut a small hole with scissors, and then use two forceps to tear a larger hole in the skin. Then use new forceps to lift the mouse peritoneum, cut it open, find the mouse spleen, carefully remove the spleen, place it in a disposable dish, and carefully remove the fat and connective tissue.

[0109] 2.5.4) After rinsing with RPMI-1640 solution, add new RPMI-1640 solution, puncture the spleen with the needle of a syringe filled with 5 ml of RPMI-1640 culture medium to carefully wash out the spleen cells, then sieve to squeeze as many spleen cells as possible through the mesh into the solution, transfer the spleen cell suspension into a centrifuge tube, centrifuge at 1100 rpm for 5 min, discard the supernatant, and centrifuge and wash twice.

[0110] 2.5.5) Gently resuspend the spleen cells in RPMI-1640 culture medium, count and set aside.

[0111] 2.6) Preparation of SP2 / 0 myeloma cell suspension

[0112] 2.6.1) Take 2 bottles of myeloma cells cultured in T75 culture flasks (change the medium one day before fusion, and the cells should be in the logarithmic growth phase during fusion) and collect them in 50 ml centrifuge tubes.

[0113] 2.6.2) Centrifuge at 1000 rpm for 5 minutes and discard the supernatant.

[0114] 2.6.3) Add 30 ml of RPMI-1640 washing solution to the precipitate, gently resuspend, mix, and wash again by centrifugation in the same way.

[0115] 2.6.4) Gently resuspend the spleen cells in 10 ml RPMI-1640 culture medium, mix evenly, count and set aside.

[0116] 2.7) Cell fusion

[0117] 2.7.1) will contain 1×10 8 A suspension of spleen cells containing 1×10 7 Mix the suspension of 100 SP2 / 0 myeloma cells in a 50ml centrifuge tube, add culture medium to 40ml, and mix thoroughly.

[0118] 2.7.2) Centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

[0119] 2.7.3) Gently tap the bottom of the centrifuge tube to loosen and evenly distribute the cell clumps into a paste.

[0120] 2.7.4) Take out the prepared 50% PEG (MW1450) and RPMI-1640 washing solution from the 4°C refrigerator and place it in a 37°C water bath to pre-warm it for later use.

[0121] 2.7.5) Use a 1 ml pipette to draw 0.8 ml of 50% PEG (MW1450) and slowly add it into the centrifuge tube while stirring for 60 seconds.

[0122] 2.7.6) Then gradually add 40 ml of preheated RPMI-1640 washing solution over 60 seconds to dilute the PEG and make it lose its fusogenic effect.

[0123] 2.7.7) Centrifuge at 1000 rpm for 5 min and discard the supernatant.

[0124] 2.7.8) Add 400 mL of HAT culture medium (purchased from Sigma, product number H0262) and gently pipette to resuspend the precipitated cells.

[0125] 2.7.9) Add the fused cell suspension into a 96-well culture plate containing feeder cells, 50 μl / well, and spread 20 culture plates in total. Then, place the culture plates in an incubator at 37°C and 5% CO2 for culture.

[0126] 2.8) Screening and cloning of positive clones

[0127] Starting from the third day after fusion, observe the cell growth in each well every day. If there is contamination, treat it with sodium azide immediately. On the 7th day after fusion, replace the medium with HT culture medium (HT, 50×, purchased from Sigma, item number H0137). The next day after the medium change, aspirate the supernatant of the well where the clone appears for specific detection. The recombinant antigen of the new coronavirus S protein was used as the coating antigen and detected by indirect ELISA. OD450 was measured with an enzyme-linked detector, and the P / N value was >2.1 for positive. The positive wells were replaced with HT culture medium again, and the ELISA test was positive again the next day. The clone wells with continuous positive results were selected for 2-3 subcloning to screen out monoclonal hybridoma cells. Specific steps of subcloning: ① Blow and mix the positive fusion well hybridoma cells and measure the cell concentration; ② Prepare feeder cells in advance and suspend them in HT culture medium, 130ul / well, and spread them into 96-well plates, and then place the culture plates in an incubator at 37°C and 5% CO2 for use; ③ Take the positive fusion well hybridoma cells and evenly distribute them into the 96-well plates in step ②; ④ Place the culture plates in an incubator at 37°C and 5% CO2 for 7 days; ⑤ Use ELISA to screen the positive single colony wells and subclone again; ⑥ After 2-3 consecutive subclones, the subclone cell colony wells can be made positive and the values ​​are similar, that is, monoclonal hybridoma cells are obtained; expand the culture of monoclonal hybridoma cells to obtain the cell culture supernatant containing monoclonal antibodies. Human Mycoplasma pneumoniae (ATCC15531, 1×10 8 Pseudomonas aeruginosa (ATCC27853, 1×10 8 1×10 8 baumannii (ATCC19606, 1×10 8 bacteria / well), Haemophilus parainfluenzae (ATCC7901, 1×10 8 bacteria / well), Legionella pneumophila (ATCC33152, 1×10 8 bacteria / well), Streptococcus pyogenes (ATCC19615, 1×10 8 bacteria / well), Staphylococcus aureus (ATCC25923, 1×10 8 bacteria / well), Haemophilus influenzae (ATCC49247, 1×10 8 bacteria / well), Klebsiella pneumoniae (ATCC 700603, 1×10 8 bacteria / well), Enterobacter cloacae (ATCC13047, 1×10 8 Escherichia coli (ATCC25922, 1×10 8 cells / well), Candida (ATCC10231, 1×10 8The ELISA plates were coated with respiratory pathogens such as influenza virus (ATCC VR-1743) and the specificity of the screened antibodies was tested by ELISA to eliminate the monoclonal antibodies that reacted positively with these pathogens. In this step, two qualified cell lines were screened.

[0128] 2.9) Ascites preparation and cell preservation

[0129] The two positive monoclonal hybridoma cells screened out at last were expanded and cultured. Monoclonal antibody ascites was prepared by conventional ascites in vivo induction method.

[0130] (1) Western blot detection of mAb: The specificity of ascites at a dilution of 1:2000 was determined using conventional Western blot assays. The results showed that both antibodies only bound to the SARS-CoV-2 S protein.

[0131] (2) Antibody purification and titer determination: Ascites was purified by Protein A affinity chromatography as follows:

[0132] Use GE-HiTrap Protein A HP prepacked column to purify the antibody according to the instructions. The specific steps are as follows:

[0133] a. Take 5 mL of hybridoma supernatant, add 0.5 mL of 1 M Tris (pH 8.0) to adjust to pH 8.0, and centrifuge at 20,000 g for 20 min to remove the precipitate.

[0134] b. After loading the column, wash with 10 column volumes of buffer A (100 mM Tris-Cl, pH 8.0), and then wash with 10 column volumes of buffer B (10 mM Tris-Cl, pH 8.0).

[0135] c. Elute IgG with approximately three column volumes of IgG elution buffer (100 mM glycine, pH 3.0). (Pre-load 0.1 mL of IgG-neutralization buffer (1 M Tris-Cl, pH 8.0) in the collection tube, and load 0.9 mL of elution buffer in each tube)

[0136] d. Dialyze with 50 volumes of PBS (8.5 g / L NaCl, 1.4 g / L Na2HPO4, 0.2 g / L NaH2PO4, pH 7.4).

[0137] e. After ultrafiltration and concentration, adjust the concentration to 1 mg / ml with PBS and store at -70°C for future use.

[0138] The purified 4 antibodies were diluted in multiples and then the titers were determined by indirect ELISA. The purity of the antibodies was above 95% by SDS-PAGE analysis, and the ELISA titers were above 1:1000000. The purified antibodies were adjusted to a concentration of 1 mg / ml and stored at -70°C for future use. The two monoclonal antibodies were named AbS1 and AbS2 respectively.

[0139] 2.10) Analysis of antibody recognition epitopes

[0140] Different concentrations (0, 30, 100, 300 and 1 000 nmol / L) of AbS1 and AbS2 monoclonal antibodies were reacted with the same concentration of S antigen. The specific steps were as follows: the AMC sensor was immersed in PBS for 60 seconds, then immersed in the antibody to be tested for solidification for 180 seconds, then immersed in PBS for 60 seconds, then immersed in antigen dilution for reaction for 120 seconds, then immersed in PBS for dissociation for 120 seconds, and finally immersed in regeneration solution (10 mmol / L glycine, pH 1.7) for regeneration for 30 seconds. The above process was detected in real time using the Octet Red96e molecular interaction instrument. At the same time, a competition experiment was used to test the antibody recognition of the antigen site. The S antigen was immobilized on the Ni-NTA sensor, and then interacted with AbS1 and AbS2 monoclonal antibodies in turn. The binding signals of each monoclonal antibody were detected to determine whether the two antibodies recognized the same epitope. A monoclonal antibody binding signal ratio of >60% indicated that there was no competition between the antibodies, and <20% indicated complete competition. The above process was detected in real time using the Octet Red96e molecular interaction instrument. The results of the binding kinetic constant analysis showed that the affinity of the two antibodies to the antigen could reach 10 9 The above results indicate that the interaction between antigen and antibody is strong. The results of binding and dissociation diagram analysis show that the binding rates of the two monoclonal antibodies are fast, the binding signal values ​​are good, and the dissociation rates are slow, indicating that the two selected monoclonal antibodies have strong binding force with the S antigen. The results of the competition experiment show that the N antigen is immobilized on the Ni-NTA sensor and interacts with AbS1 and AbS2 monoclonal antibodies in turn. According to the sensor technical manual, the binding signal ratio of AbS2 is calculated to be 75.33%, indicating that AbS1 and AbS2 monoclonal antibodies recognize different antigenic epitopes of the same antigen without steric hindrance.

[0141] The hybridoma cells were sequenced and analyzed according to conventional methods to determine the gene sequences of AbS1 and AbS2 monoclonal antibodies. AbS1 contains three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), wherein the amino acid sequence of CDR-H1 is AYSITSDYA, the amino acid sequence of CDR-H2 is ISYSGRT, the amino acid sequence of CDR-H3 is AREAFYFDY, the amino acid sequence of CDR-L1 is SSVSY, the amino acid sequence of CDR-L2 is RTS, and the amino acid sequence of CDR-L3 is HQYHSYPWT. AbS2 contains three heavy chain complementary determining regions (CDR-H1, CDR-H2 and CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2 and CDR-L3), among which the amino acid sequence of CDR-H1 is GYALTNYL, the amino acid sequence of CDR-H2 is INPGSGAP, the amino acid sequence of CDR-H3 is ALDSSGYGYALDY, the amino acid sequence of CDR-L1 is QSVLTN, the amino acid sequence of CDR-L2 is SAF, and the amino acid sequence of CDR-L3 is HQYNSYPLT.

[0142] Example 2 New Coronavirus N Antigen and S Antigen Combined Detection Reagent Strip and Preparation Method

[0143] New coronavirus N antigen and S antigen combined detection reagent strip and preparation method (such as Figure 1 ) is composed of a sample pad 1, a first conjugation pad 1, a second conjugation pad 3, a nitrocellulose membrane detection layer 4, a quality control line 5, an N antigen detection line 6, an S antigen detection line 7, a hard polyvinyl chloride backing plate 8 and a water absorbent pad 9.

[0144] The nitrocellulose membrane detection layer 4 is pasted on the hard polyvinyl chloride backing plate 8, and the second conjugation pad 3, the first conjugation pad 2 and the sample pad 1 are pasted on the left side of the nitrocellulose membrane detection layer from bottom to top, and the absorbent pad 9 is arranged on the right side of the nitrocellulose membrane detection layer 4. The first conjugation pad and the second conjugation pad are made of glass cellulose membrane, and the first conjugation pad 2 is sprayed with colloidal gold-labeled anti-new coronavirus N protein double epitope mixed antibodies AbN3 and AbN4, and the second conjugation pad 3 is sprayed with colloidal gold-labeled anti-new coronavirus S protein monoclonal antibody AbS2. On the left side of the nitrocellulose membrane detection layer are detection lines 6 and 7; the quality control line 5 is located on the right side, and the distance between the two lines is 5mm. The detection lines 6 and 7 are sprayed with anti-new coronavirus N protein double epitope mixed monoclonal antibodies AbN1 and AbN2 and anti-new coronavirus S protein monoclonal antibody AbS1 respectively; the quality control line 5 is sprayed with sheep anti-mouse IgG (purchased from Abcam (Shanghai) Trading Co., Ltd., item number ab6708).

[0145] When the sample to be tested contains the new coronavirus, the new coronavirus N and S proteins can first bind to the gold-labeled anti-new coronavirus N protein dual-epitope mixed antibody and the gold-labeled anti-new coronavirus S protein monoclonal antibody, and then bind to the antibodies on the corresponding test line under chromatography to form a visible color band after aggregation, and then make a judgment based on the color development results. If the test line 6 or 7 is colored, the quality control line is colored, which means the result is positive; conversely, if the test line 6 or 7 is not colored, the quality control line is colored, which means the result is negative; in any case, if the quality control line is not colored, it means that the test paper is invalid.

[0146] It is particularly noted that exchanging the positions of the two conjugate pads in Example 2 of the present invention, or exchanging the positions of the detection lines 6 and 7, does not affect the technical features and effects of the kit. Similar fine adjustments can be considered as equivalent replacements of the technical features of the present invention.

[0147] Example 3: Nitrocellulose membrane coated with antibodies

[0148] 1) Prepare coating buffer: add 0.15g disodium hydrogen phosphate, 0.04g sodium dihydrogen phosphate, 0.8g sodium chloride, 0.02g potassium chloride, and 5g sucrose to 80ml ultrapure water in sequence, stir evenly, adjust the pH to 7.5, and make up to 100ml.

[0149] 2) Coating antibody dilution: Two monoclonal antibodies AbN1 and AbN2 targeting different epitopes of the N protein were mixed in equal proportions to prepare an anti-novel coronavirus N protein dual-epitope mixed antibody. At the same time, the anti-S protein monoclonal antibody AbS1 was prepared. The above-mentioned coating buffer was used to dilute the anti-novel coronavirus N protein dual-epitope mixed antibody, the anti-novel coronavirus S protein antibody AbS1 and the goat anti-mouse IgG antibody to 1.0 mg / ml, 2.0 mg / ml and 1 mg / ml, respectively.

[0150] 3) Marking and coating: The marking pipeline of the Gold Standard Bio-Scribing Gold Sprayer HM3035 was cleaned with cleaning solution and pure water 20 times each; the three diluted coating antibodies (anti-novel coronavirus N protein dual-epitope mixed antibody, anti-novel coronavirus S protein antibody AbS1 and goat anti-mouse IgG antibody) were respectively aspirated into three independent marking pipelines, and the air in the pipelines was emptied; the position of each marking pen was adjusted so that the marking S antigen detection line, N antigen detection line, and quality control line were 5mm apart, and the S antigen detection line was 4mm away from the lower end of the NC membrane; the three antibody solutions were marked on the surface of the nitrocellulose membrane CN140, and the marking parameters were 1ul / cm and 100mm / s.

[0151] 4) Drying treatment: The nitrocellulose membrane after streaking is dried in a room with a relative humidity of 30% for 12 hours; the dried nitrocellulose membrane is collected in a sealed aluminum foil bag, and a bag desiccant is added to the bag;

[0152] Example 4: Sample pad and conjugate pad treatment

[0153] 1) Prepare treatment solutions: sample pad treatment solution (pH 8.7) 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, 0.3% polyvinyl pyrrolidone; binding pad treatment solution (pH 8.7) components are 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, 0.3% polyvinyl pyrrolidone. Prepare 1 L of each treatment solution according to the above formula.

[0154] 2) Soaking treatment: Pour the treatment solution into an open container, put in 2 glass fiber pads, press slightly to make the solution completely immerse the pads, soak each for about 1-2 hours, then take out and place on a grid to dry in a drying room for 12-16 hours. Perform sample pad and conjugate pad treatments separately according to the above method.

[0155] 3) Collect the dried sample pad and binding pad, and cut them into thin strips of 0.6 cm and 1.5 cm width respectively on a chopper.

[0156] 4) Collect the sample pad and conjugate pad cut in the previous step and store them in a dry sealed bag.

[0157] Example 5: Colloidal gold labeling and spray pad

[0158] 1) Preparation of colloidal gold solution:

[0159] The colloidal gold used in this product is prepared by the trisodium citrate reduction method, the specific method is as follows:

[0160] a) Pour 99 ml of ultrapure water into a 250 ml round-bottom flask, then add 1 ml of 1% chloroauric acid (final concentration 0.01%). Place the round-bottom flask in an oil bath, set the temperature to 120°C, and heat to full boiling.

[0161] b) Rapidly add 1.2 ml of 1% trisodium citrate and continue heating for 10 min. Observe the solution color change from yellow to blue, then to red, and finally to a stable red. Stop heating and cool in a water bath with stirring.

[0162] c) Scan the cooled colloidal gold solution at 400-600nm using an ultraviolet spectrophotometer to determine the maximum absorption peak wavelength and observe the peak shape and peak width of the maximum absorption peak. The colloidal gold has a single absorption peak at 520-530nm.

[0163] 2) Preparation, purification and concentration of colloidal gold-labeled anti-novel coronavirus N protein dual-epitope mixed antibodies (AbN3, AbN4) and colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody AbS2:

[0164] a) Two monoclonal antibodies AbN3 and AbN4 targeting different epitopes of the N protein were mixed in equal proportions to prepare a dual-epitope mixed antibody against the new coronavirus N protein;

[0165] b) Take three portions of 10 ml of colloidal gold solution and put them into three 50 ml round-bottom flasks with stirring bars. Add 20 ul of 0.2 M potassium carbonate solution and place it on a magnetic stirrer to stir for 5 minutes (the speed is based on no bubbles). Then slowly add the anti-novel coronavirus N protein dual-epitope mixed antibody and the anti-novel coronavirus S protein monoclonal antibody AbS2 at a final concentration of 5 ug / ml (the dripping speed is based on 1 mg of protein in 5 minutes), and stir at a low speed for 45 minutes.

[0166] c) Add 1000ul of BSA (10% concentration BSA prepared with 40mM Tris) at a constant speed, continue stirring for 15 minutes, and store at 4°C for later use.

[0167] d) Take the labeled colloidal gold solution and dispense it into 2 ml centrifuge tubes. Centrifuge at 12000g for 10 minutes, discard the supernatant, resuspend the precipitate with 1 / 5 of the original volume of the gold solution, centrifuge at 12000g for 10 minutes, discard the supernatant, and resuspend the precipitate with 1 / 10 of the original volume of the gold solution. Store at 4°C for later use. (Washing solution formula: 0.01M Tris, 1% BSA, 1% Tween-20, 5% sucrose, 0.3% polyvinyl pyrrolidone, pH 8.7).

[0168] 3) Loading of gold-labeled antibodies

[0169] The colloidal gold-labeled anti-novel coronavirus N protein dual-epitope mixed antibody (AbN3, AbN4) was sprayed onto the surface of the conjugate pad at a dosage of 8ul / cm and a speed of 100mm / s using Shanghai Jinbiao Bio-Scratch Gold Spraying Instrument HM3035 to form the first conjugate pad, which was then transferred to a room with a relative humidity below 30%, and the conjugate pad was collected after standing for 1-2 hours. The colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody AbS2 was sprayed onto the surface of the conjugate pad at a dosage of 8ul / cm and a speed of 100mm / s using Shanghai Jinbiao Bio-Scratch Gold Spraying Instrument HM3035 to form the second conjugate pad, which was then transferred to a room with a relative humidity below 30%, and the conjugate pad was collected after standing for 1-2 hours.

[0170] Example 6: Assembly and cutting

[0171] 1) Assemble a large plate on a base plate, and paste the antibody-lined nitrocellulose membrane, the sample pad, two serially connected conjugation pad strips and the absorbent pad, wherein the width of the overlapping area between the second conjugation pad and the nitrocellulose membrane is 0.8-1.2 mm, the width of the overlapping area between the first conjugation pad and the second conjugation pad is 0.8-1.2 mm, and the width of the overlapping area between the absorbent pad and the nitrocellulose membrane is 1.5-2.5 mm.

[0172] 2) Cut the assembled large plate into 3 mm wide strips on a high-speed chopper and put them into the box to obtain a reagent strip that can be used for the combined detection of the new coronavirus N and S antigens (see the specific structure for details). Figure 1 ).

[0173] Example 7: Performance test of test strips

[0174] 1) Test results of inactivated novel coronavirus culture (sensitivity test)

[0175] The inactivated novel coronavirus culture was diluted 2000 times and then diluted 2 times in a gradient manner. The diluted samples were tested as routine specimens. The results showed that at a novel coronavirus concentration of 2×10 2 CFU / ml, this kit can still detect it.

[0176] 2) Specificity test

[0177] In order to verify the specificity of the novel coronavirus N antigen and S antigen combined detection reagent strip and preparation method of the present invention, the test strip described in Example 6 was used to detect 4 large-scale popular novel coronavirus strains (the 4 strains in 2019 novel coronavirus 1-4 in Table 1 include the alpha strain numbered B.1.1.7, the beta strain numbered B.1.351, the delta strain numbered B.1.617.2, and the Omicron strain numbered B.1.1.529, which are all isolated and purified from different patients and inactivated products, and the detection process was completed by Wuhan Institute of Biological Products Co., Ltd.) and related common respiratory pathogens, as shown in Table 1. The results show that the test results of the kit of the present invention for 4 novel coronavirus strains are positive, while the test results for the other 17 common respiratory pathogens and 9 virulent viruses are all negative. The kit shows good specificity.

[0178] Table 1

[0179] Strain name Test results Strain name Test results 2019 Novel Coronavirus 1-4 Positive Legionella pneumophila ATCC 33152 Negative Mycoplasma pneumoniae ATCC 15531 Negative Streptococcus pyogenes ATCC 19615 Negative Pseudomonas aeruginosa ATCC 27853 Negative Staphylococcus aureus ATCC 25923 Negative Moraxella catarrhalis ATCC 25240 Negative Human Haemophilus influenzae ATCC 49247 Negative Acinetobacter baumannii ATCC 19606 Negative Klebsiella pneumoniae ATCC 700603 Negative Haemophilus parainfluenzae ATCC 790 Negative Enterobacter cloacae ATCC 13047 Negative Adenovirus ATCC VR-3 Negative Escherichia coli ATCC 25922 Negative Influenza A virus ATCC VR-1743 Negative Candida ATCC 10231 Negative Influenza B virus ATCC VR-790 Negative Respiratory syncytial virus ATCC VR26 Negative SARS-Cov virus inactivated antigen Negative MERS-Cov virus antigen Negative Flu H1N1 Negative Influenza H3N3 Negative Influenza BV Negative Flu Negative Poliomyelitis type 1 Negative Poliomyelitis type II Negative Poliomyelitis type III Negative Japanese encephalitis Negative EV71 Negative

[0180] Example 8: This example uses the reagent strip in Example 6 to test the secretion sample to verify the performance

[0181] 1) Sample collection

[0182] Because the new coronavirus is highly contagious (the new coronavirus used in the present invention is an inactivated virus, which is provided by Wuhan Institute of Biological Products Co., Ltd.), sample collection should be carried out by professional technicians equipped with protective equipment of corresponding levels. The collection process can refer to the following methods.

[0183] Nasopharyngeal swab: After the swab is inserted through the nostril, it slowly moves along the bottom of the lower nasal passage. When the swab reaches the back wall of the nasopharyngeal cavity, gently rotate it for a circle and then slowly remove the swab.

[0184] Oropharyngeal swab: The person being sampled slightly raises his head, opens his mouth, and makes an "ah" sound to expose the pharyngeal tonsils on both sides. The swab is repeatedly wiped on the pharyngeal tonsils on both sides for more than 3 times, and then wiped up and down the posterior pharyngeal wall for more than 3 times.

[0185] The collected swab is placed in a centrifuge tube containing 1ml sample extract (formula: 50mM Tris, 1% Triton-100, 2% Proclin300, pH8.2). The swab is repeatedly rotated and squeezed against the tube wall for about 10 times to release the specimen to the maximum extent. 100ul of the extract mixed with the sample is added to the horizontally placed test card sample well. After the sample passes through the sample pad, it infiltrates the colloidal gold marker on the binding pad and gradually releases it. The colloidal gold is chromatographed on the nitrocellulose membrane under capillary action with the liquid. If there is a new coronavirus in the sample, the corresponding antibody on the surface of the colloidal gold will recognize and bind to the virus surface protein, and be bound by the capture antibody in the nitrocellulose detection area, and colloidal gold strips will appear on the S protein detection line and the N protein detection line. The test results are interpreted 20 minutes after sample addition and chromatography: if the C line band does not appear, the test result is invalid. The appearance of the S protein test line means that the new coronavirus is positive and the viral load is high. The appearance of the N protein test line means that the new coronavirus is positive. Only one band at the quality control line means that the test result is negative.

[0186] 2) Comparison with the results of the novel coronavirus nucleic acid test

[0187] The kit of the present invention was compared with the domestic BGI Novel Coronavirus 2019-nCoV Nucleic Acid Detection Kit (Fluorescence PCR Method) on 120 positive clinical specimens to analyze and verify the detection of the novel coronavirus by the kit. The results showed that the sensitivity of the kit and the nucleic acid fluorescence PCR method compared were 100% and the specificity was 100%, indicating that the performance of the kit meets the needs of rapid screening of the novel coronavirus.

[0188] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A novel coronavirus antigen detection reagent strip, characterized in that: The novel coronavirus antigen detection reagent strip comprises a sample pad (1), a first conjugation pad (2), a second conjugation pad (3), a nitrocellulose membrane detection layer (4), a water-absorbing pad (9), and a rigid polyvinyl chloride backing plate (8); the nitrocellulose membrane detection layer (4) is laid on the upper surface of the rigid polyvinyl chloride backing plate (8); one end of the nitrocellulose membrane detection layer (4) close to the second conjugation pad (3) is coated with a dual-epitope mixed antibody against the novel coronavirus N protein and a monoclonal antibody against the novel coronavirus S protein; one end of the nitrocellulose membrane detection layer (4) close to the water-absorbing pad (9) is coated with a goat anti-mouse IgG antibody; the first conjugation pad (2) is sprayed with a colloidal gold-labeled anti-novel coronavirus antibody. The invention relates to a coronavirus N protein dual-epitope mixed antibody; the second binding pad (3) is sprayed with colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody; the sample pad (1), the first binding pad (2) and the second binding pad (3) are sequentially connected from front to back and are jointly arranged at one end of a nitrocellulose membrane detection layer (4); the absorbent pad (9) is arranged at the other end of the nitrocellulose membrane detection layer (4); the anti-novel coronavirus N protein dual-epitope mixed antibody on the nitrocellulose membrane detection layer (4) is prepared by mixing monoclonal antibody A against different epitopes of the novel coronavirus N protein and monoclonal antibody B against different epitopes of the novel coronavirus N protein in equal proportions; the different epitopes of the novel coronavirus N protein Monoclonal antibody A comprises three heavy chain complementary determining regions A-CDR-H1, A-CDR-H2 and A-CDR-H3 and three light chain complementary determining regions A-CDR-L1, A-CDR-L2 and A-CDR-L3; wherein the amino acid sequence of A-CDR-H1 is GYTFTDYS; the amino acid sequence of A-CDR-H2 is INTETGEA; the amino acid sequence of A-CDR-H3 is TRSTATDD; the amino acid sequence of A-CDR-L1 is QSVDYDGDSY; the amino acid sequence of A-CDR-L2 is TAS; the amino acid sequence of A-CDR-L3 is QQSNEDPLT; the novel coronavirus N protein The monoclonal antibody B targeting different epitopes comprises three heavy chain complementary determining regions B-CDR-H1, B-CDR-H2 and B-CDR-H3 and three light chain complementary determining regions B-CDR-L1, B-CDR-L2 and B-CDR-L3; wherein the amino acid sequence of B-CDR-H1 is GYTFTNYL; the amino acid sequence of B-CDR-H2 is INLYNDVT; the amino acid sequence of B-CDR-H3 is ARSYHGFYWYIDV; the amino acid sequence of B-CDR-L1 is EEINNYGISF; the amino acid sequence of B-CDR-L2 is AAS; the amino acid sequence of B-CDR-L3 is QQSKEVPWT; The anti-novel coronavirus S protein monoclonal antibody on the nitrocellulose membrane detection layer (4) comprises three heavy chain complementary determining regions S1-CDR-H1, S1-CDR-H2 and S1-CDR-H3 and three light chain complementary determining regions S1-CDR-L1, S1-CDR-L2 and S1-CDR-L3; wherein the amino acid sequence of S1-CDR-H1 is AYSITSDYA; the amino acid sequence of S1-CDR-H2 is ISYSGRT; the amino acid sequence of S1-CDR-H3 is AREAFYFDY; the amino acid sequence of S1-CDR-L1 is SSVSY; the amino acid sequence of S1-CDR-L2 is RTS; the amino acid sequence of S1-CDR-L3 is HQYHSYPWT; The anti-novel coronavirus N protein dual-epitope mixed antibody on the first binding pad (2) is prepared by mixing monoclonal antibody C against different epitopes of the novel coronavirus N protein and monoclonal antibody D against different epitopes of the novel coronavirus N protein in equal proportions; the monoclonal antibody C against different epitopes of the novel coronavirus N protein comprises three heavy chain complementary determining regions C-CDR-H1, C-CDR-H2 and C-CDR-H3 and three light chain complementary determining regions C-CDR-L1, C-CDR-L2 and C-CDR-L3; wherein the amino acid sequence of C-CDR-H1 is GYSFTGYN; the amino acid sequence of C-CDR-H2 is IDPYYGDT; the amino acid sequence of C-CDR-H3 is ARDGSTDGYYLDY; the amino acid sequence of C-CDR-L1 is QSVSTSSYNY; and the amino acid sequence of C-CDR-L2 is IDPYYGDT. The amino acid sequence of DR-L2 is YAS; the amino acid sequence of C-CDR-L3 is QNSWEIPLT; the monoclonal antibody D for different epitopes of the novel coronavirus N protein comprises three heavy chain complementary determining regions D-CDR-H1, D-CDR-H2 and D-CDR-H3 and three light chain complementary determining regions D-CDR-L1, D-CDR-L2 and D-CDR-L3; wherein, the amino acid sequence of D-CDR-H1 is GYTFTGYT; the amino acid sequence of D-CDR-H2 is IGYINPSSEFA; the amino acid sequence of D-CDR-H3 is ARESLDY; the amino acid sequence of D-CDR-L1 is QSLLDSGNQKNY; the amino acid sequence of D-CDR-L2 is GAS; the amino acid sequence of D-CDR-L3 is QNDHSYPLT; The anti-novel coronavirus S protein monoclonal antibody on the second conjugate pad (3) comprises three heavy chain complementary determining regions S2-CDR-H1, S2-CDR-H2 and S2-CDR-H3 and three light chain complementary determining regions S2-CDR-L1, S2-CDR-L2 and S2-CDR-L3; wherein the amino acid sequence of S2-CDR-H1 is GYALTNYL; the amino acid sequence of S2-CDR-H2 is INPGSGAP; the amino acid sequence of S2-CDR-H3 is ALDSSGYGYALDY; the amino acid sequence of S2-CDR-L1 is QSVLTN; the amino acid sequence of S2-CDR-L2 is SAF; and the amino acid sequence of S2-CDR-L3 is HQYNSYPLT.

2. A method for preparing a novel coronavirus antigen detection reagent strip as claimed in claim 1, characterized in that: The method comprises the preparation of a nitrocellulose membrane detection layer, the preparation of a first conjugate pad, the preparation of a second conjugate pad and the preparation of a water absorbent pad.

3. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 2, characterized in that: The preparation method of the nitrocellulose membrane detection layer is as follows: two monoclonal antibodies targeting different epitopes of the new coronavirus N protein are mixed in equal proportions to prepare a dual-epitope mixed antibody against the new coronavirus N protein, and monoclonal antibodies against the new coronavirus S protein are prepared at the same time. The two antibodies are coated on the membrane surface using a film drawing instrument to form T1 and T2 detection lines, respectively, and sheep anti-mouse IgG is coated on the membrane surface using a film drawing instrument to form a quality control C line.

4. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 3, characterized in that: The concentrations of the anti-novel coronavirus N protein dual-epitope mixed antibody and the anti-novel coronavirus S protein monoclonal antibody are both 0.1-3.0 mg / ml, and the streaking coating volume is 0.2-2.0 ul / cm.

5. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 4, characterized in that: The concentration of goat anti-mouse IgG coated on the nitrocellulose membrane detection layer is 0.1-3.0 mg / ml, and the streaking coating amount is 0.2-2.0 ul / cm.

6. The method for preparing a novel coronavirus antigen detection reagent strip according to any one of claims 2 to 5, characterized in that: The specific preparation method of the first conjugate pad is: prepare a conjugate pad, prepare a colloidal gold solution by a trisodium citrate reduction method, prepare a colloidal gold-labeled anti-novel coronavirus N protein double-epitope mixed antibody based on the prepared colloidal gold solution, and spray the prepared colloidal gold-labeled anti-novel coronavirus N protein double-epitope mixed antibody onto the conjugate pad surface by a biofilm gold spraying instrument to form a first conjugate pad; The specific preparation method of the second conjugate pad is: take another conjugate pad, prepare a colloidal gold solution by a trisodium citrate reduction method, prepare a colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody based on the prepared colloidal gold solution, and spray the prepared colloidal gold-labeled anti-novel coronavirus S protein monoclonal antibody onto the surface of the conjugate pad by a bio-scratch gold sprayer to form a second conjugate pad.

7. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 6, characterized in that: The preparation method of the first binding pad and the preparation method of the second binding pad both further include: transferring the prepared first binding pad and the second binding pad to a space with a relative humidity lower than 30%, and placing them for 1-2 hours for standby use.

8. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 7, characterized in that: The method for preparing the novel coronavirus antigen detection reagent strip further includes a step of pretreating the conjugate pad. The specific implementation method of the pretreating of the conjugate pad is: 1.1) Prepare a binding pad treatment solution: the content and components of the binding pad treatment solution are 0.01M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone; the pH of the binding pad treatment solution is 8.7; 1.2) Soaking treatment: Soak the two conjugate pads in the conjugate pad treatment solution for 1-2 hours and then dry them in a drying room for 12-16 hours; 1.3) Collect the dried bonding pads, cut them and store them in a dry and sealed environment for future use.

9. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 8, characterized in that: The method for preparing the novel coronavirus antigen detection reagent strip also includes preparing a sample pad, and the specific preparation method of the sample pad is: a) preparing a sample pad treatment solution: the components and contents of the sample pad treatment solution are 0.01 M Tris, 1% BSA, 1% Tween-20, 5% sucrose, and 0.3% polyvinyl pyrrolidone, and the pH of the sample pad treatment solution is 8.7; b) Soaking treatment: Soak a single sample pad in the sample pad treatment solution for 1-2 hours and then dry it in a drying room for 12-16 hours; c) Collect the dried sample pads, cut them and store them in a dry and sealed environment for future use.

10. The method for preparing a novel coronavirus antigen detection reagent strip according to claim 9, characterized in that: The binding pad is a glass fiber pad; the sample pad is a glass fiber pad or a polyester pad.

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