A novel monoclonal antibody of novel coronavirus N protein and preparation method and use thereof

Abstract

The present application relates to the field of monoclonal antibodies and in vitro diagnostic technology, and in particular to a novel coronavirus N protein monoclonal antibody and a preparation method and application thereof.The present application provides a novel coronavirus N protein monoclonal antibody, which is applied to colloidal gold immunochromatography, can be used for rapid detection of novel coronavirus antigen, has high sensitivity, and the sensitivity can reach 100 pg / ml.

Description

Technical Field

[0001] This invention relates to the fields of monoclonal antibodies and in vitro diagnostics, specifically to a monoclonal antibody against the N protein of a novel coronavirus, its preparation method, and its uses. Background Technology

[0002] The genome of the novel coronavirus (SARS-CoV-2) is a single-stranded positive RNA, belonging to the β-coronavirus genus. The novel coronavirus is mainly transmitted through direct contact and respiratory droplets, and can cause symptoms such as cough, fever, difficulty breathing, pneumonia, kidney failure, and even death. Its high infectivity, insidious nature, and the continuous emergence of mutant strains make rapid and effective detection of the novel coronavirus particularly important.

[0003] Currently, nucleic acid testing, antigen testing, and antibody testing are the main methods for detecting the invasion of the novel coronavirus into the body. Among them, nucleic acid testing has high sensitivity and specificity and is recognized as the "gold standard" for COVID-19 detection; while antigen testing serves as an important supplement to nucleic acid testing. Nucleic acid testing typically requires more specialized personnel and equipment, as well as a significant investment of time and money, for sample collection, transportation, and laboratory testing. While self-testing antigen tests have lower accuracy, they offer advantages such as short testing time, high operability, and low cost, making them suitable for early triage and rapid management of suspected cases. Therefore, this invention provides a method for preparing a monoclonal antibody against the N protein of the novel coronavirus and its application in colloidal gold immunochromatography, which can be used for the rapid detection of novel coronavirus antigens. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to provide a monoclonal antibody against the novel coronavirus N protein, a method for its preparation, and its uses. The novel coronavirus N protein monoclonal antibody, when applied to colloidal gold immunochromatography, can be used for the rapid detection of novel coronavirus antigens with high sensitivity and specificity.

[0005] A monoclonal antibody against the N protein of a novel coronavirus, comprising: monoclonal antibody N17 or monoclonal antibody N26;

[0006] The monoclonal antibody N17 comprises:

[0007] Heavy chain variable region:

[0008] (1) A CDR1 region comprising the amino acid sequence shown in SEQ ID NO.1;

[0009] (2) A CDR2 region comprising the amino acid sequence shown in SEQ ID NO.2;

[0010] (3) Contains the CDR3 region as shown in SEQ ID NO.3;

[0011] Light chain variable region:

[0012] (1) A CDR1 region containing the amino acid sequence shown in SEQ ID NO.4;

[0013] (2) Contains the CDR2 region as shown in SEQ ID NO.5;

[0014] (3) Contains the CDR3 region as shown in SEQ ID NO.6;

[0015] The monoclonal antibody N26 comprises:

[0016] Heavy chain variable region:

[0017] (1) A CDR1 region comprising the amino acid sequence shown in SEQ ID NO.7;

[0018] (2) A CDR2 region comprising the amino acid sequence shown in SEQ ID NO.8;

[0019] (3) Contains the CDR3 region with the amino acid sequence shown in SEQ ID NO.9;

[0020] Light chain variable region:

[0021] (1) A CDR1 region comprising the amino acid sequence shown in SEQ ID NO.10;

[0022] (2) A CDR2 region comprising the amino acid sequence shown in SEQ ID NO.11;

[0023] (3) The CDR3 region contains the amino acid sequence shown in SEQ ID NO.12.

[0024] Optionally, the monoclonal antibody N17 is of the IgG2a subtype with a light chain of kappa, and the monoclonal antibody N26 is of the IgG1 subtype with a light chain of kappa.

[0025] A hybridoma cell line N17, which secretes the monoclonal antibody N17; the hybridoma cell line N17 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C2022322, deposit date October 30, 2022, and address Wuhan University, Wuhan, China, 430072, China.

[0026] Optionally, the hybridoma cell line N17 uses the novel coronavirus N protein as an immunogen.

[0027] A hybridoma cell line N26, which secretes the monoclonal antibody N26; the hybridoma cell line N26 is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: C2022323, deposited on October 30, 2022, at Wuhan University, Wuhan, China, 430072, China.

[0028] Optionally, the hybridoma cell line N26 uses the novel coronavirus N protein as an immunogen.

[0029] A method for preparing a monoclonal antibody against the novel coronavirus N protein, comprising:

[0030] Ascites containing monoclonal antibody N17 or monoclonal antibody N26 was prepared by using hybridoma cell lines that secrete monoclonal antibody N17 or monoclonal antibody N26 via in vivo animal induction method.

[0031] The ascites fluid was separated and purified to obtain monoclonal antibody N17 or monoclonal antibody N26.

[0032] Optionally, the N17 hybridoma cell line that secretes monoclonal antibodies is the N17 hybridoma cell line;

[0033] The N26 hybridoma cell line that secretes monoclonal antibodies is the hybridoma cell line N26.

[0034] Optionally, the ascites preparation step includes: sensitizing mice older than 8 weeks or multiparous female mice with 0.4 ml-0.6 ml of liquid paraffin via intraperitoneal injection; 5-9 days later, injecting 0.5 × 10⁻⁶ ml of liquid paraffin into each mouse via intraperitoneal injection. 5 -1.0×10 5 Hybridoma cells were collected, and ascites fluid was collected starting 6-9 days later.

[0035] Optionally, in the separation step, the ascites fluid and saturated ammonium sulfate are mixed in equal volumes, stirred, precipitated for 18-22 hours, the supernatant is removed by centrifugation, the solution is reconstituted, and then dialyzed overnight. The dialysis molecular weight is 8-20 kDa. The antibody is collected and the volume is adjusted to obtain solution I.

[0036] Optionally, in the purification step, a protein purification chromatography column is used for purification. First, the protein purification chromatography column is rinsed with purified water, and then the protein purification chromatography column is equilibrated with buffer. Then, solution I is filtered, loaded with the sample, washed with buffer, eluted with elution buffer, the elution buffer is collected, the pH is adjusted, and then dialyzed and filtered.

[0037] Optionally, in the purification step, purification is performed using a Bio-Rad NGC or an Akta protein purifier;

[0038] Optionally, in the purification step, the column material in the protein purification chromatography column is Protein A;

[0039] Optionally, in the purification step, the volume of the protein purification chromatography column is 50-200 ml;

[0040] Optionally, in the purification step, when the purified antibody is monoclonal antibody N17, the buffer solution is a 0.01M-0.04M PB solution with a pH of 6.8-7.2.

[0041] Optionally, in the purification step, when the purified antibody is monoclonal antibody N17, the elution buffer is a 0.05M-0.2M sodium citrate solution with a pH of 2.7-4.3.

[0042] Optionally, in the purification step, when the purified antibody is monoclonal antibody N17, the pH value is adjusted to 7.0-7.4 using a 0.05M-2.0M Tris solution with pH 8.5-9.5;

[0043] Optionally, in the purification step, when the purified antibody is monoclonal antibody N26, the buffer solution is a solution containing 1-4M NaCl, 0.01-0.04M PB, and pH 6.8-7.2.

[0044] Optionally, in the purification step, when the purified antibody is monoclonal antibody N26, the elution buffer is a 0.05-0.2M sodium citrate solution with a pH of 4.3-6.5.

[0045] Optionally, in the purification step, when the purified antibody is monoclonal antibody N26, the pH value is adjusted to 7.0-7.4 using a 0.05-2.0M Tris solution with pH 8.5-9.5.

[0046] The novel coronavirus N protein monoclonal antibody, the hybridoma cell line N17, or the hybridoma cell line N26 described herein have the following uses:

[0047] (1) Use in the preparation of products for the diagnosis or detection of antibodies against the novel coronavirus;

[0048] (2) Use in the preparation of products for the isolation and purification of the novel coronavirus;

[0049] (3) Use in the preparation of products containing isolated and purified novel coronavirus N protein;

[0050] (4) Use in the preparation of products containing isolated and purified novel coronavirus antibodies;

[0051] The products include reagents, test strips, or kits.

[0052] A novel coronavirus colloidal gold test strip includes a novel coronavirus N protein monoclonal antibody, a hybridoma cell line N17, or a hybridoma cell line N26, with monoclonal antibody N17 or monoclonal antibody N26 used as the detection line coating antibody and / or colloidal gold-labeled antibody.

[0053] Optionally, it includes: a substrate and a sample pad, a gold-labeled pad, an antibody-supporting membrane, and an absorbent pad that are sequentially overlapped on the substrate; the antibody-supporting membrane is provided with a detection line and a control line spaced apart, the detection line being close to the colloidal gold adsorption pad, and the control line being close to the absorbent pad; wherein the antibody coated on the detection line is monoclonal antibody N26, and the colloidal gold adsorption pad is coated with colloidal gold-labeled monoclonal antibody N17.

[0054] A method for preparing a novel coronavirus colloidal gold test strip includes using the novel coronavirus N protein monoclonal antibody, the hybridoma cell line N17, or the hybridoma cell line N26, and coating the detection line with the monoclonal antibody N17 or the monoclonal antibody N26 and / or the colloidal gold-labeled antibody.

[0055] Optionally, this includes the preparation of a sample pad, a gold label pad, an antibody carrier membrane, and an absorbent pad, and then the sample pad, the gold label pad, the antibody carrier membrane, and the absorbent pad are sequentially overlapped on the substrate;

[0056] The preparation of the antibody carrier membrane includes: using the monoclonal antibody N26 as the detection line coating antibody, diluting the detection line coating antibody to 1.0 mg / ml-2.0 mg / ml, and performing membrane etching;

[0057] The preparation of the gold-labeled pad includes: spraying a gold-labeled pad treatment solution containing colloidal gold-labeled monoclonal antibody N17 onto the gold-labeled pad, wherein the concentration of monoclonal antibody N17 labeling is 10 μg / ml-20 μg / ml; wherein the concentration of monoclonal antibody N17 labeling refers to the final concentration of monoclonal antibody N17 in the mixture obtained after mixing monoclonal antibody N17 with colloidal gold solution.

[0058] Optionally, the monoclonal antibody N26 is used as the detection line coating antibody, and the detection line coating antibody is diluted to 1.75 mg / ml and then applied to a membrane.

[0059] Optionally, the detection line is coated with an antibody membrane with a rati value of 0.8-1.2 μl / cm;

[0060] Optionally, colloidal gold-labeled monoclonal antibody N17 is coated onto a gold-labeled pad at a concentration of 20 μg / ml.

[0061] Optionally, when the colloidal gold-labeled monoclonal antibody N17 is sprayed onto the gold-labeled pad, the rati value is 1.0-4.0 μl / cm.

[0062] Optionally, the colloidal gold particles have a size of 10nm-40nm; and / or

[0063] The pH value for colloidal gold labeling is 6.0-7.0; and / or

[0064] The preparation of the antibody carrier membrane also includes: coating the control line with goat anti-mouse IgG antibody, diluting it to 1.0 mg / ml-2.0 mg / ml, and then performing a membrane run;

[0065] It also includes the preparation of sample pads, including: treating the sample pads with a sample pad treatment solution, wherein the pH value of the sample pad treatment solution is 6.5-8.5;

[0066] Optionally, the colloidal gold particles have a size of 40 nm; and / or

[0067] Optionally, the pH value for colloidal gold labeling is 6.5; and / or

[0068] Optionally, the sample pad is treated with a sample pad treatment solution, wherein the pH value of the sample pad treatment solution is 8.0;

[0069] Optionally, the sample pad treatment solution contains 0.2 wt% S9, 0.5 wt% BSA, 0.1 v / v% Pc300, and 0.01 M PBS;

[0070] Optionally, the quality control line is coated with goat anti-mouse IgG, diluted to 1.25 mg / ml, and then scratched.

[0071] Optionally, the control line is coated with antibody for streaking, with a rati value of 0.8-1.2 μl / cm.

[0072] The technical solution of this invention has the following advantages:

[0073] 1. The present invention provides a novel coronavirus N protein monoclonal antibody. When the novel coronavirus N protein monoclonal antibody is applied to colloidal gold immunochromatography, it can be used for rapid detection of novel coronavirus antigen with high specificity and sensitivity, with a sensitivity of up to 100 pg / ml.

[0074] 2. The hybridoma cell line N17 provided by the present invention secretes the monoclonal antibody N17; the hybridoma cell line N17 has the accession number CCTCC NO: C2022322; the monoclonal antibody N17 secreted by the hybridoma cell line N17 can be used for rapid detection of novel coronavirus antigen by colloidal gold immunochromatography, with high sensitivity and specificity.

[0075] Furthermore, the hybridoma cell line N17 has a strong ability to secrete monoclonal antibody N17. Using the hybridoma cell line N17 to mass-produce monoclonal antibody N17 can significantly increase the yield of monoclonal antibody N17, with a single batch yield of 500mg-3000mg. Moreover, it has strong stability and performance consistent with small-batch purified monoclonal antibody N17.

[0076] 3. The hybridoma cell line N26 provided by the present invention secretes the monoclonal antibody N26; the hybridoma cell line N26 has the accession number CCTCC NO: C2022323; the monoclonal antibody N26 secreted by the hybridoma cell line N26 can be used for rapid detection of novel coronavirus antigen by colloidal gold immunochromatography, with high sensitivity and specificity.

[0077] Furthermore, the hybridoma cell line N26 has a strong ability to secrete monoclonal antibody N26. Using the hybridoma cell line N26 to mass-produce monoclonal antibody N26 can significantly increase the yield of monoclonal antibody N26, with a single batch yield of 500mg-3000mg. Moreover, it has strong stability and performance consistent with small-batch purified monoclonal antibody N26.

[0078] In this invention, the hybridoma cell line N17 is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: C2022322, deposit date October 30, 2022, and address Wuhan University, Wuhan, China, 430072, China.

[0079] The hybridoma cell line N26 is deposited at the China Center for Type Culture Collection (CCTCCNO: C2022323), with accession date of October 30, 2022, and address at Wuhan University, Wuhan, China, 430072, China. Attached Figure Description

[0080] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0081] Figure 1 This is the purity identification result of non-reducing SDS-PAGE electrophoresis in Experiment Example 1 of this invention;

[0082] Figure 2 This is the sensitivity detection result in Experiment Example 3 of this invention;

[0083] Figure 3 This is a schematic diagram of the structure of the colloidal gold test strip for the novel coronavirus in Embodiment 8 of the present invention.

[0084] Figure label:

[0085] 1-Sample pad, 2-Gold label pad, 3-Detection line, 4-Control line, 5-Antibody carrier membrane, 6-Absorbent pad, 7-Substrate. Detailed Implementation

[0086] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0087] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0088] The gold in the following examples is referred to as colloidal gold solution. The preparation method is as follows: prepare chloroauric acid into a 1wt% chloroauric acid solution, then take 1ml of the 1wt% chloroauric acid solution and put it into a clean beaker or conical flask, and dilute it with water to 100ml, that is, a 0.01wt% chloroauric acid solution.

[0089] The recombinant SARS-CoV-2 N protein expressed in baculovirus-insect cells and Escherichia coli was purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.

[0090] Example 1: Obtaining hybridoma cell lines N17 and N26

[0091] I. Mouse Immunization:

[0092] (1) The immunogen was derived from the recombinant SARS-CoV-2 N protein expressed by baculovirus-insect cells and Escherichia coli. The two were mixed in equal volumes to form the immunoglobulin. (2) For the first immunization, 80 μg of the recombinant SARS-CoV-2 N protein was diluted to 150 μl with physiological saline and emulsified with 150 μl of Freund's complete adjuvant. The emulsification could be done by vortexing or using an emulsifier until the emulsion turned white and opaque. The emulsion was then injected subcutaneously into mice at multiple sites. Immunization could be performed in the forelimb axilla, abdomen, and back. (3) For the second, third, and fourth immunizations, 40 μg of the recombinant SARS-CoV-2 N protein was mixed with 150 μl of Freund's incomplete adjuvant in equal volumes and emulsified. The emulsion was then injected subcutaneously into mice at multiple sites. (4) The immunization intervals were 14 days, 14 days, 14 days, 14 days, or 14 days, 14 days, 21 days, and 28 days. (4) On the 7th day after the fourth immunization, blood was collected from the ocular veins of mice, and serum was obtained for titer determination. Mice with a titer of 10,000 or higher were selected and intraperitoneal shock immunization was performed using 40 μg of recombinant protein.

[0093] II. Cell Fusion:

[0094] (1) Spleen cell preparation: After immunizing mice intraperitoneally for 3 days, they were euthanized by cervical dislocation and disinfected in 75% alcohol. Spleen cells and lymphocytes were then dissected in a clean bench and obtained. The spleen cells and lymphocytes were crushed using a grinder on a stainless steel sieve and dispersed with 10 ml of RPMI-1640 medium. After centrifugation at 1500 rad / min for 5 min, the supernatant was discarded, and 10 ml of medium was added for dispersion and counting. (2) SP2 / 0 cell preparation: SP2 / 0 cells were resuscitated approximately 2 weeks in advance for expansion culture. At confluence, the medium for SP2 / 0 cells in the logarithmic phase was discarded, and 10 ml of RPMI-1640 medium was added to each flask. SP2 / 0 cells were dispersed from the culture flask using a 5 ml pipette tip and collected into 50 ml centrifuge tubes. After centrifugation at 1500 rad / min for 5 min, the supernatant was discarded, and 10 ml of medium was added for dispersion and counting. (3) Cell fusion: Splenocytes, lymphocytes, and SP2 / 0 cells were mixed at a ratio of 5:1 (quantity), centrifuged, and the culture medium was discarded. 1 ml of preheated 37℃ 50% PEG1500 was slowly added, and the reaction was allowed to proceed for 1 min. Then, 9 ml of RPMI-1640 culture medium was added, gradually increasing the speed, and the reaction was completed within 8 min. After centrifugation at 1000 rad / min for 5 min, the supernatant was discarded, and the mixture was added to RPMI-1640 culture medium containing 1% HAT (an abbreviation for xanthine, aminopterin, and thymidin), 10% Clone Easy, and 20% FBS (fetal bovine serum). After mixing, the mixture was centrifuged at 2.5 × 10⁻⁶ cells / min. 6Add cells / well to a 96-well culture plate and incubate at 37°C in a 5% CO2 cell culture incubator. (4) Medium change treatment: Observe whether there is contamination on the second day, and observe whether the SP2 / 0 cells that have not fused with spleen cells have died on the fourth day, and whether the size and number of fused cell clusters are normal, with an average of 3-5 cells / well. On the 5th to 7th day, discard the culture medium in the 96 wells, add 200ul of RPMI-1640 medium containing 1% HT (an abbreviation for xanthine and thymidin) and 20% FBS, and incubate at 37°C in a 5% CO2 cell culture incubator for 3 days.

[0095] III. Screening of positive cell lines:

[0096] (1) Screening of positive wells: The cell supernatant from the 96 wells was diluted 10-fold with 0.01M PBS, and 100 μl was aspirated into a 96-well plate pre-coated with 1.0 μg / ml immunogen. The plate was incubated at 37°C for 1 h and washed 3 times with PBST (phosphate-Tween buffer). 100 μl of HRP-goat anti-mouse IgG (Zhongshan Jinqiao) diluted 10000-fold with 0.01M PBS was added, and the plate was incubated at 37°C for 1 h and washed 3 times with PBST. TMB chromogenic solution (Mecovander) was added for color development, and the reaction was terminated with 0.5M sulfuric acid. The OD value was read at 450 nm using a microplate reader. Positive wells with an OD value of 1.5 or higher were selected for transfection into 24-well plates. After 4 days of culture, the cell supernatant was diluted 10-fold again, and the OD value was measured. Positive cell lines with an OD value of 2.0 or higher were selected for subcloning. In the first round of subcloning, cells were diluted to 2.5 cells / ml using RPMI-1640 medium containing 10% Clone Easy (Medtec) and 20% FBS. 200 μl of each cell was added to one well of a 96-well plate, and the cells were cultured for 7 days. Cell clusters were observed under a microscope, and wells containing single cell clusters were selected for immunogen screening to obtain immunogen-positive cell lines. In the second round of subcloning, positive cell lines recognizing the recombinant SARS-CoV-2 N protein expressed in both baculovirus insect cells and *E. coli* were obtained. In the third round of subcloning, subtype detection kits (Thermo) were used to screen for subtypes, retaining positive cell lines of the IgG subtype. These positive cell lines were then expanded into larger cultures and ascites fluid was prepared. Four tubes of each positive clone were cryopreserved for later use.

[0097] IV. Ascites preparation and small-scale antibody purification:

[0098] (1) Ascites preparation: Freund's incomplete adjuvant was emulsified with an equal volume of physiological saline. Balb / c mice older than 8 weeks were selected, and each mouse was injected intraperitoneally with 0.5 ml. Seven days later, 1.0 × 10 ml of the emulsion was injected intraperitoneally. 6Cells (selected positive clones). After 7 days, ascites fluid is collected. The collected ascites fluid is centrifuged at 4000 rad / min for 5 min, the supernatant is collected and stored at -20℃ for later use. Generally, 2-5 ml can be collected per animal.

[0099] (2) Ammonium sulfate precipitation: After collecting ascites fluid, mix the ascites fluid with an equal volume of saturated ammonium sulfate for 4 hours, centrifuge at 8800g for 10 minutes, remove the supernatant, and obtain antibody precipitate, which is then reconstituted using 0.01M PBS.

[0100] (3) Affinity column purification: Antibody purification can be performed manually or by a purification instrument, with manual method being preferred.

[0101] ① Use 10ml of purified water to wash away the anhydrous ethanol in the Protein G pre-packed column (5ml), at a flow rate of 3 drops / second.

[0102] ② Equilibrate using 10 ml of 0.02 M PB (phosphate buffer), pH 7.0, at a flow rate of 3 drops / second.

[0103] ③ After reconstituted with 5ml-10ml of antibody, filter using a 0.22μm disposable filter, load the sample at a flow rate of 2 drops / second.

[0104] ④ Use 10 ml of 0.02 M PB pH 7.0 equilibration solution to wash away impurities at a flow rate of 3 drops / second.

[0105] ⑤ Elute with 5 ml of 0.1 M glycine in a pH 2.7 equilibration solution at a flow rate of 2 drops / second.

[0106] ⑥ Collect the eluent into 8-10 1.5ml centrifuge tubes. Use the A280 method to detect the concentration in each tube. After mixing the antibodies with a concentration greater than 0.25mg / ml, use 1.0M Tris, pH9.0 to adjust the pH of the antibody to 7.2.

[0107] ⑦ The purified antibody was placed in a 14KD dialysis bag and dialyzed overnight with 0.01M PBS.

[0108] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0109] V. Performance Verification:

[0110] (1) Stretching: The purified antibody (T line, test line) and goat anti-mouse IgG (C line, control line) from step four were diluted to 1.0 μg / ml with 0.01M PBS (containing 0.5 wt% sucrose). The membranes were then stretched using an automated membrane stretcher on the antibody support membrane (made of nitrocellulose membrane with a pore size of 0.45 μm). The rati (spray volume) value for both lines was 1.0 μl / cm, and the distance between the test line and the control line was 6 cm. The membranes were then dried in a 37℃ drying oven for 4 hours, and then collected in a resealable bag with desiccant for later use.

[0111] (2) Gold labeling: Add 1 / 10,000 gold (in 0.01 wt% chloroauric acid solution) to a 1.5 ml centrifuge tube. Adjust the pH by adding different volumes of 0.2 M K2CO3 or 0.1 M HCl. Then add 10 μg of antibody and mix for 15 min. The optimal pH is between two consecutive pH values ​​where the gold turns gray or purple and remains wine-red. Block with 50 μl of 20 wt% BSA (bovine serum albumin) for 15 min, centrifuge at 10000 g / min for 10 min, discard the supernatant, and reconstitute with 25 μl of 0.01 M PBS containing 5 wt% sucrose, 0.2 wt% BSA, and 0.1 v / v% Pc300 to obtain reconstituted gold. Store at 4℃ for later use.

[0112] (3) Sample pad treatment: Use the prepared sample pad treatment solution to completely wet the glass cellulose membrane to prevent air bubbles. After absorbing at room temperature for 30 minutes, place it in a 37°C drying oven and dry for about 18 hours. The sample pad treatment solution is: 0.2wt% S9, 0.5wt% BSA, 0.1v / v% Pc300, and 0.01M PBS.

[0113] (4) Gold-labeled pad treatment: Use the prepared gold-labeled pad treatment solution to completely wet the glass fiber membrane to prevent air bubbles. After absorption at room temperature for 30 minutes, place it in a 37°C drying oven and dry for about 18 hours. The gold-labeled pad treatment solution is: 0.4 wt% S9 (surfactant S9 name is TETRONIC 1307), 0.5 wt% sucrose, 0.1 v / v% Pc300, and 0.01 M PBS.

[0114] (5) The sample pad, gold label pad, antibody carrier membrane and absorbent pad are overlapped and adhered to the substrate in sequence. The overlap width is about 2.0 mm. The distance between the detection line and the edge of the gold label pad near the detection line is 8 mm. The distance between the quality control line and the edge of the absorbent pad near the quality control line is 10 mm. After assembly, the strip is cut into strips (0.4 cm × 80 cm) using a strip cutter. The reconstituted gold in step (2) is sprayed at a rate of 1.5 μl / strip (test strip) and applied manually to the gold label pad. The strip is placed in a 37°C drying oven and dried for 30 min before the test card is assembled.

[0115] (6) Use 0.01M PBS to dilute the COVID-19 N recombinant protein (the aforementioned immune protein) to 500 ng / ml as a positive sample. The blank control is 0.01M PBS. Add 100 μl of positive sample or blank control to the sample pad, and perform chromatography at room temperature for 15 min to observe the band situation. Select the antibody pairs that can be matched to enter the next round of screening.

[0116] (7) Dilute the COVID-19 N recombinant protein to 5.0 ng / ml using 0.01 M PBS; select the antibody pairs that are still detectable and then test the virus culture medium to finally obtain a pair of antibody pairs N17 and N26 that are better at recognizing the virus culture medium.

[0117] (8) The immunization intervals were 14 days, 14 days, 14 days, and 14 days, and the resulting monoclonal antibodies were derived from 13 hybridoma cell lines. The immunization intervals were 14 days, 14 days, 21 days, and 28 days, and the resulting monoclonal antibodies were derived from 13 hybridoma cell lines. The antibodies N17 and N26 were derived from the hybridoma cell lines obtained above. The secreting monoclonal antibody N17 was named hybridoma cell line N17 and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022322, deposit date October 30, 2022, address Wuhan University, Wuhan, China, 430072, China. The secreting monoclonal antibody N26 was named hybridoma cell line N26 and deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2022323, deposit date October 30, 2022, address Wuhan University, Wuhan, China, 430072, China.

[0118] VI. Determination of the variable region of antibodies

[0119] The monoclonal antibody N17, secreted by the hybridoma cell line N17, was sequenced (commissioned by Nanjing Genscript Biotech Co., Ltd.), and its variable region sequence is as follows:

[0120] Heavy chain variable region:

[0121] (1) A CDR1 region comprising the amino acid sequence shown in SEQ ID NO.1;

[0122] (2) A CDR2 region comprising the amino acid sequence shown in SEQ ID NO.2;

[0123] (3) Contains the CDR3 region as shown in SEQ ID NO.3;

[0124] Light chain variable region:

[0125] (1) A CDR1 region containing the amino acid sequence shown in SEQ ID NO.4;

[0126] (2) Contains the CDR2 region as shown in SEQ ID NO.5;

[0127] (3) Contains the CDR3 region containing the amino acid sequence shown in SEQ ID NO.6.

[0128] The monoclonal antibody N26, secreted by the hybridoma cell line N26, was sequenced (commissioned by Nanjing Genscript Biotech Co., Ltd.), and its variable region sequence is as follows:

[0129] Heavy chain variable region:

[0130] (1) A CDR1 region comprising the amino acid sequence shown in SEQ ID NO.7;

[0131] (2) A CDR2 region comprising the amino acid sequence shown in SEQ ID NO.8;

[0132] (3) Contains the CDR3 region with the amino acid sequence shown in SEQ ID NO.9;

[0133] Light chain variable region:

[0134] (1) A CDR1 region comprising the amino acid sequence shown in SEQ ID NO.10;

[0135] (2) A CDR2 region comprising the amino acid sequence shown in SEQ ID NO.11;

[0136] (3) The CDR3 region contains the amino acid sequence shown in SEQ ID NO.12.

[0137] 7. Antibody subtype identification

[0138] The monoclonal antibody N17, secreted by hybridoma cell line N17, was identified as IgG2a by Nanjing Genscript Biotech Co., Ltd. (the subtype was determined to be IgG2a with kappa light chain) after subtype identification.

[0139] The monoclonal antibody N26, secreted by hybridoma cell line N26, was identified as IgG1 by Nanjing Genscript Biotech Co., Ltd., with the light chain being kappa.

[0140] Example 2: Mass Production of Monoclonal Antibody N17

[0141] (1) Ascites preparation: 100 Balb / c mice aged 8 weeks or older were injected intraperitoneally with 0.5 ml of liquid paraffin. Seven days later, each mouse was injected intraperitoneally with 0.75 × 10⁻⁶ ml of liquid paraffin. 5Cells (hybridoma cell line N17). Ascites fluid was collected after 7 days. Three batches of ascites fluid were prepared consecutively for further purification.

[0142] (2) After mixing the saturated ammonium sulfate solution with an equal volume of ascites fluid, precipitate at 4°C for 20 h, centrifuge at 8800 g / min for 15 min, remove the supernatant, and reconstitute with 0.02 M PB (pH 7.0). Then, dialyze overnight using 0.01 M PBS in a 14 kDa dialysis bag, collect the antibody, and adjust the volume to twice the original ascites fluid volume for later use.

[0143] (3) Purify N17 antibody using the Akta protein purification instrument:

[0144] ① Load 150-170 ml of proteinA column material into a 200 ml purification column.

[0145] ② Use 500ml of purified water to wash the column material at a flow rate of 15ml / min.

[0146] ③ Use 500ml of 0.02M PB, pH 7.0 to equilibrate the column material at a flow rate of 15ml / min.

[0147] ④ Use a 0.22μm disposable filter to filter the dialyzed antibody, load about 400ml of sample at a flow rate of 10ml / min.

[0148] ⑤ Use 500ml of 0.02M PB, pH 7.0, to wash impurities at a flow rate of 15ml / min.

[0149] ⑥ Use 400 ml of 0.1 M sodium citrate at pH 3.4 to elute the antibody at a flow rate of 10 ml / min.

[0150] ⑦ Adjust the pH of the collected antibody to 7.2 using 1.0M Tris, pH 9.0. Then, place it in a 14KD dialysis bag and dialyze overnight with 0.01M PBS.

[0151] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05 v / v% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0152] Example 3: Mass Production of Monoclonal Antibody N17

[0153] (1) Ascites preparation: 200 Balb / c mice aged 8 weeks or older were injected intraperitoneally with 0.4 ml of liquid paraffin. Five days later, each mouse was injected intraperitoneally with 0.5 × 10⁻⁶ ml of liquid paraffin. 5 One cell line (hybridoma cell line N17). Ascites fluid was collected starting 6 days later. Three batches of ascites fluid were prepared consecutively for further purification.

[0154] (2) After mixing saturated ammonium sulfate with an equal volume of ascites fluid, the mixture was placed at 4°C for 18 hours to precipitate. The mixture was then centrifuged at 8800 g / min for 15 minutes to remove the supernatant. The supernatant was then reconstituted using 0.02 M PB (pH 7.0). The mixture was then dialyzed overnight using 0.01 M PBS in an 8 kDa dialysis bag. The antibody was collected and brought to a final volume twice the original ascites fluid volume for later use.

[0155] (3) Purify N17 antibody using the Akta protein purification instrument:

[0156] ① Pack proteinA column material into a 50ml purification column.

[0157] ② Use 250ml of purified water to wash the column material at a flow rate of 5ml / min.

[0158] ③ Use 250ml of 0.01M PB, pH 6.8-7.2 to equilibrate the column material at a flow rate of 5ml / min.

[0159] ④ Use a 0.22μm disposable filter to filter the dialyzed antibody, load about 100ml of sample at a flow rate of 5ml / min.

[0160] ⑤ Use 250ml of 0.01M PB, pH 6.8-7.2, to wash impurities at a flow rate of 5ml / min.

[0161] ⑥ Use 200 ml of 0.05 M sodium citrate solution, pH 2.7-4.3, to elute the antibody at a flow rate of 5 ml / min.

[0162] ⑦ After collecting the antibody, adjust its pH to 7.4 using 0.05M Tris (pH 8.5-9.5). Then, place it in a 14KD dialysis bag and dialyze overnight with 0.01M PBS.

[0163] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05 v / v% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0164] Example 4: Mass Production of Monoclonal Antibody N17

[0165] (1) Ascites preparation: 450 Balb / c mice aged 8 weeks or older were selected and injected intraperitoneally with 0.6 ml of liquid paraffin. Nine days later, each mouse was injected intraperitoneally with 1.0 × 10⁻⁶ ml of liquid paraffin. 5 Cells (hybridoma cell line N17). Ascites fluid was collected starting 9 days later. Three batches of ascites fluid were prepared consecutively for further purification.

[0166] (2) After mixing saturated ammonium sulfate with an equal volume of ascites fluid, the mixture was placed at 4°C for 22 hours to precipitate. The mixture was then centrifuged at 8800 g / min for 15 minutes to remove the supernatant. The supernatant was then reconstituted using 0.02 M PB (pH 7.0). The mixture was then dialyzed overnight using 0.01 M PBS in a 20 kDa dialysis bag. The antibody was collected and brought to a final volume twice the original ascites fluid volume for later use.

[0167] (3) Purify N17 antibody using the Akta protein purification instrument:

[0168] ① Load 50-70 ml of proteinA column material into a 100 ml purification column.

[0169] ② Use 500ml of purified water to wash the column material at a flow rate of 10ml / min.

[0170] ③ Use 500ml of 0.04M PB, pH 6.8-7.2 to equilibrate the column material at a flow rate of 10ml / min.

[0171] ④ Use a 0.22μm disposable filter to filter the dialyzed antibody, load about 200ml of sample at a flow rate of 10ml / min.

[0172] ⑤ Use 500ml of 0.04M PB, pH 6.8-7.2, to wash impurities at a flow rate of 10ml / min.

[0173] ⑥ Use 400 ml of 0.2 M sodium citrate solution, pH 2.7-4.3, to elute the antibody at a flow rate of 10 ml / min.

[0174] ⑦ After collecting the antibody, adjust its pH to 7.0 using 2.0M Tris (pH 8.5-9.5). Then, place it in a 14KD dialysis bag and dialyze overnight with 0.01M PBS.

[0175] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05 v / v% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0176] Example 5: Mass Production of Monoclonal Antibody N26

[0177] (1) Ascites preparation: 100 Balb / c mice over 8 weeks old were injected intraperitoneally with 0.5 ml of liquid paraffin. Seven days later, each mouse was injected intraperitoneally with 0.75 × 10⁻⁶ ml of liquid paraffin. 5 Cells (hybridoma cell line N26). Ascites fluid was collected after 7 days. Three batches of ascites fluid were prepared consecutively for further purification.

[0178] (2) After mixing saturated ammonium sulfate with an equal volume of ascites fluid, the mixture was placed in a container at 4°C overnight to precipitate. The mixture was then centrifuged at 8800 g / min for 15 min, the supernatant was removed, and the mixture was reconstituted using 0.01 M PB (containing 0.8 M NaCl). The mixture was then dialyzed overnight using 0.01 M PBS in a 14 kDa dialysis bag. The antibody was collected and brought to a final volume twice the original ascites fluid volume for later use.

[0179] (3) Purify N26 antibody using the AKTA protein purification instrument:

[0180] ① Load 150-170 ml of proteinA column material into a 200 ml purification column.

[0181] ② Use 500ml of purified water to wash the column material at a flow rate of 15ml / min.

[0182] ③ Use 500 ml of a solution containing 3 M NaCl and 0.02 M PB (pH 7.0) to equilibrate the column feed at a flow rate of 15 ml / min.

[0183] ④ Use a 0.22μm disposable filter to filter the dialyzed antibody, load about 400ml of sample at a flow rate of 10ml / min.

[0184] ⑤ Wash with 500 ml of 3M NaCl and 0.02 M PB solution (pH 7.0) at a flow rate of 15 ml / min.

[0185] ⑥ Use 600 ml of 0.1 M sodium citrate solution, pH 5.5, to elute the antibody at a flow rate of 10 ml / min.

[0186] ⑦ Adjust the pH of the collected antibody to 7.2 using 1.0M Tris, pH 9.0. Then, place it in a 14KD dialysis bag and dialyze overnight with 0.01M PBS.

[0187] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05 v / v% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0188] Example 6: Mass Production of Monoclonal Antibody N26

[0189] (1) Ascites preparation: 200 Balb / c mice aged 8 weeks or older were injected intraperitoneally with 0.4 ml of liquid paraffin. Five days later, each mouse was injected intraperitoneally with 0.5 × 10⁻⁶ ml of liquid paraffin. 5 One cell line (hybridoma cell line N26). Ascites fluid was collected starting 6 days later. Three batches of ascites fluid were prepared consecutively for further purification.

[0190] (2) After mixing saturated ammonium sulfate with an equal volume of ascites fluid, the mixture was placed at 4°C for 18 hours to precipitate. The mixture was then centrifuged at 8800 g / min for 15 minutes to remove the supernatant. The supernatant was then reconstituted using 0.01 M PB (containing 0.8 M NaCl). The mixture was then dialyzed overnight using 0.01 M PBS in an 8 kDa dialysis bag. The antibody was collected and brought to a final volume twice the original ascites fluid volume for later use.

[0191] (3) Purify N26 antibody using the AKTA protein purification instrument:

[0192] ① Pack proteinA column material into a 50ml purification column.

[0193] ② Use 250ml of purified water to wash the column material at a flow rate of 5ml / min.

[0194] ③ Use 250 ml of a solution containing 1 M NaCl and 0.01 M PB (pH 6.8-7.2) to equilibrate the column feed at a flow rate of 5 ml / min.

[0195] ④ Use a 0.22μm disposable filter to filter the dialyzed antibody, load about 100ml of sample at a flow rate of 5ml / min.

[0196] ⑤ Wash with 500 ml of 1M NaCl and 0.01M PB solution (pH 6.8-7.2) at a flow rate of 5 ml / min.

[0197] ⑥ Use 300 ml of 0.05 M sodium citrate solution, pH 4.3-6.5, to elute the antibody at a flow rate of 5 ml / min.

[0198] ⑦ After collecting the antibody, adjust its pH to 7.4 using 0.05M Tris (pH 8.5-9.5). Then, place it in a 14KD dialysis bag and dialyze overnight with 0.01M PBS.

[0199] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05 v / v% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0200] Example 7: Mass Production of Monoclonal Antibody N26

[0201] (1) Ascites preparation: 450 Balb / c mice aged 8 weeks or older were selected and injected intraperitoneally with 0.6 ml of liquid paraffin. Nine days later, each mouse was injected intraperitoneally with 1.0 × 10⁻⁶ ml of liquid paraffin. 5 Cells (hybridoma cell line N26). Ascites fluid was collected starting 9 days later. Three batches of ascites fluid were prepared consecutively for further purification.

[0202] (2) After mixing saturated ammonium sulfate with an equal volume of ascites fluid, the mixture was placed at 4°C for 22 hours to precipitate. The mixture was then centrifuged at 8800 g / min for 15 minutes to remove the supernatant. The supernatant was then reconstituted using 0.01 M PB (containing 0.8 M NaCl). The mixture was then dialyzed overnight using 0.01 M PBS in a 20 kDa dialysis bag. The antibody was collected and brought to a final volume twice the original ascites fluid volume for later use.

[0203] (3) Purify N26 antibody using the AKTA protein purification instrument:

[0204] ① Load 50-70 ml of proteinA column material into a 100 ml purification column.

[0205] ② Use 500ml of purified water to wash the column material at a flow rate of 15ml / min.

[0206] ③ Use 500 ml of a solution containing 4 M NaCl and 0.04 M PB (pH 6.8-7.2) to equilibrate the column feed at a flow rate of 15 ml / min.

[0207] ④ Use a 0.22μm disposable filter to filter the dialyzed antibody, load about 200ml of sample at a flow rate of 10ml / min.

[0208] ⑤ Wash with 500 ml of 4M NaCl and 0.04M PB solution (pH 6.8-7.2) at a flow rate of 15 ml / min.

[0209] ⑥ Use 600ml of 0.2M sodium citrate solution, pH 4.3-6.5, to elute the antibody at a flow rate of 10ml / min.

[0210] ⑦ After collecting the antibody, adjust the pH to 7.0 using 2.0M Tris (pH 8.5-9.5). Then, place it in a 14KD dialysis bag and dialyze overnight with 0.01M PBS.

[0211] ⑧ After dialysis, the antibody is filtered through a 0.22 μm disposable filter and 0.05 v / v% PC300 is added for preservation. After aliquoting, it is stored at -20℃ for later use.

[0212] Example 8: Novel Coronavirus Colloidal Gold Test Strip

[0213] This embodiment provides a method for preparing a colloidal gold test strip for the novel coronavirus, and the schematic diagram of the test strip structure is shown below. Figure 3 As shown, it includes the following steps:

[0214] (1) Preparation of antibody carrier membrane:

[0215] The purified monoclonal antibody N26 (T line, prepared in Example 5) was diluted to 1.75 mg / ml with 0.01 M PBS (containing 0.5 wt% sucrose), and goat anti-mouse IgG (C line) was diluted to 1.25 mg / ml with 0.01 M PBS (containing 0.5 wt% sucrose). Nitrocellulose membranes (NC membranes) were scribed using an automated scribing apparatus, with a 6 cm spacing between the T and C lines and a rati value of 1.0 μl / cm for both. The membranes were then dried in a 37°C oven for 4 hours, and then collected in resealable bags with added desiccant for later use.

[0216] (2) Bidding gold:

[0217] Add 1 ml of 0.01 wt% gold (a 0.01 wt% chloroauric acid solution) to a 1.5 ml centrifuge tube. Adjust the pH to pH 6.5 (i.e., the pH value for colloidal gold labeling) by adding different volumes of 0.2 M K₂CO₃ or 0.1 M HCl. Then add 20 μg of monoclonal antibody N17 (prepared in Example 2), i.e., the monoclonal antibody N17 labeling concentration is 20 μg / ml, and mix for 15 min. Then add 50 μl of 20 wt% BSA (bovine serum albumin) for blocking for 15 min, centrifuge at 10000 g / min for 10 min, discard the supernatant, and reconstitute using 25 μl of 0.01 M PBS containing 5 wt% sucrose, 0.2 wt% BSA, and 0.1 v / v% Pc300 to obtain reconstituted gold (colloidal gold particle size is 40 nm), and store at 4 °C for later use.

[0218] (3) Sample pad treatment:

[0219] Using the prepared sample pad treatment solution, thoroughly wet the glass fiber membrane to prevent air bubbles. After absorption at room temperature for 30 minutes, place it in a 37°C drying oven and dry for approximately 18 hours. The sample pad treatment solution consists of: 0.2 wt% S9, 0.5 wt% BSA, 0.1 v / v% Pc300, 0.01 M PBS, and a pH of 8.0.

[0220] (4) Gold-plated pad treatment:

[0221] Using the prepared gold-labeled pad treatment solution, thoroughly wet the glass fiber membrane to prevent air bubbles. After absorption at room temperature for 30 minutes, place it in a 37°C drying oven and dry for approximately 18 hours. The gold-labeled pad treatment solution consists of: 0.4 wt% S9, 0.5 wt% sucrose, 0.1 v / v% Pc300, and 0.01 M PBS.

[0222] (5) Test strip assembly:

[0223] The reconstituted gold described in step (2) was sprayed onto the gold-labeled pad at a spray rate of 2.0 μl / cm. The pad was then placed in a 37°C drying oven and dried for 30 minutes. The absorbent pad was then cut to a length and width of 30cm x 2.8cm, the sample pad to 30cm x 2.5cm, and the gold-labeled pad to 30cm x 0.8cm. An antibody-bearing membrane (NC membrane) was attached to the center of the PVC plate. Near the C-line, absorbent paper was attached parallel to the membrane, overlapping it by 0.2cm. The distance between the control line and the edge of the absorbent pad near the C-line was 10mm. Near the T-line, the sprayed gold-labeled pad was attached parallel to the membrane, overlapping it by 0.2cm. The distance between the detection line and the edge of the gold-labeled pad near the T-line was 8mm. Simultaneously, the sample pad was attached parallel to the gold-labeled pad, overlapping it by 0.2cm. After assembling the large board, cut it into strips using a strip cutter with a spacing of approximately 4mm, then assemble the test cards. Add desiccant, seal in an aluminum foil bag, and store in a cool, dry place.

[0224] Example 9: Novel Coronavirus Colloidal Gold Test Strip

[0225] The difference between this embodiment and Example 8 is that: (1) In the preparation of the antibody carrier membrane, the T-line antibody and the C-line antibody were diluted to 1.0 mg / ml and then dipped in the membrane, with a rati value of 0.8 μl / cm.

[0226] (2) In the gold labeling, the colloidal gold particles were 10 nm in size, the pH value of the colloidal gold labeling was 7.0, and the concentration of the monoclonal antibody N17 labeling was 15 μg / ml.

[0227] (3) During the sample pad treatment, the pH value of the sample pad treatment solution is 8.5.

[0228] (5) During the assembly of the test strip, the gold was reconstituted at a spray rate of 1.0 μl / cm.

[0229] Example 10: Novel Coronavirus Colloidal Gold Test Strip

[0230] The difference between this embodiment and Example 8 is that: (1) In the preparation of the antibody carrier membrane, the T-line antibody and the C-line antibody were diluted to 2.0 mg / ml and then dipped into the membrane, with a rati value of 1.2 μl / cm.

[0231] (2) In the gold labeling, the colloidal gold particles were 10 nm in size, the pH value of the colloidal gold labeling was 6.0, and the concentration of the monoclonal antibody N17 labeling was 10 μg / ml.

[0232] (3) During the sample pad treatment, the pH value of the sample pad treatment solution is 6.5.

[0233] (5) During the assembly of the test strip, the gold was reconstituted at a spray rate of 4.0 μl / cm.

[0234] Comparative Example 1

[0235] The difference between this comparative example and Example 5 is that the monoclonal antibody N26 was purified using a Bio-Rad NGC protein purifier. 20 ml of protein A column material was loaded with 40 ml of sample. The equilibration buffer was 0.02 M PB, pH 7.0, and the elution buffer was 0.1 M sodium citrate, pH 3.4. The antibody yield obtained after purification was about 50% less.

[0236] Comparative Example 2

[0237] The difference between this comparative example and Example 5 is that the equilibration solution was replaced with 3M NaCl, 0.02M PB, pH 7.0, and the eluent was 0.1M sodium citrate, pH 3.4. This resulted in a column pressure that was too high and even exceeded the equipment's specified column pressure range. In addition, a large amount of antibody precipitated during elution, which seriously affected the shelf life of the column material and the activity of the antibody.

[0238] Experimental Example 1

[0239] (1) Purity identification

[0240] The three batches of monoclonal antibody N17 obtained in Example 2 and the three batches of monoclonal antibody N26 obtained in Example 5 were subjected to non-reducing SDS-PAGE electrophoresis for purity identification. The purity of the three batches of monoclonal antibody N17 showed little difference, as did the purity of the three batches of monoclonal antibody N26. The purity of all antibodies was above 90%. See [link to relevant documentation]. Figure 1 In the figure, lanes 1-3 represent monoclonal antibody N17, and lanes 4-6 represent monoclonal antibody N26.

[0241] (2) Single batch yield testing

[0242] The concentrations of the three batches of monoclonal antibody N17 obtained in Example 2 and the three batches of monoclonal antibody N26 obtained in Example 5 were measured and the average values ​​were calculated. The average yield of a single batch of monoclonal antibody N17 was 6.8 mg / mouse, and the average yield of a single batch of monoclonal antibody N26 was 6.5 mg / mouse.

[0243] (3) Optimal pH value of elution buffer for monoclonal antibody N26

[0244] Following Example 5, the pH of the eluent was adjusted to 3.4, 4.0, 4.3, 5.0, 5.5, and 6.0. The results showed that at pH 5.5, the antibody could be eluted at low column pressure without precipitation.

[0245] Experiment Example 2

[0246] This experiment investigated the preparation process parameters of a colloidal gold test strip for the novel coronavirus. The negative samples used were nasal swabs from individuals who tested negative for COVID-19 nucleic acid. The positive samples were prepared by diluting the COVID-19 culture medium (inactivated virus culture medium provided by Nanjing Baikang Biotechnology) 1000 times. During testing, 100 μL of each sample (negative and positive) was applied to the sample pad of the test strip. Results were interpreted as follows: For positive samples, a purple-red band appeared at the test line (T) and the control line (C). For negative samples, a purple-red band appeared only at the control line (C).

[0247] (1) Film-spreading concentration

[0248] The procedure was carried out according to Example 8, except that the T-line antibody was diluted to concentrations of 1.0 mg / ml, 1.25 mg / ml, 1.5 mg / ml, 1.75 mg / ml, and 2.0 mg / ml, respectively. The concentrations of the monoclonal antibody N17 label (gold label) were 10 μg / ml, 15 μg / ml, and 20 μg / ml. The results are shown in the table below. It can be seen that the sensitivity increases with the increase of the concentration of the scratching antibody. Considering its chromatographic effect, antibody economy, and specificity, 1.75 mg / ml is preferred as the T-line scratching concentration, and the concentration of the monoclonal antibody N17 label is 20 μg / ml.

[0249] Table 1. Results of experiments determining the optimal concentrations of T-stretch antibody and gold-labeled antibody.

[0250]

[0251]

[0252] Note: "-" indicates that the color is not visible, and "+", "++", "+++", and "++++" indicate that the color ranges from light to dark.

[0253] The procedure was carried out according to Example 8, except that the C-line antibody was diluted to concentrations of 1.0 mg / ml, 1.25 mg / ml, 1.5 mg / ml, 1.75 mg / ml, and 2.0 mg / ml, respectively. The results are shown in the table below. It can be seen that the sensitivity increases with the increase of the concentration of the scratching antibody. Considering its chromatographic effect, antibody economy, and specificity, 1.25 mg / ml is preferred as the C-line scratching concentration.

[0254] Table 2C Results of Experiments Determining the Optimal Concentration of Stretched Membrane Antibody

[0255]

[0256] Note: "+" indicates a weaker color, and "++" indicates a stronger color.

[0257] (2) Colloidal gold labeling

[0258] The experiment was carried out according to Example 8, except that the colloidal gold particle size was 10 nm or 40 nm. The detection results are shown in the table below, with the 40 nm gold labeling showing better sensitivity.

[0259] Table 3. Results of the test for determining the size of colloidal gold particles

[0260] Colloidal gold particle size 10nm 40nm positive sample + ++ negative sample - -

[0261] Note: "+" indicates a weaker color, and "++" indicates a stronger color.

[0262] The procedure was carried out according to Example 8, except that the colloidal gold labeling pH values ​​were 6.0, 6.25, 6.5, 6.75, and 7.0. The test results are shown in the table below; the colloidal gold at pH 6.5 showed the best labeling efficiency.

[0263] Table 4. Results of the experiment to determine the optimal pH value for colloidal gold labeling.

[0264]

[0265]

[0266] Note: "-" indicates that the color is not visible, and "+", "++", "+++", and "++++" indicate that the color ranges from light to dark.

[0267] (3) Sample pad treatment solution

[0268] The procedure was carried out according to Example 8, except that the pH values ​​of the sample pad treatment solution were 6.5, 7.0, 7.5, 8.0, and 8.5. The results are shown in the table below. Considering its sensitivity and specificity, pH 8.0 is preferred as the optimal pH value.

[0269] Table 5. Results of the test for determining the optimal pH value of the sample pad treatment solution.

[0270] pH value 6.5 7.0 7.5 8.0 8.5 positive sample + + ++ +++ ++ negative sample - - - - -

[0271] Note: "-" indicates that the color is not visible, and "+", "++", "+++", and "++++" indicate that the color ranges from light to dark.

[0272] Experiment Example 3 Sensitivity Detection

[0273] The recombinant SARS-CoV-2 N protein was serially diluted to 5 ng / ml, 500 pg / ml, and 100 pg / ml using 0.01 M PBS as positive samples. Dilutes without the recombinant SARS-CoV-2 N protein were used as negative samples. The test strips prepared in Example 8 were used for detection. Results interpretation: For positive samples, a purple-red band was observed at the test line T and the control line C. Results are as follows... Figure 2 (The figure shows the test strip condition test results after the test card is assembled.) As can be seen, the sensitivity of the test strip of the present invention can reach 100 pg / ml.

[0274] Experimental Example 4: Clinical Application

[0275] Thirty clinical samples (pharyngeal swabs, 300 μl dilution buffer, composition: 0.5 v / v % x-100, 0.3 wt% S9, 0.5 wt% casein, and 0.1 v / v % Pc300 in 0.01 M PBS, 100 μl sample volume) were tested using an existing novel coronavirus nucleic acid detection kit (provided by Beijing Zhuocheng Huisheng Biotechnology Co., Ltd.). The test card from Example 8 was used for validation, showing a negative concordance rate of 96.7% and a positive concordance rate of 86.7%. Based on these results, testing personnel can perform multiple tests or tests at intervals of several days to reduce individual missed detections or false positives. Therefore, this test card can still be used for rapid detection of novel coronavirus antigens.

[0276] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line N17, characterized in that, The monoclonal antibody N17 is secreted by the hybridoma cell line N17; the hybridoma cell line N17 has the accession number CCTCC NO: C2022322.

2. The hybridoma cell line N17 according to claim 1, characterized in that, The hybridoma cell line N17 uses the novel coronavirus N protein as an immunogen.

3. A hybridoma cell line N26, characterized in that, The hybridoma cell line N26 secretes a monoclonal antibody N26; the hybridoma cell line N26 has the accession number CCTCC NO: C2022323.

4. The hybridoma cell line N26 according to claim 3, characterized in that, The hybridoma cell line N26 uses the novel coronavirus N protein as an immunogen.

5. A method for preparing a monoclonal antibody against the N protein of a novel coronavirus, characterized in that, include: Using the hybridoma cell line that secretes monoclonal antibody N17 or monoclonal antibody N26 as described in any one of claims 1-4, ascites containing monoclonal antibody N17 or monoclonal antibody N26 was prepared by in vivo animal induction method. The ascites fluid was separated and purified to obtain monoclonal antibody N17 or monoclonal antibody N26. The N17 hybridoma cell line that secretes monoclonal antibodies is the N17 hybridoma cell line. The N26 hybridoma cell line that secretes monoclonal antibodies is the hybridoma cell line N26.

6. The method for preparing the novel coronavirus N protein monoclonal antibody according to claim 5, characterized in that, The steps for preparing ascites include: sensitizing mice older than 8 weeks or multiparous female mice with 0.4ml-0.6ml of liquid paraffin via intraperitoneal injection; 5-9 days later, injecting 0.5×10⁻⁶ oz. of paraffin into each mouse intraperitoneally. 5 -1.0×10 5 Hybridoma cells were collected, and ascites fluid was collected starting 6-9 days later. And / or, in the separation step, the ascites fluid and saturated ammonium sulfate solution are mixed in equal volumes, stirred, precipitated for 18-22 hours, the supernatant is removed by centrifugation, the solution is reconstituted, and then dialyzed overnight. The dialysis molecular weight is 8-20 kDa. The antibody is collected and brought to a final volume to obtain solution I. And / or, in the purification step, a protein purification chromatography column is used for purification. First, the protein purification chromatography column is rinsed with purified water, and then the protein purification chromatography column is equilibrated with buffer. Then, solution I is filtered, loaded with the sample, washed with buffer, eluted with elution buffer, the elution buffer is collected, the pH is adjusted, and then dialyzed and filtered.

7. The method for preparing the novel coronavirus N protein monoclonal antibody according to claim 6, characterized in that, In the purification step, purification is performed using a Bio-Rad NGC or an Akta protein purifier; And / or, in the purification step, the column material in the protein purification chromatography column is Protein A; And / or, in the purification step, the volume of the protein purification chromatography column is 50-200 ml; And / or, in the purification step, when the purified antibody is monoclonal antibody N17, the buffer is a 0.01M-0.04M PB solution with a pH of 6.8-7.2; And / or, in the purification step, when the purified antibody is monoclonal antibody N17, the elution buffer is a 0.05M-0.2M sodium citrate solution with a pH of 2.7-4.3; And / or, in the purification step, when the purified antibody is monoclonal antibody N17, the pH is adjusted to 7.0-7.4 using a 0.05M-2.0M Tris solution with pH 8.5-9.5; And / or, in the purification step, when the purified antibody is monoclonal antibody N26, the buffer solution is a solution containing 1-4 M NaCl, 0.01-0.04 M PB, and pH 6.8-7.

2. And / or, in the purification step, when the purified antibody is monoclonal antibody N26, the elution buffer is a 0.05-0.2M sodium citrate solution with a pH of 4.3-6.5; And / or, in the purification step, when the purified antibody is monoclonal antibody N26, the pH is adjusted to 7.0-7.4 using a 0.05-2.0M Tris solution with pH 8.5-9.

5.

8. Use of the hybridoma cell line N17 according to claim 1 or 2, or the hybridoma cell line N26 according to claim 3 or 4: (1) Use in the preparation of products for detecting the novel coronavirus; (2) Use in the preparation of products containing isolated and purified novel coronavirus; (3) Use in the preparation of products containing the isolated and purified N protein of the novel coronavirus; The products include reagents, test strips, or kits.

9. A colloidal gold test strip for the novel coronavirus, characterized in that, This includes using the hybridoma cell line N17 as described in claim 1 or 2 or the hybridoma cell line N26 as described in claim 3 or 4, and using monoclonal antibody N17 or monoclonal antibody N26 as a detection line coating antibody and / or colloidal gold-labeled antibody.

10. The novel coronavirus colloidal gold test strip according to claim 9, characterized in that, include: The substrate and the sample pad, gold label pad, antibody carrier membrane and absorbent pad that are sequentially overlapped on the substrate; The antibody carrier membrane is provided with a detection line and a control line spaced apart. The detection line is close to the colloidal gold adsorption pad, and the control line is close to the absorbent pad. The detection line is coated with monoclonal antibody N26, and the colloidal gold adsorption pad is coated with colloidal gold-labeled monoclonal antibody N17.

11. A method for preparing a colloidal gold test strip for the novel coronavirus as described in claim 9 or 10, characterized in that, This includes using the hybridoma cell line N26 as described in claim 3 or 4, with monoclonal antibody N17 or monoclonal antibody N26 as the detection line coating antibody and / or colloidal gold-labeled antibody.

12. The method for preparing the novel coronavirus colloidal gold test strip according to claim 11, characterized in that, include: The sample pad, gold label pad, antibody carrier membrane and absorbent pad are prepared, and then the sample pad, gold label pad, antibody carrier membrane and absorbent pad are sequentially overlapped on the substrate; The preparation of the antibody carrier membrane includes: using the monoclonal antibody N26 as the detection line coating antibody, diluting the detection line coating antibody to 1.0 mg / ml-2.0 mg / ml, and then performing a membrane run; The preparation of the gold-labeled pad includes: spraying a gold-labeled pad treatment solution containing colloidal gold-labeled monoclonal antibody N17 onto the gold-labeled pad, wherein the concentration of monoclonal antibody N17 labeling is 10 μg / ml-20 μg / ml.

13. The method for preparing the novel coronavirus colloidal gold test strip according to claim 11 or 12, characterized in that, The monoclonal antibody N26 was used as the detection line coating antibody, and the detection line coating antibody was diluted to 1.75 mg / ml and then applied to a membrane. And / or, the detection line is coated with antibody for membrane tracing, with a rati value of 0.8-1.2 μl / cm; And / or, the colloidal gold-labeled monoclonal antibody N17 is coated onto the gold-labeled pad at a concentration of 20 μg / ml; And / or, when colloidal gold-labeled monoclonal antibody N17 is sprayed onto a gold-labeled pad, the rati value is 1.0-4.0 μl / cm.

14. The method for preparing the novel coronavirus colloidal gold test strip according to claim 13, characterized in that, The colloidal gold particles have a size of 10nm-40nm; and / or The pH value for colloidal gold labeling is 6.0-7.0; and / or The preparation of the antibody carrier membrane also includes: coating the control line with goat anti-mouse IgG antibody, diluting it to 1.0 mg / ml-2.0 mg / ml, and then performing a membrane run; It also includes the preparation of sample pads, including: treating the sample pads with a sample pad treatment solution, wherein the pH value of the sample pad treatment solution is 6.5-8.

5.

15. The method for preparing the novel coronavirus colloidal gold test strip according to claim 14, characterized in that, The colloidal gold particles have a size of 40 nm; And / or, the pH value for the colloidal gold labeling is 6.5; And / or, the sample pad is treated with a sample pad treatment solution, the pH of which is 8.0; And / or, the control line is coated with goat anti-mouse IgG, diluted to 1.25 mg / ml, and then scratched. And / or, the control line is coated with antibody and scratched, with a rati value of 0.8-1.2 μl / cm.

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