Hybridoma cell line secreting monoclonal antibody against novel coronavirus ORF8 protein, monoclonal antibody, antibody composition and application

Abstract

The present invention discloses a hybridoma cell line, a monoclonal antibody, an antibody composition and an application that secretes a monoclonal antibody against the novel coronavirus ORF8 protein. The hybridoma cell line provided by the present invention can secrete a highly specific monoclonal antibody against the novel coronavirus ORF8 protein, and at the same time, a double antibody sandwich ELISA and flow cytometry detection method are established based on this, with a low detection limit, a wide linear range, high sensitivity and strong specificity. It provides a new technical option for the detection of the novel coronavirus ORF8 protein, as well as the basic research and clinical diagnosis of the novel coronavirus, and has important promotion and application value.

Description

Technical Field

[0001] The present invention relates to the field of protein antibody detection, and specifically, to a hybridoma cell line, monoclonal antibody, antibody composition and application that secretes monoclonal antibody against novel coronavirus ORF8 protein. Background Art

[0002] The novel coronavirus is a single-stranded positive-strand RNA enveloped virus with a diameter of 100 nm. It encodes 4 structural proteins (envelope (E), membrane (M), nucleocapsid (N) and spike (S) proteins), 16 non-structural proteins (NSP1-16), and 9 auxiliary proteins. The novel coronavirus ORF8 (open reading frame 8) protein is one of the auxiliary proteins encoded by the novel coronavirus. It does not directly participate in the replication of the virus itself, but can promote the replication of the virus by regulating the pathways and gene expression of host cells. It can also regulate the host's immune response through various mechanisms and promote the immune escape of the virus.

[0003] The novel coronavirus ORF8 protein is an important target protein for studying the mechanism of novel coronavirus infection inhibition and host pathological damage: 1. ORF8 can cause endoplasmic reticulum stress response of cells, and through a series of protein interactions and other effects, it can avoid cell apoptosis, induce cell adaptation and endoplasmic reticulum reconstruction, and thus help the assembly and release of the virus; 2. ORF8 can act as a histone mimetic, promote chromatin condensation through epigenetic modification, inhibit the transcription of cellular antiviral-related pathway genes, and thus destroy the immune response to viral infection; 3. ORF8 can antagonize type I interferon production and signal transduction, and promote viral immune escape; 4. ORF8 can bind to MHC-I and promote its lysosomal degradation, thereby downregulating MHC-I molecules and protecting novel coronavirus-infected cells from CTL killing.

[0004] The novel coronavirus ORF8 protein has potential clinical detection significance: Studies have shown that ORF8 protein can be detected in the serum of patients infected with the novel coronavirus. Because ORF8 is a non-structural protein, it is only synthesized and secreted during the active replication process after the novel coronavirus infects cells, and will not enter the human body through methods such as vaccination. Therefore, if the novel coronavirus ORF8 protein is detected in the serum of the subject, it reflects the presence of active novel coronavirus infection in the body.

[0005] The novel coronavirus ORF8 protein has potential clinical therapeutic significance: Studies have shown that ORF8 is associated with poor prognosis in patients infected with the novel coronavirus. ORF8 can promote the overexpression of host inflammatory factors, leading to side effects such as cytokine storms, and its concentration is related to the severity of the patient's disease progression. Inhibiting ORF8 protein through neutralizing antibodies and other means may have a protective effect on infected patients.

[0006] Therefore, the development of antibodies with high affinity and high specificity targeting the novel coronavirus ORF8 protein has many important research significance and application value: 1. Antibodies targeting the novel coronavirus ORF8 protein can provide important research tools for basic research on the novel coronavirus ORF8 protein; 2. The application of ORF8 antibodies to develop detection methods targeting the novel coronavirus ORF8 antigen can help develop new antigen detection methods and supplement the existing novel coronavirus antigen detection technology. 3. Using ORF8 antibodies to neutralize or block ORF8 protein can inhibit its role in promoting viral replication and immune escape in novel coronavirus infection, and can provide a new treatment idea for alleviating the symptoms of patients with viral infection; however, there are few antibodies that can be used to detect the novel coronavirus ORF8 protein, and accurate detection methods have not yet been established. Summary of the invention

[0007] In order to solve the above-mentioned problems existing in the prior art, the present invention provides a hybridoma cell line, a monoclonal antibody, an antibody composition and an application that secretes a monoclonal antibody against the novel coronavirus ORF8 protein.

[0008] The first objective of the present invention is to provide a hybridoma cell line that secretes monoclonal antibodies against the novel coronavirus ORF8 protein.

[0009] The second object of the present invention is to provide the use of the hybridoma cell line in the preparation of antibodies against the new coronavirus ORF8 protein.

[0010] The third object of the present invention is to provide the use of the hybridoma cell line in the preparation of a product for detecting the novel coronavirus ORF8 protein.

[0011] The fourth object of the present invention is to provide a monoclonal antibody against the novel coronavirus ORF8 protein.

[0012] The fifth object of the present invention is to provide the use of the monoclonal antibody in the preparation of a product for detecting the novel coronavirus ORF8 protein.

[0013] The sixth object of the present invention is to provide an antibody composition for detecting the novel coronavirus ORF8 protein.

[0014] The seventh objective of the present invention is to provide the use of the antibody composition in the preparation of a product for detecting the novel coronavirus ORF8 protein.

[0015] The eighth object of the present invention is to provide an immunoassay method for detecting the novel coronavirus ORF8 protein for non-disease treatment and diagnosis purposes.

[0016] The ninth object of the invention is to provide a kit for detecting the ORF8 protein of the new coronavirus.

[0017] In order to achieve the above object, the present invention is implemented by the following scheme:

[0018] The present invention uses hybridoma cell technology to prepare a hybridoma cell strain that secretes highly specific anti-novel coronavirus ORF8 protein monoclonal antibodies, and based on this, establishes a highly sensitive novel coronavirus ORF8 antigen detection method.

[0019] The hybridoma cell line that secretes monoclonal antibodies against the novel coronavirus ORF8 protein is any one of the following hybridoma cell lines (1) to (2): the deposit number of the hybridoma cell line (1) is GDMCC No: 65043, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 22, 2024; the deposit number of the hybridoma cell line (2) is GDMCC No: 65206, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 25, 2024.

[0020] The use of the hybridoma cell line in the preparation of products for detecting the novel coronavirus and / or the novel coronavirus ORF8 protein.

[0021] The use of the hybridoma cell line in preparing antibodies against the novel coronavirus ORF8 protein.

[0022] A monoclonal antibody against the novel coronavirus ORF8 protein, secreted by the hybridoma cell line. The monoclonal antibody secreted by the hybridoma cell line (2) corresponds to the 5F1 antibody described in the present invention, and the monoclonal antibody secreted by the hybridoma cell line (1) corresponds to the 8E12 antibody described in the present invention.

[0023] The use of the monoclonal antibody in the preparation of products for detecting the new coronavirus and / or the new coronavirus ORF8 protein.

[0024] An antibody composition for detecting a novel coronavirus and / or novel coronavirus ORF8 protein, comprising a coating antibody and a detection antibody, wherein the coating antibody of the antibody composition (1) is a monoclonal antibody secreted by the hybridoma cell line (2), and the detection antibody is a monoclonal antibody secreted by the hybridoma cell line (1); the coating antibody of the antibody composition (2) is a monoclonal antibody secreted by the hybridoma cell line (1), and the detection antibody is a monoclonal antibody secreted by the hybridoma cell line (2).

[0025] More preferably, the antibody composition is the antibody composition (1).

[0026] In some specific embodiments, in order to improve the detection performance of antibodies, it is conventional to label antibodies with substances that are easy to measure and have high sensitivity, such as fluorescein, isotopes, enzymes, biotin, etc., and use the enhanced amplification effect of these markers to display the nature and content of the antigen bound to the antibody. This labeling does not affect the specific binding of the Fab functional region of the antibody to the antigen.

[0027] Preferably, the detection antibody is labeled with biotin.

[0028] The use of the antibody composition in the preparation of products for detecting the novel coronavirus and / or the novel coronavirus ORF8 protein should also be within the scope of protection of the present invention.

[0029] Preferably, the product comprises an ELISA kit or a flow cytometry kit.

[0030] An immunoassay method for detecting the novel coronavirus and / or novel coronavirus ORF8 protein for non-disease treatment and diagnosis purposes, using the monoclonal antibody or the antibody composition to perform immunoassay on the sample to be tested.

[0031] The monoclonal antibody or the antibody composition provided by the present invention can realize immunoassay including but not limited to flow cytometry, immunofluorescence, ELISA and immunoprecipitation detection methods.

[0032] Preferably, the immunoassay method is ELISA or flow cytometry.

[0033] More preferably, the ELISA is a sandwich ELISA, and the antibody composition is used to perform immunoassay on the sample to be tested.

[0034] A kit for detecting the novel coronavirus and / or the novel coronavirus ORF8 protein, comprising the hybridoma cell line, the monoclonal antibody or the antibody composition.

[0035] Preferably, it further comprises ELISA reagents or flow cytometry reagents.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] The hybridoma cell line provided by the present invention can secrete highly specific monoclonal antibodies against the novel coronavirus ORF8 protein, and at the same time, a double antibody sandwich ELISA and flow cytometry detection method is established with this, which has a low detection limit, high sensitivity, wide linear range, and strong specificity. It supplements new methods for the detection of novel coronavirus ORF8 protein in the basic research process. It also provides new ideas and technical options for clinical detection of novel coronavirus infection and antibody treatment, and has important promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the SDS-PAGE gel electrophoresis identification result after the novel coronavirus ORF8 protein was purified by nickel ion affinity chromatography. A is the result of non-reducing electrophoresis samples, in which lane 1 is Marker, lane 2 has no sample, lane 3 is the supernatant before column, lane 4 is Flow through, lane 5 is Wash buffer, lane 6 is 5 mM imidazole, lane 7 is 10 mM imidazole, lane 8 is 20 mM imidazole, and lanes 9 to 14 are 300 mM imidazole flow-through tubes 1 to 6 respectively; B is the result of reducing electrophoresis samples, in which lane 1 is Marker, lane 2 is the supernatant before column, lane 3 is Flowthrough, lane 4 is Wash buffer, lane 5 is 5 mM imidazole, lane 6 is 10 mM imidazole, lane 7 is 20 mM imidazole, and lanes 8 to 13 are 300 mM imidazole flow-through tubes 1 to 6 respectively.

[0039] Figure 2 The figure shows the further purification of the novel coronavirus ORF8 protein by ion exchange chromatography; A is a diagram of the elution process of the protein by ion exchange chromatography; B is the SDS-PAGE gel electrophoresis identification result of the protein after ion exchange chromatography, lanes 1 to 6 are the reducing electrophoresis samples of the 4th to 9th tubes of eluate of ion exchange chromatography, lane 7 is the marker; lane 8 is the blank lane; lanes 9 to 14 are the non-reducing electrophoresis samples of the 4th to 9th tubes of eluate of ion exchange chromatography.

[0040] Figure 3 These are the results of ELISA testing of the immunogenicity of ORF8 protein in mice. Numbers 1 to 4 are the numbers of the immunized mice.

[0041] Figure 4 The figure shows the SDS-PAGE gel electrophoresis identification results of the purified monoclonal antibody; A is the non-reducing electrophoresis sample result, B is the reducing electrophoresis sample result, lane 1 is the Marker, lane 2 is the supernatant before column, lane 3 is the flow through solution, lane 4 is the wash buffer flow through solution, and lanes 5 to 14 are the 1st to 10th elution tubes respectively.

[0042] Figure 5 This is the indirect ELISA test result of the binding of the purified monoclonal antibody to the new coronavirus ORF8 protein.

[0043] Figure 6 These are the detection results of the double antibody sandwich ELISA method for antibody pair combination 1 and antibody pair combination 2.

[0044] Figure 7 The results of double antibody sandwich ELISA of antibody pair combination 1 at different working concentrations.

[0045] Figure 8 The linear curve of antibody pair combination 1 in double antibody sandwich ELISA method using PBS as the matrix.

[0046] Fig. 9 This is the linear curve of antibody pair combination 1 in double antibody sandwich ELISA method using plasma as the matrix.

[0047] Fig.10 This is the specific detection result of antibody pair combination 1.

[0048] Fig.11 These are the results of double antibody sandwich ELISA testing of the supernatant of cells infected with pseudovirus of the new coronavirus.

[0049] Fig.12 The results of flow cytometry were shown.

[0050] Fig.13 The figure shows the indirect ELISA results compared with commercial antibodies.

[0051] Fig.14 The figure shows the results of double antibody sandwich ELISA compared with commercial antibodies. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0053] Example 1 Preparation of novel coronavirus ORF8 antigen protein

[0054] 1. Construction of novel coronavirus ORF8 protein expression vector and plasmid extraction

[0055] The novel coronavirus ORF8 gene (NCBI No.: 43740577; the amino acid sequence after removing the signal peptide is: AFHQECSLQSCTQHQPYVVDDPCPIHFYSKWYIRVGARKSAPLIELCVDEAGSKSPIQYIDIGNYTVSCLPFTINCQEPKLGSLVVRCSFYEDFLEYHDVRVVLDFI (SEQ ID NO.1)) was synthesized by Sangon Biotech and constructed into the eukaryotic expression vector pcDNA3.4, in which the HRV 3C restriction site, purification tag 10×His, StrepTagII and detection tag Flag were added to the N-terminus, thus obtaining the expression vector of the novel coronavirus ORF8 protein, which was recorded as pcDNA3.4-ORF8.

[0056] The pcDNA3.4-ORF8 vector was transformed into Escherichia coli competent cells according to conventional methods. After culture and identification, the pcDNA3.4-ORF8 plasmid was extracted using the Tiangen Endotoxin-Free Plasmid Extraction Kit (Enhanced) (DP120). After the concentration was determined, it was stored at -20°C for future use.

[0057] 2. Cell culture and transfection

[0058] Cell culture: Expi293F WT cells (Thermo Scientific, A14527) were used for suspension culture of cell lines, and OPM-293 CD05 medium (OPM, 81075-001) was used for suspension culture at 37°C, 5% CO2, and 110 rpm.

[0059] Transfection: Expi293F WT cells were transfected with PEI MAX (Polysciences, 24765) transfection reagent and the pcDNA3.4-ORF8 plasmid in this example, and the cells were cultured after transfection; after 5 hours, the same volume of OPM-293CD05 culture medium was added and cultured; on the third day, a glucose solution with a final concentration of 3.00 g / L was added to maintain the nutrients required for expression; on the fifth day, the cell supernatant was collected for protein purification.

[0060] 3. Protein purification

[0061] The cell culture supernatant was purified by nickel (Ni) ion affinity chromatography. The specific steps are as follows:

[0062] (1) Pretreatment: Take out 5 mL of Ni-NTA beads and load them into a 50 mL chromatography column. Wash with 5 times the volume of filler (hereinafter referred to as column volume, column volumes, CV) of ultrapure water; after the ultrapure water flows out completely, add 5 times the column volume of 100 mM EDTA to chelate nickel ions and achieve the effect of stripping nickel ions; then add 5 times the column volume of ultrapure water for washing; then use 5 times the column volume of 0.5 M NaOH for washing; then use 5 times the column volume of 1 M HCl for washing; then use 5 times the column volume of ultrapure water for washing; use 5 times the column volume of 100 mM nickel chloride to hang nickel ions on the filler, and further wash with 5 times the column volume of ultrapure water; after the ultrapure water flows out, wash 5 times the column volume of the equilibrated Ni column with Wash buffer (20 mM Tris-HCl, 500 mM NaCl, pH=8.0).

[0063] (2) Sample loading: Centrifuge the cell culture supernatant at 4°C and 1000 rpm for 30 min, discard the cells, take the supernatant and centrifuge it again at 4°C and 8000 rpm for 50 min. Collect the supernatant and add it to the equilibrated Ni column (adjust the flow rate to 100 mL / h). Place a collection tube under the Ni column to collect the Ni column flow-through, which is marked as Flow through.

[0064] (3) Washing impurities with wash buffer: Use wash buffer (20 mM Tris-HCl; 500 mM NaCl, pH = 8.0) to wash the proteins that are not bound to the Ni column. The washing volume is 5 times the column volume. Place a collection tube under the Ni column to collect the flow-through and mark it as wash buffer flow-through.

[0065] (4) 5 mM imidazole to clean the impurities: Use 5 mM imidazole (20 mM Tris-HCl; 500 mM NaCl; 5 mM imidazole, pH = 8.0) to clean the proteins that are not bound to the Ni column. The cleaning volume is 5 times the column volume. Place a collection tube under the Ni column to collect the flow-through and mark it as 5 mM imidazole flow-through.

[0066] (5) 10 mM imidazole to clean the impurities: Use 10 mM imidazole (20 mM Tris-HCl; 500 mM NaCl; 10 mM imidazole, pH = 8.0) to clean the proteins that are not bound to the Ni column. The cleaning volume is 5 times the column volume. Place a collection tube under the Ni column to collect the flow-through and mark it as 10 mM imidazole flow-through.

[0067] (6) 20 mM imidazole to clean the impurities: Use 20 mM imidazole (20 mM Tris-HCl; 500 mM NaCl; 20 mM imidazole, pH = 8.0) to clean the proteins that are not bound to the Ni column. The cleaning volume is 5 times the column volume. Place a collection tube under the Ni column to collect the flow-through and mark it as 20 mM imidazole flow-through.

[0068] (7) Elution of target protein: Elute the 2019-nCoV ORF8 protein with 300 mM imidazole (20 mM Tris-HCl; 500 mM NaCl; 300 mM imidazole, pH = 8.0). Use 5 times the volume of elution buffer to elute the ORF8 protein. Collect the flow-through at a rate of 10 mL / tube. Collect a total of 6 tubes of flow-through, which are labeled as 300 mM imidazole 1 flow-through, 300 mM imidazole 2 flow-through, 300 mM imidazole 3 flow-through, 300 mM imidazole 4 flow-through, 300 mM imidazole 5 flow-through, and 300 mM imidazole 6 flow-through.

[0069] 4. SDS-PAGE gel electrophoresis identification

[0070] The supernatant before column, flow through solution, wash buffer solution, 5 mM imidazole solution, 10 mM imidazole solution, 20 mM imidazole solution, 300 mM imidazole 1 solution, 300 mM imidazole 2 solution, 300 mM imidazole 3 solution, 300 mM imidazole 4 solution, 300 mM imidazole 5 solution, and 300 mM imidazole 6 solution were used as samples to be tested, and SDS-PAGE gel electrophoresis was used to identify the expression and purification of ORF8 protein. The specific method is as follows:

[0071] (1) Preparation of electrophoresis samples: 12 μL of each of the above-mentioned samples to be tested was mixed evenly with 3 μL of reducing (reducing) 5× Loading buffer (i.e., 250 mM Tris-HCl, pH=6.8; 10 w / v% SDS; 0.5 w / v% bromophenol blue; 50 v / v% glycerol; 2 mM DTT) or non-reducing (non-reducing) 5× Loading buffer (i.e., 250 mM Tris-HCl, pH=6.8; 10 w / v% SDS; 0.5 w / v% bromophenol blue; 50 v / v% glycerol), heated in a metal bath at 98°C for 8 min, and then centrifuged at 12,000 rpm for 1 min to obtain reducing electrophoresis samples or non-reducing electrophoresis samples, respectively.

[0072] (2) Sample loading: Add the prepared electrophoresis sample to 15% SDS-PAGE gel, adjust the electrophoresis instrument voltage to 80 V and run for 20 min, then adjust to 120 V and run for 50 min.

[0073] (3) Staining and decolorization: Use 20 mL of Coomassie Brilliant Blue solution (prepared by mixing 0.25 w / v% Coomassie Brilliant Blue G-250, 10 v / v% anhydrous acetic acid, 20 v / v% anhydrous ethanol and 70 v / v% ddH2O) to stain the SDS-PAGE gel after electrophoresis, heat it in a microwave oven at 100°C for 3 min, and then use 40 mL of decolorization solution (prepared by mixing 10 v / v% anhydrous acetic acid, 20 v / v% anhydrous ethanol and 70 v / v% ddH2O) to decolorize it at room temperature overnight.

[0074] The SDS-PAGE results are as follows Figure 1 As shown in A and B in the figure, the 2nd to 4th tubes (lanes 10 to 12) of the 300 mM imidazole flowthrough of the non-reducing electrophoresis sample have obvious bands at around 40 kDa, which is consistent with the expected size of the labeled novel coronavirus ORF8 protein dimer, and the 2nd to 4th tubes (lanes 9 to 11) of the 300 mM imidazole flowthrough of the reducing electrophoresis sample have obvious bands at around 20 kDa, which is consistent with the expected size of the labeled novel coronavirus ORF8 protein monomer. The above results show that the novel coronavirus ORF8 was successfully expressed.

[0075] 5. Use recombinant HRV 3C protease (Shanghai Biotechnology, C510303) to remove the N-terminal purification tag of the novel coronavirus ORF8 protein expressed in the previous step and collect the enzyme cleavage product.

[0076] 6. Dialysis and ion exchange chromatography

[0077] (1) Use 20 mM Tris solution (pH = 7.0) and 2KD dialysis bag (Shanghai Biotechnology, F132620) to dialyze the enzyme cleavage product obtained in the previous step, and collect the dialyzed ORF8 protein.

[0078] (2) Ion exchange chromatography: According to calculations (website: Prot pi | Protein Tool), the isoelectric point of the novel coronavirus ORF8 protein is 5.2. Therefore, the dialyzed ORF8 protein was purified using a standard process using a strong anion exchange column (HiTrap™ Capto™ QImpRes) with a column volume of 5 mL. The specific method is as follows: the dialyzed ORF8 protein sample was centrifuged at 4°C and 4700 rpm for 20 min and then loaded onto the column at a rate of 1 mL / min. After the column was completed, the novel coronavirus ORF8 protein was bound to the column. Elution buffer (20 mM Tris + 1 M NaCl, PH = 7.0) was then used to load the sample at a flow rate of 1 mL / min to perform gradient elution (gradient 0-100%) on the ORF8 protein bound to the column. The eluate was collected and the protein concentration was determined using Nanodrop. The elution process was as follows: Figure 2 As shown in Figure A, the elution process contains a main peak of the novel coronavirus ORF8 protein, with no other impurities, and the protein concentration in the eluate from tubes 4 to 9 is the highest.

[0079] The eluates from tubes 4 to 9 were collected and the purity of the ORF8 protein after the column was identified according to the method of "4. SDS-PAGE gel electrophoresis identification" in this example; the eluted samples were combined and collected, dialyzed using 1× PBS with a pH of 7.4, and quickly frozen in liquid nitrogen for later use after aliquoting.

[0080] The results of SDS-PAGE gel electrophoresis were as follows: Figure 2 As shown in Figure B, the reduced electrophoresis samples in lanes 2 to 6 have obvious bands at around 16 kDa, which is consistent with the expected size of the ORF8 protein monomer, and the non-reduced electrophoresis samples in lanes 10 to 14 have obvious bands at around 25 kDa, which is consistent with the expected size of the ORF8 protein dimer. The above results show that the novel coronavirus ORF8 has been successfully purified.

[0081] Example 2 Preparation, purification and identification of monoclonal antibodies against novel coronavirus ORF8 protein

[0082] 1. Animal immunization

[0083] The novel coronavirus ORF8 protein prepared in Example 1 was used as an antigen, and healthy BALB / c pure strain female mice (6-8 weeks old, weighing about 20 g) were immunized according to the scheme shown in Table 1. Before the first immunization, the tail vein blood of the mice was collected, and the upper light yellow serum was collected by centrifugation as the negative serum for the pre-immunization titer determination; after the first immunization, booster immunization was performed every two weeks, for a total of 4 immunizations. Before each immunization, the tail vein blood of the mice was collected, and the serum was separated as the immune serum for immune titer detection; the last booster immunization was performed 2 days before cell fusion.

[0084] Table 1 Mouse immunization scheme

[0085]

[0086] 2. Immune titer determination

[0087] The indirect ELISA method was used to determine the immune titer. The specific steps are as follows:

[0088] The purified novel coronavirus ORF8 protein prepared in Example 1 was diluted to 4.00×10 6 pg / mL, 100 μL was added to each well of the ELISA plate and coated at 4°C overnight; 300 μL PBST (containing 0.05 v / v% TWEEN-20) was added to each well and washed 3 times; 300 μL blocking solution (PBS containing 2 w / v% BSA and 3 w / v% skim milk powder) was added to each well and blocked at 37°C for 1 h; 300 μL Wash three times with PBST; dilute the mouse immune serum or negative serum at 1:500 with blocking solution, and then dilute it with blocking solution as solvent, setting 11 gradients, namely: 1:500, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000, 1:256000 and 1:512000, to obtain serum dilutions of different concentrations; add 100 μL of serum dilutions of each concentration to the ELISA plate at each well and incubate at 37°C for 1.5 h; add 300 μL of PBST to each well and wash three times; dilute anti-mouse-IgG-HRP (Sigma, A9044) 25000 times with blocking solution and use it as secondary antibody, add 100 μL / well to the ELISA plate and incubate at 37°C for 1 h; add 300 μL PBST to each well and wash five times; mix equal volumes of TMB colorimetric solution A and solution B to obtain the colorimetric agent, add 100 μL of the colorimetric agent to each well, and incubate at 37°C for 15 min; add 2 M H2SO4 at 50 μL / well to terminate the colorimetric reaction, and quickly place it on an enzyme reader for detection, setting the detection wavelength to 450 nm. The results of the immune titer test of the mouse serum after the fourth booster immunization are shown in Figure 2. Figure 3 As shown, the antibody titer of mouse serum after the fourth booster immunization reached above 1:128000, indicating that the immunization was effective and could be used for subsequent cell fusion.

[0089] 3. Cell fusion

[0090] Myeloma cells (SP2 / 0 cells) were cultured by conventional methods, and the SP2 / 0 cells were collected by centrifugation at 1000 rpm for 5 min and replaced with RPMI 1640 medium (Gibco TM , C11875500BT) were washed twice, resuspended and counted. The spleen of the immunized mouse was separated, cut into pieces and ground on a 200-mesh copper grid, spleen cells were collected, and the red blood cells were lysed with red blood cell lysis buffer, then diluted with PBS, centrifuged at 1000 rpm, and the supernatant was discarded; washed once with RPMI 1640 medium, resuspended and counted. PEG cell fusion agent (Shanghai Biotechnology, E607052) was used to induce cell fusion, in which the ratio of spleen cells and SP2 / 0 cells fused was 5:1. The fused cells were cultured in HAT medium (formula: RPMI 1640 medium + 20% FBS + HAT medium supplement (Sigma, H0262)).

[0091] 4. Hybridoma cell screening

[0092] After culturing the fused cells for about one week, the culture supernatant was taken and the activity of the antibody in the supernatant was determined by referring to the detection method of "2. Immunoassay" of this example. The cell wells with positive test results were retained and the cells were subsequently subcloned in the following steps.

[0093] The cells with positive test results were resuspended by centrifugation, counted and diluted to 1.1 cells / 100 μL / well, added to a 96-well plate, and cultured on the wall; the wells with single cell growth were retained for further culture; the activity of the antibody in the supernatant was determined by referring to the detection method of "2. Immunopotency Determination" of this Example, and the monoclonal cells that remained positive in each determination were retained.

[0094] Finally, two hybridoma cell strains that best bound to the novel coronavirus ORF8 protein were obtained, secreting monoclonal antibodies 5F1 and 8E12, respectively. Both have been deposited in the Guangdong Microbiological Culture Collection Center (GDMCC), located on the 5th floor of Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China. Among them, the hybridoma cell line secreting 5F1 antibody (recorded as 5F1 cell line) has a deposit number of GDMCC No: 65206, a deposit date of September 25, 2024, a deposit name of mouse hybridoma monoclonal cell line hSARS-CoV-2-ORF8-m-mAb-5F1, and a classification name of mouse hybridoma monoclonal cell line; the hybridoma cell line secreting 8E12 antibody (recorded as 8E12 cell line) has a deposit number of GDMCC No: 65043, a deposit date of August 22, 2024, a deposit name of mouse hybridoma monoclonal cell line hSARS-CoV-2-ORF8-m-mAb-8E12, and a classification name of mouse hybridoma monoclonal cell line.

[0095] 5. Expression of monoclonal antibodies

[0096] The 5F1 cell line and the 8E12 cell line were cultured adherently using the HybriSFM-P1B culture medium (Opumai, H081801-001) specially used for hybridoma antibody expression. When the cells adapted to the culture medium and began to proliferate, the adherent culture was switched to the suspension culture using a 200 mL suspension culture system at 37°C, 5% CO2, and 110 rpm. After culturing the cells for 4 days, the culture supernatant was collected (both the 5F1 antibody and the 8E12 antibody were secreted into the supernatant).

[0097] 6. Purification of monoclonal antibodies

[0098] The culture supernatant collected in the previous step was centrifuged at 4°C and 1000 rpm for 20 min, and the supernatant was taken. The supernatant was centrifuged again at 4°C and 8000 rpm for 40 min, and the supernatant was taken and filtered through a 0.45 μm water system membrane. Binding buffer (PH=7.0, containing a final concentration of 20 mM Na2HPO4 and 0.15 M NaCl) was added and mixed, and 50 μL was taken out and recorded as "pre-column supernatant". Then, a Protein G affinity chromatography column (GenScript, L00209) was used for column purification. The supernatant was added to the equilibrated column at 0.25 mL / min for column purification, and the flow-through was collected and recorded as "Flow through". After the column was completed, 10 CV of Wash buffer (same as Binding buffer, i.e., 20 mM Na2HPO4, 0.15 M NaCl, PH=7.0) was used to wash the impurities, and the flow-through was collected and marked as "Washbuffer flow-through". Prepare 10 1.5 mL EP tubes and add 100 μL of neutralization buffer (1 M Tris-HCl, PH=8.5) to each tube; draw 900 μL of elution buffer (0.1 M glycine, PH=2.5), elute through the column, collect the eluate with an EP tube, mix well, and collect 10 tubes of elution buffer in total.

[0099] 7. SDS-PAGE identification of monoclonal antibodies

[0100] Referring to the "SDS-PAGE gel electrophoresis identification" of Example 1, the purification results of 5F1 antibody and 8E12 antibody were identified. The identification results of 5F1 antibody are as follows: Figure 4As shown in A and B in the figure, lanes 6 to 14 of the non-reducing electrophoresis sample have an obvious band at 250 kDa, and lanes 6 to 8 with the most obvious bands have two bands between 100 kDa and 170 kDa, without other miscellaneous bands; lanes 6 to 14 of the reducing electrophoresis sample result have a heavy chain and light chain band at 50 kDa and 25 kDa respectively, without other miscellaneous bands. 8E12 antibody has similar identification results. The above results show that 5F1 antibody and 8E12 antibody have been successfully expressed and purified.

[0101] 8. Activity identification of monoclonal antibodies

[0102] The activity of the purified monoclonal antibody was identified by indirect ELISA method. The specific operation was as follows: the purified novel coronavirus ORF8 protein prepared in Example 1 was diluted to 1.00×10 5 pg / mL, the purified 5F1 antibody and 8E12 antibody obtained in the previous step were diluted to 1.67 μg / mL with blocking solution, and the detection method of "2. Immunotiter determination" of this example was used for determination. The results are as follows Figure 5 As shown, both 5F1 antibody and 8E12 antibody can effectively bind to the novel coronavirus ORF8 protein and are specific antibodies against the novel coronavirus ORF8 protein.

[0103] Example 3 Establishment of double antibody sandwich ELISA method

[0104] 1. Clarify the method of antibody combination

[0105] The 5F1 antibody and the 8E12 antibody were labeled with biotin (Thermo, 21336) to obtain biotin-5F1 antibody and biotin-8E12 antibody, respectively.

[0106] Two antibody pair combinations were established: antibody pair combination 1: 5F1 antibody was used as the coating antibody and biotin-8E12 antibody was used as the detection antibody; antibody pair combination 2: 8E12 was used as the coating antibody and biotin-5F1 antibody was used as the detection antibody. Double antibody sandwich ELISA was performed on antibody pair combinations 1 and 2 respectively to compare the differences in their effects. The steps are as follows:

[0107] The coating antibody was diluted to 1.00 μg / mL with carbonate coating buffer at pH 9.6, and 100 μL was added to each well of the ELISA plate for overnight coating at 4°C; 300 μL PBST (containing 0.05 v / v% TWEEN-20) was added to each well and washed 3 times; 300 μL blocking solution (PBS containing 2 w / v% BSA and 3 w / v% skim milk powder) was added to each well and blocked at 37°C for 1 h; 300 μL PBST was added to each well and washed 3 times; the novel coronavirus ORF8 protein prepared in Example 1 was diluted to 1.00×10 4 pg / mL, 5.00×10 3 pg / mL, 2.50×10 3 pg / mL, 1.25×10 3 pg / mL, 625 pg / mL, 313 pg / mL, 156 pg / mL, 78.1 pg / mL, 39.1 pg / mL or 19.5 pg / mL, and a blank control well without antigen was set up. Three replicate wells were set up for each concentration gradient, and 100 μL was added to the corresponding well in each well, and incubated at 37°C for 1.5 h; 300 μL PBST was added to each well and washed three times; the detection antibody was diluted to 0.500 μg / mL with blocking solution, and 100 μL was added to each well of the ELISA plate, and incubated at 37°C for 1 h; 300 μL PBST was added to each well and washed three times; SA-HRP (GenScript, M00091) was diluted with blocking solution at 1:5000, and 100 μL was added to each well of the ELISA plate, and incubated at 37°C for 1 h; 300 μL Wash five times with PBST; add 100 μL of TMB colorimetric solution to each well and incubate at 37°C for 15 min; add 50 μL / well of 2 M H2SO4 to terminate the colorimetric reaction and quickly place on a microplate reader for detection, setting the detection wavelength to 450 nm.

[0108] The results are as follows Figure 6 As shown, the linear equation of antibody pair combination 1 is Y = 0.5545*X - 1.875, R 2 is 0.9925. According to the literature such as "Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis", according to the formula LOD = Mean blank +2 *SD blankThe calculated lower limit of detection (LOD) was 16.7 pg / mL, and the linear range was 16.7 pg / mL to 1.00×10 4 pg / mL; the linear equation for antibody pair combination 2 is Y = 0.5040*X - 1.732, R 2 The calculated detection limit was 18.3 pg / mL, and the linear range was 18.3 pg / mL to 1.00×10 4 pg / mL. The detection effects of the two antibodies in combination 1 and 2 were comparable, and both were able to effectively detect the novel coronavirus ORF8 protein.

[0109] 2. Determination of the working concentration of the antibody pair combination

[0110] Antibody pair combination 1 was used to further establish a double antibody sandwich ELISA method for detecting the new coronavirus, and the working concentration of the antibody pair was determined. The specific operations are as follows:

[0111] The coating antibody was diluted to 2.50 μg / mL, 1.00 μg / mL or 0.100 μg / mL with carbonate coating buffer at pH 9.6, and 100 μL was added to each well of the ELISA plate for overnight coating at 4°C; 300 μL PBST (containing 0.05 v / v% TWEEN-20) was added to each well and washed 3 times; 300 μL blocking solution (PBS containing 2 w / v% BSA and 3 w / v% skim milk powder) was added to each well and blocked at 37°C for 1 h; 300 μL PBST was added to each well and washed 3 times; the novel coronavirus ORF8 protein prepared in Example 1 was diluted to 1.00×10 6 pg / mL (recorded as strong positive antigen solution) or 0.200×10 3 pg / mL (recorded as weak positive antigen solution), 1.00×10 6 pg / mL of strong positive antigen solution, add 0.200×10 3pg / mL weak positive antigen solution, add 100 μL of negative blocking solution to columns 7-9, incubate at 37℃ for 1.5 h; add 300 μL PBST to each well and wash three times; dilute the detection antibody to 1.00 μg / mL, 0.500 μg / mL or 0.100 μg / mL with blocking solution, add 100 μL to each well in three rows with different coating concentrations, incubate at 37℃ for 1 h; add 300 μL PBST to each well and wash three times; dilute SA-HRP (GenScript, M00091) with blocking solution at 1:5000, add 100 μL / well to the ELISA plate, and incubate at 37℃ for 1 h; add 300 μL PBST to each well and wash five times; add 100 μL TMB colorimetric solution to each well and incubate at 37℃ for 15 min; add 2 M The color development reaction was terminated with H2SO4 and the sample was quickly placed on an enzyme reader for detection, with the detection wavelength set at 450 nm.

[0112] The results are as follows Figure 7 As shown, strong positive antigen (1.00×10 6 pg / mL) and weak positive antigen (0.200×10 3 pg / mL), when the concentration of the detection antibody was the same, the detection sensitivity of the coating antibody (5F1 antibody) was equivalent when the concentration was 2.50 μg / mL and 1.00 μg / mL, while the detection sensitivity was reduced when the concentration was 0.100 μg / mL. Therefore, under the principle of saving the most antibody dosage while ensuring that the detection sensitivity was not affected, the optimal coating concentration of the coating antibody was 1.00 μg / mL; when the concentration of the coating antibody was 1.00 μg / mL, the detection sensitivity of the detection antibody (biotin-8E12 antibody) was equivalent when the concentration was 1.00 μg / mL and 0.500 μg / mL, while the detection sensitivity was reduced when the concentration was 0.100 μg / mL. Therefore, the optimal detection concentration of the detection antibody was 0.500 μg / mL.

[0113] 3. Performance evaluation of double antibody sandwich ELISA

[0114] (1) Sensitivity evaluation

[0115] The sensitivity of the double antibody sandwich ELISA method for detecting the new coronavirus established using antibody pair combination 1 was evaluated.

[0116] Double antibody sandwich ELISA test using PBS as the matrix: dilute the coating antibody to 1.00 μg / mL with carbonate coating buffer at pH = 9.6, and dilute the novel coronavirus ORF8 protein prepared in Example 1 to 1.00×10 4 pg / mL, 5.00×10 3pg / mL, 2.50×10 3 pg / mL, 1.25×10 3 pg / mL, 625 pg / mL, 313 pg / mL, 156 pg / mL, 78.1 pg / mL, 39.1 pg / mL or 19.5 pg / mL, dilute the detection antibody to 0.500 μg / mL with blocking solution, dilute SA-HRP (GenScript, M00091) with blocking solution at 1:5000, set up blank control wells without adding antigen, set up 3 replicate wells for each concentration gradient, set up 15 replicate wells for blank control wells, and perform detection according to the operating steps of the double antibody sandwich ELISA method of this embodiment.

[0117] Since the clinical test specimens are serum or plasma, in order to detect whether the proteins normally contained in the serum will non-specifically bind to the antibody pair and further determine the effectiveness of the detection method for clinical samples, the plasma of healthy volunteers was used as the matrix for double antibody sandwich ELISA detection: the plasma of healthy people (not infected with the new coronavirus) was diluted with blocking solution at a volume ratio of 1:10 to obtain plasma diluent; the blocking solution was replaced with the plasma diluent to dilute the new coronavirus ORF8 protein prepared in Example 1 to 1.00×10 4 pg / mL, 5.00×10 3 pg / mL, 2.50×10 3 pg / mL, 1.25×10 3 pg / mL, 625 pg / mL, 313 pg / mL, 156 pg / mL, 78.1 pg / mL, 39.1 pg / mL or 19.5 pg / mL, a blank control well without antigen was set up, 3 replicate wells were set up for each concentration gradient, 15 replicate wells were set up for the blank control wells, and the detection was performed according to the operating steps of the double antibody sandwich ELISA method using PBS as the matrix in this example.

[0118] According to the test results of the above two matrices, the concentration of novel coronavirus ORF8 protein was used as the horizontal axis and the OD 450 The absorbance is the ordinate, and the linear curve is fitted. Refer to the literature such as "Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis" and use the formula LOD= Mean blank +2 *SD blankThe sensitivity (i.e., the lower limit of detection (LOD)) was calculated based on the linear regression equation and coefficient of determination (R) using GraphPad Prism 8 software. 2 ), to determine the linear range of the detection method.

[0119] The results are as follows Figure 8 and Fig. 9 As shown in Table 2, the detection limit of the PBS matrix is ​​as low as 16.7 pg / mL, and the detection limit of the plasma matrix is ​​slightly increased but still can reach 28.4 pg / mL. This shows that the double antibody sandwich ELISA method established in this example using 5F1 antibody and biotin-8E12 antibody as antibody pair combination 1 has high sensitivity for the detection of the novel coronavirus ORF8 protein, a wide linear range of detection, high specificity, and little effect of complex components in plasma on the detection results.

[0120] Table 2 Detection performance of double antibody sandwich ELISA for ORF8 antigen in PBS and plasma

[0121]

[0122] (2) Specificity evaluation

[0123] In order to detect whether the antibody pairs of 5F1 antibody and 8E12 antibody non-specifically bind to other proteins with similar structures to the novel coronavirus ORF8 protein, another common novel coronavirus auxiliary protein ORF7a protein (ELYHYQECVRGTTVLLKEPCSSGTYEGNSPFHPLADNKFALTCFSTQFAFACPDGVKHVYQLRARSVSPKLFIRQEEVQELYSPIFLIVAAIVFITLCFTLKRKTE (SEQ ID NO.2)) and the novel coronavirus ORF8 protein homolog SARS virus ORF8b protein (MCLKILVRYNTRGNTYSTAWLCALGKVLPFHRWHTMVQTCTPNVTINCQDPAGGALIARCWYLHEGHQTAAFRDVLVVLNKRTN (SEQ ID NO.3)) were used as test antigens for comparative experiments. The novel coronavirus ORF7a protein and the SARS virus ORF8b protein were prepared and purified according to the method in Example 1.

[0124] The novel coronavirus ORF8 protein, novel coronavirus ORF7a protein and SARS virus ORF8b protein were diluted to 1.00×10 4pg / mL, and the double antibody sandwich ELISA method of this example was used for detection, and the results were as follows Fig.10 As shown, except for the novel coronavirus ORF8 protein, the novel coronavirus ORF7a protein and the SARS virus ORF8b protein had no effective absorbance detected. This indicates that the 5F1 antibody and the 8E12 antibody in this example have significant specific binding effects on the novel coronavirus ORF8 protein, and there is no non-specific binding with the other two proteins.

[0125] Example 4 Detection of novel coronavirus pseudovirus infection supernatant by double antibody sandwich ELISA

[0126] HEK-293 human embryonic kidney cells were infected with a pseudovirus of a novel coronavirus without replication ability (prepared with reference to the prior art "PACT inhibits theplication of SARS-CoV-2 through the blockage of GSK-3β-N-nsp3 cascade" (DOI: 10.1002 / jmv.28832)). The culture supernatant was collected 24 hours after infection to obtain the infection supernatant, and the infection supernatant was gradient diluted with a blocking solution, and 3 concentration gradients of 1:1, 1:3, and 1:9 were set, and 3 duplicate wells were set for each gradient. At the same time, the supernatant of uninfected HEK-293 cells was used as a control. The 5F1 antibody purified in Example 2 was used as the coating antibody, and the biotin-8E12 antibody prepared in Example 2 was used as the detection antibody. The detection was performed according to the operating steps of the double antibody sandwich ELISA method using PBS as the matrix in Example 3.

[0127] like Fig.11 As shown, the result of detecting ORF8 in the supernatant of cells infected with pseudovirus was substituted into the linear equation of the double antibody sandwich ELISA detection method with PBS matrix to calculate the concentration of ORF8 in the supernatant of infected cells to be 525 pg / mL. This indicates that the double antibody sandwich ELISA method established by the present invention can effectively detect the novel coronavirus ORF8 protein secreted in the supernatant of cells infected with pseudovirus.

[0128] Example 5 Flow cytometry detection of novel coronavirus ORF8 protein

[0129] After the magnetic beads (Biotech, ABL-1-71) were coated with the 5F1 antibody prepared in Example 2, different concentrations of the novel coronavirus ORF8 protein prepared in Example 1 were added for incubation (1.00×10 5 pg / mL, 3.33×10 4 pg / mL, 1.11×10 4 pg / mL, 3.70×10 3pg / mL, 1.23×10 3 pg / mL, 411 pg / mL, 137 pg / mL, 45.7 pg / mL, 15.2 pg / mL, 5.08 pg / mL, 1.69 pg / mL, 0.564 pg / mL, 0.188 pg / mL, a total of 13 concentrations), and a blank control of 0 pg / mL was set; the incubated magnetic beads were then incubated with the biotin-8E12 antibody prepared in Example 2 to form a 5F1 antibody-coated magnetic bead-ORF8-biotin-labeled 8E12 antibody sandwich complex, and finally APC-labeled streptavidin (BD Pharmingen, 554067) was added, and after binding to biotin, the fluorescence intensity was obtained by flow cytometry. Three replicates were set for each concentration gradient, and 15 replicates were set for the blank control wells.

[0130] According to the detection results of fluorescence intensity, the concentration of novel coronavirus ORF8 protein was used as the horizontal axis and the mean fluorescence intensity MFI was used as the vertical axis. The fitting curve was obtained using GraphPad Prism 8 software. The coefficient of determination (R 2 ) is as high as 0.9989. The results are as follows Fig.12 As shown in FIG. 1 , the minimum detection limit of the sandwich flow cytometry established with the 5F1 antibody and the 8E12 antibody of the present invention is 0.202 pg / ml, and the detection range is 0.202 pg / mL to 1.00×10 5 pg / mL, which is significantly better than the detection effect of the flow cytometry method in the existing technology "SARS-CoV-2 ORF8 is a viral cytokine regulating immune responses" (DOI: 10.1093 / intimm / dxac044).

[0131] Example 6 Comparative experiment with commercial antibodies

[0132] 1. Comparison of antibody detection performance alone

[0133] The indirect ELISA method was used to compare the individual detection performance of the 5F1 antibody and the 8E12 antibody prepared in Example 2 with three commercial anti-novel coronavirus ORF8 protein antibodies (mouse monoclonal antibody R&D (MAB10820), rabbit monoclonal antibody Abcam (ab283914), rabbit polyclonal antibody GeneTex (GTX135591)), and the operation was as follows:

[0134] Dilute the novel coronavirus ORF8 protein to 1.00 × 10 6pg / mL, 100 μL, overnight coating at 4℃; wash the plate 3 times with PBST; add 300 μL blocking solution and block at 37℃ for 1 h; wash the plate 3 times with PBST; dilute the three commercial antibodies and 5F1 antibody and 8E12 antibody with blocking solution to 1.00×10 3 ng / mL, and then 3-fold concentration gradient dilution was performed, with a total of 11 concentration gradients, namely 1.00×10 3 ng / mL, 333 ng / mL, 111 ng / mL, 37.0 ng / mL, 12.3 ng / mL, 4.11 ng / mL, 1.37 ng / mL, 0.457 ng / mL, 0.152 ng / mL, 0.0508 ng / mL, 0.0169 ng / mL, each concentration was repeated 3 times, 100 μL / well was added to the corresponding well, and 3 blank wells with antibody concentration of 0 were set up, incubated at 37℃ for 1.5 h; the plate was washed 3 times with PBST; anti-mouse-IgG-HRP (Sigma, A9044) or anti-rabbit-IgG-HRP (Sigma, AP307P) was diluted 1:2000 with blocking solution, 100 μL / well was added to the ELISA plate, and incubated at 37℃ for 1 h; the plate was washed 5 times with PBST; TMB colorimetric solution was added at 100 μL / well per well. μL was added and incubated at 37°C for 15 min; 2 M H2SO4 was added at 50 μL / well to terminate the color reaction, and the plate was quickly placed on an enzyme reader for detection, with the detection wavelength set to 450 nm.

[0135] The results are as follows Fig.13 As shown in Table 3, the 5F1 antibody and the 8E12 antibody were comparable to the commercial rabbit monoclonal antibody Abcam (ab283914). 50 The difference is small and the difference is at the same order of magnitude, and it is significantly better than the commercial mouse monoclonal antibody R&D (MAB10820) and rabbit polyclonal antibody GeneTex (GTX135591). Among them, the reason why the mouse monoclonal antibody R&D (MAB10820) cannot recognize ORF8 protein may be that the instruction manual of the antibody shows that this antibody is only suitable for Western Blot, Immunohistochemistry and Immunocytochemistry, and is not suitable for ELISA detection method.

[0136] Table 3 EC corresponding to different antibodies in indirect ELISA test 50

[0137]

[0138] 2. Combined detection performance

[0139] Two more antibody pair combinations were established using the rabbit monoclonal antibody Abcam (ab283914) and the biotin-5F1 antibody and biotin-8E12 antibody prepared in Example 3. Antibody combination 3: Abcam (ab283914) antibody was used as the coating antibody and biotin-8E12 antibody was used as the detection antibody. Antibody pair combination 4: Abcam (ab283914) antibody was used as the coating antibody and biotin-5F1 antibody was used as the detection antibody. Referring to the double antibody sandwich ELISA method established in Example 3, the detection performance of antibody combination 1, antibody combination 3 and antibody combination 4 were compared, wherein the working concentration of the coating antibody was 1.00 μg / mL, the working concentration of the detection antibody was 1.00 μg / mL, and the novel coronavirus ORF8 protein was set at 1.25×10 3 pg / mL, 625 pg / mL, 313 pg / mL, 156 pg / mL, 78.1 pg / mL, 39.1 pg / mL or 19.5 pg / mL, a total of 7 concentration gradients.

[0140] The results are as follows Fig.14 As shown, when the concentration of the new coronavirus ORF8 protein is low, the detection effect of antibody pair combination 1 is significantly better than that of antibody pair combination 3 and antibody pair combination 4.

[0141] The above results show that the 5F1 antibody and 8E12 antibody of the present invention, when used alone or in combination, have better detection performance than most existing novel coronavirus ORF8 protein detection antibodies.

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A hybridoma cell line secreting monoclonal antibodies against the novel coronavirus ORF8 protein, characterized in that: The following paired hybridoma cell lines (1) to (2): The deposit number of the hybridoma cell line (1) is GDMCC No: 65043, and it was deposited in Guangdong Microbiological Culture Collection Center on August 22, 2024; The deposit number of the hybridoma cell line (2) is GDMCC No: 65206, and it was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 25, 2024.

2. Use of the hybridoma cell line according to claim 1 in preparing paired antibodies against the novel coronavirus ORF8 protein.

3. Use of the hybridoma cell line according to claim 1 in the preparation of a product for detecting the novel coronavirus ORF8 protein.

4. A paired monoclonal antibody against the novel coronavirus ORF8 protein, characterized in that: The hybridoma cell line according to claim 1 is secreted.

5. Use of the paired monoclonal antibody according to claim 4 in the preparation of a product for detecting the novel coronavirus ORF8 protein.

6. An antibody composition for detecting novel coronavirus ORF8 protein, comprising a coating antibody and a detection antibody, characterized in that: Any one of the following antibody compositions (1) to (2): The coating antibody of the antibody composition (1) is a monoclonal antibody secreted by the hybridoma cell line (2) in claim 1, and the detection antibody is a monoclonal antibody secreted by the hybridoma cell line (1) in claim 1; The coating antibody of the antibody composition (2) is a monoclonal antibody secreted by the hybridoma cell line (1) in claim 1, and the detection antibody is a monoclonal antibody secreted by the hybridoma cell line (2) in claim 1.

7. Use of the antibody composition according to claim 6 in the preparation of a product for detecting the novel coronavirus ORF8 protein.

8. An immunoassay method for detecting novel coronavirus ORF8 protein for non-disease treatment and diagnosis purposes, characterized in that: The paired monoclonal antibodies according to claim 4 or the antibody composition according to claim 6 are used to perform immunoassay on the sample to be tested.

9. A kit for detecting novel coronavirus ORF8 protein, characterized in that: It comprises the hybridoma cell line according to claim 1, the paired monoclonal antibody according to claim 4 or the antibody composition according to claim 6.

10. The kit according to claim 9, characterized in that Also includes ELISA reagents or flow cytometry reagents.

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