A sars-cov-2 antibody and uses thereof

Abstract

The present disclosure relates to a SARS-Cov-2 antibody and application thereof. The antibody provided by the present disclosure has high sensitivity and good specificity, has no cross-reaction, and has an important role in the diagnosis of SARS-Cov-2 virus.

Description

[0001] Cross-references to related applications

[0002] This disclosure claims priority to Chinese Patent Application No. CN202111063235.1, filed on September 10, 2021, entitled “A SARS-CoV-2 antibody or an antigen-binding fragment thereof and its application”, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure pertains to the field of protein detection. More specifically, it relates to a SARS-CoV-2 antibody or its antigen-binding fragment and its applications. Background Technology

[0004] Coronaviruses are enveloped, single-stranded, positive-sense RNA viruses. Based on their genetic evolution, serological characteristics, and host specificity, coronaviruses are classified into four genera: α-coronaviruses, β-coronaviruses, γ-coronaviruses, and δ-coronaviruses. β-coronaviruses can be further divided into four groups: A, B, C, and D. α- and β-coronaviruses primarily infect mammals, including humans, livestock, and pets, while γ- and δ-coronaviruses primarily infect birds and other mammals. SARS-CoV-2 belongs to the β-coronavirus genus. From an evolutionary perspective, 2019-nCoV (i.e., 2019SARS-CoV-2, also known as "SARS-CoV-2") is adjacent to SARS and SARS-like viruses, sharing an outgroup of HKU9-1 coronavirus, which parasitizes fruit bats. 2019-nCoV is enveloped, with particles that are round or oval, often pleomorphic, and have a diameter of 50-200 nm.

[0005] Currently, there is a greater global demand for reagents used in screening, detecting, and diagnosing SARS-CoV-2, with higher sensitivity, specificity, and the absence of cross-reactivity. Summary of the Invention

[0006] The SARS-CoV-2 nucleocapsid protein is 419 amino acids in length, and its sequence is well known, for example, see GenBank:UGZ64655.1.

[0007] This disclosure provides an antibody that binds to amino acid fragments 44-180 of the SARS-CoV-2 nucleocapsid protein.

[0008] Furthermore, the antibody does not bind to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of the SARS-CoV-2 nucleocapsid protein.

[0009] Furthermore, the antibody binds to an epitope that is the same as the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, or the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6.

[0010] This disclosure provides an antibody combination comprising antibody 1 and antibody 2, wherein antibody 1 binds to an epitope identical to the epitope bound by an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, and antibody 2 binds to an epitope identical to the epitope bound by an antibody comprising the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6.

[0011] This disclosure also provides an antibody combination comprising antibody 3 and antibody 4, wherein antibody 3 is an antibody that binds to amino acid fragments 44-180 of the SARS-CoV-2 nucleocapsid protein but not to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of the SARS-CoV-2 nucleocapsid protein, and antibody 4 is an antibody that binds to amino acid fragments 65-103 of the SARS-CoV-2 nucleocapsid protein.

[0012] Furthermore, antibody 3 binds to an epitope that is the same as the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, or the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6.

[0013] Furthermore, antibody 4 binds to an epitope that is identical to the epitope bound by the antibody containing the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 10.

[0014] This disclosure also provides a kit comprising the antibody, or a combination of the antibodies.

[0015] This disclosure also provides the use of the antibody, or the combination of the antibody, in the preparation kit.

[0016] This disclosure also provides a method for preparing the antibody, the method comprising using a fragment of amino acids 44-180 of the SARS-CoV-2 nucleocapsid protein as an antigen, and preparing the antibody by hybridoma technology or phage display technology.

[0017] This disclosure also provides a method for preparing the antibody, the method comprising: 1) immunizing an animal with an antigen or hapten containing a fragment of amino acids 44-180 of the SARS-CoV-2 nucleocapsid protein; and 2) obtaining an antibody binding to the fragment of amino acids 44-180 of the SARS-CoV-2 nucleocapsid protein from the ascites of the animal.

[0018] In some embodiments, the method for preparing the antibody further includes screening for antibodies that do not bind to the amino acid fragments at positions 44-84, 65-103, 96-136, and 130-180 of the SARS-CoV-2 nucleocapsid protein.

[0019] This disclosure also provides the use of the antibody, or the combination of antibodies, or the kit for detecting SARS-CoV-2.

[0020] This disclosure also provides a method for detecting SARS-CoV-2, comprising: A) Under conditions sufficient for a binding reaction to occur, the antibody, or the antibody combination, or the kit, is contacted with the sample to initiate a binding reaction; and B) Detect the immune complexes produced by the binding reaction.

[0021] This disclosure also provides a method for diagnosing a subject in SARS-CoV-2 infection or SARS-CoV-2-related illness, comprising: A) Under conditions sufficient for a binding reaction to occur, the antibody, or the antibody combination, or the kit is contacted with a sample from the subject to initiate a binding reaction; and B) Detect the immune complexes produced by the binding reaction.

[0022] In some implementations, the SARS-CoV-2 infection-related illnesses include respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, and kidney failure. Detailed Implementation

[0023] SARS-CoV-2 belongs to the Coronaviridae family and is a non-segmented, single-stranded, positive-sense RNA virus. It encodes two large overlapping open reading frames (ORF1a and ORF1b), four structural proteins (S, E, M, and N proteins), and nine accessory proteins. Among these, the N protein is a core component of the viral particle; it binds to the viral genomic RNA, packaging the RNA into a ribonucleoprotein (RNP) complex. Besides assembly, the N protein plays a crucial role in viral mRNA transcription and replication, and is involved in immune regulation. Studies have reported that the N protein can bind to double-stranded RNA and possess RNAi-inhibiting activity, counteracting host RNAi-mediated antiviral responses. Furthermore, the N protein can induce humoral and cellular immune responses after infection, making it a key target for early rapid diagnosis and vaccine development.

[0024] The SARS-CoV-2 N protein is 419 amino acids long and 43-50 kDa in size. The N protein has three relatively conserved domains, one of which can intertwine with the viral genomic RNA to form the viral nucleocapsid, playing a crucial role in viral RNA synthesis and related to viral genome replication and regulation of cellular signaling pathways. The N protein is a phosphorylated protein; phosphorylation regulates its conformation, enhancing its affinity for viral RNA and its interaction with the viral protein. During nucleocapsid packaging, the N protein can interact with the M protein, promoting the packaging of the nucleocapsid into viral particles. High levels of antibodies against the N protein can be produced early in infection, allowing for the development of rapid methods for detecting 2019-nCoV serum antibodies. Therefore, the N protein is frequently used as a diagnostic tool for SARS-CoV-2 and is a core component of rapid immunological diagnostic reagents.

[0025] In conclusion, the detection of 2019-nCoV N protein plays an important role in the diagnosis of SARS-CoV-2 virus, and the development of monoclonal antibodies for the detection of 2019-nCoV N protein is of great significance.

[0026] Some embodiments of this disclosure provide an antibody that binds to amino acid segments 44-180 of the SARS-CoV-2 nucleocapsid protein. In some embodiments, the antibody may also not bind to amino acid segments 44-84, 65-103, 96-136, and 130-180 of the SARS-CoV-2 nucleocapsid protein.

[0027] As used herein, the term "antibody" is used in the broadest sense and can include full-length antibodies and their antigen-binding fragments, including monospecific, bispecific, or multispecific antibodies, as well as chimeric antibodies, provided they exhibit the desired biological activity. The term "antigen-binding fragment" is a substance containing part or all of an antibody's CDR (complementarity-determining region), lacking at least some of the amino acids present in the full-length chain but still capable of specifically binding to an antigen. Such fragments are biologically active because they bind to the antigen and can compete with other antigen-binding molecules (including intact antibodies) for binding to a given epitope. Such fragments are selected from Fab (antigen-binding fragments) (consisting of the complete light chain and Fd), Fv (consisting of VH and VL), scFv (single-chain antibody with a linker peptide connecting VH and VL), or single-domain antibodies (consisting only of VH). Such fragments can be produced using recombinant nucleic acid technology or through enzymatic or chemical cleavage of antigen-binding molecules (including intact antibodies).

[0028] In some embodiments, the antibody binds to an epitope that is the same as the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, or the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6. In some embodiments, the antibody binds to an epitope that is the same as the epitope bound by an antibody containing the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4, or the epitope bound by an antibody containing the heavy chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8.

[0029] In this disclosure, whether an antibody recognizes the same epitope as other antibodies can be confirmed through competition between the two antibodies for the epitope. Competition between antibodies can be evaluated using competitive binding assays, such as ELISA, fluorescence energy transfer assays, or fluorescence micro-assay techniques.

[0030] In some implementations, antibody binding to the amino acid fragment corresponding to the SARS-CoV-2 nucleocapsid protein means that the antibody can bind to the amino acid fragment, but the amino acid fragment is not necessarily the minimum binding fragment.

[0031] In some embodiments, this disclosure provides an antibody combination comprising antibody 1 and antibody 2, wherein antibody 1 binds to an epitope identical to the epitope bound by an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, and antibody 2 binds to an epitope identical to the epitope bound by an antibody comprising the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6.

[0032] In some embodiments, this disclosure provides an antibody combination comprising antibody 1 and antibody 2, wherein antibody 1 binds to an epitope identical to the epitope bound by an antibody comprising the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4, and antibody 2 binds to an epitope identical to the epitope bound by an antibody comprising the heavy chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8.

[0033] In some embodiments, this disclosure also provides an antibody combination comprising antibody 3 and antibody 4.

[0034] In some embodiments, antibody 3 binds to amino acid fragments 44-180 of the SARS-CoV-2 nucleocapsid protein. In some embodiments, antibody 3 may not bind to amino acid fragments 44-84, 65-103, 96-136, and 130-180 of the SARS-CoV-2 nucleocapsid protein. In some embodiments, antibody 3 binds to an epitope that is the same as the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, or the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6; antibody 4 binds to amino acid fragments 65-103 of the SARS-CoV-2 nucleocapsid protein.

[0035] In some embodiments, antibody 4 binds to an epitope that is identical to the epitope bound by an antibody containing the heavy chain variable region of SEQ ID NO: 9 and the light chain variable region of SEQ ID NO: 10. Antibody 4 also binds to an epitope that is identical to the epitope bound by an antibody containing the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 12.

[0036] In some embodiments, this disclosure also provides a kit comprising the antibody, the antibody combination.

[0037] In some embodiments, the effectiveness of the disclosed antibodies, such as binding activity and / or cross-reactivity, can be detected using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc. In some embodiments, the assay may include, for example, ELISA (enzyme-linked immunosorbent assay), FACS (flow cytometry fluorescence sorting) binding assay, Biacore, competitive binding assay, etc. In some embodiments, the reactivity of the disclosed antibodies to antigens (antigen peptides) is characterized, for example, in an ELISA, such as by reading a reaction value ≥0.5 at 405 nm using a peroxidase-labeled ELISA method to determine good reactivity, which can be used for immunoassay.

[0038] In some embodiments, the antibody is optionally labeled with a detectable marker. In some embodiments, the detectable marker includes, for example, colloidal gold, radioactive markers, luminescent substances, colored substances, enzymes such as fluorescent markers, chromophore markers, electron-dense markers such as radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / antibiotin protein, and spin-labeled markers.

[0039] In some embodiments, this disclosure also provides the use of the antibody, or the antibody combination, in the preparation kit.

[0040] In some embodiments, this disclosure also provides a method for preparing the antibody.

[0041] The antibody was prepared using amino acid fragments 44-180 of the SARS-CoV-2 nucleocapsid protein as an antigen via hybridoma technology or phage display technology.

[0042] This disclosure provides a method for preparing the antibody, which is a monoclonal antibody, and the method includes preparing the monoclonal antibody using a fragment of amino acids 44-180 of the SARS-CoV-2 nucleocapsid protein as an antigen.

[0043] Some embodiments of this disclosure also provide a method for preparing the antibody, the method comprising: 1) immunizing an animal with an antigen or hapten containing a fragment of amino acids 44-180 of the SARS-CoV-2 nucleocapsid protein; and 2) obtaining an antibody binding to the fragment of amino acids 44-180 of the SARS-CoV-2 nucleocapsid protein from the ascites of the animal.

[0044] Some embodiments of this disclosure also provide the use of the antibody, or the antibody combination, or the kit for detecting SARS-CoV-2.

[0045] Some embodiments of this disclosure also provide a method for detecting SARS-CoV-2, including: A) Under conditions sufficient for a binding reaction to occur, the antibody, or the antibody combination, or the kit, is contacted with the sample to initiate a binding reaction; and B) Detect the immune complexes produced by the binding reaction.

[0046] Some embodiments of this disclosure also provide a method for diagnosing whether a subject is infected with SARS-CoV-2 or has a disease related to SARS-CoV-2 infection, including: A) Under conditions sufficient for a binding reaction to occur, the antibody, or the antibody combination, or the kit is contacted with a sample from the subject to initiate a binding reaction; and B) Detect the immune complexes produced by the binding reaction.

[0047] In some implementations, the SARS-CoV-2 infection-related illness includes at least one of respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, and renal failure.

[0048] The antibodies disclosed herein have high sensitivity, good specificity, and no cross-reactivity, and play an important role in the diagnosis of SARS-CoV-2 virus.

[0049] Example The implementation schemes of this disclosure will now be described in detail with reference to the embodiments.

[0050] Example 1: Preparation of SARS-CoV-2N protein monoclonal antibody 1. Immunized animals BALB / c mice aged 8–12 weeks, from the same line as myeloma cells, were injected intraperitoneally with a mixture of 100 μg / mouse of 2019-nCoV N protein recombinant antigen and an equal volume of Freund's complete adjuvant. Every two weeks, 100 μg / mouse of 2019-nCoV N protein recombinant antigen and an equal volume of Freund's incomplete adjuvant (FIA) were injected intraperitoneally multiple times to boost immunization. Mice with serum titers above 1:2000 (indirect ELISA) were eligible for fusion. Three days prior to fusion, mice were given a second intraperitoneal booster immunization at a dose of 50 μg / mouse.

[0051] 2. Preparation of feeder cells BALB / c mouse peritoneal macrophages were used as feeder cells. One day before fusion, BALB / c mice were euthanized by cervical retraction, immersed in 75% alcohol, and under aseptic conditions in a laminar flow hood, the abdominal skin was cut open with scissors to expose the peritoneum. 5 mL of RPMI 1640 basal culture medium was injected into the peritoneal cavity using a syringe. The cells were repeatedly rinsed, and the rinsing fluid was collected. The cells were centrifuged at 1000 rpm for 5 minutes, and the pellet was resuspended in RPMI 1640 selection medium (RPMI 1640 complete culture medium containing HAT). The cell concentration was adjusted to 1 × 10⁻⁶ cells / mL. 5 Cells / mL, add 150 μL / well to a 96-well plate, and incubate overnight at 37°C with 5% CO2.

[0052] 3. Preparation of immune spleen cells Three days after the last immunization of mice, the spleen was removed under sterile conditions, placed in a petri dish, rinsed once with RPMI 1640 basal culture medium, and then ground and filtered through a nylon mesh in a small beaker to prepare a cell suspension. The suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in RPMI 1640 basal culture medium. This process was repeated three times, and the cells were counted.

[0053] 4. Cell fusion (1) Take 40 mL of HAT culture medium, 15 mL of DMEM serum-free culture medium and 1 mL of 50% PEG (M12000) and place them in a 37℃ water bath for preheating; (2) Take mouse myeloma cells Sp2 / 0 (preserved by Feipeng Biotechnology Co., Ltd.) (2-5 × 10⁻⁶) 7 ), the above immune spleen cells (10 8 Add the suspension to a 50mL centrifuge tube and mix well. Add DMEM serum-free culture medium to a final volume of 40mL. Centrifuge for 10 minutes, discard the supernatant, and mix well. (3) Place the centrifuge tube in pre-warmed water at 37°C, take 0.7 mL of pre-warmed 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of pre-warmed serum-free culture medium at 37°C; (4) Add DMEM serum-free culture medium to 40 mL, centrifuge for 10 minutes, and discard the supernatant. Add 40 mL of HAT culture medium containing 15%–20% (v) fetal bovine serum. Mix well with a pipette and add 2 drops to each of the 4 wells of a 96-well cell culture plate containing feeder cells. Incubate at 37°C and 7% CO2.

[0054] 5. Selective culture of hybridoma cells Immunized mouse spleen cells and mouse myeloma cells, after PEG treatment, form a mixture of various cellular components, including unfused myeloma cells and immune spleen cells; conjugates of myeloma cells and immune spleen cells; and heterokaryotic bodies of myeloma cells and immune spleen cells. Only the latter can form hybridoma cells. Therefore, it is necessary to remove unfused cells and conjugate bodies from this mixture and select for true hybrid cells. Thus, the cells are cultured in HAT medium as described above on days 1, 3, 5, and 7 after fusion.

[0055] 6. Detection of specific antibodies and cloning of hybridoma cells Supernatant was collected from each culture well, and the wells containing antibodies specifically recognizing the 2019-nCoV N protein recombinant antigen were identified using indirect ELISA. Cross-reactivity of the cell culture supernatant was assessed using indirect ELISA. 96-well plates were coated with the 2019-nCoV N protein recombinant antigen, blocked, and incubated with hybridoma cell culture supernatant. Secondary antibody was added, and the reaction value at 405 nm was measured. Cell lines with higher reaction values ​​(above 0.5) were selected to prepare antibodies for the next round of screening experiments. The following antibodies with good reactivity were obtained through screening.

[0056] Example 2: Identification of antibody-binding fragments Different fragments of the 2019-nCoV N protein recombinant antigen were used to coat microwells. The monoclonal antibodies were diluted to a primary antibody concentration of 1 μg / ml using PBS + 20% NBS (N-bromosuccinimide). Goat anti-mouse IgG-HRP (horseradish peroxidase) was used as the secondary antibody. The monoclonal antibody fragments were determined based on the responses of each antibody to different antigens. Statistical analysis revealed that the selected antibodies targeted the following fragments: Table 1

[0057] The responses of eleven antibodies that bind to the 44-180aa fragment to the fragment are summarized in Table 2: Table 2

[0058] Table 2 shows reactivity, i.e., OD. 405 Reaction value.

[0059] Example 3: Pairing and Screening The above antibodies were used for coating and labeling, respectively, and the experimental procedure is as follows: 1. SARS-CoV-2N antibody labeling: Take 5 ml of colloidal gold with a concentration of 4 / 10,000, add 30-40 μl of 0.2 M K2CO3, stir for 5 min, add SARS-CoV-2N labeled antibody (antibody volume = 50 μg / antibody concentration), stir for 5 min, then add 50 μl of 10% BSA (bovine serum albumin) for blocking and termination labeling; centrifuge at 10,000 rpm for 7 min, remove the supernatant, reconstitute the precipitate with gold reconstitution solution, and finally adjust the volume to 0.5 ml (i.e., 1 / 10 of the colloidal gold solution volume) with gold reconstitution solution.

[0060] 2. Preparation of gold working solution: Prepare gold working solution by diluting the concentrated gold of SARS-Cov-2N labeled antibody with gold reconstitution solution at a ratio of 20%, and spread it on glass fiber.

[0061] 3. Prepare dried gold: Place the spread gold in a freeze dryer for freeze drying (1-2 hours) or dry it overnight in a 37°C drying room.

[0062] 4. SARS-Cov-2N antibody coating: Assemble the nitrocellulose membrane and PVC base plate for later use; dilute the SARS-Cov-2N coating antibody to 1.0-1.5 mg / ml, and use a gold spraying membrane spectrometer to evenly draw lines on the NC membrane, and place it in a 37℃ incubator for 60 min.

[0063] 5. Preparation of gold bar: Cut the gold bar into strips of the required width using a strip cutter, assemble them, and then add samples for testing.

[0064] 6. Testing (1) Quality control material: 2019-nCoV N protein recombinant antigen, diluted to 1 ng / ml with PBS for preliminary antibody pairing screening; (2) Detection method: According to the color card comparison, the color reading value is judged by visual inspection.

[0065] 7. Results (Each segment shows the detection result of one antibody): Table 3-1 Detection results of 3B8 as coating antibody

[0066] Table 3-2 Detection results of 5C3 as coating antibody

[0067] Table 3-3 Detection results of 4B7 as coating antibody

[0068] Table 3-4 Detection results of 1B5 as a coating antibody

[0069] Table 3-5 Detection results of 4C6 as coating antibody

[0070] In Tables 3-1, 3-2, 3-3, 3-4, and 3-5, the letter B represents no color development (not detected), and the number after the letter C represents color development. The larger the number, the weaker the color development (the lower the activity).

[0071] The test results showed that the antibody binding to the 44-180aa segment exhibited high activity in detection performance, so further fragment identification of the antibody in this segment was carried out.

[0072] Example 4: Further Fragment Identification of Dominant Antibody The 44-180aa fragment of the 2019-nCoV N protein recombinant antigen was expressed as a small fragment. Different fragments of the 2019-nCoV N protein recombinant antigen were used to coat microwells. Monoclonal antibodies were diluted to a primary antibody concentration of 1 μg / ml using PBS + 20% NBS as dilution buffer. Goat anti-mouse IgG-HRP was used as the secondary antibody. The specific monoclonal antibody fragment was determined based on the response of each antibody to different antigens. The detailed data are as follows: Table 4-1

[0073] Table 4-2

[0074] Based on the results above, the antibodies 9D2, 7R1, and 5S7 further recognized and bound 65-103 aa, while the other antibodies only recognized 44-180 aa.

[0075] Example 5 further validates the pairing performance of the dominant antibody. For the preparation process of antibody labeling, coating and assembly, please refer to steps 1-5 of Example 3.

[0076] Detection (1) Quality control materials: 2019-nCoV N protein recombinant antigen, diluted with PBS to 25ng / ml, 2ng / ml, 500pg / ml, 100pg / ml, 25pg / ml and 10pg / ml; (2) Detection method: According to the color card comparison, the color reading value is judged by visual inspection.

[0077] result 1. Activity: The following examples show that the recombinant antigen expressed in Escherichia coli is clearly detectable at 100 pg / ml (C7+ color development) and at 25 pg / ml (C8 or C8+ color development), with a detection limit as low as 10 pg / ml.

[0078] Table 5

[0079] 2. Specificity: The three sample pairs described above were used to perform colloidal gold staining tests on 300 nasal swabs, 300 pharyngeal swabs, and 300 saliva samples collected from normal individuals, respectively, and the specificity of each test was 100%.

[0080] 3. Cross-reaction: Multiple pathogens, including adenovirus, cytomegalovirus, Epstein-Barr virus, measles virus, mumps virus, mycoplasma pneumoniae, parainfluenza virus, respiratory and cytotoxic viruses, rotavirus, varicella-zoster virus, and influenza A / B virus, were diluted to different concentrations and tested in pairs with the three examples mentioned above. No cross-reactivity was found.

[0081] 4. Pairing: By swapping the labeled antibody and the coated antibody in the three example pairings described above, the pairing performance remained comparable.

[0082] Among them, 5C3, 1E5, and 7R1 were sequenced, as follows.

[0083] 5C3 Heavy chain variable region SEQ NO.1: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSALLRSYFDYWGQGTTLTVSS. Light chain variable region SEQ NO.2: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWCQQKPGQSPKLLIYWASTRHTGVPDRFTGIRSGTDYTLTISSVQAEDLALYYCQQHYSTPLTFGAGTKLELKR. Heavy chain SEQ NO. 3: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSALLRSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK. Light chain SEQ NO. 4: DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWCQQKPGQSPKLLIYWASTRHTGVPDRFTGIRSGTDYTLTISSVQAEDLALYYCQQHYSTPLTFGAGTKLELKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC. 1E5 Heavy chain variable region SEQ NO. 5: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSS. Light chain variable region SEQ NO. 6: DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK. Heavy chain SEQ ID NO.7: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG. Light chain SEQ ID NO.8: DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC 7R1 Heavy chain variable region SEQ ID NO.9: DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSGSNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSS. Light chain variable region SEQ NO.10: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK. Heavy chain SEQ NO.11: DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSGSNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK. Light chain SEQ NO.12: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC. 5. Verification of detection performance of antibodies that bind to the same epitope as 5C3, 1E5 or 7R1 Single-point mutations were performed on the variable region of the 5C3 heavy chain to obtain variant 5C3-1, which binds to the same epitope as 5C3; single-point mutations were performed on the variable regions of the heavy and light chains of 1E5 to obtain variant 1E5-1, which binds to the same epitope as 1E5; single-point mutations were performed on the variable regions of the heavy and light chains of 7R1 to obtain variant 7R1-1, which binds to the same epitope as 7R1. Replacing 5C3, 1E5, or 7R1 in the original pair with 5C3-1, 1E5-1, or 7R1 respectively yielded comparable detection efficacy against SARS-CoV-2 recombinant antigen. Epitope experiments confirmed that the replaced antibody had the same epitope as the original antibody. The variant sequences are as follows.

[0084] 5C3-1 Heavy chain variable region SEQ NO. 13: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGLLRSYFDYWGQGTTLTVSS. Heavy chain SEQ NO.14: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGLLRSYF DYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPR DCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKT ISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK. 1E5-1 Heavy chain variable region SEQ NO.15: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSS. Light chain variable region SEQ NO.16: DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK. Heavy chain SEQ NO.17: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG. Light chain SEQ NO.18: DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC. 7R1-1 Heavy chain variable region SEQ NO. 19: DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSASNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSS. Light chain variable region SEQ NO. 20: DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK. Heavy chain SEQ NO. 21: QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG. Light chain SEQ NO. 22: DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC. Example 6: Re-preparation of dominant antibodies This disclosure also describes the antibody preparation method described above, using the 44-180 amino acid fragment of the SARS-CoV-2 nucleocapsid protein as an immunogen to prepare antibodies that bind to the 44-180 amino acid fragment but do not bind to the 44-84 amino acid fragment, the 65-103 amino acid fragment, the 96-136 amino acid fragment, and the 130-180 amino acid fragment. These antibodies can be screened for reactivity and used in ELISA to evaluate OD. 405 Antibodies with a value ≥0.5, such as 1COV19-8 and 3COV19-17, also meet the requirements of high sensitivity, good specificity, and no cross-reactivity.

[0085] The above embodiments are optional implementations of this disclosure, but the implementation of this disclosure is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of this disclosure shall be considered equivalent substitutions and shall be included within the protection scope of this disclosure.

Claims

1. An antibody combination for detecting SARS-CoV-2, characterized in that, Includes antibody 3 and antibody 4; The antibody 3 comprises: an antibody of the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2, or an antibody of the heavy chain variable region of SEQ ID NO: 5 and the light chain variable region of SEQ ID NO: 6; The antibody 4 comprises a heavy chain variable region of SEQ ID NO: 9 and a light chain variable region of SEQ ID NO:

10.

2. A reagent kit, characterized in that, The kit contains the antibody combination for detecting SARS-CoV-2 as described in claim 1.

3. The use of the antibody combination for detecting SARS-CoV-2 as described in claim 1 in the preparation of a kit for detecting SARS-CoV-2.

4. A method for detecting SARS-CoV-2 for non-disease diagnostic and therapeutic purposes, comprising: A) Under conditions sufficient for a binding reaction to occur, contact the antibody combination for detecting SARS-CoV-2 as described in claim 1 or the kit as described in claim 2 with the sample to initiate a binding reaction; and B) Detect the immune complexes produced by the binding reaction.

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