A method and kit for detecting SARS-Cov-2

Abstract

The present application relates to a SARS-Cov-2 detection method and kit. The present application provides a SARS-Cov-2 detection kit, which comprises a first group and a second group of antibodies for detecting SARS-Cov-2 from a sample of a subject. The method and kit of the present application can improve the detection sensitivity and detection rate, and can detect new crown mutant strains in particular.

Description

[0001] Priority Statement

[0002] This application claims priority to Chinese Patent Application No. 202111064565.2, filed on September 10, 2021, entitled "A SARS-CoV-2 Detection Method and Kit", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of protein detection. Specifically, this invention relates to methods and kits for detecting SARS-CoV-2. Background Technology

[0004] Currently, the main detection methods for diagnosing novel coronavirus infection include nucleic acid testing and antigen-antibody immunoassay. Nucleic acid testing primarily uses fluorescent RT-PCR technology, while antigen-antibody immunoassay methods are divided into detection of viral antibodies and detection of viral antigens. Antibody test reagents detect IgM or IgG antibodies produced in the body after the virus enters the serum; IgM antibodies appear earlier, and IgG antibodies appear later. Viral antigen detection mainly detects certain proteins on the viral surface, such as the S protein. Finding specific antigenic epitope peptides is crucial for developing antibody-based COVID-19 detection methods.

[0005] Nucleic acid testing is characterized by early diagnosis, high sensitivity, and high specificity, making it the "gold standard" for confirming novel coronavirus infection. However, due to the high requirements for sample collection, RNA extraction, and testing equipment, false negatives are prone to occur. Therefore, there is an urgent need for rapid immunological testing methods to supplement nucleic acid testing. Studies have shown that the combined detection of total antibodies and nucleic acid can significantly improve the sensitivity of early diagnosis of COVID-19 infection, effectively compensating for the shortcomings of nucleic acid testing, which is prone to false negatives and missed diagnoses.

[0006] The SARS-CoV-2 structural genome is 29,883 bp long and includes 14 ORF frames. Larger protein molecules include the following regions: N: nucleocapsid protein (N protein); S: Spike protein; M: membrane protein; HE: hemagglutinin esterase; E: envelope protein. The N protein is also the most abundant structural protein in the SARS-CoV-2 infection process. After infection, human cells produce a strong immune response, and its gene sequence is relatively conserved and stable. Compared with the S protein, the N protein undergoes fewer mutations over time. Due to its good stability, domestic and international IVD companies have developed antigen detection kits with the N protein as the main target for case screening and epidemic monitoring. However, because site mutations have also appeared on the N protein in circulating mutant strains, these mutations may affect the detection rate of the kits, which are the main targets of SARS-CoV-2 antigen detection kits. Therefore, supplementing the target site, rather than the segment site of the mutant strain, is crucial. Currently, site mutations in circulating mutant strains do not affect the main target site of the kit and can even supplement sensitivity, improving the virus detection rate. Summary of the Invention

[0007] The present invention provides an antibody combination comprising a first group of antibodies and a second group of antibodies. The first group of antibodies includes antibody 1 selected from amino acid fragments 44-180 of the N protein that bind to SARS-CoV-2, and the second group of antibodies includes antibody 2 selected from amino acid fragments 44-180 of the N protein that bind to SARS-CoV-2. The antibody 1 and the antibody 2 may be the same antibody or different antibodies.

[0008] Optionally, the first group of antibodies may also include at least one of antibody 3 to antibody 6.

[0009] Optionally, the second group of antibodies also includes at least one of antibody 3 to antibody 6.

[0010] Optionally, the first group of antibodies may further include antibody 4 or antibody 5, and the second group of antibodies may further include antibody 6.

[0011] Antibody 3: Binds to 50-107 amino acid fragments of the N protein;

[0012] Antibody 4: binds to amino acid fragments 1-43 of the N protein;

[0013] Antibody 5: binds to amino acid fragments 248-361 of the N protein;

[0014] Antibody 6: binds to amino acid fragments 74-105 of the N protein.

[0015] Optionally, the antibody has a reaction titer higher than 10. 5 .

[0016] Optionally, the first group of antibodies is used for labeling, and the second group of antibodies is used for coating; or the second group of antibodies is used for labeling, and the first group of antibodies is used for coating.

[0017] Optionally, the antibody used for labeling is labeled with a detectable marker, such as metal particles like colloidal gold, fluorescent markers, chromophore markers, electron-dense markers, chemiluminescent markers, radioactive markers, enzyme markers such as radioisotopes, fluorophores, rhodamine, luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, glucose-6-phosphate dehydrogenase, biotin / avidin, spin markers, phage markers such as acridinium ester markers, or fluorescent markers such as acridinium ester markers added via a linker such as biotin-avidin.

[0018] Optionally, the antibody used for coating is linked to a solid phase, such as magnetic particles, latex particles, ELISA plates, microtiter plates, nitrocellulose membranes, or microfluidic chips.

[0019] Optionally, the heavy chain complementarity-determining region (VH-CDR1) of antibody 1 includes the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:13, the heavy chain complementarity-determining region (VH-CDR2) includes the amino acid sequence shown in SEQ ID NO:8, and the heavy chain complementarity-determining region (VH-CDR3) includes the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:14; the light chain complementarity-determining region (VL-CDR1) of antibody 1 includes the amino acid sequence shown in SEQ ID NO:10, the light chain complementarity-determining region (VL-CDR2) includes the amino acid sequence shown in SEQ ID NO:11, and the light chain complementarity-determining region (VL-CDR3) includes the amino acid sequence shown in SEQ ID NO:12.

[0020] Optionally, the heavy chain variable region of antibody 1 includes the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16, and the light chain variable region of antibody 1 includes the amino acid sequence shown in SEQ ID NO:17.

[0021] Optionally, the heavy chain of antibody 1 comprises the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:19, and the light chain of antibody 1 comprises the amino acid sequence shown in SEQ ID NO:20.

[0022] Optionally, the antibody 1 binds to epitope 1, wherein epitope 1 is the same epitope that binds to an antibody containing the heavy chain variable region of SEQ ID NO:15 or SEQ ID NO:16 and the light chain variable region of SEQ ID NO:17, or to an antibody containing the heavy chain of SEQ ID NO:18 or SEQ ID NO:19 and the light chain of SEQ ID NO:20.

[0023] Optionally, the heavy chain complementarity-determining region (VH-CDR1) of the antibody 2 includes the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:27, the heavy chain complementarity-determining region (VH-CDR2) includes the amino acid sequence shown in SEQ ID NO:22, and the heavy chain complementarity-determining region (VH-CDR3) includes the amino acid sequence shown in SEQ ID NO:23; the light chain complementarity-determining region (VL-CDR1) of the antibody 2 includes the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:28, the light chain complementarity-determining region (VL-CDR2) includes the amino acid sequence shown in SEQ ID NO:25, and the light chain complementarity-determining region (VL-CDR3) includes the amino acid sequence shown in SEQ ID NO:26.

[0024] Optionally, the antibody 2 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:30, and the antibody 2 light chain variable region includes the amino acid sequence shown in SEQ ID NO:31 or SEQ ID NO:32.

[0025] Optionally, the antibody 2 heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34, and the antibody 2 light chain comprises the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36.

[0026] Optionally, the antibody 2 binds to epitope 2, wherein the epitope 2 is the same epitope that binds to an antibody containing the heavy chain variable region of SEQ ID NO:29 or SEQ ID NO:30 and the light chain variable region of SEQ ID NO:31 or SEQ ID NO:32, or to an antibody containing the heavy chain of SEQ ID NO:33 or SEQ ID NO:34 and the light chain of SEQ ID NO:35 or SEQ ID NO:36.

[0027] Optionally, the heavy chain complementarity-determining region (VH-CDR1) of the antibody 3 includes the amino acid sequence shown in SEQ ID NO:37, the heavy chain complementarity-determining region (VH-CDR2) includes the amino acid sequence shown in SEQ ID NO:38 or SEQ ID NO:43, and the heavy chain complementarity-determining region (VH-CDR3) includes the amino acid sequence shown in SEQ ID NO:39; the light chain complementarity-determining region (VL-CDR1) of the antibody 3 includes the amino acid sequence shown in SEQ ID NO:40, the light chain complementarity-determining region (VL-CDR2) includes the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:44, and the light chain complementarity-determining region (VL-CDR3) includes the amino acid sequence shown in SEQ ID NO:42.

[0028] Optionally, the antibody 3 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:45 or SEQ ID NO:46, and the antibody 3 light chain variable region includes the amino acid sequence shown in SEQ ID NO:47 or SEQ ID NO:48.

[0029] Optionally, the antibody triple heavy chain comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:50, and the antibody triple light chain comprises the amino acid sequence shown in SEQ ID NO:51 or SEQ ID NO:52.

[0030] Optionally, the antibody 3 binds to epitope 3, wherein the epitope 3 is the same epitope that binds to an antibody containing the heavy chain variable region of SEQ ID NO:45 or SEQ ID NO:46 and the light chain variable region of SEQ ID NO:47 or SEQ ID NO:48, or to an antibody containing the heavy chain of SEQ ID NO:49 or SEQ ID NO:50 and the light chain of SEQ ID NO:51 or SEQ ID NO:52.

[0031] Optionally, antibody 1 or antibody 2 binds to a conformation-dependent epitope of the N protein of SARS-CoV-2.

[0032] Optionally, antibody 1 or antibody 2 does not bind to any polypeptide consisting of six consecutive amino acids of the N protein of SARS-CoV-2. The present invention also provides a SARS-CoV-2 antigen detection kit comprising the antibody combination described above.

[0033] The present invention also provides a SARS-CoV-2 antigen detection kit for detecting SARS-CoV-2 N protein from subject samples, comprising the antibody combination described above.

[0034] Optionally, the kit is an immunochromatographic test strip, an enzyme-linked immunosorbent assay (ELISA) reagent, or a chemiluminescent reagent.

[0035] Optionally, the sample may include biological tissues, cells, or body fluids in a healthy or pathological state, such as saliva or nasopharyngeal swabs.

[0036] Optionally, the present invention provides a SARS-CoV-2 immunochromatographic test strip, the immunochromatographic test strip comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad, wherein the nitrocellulose membrane is provided with T lines and C lines, and the conjugate pad is provided with a second group of antibodies, wherein the T lines are coated with a first group of antibodies; or the conjugate pad is provided with a first group of antibodies, and the T lines are coated with a second group of antibodies.

[0037] Optionally, the present invention provides an immunochromatographic test strip for detecting SARS-CoV-2 antigen.

[0038] Optionally, the cellulose membrane may be one of nitrocellulose membrane and glass cellulose membrane.

[0039] This invention provides the application of an antibody combination in the preparation of a kit for detecting SARS-CoV-2.

[0040] The beneficial effect of the present invention is that the antibody combination or the detection kit or immunochromatographic test strip provided by the present invention has higher sensitivity and detection rate when detecting SARS-CoV-2, which can effectively improve the problem of missed detection of mutant strains. Detailed Implementation

[0041] Terminology Definition

[0042] As used herein, the term "antibody" is used in the broadest sense and can include full-length monoclonal antibodies, polyclonal antibodies, bispecific or multispecific antibodies, chimeric antibodies, and antibody fragments, provided they exhibit the desired biological activity. An "antibody fragment" includes a portion of a full-length antibody, optionally its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), bivalent antibodies, or domain antibodies.

[0043] As used herein, the term "complementarity determining region (CRD)" refers to: a complete or whole antibody comprising two heavy chains and two light chains; each heavy chain containing a variable region (VH) and a constant region (CH); each light chain containing a variable region (VL) and a constant region (CL); the antibody having a "Y" shape, the stem of which consists of the second and third constant regions of the two heavy chains linked together by disulfide bonds; each arm of the Y including a variable region and a first constant region of a single heavy chain bound to the variable and constant regions of a single light chain; the variable regions of the light and heavy chains are responsible for antigen binding; the variable regions of the two chains typically contain three highly variable regions, called complementarity determining regions.

[0044] As used in this article, the term "constant region" refers to the region of the light and heavy chains of an antibody molecule that is relatively stable near the C-terminal amino acid sequence.

[0045] As used in this article, the term "variable region" refers to the region in the light and heavy chains of an antibody molecule that exhibits significant changes in the amino acid sequence near the N-terminus.

[0046] As used in this article, “aa” represents the amino acid at position ... of the peptide.

[0047] As used herein, the term "subject" refers to a vertebrate, optionally a mammal, and optionally a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, racing animals, and pets. Tissues, cells, and their progeny from biological entities obtained in vivo or cultured in vitro are also included. It should be understood that the terminology used herein is for the purpose of describing the context of the invention, such as embodiments and examples, and is not intended to limit the scope of the invention.

[0048] As described in this article, the term "homon" refers to a hybrid cell formed by the fusion of cells with the same genotype.

[0049] Some embodiments of the present invention provide an antibody combination comprising a first group of antibodies and a second group of antibodies. The first group of antibodies includes antibody 1 selected from the amino acid fragment of the N protein at positions 44-180 of SARS-CoV-2, and the second group of antibodies includes antibody 2 selected from the amino acid fragment of the N protein at positions 44-180 of SARS-CoV-2. Antibody 1 and antibody 2 may be the same antibody or different antibodies.

[0050] In some embodiments, the first group of antibodies may also include at least one of antibody 3, antibody 4, antibody 5, or antibody 6.

[0051] In some embodiments, the second group of antibodies may also include at least one of antibody 3, antibody 4, antibody 5 or antibody 6.

[0052] In some implementations, the first group of antibodies or the second group of antibodies may also include antibody 3.

[0053] In some implementations, the first group of antibodies further includes antibody 4 or antibody 5, and the second group of antibodies further includes antibody 6.

[0054] In some embodiments, antibody 3 binds to the N protein amino acid fragment at positions 50-107;

[0055] Antibody 4 binds to the N protein amino acid fragment at positions 1-43;

[0056] Antibody 5 binds to the N protein amino acid fragment at positions 248-361;

[0057] Antibody 6 binds to the N protein amino acid fragment at positions 74-105.

[0058] In some embodiments, the kit of the present invention includes a SARS-CoV-2 test strip. In some embodiments, colloidal gold lateral chromatography technology can be used to label antibodies onto a detectable marker such as colloidal gold, and the double-antibody sandwich method can be used to detect whether the subject is infected with the novel coronavirus SARS-CoV-2. In some embodiments, the method and kit of the present invention improve the detection rate and can overcome the problem of missed detection of mutant strains.

[0059] In some implementations, the present invention addresses the technical problems of low detection rate and inability to quickly detect whether or not a person is infected with the novel coronavirus, which are present in most existing technologies. The present invention provides a detection method and kit that can improve the detection rate and reduce the risk of missed detection.

[0060] In some embodiments, the present invention may be performed by or include reagents for colloidal gold immunochromatography. In some embodiments, immunochromatography may be performed by a test strip using a double-antibody sandwich method. In some embodiments, there are no particular limitations on the reagents and apparatus used for the double-antibody sandwich method, and any suitable reagents and / or apparatus may be used. For example, the immunochromatographic test strip may include a sample pad, a conjugate pad, an NC membrane, and an absorbent pad. In some embodiments, the conjugate pad may be coated with a colloidal gold-antibody complex containing multiple epitopes; the NC membrane may be coated with two lines, such as a T line and a C line; antibodies containing multiple epitopes may be mixed on the T line to capture the colloidal gold-antibody complex from the conjugate pad to form a double-antibody sandwich complex, thereby immobilizing the colloidal gold on the T line; the C line may be coated with an antibody capable of capturing free colloidal gold-antibody complexes. In some implementations, the sample to be tested is added to the sample dispensing port of the test kit. Under the action of the lateral capillary, the sample first passes through the conjugation pad, where it undergoes specific immunobinding with different colloidal gold-antibody complexes, forming antigen-antibody colloidal gold complexes, which are then immobilized in the T line. When the free colloidal gold-antibody complex passes through the C line, it can undergo specific immunobinding with the antibody in the C line, thus being immobilized in the C line. The color intensity of the control band (C) is inversely proportional to that of the test band (T), thereby correcting for individual differences in samples.

[0061] In some embodiments, the present invention can utilize multi-epitope antibodies to recognize multiple epitopes on an antigen, thereby reducing the risk of missed detection and improving the detection rate.

[0062] In some embodiments, the present invention can achieve a wide coverage by combining antigenic epitopes, and utilize multi-epitope antibody pairing to identify multiple epitopes on the antigen, thereby reducing the risk of missed detection and improving the detection rate.

[0063] In some embodiments, the labeled antibodies of the present invention can be mixed with antibody epitopes of 44-180 aa, 50-107 aa, or 74-105 aa of the N protein of SARS-CoV-2, and the coating antibodies can be mixed with antibody epitopes of 44-180 aa, 1-43 aa, or 248-361 aa. Alternatively, the coating and labeling can be interchanged.

[0064] In this document, unless otherwise stated, the N protein epitopes or fragments are numbered according to the corresponding positions in SEQ ID NO:1.

[0065] In some embodiments, the present invention provides a method and kit for detecting SARS-CoV-2. The kit of the present invention has improved sensitivity, increased detection rate, and overcomes the problem of missed detection of mutant strains.

[0066] In some embodiments, the first group of antibodies and / or the second group of antibodies of the present invention may be monoclonal antibodies.

[0067] In some embodiments, the antibodies of the present invention can be prepared using methods known in the art, such as the first group of antibodies and / or the second group of antibodies.

[0068] In some embodiments, antibodies of the present invention can be prepared by immunizing animals with antigens containing the amino acid fragments described herein, such as group one antibodies and / or group two antibodies. To increase immunogenicity, larger antigenic compounds (including but not limited to BSA (bovine serum albumin), ovalbumin, KLH (keyhole hemocyanin), etc.) can be conjugated to epitope peptides. The immunogenic portion can include a variety of proteins or peptides that can act as immunogenic carriers. These types of peptides include albumins, serum proteins, globulins, lens proteins, lipoproteins, and / or fragments thereof.

[0069] In some embodiments, synthetic peptides may be used. The immunogen portion or carrier may also be a particle or microparticle. Immunogen particles may be organic or inorganic, porous or non-porous. Immunogen particles may be biological materials such as cells and microorganisms, and may also include organic and inorganic polymers, liposomes, latex, phospholipid vesicles, cationic liposomes, anionic liposomes, lipoproteins, lipid polymers, etc. Therefore, the epitope peptides described in this invention can be used to generate antibodies with affinity for N proteins.

[0070] In some embodiments, the effectiveness of the antibodies of the present invention, 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 (Fluorescence-Activated Cell Sorting) binding assay, Biacore (Biomolecule Interaction Analysis core technique), competitive binding assay, etc. In some embodiments, for example, the EC50 value of the antibody (or its antigen-binding fragment) of the present invention binding to the antigen in ELISA or FACS may be, for example, 1 μM-1 pM, 1 nM-1 pM, or 100 pM-1 pM. In some embodiments, the titer of the antibody of the present invention for detecting the N protein antigen is higher than 10. 5 including, for example, above 10 6 For example, higher than 10 7 .

[0071] In some embodiments, the first group of antibodies and / or the second group of antibodies in the kit can be used as a coating antibody (or capture antibody) and a labeling antibody, respectively. For example, the first group of antibodies is a coating antibody and the second group of antibodies is a labeling antibody, or the first group of antibodies is a labeling antibody and the second group of antibodies is a coating antibody. Optionally, in some embodiments, the first group of antibodies is a labeling antibody and the second group of antibodies is a coating antibody (or capture antibody).

[0072] In some embodiments, the coated antibody binds to a solid phase. In some embodiments, the coated antibody may be used to coat a solid-phase support. In some embodiments, the solid-phase support is not particularly limited and may be, for example, magnetic particles, latex particles, microtiter plates, nitrocellulose membranes, or microfluidic chips; optionally, the kit comprises or is prepared as an immunochromatographic test strip or test card. In some embodiments, the labeled antibody is labeled with a detectable marker, such as a fluorescent marker, for example, via a linker such as biotin-avidin to the fluorescent marker.

[0073] Some embodiments of the present invention provide a SARS-CoV-2 immunochromatographic test strip, the immunochromatographic test strip comprising a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane and an absorbent pad, wherein the nitrocellulose membrane is provided with T lines and C lines, and the conjugate pad is provided with the second group of antibodies described above, wherein the T lines are coated with the first group of antibodies described above; or the conjugate pad is provided with the first group of antibodies described above, and the T lines are coated with the second group of antibodies described above.

[0074] In some embodiments, the detectable markers used in this invention are not particularly limited. In some embodiments, the markers may include, but are not limited to, metal particles such as colloidal gold, fluorescent markers, chromophore markers, electron-dense markers, chemiluminescent markers, and radioactive markers, as well as indirect markers such as enzymes or ligands, for example, for indirect detection through enzymatic reactions or molecular interactions. In some embodiments, exemplary markers include, but are not limited to, radioisotopes, fluorophores, rhodamine and its derivatives, luciferase, luciferin, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin / antibiotin protein, spin markers, bacteriophage markers, etc.

[0075] In some embodiments, the heavy chain complementarity-determining region (VH-CDR1) of antibody 1 includes the amino acid sequence shown in SEQ ID NO:7 or SEQ ID NO:13, the heavy chain complementarity-determining region (VH-CDR2) includes the amino acid sequence shown in SEQ ID NO:8, and the heavy chain complementarity-determining region (VH-CDR3) includes the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:14; the light chain complementarity-determining region (VL-CDR1) of antibody 1 includes the amino acid sequence shown in SEQ ID NO:10, the light chain complementarity-determining region (VL-CDR2) includes the amino acid sequence shown in SEQ ID NO:11, and the light chain complementarity-determining region (VL-CDR3) includes the amino acid sequence shown in SEQ ID NO:12.

[0076] In some embodiments, the variable region of antibody 1 heavy chain includes the amino acid sequence shown in SEQ ID NO:15 or SEQ ID NO:16, and the variable region of antibody 1 light chain includes the amino acid sequence shown in SEQ ID NO:17.

[0077] In some embodiments, the heavy chain of antibody 1 comprises the amino acid sequence shown in SEQ ID NO:18 or SEQ ID NO:19, and the light chain of antibody 1 comprises the amino acid sequence shown in SEQ ID NO:20.

[0078] In some embodiments, antibody 1 binds to epitope 1, which is the same epitope that binds to an antibody containing the heavy chain variable region of SEQ ID NO:15 or SEQ ID NO:16 and the light chain variable region of SEQ ID NO:17, or to an antibody containing the heavy chain of SEQ ID NO:18 or SEQ ID NO:19 and the light chain of SEQ ID NO:20. Antibody 1 binds to amino acid regions from positions 44 to 180 of the N protein amino acid sequence of SARS-CoV-2 shown in SEQ ID NO:1.

[0079] SEQ ID NO:7: NYGMN. SEQ ID NO: 13: NYGWN.

[0080] SEQ ID NO:8: WINTYTGEPTYADDFKG.

[0081] SEQ ID NO:9: SALLRSYFDY.

[0082] SEQ ID NO:14:SGLLRSYFDY.

[0083] SEQ ID NO:10: KASQDVSTAVA.

[0084] SEQ ID NO:11:WASTRHT。

[0085] SEQ ID NO:12:QQHYSTPLT。

[0086] SEQ ID NO:15:

[0087] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSALLRSYFDYWGQGTTLTVSS。

[0088] SEQ ID NO:16:

[0089] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGLLRSYFDYWGQGTTLTVSS。

[0090] SEQ ID NO:17:

[0091] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWCQQKPGQSPKLLIYWASTRHTGVPDRFTGIRSGTDYTLTISSVQAEDLALYYCQQHYSTPLTFGAGTKLELKR。

[0092] SEQ ID NO:18:

[0093] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSALLRSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

[0094] SEQ ID NO:19:

[0095] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARSGLLRSYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

[0096] SEQ ID NO:20:

[0097] DIVMTQSHKFMSTSVGDRVSITCKASQDVSTAVAWCQQKPGQSPKLLIYWASTRHTGVPDRFTGIRSGTDYTLTISSVQAEDLALYYCQQHYSTPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0098] In some embodiments, the heavy chain complementarity-determining region (VH-CDR1) of antibody 2 includes the amino acid sequence shown in SEQ ID NO:21 or SEQ ID NO:27, the heavy chain complementarity-determining region (VH-CDR2) includes the amino acid sequence shown in SEQ ID NO:22, and the heavy chain complementarity-determining region (VH-CDR3) includes the amino acid sequence shown in SEQ ID NO:23; the light chain complementarity-determining region (VL-CDR1) of antibody 2 includes the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:28, the light chain complementarity-determining region (VL-CDR2) includes the amino acid sequence shown in SEQ ID NO:25, and the light chain complementarity-determining region (VL-CDR3) includes the amino acid sequence shown in SEQ ID NO:26.

[0099] In some embodiments, the antibody 2 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:30, and the antibody 2 light chain variable region includes the amino acid sequence shown in SEQ ID NO:31 or SEQ ID NO:32.

[0100] In some embodiments, the antibody 2 heavy chain comprises the amino acid sequence shown in SEQ ID NO:33 or SEQ ID NO:34, and the antibody 2 light chain comprises the amino acid sequence shown in SEQ ID NO:35 or SEQ ID NO:36.

[0101] In some embodiments, antibody 2 binds to epitope 2, which is the same epitope that binds to an antibody containing the heavy chain variable region of SEQ ID NO:29 or SEQ ID NO:30 and the light chain variable region of SEQ ID NO:31 or SEQ ID NO:32, or to an antibody containing the heavy chain of SEQ ID NO:33 or SEQ ID NO:34 and the light chain of SEQ ID NO:35 or SEQ ID NO:36. Antibody 2 binds to amino acid regions from positions 44 to 180 of the N protein amino acid sequence of SARS-CoV-2 shown in SEQ ID NO:1.

[0102] SEQ ID NO:21: NYGMN.

[0103] SEQ ID NO:27: NYGWN.

[0104] SEQ ID NO:22: WINTYTGEPTYADDFKG.

[0105] SEQ ID NO:23: KGNWDEENAMDY.

[0106] SEQ ID NO:24: RASENIYSNLA.

[0107] SEQ ID NO:28:RGSENIYSNLA.

[0108] SEQ ID NO:25: TATDLPD.

[0109] SEQ ID NO:26: QHFWGTPWT.

[0110] SEQ ID NO:29:

[0111] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSS.

[0112] SEQ ID NO:30:

[0113] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSS.

[0114] SEQ ID NO:31:

[0115] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK。

[0116] SEQ ID NO:32:

[0117] DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK。

[0118] SEQ ID NO:33:

[0119] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGMNWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG。

[0120] SEQ ID NO:34:

[0121] QIQLVQSGPELKKPGETVKISCKASGYTFTNYGWNWVKQAPGKGLKWMGWINTYTGEPTYGDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARKGNWDEENAMDYWGQGTSVTVSSAKTTPPSVYPLPPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGAHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG。

[0122] SEQ ID NO:35:

[0123] DIQMTQSPASLSVSVGETVTITCRASENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0124] SEQ ID NO:36:

[0125] DIQMTQSPASLSVSVGETVTITCRGSENIYSNLAWYQQKQGKSPQLLVYTATDLPDGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPWTFGGGTKLEIK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0126] In some embodiments, the heavy chain complementarity-determining region (VH-CDR1) of antibody 3 includes the amino acid sequence shown in SEQ ID NO:37, the heavy chain complementarity-determining region (VH-CDR2) includes the amino acid sequence shown in SEQ ID NO:38 or SEQ ID NO:43, and the heavy chain complementarity-determining region (VH-CDR3) includes the amino acid sequence shown in SEQ ID NO:39; the light chain complementarity-determining region (VL-CDR1) of antibody 3 includes the amino acid sequence shown in SEQ ID NO:40, the light chain complementarity-determining region (VL-CDR2) includes the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:44, and the light chain complementarity-determining region (VL-CDR3) includes the amino acid sequence shown in SEQ ID NO:42.

[0127] In some embodiments, the antibody 3 heavy chain variable region includes the amino acid sequence shown in SEQ ID NO:45 or SEQ ID NO:46, and the antibody 3 light chain variable region includes the amino acid sequence shown in SEQ ID NO:47 or SEQ ID NO:48.

[0128] In some embodiments, the antibody triplet chain comprises the amino acid sequence shown in SEQ ID NO:49 or SEQ ID NO:50, and the antibody triplet light chain comprises the amino acid sequence shown in SEQ ID NO:51 or SEQ ID NO:52.

[0129] In some embodiments, antibody 3 binds to epitope 3, which is the same epitope that binds to an antibody containing the heavy chain variable region of SEQ ID NO:45 or SEQ ID NO:46 and the light chain variable region of SEQ ID NO:47 or SEQ ID NO:48, or to an antibody containing the heavy chain of SEQ ID NO:49 or SEQ ID NO:50 and the light chain of SEQ ID NO:51 or SEQ ID NO:52. Antibody 3 binds to amino acid regions from positions 50 to 107 of the N protein amino acid sequence of SARS-CoV-2 shown in SEQ ID NO:1.

[0130] SEQ ID NO:37:TFGMH.

[0131] SEQ ID NO:38:YINSGSNIIYYADTVKG。

[0132] SEQ ID NO:43: YINSASNIIYYADTVKG

[0133] SEQ ID NO:39:HAMDY.

[0134] SEQ ID NO:40:KASQSVDYDGDSYMN。

[0135] SEQ ID NO:41: AASNLES.

[0136] SEQ ID NO:44: DASNLES.

[0137] SEQ ID NO:42: QQSNEDPYT

[0138] SEQ ID NO:45:

[0139] DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSGSNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSS。

[0140] SEQ ID NO:46:

[0141] DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSASNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSS。

[0142] SEQ ID NO:47:

[0143] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIK。

[0144] SEQ ID NO:48:

[0145] DIVLTQSPASLAVSLGQRATICSKASQSVDYDGDSYMNWFQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEDAATYYCQQSNEDPYTFGGGTKLEIK。

[0146] SEQ ID NO:49:

[0147] DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSGSNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETTVTCNVAHPASSTKVDKKIVPRDCGCK PCICTVPEVSSVFIFPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK。

[0148] SEQ ID NO:50:

[0149] DVQLVESGGGLVQPGGSRKLSCAASGFTFSTFGMHWVRQAPEKGLEWVAYINSASNIIYYADTVKGRFTISRDNPKNTLFLQMTSLRSEDTAMYYCARHAMDYWGQGTSVTVSSAKTTPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPTVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK.

[0150] SEQ ID NO:51:

[0151] DIVLTQSPASLAVSLGQRATICSKASQSVDYDGDSYMNWFQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC。

[0152] SEQ ID NO:52:

[0153] DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWFQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLE IKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

[0154] In some implementations, antibody 1 or antibody 2 binds to a conformation-dependent epitope of the N protein of SARS-CoV-2.

[0155] In some embodiments, the conformational epitope consists of a discontinuous portion of the antigen amino acid sequence.

[0156] In some implementations, antibodies 1 and 2 are analyzed using PepScan. The synthetic peptide consists of six consecutive amino acids, each shifted by one amino acid, for example: 44-49, 45-50, and so on, up to 175-180. The binding state of the synthetic peptide to the antibody is analyzed, specifically using HRP-labeled secondary antibody to detect the binding of the synthetic peptide to the antibody. The detection results show that both antibody 1 and antibody 2 are conformational epitopes.

[0157] In some embodiments, antibody 1 or antibody 2 does not bind to any polypeptide consisting of six consecutive amino acids of the N protein of SARS-CoV-2. In some embodiments, the kit of the present invention includes reagents suitable for performing immunoassays. In some embodiments, the kit of the present invention can be used for performing immunoassays, such as ELISA, indirect immunofluorescence assay (IFA), radioimmunoassay (RIA), and other non-enzyme-linked antibody binding assays or methods.

[0158] In some embodiments, such as in chemiluminescence assays, N protein antibodies can be coated onto a solid phase, such as magnetic beads, to capture the N protein antigen in the sample. The labeled antibody then re-binds to the antigen bound to the magnetic beads, and the result is read after adding a luminescent substrate. In some embodiments, the N protein antibody of the present invention can be used to coat a solid phase, such as magnetic beads, or as a second group of labeled antibodies. In some embodiments, such as in ELISA assays, the antibody or its antigen-binding fragment is immobilized on a surface, such as a solid support, like a plastic plate or ELISA plate. The sample from the subject comes into contact with the solid support and then with an antibody indicator bearing a detectable label for color development. In some embodiments, blocking agents such as bovine serum albumin, milk powder solution, gelatin, PVP, or Superblock can be used to block nonspecific sites, thus reducing background caused by nonspecific binding. In some embodiments, diluents such as BSA and phosphate-buffered saline (PBS) / Tween can be used to help reduce nonspecific background.

[0159] In this article, samples from subjects may include biological tissues, cells, or body fluids in healthy or pathological states, such as saliva or nasopharyngeal swabs.

[0160] In some embodiments, the present invention provides combinations of a first group of antibodies and a second group of antibodies for detecting the N protein, and provides their use in the preparation of a kit for detecting SARS-CoV-2. In some embodiments, the present invention provides combinations of immunogenic peptides comprising the amino acid fragment epitopes described herein, and their use in the preparation of antibodies for detecting the N protein. In some embodiments, the present invention provides a method for preparing antibodies for detecting the N protein, the method comprising immunizing animals with immunogenic peptides comprising the amino acid fragment epitopes described herein, thereby preparing antibodies for detecting the N protein, such as monoclonal antibodies or polyclonal antibodies. Monoclonal antibodies or polyclonal antibodies can be prepared by methods known in the art. In some embodiments, the epitopes identified by the present invention (which can be artificially synthesized, for example, by chemical methods) can be linked to a suitable carrier protein for immunizing animals to prepare antibodies, such as monoclonal antibodies. In some embodiments, suitable carrier proteins are known to those skilled in the art and may be, for example, KLH and BSA. In some embodiments, the kits of the present invention may comprise the aforementioned first group of antibodies and second group of antibodies.

[0161] In some embodiments, the present invention provides the use of immunogenic peptides comprising the amino acid fragment epitopes described herein in the preparation of reagents or kits for detecting SARS-CoV-2.

[0162] In some embodiments, the method may include contacting a sample with at least one N protein antibody or fragment thereof coated on a solid phase to form an immune complex; detecting the presence of the complex to determine the presence of N protein in the sample. In some embodiments, the method may include contacting a sample with at least one N protein antibody or fragment thereof coated on a solid phase to form an immune complex; adding a second N protein antibody linked to a detectable label to the generated complex; and detecting the generated signal to determine the presence and / or content of N protein in the sample. In some embodiments, the present invention provides a kit for the method comprising 1) a container containing at least one N protein antibody coated on a solid phase. In some embodiments, the kit may optionally comprise a second N protein antibody linked to a detectable label. In some embodiments, the at least one N protein antibody is a monoclonal antibody of N protein. In some embodiments, the present invention provides a kit comprising magnetic beads or test strips. In some embodiments, the kit of the present invention may include reagents suitable for mechanoluminescence detection. In some embodiments, the kit of the present invention can utilize fully automated chemiluminescence instruments for high-throughput, rapid, and accurate detection of N protein, shortening detection time and providing rapid test results.

[0163] In some embodiments, the present invention includes antibodies labeled on magnetic beads or test strips. In some embodiments, the kit of the present invention can utilize magnetic beads or test strips as a solid phase, directly coating antibodies onto the magnetic beads or test strips, and employing the double-antibody sandwich method to detect N protein, thereby improving the detection rate.

[0164] Example

[0165] The following mainly focuses on specific embodiments.

[0166] The present invention will be described in further detail below. The following examples are provided to illustrate embodiments of the invention and are not intended to limit the invention. The invention may optionally include embodiments not shown in the examples.

[0167] Preparation of monoclonal antibodies against N protein

[0168] 1. Immunized animals

[0169] BALB / c mice aged 8–12 weeks, of the same strain as myeloma cells, were used. A mixture of 100 μg / mouse N protein antigen and an equal volume of Freund's complete adjuvant was injected intraperitoneally. Every two weeks, 100 μg / mouse N protein antigen was mixed with an equal volume of Freund's incomplete adjuvant and injected intraperitoneally multiple times to boost immunization. Mice with serum titers above 1:2000, as determined by 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.

[0170] N protein antigen sequence: SEQ ID NO:1.

[0171] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPAR MAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAF FGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAAADLDDFSKQLQQSMSSADSTQA

[0172] 2. Preparation of feeder cells

[0173] 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 Add 150 μL / well to a 96-well plate and incubate overnight at 37°C with 5% CO2.

[0174] 3. Preparation of immune spleen cells

[0175] 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, ground and filtered on 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.

[0176] 4. Cell fusion

[0177] (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;

[0178] (2) Take mouse myeloma cells Sp2 / 0 (preserved by Phytobio 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.

[0179] (3) Place the centrifuge tube in water preheated to 37°C, take 0.7 mL of preheated 50% PEG solution, and let it stand for 90 seconds. Immediately add 15 mL of preheated serum-free culture medium at 37°C.

[0180] (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% fetal bovine serum. Mix well with a pipette and add 2 drops to each of the four 96-well cell culture plates containing feeder cells. Incubate at 37°C and 7% CO2.

[0181] 5. Selective culture of hybridoma cells

[0182] 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; co-nuclei of myeloma cells and immune spleen cells; and heteronuclei of myeloma cells and immune spleen cells. Only the latter can form hybridoma cells. Therefore, unfused cells and co-nuclei of the same type must be removed from this mixture, and true hybrid cells must be selected. Thus, the cells were cultured in HAT medium as described above on days 1, 3, 5, and 7 after fusion.

[0183] 6. Detection of specific antibodies and cloning of hybridoma cells

[0184] The supernatant from each culture well was collected, and the wells containing specific antibodies against the N protein were detected by indirect ELISA. Hybridoma cells were cloned using a limiting dilution method. After culture, individual cells proliferated into homologous cell clones; through reactive screening, 40 cell lines stably secreting anti-N protein monoclonal antibodies were obtained (see Table 1 below), with titers all above 10. 5 ~10 7 between.

[0185] Table 1. Cell lines that stably secrete anti-N protein monoclonal antibodies obtained through screening.

[0186]

[0187]

[0188] Reactive screening

[0189] At room temperature, microtiter plates (Nunc, Maxisorb) were coated for 1 hour with 100 μl / well of coating buffer containing 2.5 μg / ml N protein (as antigen) for 1 hour with stirring. Post-coating treatment involved incubation in PBS buffer and 1% monoclonal antibody for 30 minutes. Subsequently, the plates were washed with washing buffer. 100 μl / well of antibody sample was incubated for 1 hour at room temperature with stirring. The plates were then washed twice more with washing solution. Next, at room temperature with stirring, the plates were incubated with 100 μl / well of goat anti-mouse IgG conjugated with the detection antibody peroxidase, diluted 1:40000 with PBS buffer, for 1 hour. After washing again with washing buffer, peroxidase activity was measured using standard methods (e.g., changes in extinction value at 405 nm, measured in mU, using an ELISA reader after 30 minutes at room temperature). Antibodies secreted by the 40 monoclonal cell lines showed good reactivity.

[0190] It should be noted that the cell lines in the table above are cell lines preserved after screening by Phytobio Co., Ltd., and were obtained through reactivity screening. Those skilled in the art can also obtain functionally similar cell lines using this method or similar techniques such as ELISA, immunofluorescence, or Western blotting. The selected cell lines are those with good reactivity and stably secrete anti-N protein monoclonal antibodies. The preparation methods and selection of the above cell lines do not limit the scope of this application in any way, but are merely illustrative examples.

[0191] Identification of the binding epitope of the two antibodies

[0192] Different short peptide antigens of the N protein were used to coat microwells. The monoclonal antibodies were diluted to a primary antibody concentration of 1 μg / ml using PBS + 20% NBS as the diluent. Goat anti-mouse IgG-HRP was used as the secondary antibody. The epitopes of the monoclonal antibodies were determined based on their responses to different antigens. Statistical analysis showed that the 41 antibodies targeted the following segments of the N protein antigen (see Table 2 below):

[0193] Table 2. Antibodies and segments of antibody-targeted N protein antigens

[0194]

[0195]

[0196] "+" indicates a response, and "-" indicates no response.

[0197] The segment sequence of the N protein antigen is as follows:

[0198] The sequence of fragment 1-43 (i.e., amino acid fragments of the N protein at positions 1-43 of SARS-CoV-2) is shown in SEQ ID NO:2:

[0199] MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQ

[0200] The sequence of fragment 44-180 (i.e., the N protein amino acid fragment of SARS-CoV-2 from position 44 to 180) is shown in SEQ ID NO:3:

[0201] GLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGS

[0202] The sequence of fragment 248-361 (i.e., the N protein amino acid fragment of SARS-CoV-2 from position 248 to 361) is shown in SEQ ID NO:4:

[0203] KKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYK

[0204] The sequence of fragment 50-107 (i.e., the N protein amino acid fragment of SARS-CoV-2 from position 50 to 107) is shown in SEQ ID NO:5:

[0205] ASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPR

[0206] The sequence of fragment 74-105 (i.e., the N protein amino acid fragment of SARS-CoV-2 from position 74 to 105) is shown in SEQ ID NO:6:

[0207] INTNSSPDDQIGYYRRATRRIRGGDGKMKDLS

[0208] Anti-N protein antibody epitope pairing: The antibody with the highest titer against different epitopes was used as the pairing combination. The ELISA double antibody sandwich detection method was used. 100 nucleic acid positive samples were used as test samples. A detection rate of ≥95% was considered a good pairing effect. See Table 3 for details.

[0209] Table 3 Anti-N protein antibody epitope pairing

[0210]

[0211]

[0212] In the above test results, the detection rates of paired 6-8 and paired 12-14 were both higher than 95%. The factors contributing to the increased detection rate were that antibodies binding to the 44-180aa (16D8) segment were used in both coating and labeling.

[0213] Using the above pairing combinations, the mutant strains were detected using the ELISA double-antibody sandwich assay to determine whether there were any missed detections. See Table 4 for details.

[0214] Table 4 shows the detection of mutant strains using the ELISA double-antibody sandwich assay.

[0215]

[0216] "+" indicates detection, and "-" indicates no detection.

[0217] In the above test results, pairs 6-8 and 12-14 were able to effectively detect D103Y and P13L+R203K+G204R+G214C mutant strains at concentrations up to 0.2 ng / ml, and some pairs could also be detected at concentrations as low as 50 pg / ml.

[0218] In addition, for the matching of identical epitopes (pairs 6-8 and pairs 12-14), random substitution of antibodies from different strains was performed using the ELISA double-antibody sandwich detection method, with 100 nucleic acid-positive samples as the test sample detection rate. The results are shown in Table 5 below:

[0219] Table 5 Detection rate of test samples

[0220] Wrapped mark Detection rate Pair 15 4B5+3A7 9F5 97％ Pair 16 12H3+13B5 15A3+15C8 96％ Pair 17 13B5+13F1 5A1+1D9 96％ Pair 18 3B4+10A3 15A3+2A5 96％ Pairing 19 9F5+4F1 11G2+3G10 96％ Pair 20 11G2 9F5+14H2 96％ Pair 21 4B5+2C9 6B8+9F5 98％ Pair 22 11G2+14D9 4B5+14E4 96％ Pair 23 3B4+15C8 4F1+13B5 98％ Pair 24 11G2+3A7 4B5 97％ Pair 25 4B5+13F1 13B5+11A5 96％ Pair 26 15A3+2A5 9F5+14D9 96％ Pair 27 9F5+1D9 15A3+4F1 96％ Pair 28 3B4+14H2 11G2 97％ Pairing 29 13B5 13B5+4C10 96％ Pair 30 3B4+14C2 6B8+4B5 98％ Pair 31 9F5+3G10 13B5+12H3 96％ Pair 32 5A1+1B6 12H3+3B4 98％ Pair 33 15A3 13B5+10C6 96％ Pair 34 11G2+13F1 9F5+4G3 96％

[0221] The above pairings still maintain a high detection rate even after replacing other antibody strains.

[0222] In the above test results, the detection rate of pairs 15 to 34 was all above 95%. The reason for the high detection rate is that both the coating antibody and the labeling antibody in the above pairing schemes include antibodies that bind to the 44-180aa region. The above data also demonstrates that only 3 antibodies need to be selected in the above scheme to achieve the technical effect of using 4 antibodies in combination.

[0223] Taking the antibodies 9F5, 4B5, and 3A7 in the above pairing scheme 15 as an example; where 9F5 is antibody 1, 4B5 is antibody 2, and 3A7 is antibody 3.

[0224] After sequencing, the heavy chain, light chain, and CDR of the antibody sequence are shown in Table 6 below:

[0225] Table 6. Heavy chain, light chain, and CDR division of antibody sequences.

[0226]

[0227] Individual mutations were performed on the CDR regions of the above antibodies to obtain the following antibody sequences:

[0228]

[0229] Note: The heavy chain, light chain, and CDR numbers correspond to the sequence numbers on SEQ ID NO:, for example, 7 corresponds to SEQ ID NO:7.

[0230] When the above antibodies were used to replace the corresponding antibodies in the original pair 15, the detection rate was over 96%.

[0231] III. Identification of Epitopes in Monoclonal Antibodies:

[0232] Dilute the purified monoclonal antibody to be identified with 0.06M pH 9.6 carbonate buffer to a final concentration of 1 μg / mL. Add 0.1 mL to each well of a 96-well polystyrene plate and incubate at 37°C for 2 hours or at 4°C overnight. The next day, block with 0.15 mL / well of 0.02M pH 7.2 PBS containing 10% fetal bovine serum or 1% skim milk powder at 37°C for 2 hours. Add 2000-fold diluted horseradish peroxidase-labeled antigen of each epitope segment of the SARS-CoV-2 N protein, incubate at 37°C for 30 minutes, wash 5 times with PBST, blot dry, add 100 μL of citrate-phosphate buffer containing 0.1% (M / V) o-phenylenediamine, 0.1% (V / V) hydrogen peroxide, and pH 5.0 to each well, incubate at 37°C for 15 minutes, add 50 μL of dilute sulfuric acid solution to each well, and measure the absorbance at 450 nm to identify epitopes based on the reaction.

[0233] Whether an antibody recognizes the same epitope as other antibodies can be confirmed by the competition between them for that epitope. Competition between antibodies can be evaluated using competitive binding assays, such as ELISA, fluorescence energy transfer assays, or fluorescence micro-assays. Identification revealed that the mutant antibodies corresponding to 9F5, 4B5, and 3A7 bind to the same epitopes as the non-mutated antibodies.

[0234] IV. SARS-CoV-2 Colloidal Gold Immunochromatographic Test Strip and its Preparation:

[0235] In this embodiment, the immunochromatographic test strip includes a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has T lines and C lines, and the conjugate pad has a second group of antibodies. The T lines are coated with a first group of antibodies.

[0236] The preparation of the immunochromatographic test strip includes the following steps: First, fix the nitrocellulose membrane in the center of the base plate. Press one end of the treated colloidal gold conjugate pad (with the second group of antibodies labeled with colloidal gold sprayed onto the conjugate pad) against the detection T-line end of the nitrocellulose membrane (the T-line is sprayed with the first group of antibodies), overlapping by 1 mm. Press one end of the treated sample pad against the other end of the colloidal gold conjugate pad, overlapping by 2 mm. Attach the absorbent pad to the base plate, with one end pressing against the control C-line end of the nitrocellulose membrane, overlapping by 2 mm. Cut the assembled test strip test piece into 4 mm wide pieces using a strip cutter, place them into the groove of the plastic clip base, and close the plastic cap tightly.

[0237] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0238] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. An antibody combination for detecting SARS-CoV-2, characterized in that, It includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies consists of antibody 1, and the second group of antibodies consists of antibody 2 and antibody 3; The heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 1 have the amino acid sequences shown in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively; the light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 1 have the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively. The heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 2 have the amino acid sequences shown in SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23, respectively, and the light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 2 have the amino acid sequences shown in SEQ ID NO:24, SEQ ID NO:25, and SEQ ID NO:26, respectively. The heavy chain complementarity-determining regions VH-CDR1, VH-CDR2, and VH-CDR3 of antibody 3 have amino acid sequences as shown in SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively; the light chain complementarity-determining regions VL-CDR1, VL-CDR2, and VL-CDR3 of antibody 3 have amino acid sequences as shown in SEQ ID NO:40, SEQ ID NO:41, and SEQ ID NO:42, respectively.

2. An antibody combination for detecting SARS-CoV-2, characterized in that, It includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies consists of antibody 1, and the second group of antibodies consists of antibody 2 and antibody 3; The heavy chain variable region of antibody 1 has the amino acid sequence shown in SEQ ID NO: 15, and the light chain variable region of antibody 1 has the amino acid sequence shown in SEQ ID NO:

17. The heavy chain variable region of antibody 2 has an amino acid sequence as shown in SEQ ID NO: 29, and the light chain variable region of antibody 2 has an amino acid sequence as shown in SEQ ID NO:

31. The variable region of the antibody 3 heavy chain has the amino acid sequence shown in SEQ ID NO: 45, and the variable region of the antibody 3 light chain has the amino acid sequence shown in SEQ ID NO:

47.

3. An antibody combination for detecting SARS-CoV-2, characterized in that, It includes a first group of antibodies and a second group of antibodies, wherein the first group of antibodies consists of antibody 1, and the second group of antibodies consists of antibody 2 and antibody 3; The heavy chain of antibody 1 has the amino acid sequence shown in SEQ ID NO: 18, and the light chain of antibody 1 has the amino acid sequence shown in SEQ ID NO:

20. The antibody 2 heavy chain has the amino acid sequence shown in SEQ ID NO: 33, and the antibody 2 light chain has the amino acid sequence shown in SEQ ID NO: 35; The antibody heavy chain 3 has the amino acid sequence shown in SEQ ID NO: 49, and the antibody light chain 3 has the amino acid sequence shown in SEQ ID NO:

52.

4. The antibody combination according to any one of claims 1 to 3, characterized in that, The first group of antibodies is used for labeling, and the second group of antibodies is used for coating; or the second group of antibodies is used for labeling, and the first group of antibodies is used for coating.

5. The antibody combination according to claim 4, characterized in that, The antibody used for labeling is labeled with a detectable marker, which includes one or more of the following: metal particles, fluorescent markers, chromophore markers, electron-dense markers, chemiluminescent markers, radioactive markers, enzyme markers, spin markers, and phage markers.

6. The antibody combination according to claim 5, characterized in that, The markers include any one or more of the following: colloidal gold, radioisotopes, fluorophores, luciferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucosylamylase, lysozyme, carbohydrate oxidase, glucose-6-phosphate dehydrogenase, biotin / avidin, and acridinium ester markers.

7. The antibody combination according to claim 5, characterized in that, The markers include any one or more of rhodamine and fluorescein.

8. The antibody combination according to claim 5, characterized in that, The markers include any one or more of glucose oxidase and galactose oxidase.

9. The antibody combination according to claim 4, characterized in that, The antibody used for coating is connected to a solid phase; the solid phase is one of the following: magnetic particles, latex particles, ELISA plates, microtiter plates, nitrocellulose membranes, or microfluidic chips.

10. A reagent kit, characterized in that, Includes the antibody combination as described in any one of claims 1-9.

11. The reagent kit according to claim 10, characterized in that, The kit is one of the following: immunochromatographic test strips, enzyme-linked immunosorbent assay (ELISA) reagents, and chemiluminescent reagents.

12. A SARS-CoV-2 immunochromatographic test strip, characterized in that, The immunochromatographic test strip includes a base plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The nitrocellulose membrane has T lines and C lines. The conjugate pad has a second group antibody as described in any one of claims 1-9, wherein the T lines are coated with a first group antibody as described in any one of claims 1-9; or, the conjugate pad has a first group antibody as described in any one of claims 1-9, and the T lines are coated with a second group antibody as described in any one of claims 1-9.

13. The use of an antibody combination according to any one of claims 1-9 in the preparation of a kit.

Images