Specific antibody of novel coronavirus n protein and application thereof

Abstract

This invention relates to antibodies or antigen-binding fragments thereof that specifically bind to the N protein of the novel coronavirus, and their applications. The antibodies or antigen-binding fragments of this invention exhibit improved detection sensitivity and specificity, and the colloidal gold test strips involved are convenient for rapid detection and operation, have low detection costs, and are widely applicable.

Description

Technical Field

[0001] This invention relates to the field of antibodies, specifically to an antibody against the N protein of a novel coronavirus and its preparation and application. Background Technology

[0002] The main structural proteins of the novel coronavirus (SARS-CoV-2) include the spike protein (S), envelope protein (E), nucleocapsid protein (N), and membrane protein (M). Much attention in COVID-19 drug and vaccine research has focused on the spike protein (S), which directly mediates the virus's infection of human cells. Furthermore, current antibody preparation, especially the preparation of traditional human antibodies, largely employs hybridoma technology, which generally suffers from long preparation cycles and low efficiency.

[0003] Currently, the market offers various detection methods for the novel coronavirus, including nucleic acid-based RT-PCR and NGS sequencing, antigen-based ELISA or colloidal gold immunochromatography, and virus-based cell culture. However, these methods have some limitations. For example, while RT-PCR is highly sensitive, it typically requires complex equipment and a long testing time, making it unsuitable for large-scale rapid screening and on-site testing. ELISA, while offering good quantitative capabilities, has a complex procedure requiring professional analysis, resulting in high costs. Most existing rapid test kits are based on immunochromatography or colloidal gold methods. Although convenient, they often suffer from insufficient sensitivity and specificity, especially due to differences in species, antibody conformation, and epitopes, frequently leading to inaccurate results and limiting their practical application. With changes in viral mutation and transmission speed, the demand for faster, more accurate, and simpler detection methods is constantly increasing. Summary of the Invention

[0004] To address one of the aforementioned technical problems in existing technologies, particularly the long cycle and low efficiency of traditional human antibody acquisition, this invention develops a new technique to isolate memory B cells targeting the SARS-CoV-2 N protein from the peripheral blood of COVID-19 patients. High-affinity human antibodies were successfully screened using phage surface display technology. These antibodies will serve as important raw materials for developing colloidal gold reagent kits to detect the SARS-CoV-2 N protein. Furthermore, through optimization and improvement of the traditional antibody preparation process, the preparation cycle of human antibodies has been significantly shortened to less than one month, greatly improving the efficiency of antibody screening and preparation. The N protein, as the most abundant viral structural protein during SARS-CoV-2 infection, is expressed in large quantities after viral infection of human cells, triggering a strong immune response. In addition, the N protein sequence is relatively conserved and stable, sharing approximately 90% homology with the SARS-CoV N protein, its closest lineage member; compared to the S protein, the N protein undergoes fewer mutations over time. Therefore, the N protein-based detection method not only helps in the early diagnosis of SARS-CoV-2 infection but also provides fundamental data for epidemiological research. Meanwhile, the new technical approach of this invention not only efficiently screens high-quality human antibodies with high affinity and stability, but also provides a foundation for subsequent antibody pairing. This invention utilizes this technical approach to develop a high-performance diagnostic kit for the SARS-CoV-2 N protein, providing a novel rapid detection kit for the novel coronavirus based on colloidal gold technology. Antibodies expressed using IgM as a carrier can bind more viral antigens, thereby efficiently and accurately detecting the SARS-CoV-2 N protein, greatly improving the sensitivity and specificity of the detection. The detection kit of this invention features a simple operating procedure and low production cost, facilitating applications such as on-site testing and large-scale screening.

[0005] The human antibody screening technology involved in this invention also shows broad application prospects in antibody drug development, biomedical research and other fields. It is expected to accelerate scientific research progress and achievement transformation in related fields, thereby promoting the development and clinical application of new treatment options and contributing to public health security.

[0006] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the N protein of the novel coronavirus (SARS-CoV-2), comprising: a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2 and HCDR3, and a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2 and LCDR3.

[0007] In some embodiments, the antibody or its antigen-binding fragment recognizes an antigenic epitope containing any one or a combination of amino acid residues R14, F17, G18, G19, S26, N27, R32, and S33 in the novel coronavirus N protein (amino acid sequence as shown in SEQ ID NO: 115).

[0008] In some embodiments, the antibody or its antigen-binding fragment recognizes an antigenic epitope containing amino acid residues R14 and F17 in SEQ ID NO: 115.

[0009] In some embodiments, the antibody or its antigen-binding fragment recognizes an antigenic epitope containing amino acid residues G18 and G19 in SEQ ID NO: 115.

[0010] In some embodiments, the HCDR1 is selected from: HCDR1 containing the amino acid sequence of SEQ ID NO:2, HCDR1 containing the amino acid sequence of SEQ ID NO:10, HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR1 containing the amino acid sequence of SEQ ID NO:26, HCDR1 containing the amino acid sequence of SEQ ID NO:34, HCDR1 containing the amino acid sequence of SEQ ID NO:42, HCDR1 containing the amino acid sequence of SEQ ID NO:50, HCDR1 containing the amino acid sequence of SEQ ID NO:58, HCDR1 containing the amino acid sequence of SEQ ID NO:66, and HCDR1 containing the amino acid sequence of SEQ ID NO:74.

[0011] In some embodiments, the HCDR2 is selected from: HCDR2 containing the amino acid sequence of SEQ ID NO:3, HCDR2 containing the amino acid sequence of SEQ ID NO:11, HCDR2 containing the amino acid sequence of SEQ ID NO:19, HCDR2 containing the amino acid sequence of SEQ ID NO:27, HCDR2 containing the amino acid sequence of SEQ ID NO:35, HCDR2 containing the amino acid sequence of SEQ ID NO:43, HCDR2 containing the amino acid sequence of SEQ ID NO:51, HCDR2 containing the amino acid sequence of SEQ ID NO:59, HCDR2 containing the amino acid sequence of SEQ ID NO:67, and HCDR2 containing the amino acid sequence of SEQ ID NO:75.

[0012] In some embodiments, the HCDR3 is selected from: HCDR3 containing the amino acid sequence of SEQ ID NO:4, HCDR3 containing the amino acid sequence of SEQ ID NO:12, HCDR3 containing the amino acid sequence of SEQ ID NO:20, HCDR3 containing the amino acid sequence of SEQ ID NO:28, HCDR3 containing the amino acid sequence of SEQ ID NO:36, HCDR3 containing the amino acid sequence of SEQ ID NO:44, HCDR3 containing the amino acid sequence of SEQ ID NO:52, HCDR3 containing the amino acid sequence of SEQ ID NO:60, HCDR3 containing the amino acid sequence of SEQ ID NO:68, and HCDR3 containing the amino acid sequence of SEQ ID NO:76.

[0013] In some embodiments, at least one of HCDR1, HCDR2 and HCDR3 contains a mutation, which is a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of one, two or three amino acids or any combination thereof).

[0014] In some embodiments, the LCDR1 is selected from:

[0015] LCDR1 containing the amino acid sequence of SEQ ID NO:6, LCDR1 containing the amino acid sequence of SEQ ID NO:14, LCDR1 containing the amino acid sequence of SEQ ID NO:22, LCDR1 containing the amino acid sequence of SEQ ID NO:30, LCDR1 containing the amino acid sequence of SEQ ID NO:38, LCDR1 containing the amino acid sequence of SEQ ID NO:46, LCDR1 containing the amino acid sequence of SEQ ID NO:54, LCDR1 containing the amino acid sequence of SEQ ID NO:62, LCDR1 containing the amino acid sequence of SEQ ID NO:70, and LCDR1 containing the amino acid sequence of SEQ ID NO:78.

[0016] In some embodiments, the LCDR2 is selected from:

[0017] LCDR2 containing the amino acid sequence AAS, LCDR2 containing the amino acid sequence RNK, LCDR2 containing the amino acid sequence GAS, LCDR2 containing the amino acid sequence QNN, LCDR2 containing the amino acid sequence SNN, LCDR2 containing the amino acid sequence KAS, LCDR2 containing the amino acid sequence DVT, LCDR2 containing the amino acid sequence ATS, LCDR2 containing the amino acid sequence VGS, and LCDR2 containing the amino acid sequence NNN.

[0018] In some embodiments, the LCDR3 is selected from:

[0019] LCDR3 containing the amino acid sequence of SEQ ID NO:8, LCDR3 containing the amino acid sequence of SEQ ID NO:16, LCDR3 containing the amino acid sequence of SEQ ID NO:24, LCDR3 containing the amino acid sequence of SEQ ID NO:32, LCDR3 containing the amino acid sequence of SEQ ID NO:40, LCDR3 containing the amino acid sequence of SEQ ID NO:48, LCDR3 containing the amino acid sequence of SEQ ID NO:56, LCDR3 containing the amino acid sequence of SEQ ID NO:64, LCDR3 containing the amino acid sequence of SEQ ID NO:72, and LCDR3 containing the amino acid sequence of SEQ ID NO:80.

[0020] In some embodiments, at least one of LCDR1, LCDR2 and LCDR3 contains a mutation, which is a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of one, two or three amino acids or any combination thereof).

[0021] In some embodiments, HCDR1, HCDR2, and HCDR3 are selected from:

[0022] (1) HCDR1 containing the amino acid sequence of SEQ ID NO:2, HCDR2 containing the amino acid sequence of SEQ ID NO:3, and HCDR3 containing the amino acid sequence of SEQ ID NO:4;

[0023] (2) HCDR1 containing the amino acid sequence of SEQ ID NO:10, HCDR2 containing the amino acid sequence of SEQ ID NO:11, and HCDR3 containing the amino acid sequence of SEQ ID NO:12;

[0024] (3) HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR2 containing the amino acid sequence of SEQ ID NO:19, and HCDR3 containing the amino acid sequence of SEQ ID NO:20;

[0025] (4) HCDR1 containing the amino acid sequence of SEQ ID NO:26, HCDR2 containing the amino acid sequence of SEQ ID NO:27, and HCDR3 containing the amino acid sequence of SEQ ID NO:28;

[0026] (5) HCDR1 containing the amino acid sequence of SEQ ID NO:34, HCDR2 containing the amino acid sequence of SEQ ID NO:35, and HCDR3 containing the amino acid sequence of SEQ ID NO:36;

[0027] (6) HCDR1 containing the amino acid sequence of SEQ ID NO:42, HCDR2 containing the amino acid sequence of SEQ ID NO:43, and HCDR3 containing the amino acid sequence of SEQ ID NO:44;

[0028] (7) HCDR1 containing the amino acid sequence of SEQ ID NO:50, HCDR2 containing the amino acid sequence of SEQ ID NO:51, and HCDR3 containing the amino acid sequence of SEQ ID NO:52;

[0029] (8) HCDR1 containing the amino acid sequence of SEQ ID NO:58, HCDR2 containing the amino acid sequence of SEQ ID NO:59, and HCDR3 containing the amino acid sequence of SEQ ID NO:60;

[0030] (9) HCDR1 containing the amino acid sequence of SEQ ID NO:66, HCDR2 containing the amino acid sequence of SEQ ID NO:67, and HCDR3 containing the amino acid sequence of SEQ ID NO:68; and

[0031] (10) HCDR1 containing the amino acid sequence of SEQ ID NO:74, HCDR2 containing the amino acid sequence of SEQ ID NO:75, and HCDR3 containing the amino acid sequence of SEQ ID NO:76;

[0032] (11) HCDR1, HCDR2 and HCDR3 as described in (1) to (10), wherein at least one CDR contains a mutation, said mutation being a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of one, two or three amino acids or any combination thereof).

[0033] In some embodiments, LCDR1, LCDR2, and LCDR3 are selected from:

[0034] (12) LCDR1 containing the amino acid sequence of SEQ ID NO:6, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:8;

[0035] (13) LCDR1 containing the amino acid sequence of SEQ ID NO:14, LCDR2 containing the amino acid sequence RNK, and LCDR3 containing the amino acid sequence of SEQ ID NO:16;

[0036] (14) LCDR1 containing the amino acid sequence of SEQ ID NO:22, LCDR2 containing the amino acid sequence GAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:24;

[0037] (15) LCDR1 containing the amino acid sequence of SEQ ID NO:30, LCDR2 containing the amino acid sequence QNN, and LCDR3 containing the amino acid sequence of SEQ ID NO:32;

[0038] (16) LCDR1 containing the amino acid sequence of SEQ ID NO:38, LCDR2 containing the amino acid sequence SNN, and LCDR3 containing the amino acid sequence of SEQ ID NO:40;

[0039] (17) LCDR1 containing the amino acid sequence of SEQ ID NO:46, LCDR2 containing the amino acid sequence KAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:48;

[0040] (18) LCDR1 containing the amino acid sequence of SEQ ID NO:54, LCDR2 containing the amino acid sequence DVT, and LCDR3 containing the amino acid sequence of SEQ ID NO:56;

[0041] (19) LCDR1 containing the amino acid sequence of SEQ ID NO:62, LCDR2 containing the amino acid sequence ATS, and LCDR3 containing the amino acid sequence of SEQ ID NO:64;

[0042] (20) LCDR1 containing the amino acid sequence of SEQ ID NO:70, LCDR2 containing the amino acid sequence VGS, and LCDR3 containing the amino acid sequence of SEQ ID NO:72;

[0043] (21) LCDR1 containing the amino acid sequence of SEQ ID NO:78, LCDR2 containing the amino acid sequence NNN, and LCDR3 containing the amino acid sequence of SEQ ID NO:80;

[0044] (22) (12) to (21) LCDR1, LCDR2 and LCDR3, wherein at least one CDR contains a mutation, the mutation being a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g. a substitution, deletion or addition of one, two or three amino acids or any combination thereof).

[0045] In some embodiments, the antibody or its antigen-binding fragment comprises: a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3, wherein:

[0046] HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 are selected from:

[0047] (1) HCDR1 containing the amino acid sequence of SEQ ID NO:2, HCDR2 containing the amino acid sequence of SEQ ID NO:3, HCDR3 containing the amino acid sequence of SEQ ID NO:4, LCDR1 containing the amino acid sequence of SEQ ID NO:6, LCDR2 containing the amino acid sequence AAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:8.

[0048] (2) HCDR1 containing the amino acid sequence of SEQ ID NO:10, HCDR2 containing the amino acid sequence of SEQ ID NO:11, HCDR3 containing the amino acid sequence of SEQ ID NO:12, LCDR1 containing the amino acid sequence of SEQ ID NO:14, LCDR2 containing the amino acid sequence RNK, and LCDR3 containing the amino acid sequence of SEQ ID NO:16.

[0049] (3) HCDR1 containing the amino acid sequence of SEQ ID NO:18, HCDR2 containing the amino acid sequence of SEQ ID NO:19, HCDR3 containing the amino acid sequence of SEQ ID NO:20, LCDR1 containing the amino acid sequence of SEQ ID NO:22, LCDR2 containing the amino acid sequence GAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:24.

[0050] (4) HCDR1 containing the amino acid sequence of SEQ ID NO:26, HCDR2 containing the amino acid sequence of SEQ ID NO:27, HCDR3 containing the amino acid sequence of SEQ ID NO:28, LCDR1 containing the amino acid sequence of SEQ ID NO:30, LCDR2 containing the amino acid sequence QNN, and LCDR3 containing the amino acid sequence of SEQ ID NO:32.

[0051] (5) HCDR1 containing the amino acid sequence of SEQ ID NO:34, HCDR2 containing the amino acid sequence of SEQ ID NO:35, HCDR3 containing the amino acid sequence of SEQ ID NO:36, LCDR1 containing the amino acid sequence of SEQ ID NO:38, LCDR2 containing the amino acid sequence SNN, and LCDR3 containing the amino acid sequence of SEQ ID NO:40.

[0052] (6) HCDR1 containing the amino acid sequence of SEQ ID NO:42, HCDR2 containing the amino acid sequence of SEQ ID NO:43, HCDR3 containing the amino acid sequence of SEQ ID NO:44, LCDR1 containing the amino acid sequence of SEQ ID NO:46, LCDR2 containing the amino acid sequence KAS, and LCDR3 containing the amino acid sequence of SEQ ID NO:48.

[0053] (7) HCDR1 containing the amino acid sequence of SEQ ID NO:50, HCDR2 containing the amino acid sequence of SEQ ID NO:51, HCDR3 containing the amino acid sequence of SEQ ID NO:52, LCDR1 containing the amino acid sequence of SEQ ID NO:54, LCDR2 containing the amino acid sequence DVT, and LCDR3 containing the amino acid sequence of SEQ ID NO:56.

[0054] (8) HCDR1 containing the amino acid sequence of SEQ ID NO:58, HCDR2 containing the amino acid sequence of SEQ ID NO:59, HCDR3 containing the amino acid sequence of SEQ ID NO:60, LCDR1 containing the amino acid sequence of SEQ ID NO:62, LCDR2 containing the amino acid sequence ATS, and LCDR3 containing the amino acid sequence of SEQ ID NO:64.

[0055] (9) HCDR1 containing the amino acid sequence of SEQ ID NO:66, HCDR2 containing the amino acid sequence of SEQ ID NO:67, HCDR3 containing the amino acid sequence of SEQ ID NO:68, LCDR1 containing the amino acid sequence of SEQ ID NO:70, LCDR2 containing the amino acid sequence VGS, and LCDR3 containing the amino acid sequence of SEQ ID NO:72.

[0056] (10) HCDR1 containing the amino acid sequence of SEQ ID NO:74, HCDR2 containing the amino acid sequence of SEQ ID NO:75, HCDR3 containing the amino acid sequence of SEQ ID NO:76, LCDR1 containing the amino acid sequence of SEQ ID NO:78, LCDR2 containing the amino acid sequence NNN, and LCDR3 containing the amino acid sequence of SEQ ID NO:80.

[0057] (11) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in (1) to (10), wherein at least one CDR contains a mutation, the mutation being a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., substitution, deletion or addition of 1, 2 or 3 amino acids or any combination thereof); the antibody containing the mutation or its antigen-binding fragment can still specifically bind to the novel coronavirus N protein.

[0058] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL):

[0059] The heavy chain variable region contains an amino acid sequence selected from any one of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions, or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids, or any combination thereof), or an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it, and / or

[0060] The light chain variable region contains an amino acid sequence selected from any one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, or an amino acid sequence having one or more amino acid substitutions, deletions, or additions or any combination thereof (e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids or any combination thereof), or an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with it.

[0061] In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region (VH) and the light chain variable region (VL) are selected from:

[0062] (1) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:1 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:5;

[0063] (2) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:9 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:13;

[0064] (3) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:17 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:21;

[0065] (4) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:25 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:29;

[0066] (5) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:33 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:37;

[0067] (6) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:41 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:45;

[0068] (7) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:49 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:53;

[0069] (8) Containing a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:57 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:61;

[0070] (9) Containing a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:65 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:69; and

[0071] (10) Contains a heavy chain variable region (VH) of the amino acid series shown in SEQ ID NO:73 and a light chain variable region (VL) of the amino acid series shown in SEQ ID NO:77;

[0072] Or, the VH and / or VL contained in the antibody or its antigen-binding fragment have one or more amino acid substitutions, deletions or additions or any combination thereof (e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids or any combination thereof) compared to the VH and / or VL described in (1) to (10); preferably, the substitution is a conservative substitution.

[0073] Or, the amino acid sequence of VH and / or VL contained in the antibody or its antigen-binding fragment has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of VH and / or VL described in (1) to (10).

[0074] In some embodiments, the antigen-binding fragment is selected from Fab, Fab', F(ab)2, F(ab')2, scFv, disulfide-linked Fv (dsFv), or single-domain antibodies. In some preferred embodiments, the antigen-binding fragment is scFv.

[0075] In some embodiments, the antibody or its antigen-binding fragment may be a full-length antibody, such as immunoglobulin G (IgG), IgM, IgE, IgA, or IgD.

[0076] In some embodiments, the antibody or its antigen-binding fragment comprises a constant region of IgG1, IgG2, IgG3, or IgG4, or a variant thereof.

[0077] In some embodiments, the antibody or its antigen-binding fragment comprises:

[0078] (1) The CH (heavy chain constant region) of human immunoglobulin or a variant thereof, said variant having one or more amino acid substitutions, deletions, or additions compared to its derived wild-type sequence (e.g., substitutions, deletions, or additions of up to 20, 15, 10, or 5 amino acids; e.g., substitutions, deletions, or additions of 1, 2, 3, 4, or 5 amino acids); and / or,

[0079] (2) The CL (light chain constant region) of human immunoglobulin or a variant thereof, the variant having one or more amino acid substitutions, deletions or additions compared to the wild-type sequence from which it is derived (e.g., substitutions, deletions or additions of up to 20, up to 15, up to 10 or up to 5 amino acids; e.g., substitutions, deletions or additions of 1, 2, 3, 4 or 5 amino acids).

[0080] In some implementations, CH is an IgM heavy chain constant region, such as the IgM1 or IgM2 heavy chain constant region.

[0081] In some embodiments, the antibody comprises the heavy chain constant region of human IgM1.

[0082] In some embodiments, the antibody comprises the J-chain region of human IgM.

[0083] In some embodiments, the antibody comprises the amino acid sequence shown in SEQ ID NO: 90.

[0084] In some embodiments, the antibody comprises a signal peptide having the amino acid sequence shown in SEQ ID NO: 89.

[0085] In some implementations, the CL is a constant region of the κ or λ light chain.

[0086] In some embodiments, the antibody or its antigen-binding fragment is a fully human antibody.

[0087] In a second aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a heavy chain variable region and / or a light chain variable region encoding an antibody or an antigen-binding fragment thereof as described in the first aspect.

[0088] In some embodiments, the nucleic acid molecule comprises:

[0089] (i) A nucleotide sequence containing SEQ ID NO: 91 and a nucleotide sequence containing SEQ ID NO: 92;

[0090] (ii) A nucleotide sequence containing SEQ ID NO: 93 and a nucleotide sequence containing SEQ ID NO: 94;

[0091] (iii) A nucleotide sequence containing SEQ ID NO: 95 and a nucleotide sequence containing SEQ ID NO: 96;

[0092] (iv) The nucleotide sequence containing SEQ ID NO: 97 and the nucleotide sequence containing SEQ ID NO: 98;

[0093] (v) A nucleotide sequence containing SEQ ID NO: 99 and a nucleotide sequence containing SEQ ID NO: 100;

[0094] (vi) A nucleotide sequence containing SEQ ID NO: 101 and a nucleotide sequence containing SEQ ID NO: 102;

[0095] (vii) The nucleotide sequence containing SEQ ID NO: 103 and the nucleotide sequence containing SEQ ID NO: 104;

[0096] (viii) A nucleotide sequence containing SEQ ID NO: 105 and a nucleotide sequence containing SEQ ID NO: 106;

[0097] (ix) The nucleotide sequence containing SEQ ID NO: 107 and the nucleotide sequence containing SEQ ID NO: 108;

[0098] (x) Contains a nucleotide sequence of SEQ ID NO: 109 and a nucleotide sequence of SEQ ID NO: 110; or

[0099] (xi) The nucleic acid molecule contains a nucleotide sequence that has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the nucleotide sequence described in (ix).

[0100] Thirdly, the present invention provides an expression vector comprising the isolated nucleic acid molecules described in the second aspect.

[0101] Fourthly, the present invention provides a host cell comprising the isolated nucleic acid molecule described in the second aspect and the vector described in the third aspect.

[0102] Fifthly, the present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described in the first aspect, the nucleic acid molecule as described in the second aspect, the expression vector as described in the third aspect, the host cell as described in the fourth aspect, and a pharmaceutically acceptable carrier.

[0103] In a sixth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect, the nucleic acid molecule described in the second aspect, the expression vector described in the third aspect, the host cell described in the fourth aspect, and the pharmaceutical composition described in the fifth aspect in the preparation of a medicament for use in neutralizing SARS-CoV-2 virus in a subject, preventing and / or treating SARS-CoV-2 infection or diseases associated with SARS-CoV-2 infection.

[0104] In some embodiments, the SARS-CoV-2 includes mutant strains. In some embodiments, the N protein of the mutant strain may contain mutations, such as one or more (e.g., 1, 2, 3, 4, or 5) amino acid substitutions, deletions, or additions. In some embodiments, the mutant strain is selected from Delta strains (B.1.617.2), Alpha strains (e.g., B.1.1.7), Beta strains (e.g., B.1.351), Gamma strains (e.g., P.1), Delta strains (e.g., B.1.617.2), Omicron strains (e.g., B.1.1.529, BA.5.2.1, BA.2.86, BQ.1, KP.3, XDV, LB.1), or any combination thereof.

[0105] In some implementations, the subject is a mammal, such as a human.

[0106] In some embodiments, the antibody or its antigen-binding fragment or pharmaceutical composition is used alone or in combination with other pharmaceutically active agents (e.g., other antiviral agents such as interferon, lopinavir, ritonavir, remdesivir, dexamethasone, etc.).

[0107] In some implementations, the disease associated with SARS-CoV-2 infection is novel coronavirus infection.

[0108] In some implementations, the disease associated with SARS-CoV-2 infection is severe acute respiratory syndrome.

[0109] In a seventh aspect, the present invention provides a reagent for detecting the presence or level of SARS-CoV-2 in biological samples.

[0110] In some embodiments, the reagent comprises the antibody or antigen-binding fragment thereof described in the first aspect.

[0111] In some embodiments, the biological samples are selected from nasal and pharyngeal swab test samples, as well as liquid samples and tissue specimens such as serum, whole blood, sputum, oral / nasopharyngeal secretions or washings, urine, feces, pleural and peritoneal effusions, and cerebrospinal fluid.

[0112] In some embodiments, the reagent comprises at least two antibodies or antigen-binding fragments thereof as described in the first aspect.

[0113] In some embodiments, the reagent comprises an antigen capture agent and / or an antigen detection agent, each independently selected from the antibodies or antigen-binding fragments thereof described in the first aspect.

[0114] In some embodiments, the reagent comprises an antigen capture agent and an antigen detection agent selected from:

[0115] (a) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 65 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 69 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 1 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 5 as the light chain variable region (VL);

[0116] (b) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 65 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 69 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 33 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 37 as the light chain variable region (VL);

[0117] (c) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 65 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 69 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 73 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 77 as the light chain variable region (VL).

[0118] (d) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 57 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 61 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 73 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 77 as the light chain variable region (VL).

[0119] (e) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 1 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 5 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 33 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 37 as the light chain variable region (VL); or

[0120] (f) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 1 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 5 as the light chain variable region (VL), and an antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 41 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 45 as the light chain variable region (VL).

[0121] In some embodiments, the antigen detection agent comprises a detectable marker, preferably selected from magnetic beads, fluorescent dyes, radioactive isotopes, enzymes, biotin, metals, or any combination thereof.

[0122] In some embodiments, the reagent is an immune sandwich assay reagent or kit.

[0123] In some embodiments, the reagent further includes a solid support, and the antigen capture agent is attached to the solid support.

[0124] In some embodiments, the solid support is selected from, for example, well plates, test tubes, dip rods, microcentrifuge tubes, beads, centrifugable discs, permeable or semi-permeable membranes, etc. The beads are selected from magnetic beads, plastic beads, ceramic beads, glass beads, polystyrene beads, methylstyrene beads, acrylic polymer beads, carbon graphite beads, titanium dioxide beads, latex or cross-linked dextran, cellulose beads, nylon beads, or cross-linked micelles. Suitable solid support materials include glass, plastics (e.g., polyethylene, PVC, polypropylene, polystyrene, etc.), proteins, paper, carbohydrates, lipid monolayers or supported lipid bilayers, films, or any combination thereof. Other materials that can be used include ceramics, metals, metalloids, semiconductor materials, etc.

[0125] In some embodiments, the reagent is an enzyme-linked immunosorbent assay (ELISA) reagent comprising an antigen capture agent and / or an enzyme-labeled antigen detection agent. The antigen capture agent and / or enzyme-labeled antigen detection agent are independently selected from one or more of the antibodies or antigen-binding fragments thereof described in the first aspect.

[0126] In some embodiments, the labeling enzyme on the enzyme-labeled antibody is selected from horseradish peroxidase, alkaline phosphatase, and glucose oxidase, or any combination thereof.

[0127] In some embodiments, the reagent is a lateral flow immunochromatographic plate comprising an antigen capture agent and / or a metal-labeled antigen detection agent, wherein the antigen capture agent and / or the metal-labeled antigen detection agent are attached to a membrane.

[0128] In some embodiments, the membrane is made of a material selected from mixed cellulose, nitrocellulose, nitrocellulose, glass cellulose, cotton, woven web, nonwoven material, porous plastic, polymer, polyester, or any combination thereof.

[0129] Eighthly, the present invention provides a method for detecting the presence or level of SARS-CoV-2 in biological samples.

[0130] In some embodiments, the method includes contacting the biological sample with the antibody or antigen-binding fragment described in the first aspect, and detecting the binding of the antibody or antigen-binding fragment to SARS-CoV-2, thereby detecting the presence or level of SARS-CoV-2 in the biological sample.

[0131] In some embodiments, the method uses the reagents described in the seventh aspect.

[0132] In some embodiments, the biological samples are selected from nasal and pharyngeal swab test samples, as well as liquid samples and tissue specimens such as serum, whole blood, sputum, oral / nasopharyngeal secretions or washings, urine, feces, pleural and peritoneal effusions, and cerebrospinal fluid.

[0133] In some implementations, the method is an immunological detection method.

[0134] In some embodiments, the method is selected from: ELISA, liquid microarray, immunofluorescence, immunohistochemistry, immunochromatography, flow cytometry, cell sorting, radioimmunoassay, immunodiffusion, immunoprecipitation, and / or Western blotting.

[0135] In some preferred embodiments, the method is immunochromatography.

[0136] In a ninth aspect, the present invention provides the use of the antibody or antigen-binding fragment thereof described in the first aspect in the preparation of a reagent for detecting the presence or level of SARS-CoV-2 in a biological sample.

[0137] In a tenth aspect, the present invention provides a method for screening antibodies or antigen-binding fragments thereof that specifically bind to the novel coronavirus (SARS-CoV-2), comprising:

[0138] An antibody display library against the novel coronavirus and the N protein of SARS-CoV-2 are provided. The antibody display library is contacted with the N protein, and antibodies or antigen-binding fragments that specifically bind to the novel coronavirus (SARS-CoV-2) are screened for results.

[0139] In some embodiments, the method includes obtaining peripheral blood mononuclear cells (PBMCs) from a person infected with the novel coronavirus and generating an antibody display library expressing antibody fragments using cDNA derived from the PBMCs.

[0140] In some embodiments, the antibody display library is selected from phage display libraries, yeast display libraries, ribosome display libraries, and mammalian cell display libraries.

[0141] In some preferred embodiments, the antibody display library is a phage display library.

[0142] In some embodiments, the antigen-binding fragment includes a variable region fragment of the scFv heavy chain and / or light chain.

[0143] In some embodiments, the method includes amplifying cDNA derived from the PBMC to obtain a heavy chain variable region amplification product, a light chain λ variable region amplification product, and a light chain κ variable region amplification product, and ligating the heavy chain variable region amplification product and the light chain variable region amplification product to an expression vector to obtain an expression vector for the scFv fragment.

[0144] In some embodiments, the expression vector is selected from phage vectors.

[0145] Eleventhly, the present invention provides a method for preparing human IgM antibodies that specifically bind to the novel coronavirus (SARS-CoV-2), comprising:

[0146] The antigen-binding fragment obtained from the tenth screening is provided, and the antigen-binding fragment is expressed in a human IgM expression vector to obtain a human IgM antibody that specifically binds to the novel coronavirus (SARS-CoV-2).

[0147] In some embodiments, the method includes obtaining an amplified product of the heavy chain variable region of the antigen-binding fragment, ligating it into an IgM expression vector to obtain an IgM antibody heavy chain expression vector.

[0148] In some embodiments, the method includes co-transferring an IgM antibody heavy chain variable region expression vector and an antibody light chain variable region expression vector into host cells for expression to obtain a pentamer IgM antibody.

[0149] In some embodiments, the IgM antibody is a human antibody.

[0150] Beneficial effects of the present invention

[0151] 1. Improve detection sensitivity:

[0152] Based on high-affinity human antibody IgM, it can effectively capture the N protein of the SARS-CoV-2 virus, achieving accurate detection even at low concentrations. This will significantly improve the ability to screen for SARS-CoV-2 virus infection at an early stage, thereby helping to intervene and control potential infection spread in a timely manner.

[0153] 2. High detection specificity:

[0154] Choosing antibodies targeting different epitopes of the N protein reduces the risk of cross-reactivity and improves the specificity of the test. This ensures the reliability of the test results and reduces the occurrence of false positives.

[0155] 3. Rapid detection and easy operation:

[0156] The colloidal gold test strip design makes the entire testing process simple and intuitive, providing results in just a few minutes, making it suitable for field applications. This feature greatly improves testing efficiency.

[0157] 4. Cost-effectiveness:

[0158] Compared to traditional RT-PCR and ELISA methods, the colloidal gold test kit produced using the antibodies of this invention offers a more cost-effective testing option, reducing the cost per test, which is particularly important for large-scale screening. This will help public health departments implement effective surveillance with limited resources.

[0159] 5. Wide applicability:

[0160] The reagents of this invention are not limited to laboratory environments and can be applied to various scenarios such as hospitals, clinics, community testing sites, and epidemiological investigations, thereby improving the flexibility and accessibility of public health monitoring.

[0161] 6. Support for follow-up research:

[0162] The high-quality human antibodies screened during the development of this invention are not only applicable to the detection kit of this invention, but can also provide a foundation for the detection of other viruses (such as variants), the development of therapeutic drugs and vaccines, and promote technological progress and achievement transformation in related fields.

[0163] 7. Meet regulatory requirements:

[0164] This invention conforms to international standards in its design and production, ensuring the safety and effectiveness of the reagent kit and facilitating its market approval. This will further expand the product's market application and promotion. Attached Figure Description

[0165] Figure 1 A flowchart for antibody pair sequence discovery is shown.

[0166] Figure 2 The diagram shows the roadmap for constructing scFv library fragments.

[0167] Figure 3 The image shows a PCR electrophoresis diagram of the antibody variable region gene.

[0168] Figure 4 An electrophoresis image of a portion of the G4S Linker PCR fragment is shown.

[0169] Figure 5 The electrophoresis diagram of the scFv overlap extension PCR fragment is shown.

[0170] Figure 6 The diagram shows the amplified phage particles and the detection of their potency.

[0171] Figure 7 The results of partial phage monoclonal ELISA and sequencing analysis are shown.

[0172] Figure 8 Maps of mammalian cell IgG1 and IgK expression vectors are shown.

[0173] Figure 9 The results of SDS-PAGE electrophoresis of the purified different IgG antibodies are shown.

[0174] Figure 10 The results of ELISA testing show the affinity of some screening antibodies for the SARS-CoV-2 N protein antigen.

[0175] Figure 11 The results of BLI assays show the affinity of some screening antibodies for the SARS-CoV-2 N protein antigen.

[0176] Figure 12 The results of the verification of the colloidal gold test strip for detecting COVID-19 N1 virus prepared by screening antibodies are shown.

[0177] Figure 13 A map of mammalian cell IgM expression vectors is shown.

[0178] Figure 14 The SDS-PAGE electrophoresis results of the purified IgM antibodies are shown.

[0179] Figure 15 The results of the comparative detection of affinity between IgG antibody and IgM antibody and SARS-CoV-2 N antigen are shown.

[0180] Figure 16 The results of the comparative detection of IgM capture antibody and IgG capture antibody are shown. Detailed Implementation

[0181] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0182] definition

[0183] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0184] Unless the context clearly indicates otherwise, references to a specific quantity herein include their plural forms. For example, the term "cell" includes one or more such cells and equivalents known to those skilled in the art, etc.

[0185] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0186] The term "novel coronavirus (SARS-CoV-2)" as used in this article, abbreviated as COVID-19, refers to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), including its variants, as defined by the World Health Organization (WHO). SARS-CoV-2 belongs to the genus *Betacoronavirus*, subgenus *Sarbecovirus*, within the family Coronaviridae. It is an enveloped, non-segmented, positive-sense, single-stranded RNA virus. The SARS-CoV-2 genome encodes four major structural proteins: spike protein (S), envelope protein (E), nucleoprotein (N), and membrane protein (M). The spike glycoprotein binds to the ACE-2 receptor to enter cells.

[0187] As used in this article, "novel coronavirus infection" refers to the infectious respiratory illness caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Common symptoms of novel coronavirus infection may include fever, cough, fatigue, shortness of breath, and loss of smell and taste. Some symptoms can develop into viral pneumonia, multiple organ failure, or a cytokine storm. The disease is primarily transmitted through close person-to-person contact, such as through droplets produced by coughing, sneezing, and talking.

[0188] The "N protein of the novel coronavirus" used in this article refers to the core protein of SARS-CoV-2. As one of the most important proteins within the viral nucleocapsid, the N protein is primarily responsible for RNA replication. The N protein intertwines with the viral genomic RNA to form the viral nucleocapsid, playing a crucial role in viral RNA synthesis. Furthermore, the N protein is relatively conserved, constitutes the largest proportion of the virus's structural proteins, and the body can produce high levels of antibodies against the N protein in the early stages of infection.

[0189] Antibody or its antigen-binding fragment

[0190] As used in this article, "antibody" refers to an antibody and any antigen-binding fragment or its single chain. Antibodies typically consist of a glycoprotein containing one or more heavy chains (H) linked by disulfide bonds and one or more light chains (L), or its antigen-binding portion. Immunoglobulin light chains are generally classified as κ or λ. Immunoglobulin heavy chains are classified as γ, μ, α, δ, or ε, thus determining the immunoglobulin type, which is IgG, IgM, IgA, IgD, and IgE, respectively. Several major antibody classes are further subdivided into subclasses, such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). The basic four-chain IgG antibody unit is a heterotetrameric glycoprotein, typically approximately 150 kDa. IgM antibodies consist of five basic heterotetrameric units along with an additional polypeptide chain called the J chain, and contain 10 antigen-binding sites.

[0191] The term "variable region" as used in this article refers to a segment of the IgG chain whose sequence varies between different antibodies. The variable region of the heavy chain can be referred to as "VH," and the variable region of the light chain as "VL." Typically, the variable regions of both the heavy and light chains contain three hypervariable regions, also known as complementarity-determining regions (CDRs), located within relatively conserved frame regions (FRs). These CDRs are usually arranged within the frame regions to enable the binding of specific epitopes. Typically, from the N-terminus to the C-terminus, the variable domains of both the light and heavy chains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0192] As used herein, "complementarity-determining regions (CDRs)" refer to the amino acid residues in the variable region of an antibody responsible for antigen binding. A CDR is one of three hypervariable regions (H1, H2, or H3) within the non-framework region of the antibody VH β-sheet frame, or one of three hypervariable regions (L1, L2, or L3) within the non-framework region of the antibody VL β-sheet frame. Therefore, a CDR is a variable region sequence scattered within the framework region sequence. The definition of a CDR region is known to those skilled in the art and has been defined, for example, by Kabat as the most highly variable region within the antibody variable domain. Chothia also structurally defines a CDR region sequence as those residues that are not conserved β-sheet frame portions and are therefore adaptable to different conformations. Both definitions are known in the art. IMGT has also defined CDR region sequences. The relationships between numbering systems, including, for example, the Kabat numbering and the IMGT numbering systems, are well known to those skilled in the art. In some embodiments, the CDR region sequence is defined by the Kabat numbering system. In some embodiments, the CDR region sequence is defined by the Chothia numbering system. In some implementations, the CDR region sequence is preferably defined by the IMGT numbering system.

[0193] The term "frame" or "frame region" as used in this paper refers to the region remaining after subtracting the CDR from the variable region. Because the precise definition of the CDR sequence can be determined using different systems, the meaning of the frame sequence requires correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 for the light chain and CDR-H1, CDR-H2, and CDR-H3 for the heavy chain) further divide the frame region on both the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain. CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. As used in this paper, FR represents one of the four sub-regions, or FR represents two or more of the four sub-regions that constitute the frame region.

[0194] The antibodies of this invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies (scFv), scFv dimers, multispecific antibodies formed from a portion of an antibody containing one or more CDRs, camelified single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, humanized antibodies and human antibodies, or any other antibody fragment capable of binding to the same antigen as the parent antibody or a fragment of the parent antibody (e.g., the parental scFv) but not containing a complete antibody structure.

[0195] As used herein, the term "antigen-binding moiety" (or "antigen-binding fragment") of an antibody refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (e.g., SARS-CoV-2). It has been shown that fragments of full-length antibodies can perform the antigen-binding function. Examples of binding fragments encompassed within the "antigen-binding moiety" of an antibody include: (i) Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab′)2 fragments, which are bivalent fragments comprising two Fab fragments linked by disulfide bonds in the hinge region; (iii) Fd fragments, which consist of VH and CH1 domains; (iv) Fv fragments, which consist of VL and VH domains on a single arm of the antibody; (v) a single domain or dAb fragment consisting of a VH domain; and (vi) separate complementarity-determining regions (CDRs), or (vii) combinations of two or more separate CDRs optionally linked by synthetic linkers. These antibody fragments can be obtained using conventional techniques known to those skilled in the art, and can be screened for utilization in the same manner as intact antibodies. Antigen-binding moieties can be prepared using recombinant DNA technology or by enzymatic or chemical cleavage of intact immunoglobulins.

[0196] As used herein, "Fv" refers to a dimer (VH-VL dimer) composed of a tightly, non-covalently linked heavy chain and a light chain variable region, wherein the variable regions may be identical or different, and the heavy chain variable region is linked, bound, fused, or covalently connected to the light chain variable region. An Fv is the smallest antibody fragment containing all antigen recognition and binding sites. An Fv can be a single-chain Fv (scFv) or a disulfide-stabilized Fv (dsFv). In some embodiments, the antigen-binding fragment may be a single-chain antibody (scFv). scFv antibodies may include a heavy chain (VH) fragment and a light chain (VL) fragment, which may be directly linked or linked via flexible peptide linkers (e.g., 5, 10, 15, 20, 25 amino acids), the linkers typically being glycine-rich for flexibility and serine or threonine-rich for solubility. In some specific embodiments, the linker is a G4S linker. In some embodiments, the scFv antibody may be VH→VL oriented (N-terminus to C-terminus). In some other embodiments, the scFv antibody may be VL→VH oriented (N-terminus to C-terminus). In some embodiments, the three CDRs in each variable region interact to define the antigen-binding site on the surface of the VH-VL dimer. Therefore, the scFv antibody may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH.

[0197] The six CDRs collectively confer antibody antigen-binding specificity. In some embodiments, the heavy chain of any anti-N protein antibody described herein may further include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or combinations thereof). Alternatively or additionally, the light chain of any anti-N protein antibody described herein may further include a light chain constant region (CL), which may be any CL known in the art. In some embodiments, the CL is a κ light chain. In other embodiments, the CL is a λ light chain. The antibody heavy chain and light chain constant region have meanings well known in the art.

[0198] As used herein, "whole antibody" refers to an antibody composed of two "heavy chains" and two "light chains." The "heavy chain" consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain in the direction from the N-terminus to the C-terminus; and, optionally, when the whole antibody is an IgE isotype, it also includes a heavy chain constant region CH4 domain. Preferably, the "heavy chain" is a polypeptide chain composed of VH, CH1, HR, CH2, and CH3 in the direction from the N-terminus to the C-terminus. The "light chain" is a polypeptide chain composed of a light chain variable region (VL) and a light chain constant region (CL) in the direction from the N-terminus to the C-terminus. The two pairs of full-length antibody chains are linked together by disulfide bonds between CL and CH1 and between the HRs of the two full-length heavy chains. The whole antibody of the present invention can be derived from a single species, such as humans; it can also be a chimeric antibody or a humanized antibody.

[0199] As used herein, "human antibody" includes antibodies having variable and constant regions derived from human immunoglobulin sequences. The human antibodies of this invention may, for example, include amino acid residues not encoded by human germline immunoglobulin sequences in the CDR and CDR3 (e.g., mutations introduced through random or site-directed mutagenesis in vitro or through somatic mutation in vivo). In some embodiments, the anti-SARS-CoV-2 human antibody is a recombinant human antibody. In this case, the human antibody refers to an antibody recombinantly expressed in vitro using mammalian cells by transferring a human antibody-encoding gene into a genetically engineered antibody expression vector using genetic engineering techniques. All parts of the antibodies of this invention (including the variable and constant regions) are encoded by human-derived genes. Human antibodies can significantly reduce the immune side effects caused by heterologous antibodies in humans. Methods for obtaining human antibodies in the art include phage display technology, transgenic mouse technology, ribosome display technology, and RNA-peptide technology. In some embodiments, the human antibody also contains at least a portion of the immunoglobulin constant region (Fc), typically a portion of a human immunoglobulin. Generally, antibodies comprise a light chain and at least a variable region of a heavy chain. Antibodies may also contain CH1, hinge, CH2, CH3 and / or CH4 regions of the heavy chain. Human antibodies may be selected from any immunoglobulin type, including but not limited to IgM, IgG, IgD, IgA and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3 and IgG4.

[0200] The amino acid residues used in this article are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0201] The “conserved amino acid substitutions” used in this article include: (1) glycine, alanine, valine, leucine and isoleucine, (2) phenylalanine, tyrosine and tryptophan, (3) serine and threonine, (4) aspartic acid and glutamic acid, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.

[0202] As used herein, “identical” or “percentage of identity” has the meaning known in the art and refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by sequence comparison. When comparing and aligning for maximum consistency, two or more sequences or subsequences are identical or have a specified percentage of the same amino acid residues or nucleotides, as determined by one of the following sequence comparison algorithms or by visual measurement. Methods typically used to determine identity are encoded in computer programs. Preferred computer methods for determining identity between two sequences include, but are not limited to, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

[0203] In some embodiments, the anti-N protein antibody may include, individually or collectively, a heavy chain CDR having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity compared to the VH CDR of the exemplary antibody described herein. Alternatively or additionally, the anti-N protein antibody may include, individually or collectively, a light chain CDR having at least 80% (e.g., 85%, 90%, 95%, or 98%) sequence identity compared to the VL CDR of the exemplary antibody described herein. As used herein, “individually” means the sequence identity of one CDR of an antibody relative to the corresponding CDR of the exemplary antibody. “Collectively” means the sequence identity of the three VH or VL CDRs of the combined antibody relative to the corresponding three VH or VL CDRs of the combined exemplary antibody.

[0204] As used herein, an "epitaph" refers to an antigenic region bound by an antibody or its antigen-binding fragment. In some embodiments, epitope determinants include chemically active surface groups of a molecule (such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups). In some embodiments, specific three-dimensional structural features and / or mass-charge ratio features may be present.

[0205] As used herein, "neutralization" generally refers to the neutralizing activity of an antibody or its antigen-binding fragment, meaning that the antibody or its antigen-binding fragment can prevent and / or neutralize the biochemical activity of its corresponding antigen. In some cases, antibodies or their antigen-binding fragments possessing the aforementioned neutralizing activity can resist antigens that attack the immune system (e.g., SARS-CoV-2) and render them inactive. In some cases, antibodies or their antigen-binding fragments possessing the aforementioned neutralizing activity do not require the participation of leukocytes to neutralize the biochemical activity of their corresponding antigens.

[0206] As used herein, "specific binding" refers to a non-random binding reaction between two molecules. For example, an antibody that specifically binds to a target (which may be an antigenic epitope) has a greater affinity, stronger affinity, easier binding, and / or longer duration of binding to that target than it binds to other targets. An antibody is said to "specifically bind" to an epitope when it binds to the epitope more easily through its antigen-binding domain than to a random, unrelated epitope. The antibodies or their antigen-binding portions described herein have a KD 10 -5 M (10000nM) or lower, such as 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 - 10 M, 10 -11 M, 10 -12 The ability of M or a lower dissociation constant to bind to the N protein of the novel coronavirus.

[0207] The specific binding properties between molecules can be determined using methods known in the art. In some embodiments, the strength or affinity of the specific binding interaction is expressed using the equilibrium dissociation constant (KD). The KD value can be measured using any effective method. As used herein, the term "KD" refers to the dissociation constant of the interaction between the antibody or its antigen-binding fragment and the antigen, which describes the binding affinity between the antibody or its antigen-binding fragment and the antigen. The smaller the equilibrium dissociation constant, the tighter the binding, and the higher the affinity between the antibody or its antigen-binding fragment and the antigen. The dissociation constant can be measured using surface plasmon resonance (SPR), or alternatively, biomembrane interferometry or KinExA. In some embodiments, affinity is measured by competitive radioimmunoassay. In some embodiments, affinity is determined by ELISA. In some embodiments, the affinity KD is measured using surface plasmon resonance (SPR).

[0208] As used herein, a "library" refers to a collection of antibody or antibody fragment sequences, or nucleic acids encoding these sequences. In some embodiments, an antibody sequence library may contain at least 10 antibody sequences within a given antibody collection. 6 10 7 10 8 10 9 A library may contain one or more different antibody fragment sequences. The "library" may contain polynucleotides of a common class. For example, the class may be a polynucleotide encoding a type and class of immunoglobulin subunit polypeptide. In some embodiments, the library may encode antibody μ, γ1, γ2, γ3, γ4, α1, α2, ε, or δ heavy chains. In some embodiments, the library may encode antibody κ or λ light chains.

[0209] Antibody display

[0210] Antibody display technology is a class of molecular biology methods used for screening and engineering antibodies, and is widely applied in antibody discovery and optimization. It includes phage display, mRNA and DNA display, ribosome display, eukaryotic virus display, bacterial display, yeast display, and mammalian cell display to screen combinatorial libraries of recombinant proteins for desired characteristics. These display technologies have been widely used for antibody screening to identify antibodies with improved stability and desired binding affinity and activity, and have been applied to various applications, including directed evolution, affinity maturation, antibody engineering, biofuel production, and epitope localization.

[0211] Phage display technology

[0212] The "phage display" used in this article is a technique that displays antibody fragment sequences as fusion proteins with at least a portion of the coat protein on the surface of phage particles (e.g., filamentous phages). Phage display allows for the rapid and efficient screening of target sequences that bind to target antigens with high affinity from randomized antibody sequence libraries. Immunoglobulin VH or VL genes are amplified by PCR, ligated, and cloned into a vector containing the encoding phage. These genes are incorporated into the phage particles during phage assembly. Each recombinant phage contains a gene in its genome that displays a different antibody molecule on the phage surface. The phage library is panned in antigen-coated microculture wells; non-specific phages are washed away, and antigen-bound phages are eluted. The genomes from antigen-specific clones are isolated, and VH and VL sequences are obtained, allowing antibodies to be expressed as human antibodies in soluble scFv form for further identification. Phage display libraries (also known as phage peptide / antibody libraries, phage libraries, or peptide / antibody libraries) contain a large number of phages (10^6 phages). 8 (or more), each phage particle displays a different peptide or polypeptide sequence. These peptide or polypeptide fragments can be constructed in various lengths. The source of the displayed peptides or polypeptides includes, but is not limited to, human antibody heavy or light chains.

[0213] Several commonly used phage display systems have been developed. For example, the filamentous phage display system utilizes the unique life cycle of M13 phage and several phage proteins it expresses to screen for ligands with high affinity and high copy number released in secretory form. Furthermore, filamentous phages have excellent immunogenicity and are of great value in the research and development of biological vaccines.

[0214] The vectors for phage display are mainly divided into two categories: phage particle display vectors and phage display vectors.

[0215] Phage vectors are commonly used in genetic engineering. They are formed by replacing or inserting foreign genes into the phage genome. Most phage vectors have multiple protein display sites on their surface, typically multivalent display. Generally, multivalent display can lead to false positives in weakly binding clones, making it difficult to screen for highly specific clones. However, when using phages to display peptides, because peptides have weaker binding affinity to antigens, multivalent display can actually be more beneficial for screening target clones. Conversely, monovalent display can increase the likelihood of screening for high-affinity clones. Therefore, phage particles are chosen as vectors for monovalent display to screen for high-affinity clones.

[0216] Phagemid vectors are a type of plasmid vector containing a phage replication origin. They cleverly combine the characteristics of plasmids and phages, and are artificially constructed vectors containing single-stranded phage packaging sequences, replicons, plasmid replicons, cloning sites, and marker genes. Phagemids require helper phages to provide the proteases and coat proteins needed for replication and packaging. When helper phages are present in a bacterial cell, they can be induced into single-stranded DNA phagemids, which can replicate like phages or plasmids.

[0217] Phage particles have the following characteristics: 1. Double-stranded DNA is both stable and highly productive, exhibiting characteristics of conventional plasmids; 2. They eliminate the tedious and time-consuming step of subcloning foreign DNA fragments from plasmids into phage vectors; 3. Due to the sufficiently small size of the vector, single-stranded foreign DNA segments up to 10 kb can be obtained. Among them, Lerner's laboratory constructed pCBAKS, pComb3, and plomb8 vectors based on pBluescript, primarily for constructing Fab antibody libraries; the pHEN vector based on PUC was used for constructing scFv antibody libraries; and Winter's laboratory constructed the pCANTAB series of vectors based on PUC119.

[0218] The pCANTAB5E vector is a phage vector constructed by Jackson et al. of Pharmacia in 1992. It contains the ampicillin resistance gene (Amp), the Plac promoter, and the M13 phage spacer fragment. It is used to clone the Sfi I and Not I cloning sites of the ScFv gene, as well as the E-tag sequence and the termination code TAG. This vector has been widely used in the construction of phage antibody libraries.

[0219] Yeast cell display technology

[0220] Similar to phage display, in yeast display, antibody sequence fragments are displayed on the surface of yeast cells as fusions with the Aga2p protein, protruding the fusion protein away from the cell surface. Flow cytometry can then be used to select yeast cells expressing target antibody sequences that respond to the target antigen. Once identified, the expressed heavy chain V region and / or light chain V region genes are recovered and inserted into full-length heavy chain and / or light chain expression vectors. Cultured host cells transfected with such vectors allow for the production of large quantities of antibodies or their antigen-binding fragments.

[0221] Mammalian cell display technology

[0222] Mammalian cell display technology refers to techniques for expressing and displaying proteins, antibodies, or fragments on the surface of mammalian host cells for screening purposes. For example, fluorescence-activated cell sorting is used to screen for specific antigen binding. On one hand, mammalian expression vectors are used to express immunoglobulins simultaneously in secretory and cell-surface-bound forms. On the other hand, viral vectors or antibody fragment libraries encoding antibody libraries, when expressed in cells, display antibody fragments on the cell membrane.

[0223] The terms "isolated nucleic acid molecule" and "isolated polynucleotide" are used interchangeably throughout this document and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), DNA or RNA analogs produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally produced nucleotide analogs), and their hybrids. Nucleic acid molecules can be single-stranded or double-stranded. In some embodiments, the nucleic acid molecule contains consecutive open reading frames encoding the antibody or fragments, derivatives, mutant proteins, or variants thereof. The isolated nucleic acid molecule may not be associated with all or part of a polynucleotide found in nature, or may be linked to a polynucleotide to which it is not linked in nature.

[0224] The “expression vector” as used herein contains necessary regulatory sequences encoding the nucleic acid molecule and coding sequences for transcription and translation incorporated into a suitable host cell. Plasmids are typically used as expression vectors. Suitable plasmids are well-known and commercially available. The selection of the regulatory sequences in this invention depends on the type of host cell and can be readily performed by those skilled in the art. Examples of such regulatory sequences are transcription promoters and enhancers or RNA polymerase-binding sequences that include a transcription initiation signal preceding the inserted coding sequence, a ribosome-binding sequence, and a transcription termination sequence following the inserted coding sequence. Furthermore, depending on the host cell and vector used, other sequences may be introduced into the expression vector, such as replication initiators, additional DNA restriction sites, enhancers, and sequences that allow induction of transcription. The expression vector also contains a marker gene sequence that confers a specific phenotype on the transformed cells and enables specific selection of transformed cells. Additionally, the vector may contain a second marker sequence that allows differentiation between cells transformed with a recombinant plasmid containing the inserted target protein sequence and cells that have taken up a plasmid without the insert. Typically, conventional antibiotic resistance markers are used; however, any other reporter gene known in the art may be used, whose presence in cells (in vivo) can be readily determined using autoradiography, spectrophotometry, or bioluminescence and chemiluminescence methods. For example, depending on the host cell, reporter genes such as β-galactosidase, β-glucuronidase, luciferase, chloramphenicol acetyltransferase, or green fluorescent protein may be used.

[0225] In addition, the expression vector may contain a signal sequence that transports the protein to a suitable cellular compartment, such as the periplasm, where it promotes folding. Additionally, sequences encoding markers / tags, such as a His-Tag linked to the N-terminus or a GST linked to the C-terminus, may be present to facilitate subsequent affinity chromatography purification of the resulting protein using a nickel column. In some embodiments, the signal sequence comprises the amino acid sequence shown in SEQ ID NO: 89. In some embodiments, the expression vector may also contain additional sequences that protect the protein from proteolytic degradation in the host cell and sequences that enhance its solubility.

[0226] As used herein, "host cell" refers to a cell that can be used to express nucleic acids, such as those of this invention. The host cell can be a prokaryote, for example, *Escherichia coli* (E. coli). E. coliThe host cell can be a eukaryote, such as a single-celled eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or a hybridoma. Examples of host cells include the COS-7 line of monkey kidney cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells or derivatives thereof, HeLa cells, BHK cell lines, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1, human embryonic kidney cells such as 293, 293EBNA, or MSR293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from primary tissues cultured in vitro, primary explants, HL-60, U937, HaK, or Jurkat cells. Typically, the host cell is a cultured cell transformed or transfected with a peptide-encoded nucleic acid, which can then be expressed in the host cell. "Recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with the nucleic acid to be expressed. In this invention, a suitable host cell can be transformed or transfected with DNA and can be used to express and / or secrete the antibody.

[0227] As used herein, “transformation,” “transfection,” and “transduction” refer to any method or means by which nucleic acids are introduced into a cell or host organism, and are used interchangeably to convey the same meaning. Such methods include, but are not limited to, transfection, electroporation, microinjection, and PEG fusion. The introduced nucleic acid may be integrated (covalently linked) or not integrated into the nucleic acid of the recipient cell or organism. For example, in bacterial, yeast, plant, and mammalian cells, the introduced nucleic acid may be maintained as a free element or an independent replicon such as a plasmid. Alternatively, the introduced nucleic acid may become integrated into the nucleic acid of the recipient cell or organism and be stably maintained in that cell or organism, and further transferred or inherited by the recipient cell or organism's progeny cells or organisms. Finally, the introduced nucleic acid may exist only transiently in the recipient cell or host organism.

[0228] application

[0229] As used herein, "biological sample" refers to a biological sample typically obtained from a subject that contains or is suspected of containing an infectious agent of interest, such as SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment may be used for diagnostic applications, such as for detecting samples to provide diagnostic information. And / or methods, for example, may be used to detect samples (e.g., nasal or pharyngeal swab samples, and fluid samples and tissue specimens such as serum, whole blood, sputum, oral / nasopharyngeal secretions or washes, urine, feces, pleural or peritoneal effusions, cerebrospinal fluid, etc.) from subjects (e.g., patients suspected of being infected with SARS-CoV-2, or already infected with SARS-CoV-2) as indicators of therapeutic efficacy and whether the sample is infectious and requires isolation. In some embodiments, the antibody or antigen-binding fragment and / or method may provide a monitoring protocol for therapeutic interventions.

[0230] In some embodiments, the present invention utilizes a sandwich immunoassay of at least two epitope-compatible antibodies, comprising at least one antigen capture agent and at least one antigen detection agent. The at least one antigen capture agent and the at least one antigen detection agent typically bind to different epitopes and therefore do not significantly compete with each other for antigen binding. In some embodiments, the KD of the capture and antigen detection agents for SARS-CoV-2 is preferably less than or about 1 nM, for example, not exceeding 0.5 nM, not exceeding 0.15 nM, or not exceeding 0.1 nM.

[0231] The compatibility of two different antibodies functioning in a sandwich immunoassay can be determined, for example, by using one or more antigen capture agents in a sandwich immunoassay combining one or more of the at least one antigen detection agent. The assay may include, for example, contacting a known amount of target antigen with the at least one antigen capture agent (attached to a solid support) under conditions allowing the target to bind to the at least one antigen capture agent, optionally washing away excess target from the solid support, and then contacting the at least one antigen detection agent with the bound target. The presence, absence, or amount of the at least one antigen detection agent will indicate the compatibility of the at least one antigen capture agent and the at least one antigen detection agent. Problems may arise, for example, when two antibodies compete to bind to the same epitope or when the binding of the at least one antigen capture agent does not allow for the availability of the target epitope of the at least one antigen detection agent. Therefore, by comparing at least one candidate antigen capture agent with at least one candidate antigen detection agent in a sandwich immunoassay, it is possible to rapidly and efficiently identify which antibody pair is compatible and, optionally, which pair is "best," i.e., providing optimal detection or accurate determination of the target amount.

[0232] Numerous sandwich immunoassays are known and can be used to determine the compatibility of two antibodies. In a typical microtiter plate sandwich immunoassay, at least one antigen capture agent is adsorbed onto a plastic microtiter plate. When a test sample is added to the plate, the antibody on the plate binds to the target antigen from the sample and remains in the plate. When the at least one antigen detection agent is added in the next step, it also binds to the target antigen (which has already bound to the at least one antigen capture agent on the plate), thereby forming an antigen “sandwich” between two different antibodies. This binding reaction can then be measured by any method known in the art.

[0233] Immunoassays often use labeled reagents that specifically bind to and label a complex formed from an antibody and an antigen. The labeled reagent itself can be one of the structural motifs contained within the antibody / antigen complex. Therefore, the labeled reagent can be a polypeptide with a detectable label that binds to the target protein, or a tagged (e.g., biotinylated) antibody. Alternatively, the detectable label can be a third structural motif, such as a secondary antibody that specifically binds to the antibody / antigen complex (secondary antibodies are typically specific to antibodies from the species from which the primary antibody originates). Other proteins capable of specifically binding to immunoglobulin constant regions, such as protein A or protein G, can also be used as labeling reagents. The labeled reagent can be modified with a detectable structural motif, such as biotin, to which another molecule, such as avidin or streptavidin, can specifically bind. A variety of detectable structural motifs are well known to those skilled in the art.

[0234] The specific markers or detectable markers used in the assay are well known to those skilled in the art, provided they do not significantly interfere with the specific binding of the antibody used in the assay. Detectable markers can be any substance having detectable physical or chemical properties. Such detectable markers have been well developed in the field of immunoassays, and generally, almost any marker useful in this method can be applied to the present invention. Therefore, the marker is any composition detectable by methods such as spectrophotometry, photochemical methods, biochemical methods, immunochemical methods, electrical methods, optical methods, or chemical methods. Detectable markers useful in the present invention include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive markers, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), and colorimetric markers such as colloidal gold or colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.).

[0235] The marker can be directly or indirectly linked to the desired component to be measured (e.g., the at least one antigen detection agent) using methods known in the art. A variety of detectable markers can be used, wherein the selection of the marker depends on the required sensitivity, ease of conjugation to the compound, stability requirements, available equipment, and processing specifications.

[0236] Non-radioactive labels are typically linked via indirect methods. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to another molecule (e.g., streptavidin), which is itself detectable or covalently bound to a signaling system, such as a detectable enzyme, fluorescent compound, or chemiluminescent compound. The ligands and their targets can be used in any suitable combination with antibodies that recognize the target peptide or secondary antibodies that recognize antibodies that bind to the peptide.

[0237] The molecules can also bind directly to signal-generating compounds, for example, by binding to enzymes or fluorophores. The target enzymes used as labels will primarily be hydrolases, especially phosphatases, esterases, and glycosidases, or oxidases, especially peroxidases. Fluorescent compounds include luciferin and its derivatives, rhodamine and its derivatives, dansyl, 7-hydroxycoumarin, etc. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinediones, such as luminol.

[0238] Methods for detecting markers are well known to those skilled in the art. Therefore, for example, when the marker is a radioactive marker, detection methods include scintillation counters or photographic film, such as in autoradiography. When the marker is a fluorescent marker, it can be detected by exciting a fluorescent dye with light of a suitable wavelength and detecting the resulting fluorescence. Fluorescence can be visually detected using photographic film with an electronic detector such as a charge-coupled device (CCD) or a photomultiplier tube. Similarly, enzyme markers can be detected by providing the enzyme with a suitable substrate and detecting the resulting reaction product. Finally, simple colorimetric markers can be detected by observing the color associated with the marker. Thus, in various test strip assays, conjugated gold typically appears pink.

[0239] Some assays do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of target antibodies. In this assay, no labeling of the components is required, and the presence of the target antigen is detected by simple visual inspection.

[0240] In some embodiments, the reagent is a lateral flow immunochromatographic strip comprising an antigen-capturing agent and / or a metal-labeled antigen detection agent. This invention utilizes IgM antibodies to provide a lateral flow immunochromatographic strip with improved detection sensitivity and specificity. In some embodiments, the novel coronavirus N protein is separated from other proteins in the sample by applying a sample to one end of the chromatographic strip and allowing the antigen and antigen-capturing agent complex to migrate via capillary action or lateral flow. Methods and apparatus for lateral flow separation, detection, and quantification are known in the art. In these embodiments, the chromatographic strip comprises, from proximal to distal, a region for sample loading (sample loading area) and a test region containing an antigen-capturing agent, which may be the antigen of this invention. When a sample is loaded onto the sample loading area, the novel coronavirus N protein is captured by the antigen-capturing agent in the first test region. In some embodiments, the detection of the captured N protein is performed using a metal-labeled antigen detection agent. In some embodiments, depending on the specific selection of the detectable marker, the detection or observation of the control area (e.g., a control line) can be performed by any convenient means, such as, but not limited to, visual, fluorescent, reflective, radiographic, etc.

[0241] Example

[0242] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0243] Figure 1 This diagram illustrates the flowchart for antibody pair sequence discovery in this invention. A: 100 mL of blood is drawn from a COVID-19 patient's vein and peripheral blood mononuclear cells (PBMCs) are obtained by Ficoll density gradient centrifugation. B: RNA is reverse transcribed from the extracted PBMCs to synthesize complementary DNA (cDNA). C: The genes of the VH and VL variable regions are amplified by PCR and ligated into a phage display vector. After 3-4 rounds of phage amplification, panning, and positive clone identification, the antibody sequence is obtained. D: The plasmid is transfected into HEK293F cells to express and purify the human antibody. ELISA and BLI are used to verify antigen-antibody affinity, obtaining antibodies with high affinity. E: Antibody pairs are screened using an ELISA sandwich method. F: Development of colloidal gold test strips.

[0244] Example 1. Isolation of PBMC cells

[0245] In this embodiment, 100 mL of peripheral blood was collected from a patient infected with the novel coronavirus (SARS-CoV-2). The blood was centrifuged using Ficoll separation buffer at 2000 rpm for 15 minutes, and the supernatant was removed to obtain the intermediate layer of cells, namely peripheral blood mononuclear cells (PBMCs). The PBMCs were washed with 20 mL of DPBS, centrifuged at 1500 rpm for 10 minutes, and the cell pellet was collected and counted, yielding approximately 7 × 10⁶ cells. 7 Each cell.

[0246] Peripheral blood mononuclear cells (PBMCs) in peripheral blood mainly consist of lymphocytes (such as T cells, B cells, and natural killer cells) and monocytes, which play an important role in the immune response. By utilizing the density differences between cells and employing density gradient centrifugation, lymphocytes can be effectively separated from other blood components (such as erythrocytes and granulocytes), thereby obtaining PBMCs rich in immune cells. This process not only helps obtain high-quality PBMCs but also lays the foundation for subsequent antibody library construction.

[0247] Example 2. RNA extraction

[0248] 2.1 Methods

[0249] Using a microRNA extraction kit (Omega BIO-TEK, Cat# R6831-01), at a rate of 5-10 × 10⁻⁶ ppm. 5 Add cell lysis buffer to the collected mononuclear cell pellet at a ratio of 350 μL per cell. Lyse on ice for 10 minutes, gently mixing every 2-3 minutes to ensure complete cell lysis. Centrifuge at 12,000 rpm for 5 minutes at 4°C. Gently aspirate the supernatant and add an equal volume of 70% ethanol, mixing thoroughly by inverting. Add the supernatant sequentially (700 μL / time) to an RNA recovery column, allowing it to stand at room temperature for 2-5 minutes to allow RNA to fully adsorb onto the column. Centrifuge at 12,000 rpm for 15 seconds and discard the filtrate. Dissolve the RNA in preheated DEPC water (37°C) and centrifuge to obtain 30-50 μL of RNA.

[0250] 2.2 Results

[0251] The concentration measured by Nanodrop 2000 was approximately 400 ng / μL. The total RNA obtained was then used for reverse transcription to generate cDNA, or stored in a -80°C freezer.

[0252] MicroRNA extraction kits enable rapid and efficient isolation and purification of intracellular RNA, effectively removing DNA, proteins, and other impurities, thus ensuring high purity and integrity of the extracted RNA. Compared to traditional methods using chemical reagents such as chloroform or TRIzol for RNA extraction, these kits provide RNA with superior purity and integrity. High-purity and high-concentration RNA serves as a template for cDNA synthesis, laying a solid foundation for subsequent DNA library construction.

[0253] Example 3. cDNA Synthesis

[0254] Using RNA as a template, cDNA is synthesized under the action of reverse transcriptase.

[0255] The SuperScript II reverse transcriptase kit (Thermo Fisher Scientific, Cat#18064014) was used to reverse transcribe 4 μg of RNA into cDNA. The reaction system configurations are shown in Tables 1 and 2. Reverse transcription reaction system 1 was prepared as shown in Table 1.

[0256] Table 1

[0257]

[0258] Mix reaction component 1 thoroughly, react at 72°C for 3 minutes, and then immediately place on ice.

[0259] Table 2

[0260]

[0261] Mix reaction components 1 and 2 thoroughly to a total volume of 50 μL. Place in a PCR instrument and set the reaction conditions as follows: 25℃ for 5 minutes; 42℃ for 60 minutes; 50℃ for 30 minutes; 70℃ for 10 minutes, then maintain at 4℃. After PCR, the synthesized cDNA can be used directly for subsequent experiments or stored at -20℃.

[0262] Example 4. Construction of scFv phage display library

[0263] The construction route of scFv phage library fragments is as follows: Figure 2 As shown.

[0264] 4.1 Methods

[0265] 1) Design specific primers, configure the PCR reaction system according to Table 3, and set the PCR reaction program according to Table 4 to perform PCR amplification reaction. Through PCR reaction, amplify the variable region genes of IgG / IgM antibody heavy chain, kappa light chain and lambda light chain (approximately 350bp and 330bp respectively).

[0266] Table 3

[0267]

[0268] Table 4

[0269]

[0270] 2) Design primers to prepare a G4S linker (approximately 100 bp) with an overlap fragment at the 3' end of the heavy chain variable region and the 5' end of the light chain variable region. Configure the PCR reaction system according to Table 5 and set the PCR reaction program according to Table 6 for PCR amplification.

[0271] Table 5

[0272]

[0273] Table 6

[0274]

[0275] 3) After gel extraction and recovery of the antibody variable region gene fragment and linker fragment, VH, G4S Linker, and Vκ / Vλ were ligated using overlap extension PCR technology. The scFv library fragment (approximately 900 bp) was obtained through two-step PCR and gel recovery, and its concentration was measured. The overlap extension PCR reaction system was configured according to Table 7, and the PCR reaction program was set according to Table 8 for the first step of PCR amplification. The second overlap extension PCR reaction system was configured according to Table 9, and the PCR reaction program was set according to Table 10 for the second step of PCR amplification.

[0276] Table 7

[0277]

[0278] Table 8

[0279]

[0280] Table 9

[0281]

[0282] Table 10

[0283]

[0284] 4) The phage vector pCANTAB-5E was double-digested with NcoI and NotI restriction endonucleases, and the linear vector (4475bp) was prepared by gel recovery.

[0285] 5) Using homologous recombination technology, the pCANTAB-5E linear vector and scFv library fragment were added at a molar ratio of 1:4, with a total DNA amount of 1 μg. Homologous recombination was performed using 2 × SeamLess Mix (Biomed, Cat#CL117-01). After reacting at 50℃ for 30 min, the ligation product was recovered using a standard PCR product recovery kit and the concentration was measured.

[0286] 4.2 Results

[0287] 4.2.1 Electrophoresis results of the amplification products of the variable region genes of the IgG antibody heavy chain (approximately 350 bp), kappa light chain (κ light chain), and lambda light chain (λ light chain) (approximately 350 bp and 330 bp, respectively) are as follows: Figure 3 As shown in the figure, where m is the DNA Ladder 2000 marker. The figure shows that the size of the antibody heavy chain VH band, as well as the sizes of the antibody light chain Vκ and Vλ bands, are consistent with the design.

[0288] 4.2.2 The amplification results of the G4S Linker (approximately 100 bp) containing a fragment overlapping the 3' end of the heavy chain variable region and the 5' end of the light chain variable region are as follows: Figure 4 As shown. Lanes 1-13 are G4S Linker PCR fragments, approximately 100 bp in size, containing different sequences at the 3' end of the heavy chain variable region and the 5' end of the light chain variable region. From Figure 4 It can be seen that the fragment size of the G4S Linker PCR product is consistent with the design.

[0289] 4.2.3 Electrophoresis results of the recovered scFv library fragment (approximately 900 bp) are as follows: Figure 5 As shown in the figure, Lane 1 is VH-Vκ scFv, Lane 2 is VH-Vλ scFv, and m is a 5K DNA Marker. The figure shows that the scFv overlapextension PCR fragment size is consistent with the design, approximately 900 bp; the measured product concentration is approximately 200 ng / μL.

[0290] 4.2.4 The concentration of the linear carrier (4475bp) prepared by gel recovery was measured to be approximately 100 ng / μL.

[0291] 4.2.5 Using homologous recombination technology, the concentration of the product after ligating the pCANTAB-5E linear vector with the scFv library fragment was measured to be approximately 50 ng / μL.

[0292] This embodiment utilizes phage display technology to construct an antibody library containing variable regions of heavy chain (VH) and light chain (VL). Single-chain variable fragments (scFvs) are recombinant antibodies formed by linking the VH and VL regions of an antibody with a linker peptide (such as G4S). They possess the specificity and affinity of intact antibodies, and their small molecular size makes them more convenient for screening, expression, and modification. This technology can display a large number of diverse scFvs on the surface of phages, enabling efficient screening of antibodies that specifically bind to specific antigens, thus providing an important tool for targeted therapy, diagnostic reagent development, and basic research.

[0293] Example 5. Phage Display and Screening

[0294] In this embodiment, the constructed scFv phage library was introduced into TG1 competent cells via electroporation. After successful transformation, these phages displayed specific antibody fragments on the cell surface. A solid-phase screening method was used to bind the specific antibodies displayed on the phage surface to the target antigen. To improve the accuracy and specificity of the screening, 3-4 rounds of panning were required, each round including washing, elution, and phage amplification. In each round, unbound or weakly bound phages were continuously removed, ultimately yielding high-affinity positive clones.

[0295] 5.1 Methods

[0296] 5.1.1 Display

[0297] 1) The recovered homologous recombination ligation product was diluted with sterile water to 40 ng / μL. 5 μL of ligation product was electrotransfused per competent cell. Two TG1 cells (Zhuangmeng Biotechnology, Cat. NO. ZC1018D-2) were electrotransfused with κ and λ libraries respectively.

[0298] 2) Before electroporation, preheat 10 mL of SOC medium at 37°C, prepare eight 15 cm² × YT-GA plates (containing 2% glucose and 100 μg / mL ampicillin), and four 90 mm agar plates. 2 2 × YT-GA flat plates, pre-cooled 0.1cm electroporation cup and connecting products on ice, and 4 TG1 electroporation competent cells melted on ice.

[0299] 3) Transfer TG1 cells to an electroporation cuvette, add 5 μL of ligation product and perform electroporation with the following parameters: voltage 1.8 kV, pulse 10 μF, and resistance 600 Ω.

[0300] 4) After electroporation, transfer TG1 cells to SOC medium and incubate at 37°C and 250 rpm for 1 hour. After centrifugation at 4000 rpm for 10 min, discard the supernatant, resuspend the cell pellet in 1 mL of medium, and then serially dilute 1 μL of the bacterial culture 10-fold to 10⁻⁶. -1 10 -2 10 -3 10 -4 10 -5 10 -6 .

[0301] 5) Place 10 -3 10 -4 10 -5 10 -6 100 μL of each of the four bacterial cultures was spread onto a 90 mm thick plate. 2 The volume of the culture was determined using 2 × YT-GA plates. 1 mL of culture medium was added to the remaining bacterial culture, and then 250 μL was spread onto 15 cm 2 × YT-GA plates. The plates were placed in an incubator at 37°C and incubated upside down overnight.

[0302] 6) On the second day, from 90 mm 2 Twenty colonies were picked from a 2 × YT-GA plate and cultured in 2 × YT-Amp medium, and sequenced to detect sequence diversity, yielding an actual library volume of 1.2 × 10⁻⁶. 8 Human scFv phage display bacterial library.

[0303] 7) Collect all colonies on a 15cm 2×YT-GA plate using 2×YT-Amp medium and measure the OD. 600 value.

[0304] 8) Transfer 5 OD of bacterial culture to 50 mL of 2 × YT-GA medium and incubate at 37℃ and 250 rpm until OD reaches 100 oz. 600 ≈0.6, add approximately 6 × 10 11 PFU M13K07 helper phage particles (bacteria: helper phage = 1:10~1:20) were incubated at 37℃ and 250 rpm for 30 min and then transferred to 50 mL centrifuge tubes.

[0305] 9) After centrifuging at 4000 rpm for 10 min, remove the supernatant, resuspend the cells in 50 mL of 2 × YT-Amp-Kan medium in a 250 mL culture flask, and express the cells overnight at 30℃ and 250 rpm.

[0306] 10) On the third day, the expressed phage particles were collected using PEG / NaCl precipitation technology, and 1 μL of each particle was serially diluted and then inoculated with TG1 to detect the gradient.

[0307] 5.1.2 Selection

[0308] Using solid-phase panning, the amplified phage particles were panned three times. The concentration of recombinant N protein (self-made) used in each panning round was 2.2 mg / mL, which was then diluted to 15 μg / mL, 7.5 μg / mL, and 2.5 μg / mL respectively.

[0309] 1) The amount of bacteriophage particles introduced per round is approximately 1 × 10⁻⁶ 12 PFU. During screening, dilute the SARS-CoV-2 nucleoprotein N (His-Tag) to the screening concentration with coating buffer, and coat 100 μL / well onto an ELISA plate (coat 10 wells per round, and prepare 2 negative wells with PBS as controls), and coat overnight at 4°C.

[0310] 2) On the second day, block with 5% PBSM (Skim-Milk) for 2 hours, approximately 1 × 10⁻⁶ 12 Pfu phage was diluted to 1.2 mL with PBSM and added to an ELISA plate at a rate of 100 μL / well. The plate was incubated at 37 °C for 1 h to capture phage particles.

[0311] 3) Discard the unbound phage liquid, wash with 0.05% PBST (10 washes in the first round, 15 washes in the second round, and 20 washes in the third round), pat dry, add 100 μL of 0.1 M glycine (pH 2.2) elution buffer to each well, incubate at room temperature for 10 min, collect the elution buffer into a 1.5 mL centrifuge tube, and add 70 μL of 1 M Tris-HCl (pH 9.0) to each 1 mL of elution buffer for neutralization.

[0312] 4) Prepare OD on the same day 600 ≈0.6% fresh TG1 cells were used. 10 μL of elution buffer was taken and serially diluted 10-fold to infect TG1 cells to test the elution titer. 900 μL of elution buffer was used to infect 5 mL of TG1 cells and spread onto 15 cm2 × YT-GA plates. The plates were placed in an incubator at 37°C and incubated upside down overnight.

[0313] 5) On the second day, proceed with steps (6-10) in 5.1.1 to carry out the next round of phage amplification and expression.

[0314] 5.2 Results

[0315] Figure 6 This is a display diagram showing the amplified phage particles and their titer detection. As shown in A, the amplified phage titer is approximately 3 × 10⁻⁶. 13 pfu / mL; B shows the output titer after a partial third round of elution, approximately 3 × 10⁻⁶ pfu / mL. 6pfu / mL (N-R3-OP), blank control titer less than 5 × 10 4 pfu / mL (N-R3-Blank).

[0316] Example 6. Identification of positive clones and sequence extraction

[0317] In this embodiment, the phage elution buffer from the second or third round of washing in Example 5 was used to inoculate phages. Single clones were selected for expression, and their affinity for the target antigen was verified. The antigen-antibody complex was detected by ELISA to confirm their binding ability and affinity. After confirming its specificity, the phages of positive clones were amplified, and their DNA was extracted and subjected to Sanger sequencing to obtain the scFv gene sequence.

[0318] 6.1 Methods

[0319] 1) The second and third rounds of elution buffer were serially diluted and used to inoculate TG1 cells and spread onto 2 × YT-GA plates. On the second day, single clones were selected and transferred to 250 μL of 2 × YT-Amp medium (96-well deep plate). After culturing at 37°C and 250 rpm for 3 hours, 50 μL of bacterial culture was transferred for storage.

[0320] 2) Add approximately 3 × 10⁻⁶ to the remaining 200 μL of bacterial culture. 9 Add 100 μL of PFU M13K07 helper phage (diluted with 2 × YT-Amp medium and added to each well) and incubate at 37 °C and 250 rpm for 30 min.

[0321] 3) Then add kan+ antibiotic to the bacterial culture to a final concentration of 50 μg / mL (100 μL / well after dilution with 2 × YT medium) and express overnight at 30℃ and 250 rpm.

[0322] 4) On the third day, after centrifugation at 4000 rpm for 10 min, the supernatant of the expressed phage was collected and cloned using methods such as ELISA.

[0323] 5) Select the experimental group OD 450 The value is greater than 0.5, and the OD of the negative control is also greater. 450 A negative result indicates that the phage can specifically bind to the SARS-CoV-2 nucleoprotein (His-Tag) in the cloning culture. The antibody sequence is then extracted and analyzed using next-generation sequencing.

[0324] 6.2 Results

[0325] In this embodiment, a total of 150 experimental groups of OD were selected. 450 The value is greater than 0.5, and the OD value of the negative control group is also higher. 450Phage clones with a ELISA value less than 0.2 were sequenced. Analysis and comparison of the sequencing results yielded 50 unique antibody sequences, which were then used for further expression and characterization validation. Some ELISA data and sequencing results are shown below. Figure 7 As shown in Table 11.

[0326] Table 11

[0327]

[0328] Example 7. Subcloning of positive antibody sequences into human IgG1 and IgK expression vectors

[0329] In this embodiment, the VH and VL sequences obtained from the positive clone were cloned into expression vectors for the human IgG1 heavy chain and IgK light chain, respectively (expression vector maps are shown below). Figure 8 As shown in the figure.

[0330] After successful sequencing, plasmids were prepared using an endotoxin-free plasmid extraction kit (Tiangen Biotech, Cat#DP118-02).

[0331] Example 8. Antibody expression and purification in HEK293F cells

[0332] In this embodiment, plasmids encoding the heavy and light chains were mixed in a 1:1 ratio and co-transfected into HEK293F cells using PEI transfection reagent. After transfection, the cells were cultured at 37°C and 5% CO2. After 72 hours, the supernatant was collected to obtain the expressed recombinant antibody. Antibody purification was performed using an affinity chromatography column packed with Protein A, which specifically binds to human IgG antibodies. Unbound impurities were removed in the washing buffer, and the antibody was eluted with an appropriate elution buffer (e.g., Glycine / Tris-HCl buffer). Finally, the purified antibody was analyzed by SDS-PAGE to assess its purity and molecular size, thereby verifying its quality and function.

[0333] 8.1 Methods

[0334] 1) Remove HEK293F cells from the liquid nitrogen container, rapidly resuscitate them in a 37°C water bath, transfer them to 10 mL of preheated 37°C OPM-293 CD05 medium, centrifuge at 200 × g for 5 min, discard the supernatant, resuspend the cells, count them, and perform the cell count at 0.5 × 10⁻⁶. 6 The cells were seeded at a density of 100 cells / mL into shake flasks and incubated at 37°C, 130 rpm, and 8% CO2.

[0335] 2) Three to four days after cell resuscitation, perform dilution passages, with a seeding density of 0.3–0.5 × 10⁶ cells per passage.6 Cells / mL, after three passages, cell viability recovered to over 95%, and transient expression was performed.

[0336] 3) One day before transfection (D-1), based on the cell count results, take an appropriate amount of cell suspension, centrifuge at 200 × g for 5 min, discard the supernatant, and resuspend the cell pellet in fresh culture medium to a final volume of 0.9 × 10⁻⁶. 6 Continue culturing at cells / mL.

[0337] 4) On the day of transfection (D0), according to the volume of the cell suspension, prepare DNA-PEI suspension with PBS, incubate at room temperature for 5-10 min, add it dropwise to the cell suspension, mix well, and continue expression at 37℃, 130 rpm, 8% CO2. The transfection system is shown in Table 12 (taking 40 mL of cell suspension as an example).

[0338] Table 12

[0339]

[0340] 5) 48 to 72 hours after transfection (D2~D3), add 5% OPM-293 ProFeed to the cell suspension and continue expression until D5. Centrifuge the cell suspension at 4000 rpm for 20 min and collect the supernatant.

[0341] 6) The collected supernatant was purified by gravity column chromatography using Protein A affinity chromatography media. The antibody, captured by Protein A and washed with PBS, was eluted with 0.1M Glycine elution buffer (pH 3.4) and immediately neutralized with 1M Tris-HCl (pH 8.5). The purified antibody was then subjected to SDS-PAGE after replacing the Glycine buffer with PBS using an Amicon® Ultra filter (MWCO = 30 kDa) to measure its concentration with Nanodrop.

[0342] 8.2 Results

[0343] SDS-PAGE electrophoresis results are as follows: Figure 9 As shown. From Figure 9 As can be seen, the purified intact antibody is approximately 180 kDa in size, while the reduced antibody heavy chain and light chain are approximately 50 kDa and 25 kDa, respectively, with a purity of ≥90%. This is consistent with the expected results, indicating successful antibody expression and purification.

[0344] Example 9. Antibody Affinity Detection

[0345] Antibody affinity testing is an important indicator for assessing the ability of an antibody to bind to its target (antigen), and is usually expressed as the dissociation constant (Kd). A lower Kd value indicates a higher affinity between the antibody and the antigen, and vice versa. Different types of antibodies may have different affinity ranges; generally, high-affinity antibodies typically have a Kd value around 10. -10 Up to 10 -12 The M and M proteins exhibit a strong binding affinity. Commonly used affinity detection methods include enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), and biomembrane interferometry (BLI). These techniques can effectively monitor the kinetics and thermodynamics of antibody-antigen interactions. This embodiment uses ELISA and BLI methods to detect the affinity of screened antibodies for the SARS-CoV-2 N protein.

[0346] 9.1 ELISA assay for antigen-antibody kinetics

[0347] 9.1.1 Method

[0348] 1) Dilute the SARS-CoV-2 N protein to 1 μg / mL with coating buffer, add 100 μL / well to a 96-well ELISA plate, and incubate overnight at 4°C.

[0349] 2) On the second day, discard the coating solution, wash three times with 0.05% PBST, add 200 μL / well of 5% PBSM, and block at 37°C for 2 hours.

[0350] 3) After discarding the blocking solution and washing, add 100 μL / well of serially diluted antibody, incubate at 37°C for 1 hour, discard the sample and wash with PBST to remove unbound antibody 5 times, add 100 μL / well of detection secondary antibody anti-human IgG Fc (HRP) diluted 1:10000, and incubate at 37°C for 1 hour.

[0351] 4) Discard the secondary antibody and wash the unbound antibody 5 times with PBST. Add 100 μL / well of TMB chromogenic buffer and incubate at room temperature in the dark for about 10 min. Add 50 μL / well of stop solution and read the OD using a microplate reader. 450 value.

[0352] 5) Use GraphPadPrism8 software to perform 4-parameter nonlinear processing on the data and calculate the antibody affinity.

[0353] 9.1.2 Results

[0354] Determining antigen-antibody affinity, such as Figure 10 As shown, from Figure 10 It can be seen that the antigen-antibody affinity measured by the screening antibody is approximately 10. 12 M.

[0355] 9.2 BLI detection for antigen-antibody kinetics

[0356] 9.2.1 Method

[0357] 1) The Ni-NTA biosensor used in the Octet 8R device. According to the manufacturer's instructions, the SARS-CoV-2 N antigen (capture) to be tested was diluted to 100 nM with PBST.

[0358] 2) Serially dilute the antibody to be tested to 100 nM, 25 nM, 6.25 nM, 1.56 nM, and 0 nM, and add 200 μL / well to a black 96-well plate. At the same time, add 200 μL / well of equilibration buffer, dissociation buffer, neutralization buffer (all PBST), regeneration buffer (pH 1.5, 10 mM glycine buffer), and 50 mM NiCl2 solution.

[0359] 3) After balancing the biosensor to baseline, capture the antigen to be tested until the signal value is 1.0, and then balance it again (to assess whether the antigen capture is stable).

[0360] 4) The antigen-antibody affinity was sequentially bound to the antibody to be tested for 180s, then dissociated in PBST solution for 300s, and finally regenerated to obtain the antigen-antibody affinity.

[0361] 9.2.2 Results

[0362] BLI assays for affinity such as Figure 11 As shown. From Figure 11 It can be seen that some of the selected antibodies, B1, H1, and B12, exhibit good affinity for the antigen.

[0363] The results of antigen-antibody kinetic assays using both ELISA and BLI showed that the selected antibodies M4F3, 2C7, 5D6, B1, M3A8, H1, 2E1, E1, M4B11, 2D12, and B12 all exhibited an affinity of approximately 10 for the SARS-CoV-2 N protein. 12 M. The amino acid and nucleotide sequences of the variable region of the antibody are available in the sequence listing and have the SEQ ID NO as shown in Table 13 below. In these sequences, the CDR is determined based on the IMGT numbering system.

[0364] Table 13

[0365]

[0366]

[0367] Example 10. Antibody Pairing

[0368] Antibody pairing employs a double-sandwich method to screen antibody pairs targeting different epitopes of the same antigen. Typically, one antibody acts as a capture antibody, and the other as a detection antibody. In this process, the capture antibody is first immobilized on a solid support (such as a microplate) to capture the target antigen in the sample. Subsequently, the detection antibody is added; this antibody specifically binds to the captured antigen, forming an antigen-antibody complex. This pairing method amplifies the signal and improves detection sensitivity because the detection antibody is usually linked to an enzyme or fluorescent label, enhancing the intensity of the measurable signal. Furthermore, antibody pairing plays a crucial role in antibody development and application, directly affecting antibody performance, specificity, and function. By rationally designing and optimizing antibody pairings, the sensitivity and accuracy of experiments can be improved, leading to more reliable results in various biomedical research and clinical diagnostic applications. Therefore, selecting appropriate antibody pairings not only helps improve experimental efficiency but also provides a solid foundation for subsequent treatment and research.

[0369] In this embodiment, a double-antibody sandwich ELISA method was used to screen for pairing different antibodies.

[0370] 10.1 Method

[0371] 1) The double-antibody sandwich ELISA assay was used to pair the obtained human antibody with the biotinylated antibody.

[0372] 2) Coat the human antibody onto a 96-well plate at a concentration of 3 μg / mL, 100 μL / well, and incubate overnight at 4°C.

[0373] 3) On the second day, remove the coating solution and wash three times with 0.05% PBST. Add 200 μL of 5% PBSM to each well and block at 37°C for 2 hours.

[0374] 4) After removing the blocking solution and washing, add antigen at a concentration of 100 ng / mL and incubate at 37°C for 1 hour.

[0375] 5) Discard the sample and wash the unbound antibody 5 times with 0.05% PBST. Add biotinylated antibody at a concentration of 20 μg / mL and incubate at 37°C for 1 hour.

[0376] 6) Remove the sample and wash with 0.05% PBST five times to remove unbound antibodies. Add 100 μL / well of the detection secondary antibody anti-human IgG Fc (HRP) diluted 1:500 and incubate at 37°C for 1 hour.

[0377] 7) Remove the secondary antibody and wash the unbound antibody 5 times with 0.05% PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for about 10 minutes.

[0378] 8) Add 50 μL of stop solution to each well and read the OD value using a microplate reader. 450 value.

[0379] 10.2 Results

[0380] Data were analyzed using GraphPadPrism8 software, and six antibody pairs with relatively good pairings were selected: M4B11-2C7, M4B11-2E1, M4B11-2D12, E1-2D12, 2C7-2E1, and 2C7-B12. Subsequent immunochromatographic test strip experiments were then conducted.

[0381] Example 11. Colloidal gold test strip

[0382] Immunochromatography utilizes gold nanoparticles to form colloidal gold complexes with specific antibodies or ligands to capture target molecules. The sample to be tested (such as blood, urine, or other biological fluids) is applied to the sample window of the test strip, where it flows downwards via capillary action. During this process, the target molecules in the sample bind to the colloidal gold-labeled antibody, forming a visible result line on the test strip. Results are typically obtained within minutes, making it suitable for rapid diagnostic applications. This technology requires no complex equipment, is simple to operate, and is suitable for on-site testing. Furthermore, the high sensitivity and affordability of colloidal gold test strips have led to their widespread use in medical diagnostics, food safety, and environmental monitoring.

[0383] This embodiment uses colloidal gold test strips to verify the detection performance of paired antibodies.

[0384] 11.1 Method

[0385] 1) M4B11, E1 and 2C7 were used as capture antibodies, respectively, and were immobilized on nitrocellulose membranes. The membrane concentration was 1.5 mg / mL and the membrane speed was 100 mm / s.

[0386] 2) Select 2C7, 2E1, 2D12 and B12 as detection antibodies respectively, and prepare detection probes by coupling with colloidal gold.

[0387] 3) Immunochromatographic test strips with different paired antibodies were obtained: M4B11-2C7, M4B11-2E1, M4B11-2D12, E1-2D12, 2C7-2E1 and 2C7-B12.

[0388] 4) Add the antigen to be tested, namely SARS-CoV-2, with a titer of 10. 5 pfu / mL. Add lysis buffer to the Blank group.

[0389] 11.2 Results

[0390] The results are as follows Figure 12 As shown, colloidal gold test strips prepared from the selected antibody pairs M4B11-2C7, M4B11-2E1, M4B11-2D12, E1-2D12, 2C7-2E1, and 2C7-B12 can all detect the novel coronavirus.

[0391] Example 12. Expression of pentamer IgM subtype antibody

[0392] 12.1 Construction of expression vector for pentamer IgM subtype antibody

[0393] In this embodiment, specific primers were designed, and the VH sequence obtained from the positive clone was subcloned into a mammalian cell expression vector with an IgM backbone (e.g., PCR and homologous recombination techniques) using PCR and homologous recombination techniques. Figure 13 The nucleotide sequence of the human IgM signal peptide is shown in SEQ ID NO: 113, and the nucleotide sequence of the IgM backbone is shown in SEQ ID NO: 114.

[0394] After the recombinant plasmid was confirmed to be correct by sequencing, the plasmid was prepared using the Tiangen endotoxin-free plasmid extraction kit (Tiangen Biochemistry, Cat#DP118-02) for subsequent expression.

[0395] 12.2 Expression and purification of pentamer IgM subtype antibodies

[0396] 12.2.1 Method

[0397] The antibody was expressed and crude material was collected according to the method in Example 8.

[0398] 1) Prepare the transfection system as shown in Table 14 (taking 40 mL of cell suspension as an example).

[0399] Table 14

[0400]

[0401] 2) The collected supernatant was purified by gravity column chromatography using Protein L affinity chromatography media. During this process, the antibody was captured by Protein L and washed with 20 mM PBS buffer (pH 7.0) to remove non-specifically bound impurities. Subsequently, the bound antibody was eluted with 0.1 M glycine elution buffer (pH 2.5) and immediately neutralized with 1 M Tris-HCl buffer (pH 8.5) to prevent antibody denaturation at low pH.

[0402] 3) The purified antibody was processed by Amicon. ®An ultrafiltration filter (MWCO = 50 kDa) was used to replace the glycine buffer with PBS. Antibody concentration was then measured using Nanodrop, and SDS-P... AGE Electrophoretic identification

[0403] 12.2.2 Results

[0404] Electrophoresis results as follows Figure 14 As shown in the figure, the antibody reduced to approximately 75 kDa for the heavy chain and 25 kDa for the light chain, consistent with the expected results, thus verifying the successful expression and purification of the antibody.

[0405] 12.3 ELISA detection of antigen-IgM antibody binding affinity

[0406] 12.3.1 Method

[0407] 1) Dilute the SARS-CoV-2 N protein to 1 μg / mL with coating buffer, add 100 μL / well to a 96-well ELISA plate, and incubate overnight at 4°C.

[0408] 2) On the second day, discard the coating solution, wash three times with 0.05% PBST, add 200 μL / well of 5% PBSM, and block at 37°C for 2 hours.

[0409] 3) After discarding the blocking solution and washing, add 100 μL of serially diluted antibody per well and incubate at 37°C for 1 hour.

[0410] 4) Discard the sample and wash with PBST to remove unbound antibodies 5 times. Add 100 μL / well of the detection secondary antibody Rabbit Anti-Kappa Light Chain / IGKC Antibody (HRP) at a dilution ratio of 1:5000 and incubate at 37°C for 1 hour.

[0411] 5) Discard the secondary antibody and wash the unbound antibody 5 times with PBST. Add 100 μL of TMB chromogenic solution to each well and incubate at room temperature in the dark for about 10 minutes. Add 50 μL of stop solution to each well and read the OD value using a microplate reader. 450 value.

[0412] 6) Use GraphPad Prism8 software to perform 4-parameter nonlinear processing on the data and calculate the antibody affinity.

[0413] 12.3.2 Results

[0414] The results are as follows Figure 15 As shown in the figure, the pentamer IgM antibody exhibits a stronger antigen capture ability compared to the IgG antibody at the same concentration.

[0415] Example 13. Pentamer IgM subtype antibody used for detection on colloidal gold test strips

[0416] 13.1 Methods

[0417] 1) IgM E1 and IgG E1 were used as capture antibodies, respectively, and were immobilized on nitrocellulose membranes at a concentration of 1.5 mg / mL and a scratching speed of 100 mm / s.

[0418] 2) 2D12 was selected as the detection antibody and coupled with colloidal gold to prepare the detection probe.

[0419] 3) Prepare immunochromatographic test strips by combining IgM E1 and IgG E1 capture antibodies with 2D12 detection antibodies to obtain immunochromatographic test strips with different paired antibodies: IgM E1-2D12 and IgG E1-2D12.

[0420] 4) SARS-CoV-2 N protein was added at concentrations of 0 ng / mL, 50 ng / mL, 100 ng / mL, and 500 ng / mL. Lysis buffer was added to the Blank group.

[0421] 13.2 Results

[0422] The results are as follows Figure 16 As shown, at the same antigen concentration, the IgM antibody test strip produces a darker color and a stronger signal than the IgG antibody test strip, thus the IgM antibody test strip has higher detection sensitivity.

[0423] The partial light chain complementarity determination region sequences are shown in the table below.

[0424]

[0425] This invention aims to address the technical challenges in current antibody preparation processes, particularly the long lead time and low efficiency of traditional human antibody production. Utilizing phage surface display technology, high-affinity human antibodies were successfully screened. Furthermore, through optimization and improvement of the traditional antibody preparation process, the entire human antibody preparation cycle was significantly shortened to less than one month. This innovation greatly improves the efficiency of antibody screening and preparation. Simultaneously, this new technical approach not only efficiently screens high-affinity and stable high-quality human antibodies but also lays the foundation for subsequent antibody pairing, leading to the development of a high-performance diagnostic kit for the COVID-19 N protein.

[0426] Furthermore, the human antibody screening technology of this invention also shows broad application prospects in antibody drug development, biomedical research and other fields, and is expected to accelerate scientific research progress and achievement transformation in related fields, thereby promoting the development and clinical application of new treatment options and contributing to public health security.

[0427] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the N protein of the novel coronavirus SARS-CoV-2, characterized in that, The antibody or its antigen-binding fragment comprises: a heavy chain variable region containing heavy chain complementarity-determining regions HCDR1, HCDR2, and HCDR3, and a light chain variable region containing light chain complementarity-determining regions LCDR1, LCDR2, and LCDR3. The heavy chain complementarity determination region includes: (a) HCDR1, whose amino acid sequence is shown in SEQ ID NO: 2; (b) HCDR2, whose amino acid sequence is shown in SEQ ID NO: 3; (c) HCDR3, whose amino acid sequence is shown in SEQ ID NO: 4; The light chain variable region includes: (e) LCDR1, whose amino acid sequence is shown in SEQ ID NO: 6; (f) LCDR2, whose amino acid sequence is AAS; (g)LCDR3, whose amino acid sequence is shown in SEQ ID NO:

8.

2. The antibody or its antigen-binding fragment according to claim 1, characterized in that, The heavy chain variable region contains the amino acid sequence shown in SEQ ID NO: 1, and The light chain variable region contains the amino acid sequence shown in SEQ ID NO:

5.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antigen-binding fragment is selected from Fab, Fab', F(ab)2, F(ab')2, scFv or disulfide-linked Fv (dsFv).

4. The antibody or its antigen-binding fragment according to claim 3, characterized in that, The antigen-binding fragment is scFv.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody is immunoglobulin G (IgG), IgM, IgE, IgA, or IgD.

6. The antibody or its antigen-binding fragment according to claim 5, characterized in that, The antibody contains a constant region of IgG1, IgG2, IgG3, IgG4, or IgM.

7. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody contains the amino acid sequence shown in SEQ ID NO:

89.

8. The antibody or its antigen-binding fragment according to claim 6, characterized in that, The antibody contains the amino acid sequence shown in SEQ ID NO:

90.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1-4, characterized in that, The antibody in question is a human antibody.

10. An isolated nucleic acid molecule encoding a heavy chain variable region and a light chain variable region of an antibody or antigen-binding fragment thereof as described in any one of claims 1-9.

11. The nucleic acid molecule according to claim 10, characterized in that, The nucleotide sequence encoding the heavy chain variable region amino acid sequence of the nucleic acid molecule is shown in SEQ ID NO: 91, and the nucleotide sequence encoding the light chain variable region amino acid sequence is shown in SEQ ID NO:

92.

12. An expression vector comprising the nucleic acid molecule of claim 10 or 11.

13. A host cell comprising the nucleic acid molecule of claim 10 or 11 or the expression vector of claim 12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1-9, a nucleic acid molecule as described in claim 10 or 11, an expression vector as described in claim 12, a host cell as described in claim 13, and a pharmaceutically acceptable carrier.

15. The use of the antibody or antigen-binding fragment thereof of any one of claims 1-9, the nucleic acid molecule of claim 10 or 11, the expression vector of claim 12, the host cell of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a diagnostic kit, characterized in that, The test kit is used to detect the presence or level of SARS-CoV-2 in biological samples.

16. A reagent for detecting the presence or level of SARS-CoV-2 in a biological sample, comprising an antibody or antigen-binding fragment thereof as any one of claims 1-9 as an antigen capture agent and / or antigen detection agent.

17. The reagent according to claim 16, characterized in that, The reagent comprises an antigen capture agent and an antigen detection agent, wherein the antigen capture agent and the antigen detection agent are selected from: (1) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 65 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 69 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 1 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 5 as the light chain variable region (VL); (2) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 1 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 5 as the light chain variable region (VL); and antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 33 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 37 as the light chain variable region (VL); or (3) Antigen capture agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 1 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 5 as the light chain variable region (VL), and an antigen detection agent: comprising an antibody or an antigen-binding fragment thereof containing the amino acid sequence shown in SEQ ID NO: 41 as the heavy chain variable region (VH) and the amino acid sequence shown in SEQ ID NO: 45 as the light chain variable region (VL).

18. The reagent according to claim 16 or 17, characterized in that, The antigen detection reagent contains a detectable marker; The detectable marker is selected from magnetic beads, fluorescent dyes, radioactive isotopes, biotin, metals, or any combination thereof.

19. The reagent according to claim 16 or 17, characterized in that, The reagent is an immune sandwich assay reagent or kit.

20. The reagent according to claim 19, characterized in that, The reagent also includes a solid support, and the antigen capture agent is attached to the solid support, which is selected from plates or beads.

21. The reagent according to claim 20, characterized in that, The plate is a perforated plate or a test strip.

22. The reagent according to claim 19, characterized in that, The antigen detection reagent contains a metal marker.

23. The reagent according to claim 22, characterized in that, The reagent is a lateral flow immunochromatographic slide containing an antigen capture agent and a metal-labeled antigen detection agent.

24. A method for detecting the presence or level of SARS-CoV-2 in a biological sample for non-therapeutic or diagnostic purposes, characterized in that, The method uses the antibody or antigen-binding fragment thereof as described in any one of claims 1-9 or the reagent as described in any one of claims 16-23.

25. The method according to claim 24, characterized in that, The method described is an immunological detection method.

26. The method according to claim 25, characterized in that, The method is selected from ELISA, liquid microarray, immunofluorescence, immunohistochemistry, immunochromatography, flow cytometry, cell sorting, radioimmunoassay, immunodiffusion, immunoprecipitation, or Western blotting.

27. The method according to claim 26, characterized in that, The method described is immunochromatography.

28. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-9 in the preparation of a reagent for detecting the presence or level of SARS-CoV-2 in a biological sample.

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