阅读说明：本技术 新型冠状病毒抗体和新型冠状病毒抗体的elisa检测试剂盒 (Novel coronavirus antibody and ELISA detection kit for same ) 是由 张黎 高行素 郑滨洋 孟繁岳 王文娟 郭喜玲 梁祁 朱凤才 于 2020-05-27 设计创作，主要内容包括：本发明公开了新型冠状病毒抗体和新型冠状病毒抗体的ELISA检测试剂盒,所述抗体包含SEQ ID NO.1所示的CDR1、SEQ ID NO.2所示的CDR2、SEQ ID NO.3所示的CDR3以及SEQ ID NO.5所示的CDR1、SEQ ID NO.6所示的CDR2、SEQ ID NO.7所示的CDR3。本发明的抗体可用于新型冠状病毒检测或新型冠状病毒感染诊断。(The invention discloses a novel coronavirus antibody and an ELISA detection kit for the novel coronavirus antibody, wherein the antibody comprises CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2, CDR3 shown in SEQ ID NO.3, CDR1 shown in SEQ ID NO.5, CDR2 shown in SEQ ID NO.6 and CDR3 shown in SEQ ID NO. 7. The antibodies of the invention are useful for novel coronavirus detection or novel coronavirus infection diagnosis.)

1. A binding protein of a novel coronavirus NP protein, which comprises CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO.2 and CDR3 shown in SEQ ID NO. 3.

2. The binding protein of claim 1, comprising the heavy chain variable region set forth in SEQ ID No. 4.

3. The binding protein of claim 1, further comprising the CDR1 of SEQ ID No.5, the CDR2 of SEQ ID No.6, and the CDR3 of SEQ ID No. 7.

4. The binding protein of claim 3, comprising the light chain variable region set forth in SEQ ID No. 8.

5. An isolated, recombinant or synthetic DNA molecule encoding the binding protein of any one of claims 1-4; preferably, it comprises the sequences shown in SEQ ID NO.17 and 18.

6. A vector comprising the DNA molecule of claim 5.

7. A host cell comprising the vector of claim 6.

8. A method for producing a binding protein for a novel coronavirus NP protein, comprising culturing the host cell of claim 7 under conditions suitable for the host cell to produce the binding protein for the novel coronavirus NP protein.

9. A composition or kit comprising the binding protein of any one of claims 1-4; preferably, the composition or kit further comprises a second binding protein comprising heavy chain variable region CDR1, heavy chain variable region CDR2, heavy chain variable region CDR3, light chain variable region CDR1, light chain variable region CDR2, light chain variable region CDR 3; wherein the content of the first and second substances,

heavy chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 9;

heavy chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 10;

heavy chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 11;

light chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 13;

light chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 14;

light chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 15;

more preferably, the second binding protein comprises a heavy chain variable region, a light chain variable region; wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO.12, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 16.

10. A use comprising the use of any one of:

(1) use of a binding protein according to any one of claims 1 to 4 for the preparation of a novel coronavirus detection product;

(2) use of a binding protein according to any one of claims 1 to 4 for the preparation of a novel diagnostic product for coronavirus infection;

(3) use of a composition according to claim 9 for the preparation of a product for the detection of a novel coronavirus.

Technical Field

The invention belongs to the fields of cellular immunology and molecular biology, and relates to a novel coronavirus antibody and an ELISA (enzyme-Linked immunosorbent assay) detection kit for the novel coronavirus antibody.

Background

The international committee for viral classification named the novel coronavirus SARS-CoV-2 and the world health organization named the pneumonia caused by infection with this virus COVID-19. The virus has strong infectivity and wide transmission path. The virus can adapt to the environment of human body rapidly, has transmission capability in latent period after infection, and reports by some asymptomatic infectors that virus nucleic acid is detected even in various animals. These factors complicate the control of the virus and no effective therapeutic drugs and vaccines are currently on the market.

SARS-CoV-2 belongs to the genus Coronavirus, is a single-stranded positive-strand RNA virus, has a size of about 30kb, has a similarity of 79% to SARS-CoV, and has a similarity of up to about 88% to a Coronavirus (CoV) isolated from Bats. SARS-CoV-2 has typical coronavirus characteristics, and the virus envelope has typical spinous processes, which are shaped like coronages. The Nucleocapsid is of a spiral symmetrical type, the main structural protein is Nucleocapsid Protein (NP), and the total length of the NP is 420 amino acids. The NP has the most content in virus structural protein, is expressed in a large amount in the early stage of host infection, has stronger immunogenicity, and can cause strong immune response of a host. Thus, NP can be used as the main target antigen for serological diagnosis of SARS-CoV-2 infection.

Because specific therapeutic drugs and effective vaccines are not developed successfully, early diagnosis becomes an important measure for preventing and controlling epidemic situations, and early nucleic acid diagnosis and clinical diagnosis become important basis for accurate diagnosis. Although the nucleic acid diagnosis speed is high, the influence of the quality of the sampling is large, false positive and false negative exist, and the implementation of the prevention and control measures is influenced. Nucleic acid detection of part of asymptomatic infected persons is negative in the late stage of the disease process, and missed diagnosis is easy to occur only by nucleic acid detection. Serological diagnosis is to detect the immune response of an organism after pathogen infection, the duration is long, the immune response is stable, and the immune response shows a dynamic change trend along with the progress of the disease course. Serodiagnosis is therefore also an important tool for early diagnosis and assessment of the current state of infection.

Disclosure of Invention

The present invention provides a solution to these and other problems by providing binding proteins (e.g., isolated, recombinant, or synthetic antibodies, or fragments or derivatives thereof) that specifically bind to a novel coronavirus NP protein.

The antibody molecules of the invention may be of any class (e.g., IgG, IgE, IgM, IgD or IgA) or subclass of immunoglobulin molecule. The constant region domains of the antibody (if present) may be selected based on the recommended antibody molecule function. For example, the constant region domain may be a human IgA, IgD, IgE, IgG or IgM domain. In particular, human IgG constant region domains, particularly IgG1, IgG2, IgG3, and IgG4, may be used.

Antibody fragments include, for example, Fab, F (ab)2, Fab ', F (ab ') 2, F (ab ') 3, F (v), Fd, dAb, diabodies, miniantibodies, nanobody fragments, fragments of a single variable domain (e.g., a VH or VL domain), or fragments containing only heavy or light chain domains.

The antibodies of the invention, including fragments and derivatives thereof, against the novel coronavirus NP protein may be monoclonal, polyclonal, murine, chimeric, primatized, humanized or fully human antibodies. The antibodies of the invention may be multimeric, heterodimeric, hemi-dimeric (hemmidimeric), monovalent, bivalent, tetravalent, bispecific, and may include single chain antibodies; and derivatives of these.

In one embodiment of the invention, the antibody against the novel coronavirus NP protein refers to a fully human antibody. Fully human antibodies comprise antibody polypeptides or immunoglobulin variable domains having sequences derived from human immunoglobulins (e.g., from human immunoglobulin coding sequences). The term "human" as applied herein to antibodies or fragments (e.g., variable domains) is free of antibodies from another species (e.g., a mouse) that have been "humanized" by grafting human constant region sequences to the antibody polypeptide (i.e., replacing non-human constant regions with human constant regions) or by grafting human V region framework sequences to immunoglobulin variable domains from a non-human mammal (i.e., replacing the non-human framework regions of the V domains with human framework regions).

The antibodies of the invention comprise synthetic antibodies or recombinant antibodies produced using recombinant DNA techniques, such as antibodies expressed by phage. It should also be construed to include antibodies that have been produced by synthesizing a DNA molecule encoding an antibody that expresses an antibody protein, or determining the amino acid sequence of an antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence techniques available and well known in the art.

In certain embodiments, the invention provides the following binding proteins (e.g., antibodies): it specifically binds to a novel coronavirus NP protein and comprises or consists of more than one CDR heavy chain (H) sequence selected from the group consisting of CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and CDR-H3(SEQ ID NO. 3).

In a further embodiment, the binding protein or antibody of the novel coronavirus NP protein comprises at least 2 CDRs selected from the group consisting of CDR-H1(SEQ ID No.1), CDR-H2(SEQ ID No.2) and CDR-H3(SEQ ID No.3), or consists of at least 2 CDRs selected from the group consisting of CDR-H1(SEQ ID No.1), CDR-H2(SEQ ID No.2) and CDR-H3(SEQ ID No. 3).

In a still further embodiment, the binding protein or antibody contains or consists of all 3 of the following CDR H sequences, CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and CDR-H3(SEQ ID NO. 3).

In some embodiments, the invention provides the following binding proteins (e.g., antibodies): it specifically binds to a novel coronavirus NP protein and contains or consists of one or more CDR light chain (L) sequences selected from the group consisting of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO. 7).

In a further embodiment the novel coronavirus NP protein or antibody comprises at least 2 CDRs selected from the group consisting of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO.7), or consists of at least 2 CDRs selected from the group consisting of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO. 7).

In a still further embodiment, the binding protein or antibody of the novel coronavirus NP protein comprises or consists of all 3 CDR L sequences which are CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO. 7).

In certain embodiments, the invention provides the following binding proteins (e.g., antibodies): it specifically binds to a novel coronavirus NP protein and comprises or consists of CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO.7) and/or CDR-L1(SEQ ID NO.5), CDR-L2(SEQ ID NO.6) and CDR-L3(SEQ ID NO.7) and wherein said binding protein further comprises or consists of CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and/or CDR-H3(SEQ ID NO.3) and/or CDR-H1(SEQ ID NO.1), CDR-H2(SEQ ID NO.2) and/or CDR-H3(SEQ ID NO. 3).

In certain embodiments, the present invention provides binding proteins (e.g., antibodies) that specifically bind to a novel coronavirus NP protein, wherein the binding protein or antibody of the novel coronavirus NP protein comprises or consists of the VH sequence of SEQ ID No. 4.

In certain embodiments, the present invention provides binding proteins (e.g., antibodies) that specifically bind to a novel coronavirus NP protein, wherein the binding protein or antibody of the novel coronavirus NP protein comprises or consists of the VL sequence of SEQ ID No. 8.

In certain embodiments, the present invention provides binding proteins (e.g., antibodies) that specifically bind to a novel coronavirus NP protein, wherein the binding protein or antibody of the novel coronavirus NP protein comprises or consists of the VL sequence of SEQ ID No.8 and the VH sequence of SEQ ID No. 4.

Binding proteins (e.g., antibodies) of the novel coronavirus NP proteins of the invention can be pegylated. Wherein the antibody may be pegylated on the heavy chain, the light chain, or both chains.

The invention also provides isolated, recombinant and/or synthetic DNA molecules encoding the binding proteins described above.

The DNA sequences of the present invention may comprise, for example, synthetic DNA, cDNA, genomic DNA produced by chemical treatment, or any combination thereof.

In a particular embodiment of the invention, the DNA molecule comprises the amino acid sequences shown in SEQ ID NO.17 and SEQ ID NO. 18.

The invention also provides a vector comprising a DNA molecule as described above.

The invention also provides a host cell comprising a DNA sequence or vector of the invention. In certain aspects, the invention relates to a method for producing a binding protein (e.g., an antibody) as described above, comprising culturing a host cell containing any of the vectors described above under conditions suitable for production of the binding protein by the host cell. In some embodiments, the method comprises recovering the binding protein from the host cell culture.

The host cell may be, for example, a prokaryotic cell, such as E.coli, or other microbial cell, or a eukaryotic cell including, but not limited to, a mammalian cell, such as a human, mouse, monkey, rabbit, goat, hamster, or rat cell, an insect cell, an avian cell, a plant cell, and a lower eukaryotic cell, such as a fungal cell.

In some embodiments of the invention, host cells useful in the practice of the invention can be, for example, (1) bacterial cells such as E.coli, Bacillus crescentus, Streptococcus, Staphylococcus, Streptomyces and Bacillus subtilis cells, and Salmonella typhimurium; (2) fungal Cells and Aspergillus (Aspergillus) Cells, yeast Cells, such as Saccharomyces cerevisiae (Saccharomyces cerevisiae), Schizosaccharomyces pombe (Schizosaccharomyces pombe), Pichia pastoris (Pichia pastoris), Pichia methanolica (Pichia methanolica), other Pichia species, Kluyveromyces lactis (K.lactis), (3) insect cell lines, such as Cells from Spodoptera frugiperda (Spodoptera frugiperda) (e.g., Sf9 and Sf21 cell lines, and expressSFTM Cells (Protein Sciences Corp., Meriden, CT, USA)), Drosophila S2 Cells, and Trichoplusia ni Hiitrogen (Carlsbad, Calif., USA); (4) mammalian cells, or (5) plant cells.

Typical mammalian cells include COS1 and COS7 cells, Chinese Hamster Ovary (CHO) cells, NSO myeloma cells, NIH3T3 cells, 293 cells, H Ε PG2 cells, HeLa cells, C127, 3T3, BHK, Bowes melanoma cells, L cells, MDCK, HEK293, WI38, murine ES cell lines (e.g., from lines 129/SV, C57/BL6, DBA-1, 129/SVJ), K562, Jurkat cells, and BW 5147.

In certain embodiments, the binding proteins, or nucleic acids, of the invention are labeled with a detectable label, which may be a radioisotope, an enzyme, a dye, or biotin.

In yet other embodiments, the antibodies of the invention are conjugated to an imaging agent, which may be a labeling moiety. The labeling agent may be biotin, a fluorescent or luminescent moiety, a radioactive moiety, a histidine tag, or a peptide tag.

The invention also relates to sequence variants of the binding proteins (e.g., antibodies) described herein, as well as nucleic acid sequences encoding the same. The sequence variants of the invention preferably share at least 90%, 91%, 92%, 93% or 94% identity with the polypeptides of the invention or the nucleic acid sequences encoding them. More preferably, sequence variants share at least 95%, 96%, 97% or 98% identity at the amino acid or nucleic acid level. Most preferably, the sequence variant shares at least 99%, 99.5%, 99.9% or more identity with the polypeptide of the invention or the nucleic acid sequence encoding it.

In certain embodiments, the antibody polypeptides of the invention against the novel coronavirus NP protein are, for example, dabs, fabs, Fab', scFv, Fv, disulfide-bonded fvs, or comprise a single immunoglobulin variable domain, for example a VH or VL domain, that is specific for and monovalent for binding of the novel coronavirus NP protein. Certain binding protein polypeptides of the invention comprise 1, 2 or more CDRs of the invention and an alternative scaffold or universal framework sequence of the invention.

In certain embodiments, the binding protein or antibody of the invention comprises a single variable domain selected from the group consisting of a variable heavy chain (VH) and a variable light chain (VL).

The invention also provides compositions comprising the binding proteins of the invention as described above.

The composition can further comprise a second binding protein comprising a heavy chain variable region CDR1, a heavy chain variable region CDR2, a heavy chain variable region CDR3, a light chain variable region CDR1, a light chain variable region CDR2, a light chain variable region CDR 3; wherein the content of the first and second substances,

heavy chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 9;

heavy chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 10;

heavy chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 11;

light chain variable region CDR1 comprises the amino acid sequence shown in SEQ ID NO. 13;

light chain variable region CDR2 comprises the amino acid sequence shown in SEQ ID NO. 14;

light chain variable region CDR3 comprises the amino acid sequence shown in SEQ ID NO. 15;

more preferably, the second binding protein comprises a heavy chain variable region, a light chain variable region; wherein the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO.12, and the light chain variable region comprises an amino acid sequence shown in SEQ ID NO. 16.

An example of such a second binding protein is a monoclonal antibody.

The composition may further comprise additional bioactive or diagnostic agents.

The antibodies of the invention or compositions containing them may be included in a container, package, or dispenser, alone or as part of a kit, together with a label and instructions for administration.

The diagnostic agent includes a detectable substance, examples of which include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radionuclides, positron emitting metals (for use in positron emission tomography), and non-detectable substancesAs regards the metal ions which can be conjugated to antibodies for use as diagnostic agents, see generally U.S. Pat. No.4,741,900, suitable enzymes include horseradish peroxidase, alkaline phosphatase, β -galactosidase, or acetylcholinesterase, suitable prosthetic groups include streptavidin, avidin and biotin, suitable fluorescent materials include umbelliferone, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin, suitable luminescent materials include luminol, suitable bioluminescent materials include luciferase, fluorescein and aequorin, radioisotopes such as, for example, luciferase, luciferin and aequorin¹²⁵I、¹³¹I、¹¹¹In and⁹⁰Y、Lu¹⁷⁷bismuth, bismuth²¹³Californium²⁵²Iridium (III)¹⁹²And tungsten¹⁸⁸Rhenium¹⁸⁸、²¹¹Astatine, astatine,⁹⁹Tc。

The terms "pegylated," "polyethylene glycol," or "PEG" include polyalkylene glycol compounds or derivatives thereof, with or without a coupling agent or derivatization with a coupling or activating moiety, such as with a thiol, triflate, tresylate, aziridine, ethylene oxide, or preferably with a maleimide moiety, such as PEG-maleimide. Other suitable polyalkylene glycol compounds include, but are not limited to, maleimide monomethoxy PEG, activated PEG polypropylene glycol, and the following types of charged or neutral polymers: dextrose, polyacetyl neuraminic acid, or other carbohydrate-based polymers, amino acid polymers, and biotin and other affibody derivatives.

The invention also provides methods for producing binding proteins (e.g., antibodies) of the invention, including methods using immortalized cell lines, artificial synthesis, recombinant expression, or phage display techniques. In a particular embodiment, the method of the invention comprises the step of culturing the host cell as described above.

The invention provides an application, which comprises the following applications:

(1) the use of the binding protein as hereinbefore described for the preparation of a novel coronavirus detection product;

(2) use of the binding protein as hereinbefore described for the preparation of a novel diagnostic product for coronavirus infection;

(3) use of a composition as hereinbefore described for the preparation of a test product for a novel coronavirus.

The test product or diagnostic product comprises the kit as described above.

Drawings

FIG. 1 shows a SDS-PAGE pattern of the recombinant SARS-CoV 2NP protein of the present invention;

FIG. 2 is a graph showing the results of detection of antibody titer by indirect ELISA;

FIG. 3 is a graph showing the results of detecting the binding of an antibody to an antigen using WB;

FIG. 4 shows the results of the affinity activity of JS01 detected by SPR;

FIG. 5 shows the results of the affinity activity of JS02 detected by SPR;

FIG. 6 is a graph showing the results of detecting the affinity activity of JS03 using SPR;

FIG. 7 is a graph showing the results of detecting the affinity activity of JS04 using SPR;

FIG. 8 is a graph showing the results of detecting the affinity activity of JS05 using SPR;

FIG. 9 is a graph showing the results of detecting the affinity activity of JS06 using SPR;

FIG. 10 is a graph showing the results of detecting the affinity activity of JS07 using SPR;

FIG. 11 is a graph showing the results of detecting the affinity activity of JS08 using SPR;

FIG. 12 is a graph showing the results of detecting the affinity activity of JS09 using SPR;

FIG. 13 is a graph showing the results of detecting the affinity activity of JS10 using SPR;

FIG. 14 is a graph showing the results of detecting the affinity activity of JS11 using SPR;

FIG. 15 is a graph showing the results of detecting the affinity activity of JS12 using SPR;

FIG. 16 is a graph showing the results of detecting the affinity activity of JS13 using SPR;

FIG. 17 is a graph showing the results of detecting the affinity activity of JS14 using SPR;

FIG. 18 is a graph showing the results of detecting the affinity activity of JS15 using SPR;

FIG. 19 is a graph showing the results of detecting the affinity activity of JS16 using SPR;

FIG. 20 is a graph showing the results of measuring the antibody coating concentration by the double antibody sandwich method;

FIG. 21 is a graph showing the results of detection sensitivity of antibodies by the double antibody sandwich method;

FIG. 22 is a graph showing the detection effect of the antigen detection chromatographic strip of the present invention.

Detailed Description

The invention is further illustrated by the figures and examples. It should be understood that the examples of the present invention are for illustrative purposes and not intended to limit the present invention. Simple modifications of the invention in accordance with its spirit fall within the scope of the claimed invention.