阅读说明：本技术 用于新型冠状病毒检测的试剂盒 (Kit for detecting novel coronavirus ) 是由 张黎 郑滨洋 高行素 潘红星 金鹏飞 史云凤 朱凤才 于 2020-05-27 设计创作，主要内容包括：本发明公开了用于新型冠状病毒检测的试剂盒,所述试剂盒包含单克隆抗体,所述单克隆抗体特异性结合新型冠状病毒NP蛋白。本发明的单克隆抗体包含含有SEQ ID NO.1-3的氨基酸序列的重链可变区,以及含有SEQ ID NO.5-7的氨基酸序列的轻链可变区。本发明的单克隆抗体可用于检测新型冠状病毒的存在。(The invention discloses a kit for detecting novel coronavirus, which comprises a monoclonal antibody, wherein the monoclonal antibody is specifically combined with novel coronavirus NP protein. The monoclonal antibody of the present invention comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID nos. 1 to 3, and a light chain variable region comprising the amino acid sequence of SEQ ID nos.5 to 7. The monoclonal antibodies of the invention can be used to detect the presence of novel coronaviruses.)

1. An isolated monoclonal antibody, or antigen-binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences; wherein CDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.1 or a conservatively modified form thereof; CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.2 or a conservatively modified form thereof; CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.3 or a conservatively modified form thereof; CDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID No.5 or a conservatively modified form thereof; CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID No.6 or a conservatively modified form thereof; CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID No.7 or a conservatively modified form thereof.

2. The monoclonal antibody, or antigen-binding portion thereof, of claim 1, wherein the heavy chain variable region of the monoclonal antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID No.4, and the light chain variable region of the monoclonal antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID No. 8.

3. A bispecific molecule comprising the monoclonal antibody or antigen-binding portion thereof of claim 1 or 2 linked to a second functional moiety having a different binding specificity than said monoclonal antibody or antigen-binding portion thereof.

4. An isolated nucleic acid molecule encoding the monoclonal antibody or antigen binding portion thereof of claim 1 or 2.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the expression vector of claim 5.

7. A composition comprising the monoclonal antibody or antigen-binding portion thereof of claim 1 or 2; preferably, the composition further comprises a second monoclonal antibody or antigen binding portion thereof 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;

most preferably, the second antibody comprises a heavy chain variable region, a light chain variable region; wherein, the heavy chain variable region contains an amino acid sequence shown by SEQ ID NO.12, and the light chain variable region contains an amino acid sequence shown by SEQ ID NO. 16.

8. The composition of claim 7, wherein the composition further comprises a diagnostic agent comprising: radionuclides, contrast agents, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitizing diagnostic agents.

9. A product for detecting a novel coronavirus, which comprises the monoclonal antibody or an antigen-binding portion thereof according to claim 1 or 2; preferably, the product comprises a product for detecting antigen-antibody binding by enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescence immunoassay, colloidal gold immunochromatography, agglutination, immunoturbidimetry;

more preferably, the product further comprises a second monoclonal antibody or antigen binding portion thereof 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;

most preferably, the second antibody comprises a heavy chain variable region, a light chain variable region; wherein, the heavy chain variable region contains an amino acid sequence shown by SEQ ID NO.12, and the light chain variable region contains an amino acid sequence shown by SEQ ID NO. 16.

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

(1) use of the monoclonal antibody or antigen-binding portion thereof of claim 1 or 2 for the preparation of a novel coronavirus detection product;

(2) use of the monoclonal antibody or antigen-binding portion thereof of claim 1 or 2 for the preparation of a novel diagnostic product for coronavirus infection;

(3) use of a composition according to claim 7 or 8 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 kit for detecting a novel coronavirus.

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 an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences; and a light chain variable region comprising the CDR1, CDR2, and CDR3 sequences; wherein CDR1 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.1 or a conservatively modified form thereof; CDR2 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.2 or a conservatively modified form thereof; CDR3 of the heavy chain variable region comprises the amino acid sequence shown in SEQ ID No.3 or a conservatively modified form thereof; CDR1 of the light chain variable region comprises the amino acid sequence shown in SEQ ID No.5 or a conservatively modified form thereof; CDR2 of the light chain variable region comprises the amino acid sequence shown in SEQ ID No.6 or a conservatively modified form thereof; CDR3 of the light chain variable region comprises the amino acid sequence shown in SEQ ID No.7 or a conservatively modified form thereof.

The heavy chain variable region of the monoclonal antibody or antigen-binding portion thereof of the present invention comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID No.4, and the light chain variable region of the monoclonal antibody or antigen-binding portion thereof of the present invention comprises an amino acid sequence that is at least 80% homologous to the amino acid sequence set forth in SEQ ID No. 8.

The invention also provides bispecific molecules comprising a monoclonal antibody or antigen-binding portion thereof as described above linked to a second functional module having a different binding specificity to the monoclonal antibody or antigen-binding portion thereof.

The invention also provides compositions comprising a monoclonal antibody, or antigen-binding portion thereof, or a bispecific molecule of the invention.

The invention also encompasses nucleic acid molecules encoding the monoclonal antibodies of the invention, or antigen binding portions thereof, as well as expression vectors comprising such nucleic acids and host cells comprising such expression vectors.

"antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

The term "antigen-binding portion" as used herein refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab')2 fragment comprising a bivalent fragment of two Fab fragments connected by a hinge region disulfide bridge; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) fv fragments, consisting of the VL and VH domains of a single arm of an antibody; (v) dAb fragments (Ward et al (1989) Nature 341:544-546) consisting of a VH domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombinant methods, enabling them to be prepared as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments can be obtained using conventional techniques well known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies.

An "isolated monoclonal antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. Furthermore, the isolated antibody may be substantially free of other cellular material and/or chemicals.

"monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

Homologous antibodies

The antibodies of the invention comprise variable regions of the heavy and light chains comprising amino acid sequences that are homologous to the amino acid sequences of preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-novel coronavirus antibodies of the invention.

For example, the invention provides an isolated monoclonal antibody, or antigen binding portion thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

(a) the heavy chain variable region comprises an amino acid sequence which is at least 80% homologous to the amino acid sequence shown in SEQ ID No. 4;

(b) the light chain variable region comprises an amino acid sequence which is at least 80% homologous to the amino acid sequence shown in SEQ ID NO. 8.

In other embodiments, the VH and/or VL amino acid sequences may be 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous to the sequences described above. Antibodies having high (i.e., 80% or greater) homology of the VH and VL regions to the VH and VL regions of the sequences described above can be obtained by mutagenesis (e.g., site-directed mutagenesis or PCR-mediated mutagenesis) of the nucleic acid molecule encoding the amino acid sequence.

As used herein, the percent homology between two amino acid sequences is equal to the percent identity between the two sequences. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e.,% homology is the number of identical positions/total number of positions x 100), taking into account the number of gaps that need to be introduced and the length of each gap to produce an optimal alignment of the two sequences. As shown in the following non-limiting examples, comparison of sequences and determination of percent identity between two sequences can be accomplished using mathematical algorithms.

The percent identity between two amino acid sequences can be determined using the algorithm of e.meyers and w.miller (comput.appl.biosci.,4:11-17(1988)) which has been incorporated into the ALIGN program (version 2.0) using a PAM120 residue weight table with a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (J.mol.biol.48: 444-.

Additionally or alternatively, the protein sequences of the invention may further be used as "query sequences" to search public databases, for example to identify related sequences. Such searches can be performed using the XBLAS program (version 2.0) of Altschul et al (1990) J.mol.biol.215: 403-10. BLAST protein searches can be performed using the XBLAST program to score 50 and the word length 3 to obtain amino acid sequences homologous to the antibody molecules of the present invention. To obtain gapped alignments for comparison, gappedBLAST was used as described in Altschul et al (1997) Nucleic Acids Res.25(17): 3389-3402. When BLAST and Gapped BLAST programs are used, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. (see www.ncbi.nlm.nih.gov).

Antibodies with conservative modifications

In certain embodiments, the antibodies of the invention comprise a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprises a particular amino acid sequence or conservative modifications thereof based on the preferred antibodies described herein, and wherein the antibodies retain the desired functional properties of the anti-novel coronavirus antibodies of the invention.

As used herein, the term "conservative sequence modification" is intended to mean that the amino acid modification does not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated advantages. Conservative amino acid substitutions refer to the replacement of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been described in detail in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues in a CDR region of an antibody of the invention can be replaced with other amino acid residues from the same side chain family.

Engineered and modified antibodies

The antibodies of the invention may further be prepared using antibodies having one or more of the VH and/or VL sequences disclosed herein as starting materials to engineer modified antibodies, wherein the modified antibodies may have different properties than the starting antibodies. Antibodies can be engineered by modifying one or more residues in one or both variable regions (i.e., VH and/or VL), e.g., in one or more CDR regions and/or in one or more framework regions. Additionally or alternatively, antibodies may be engineered by modifying residues in the constant region, for example, to alter the effector function of the antibody.

One type of variable region engineering that can be performed is CDR grafting. Antibodies interact with the target antigen primarily through amino acid residues located in the six heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the difference in amino acid sequence in CDR among individual antibodies is larger than that of the sequence outside CDR. Because the CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing an expression vector that contains CDR sequences from the particular naturally occurring antibody grafted onto framework sequences from different antibodies with different properties (see, e.g., Riechmann, L. et al (1998) Nature 332: 323-327; Jones, P. et al (1986) Nature321: 522-525; Queen, C. et al (1989) Proc. Natl. Acad. See. U.S. A.86: 10029-10033; Winter U.S. Pat. No.5,225,539 and Queen et al U.S. Pat. No.5,530,101; 5,585,089; 5,693,762 and 6,180,370).

Another type of variable region modification is mutation of amino acid residues in the VH and/or VK CDR1, CDR2, and/or CDR3 regions to improve one or more binding properties (e.g., affinity) of the antibody of interest. Mutations can be introduced by site-directed mutagenesis or PCR-mediated mutagenesis. Preferably, conservative modifications (as described above) are introduced. The mutation may be a substitution, addition or deletion of an amino acid, but is preferably a substitution. In addition, the residues in the CDR regions typically vary by no more than one, two, three, four or five.

Engineered antibodies of the invention include those in which framework residues in the VH and/or VK are modified, e.g., to improve antibody properties. Such framework modifications are typically made to reduce the immunogenicity of the antibody. For example, one approach is to "back mutate" (back mutation) one or more framework residues into the corresponding germline sequence. More specifically, an antibody in which somatic mutations occur may contain framework residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the antibody framework sequence to the germline sequence from which the antibody was derived.

In addition or alternatively to modifications made in the framework or CDR regions, antibodies of the invention can be engineered to include modifications in the Fc region, which are typically used to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antibody-dependent cellular cytotoxicity. In addition, the antibodies of the invention may be chemically modified (e.g., by attaching one or more chemical moieties to the antibody) or modified to alter glycosylation thereof, again for altering one or more functional properties of the antibody. The numbering of the residues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This process is further described in U.S. Pat. No.5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another embodiment, the Fc hinge region of the antibody is mutated to shorten the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH 2-CH 3 domain interface region of the Fc-hinge fragment such that the antibody has impaired staphylococcal protein a (SpA) binding relative to native Fc-hinge domain SpA binding. This method is further described in detail in U.S. Pat. No.6,165,745 to Ward et al.

In another embodiment, the antibody is modified to increase its biological half-life. There are several ways that are possible. For example, as described in U.S. Pat. No.6,277,375 to Ward, one or more of the following mutations are introduced: T252L, T254S, T256F. Alternatively, to extend the biological half-life, antibodies may be altered in the CH1 or CL region to include a salvage receptor (salvage receptor) binding epitope taken from both loops of the CH2 domain of the Fc region of IgG, as described in Presta et al, U.S. patent nos.5,869,046 and 6,121,022.

In yet another embodiment, the glycosylation of the antibody is modified. For example, aglycosylated (i.e., antibodies lacking glycosylation) antibodies may be prepared. Glycosylation can be altered, for example, to increase the affinity of an antibody for an antigen. Such carbohydrate modifications can be achieved, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more amino acid substitutions are made to remove one or more variable region framework glycosylation sites, thereby removing glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for the antigen. This process is described in further detail in U.S. Pat. Nos.5,714,350 and 6,350,861 to Co et al.

Another modification of the antibodies herein contemplated by the present invention is pegylation. The antibody can be pegylated, for example, to extend the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions such that one or more PEG groups are attached to the antibody or antibody fragment. Preferably, pegylation can be performed by acylation or alkylation with a reactive PEG molecule (or similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to include any form of PEG that has been used to derivatize other proteins, such as mono (C1-C10) alkoxy-or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods of PEGylating proteins are known in the art and can be used with the antibodies of the invention. See, for example, EP 0154316 to Nishimura et al and EP 0401384 to Ishikawa et al.

Nucleic acid molecules encoding the antibodies of the invention

Another aspect of the invention relates to a nucleic acid molecule encoding an antibody of the invention. The nucleic acid may be present in an intact cell, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "rendered substantially pure" when purified of other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkali/SDS treatment, CsCl banding (banding), column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, e.g., Ausubel et al (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York. The nucleic acids of the invention may be, for example, DNA or RNA, and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

The nucleic acids of the invention can be obtained using standard molecular biology techniques. Once the DNA fragments encoding the VH and VL segments are obtained, these are further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes to full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a DNA fragment encoding a VL or VH is operably linked to another DNA fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked" as used herein is intended to mean that two DNA fragments are linked such that the amino acid sequences encoded by the two DNA fragments remain in frame (in-frame).

Isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operably linking the DNA encoding the VH to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH 3). The sequence of the human heavy chain constant region gene is known in the art.

The isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as the Fab light chain gene) by operably linking the DNA encoding the VL to another DNA molecule encoding the light chain constant region CL. The sequence of the human light chain constant region gene is known in the art.

Bispecific molecules

The invention encompasses bispecific molecules against the novel coronavirus antibodies of the invention or fragments thereof.

The antibodies of the invention or antigen-binding portions thereof can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand to a receptor) to generate a bispecific molecule that binds to at least two different binding sites or target molecules. The antibodies of the invention may in fact be derivatized or linked to one or more other functional molecules to generate multispecific molecules that bind to two or more different binding sites and/or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule of the invention, an antibody of the invention can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent binding, or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, thereby producing a bispecific molecule.

The invention also provides an expression vector comprising the nucleic acid molecule as described above.

The present invention also provides a host cell comprising the expression vector as described above.

The invention also provides compositions comprising the aforementioned monoclonal antibodies or antigen-binding portions thereof.

Further, the composition further comprises a second monoclonal antibody or antigen binding portion thereof 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;

preferably, the second antibody comprises a heavy chain variable region, a light chain variable region; wherein, the heavy chain variable region contains an amino acid sequence shown by SEQ ID NO.12, and the light chain variable region contains an amino acid sequence shown by SEQ ID NO. 16.

Still further, the composition further comprises a diagnostic agent.

Diagnostic agent

The diagnostic agent useful in the present invention includes: radionuclides, contrast agents, fluorescent agents, chemiluminescent agents, bioluminescent agents, paramagnetic ions, enzymes, and photosensitizing diagnostic agents.

The radionuclide comprises¹¹⁰In、¹¹¹In、¹⁷⁷Lu、¹⁸F、⁵²Fe、⁶²Cu、⁶⁴Cu、⁶⁷Cu、⁶⁷Ga、⁶⁸Ga、⁸⁶Y、⁹⁰Y、⁸⁹Zr、^94mTc、⁹⁴Tc、^99mTc、¹²⁰I、¹²³I、¹²⁴I、¹²⁵I、¹³¹I、^154-158Gd、³²F、¹¹C、¹³N、¹⁵O、¹⁸⁶Re、¹⁸⁸Re、⁵¹Mn、^52mMn、⁵⁵Co、⁷²As、⁷⁵Br、⁷⁶Br、^82mRb、⁸³Sr or other gamma emitters, β emitters or positron emitters.

Paramagnetic ions include: chromium (III), manganese (II), iron (III), iron (II), cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III), ytterbium (III), gadolinium (III), vanadium (II), terbium (III), dysprosium (III), holmium (III) and erbium (III).

The fluorescent labeling compound comprises fluorescein isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

Chemiluminescent labeling compounds include luminol, isoluminol, aromatic acridinium esters, imidazoles, acridinium salts, and oxalate esters.

Bioluminescent compounds include luciferin, luciferase and aequorin.

The present invention provides a product for detecting a novel coronavirus which comprises the monoclonal antibody or antigen-binding portion thereof as described above.

Further, the kit may further comprise a second monoclonal antibody or antigen binding portion thereof as described above.

Still further, the products include products for detecting antigen-antibody binding by enzyme-linked immunosorbent assay, immunofluorescence assay, radioimmunoassay, luminescence immunoassay, colloidal gold immunochromatography, agglutination, immunoturbidimetry.

The invention provides the use of a monoclonal antibody or an antigen-binding portion thereof as hereinbefore described in the preparation of a novel coronavirus detection product.

The invention provides the use of a monoclonal antibody or an antigen-binding portion thereof as hereinbefore described in the manufacture of a novel diagnostic product for coronavirus infection.

The invention provides the use of a composition as hereinbefore described in the preparation of a product for the detection of a novel coronavirus.

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.