CN113004395A - Monoclonal antibody for resisting novel coronavirus and application thereof in immunoassay - Google Patents
Monoclonal antibody for resisting novel coronavirus and application thereof in immunoassay Download PDFInfo
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- CN113004395A CN113004395A CN202010463552.1A CN202010463552A CN113004395A CN 113004395 A CN113004395 A CN 113004395A CN 202010463552 A CN202010463552 A CN 202010463552A CN 113004395 A CN113004395 A CN 113004395A
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Abstract
The invention discloses a monoclonal antibody for resisting novel coronavirus and application thereof in immunodetection. 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.
Description
Technical Field
The invention belongs to the fields of cellular immunology and molecular biology, and relates to a monoclonal antibody for resisting novel coronavirus and application thereof in immunodetection.
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, Gapped BLAST 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) Nature 321: 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 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.
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, beta 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.
Example 1 antibody screening
Expression of recombinant SARS-CoV2 Nucleoprotein (NP)
1.1 Primary reagents
The SARS-CoV 2NP gene sequence (GenBank sequence number: MT066176.1) and the related primer synthesis and sequencing are all completed by general biological systems (Anhui) limited company; coli DH5 α, BL21(DE3) competent cells were purchased from general biosystems (anhui) ltd; BamHI and NotI endonucleases were purchased from New England Biolabs (NEB); EX Taq enzyme was purchased from TaKaRa; HRP-labeled anti-human Fc antibody was purchased from Sigma; other chemical reagents are domestic analytical pure reagents; serum of 2019-nCoV infected patients is collected and stored by the center, and all cases are Jiangsu cases.
1.2 prokaryotic expression plasmid construction
Designing a prokaryotic expression primer of the NP gene, wherein an upstream primer is provided with a BamH I restriction site, and a downstream primer is provided with a Not I restriction site. The primer sequence is as follows: cov 2-NP-F: CGGGATCCTCTGATAATGGACCCCAAAATC; cov2-NP-R: ATAAGAATGCGGCCGCAGGCCTGAGTTGAGTCAGCAC. The NP gene was amplified using EX Taq enzyme, and the PCR reaction program was: 3min at 94 ℃; 30 cycles of 94 ℃ for 30s,58 ℃ for 30s and 72 ℃ for 80 s; 10min at 72 ℃. And recovering a 1300 bp target fragment from the PCR product by using glue, performing double enzyme digestion on the PCR product by using BamH I and Not I, connecting the PCR product with a pET28a vector, and transforming E.coli DH5 alpha competent cells. After single colony is selected the next day and sequenced correctly, the quality-improved particles are transformed into prokaryotic expression bacterium E.coli BL21(DE3) competent cells.
1.3NP expression and purification
Culturing NP expressing strain until OD600 is 0.6, adding IPTG with final concentration of 0.5mmol/L, inducing at 16 deg.C for 6h, collecting thallus, ultrasonic crushing, and centrifuging to collect inclusion body. The inclusion bodies were dissolved in 8mol/L urea and then purified by nickel column affinity chromatography. After purification, the urea content is reduced in a gradient manner, the protein is dialyzed and renatured into PBS, and finally the protein expression and purification effects are detected by SDS-PAGE. After the small amount of fermentation is finished, the mixture is put into a 100L fermentation tank for mass fermentation, the fermentation medium is a TB medium (1% glycerol), the fermentation parameter is 280 rpm, the aeration ratio is 0.5vvm (15L/min), the pH is controlled to be 6.8-7.2, the tank pressure is 0.06 MPa-0.1 MPa, the fermentation temperature is 16 ℃, and the mixture is cultured for 24 hours.
The SDS-PAGE results showed: the total length of the NP plus His tag and other additional amino acids in the vector predicted that the protein had a relative molecular mass of about 50X 103. The expression strain is found to be 50 multiplied by 10 after being induced by IPTG3There is a clear band around, consistent with the expected molecular weight size (FIG. 1A). After the inclusion body is dissolved, the inclusion body is purified by a nickel column, and an obvious elution peak is obtained when the concentration is 150 mmol/L imidazole. After the proteins were renatured by dialysis, a single protein band was found to appear at the same position by SDS-PAGE (FIG. 1B). This indicates that the NP was successfully induced and purified to a higher degree. Note: in the figure, M: proteins, Makers; 1: uninduced pET28a-NP expressing bacteria; 2: pET28a-NP recombinant expression bacteria after IPTG induction; 3: and (4) purifying to obtain the recombinant nucleocapsid protein.
Second, phage library construction
1. Collecting peripheral blood of patient with COVID-19 in convalescent period, and separating mononuclear cells (PBMC) from the peripheral blood
In the project, 20ml of peripheral blood of 5 COVID-19 patients before discharge is collected from Jiangsu province, a certain city and after informed consent, on 14 days 2 and 14 months in 2020. 5 patients are in the same transmission chain, 5 patients are not severe, and after treatment, the patients are respectively isolated from 2 months, 15 days to 22 days of hospital discharge and home. Mononuclear Cells (PBMC) were separated from 20ml of heparin anticoagulated using GE Ficoll-Paque PLUS by density gradient centrifugation.
2. Extraction of RNA and cDNA Synthesis in PBMC
PBMC cellular RNA was extracted using the RNeasy Mini Kit from QIAGEN, and then the RNA was reverse-transcribed into cDNA using the First Strand synthesis Kit from Roche (Transcriptor First Strand cDNAsynthesis Kit, Roche, Cat No.: 04896866001).
3. PCR amplification of VK, VL and VH (EX Taq, Takara, Cat No.: DRR001A)
(1) The amplification VK & VL system is shown in Table 1.
TABLE 1 amplification VK & VL system
Solutions or compositions | Volume (μ L) |
|
1 |
EX Buffer(10x) | 5 |
dNTPs(10mM each) | 4 |
P1(10μM) | 2 |
P2(10μM) | 2 |
EX Taq 1U/μl | 0.3 |
dH2O | 35.7 |
(2) The amplified heavy chain Fd fragment system is shown in Table 2.
TABLE 2 amplification of heavy chain Fd segment systems
Solutions or compositions | Volume (μ L) |
|
2 |
EX Buffer(10x) | 10 |
dNTPs(10mM each) | 8 |
P1(10μM) | 2 |
P2(10μM) | 2 |
EX Taq 1U/μl | 0.6 |
dH2O | 75.4 |
(3) The reaction sequence is shown in table 3.
TABLE 3 reaction procedure
The PCR product was electrophoresed through 2% agarose gel, and a fragment of about 750bp was recovered.
4. Cloning of the light chain (cloning VK and VL into pComb3H vector)
VK and VL were digested with XbaI and SacI and ligated with pComb3H vector, which was also digested with XbaI and SacI, and the ligation product was recovered and then transfected into XL1-Blue competent cells.
And (3) coating the electric shock bacterium liquid on a 15cm large plate, scraping the bacterium the next day, and obtaining the quality-improved particles, namely the light chain library. The recombinant plasmids were pComb3H-VK and pComb3H-VL at this time.
5. Heavy chain cloning (cloning VH Gene into pComb3H-VK and pComb3H-VL light chain Bank)
The light chain library pComb3-L and Fd fragments are respectively subjected to double enzyme digestion by XhoI and SpeI, are connected with pComb3H-VK and pComb3H-VL which are also subjected to double enzyme digestion by XhoI and SpeI, and are then electrically transformed to obtain the antibody library.
6. Packaging of antibody libraries
(1) Taking out the antibody library from a refrigerator at the temperature of-80 ℃, melting on ice, adding 1ml of the antibody library into 10ml of A + (20 mu g/ml)2YT culture medium, and shaking at the temperature of 37 ℃ and 200rpm for 1 hour;
(2) adding 100ml of A + (100. mu.g/ml), T + (20. mu.g/ml) 2YT medium, and shaking at 200rpm for 1 hour;
(3) plus 1012pfu VCSM13 helper phage, standing at 37 deg.C for 20min, shaking at 200rpm for 2 hr;
(4) adding 70 mu g/ml kanamycin at 30 ℃ and shaking at 200rpm overnight;
(5) centrifuging at 6000rpm for 20min the next day, pouring out the supernatant, adding 4% PEG8000(4g) and 3% NaCl (3g), mixing, and placing on ice for more than 30 min;
(6) and subpackaging in a 50ml centrifuge tube, centrifuging at 9000rpm for 25min, removing supernatant, draining, and resuspending the precipitate with 1ml PBS to obtain the packaged library.
Screening of phage library
1. The recombinant SARS-CoV2 Nucleoprotein (NP) was coated in an immune tube, 3 tubes were coated at 50. mu.g/tube, and left overnight at 4 ℃ with 2% skim milk for the next day to block the immune tube for 1 h.
2. 1.75ml of PBS containing 2% skim milk and 250. mu.l of the phage library were added to the tube, shaken at 37 ℃ for 1h, and then allowed to stand at 37 ℃ for 1 h.
3. The phage library was inverted and washed 20 times with PBST, 5min each.
4. The tube was eluted with 1ml Gly-HCl pH 2.2, left to stand at room temperature for 5min, shaken at 37 ℃ for 5min, then pipetted into a 1.5ml EP tube and neutralized to pH 7 with 57 μ l 2M Tris.
5. The eluate was transferred to a new 50ml centrifuge tube and 10ml of OD 1 fresh XL1-Blue was added immediately, mixed well and incubated at 37 ℃ for 30min, 10ml of 2YT (Amp 100. mu.g/ml, Tet 20. mu.g/ml) was added.
6. Mu.l of the broth was used to determine the volume of the elution pool, and 20ml of the remaining medium was poured into a 500ml Erlenmeyer flask and shaken at 230rpm for 1 hour.
7. 130ml of 2YT (Amp 100ug/ml, Tet 20. mu.g/ml) were added, shaken at 230rpm for 1 h.
8. The helper phage with MOI 20 was added and incubated at 37 ℃ for 30 min.
9. Centrifuge at 3000g for 10min, resuspend pellet into 150ml 2YT (Amp 100. mu.g/ml, Tet 20. mu.g/ml), shake at 37 ℃ at 230rpm for 2 h.
10. 110. mu.l of 70mg/ml kanamycin was added, and 30 ℃ overnight at 230 rpm. Adding 1/5 volume of PEG-NaCl (40ml) the next day, mixing, ice-cooling for at least 1h, centrifuging at 10000g and 4 deg.C for 20min, suspending the precipitate in 2-3ml PBS, centrifuging instantaneously to remove bacteria, and filtering with 0.45 μm filter for the next round of screening.
11. Repeating the screening step for 3 times to achieve the purpose of enriching and screening the phage library.
12. After the third round of enrichment, 2 x 96 clones were picked. After IPTG induction, ELSA detection was performed the next day.
Four, ELISA detection of 2 x 96 clones binding specificity
1.2 pieces of anti-human Fab antibody (1:3000) and 2 pieces of NP protein (2. mu.g/ml) were coated separately and left overnight at 4 ℃.
2. The next day was blocked with 3% skim milk for 1h, then 50. mu.l of induction supernatant and 50. mu.l of skim milk were added, incubated at 37 ℃ for 1h, and washed with PBST.
3. HRP-labeled anti-human Fab antibody (1:3000) was added to each of the 4 plates, incubated at 37 ℃ for 1h, washed with PBST, and then TMB developed.
178 phage antibody fragments which can be specifically combined with NP are obtained through screening, and the fragments are Fab fragments of human origin, including full-length light chain and Fd fragment of heavy chain. 178 single colonies were amplified and sequenced to obtain 159 strains of complete and qualified sequences.
Example 2 expression of full antibodies and related functional validation
Finally selecting 16 antibodies from the 159 antibodies for expression of the whole antibody and relevant function verification, and naming the 16 antibodies as JS01-JS 16.
Wherein the JS06 antibody sequence is shown as follows:
the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO. 1;
the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO. 2;
the amino acid sequence of the heavy chain variable region CDR3 is shown in SEQ ID NO. 3;
the amino acid sequence of CDR1 in the variable region of the light chain is shown in SEQ ID NO. 5;
the amino acid sequence of CDR2 in the variable region of the light chain is shown in SEQ ID NO. 6;
the amino acid sequence of CDR3 in the variable region of the light chain is shown in SEQ ID NO. 7;
the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.4, and the nucleic acid sequence is shown as SEQ ID NO. 17; the amino acid sequence of the light chain variable region is shown as SEQ ID NO.8, and the nucleic acid sequence is shown as SEQ ID NO. 18.
The JS08 antibody sequence is shown below:
the amino acid sequence of the heavy chain variable region CDR1 is shown in SEQ ID NO. 9;
the amino acid sequence of the heavy chain variable region CDR2 is shown in SEQ ID NO. 10;
the amino acid sequence of CDR3 in the heavy chain variable region is shown in SEQ ID NO. 11;
the amino acid sequence of CDR1 in the variable region of the light chain is shown in SEQ ID NO. 13;
the amino acid sequence of CDR2 in the variable region of the light chain is shown in SEQ ID NO. 14;
the amino acid sequence of CDR3 in the variable region of the light chain is shown in SEQ ID NO. 15.
The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 12; the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 16.
1. Full antibody expression
The 16-strain humanized antibody is constructed into an IgG-type humanized whole molecule antibody, expressed in 293F cells and purified by Protein A for later use.
2. ELISA (enzyme-Linked immuno sorbent assay) for detecting binding specificity of 16-strain antibody and recombinant NP
Recombinant NPs were coated onto ELISA plates with PBS at a concentration of 1. mu.g/ml, all antibody concentrations were diluted to 1mg/ml, then diluted in multiples starting at 1:10000 and incubated at 37 ℃ for 30 min. Then PBST was washed 3 times, HRP-labeled anti-human IgG (1:5000) was added, and after incubation at 37 ℃ for 30min, PBST was washed 3 times, then TMB was developed, and OD450 absorbance values were read after termination.
The dilution titer of the 16 NP antibody was measured by indirect ELISA, and the average OD value of the negative control was 0.119 with a standard deviation of 0.132, so that the cutoff value was defined asThe detection titer of the 16-strain antibody was judged to be between 1:80000 and 1:1280000 (FIG. 2).
3. Western Blot results of 16 antibodies and purified NP
Mu.g of the recombinant NP was electrophoresed by SDS-PAGE, transferred to a PVDF membrane, incubated with the above 16 antibodies (0.5. mu.g/ml) at 37 ℃ for 1h, washed 3 times with PBST, then incubated with HRP-labeled anti-human IgG (1:5000) for 30min, washed 3 times with PBST, and then developed on the membrane with DAB.
WB experimental results showed that 16 antibodies were able to specifically bind to recombinantly expressed Nucleoprotein (NP) and a distinct band of color appeared at 50kDa, suggesting that the group of antibodies were all linear epitope antibodies (fig. 3).
4. Antibody affinity activity detection
The antibody affinity determination is completed by a Biacore T200 workstation and is carried out according to the following steps: the CM5 chip was first activated with amino-coupled activators NHS and EDC at 10. mu.l/min for 300s, then the recombinantly expressed SARS-CoV-2NP was diluted to 1ug/mL with 10mM sodium acetate buffer (pH5.5), the Response values (RUs) were brought to around 600 by flowing 10. mu.l/min through the chip for 30s, and finally 10. mu.l/min, 420s were set, and the remaining activated sites on the chip surface were blocked with ethanolamine. Serially diluted antibodies were sequentially injected at 25 ℃ at a flow rate of 30. mu.l/min, and after each concentration measurement, CM5 chips were regenerated with glycine-hydrochloric acid of pH 2.0, and then subjected to the next concentration measurement. After the experiment was completed, binding affinity was obtained by global fitting of the curve using Biacore T200 Evaluation Software.
The experimental results are shown in FIGS. 4-19, JS01-JS16 can efficiently bind to SARS-CoV-2NP protein, and the parameters related to the affinity activity are shown in Table 4.
TABLE 4 antibody affinity parameters
Name of antibody | Amount of ligand coupling | ka(1/Ms) | Kd(1/s) | KD(M) |
JS01 | 102RU | 2.18E+06 | 4.79E-04 | 2.20E-10 |
JS02 | 162RU | 9.12E+05 | 5.83E-05 | 6.39E-11 |
JS03 | 102RU | 8.97E+05 | 2.12E-04 | 2.36E-10 |
JS04 | 162RU | 7.15E+04 | 1.91E-04 | 2.67E-09 |
JS05 | 162RU | 5.94E+05 | 2.43E-04 | 4.09E-10 |
JS06 | 136RU | 1.61E+05 | 0.002576 | 1.61E-08 |
JS07 | 110RU | 1.44E+06 | 1.78E-04 | 1.24E-10 |
JS08 | 162RU | 3.03E+04 | 5.77E-06 | 1.90E-10 |
JS09 | 129RU | 6.89E+05 | 4.79E-05 | 6.94E-11 |
JS10 | 136RU | 1.47E+06 | 2.99E-04 | 2.04E-10 |
JS11 | 110RU | 1.93E+05 | 5.62E-05 | 2.92E-10 |
JS12 | 110RU | 4.88E+05 | 7.33E-05 | 1.50E-10 |
JS13 | 110RU | 8.05E+05 | 9.66E-05 | 1.20E-10 |
JS14 | 136RU | 1.24E+06 | 2.21E-04 | 1.78E-10 |
JS15 | 110RU | 7.72E+04 | 1.22E-04 | 1.58E-09 |
JS16 | 136RU | 2.68E+05 | 4.01E-05 | 1.50E-10 |
5. Antibody pairing assay
5.1 determination of antibody coating concentration
(1) Mu.l of JS12 antibody was diluted from 5. mu.g/ml to 0.0024. mu.g/ml for 12 dilutions before being coated in ELISA plates. Coating at 4 deg.C overnight, blocking with 1% BSA for 2h, and washing with PBST for 3 times.
(2) 50ng of recombinant NP was added to the first well of each coating concentration, then diluted in multiples to 0.39 ng/well for 8 dilutions, incubated for 1h at 37 ℃ and washed 3 times with PBST.
(3) Adding HRP marked JS08 diluted at 1:1000, incubating for 1h at 37 ℃, PBST washing for 3 times, and reading the OD450nm absorbance value after TMB color development.
As can be seen from the graph in FIG. 20, the amount of the coated antibody has an effect on the detection sensitivity, and the amount of the coating from 5. mu.g/ml to 0.00245. mu.g/ml is not so much affected, and the sensitivity for detecting the NP antigen is less than 3.9 ng/ml. Therefore, in all subsequent pairing experiments, we chose a concentration of 2. mu.g/ml as the antibody coating and 1:4000 as the dilution of the enzyme-labeled antibody.
5.2 double antibody Sandwich method for detecting NP
(1) 16 NP antibodies JS01-JS16 were coated on ELISA plates at 2. mu.g/ml, coated overnight at 4 ℃, blocked with 1% BSA for 2h the next time, and washed with PBST for 3 times.
(2) 0.1. mu.g/ml recombinant NP protein was added, then diluted in multiples to 0.78ng/ml for 8 dilutions, incubated at 37 ℃ for 1h, and washed 3 times with PBST.
(3) HRP-labeled JS08(1:1000) was added, incubated at 37 ℃ for 1h, PBST washed 3 times, TMB developed, and OD450nm absorbance values were read.
As can be seen from FIG. 21, enzyme-labeled JS08 cannot pair with JS06, JS11 and JS08 per se, but can pair with other 13 NP antibodies for double antibody sandwich NP detection. The JS08 and JS16 have the best matching effect, the detection limit can reach below 0.78ng/ml, and the detection limit of other matched antibodies is 12.5-1.56 ng/ml.
6. Sensitivity of double-antibody sandwich immunochromatography for detecting recombinant NP
The anti-JS 08 monoclonal antibody is coated on a nitrocellulose membrane to form a T line, and the anti-human IgG antibody is marked to the C line. After the NP protein is diluted in series, 50 mu L of the NP protein is added into a sample hole, JS01-JS16 antibodies of the labeled colored microspheres on a binding pad under the sample hole and the NP form an immune complex, then the immune complex is migrated to a T line through chromatography, and the T line is combined and fixed with the labeled antibodies to form a colored T line. And the redundant humanized monoclonal antibodies are continuously transferred to the C line and combined with the anti-human antibodies to form the C line. This was used to determine the binding sensitivity to NP.
Respectively matching the JS08 antibody marked by the colored microspheres with the 13 strains of antibodies to prepare the antigen detection chromatographic strip. When the test strip is verified by using 2ng/ml of recombinant NP, all chromatographic strips can see a remarkable detection T line and a quality control C line is also very remarkable (FIG. 22). This indicates that both 13 pairs of antibody combinations can be used to detect nucleoproteins of the novel coronavirus with a limit of detection of less than 2 ng/ml.
Although only specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are within the scope of the invention.
Sequence listing
<110> center for disease prevention and control in Jiangsu province (public health research institute in Jiangsu province)
<120> monoclonal antibody against novel coronavirus and application thereof in immunoassay
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gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag 240
cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcacctat attcactttc 300
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Claims (10)
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 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.
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 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 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.
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