CN118440188B

CN118440188B - Antibodies against the novel coronavirus or its N protein, reagents and kits for detecting the novel coronavirus or its N protein

Info

Publication number: CN118440188B
Application number: CN202310076686.1A
Authority: CN
Inventors: 孟媛; 钟冬梅; 唐丽娜; 周俊; 游辉
Original assignee: Dongguan Pengzhi Biotechnology Co Ltd
Current assignee: Dongguan Pengzhi Biotechnology Co Ltd
Priority date: 2023-02-03
Filing date: 2023-02-03
Publication date: 2025-04-08
Anticipated expiration: 2043-02-03
Also published as: CN118440188A

Abstract

The invention discloses an antibody for resisting novel coronavirus or N protein thereof or a reagent and a kit for detecting novel coronavirus or N protein thereof, and relates to the field of antibodies. The anti-novel coronavirus or N protein antibody thereof disclosed by the invention comprises a heavy chain complementarity determining region and a light chain complementarity determining region, provides an important raw material source for the detection of novel coronavirus or N protein thereof, and has improved affinity or activity.

Description

Anti-novel coronavirus or N protein antibody thereof, reagent for detecting novel coronavirus or N protein thereof and kit

Technical Field

The invention relates to the technical field of antibodies, in particular to an anti-novel coronavirus or N protein antibody thereof, a reagent for detecting novel coronavirus or N protein thereof and a kit.

Background

Structural proteins of the novel coronaviruses 2019-nCoV are classified into spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein), which include a plurality of epitopes. N protein and viral genome RNA are intertwined to form a viral nucleocapsid, which plays an important role in the synthesis process of viral RNA. Meanwhile, N protein is relatively conserved, the proportion of the N protein in structural proteins of viruses is the largest, and the organism can generate high-level antibodies against the N protein in early infection. Finally, the N protein is an important marker protein of the novel coronavirus, and the antigen can be detected through the N protein monoclonal antibody by utilizing the principle of specific binding of the antigen and the antibody, so that the sample is directly proved to contain the novel coronavirus, and the detection of the novel coronavirus is realized.

Antibodies detected are largely classified into IgM and IgG classes. There is currently no systematic study of the generation and duration of these two classes of antibodies for the novel coronaviruses. In general, igM antibodies are produced early, once infected, and are produced rapidly, the maintenance time is short, the disappearance is rapid, and detection positivity in blood can reflect that the organism is in an acute infection state and can be used as an index of early infection. Compared with the nucleic acid detection method, the antibody detection sample is serum or plasma, is less influenced by sample sampling, is favorable for early detection and elimination of suspicious cases, and is rapid and convenient to detect and suitable for large-scale screening.

The detection of novel coronavirus or N protein antigen thereof adopts a serological detection method, mainly comprises a double-antibody sandwich ELISA method, an immune complex cleavage detection method, a hypersensitive EIA method, an enzyme-linked immunofluorescence method and the like. Currently, a double-antibody sandwich method is generally used for detecting novel coronaviruses or N protein antigens thereof, and obtaining antibodies against the novel coronaviruses or N proteins thereof is a key for realizing the double-antibody sandwich method. Thus, there is a strong need in the art for antibodies that bind efficiently to and detect novel coronaviruses or their N proteins.

Disclosure of Invention

The application provides an antibody or a functional fragment thereof, which provides an important raw material source for detecting novel coronaviruses or N proteins thereof.

In order to achieve the above object, according to one aspect of the present invention, there is provided an antibody or a functional fragment thereof, which comprises HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, wherein the HCDR1, HCDR2, HCDR3 comprises/is an amino acid sequence identical to HCDR1, HCDR2, HCDR3 of the heavy chain variable region shown in SEQ ID NO:17, and wherein the LCDR1, LCDR2, LCDR3 comprises/is an amino acid sequence identical to LCDR1, LCDR2, LCDR3 of the light chain variable region shown in SEQ ID NO: 18.

In order to achieve the above object, according to a second aspect of the present invention, there is provided an antibody or a functional fragment thereof, comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 17 and/or a light chain variable region having an amino acid sequence shown in SEQ ID NO. 18.

In order to achieve the above object, according to a third aspect of the present invention, there is provided an antibody or a functional fragment thereof, comprising a heavy chain having an amino acid sequence shown in SEQ ID NO. 19 and/or a light chain having an amino acid sequence shown in SEQ ID NO. 20.

In order to achieve the above object, according to a fourth aspect of the present invention, there is provided an antibody conjugate comprising the above antibody or a functional fragment thereof.

In order to achieve the above object, according to a fifth aspect of the present invention, there is provided a reagent or kit comprising the above antibody or a functional fragment thereof or the above antibody conjugate.

In order to achieve the above object, according to a sixth aspect of the present invention there is provided a method for detecting a novel coronavirus or N protein thereof comprising a) contacting an antibody or functional fragment thereof, antibody conjugate, or reagent or kit as described above with a novel coronavirus or N protein antigen thereof in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex, and b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said antigen in said test sample.

In order to achieve the above object, according to a seventh aspect of the present invention, there is provided a nucleic acid encoding the above antibody or a functional fragment thereof.

In order to achieve the above object, according to an eighth aspect of the present invention, there is provided a vector comprising the nucleic acid described above.

In order to achieve the above object, according to a ninth aspect of the present invention, there is provided a cell comprising the above nucleic acid, vector or antibody or functional fragment thereof expressed as above.

In order to achieve the above object, according to a tenth aspect of the present invention, there is provided a method for producing the above antibody or a functional fragment thereof, the method comprising culturing the above cell.

In order to achieve the above object, according to an eleventh aspect of the present invention, there is provided the use of an antibody or a functional fragment, an antibody conjugate, a reagent or a kit as described above for detecting or preparing a novel coronavirus or an N protein product thereof.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the results of reducing SDS-PAGE of Anti-COVN C10 mut.

Detailed Description

In a first aspect, embodiments of the present invention provide an antibody or functional fragment thereof, the antibody or functional fragment thereof comprising HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, the HCDR1, HCDR2, HCDR3 comprising/being an amino acid sequence identical to HCDR1, HCDR2, HCDR3 of the heavy chain variable region of SEQ ID NO:17, the LCDR1, LCDR2, LCDR3 comprising/being an amino acid sequence identical to LCDR1, LCDR2, LCDR3 of the light chain variable region of SEQ ID NO: 18.

In the present invention, the term "antibody" is used in the broadest sense and may include full length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies so long as they exhibit the desired biological activity.

In the present invention, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, and refer to regions comprising one or more or even all of the major amino acid residues responsible for the binding of an antibody or antigen-binding fragment to the antigen or epitope recognized by it. In a specific embodiment of the invention, CDRs refer to the highly variable regions of the heavy and light chains of the antibody.

In the present invention, the heavy chain complementarity determining region is represented by HCDR and includes HCDR1, HCDR2 and HCDR3, and the light chain complementarity determining region is represented by LCDR and includes LCDR1, LCDR2 and LCDR3.

CDR definition methods are well known in the art and include Kabat definition, chothia definition, IMGT definition, contact definition and AbM definition. As used herein, "Kabat definition" refers to the definition system described by Kabat et al, U.S. Dept. Of HEALTH AND Human Services, "Sequence of Proteins of Immunological Interest" (1983). "Chothia definition" see Chothia et al, J Mol Biol 196:901-917 (1987). Still other CDR definition methods may not strictly follow one of the above schemes, but still overlap at least a portion of the Kabat-defined CDR regions, although they may be shortened or lengthened depending on the predicted or experimental outcome of a particular residue or group of residues. Exemplary defined CDRs are listed in table 1 below. Given the variable region amino acid sequence of an antibody, one of skill in the art can routinely determine which residues comprise a particular CDR.

TABLE 1 CDR definition ¹

CDR	Kabat	AbM²	IMGT
				HCDR1	31~35	26-35	26-35
HCDR2	50~65	50-58	51-56
				HCDR3	95~102	95-102	93-102
LCDR1	24~34	24-34	27-32
				LCDR2	50~56	50-56	50-51
LCDR3	89~97	89-97	89-97

¹ The numbering of all CDR definitions in table 1 is according to the Kabat numbering system (see below).

² The "AbM" as used in table 1 has a lower case "b" referring to CDRs defined by the "AbM" antibody modeling software of OxfordMolecular.

Kabat et al also propose a numbering system for the variable region sequences of any antibody. The Kabat numbering system can be specifically mapped to any variable region sequence by one of ordinary skill in the art without relying on any experimental data outside of the sequence itself.

The invention adopts Kabat system to define CDR region, and can pass through network http:// www.abysis.org/abysis/sequence_input +.

The key_registration/key_registration.cgi yields the CDR regions defined by the Kabat system. CDR regions defined by other methods are also within the scope of the present invention.

In an alternative embodiment, the present examples provide an antibody or functional fragment thereof comprising the following complementarity determining regions:

HCDR1, comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 1.

HCDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 2.

HCDR3, comprising or consisting of the amino acid sequence set forth in SEQ ID NO. 3.

LCDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 4.

LCDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 5.

LCDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO. 6.

In the present invention, a "framework region" or "FR" region includes a heavy chain framework region and a light chain framework region, which refers to regions of an antibody heavy chain variable region and a light chain variable region other than CDRs, wherein the heavy chain framework region can be further subdivided into contiguous regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions, and the light chain framework region can be further subdivided into contiguous regions separated by CDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions.

In the present invention, the heavy chain variable region is obtained by ligating the CDRs numbered from HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4 with the FRs in a combinatorial arrangement, and the light chain variable region is obtained by ligating the CDRs numbered from LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4 with the FRs in a combinatorial arrangement.

In alternative embodiments, the antibody or functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, and LFR 4;

the HFR1 comprises/has an amino acid sequence as shown in SEQ ID NO 7 or having at least 80% identity thereto;

the HFR2 comprises/has an amino acid sequence as shown in SEQ ID NO 8 or having at least 80% identity thereto;

the HFR3 comprises/has as SEQ ID NO 9 or an amino acid sequence having at least 80% identity thereto;

The HFR4 comprises/has an amino acid sequence as shown in SEQ ID NO 10 or having at least 80% identity thereto;

the LFR1 comprises/is as SEQ ID No. 11 or an amino acid sequence having at least 80% identity thereto;

the LFR2 comprises/is as SEQ ID No. 12 or an amino acid sequence having at least 80% identity thereto;

the LFR3 comprises/is as SEQ ID No. 13 or an amino acid sequence having at least 80% identity thereto;

the LFR4 comprises/is as SEQ ID No. 14 or an amino acid sequence having at least 80% identity thereto;

in other embodiments, each of the framework region amino acid sequences of the antibodies or functional fragments thereof provided herein may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the corresponding framework region (SEQ ID NO:7, 8, 9, 10, 11, 12, 13 or 14) described above.

In alternative embodiments, the antibody or functional fragment thereof binds to the novel coronavirus or N protein thereof with an affinity of KD <9.27 x 10 ^-9 M.

In alternative embodiments, the antibody or functional fragment thereof binds to a novel coronavirus or N protein thereof with an affinity of KD.ltoreq.10 10 ^-9 M or KD.ltoreq.10 10 ^-10 M.

In alternative embodiments, the antibody or functional fragment thereof binds to a novel coronavirus or N protein thereof with an affinity of KD.ltoreq.4.14X10 ^-10 M.

Antibody affinity (KD) assays are widely varied and can be classified into thermodynamic, kinetic and dynamic equilibrium assays based on the principle of detection. Among them, thermodynamic detection methods are commonly known as Isothermal Titration Calorimetry (ITC), kinetic detection methods are commonly known as Surface Plasmon Resonance (SPR) and biological membrane light interferometry (BLI), and dynamic equilibrium detection methods are commonly known as enzyme-linked immunosorbent assay (ELISA).

In alternative embodiments, the KD is determined using kinetic detection methods, alternatively surface plasmon resonance methods, for example, by using techniques such asA biosensor system of the system.

In a second aspect, embodiments of the invention provide an antibody or functional fragment thereof comprising a heavy chain variable region having an amino acid sequence shown in SEQ ID NO. 17 and/or a light chain variable region having an amino acid sequence shown in SEQ ID NO. 18.

In alternative embodiments, the antibody or functional fragment thereof further comprises a constant region.

In alternative embodiments, the constant region comprises a heavy chain constant region and/or a light chain constant region.

In alternative embodiments, the heavy chain constant region is selected from the group consisting of an IgG1, igG2, igG3, igG4, igA, igM, igE, or IgD heavy chain constant region, and the light chain constant region is selected from the group consisting of kappa-type or lambda-type light chain constant regions.

In alternative embodiments, the constant region is of a species derived from a cow, horse, cow, pig, sheep, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, turkey, chicken, or human.

In an alternative embodiment, the constant region is of murine species origin.

In an alternative embodiment, the heavy chain constant region sequence (CH) is shown in SEQ ID NO. 15 and the light chain constant region (CL) sequence is shown in SEQ ID NO. 16.

In other embodiments, the constant region sequence may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the constant region (SEQ ID NO:15 or 16) described above.

In alternative embodiments, the functional fragment is selected from any one of F (ab ') 2, fab', fab, fv, and scFv of the antibody.

The functional fragments of the above antibodies generally have the same binding specificity as the antibody from which they were derived. It will be readily appreciated by those skilled in the art from the disclosure herein that functional fragments of the above antibodies may be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or by methods of chemical reduction cleavage of disulfide bonds. The above functional fragments are readily available to those skilled in the art based on the disclosure of the structure of the intact antibodies.

Functional fragments of the above antibodies may also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, automated peptide synthesizers such as those sold by Applied BioSystems and the like.

In a third aspect, the invention provides an antibody or functional fragment thereof, comprising a heavy chain having an amino acid sequence as shown in SEQ ID NO. 19 and/or a light chain having an amino acid sequence as shown in SEQ ID NO. 20.

In a fourth aspect, the invention provides an antibody conjugate comprising an antibody or functional fragment thereof as described above.

In an alternative embodiment, the above antibody conjugate further comprises biotin or a biotin derivative conjugated to the antibody or a functional fragment thereof.

In alternative embodiments, the antibody conjugate further comprises a label conjugated to the antibody or functional fragment thereof.

In an alternative embodiment, the above-mentioned marker refers to a substance having a property such as luminescence, color development, radioactivity, etc., which can be directly observed by naked eyes or detected by an instrument, by which qualitative or quantitative detection of the corresponding target can be achieved.

In alternative embodiments, the labels include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

In the actual use process, a person skilled in the art can select a suitable marker according to the detection conditions or actual needs, and no matter what marker is used, the marker belongs to the protection scope of the invention.

In alternative embodiments, the fluorescent dyes include, but are not limited to, fluorescein-based dyes and derivatives thereof (including, but not limited to, fluorescein Isothiocyanate (FITC) hydroxy-light (FAM), tetrachlorolight (TET), and the like, or analogs thereof), rhodamine-based dyes and derivatives thereof (including, but not limited to, red Rhodamine (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), and the like, or analogs thereof), cy-based dyes and derivatives thereof (including, but not limited to, cy2, cy3B, cy3.5, cy5, cy5.5, cy3, and the like, or analogs thereof), alexa-based dyes and derivatives thereof (including, but not limited to, alexa fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, and the like, or analogs thereof), and protein-based dyes and derivatives thereof (including, but not limited to, for example, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polyazosin (preCP), and the like).

In alternative embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose 6-phosphate deoxygenase.

In alternative embodiments, the radioisotope includes, but is not limited to 212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu and 18F.

In alternative embodiments, the chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, ruthenium bipyridine and its derivatives, acridinium esters and its derivatives, dioxane and its derivatives, lomustine and its derivatives, and peroxyoxalate and its derivatives.

In alternative embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

In alternative embodiments, the colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

In alternative embodiments, the colloidal metals include, but are not limited to, colloidal gold, colloidal silver, and colloidal selenium.

In an alternative embodiment, the colloidal metal is colloidal gold.

In an alternative embodiment, the antibody conjugate described above further comprises a solid support coupled to the antibody or functional fragment thereof.

In alternative embodiments, the solid support is selected from the group consisting of microspheres, plates, and membranes.

In alternative embodiments, the solid support includes, but is not limited to, magnetic microspheres, plastic microparticles, microplates, glass, capillaries, nylon, and nitrocellulose membranes.

In a fifth aspect, the invention provides a reagent or kit comprising an antibody or functional fragment thereof as described above or an antibody conjugate as described above.

As previously mentioned, the antibodies or functional fragments thereof of some embodiments or examples of the present invention are capable of efficiently binding to a novel coronavirus or an N-protein antigen thereof, and thus, reagents or kits comprising the novel coronavirus or N-protein antibody or functional fragment thereof are capable of efficiently performing qualitative or quantitative detection of the novel coronavirus or N-protein thereof. The reagent or the kit provided by the invention can be used for detecting specific binding performance of novel coronaviruses or N proteins thereof and antibodies thereof, such as immunoblotting, immunoprecipitation and the like. As previously mentioned, the antibodies or functional fragments thereof in some embodiments or examples of the present invention have higher binding activity or affinity to the novel coronavirus or N protein thereof, and thus the reagents or kits comprising the antibodies or functional fragments thereof have higher detection sensitivity or specificity.

In a sixth aspect, the present invention provides a method of detecting a novel coronavirus or N protein thereof comprising a) contacting an antibody or functional fragment thereof, antibody conjugate, reagent or kit as described above with a novel coronavirus or N protein antigen thereof in a sample to be detected under conditions sufficient for an antibody/antigen binding reaction to occur to form an immune complex, and b) detecting the presence of said immune complex, the presence of said complex being indicative of the presence of said antigen in said test sample;

in an alternative embodiment, the immune complex further comprises a second antibody, which binds to the antibody or a functional fragment thereof.

In alternative embodiments, the immune complex further comprises a second antibody that binds to a novel coronavirus or an N protein antigen thereof.

In a seventh aspect, the invention provides a nucleic acid molecule encoding an antibody or functional fragment thereof as described above.

In an eighth aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In a ninth aspect, the present invention provides a cell comprising the vector described above.

In a tenth aspect, the invention provides a method of producing an antibody or functional fragment thereof comprising culturing a cell as described above.

In an eleventh aspect, the present invention provides the use of an antibody or functional fragment thereof, an antibody conjugate or a reagent or kit as described above for detecting a novel coronavirus or an N protein thereof or for preparing a product for detecting a novel coronavirus or an N protein thereof.

On the basis of the present invention, which discloses the amino acid sequence of an antibody or a functional fragment thereof, it is easy for a person skilled in the art to prepare the antibody or the functional fragment thereof by genetic engineering techniques or other techniques (chemical synthesis, recombinant expression), for example, by separating and purifying the antibody or the functional fragment thereof from a culture product of recombinant cells capable of recombinantly expressing the antibody or the functional fragment thereof according to any one of the above, and on the basis of this, it is within the scope of the present invention to prepare the antibody or the functional fragment thereof by any technique.

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of formulations or unit doses herein, some methods and materials are now described. Unless otherwise indicated, techniques employed or contemplated herein are standard methods. The materials, methods, and examples are illustrative only and not intended to be limiting.

Unless otherwise indicated, practice of the present invention will employ conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the ability of a person skilled in the art. Such techniques are well explained in the literature, e.g., in the molecular cloning laboratory Manual (Molecular Cloning: A Laboratory Manual), second edition (Sambrook et al, 1989), oligonucleotide Synthesis (Oligonucleotide Synthesis) (M.J.Gait et al, 1984), animal cell Culture (ANIMAL CELL Culture) (R.I. Freshney, 1987), enzymatic methods (Methods in Enzymology) (academic Press Co., ltd. (ACADEMIC PRESS, inc.), experimental immunology Manual (Handbook of Experimental Immunology) (D.M.Weir and C.Blackwell, inc.), mammalian cell gene transfer Vectors (GENE TRANSFER Vectors for MAMMALIAN CELLS) (J.M.Miller and M.P.Calos, 1987), contemporary molecular biology methods (F.M.Ausubel et al, 1987), polymerase chain reactions (28) (J.M.Weir. And C.Blackwell, inc.), PCR methods (J.34.J.37, J.J.37, J.F.37) and PCR methods (J.37, J.F.37, J.J.F.37, J.J.J.J.F.37, J.J.J.J.J.J.J.J.F.J.J.J.J.J.F.J.J.J.L).

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1 preparation of Anti-COVN-14C10 monoclonal antibodies

Restriction enzymes, PRIME STAR DNA polymerase in this example were purchased from Takara. MagExtractor-RNA extraction kit was purchased from TOYOBO company. BD SMART ^TM RACE cDNA Amplification Kit kit was purchased from Takara. pMD-18T vector was purchased from Takara. Plasmid extraction kits were purchased from Tiangen. Primer synthesis and gene sequencing were accomplished by Invitrogen corporation. The hybridoma cell strain secreting the Anti-COVN C10 monoclonal antibody is an existing hybridoma cell strain, and is recovered for later use.

(1) Antibody Gene production

MRNA is extracted from hybridoma cell strain secreting Anti-COVN C10 monoclonal antibody, DNA product is obtained by RT-PCR method, the product is inserted into pMD-18T vector after adding A reaction by rTaq DNA polymerase, and is transformed into DH5 alpha competent cells, HEAVY CHAIN and LIGHT CHAIN gene clones are respectively taken after colony growth, and each 4 clones are sent to gene sequencing company for sequencing.

(2) Sequence analysis of Anti-COVN C10 antibody variable region Gene

The gene sequence obtained by sequencing is placed in a kabat antibody database for analysis, and VNTI 11.5.5 software is used for analysis to determine that the amplified genes of the heavy chain and light chain primer pair are correct, wherein the VL gene sequence in the LIGHT CHAIN amplified gene fragment is 324bp, the front of the VL gene sequence is 57bp leader peptide sequence, and the VH gene sequence in the HEAVY CHAIN primer pair amplified gene fragment is 366bp, belongs to the VH1 gene family, and the front of the VL gene fragment is 57bp leader peptide sequence.

(3) Construction of recombinant antibody expression plasmids

pcDNA^TM3.4Vector is a constructed eukaryotic expression vector of recombinant antibody, which has been introduced with HindIII, bamHI, ecoRI and other polyclonal enzyme cutting sites and named pcDNA3.4A expression vector, 3.4A expression vector, VL and VH gene specific primers of the antibody are designed according to the result of gene sequencing of the antibody variable region in pMD-18T, and both ends have HindIII, ecoRI enzyme cutting sites and protective bases respectively, and a LIGHT CHAIN gene fragment of 0.73kb and a HEAVY CHAIN gene fragment of 1.43kb are amplified by a PCR amplification method.

The HEAVY CHAIN and LIGHT CHAIN gene fragments are respectively cut by HindIII/EcoRI double enzyme, the 3.4A vector is cut by HindIII/EcoRI double enzyme, the HEAVY CHAIN gene and LIGHT CHAIN gene after the fragments and the vector are purified and recovered are respectively connected with the 3.4A expression vector, and recombinant expression plasmids of HEAVY CHAIN and LIGHTCHAIN are respectively obtained.

2. Recombinant antibody production

Recovering HEK293 cells in advance, subculturing to a 200ml system to enable the cell density to reach 3-5 cell density to achieve the concentration of selected antibody and cell viability of more than 95%, centrifugally cleaning the cells, re-dissolving the cells by using a culture medium, adjusting the cell density to 2.9, washing the cells, re-dissolving the cells by using the culture medium, and simultaneously, taking the cells as a cell diluent. The medium was used to prepare dilutions of plasmid DNA and transfection reagent, respectively. Adding transfection reagent diluent into plasmid DNA diluent, mixing uniformly, standing at room temperature for 15min, slowly adding the mixture into cell diluent within 1min, mixing uniformly, sampling, counting, recording and observing activity of transfected cells, culturing in a 35 ℃ constant temperature incubator at a rotating speed 120rmp and a CO2 content of 8%, and centrifuging and collecting samples after 13 days. The supernatant was affinity purified using a proteona affinity column. 6ug of the purified antibody was subjected to reducing SDS-PAGE, and the electrophoresed pattern was as shown. Two bands were shown after reducing SDS-PAGE, 1 Mr was 50KD (heavy chain) and the other Mr was 28KD (light chain).

Example 2 affinity and Activity optimization

The Anti-COVN-14C10 monoclonal antibody obtained in example 1, although having the ability to bind to a novel coronavirus or its N protein, was not ideal in affinity and antibody activity, and thus the applicant had performed directed mutation of the variable region of the antibody. The method comprises the steps of performing structural simulation of an antibody variable region, structural simulation of an antigen-antibody variable region acting complex, analysis of key amino acids of an antibody and mutation design by using a computer, designing and synthesizing a two-way primer covering a mutation site according to a mutation scheme, synthesizing primers at two ends of target DNA, performing high-fidelity PCR reaction, cloning a PCR product to a vector, and preparing the mutant antibody according to the method described in the example 1. Monoclonal antibodies with significantly improved affinity and antibody activity were obtained by screening and named Anti-COVN-14C10mut. The heavy and light chain amino acid sequences of Anti-COVN-14C10mut are shown as SEQ ID NO. 19 and SEQ ID NO. 20, respectively.

Example 3 detection of Performance of antibodies

1. Affinity analysis

The antigen-antibody binding dissociation curve is tested on Biacore 8K+ equipment by utilizing a CM5 chip which is coupled with the antigen in advance, and an instrument is automatically fitted to obtain an affinity constant, a binding rate and a dissociation rate. (KD represents equilibrium dissociation constant, i.e., affinity constant; ka represents binding rate; KD represents dissociation rate).

Table 2 affinity data

2. Activity assay

The novel coronavirus N protein recombinant antigen (purchased from the Phpeng organism) was diluted to 1ug/ml by the coating solution (NaHCO 3 as a main component), washed 2 times per well at 100uL and 4 ℃ overnight, and then dried by shaking, adding a blocking solution (Na2HPO4+Nacl as a main component) to 120uL,37 ℃ and 1H per well, adding the diluted purified and control antibodies, 100 uL/well, 37 ℃ and 30min, washing 5 times per well, drying by shaking, adding goat anti-mouse IgG-HRP, 100uL,37 ℃ and 30min per well, washing 5 times per well, drying by shaking, adding a developing solution A (50 uL/well, containing 1.05g/L citric acid, 0.186 g/LEDTA.1, 0.45g/L TMB and 0.2ml/L HCl), adding a developing solution B (50 uL/well, containing 1.05 g/62.45 g citric acid, 53.45 g/L, 0.45 ml/2 ml HCl), stopping reading by shaking, and stopping reading by a standard reader (10.45 mg/ml) to 10 mg/L of HCl (standard solution, 10 mg/75 mg/ml). The results are shown in the following table:

TABLE 3 Activity data

Sample concentration (ng/ml)	125.00	62.50	31.25	15.63	7.81	0.00
							Control	1.616	1.302	0.859	0.338	0.156	0.014
Anti-COVN-14C10mut	2.153	1.793	1.175	0.770	0.462	0.036

3. Stability assessment

Placing the antibody at 4 ℃, -80 ℃ (refrigerator) and 37 ℃ (incubator) for 21 days, taking 7 days, 14 days and 21 days for state observation, and detecting the activity of the 21 days, wherein the results show that no obvious protein state change is seen in the antibody placed for 21 days under three examination conditions, and the activity is not in a descending trend along with the increase of the examination temperature, thus indicating that the antibody is stable. Table 4 below shows the results of the detection of OD after 21 days of antibody examination.

Table 4 stability data

Sample concentration (ng/ml)	31.25	15.63	0.00
				4 ℃ 21 Day sample	1.135	0.758	0.028
-80 ℃,21 Day sample	1.166	0.763	0.026
				37 ℃ 21 Day sample	1.152	0.779	0.023

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

The partial amino acid sequence related to the application is as follows:

Claims

1. An antibody against the novel coronavirus or its N protein, the antibody comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising HCDR1, HCDR2, and HCDR3, the light chain variable region comprising LCDR1, LCDR2, and LCDR3, characterized in that the HCDR1, HCDR2, and HCDR3 are respectively the same amino acid sequences as the HCDR1, HCDR2, and HCDR3 of the heavy chain variable region as shown in SEQ ID NO: 17; the LCDR1, LCDR2, and LCDR3 are respectively the same amino acid sequences as the LCDR1, LCDR2, and LCDR3 of the light chain variable region as shown in SEQ ID NO: 18;

The HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3 are defined by the Kabat, Chothia, IMGT, AbM or Contact systems.

2. An antibody against a novel coronavirus or its N protein, the antibody comprising the following complementary determining regions:

HCDR1, which consists of the amino acid sequence shown in SEQ ID NO: 1;

HCDR2, which consists of the amino acid sequence shown in SEQ ID NO: 2;

HCDR3, which consists of the amino acid sequence shown in SEQ ID NO: 3;

LCDR1, which consists of the amino acid sequence shown in SEQ ID NO:4;

LCDR2, which consists of the amino acid sequence shown in SEQ ID NO:5;

LCDR3 consists of the amino acid sequence shown in SEQ ID NO:6.

3. The antibody according to any one of claims 1-2, characterized in that the antibody further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4;

The HFR1 comprises SEQ ID NO:7 or an amino acid sequence having at least 80% identity thereto;

The HFR2 comprises SEQ ID NO: 8 or an amino acid sequence having at least 80% identity thereto;

The HFR3 comprises SEQ ID NO:9 or an amino acid sequence having at least 80% identity thereto;

The HFR4 comprises SEQ ID NO: 10 or an amino acid sequence having at least 80% identity thereto;

The LFR1 comprises SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto;

The LFR2 comprises SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto;

The LFR3 comprises SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto;

The LFR4 comprises SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

4. The antibody according to any one of claims 1-2, characterized in that the antibody binds to the new coronavirus or its N protein with an affinity of KD <9.27×10 ^-9 M.

5. An antibody against the new coronavirus or its N protein, comprising a heavy chain variable region and a light chain variable region, characterized in that the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 17; the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 18.

6. The antibody according to any one of claims 1-2 and 5, characterized in that the antibody further comprises a constant region.

7. The antibody according to claim 6, characterized in that the constant region comprises a heavy chain constant region and/or a light chain constant region.

8. The antibody according to claim 7, characterized in that the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; and the light chain constant region is selected from the κ type or λ type light chain constant region.

9. The antibody according to claim 6, characterized in that the species of origin of the constant region is cattle, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

10. The antibody according to claim 6, characterized in that the species origin of the constant region is mouse.

11. The antibody according to claim 7, characterized in that the heavy chain constant region sequence is as shown in SEQ ID NO: 15 or has at least 80% identity thereto.

12. The antibody according to claim 7, characterized in that the light chain constant region sequence is as shown in SEQ ID NO: 16 or has at least 80% identity thereto.

13. A functional fragment of an antibody against the novel coronavirus or its N protein, characterized in that the antibody is the antibody according to any one of claims 1 to 12, and the functional fragment is selected from any one of F(ab') ₂ , Fab', Fab, Fv and scFv of the antibody.

14. An antibody against the new coronavirus or its N protein, comprising a heavy chain and a light chain, characterized in that the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain is shown in SEQ ID NO: 20.

15. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody according to any one of claims 1 to 12 and 14 or a functional fragment of the antibody according to claim 13.

16 . The antibody conjugate according to claim 15 , characterized in that the antibody conjugate further comprises biotin or a biotin derivative coupled to the antibody or a functional fragment of the antibody.

17 . The antibody conjugate according to claim 15 , characterized in that the antibody conjugate further comprises a label coupled to the antibody or the functional fragment of the antibody.

18. The antibody conjugate according to claim 17, characterized in that the label is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent agents and nanoparticle labels.

19. The antibody conjugate according to claim 15, characterized in that the antibody conjugate further comprises a solid phase carrier coupled to the antibody or the functional fragment of the antibody.

20. The antibody conjugate according to claim 19, characterized in that the solid phase carrier is selected from microspheres, plates and membranes.

21. A reagent or a kit, characterized in that the reagent or the kit comprises the antibody according to any one of claims 1-12 and 14, or the functional fragment of the antibody according to claim 13, or the antibody conjugate according to any one of claims 15-20.

22. Use of the antibody according to any one of claims 1 to 12 and 14, the functional fragment of the antibody according to claim 13, the antibody conjugate according to any one of claims 15 to 20, or the reagent or kit according to claim 21 in the preparation of a product for detecting a novel coronavirus or its N protein, characterized in that it comprises:

a) contacting the antibody of any one of claims 1 to 12 and 14, the functional fragment of the antibody of claim 13, the antibody conjugate of any one of claims 15 to 20, or the reagent or kit of claim 21 with the novel coronavirus or its N protein antigen in the sample to be detected to form an immune complex under conditions sufficient for an antibody/antigen binding reaction to occur; and

b) detecting the presence of the immune complex, the presence of the complex indicating the presence of the antigen in the test sample.

23. The use according to claim 22, characterized in that the immune complex further comprises a second antibody, and the second antibody binds to the antibody or a functional fragment of the antibody.

24. The use according to claim 22 is characterized in that the immune complex also includes a second antibody, which binds to the new coronavirus or its N protein antigen.

25. A nucleic acid, characterized in that it encodes the antibody according to any one of claims 1 to 12 and 14 or a functional fragment of the antibody according to claim 13.

A vector, characterized in that it contains the nucleic acid according to claim 25.

27. A cell, characterized in that it contains the nucleic acid according to claim 25 or the vector according to claim 26.

28. A method for preparing the antibody according to any one of claims 1 to 12 and 14 or the functional fragment of the antibody according to claim 13, characterized in that it comprises: culturing the cell according to claim 27.

29. Use of the antibody of any one of claims 1-12 and 14, the functional fragment of the antibody of claim 13, the antibody conjugate of any one of claims 15-20, or the reagent or kit of claim 21 in the preparation of a product for detecting a novel coronavirus or its N protein.