WO2023131318A1 - Antibody against covid-19, reagent and kit for detecting covid-19 - Google Patents

Abstract

The present disclosure relates to the technical field of antibodies, and provides an antibody against COVID-19, a reagent and a kit for detecting COVID-19. The antibody against COVID-19 provided by the present disclosure comprises a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has a good affinity to the N protein of COVID-19, and the antibody is highly sensitive and specific for the detection of COVID-19. The present disclosure provides a superior antibody selection for the detection of COVID-19.

Description

Antibodies against novel coronavirus, reagents and kits for detecting novel coronavirus

Cross References to Related Applications

This disclosure claims the priority of the Chinese patent application with the application number CN202210022971.0 and titled "antibodies against novel coronavirus, reagents and kits for detecting novel coronavirus" submitted to the China Patent Office on January 10, 2022. The entire contents are incorporated by reference in this disclosure.

technical field

The present disclosure relates to the technical field of antibodies, in particular, to an anti-new coronavirus antibody, a reagent and a kit for detecting the new coronavirus.

Background technique

Since the outbreak of the novel coronavirus 2019-nCoV pneumonia, it has spread globally, seriously threatening human life and health. Respiratory droplets and close contact transmission are the main routes of transmission of novel coronavirus pneumonia, and there is a possibility of aerosol transmission in relatively closed environments exposed to high concentrations of aerosols for a long time. 2019-nCoV is highly contagious, and most patients develop clinical symptoms after infection, but some patients are asymptomatic. Common signs after a person is infected with a coronavirus include respiratory symptoms, fever, cough, shortness of breath, and dyspnea. In more severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. Although there is currently no specific treatment for the disease caused by the new coronavirus, the treatment of mild or asymptomatic patients can greatly improve the cure rate and shorten the treatment time. Therefore, the detection or identification of patients becomes particularly important.

At present, nucleic acid testing and virus gene sequencing are mainly used as the evidence of etiological confirmation of the new coronavirus. Due to the influence of various factors such as sampling, operation, and reagents, nucleic acid testing may produce false negative results. The positive detection rate of viral nucleic acid in patients with highly suspected 2019 novel coronavirus (2019-nCoV) infection is only 30% to 50%. In addition, nucleic acid detection has high requirements on equipment, testing sites and environmental conditions, and has disadvantages such as long detection time and low throughput, which is not convenient for large-scale detection of people under the current epidemic situation. Compared with nucleic acid detection methods, antibody detection samples are serum or plasma, which is less affected by sample sampling, which is conducive to early diagnosis and exclusion of suspicious cases. At the same time, the detection is fast, convenient, and suitable for large-scale screening. Therefore, antibody detection kits become more important.

Therefore, there is an urgent need to develop a rapid and convenient detection kit for clinical detection, so as to isolate infected people as soon as possible to block the spread of the virus.

The structural proteins of the new coronavirus 2019-nCoV are divided into: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein) and nucleocapsid protein (N protein), these proteins include multiple antigenic epitopes. The N protein and the viral genome RNA are intertwined to form the viral nucleocapsid, which plays an important role in the synthesis of viral RNA. At the same time, the N protein is relatively conservative and accounts for the largest proportion of the structural proteins of the virus. The body can produce high-level antibodies against the N protein in the early stage of infection. Finally, the N protein is an important marker protein of the new coronavirus. Using the principle of specific combination of antigen and antibody, the presence of the antigen can be detected by the N protein monoclonal antibody, so as to directly prove that the sample contains the new coronavirus and realize the detection of the new coronavirus. detection.

At present, there are few 2019-nCoV N protein monoclonal antibody products, and there are differences in performance.

Contents of the invention

The present disclosure provides an anti-new coronavirus or its N protein antibody or functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, and the HCDR1-HCDR3 include the duplicates shown in SEQ ID NO: 15 The amino acid sequence consistent with HCDR1-HCDR3 of the chain variable region; the LCDR1-LCDR3 includes an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.

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

Optionally, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 include or are shown in sequence as SEQ ID NO: 1-6.

The present disclosure also provides an anti-new coronavirus or its N protein antibody or its functional fragment, said antibody or its functional fragment includes HCDR1, HCDR2, HCDR3 as shown in the amino acid sequence of SEQ ID NO: 1-3 , and the amino acid sequence LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4-6.

Optionally, the antibody or functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

Optionally, the amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequentially include SEQ ID NO: 7-14 or a sequence having at least 80% homology with SEQ ID NO: 7-14 Amino acid sequence, or sequentially as shown in SEQ ID NO: 7-14 or an amino acid sequence having at least 80% homology to SEQ ID NO: 7-14.

Optionally, the antibody or its functional fragments also have amino acid sequences such as HFR1, HFR2, HFR3, HFR4 shown in SEQ ID NO: 7-10, and amino acid sequences such as LFR1 shown in SEQ ID NO: 11-14 , LFR2, LFR3 and LFR4, or an amino acid sequence having at least 80% homology to each of said sequences.

Optionally, the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K _D ≤ 10 ^-9 M.

Optionally, the antibody or a functional fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 15, and a light chain variable region shown in SEQ ID NO: 16.

The present disclosure also provides an antibody or functional fragment thereof against novel coronavirus or its N protein, the antibody or functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain The amino acid sequence of the variable region is shown in SEQ ID NO: 15;

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

The present disclosure also provides an antibody or functional fragment thereof against novel coronavirus or its N protein, the antibody or functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain The variable region contains the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the HCDR1, The amino acid sequence of HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of the above-mentioned HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, and the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 It is the amino acid sequence of the above-mentioned HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4.

Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15;

The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

Optionally, the antibody or functional fragment thereof further comprises a constant region.

Optionally, the constant region comprises a heavy chain constant region and/or a light chain constant region.

Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region .

Optionally, the species source of the constant region is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human.

Optionally, the species source of the constant region is mouse.

Optionally, the sequence of the heavy chain constant region is shown in SEQ ID NO: 17 or has at least 80% homology with it, and the sequence of the light chain constant region is shown in SEQ ID NO: 18 or has at least 80% homology with it 80% homology.

Optionally, the antibody or a functional fragment thereof comprises a heavy chain shown in SEQ ID NO: 19 and a light chain shown in SEQ ID NO: 20.

Optionally, the functional fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody.

The present disclosure also provides an antibody against a new type of coronavirus or its N protein, including a heavy chain and/or a light chain, the heavy chain including the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain The chain comprises the light chain variable region described above and the light chain constant region described above.

Optionally, 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.

The present disclosure also provides an antibody against novel coronavirus or its N protein, including heavy chain and/or light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 19; the amino acid sequence of the light chain As shown in SEQ ID NO:20.

The present disclosure also provides an antibody conjugate, which includes the above-mentioned antibody or a functional fragment thereof.

Optionally, the antibody or functional fragment thereof is labeled with a label.

Optionally, the label is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle-based labels.

Optionally, the fluorescent dye is selected from the group consisting of fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, protein dyes and derivatives thereof thing.

Optionally, the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and glucose-6-phosphate deoxygenase.

Optionally, the radioactive isotope is selected from ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi, ³² P, ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ^177Lu , ^172Lu and ^18F .

Optionally, the chemiluminescence reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives, dioxane Ethane and its derivatives, lophine and its derivatives, and peroxalate and its derivatives.

Optionally, the nanoparticle marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles.

Optionally, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex.

Optionally, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium.

Optionally, the antibody or functional fragment thereof is coated onto a solid phase.

Optionally, the solid phase is selected from microspheres, plates and membranes.

Optionally, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

Optionally, the solid phase is selected from magnetic microspheres.

The present disclosure also provides a reagent or kit, which includes the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate.

Optionally, the reagent or kit is a reagent or kit for detecting novel coronavirus or its N protein.

The present disclosure also provides a nucleic acid encoding the antibody or a functional fragment thereof of any one of the above.

The present disclosure also provides a vector comprising a nucleic acid fragment of any one of the above-mentioned antibodies or functional fragments thereof.

The present disclosure also provides a recombinant cell containing the vector.

The present disclosure also provides a method for preparing the antibody or functional fragment thereof according to any one of the above, which comprises: culturing the recombinant cells.

The present disclosure also provides an antibody or its functional fragment, antibody, antibody conjugate, or reagent or kit described in any one of the above, which is used for detecting novel coronavirus or its N protein and diagnosing novel coronavirus infection Or use in diseases related to novel coronavirus infection.

The present disclosure also provides a method for diagnosing a subject infected with a novel coronavirus or a disease related to a novel coronavirus infection, including:

A) contacting the antibody or functional fragment thereof, antibody, antibody conjugate, or reagent or kit of any of the above with a sample from said subject under conditions sufficient for a binding reaction to occur to carry out a binding reaction; and

B) Detection of immune complexes generated by the binding reaction.

The present disclosure also provides a method for detecting novel coronavirus or its N protein in a test sample, comprising:

a) Under conditions sufficient for an antibody/antigen binding reaction to occur, coupling the novel coronavirus or its N protein antigen in the test sample to the antibody or its functional fragment, antibody, or antibody described in any one of the above substances, or reagents or kits, to form immune complexes; and

b) detecting the presence of said immune complex indicative of the presence of said antigen in said test sample.

Optionally, in step a), the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment.

Optionally, in step a), the immune complex further includes a second antibody that binds to the novel coronavirus or its N protein antigen.

Optionally, the diseases associated with novel coronavirus infection include respiratory symptoms, fever, cough, hypoxemia, pneumonia, acute respiratory distress syndrome, severe acute respiratory syndrome, renal failure or septic shock.

Description of drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the following will briefly introduce the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present disclosure, and therefore are not It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.

Fig. 1 is the result of reducing SDS-PAGE of the anti-new coronavirus antibody of embodiment 1.

Detailed ways

In this disclosure, as used herein, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies, bispecific or multispecific antibodies, and chimeric antibodies, so long as they exhibit the desired biological active.

As used herein, the terms "complementarity determining regions", "CDRs" or "CDRs" refer to the hypervariable regions of the heavy and light chains of immunoglobulins, which contain one or more, or even all, of the antibodies or The region of major amino acid residues responsible for the binding affinity of an antigen-binding fragment to the antigen or epitope it recognizes. In particular embodiments of the present disclosure, CDRs refer to the hypervariable regions of the heavy and light chains of antibodies.

In this disclosure, the heavy chain complementarity determining region is represented by "HCDR", which includes HCDR1, HCDR2 and HCDR3; the light chain complementarity determining region is represented by "LCDR", which includes LCDR1, LCDR2 and LCDR3.

Embodiments of the present disclosure provide an anti-new coronavirus or its N protein antibody or a functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, HCDR1～HCDR3 including and shown in SEQ ID NO: 15 The amino acid sequence consistent with HCDR1-HCDR3 of the heavy chain variable region; LCDR1-LCDR3 includes an amino acid sequence consistent with LCDR1-LCDR3 of the light chain variable region shown in SEQ ID NO:16.

Commonly used CDR labeling methods in the field include: Kabat numbering scheme, IMGT numbering scheme, Chothia and Lesk numbering scheme, and a new standardized numbering system introduced by Lefranc et al. in 1997 for all protein sequences of the immunoglobulin superfamily. Kabat et al. were the first to propose a standardized numbering scheme for immunoglobulin variable regions. The accumulation of sequences over the past few decades has led to the creation of the KABATMAN database, and the Kabat numbering scheme is generally considered the widely adopted standard for numbering antibody residues.

The system for defining CDR is not particularly limited, and CDR sequences defined by conventional systems in the art are within the protection scope of the present application. For example, the CDR definition method is referred to such as Kabat et al., U.S.Dept.of Health and Human Services, Sequences of Proteins of Immunological Interest (1983) or Chothia et al., J Mol Biol 196:901-917 (1987). 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 Numbering of all CDR definitions in Table 1 is according to the Kabat numbering system (see below).

² "AbM" as used in Table 1 has a lowercase "b" and refers to the CDRs defined by Oxford Molecular's "AbM" antibody modeling software.

Kabat et al. also defined a numbering system applicable to the variable region sequences of any antibody. One of ordinary skill in the art can unambiguously map this Kabat numbering system to any variable region sequence, without reliance on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., U.S. Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983). The polypeptide sequences in the sequence listing are not numbered according to the Kabat numbering system. However, those of ordinary skill in the art are fully able to convert the sequence numbers of the sequence listing into Kabat numbers.

In an optional embodiment, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 (CDRs for short) are defined by Kabat, Chothia, IMGT, AbM or Contact system.

In an optional embodiment, HCDR1, HCDR2 and HCDR3 sequentially comprise the 31-35, 50-65, and 95-100F amino acid sequences of the heavy chain variable region, or sequence such as 31-35 of the heavy chain variable region Position, 50-65, 95-100F amino acid sequence shown;

LCDR1, LCDR2 and LCDR3 sequentially comprise the 24-34, 50-56, 89-95 amino acid sequences of the light chain variable region, or such as the 24-34, 50-56, 89 ~95th amino acid sequence; and, the numbering of the amino acid positions is based on the Kabat numbering system. In an optional embodiment, the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 include or are shown in sequence as SEQ ID NO: 1-6.

Embodiments of the present disclosure also provide an antibody against novel coronavirus or its N protein or a functional fragment thereof, the antibody or a functional fragment thereof comprising amino acid sequences such as HCDR1 shown in SEQ ID NO: 1-3, HCDR2, HCDR3, and LCDR1, LCDR2 and LCDR3 of amino acid sequence as shown in SEQ ID NO:4-6.

In the present disclosure, "framework region" or "FR" region includes the heavy chain framework region and the light chain framework region, and refers to the region except the CDR in the heavy chain variable region and the light chain variable region of the antibody; wherein, heavy Chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising HFR1, HFR2, HFR3, and HFR4 framework regions; light chain framework regions can be further subdivided into contiguous regions separated by CDRs, comprising LFR1 , LFR2, LFR3 and LFR4 framework regions.

In the present disclosure, the heavy chain variable region is obtained by connecting the following numbered CDRs and FRs in the following combinations: HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is composed of the following numbered CDRs and FRs Arrange and connect as follows: LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

In an optional embodiment, the antibody or a functional fragment thereof further has at least one of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4.

In an alternative embodiment, the amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequentially comprise SEQ ID NO:7-14 or have at least 80% homology with SEQ ID NO:7-14 A homologous amino acid sequence, or sequentially as shown in SEQ ID NO: 7-14 or an amino acid sequence having at least 80% homology with SEQ ID NO: 7-14.

In an optional embodiment, the antibody or its functional fragment also has an amino acid sequence such as HFR1, HFR2, HFR3, HFR4 as shown in SEQ ID NO:7-10, and an amino acid sequence as shown in SEQ ID NO:11-14 LFR1, LFR2, LFR3 and LFR4, or an amino acid sequence having at least 80% homology to each sequence.

It should be noted that, in other embodiments, the amino acid sequence of each framework region of the antibody or its functional fragment provided by the present disclosure may be the same as the corresponding framework region (SEQ ID NO: 7, 8, 9, 10, 11, 12, 13 or 14) have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology.

In an optional embodiment, the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K _D ≤ 10 ⁻⁹ M.

In an optional embodiment, the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K _D ≤ 10 ^-10 M;

The detection of _KD is carried out with reference to the method in the embodiments of the present disclosure.

In an alternative embodiment, the antibody or functional fragment thereof comprises a heavy chain variable region set forth in SEQ ID NO:15, and a light chain variable region set forth in SEQ ID NO:16.

An embodiment of the present disclosure provides an antibody or a functional fragment thereof against a novel coronavirus or its N protein, the antibody or a functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region The amino acid sequence of the region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.

An embodiment of the present disclosure provides an antibody or a functional fragment thereof against a novel coronavirus or its N protein, the antibody or a functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain variable region The region contains the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, HCDR1, HCDR2, HCDR3, LCDR 1 The amino acid sequences of LCDR2 and LCDR3 are the above-mentioned amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 are the above-mentioned HFR1, HFR2, Amino acid sequences of HFR3, HFR4, LFR1, LFR2, LFR3, LFR4.

In an alternative embodiment, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 15.

In an alternative embodiment, the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

In alternative embodiments, the antibody 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 an optional embodiment, 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 .

In an optional embodiment, the species source of the constant region is cow, horse, dairy cow, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose , Turkey, Fighting Cock Or Man.

In an alternative embodiment, the species source of the constant region is mouse.

In an optional embodiment, the sequence (CH) of the heavy chain constant region is shown in SEQ ID NO: 17, and the sequence of the light chain constant region (CL) is shown in SEQ ID NO: 18.

It should be noted that, in other embodiments, the sequence of the constant region 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% homology.

In an alternative embodiment, the antibody or functional fragment thereof comprises a heavy chain set forth in SEQ ID NO:19 and a light chain set forth in SEQ ID NO:20.

In an optional embodiment, the functional fragment is selected from any one of VHH, F(ab')2, Fab', Fab, Fv and scFv of the antibody.

Functional fragments of the above-mentioned antibodies generally have the same binding specificity as the antibody from which they were derived. Those skilled in the art can easily understand from the content of the present disclosure that the functional fragments of the above-mentioned antibodies can be obtained by methods such as enzymatic digestion (including pepsin or papain) and/or methods of splitting disulfide bonds by chemical reduction. On the basis of the structure of the complete antibody provided in the present disclosure, those skilled in the art can easily obtain the above-mentioned functional fragments.

Functional fragments of the above-mentioned antibodies can also be synthesized by recombinant genetics techniques also known to those skilled in the art or by, for example, automatic peptide synthesizers such as those sold by Applied BioSystems.

Embodiments of the present disclosure also provide an antibody against a new type of coronavirus or its N protein, including a heavy chain and/or a light chain, the heavy chain including the above-mentioned heavy chain variable region and the above-mentioned heavy chain constant region; the light chain includes The above-mentioned light chain variable region and the above-mentioned light chain constant region.

In an alternative embodiment, 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.

Embodiments of the present disclosure also provide an antibody against novel coronavirus or its N protein, including heavy chain and/or light chain, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19; the amino acid sequence of the light chain is as shown in SEQ ID NO:19 ID NO:20.

An embodiment of the present disclosure provides an antibody conjugate, which includes the above-mentioned antibody or a functional fragment thereof.

In an optional embodiment, the antibody or its functional fragment in the above antibody conjugate is labeled with a label.

In an optional embodiment, the aforementioned markers refer to a class of substances that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., through which the corresponding target can be detected. Qualitative or quantitative detection of substances.

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

During actual use, those skilled in the art can select appropriate markers according to detection conditions or actual needs, and no matter what kind of markers are used, they all fall within the protection scope of the present disclosure.

In an optional embodiment, the fluorescent dyes include but are not limited to fluorescein dyes and their derivatives (for example including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET ), etc. or their analogues), rhodamine dyes and their derivatives (such as including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogues) , Cy series dyes and derivatives thereof (such as including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and derivatives thereof (such as including but not limited to limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (such as including but not limited to Phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinoxanthin-chlorophyll protein (preCP), etc.).

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

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

In alternative embodiments, chemiluminescent reagents include, but are not limited to, luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives , dioxetane and its derivatives, lophine and its derivatives, and peroxyoxalate and its derivatives.

In an optional embodiment, 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, colloids include, but are not limited to, colloidal metals, disperse dyes, dye-labeled microspheres, and latex.

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

In an optional embodiment, the antibodies or functional fragments thereof in the above-mentioned antibody conjugates are coated on a solid phase.

In an alternative embodiment, the solid phase is selected from microspheres, plates and membranes.

In alternative embodiments, solid phases include, but are not limited to, magnetic microspheres, plastic microspheres, microplastics, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes.

In an alternative embodiment, the solid phase is magnetic microspheres.

Embodiments of the present disclosure also provide a reagent or kit for detecting novel coronavirus or its N protein, the reagent or kit includes the above-mentioned antibody or its functional fragment or the above-mentioned antibody conjugate. Embodiments of the present disclosure also provide a nucleic acid molecule encoding the above antibody or a functional fragment thereof.

Embodiments of the present disclosure also provide vectors comprising the nucleic acid molecules described above.

Embodiments of the present disclosure also provide recombinant cells containing the above vectors.

Embodiments of the present disclosure also provide a method for preparing an antibody or a functional fragment thereof, comprising: culturing the above-mentioned recombinant cells.

Embodiments of the present disclosure also provide the above-mentioned antibodies or functional fragments thereof, antibodies, antibody conjugates, or reagents or kits for detecting novel coronaviruses or their N proteins, diagnosing novel coronavirus infections or interacting with novel coronaviruses Use in infection-related diseases.

Embodiments of the present disclosure also provide the use of the above reagent or kit in the detection of novel coronavirus or its N protein.

Embodiments of the present disclosure also provide a method for diagnosing a subject infected with a new coronavirus or a disease related to a new coronavirus infection, including:

A) contacting the above-mentioned antibody or functional fragment thereof, antibody, antibody conjugate, or reagent or kit with a sample from the subject under conditions sufficient for the binding reaction to occur; and

B) Detection of immune complexes generated by the binding reaction.

Embodiments of the present disclosure also provide a method for detecting novel coronavirus or its N protein in a test sample, comprising:

a) Under conditions sufficient for an antibody/antigen binding reaction to occur, contact the novel coronavirus or its N protein antigen in the test sample with the above-mentioned antibody or its functional fragment, antibody, antibody conjugate, or reagent or kit to form immune complexes; and

b) detecting the presence of said immune complex indicative of the presence of said antigen in said test sample.

In an optional embodiment, in step a), the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment.

In an optional embodiment, in step a), the immune complex further includes a second antibody that binds to the novel coronavirus or its N protein antigen.

In an optional embodiment, diseases associated with novel coronavirus infection include respiratory symptoms, fever, cough, hypoxemia, pneumonia, acute respiratory distress syndrome, severe acute respiratory syndrome, renal failure or septic shock .

As used herein, the term "subject" refers to a vertebrate, preferably a mammal, most preferably a human. Mammals include, but are not limited to, rodents, apes, humans, livestock, sport animals, and pets. Also included are tissues, cells and progeny of biological entities obtained in vivo or cultured in vitro.

The disclosure provides an anti-new coronavirus antibody, a reagent and a kit for detecting the new coronavirus, the antibody can specifically bind to the N protein of the new coronavirus, and has a high affinity for it, and the antibody can be used to detect the new coronavirus It has good sensitivity and specificity. The present disclosure provides a richer selection of antibodies for the detection of novel coronaviruses.

On the basis of the amino acid sequence of the antibody or its functional fragment provided in the present disclosure, those skilled in the art can easily imagine that the antibody or its functional fragment can be prepared by using genetic engineering technology or other techniques (chemical synthesis, recombinant expression), for example, from It is easy for those skilled in the art to separate and purify the antibody or its functional fragment from the culture product of recombinant cells capable of recombinantly expressing the antibody or its functional fragment as described in any one of the above. Based on this, no matter Which technology is used to prepare the antibody or its functional fragments of the present disclosure belongs to the protection scope of the present disclosure.

In order to make the purpose, technical solutions and advantages of the embodiments of the present disclosure clearer, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all conventional products that could be purchased from the market.

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 dosages herein, some methods and materials are now described. Unless otherwise stated, techniques employed or considered herein are standard methods. The materials, methods, and examples are illustrative only and not limiting.

The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry and immunology, which are within the skill of the art. This technique is fully explained in the literature, e.g., Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989); Oligonucleotide Synthesis ( M.J.Gait edited, 1984); "Animal Cell Culture (Animal Cell Culture)" (R.I.Freshney edited, 1987); "Methods in Enzymology" (Academic Press, Inc.); " "Handbook of Experimental Immunology" (D.M.Weir and C.C.Blackwell edited); "Gene Transfer Vectors for Mammalian Cells" (J.M.Miller and M.P.Calos edited, 1987); "Contemporary Methods in Molecular Biology (Current Protocols in Molecular Biology)" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction)" (Mullis et al., eds., 1994); and Contemporary Current Protocols in Immunology (J.E. Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

Example

The features and performances of the present disclosure will be described in further detail below in conjunction with the examples.

Preparation of Example 1 Antibody 9B54

In this example, the restriction endonuclease and Prime Star DNA polymerase were purchased from Takara Company. MagExtractor-RNA extraction kit was purchased from TOYOBO Company. BD SMART ^TM RACE cDNA Amplification Kit was purchased from Takara Company. The pMD-18T vector was purchased from Takara Company. Plasmid extraction kit was purchased from Tiangen Company. Primer synthesis and gene sequencing were performed by Invitrogen.

1 Construction of recombinant plasmids

(1) Antibody gene preparation

The mRNA was extracted from the hybridoma cell line that secretes the antibody against the N protein of the new coronavirus prepared in our laboratory, and the DNA product was obtained by RT-PCR method, and the product was inserted into pMD-18T after adding A reaction with rTaq DNA polymerase The vector was transformed into DH5α competent cells, and after the colonies grew out, heavy chain (Heavy Chain) and light chain (Light Chain) gene clones were taken respectively, and each 4 clones were sent to a gene sequencing company for sequencing.

(2) Sequence analysis of the variable region gene of the 9B54 antibody

Put the gene sequence obtained by the above sequencing into the kabat antibody database for analysis, and use VNTI11.5 software to analyze and confirm that the genes amplified by the heavy chain and light chain primer pairs are correct, and the genes amplified by the light chain In the fragment, the gene sequence of VL (variable region of light chain) is 321bp, and there is a leader peptide sequence of 57bp in front of it; in the gene fragment amplified by the heavy chain primer pair, the gene sequence of VH (variable region of heavy chain) is 369bp, belonging to the VH1 gene family, with a 57bp leader peptide sequence in front of it (the sequences of the heavy and light chains of 9B54 are shown in SEQ ID NO: 19, 20, respectively).

(3) Construction of recombinant antibody expression plasmid

pcDNA ^™ 3.4

Vector is a recombinant antibody eukaryotic expression vector constructed. The expression vector has introduced multiple cloning restriction sites such as HindIII, BamHI, and EcoRI, and named it pcDNA3.4A expression vector, which will be referred to as 3.4A expression vector in the future; according to the above pMD-18T According to the sequencing results of the variable region gene of the antibody, the VL and VH gene-specific primers of the antibody were designed, with HindIII, EcoRI restriction sites and protective bases at both ends, and a 0.72kb light chain was amplified by PCR amplification Gene fragment and 1.40kb heavy chain gene fragment.

The heavy chain and light chain gene fragments were respectively digested with HindIII/EcoRI double enzymes, and the 3.4A vector was digested with HindIII/EcoRI double enzymes. After the fragments and vectors were purified and recovered, the heavy chain genes and light chain genes were respectively connected to the 3.4A expression vectors, respectively. Recombinant expression plasmids for the heavy and light chains were obtained.

2 Stable cell line screening

(1) Transient transfection of recombinant antibody expression plasmid into CHO cells to determine the activity of the expression plasmid

Dilute the plasmid with ultrapure water to 40 μg/100 μL, adjust CHO cells to 1.43×10 ⁷ cells (cells)/mL in a centrifuge tube, mix 100 μL plasmid with 700 μL cells, transfer to electroporation cup, electroporation, 3rd, 5th, 7th Sampling and counting on the first day, and receiving samples for testing on the seventh day.

Dilute 2019-nCoV N protein antigen to 1 μg/mL in coating solution, 100 μL per well, overnight at 4°C; the next day, wash twice with washing solution and pat dry; add blocking solution (20% BSA + 80% PBS) to each well 120 μL, 37°C, 1h, pat dry; add diluted cell supernatant, 100μL/well, 37°C, 30min (partial supernatant 1h); wash with washing solution 5 times, pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, 37°C, 30 min; wash with washing solution 5 times, pat dry; add chromogenic solution A (50 μL/well, containing citric acid + sodium acetate + acetanilide + urea peroxide), add chromogenic solution B (50μL/well, containing citric acid+EDTA·2Na+TMB+concentrated HCL), 10min; add stop solution (50μL/well, containing EDTA·2Na+concentrated H ₂ SO ₄ ); read at 450nm (refer to 630nm) on a microplate reader OD value. The results showed that after the cell supernatant was diluted 1000 times, the reaction OD was still greater than 1.0, and the reaction OD of the wells without cell supernatant was less than 0.1, indicating that the antibody produced after plasmid transient transfer was active against the 2019-nCoV N protein antigen.

(2) Linearization of recombinant antibody expression plasmid

Prepare the following reagents: buffer (Buffer) 50 μL, DNA 100 μg/tube, Puv I enzyme 10 μL, sterile water to 500 μL, 37 ° C water bath enzyme digestion overnight; first use an equal volume of phenol/chloroform/isoamyl alcohol (lower layer) 25 : 24:1 (volume ratio), and then sequentially extracted with chloroform (water phase); 0.1 times volume (water phase) of 3M sodium acetate and 2 times volume of ethanol were precipitated on ice, rinsed with 70% ethanol to remove the organic solvent, After the ethanol volatilizes completely, rethaw with an appropriate amount of sterilized water, and finally measure the concentration.

(3) Stable transfection of recombinant antibody expression plasmids, pressurized selection of stable cell lines

Dilute the plasmid with ultrapure water to 400ng/mL, adjust CHO cells to 1.43× ¹⁰⁷ cells/mL in a centrifuge tube, mix 100 μL plasmid with 700 μL cells, transfer to an electroporation cup, electroporation, and count the next day; 25 μmol/L MSX 96 The wells were cultured under pressure for about 25 days.

Observe and mark the clone wells with cells under a microscope, and record the confluence; take the culture supernatant and send samples for detection; select cell lines with high antibody concentration and relative concentration and transfer to 24 wells, and transfer to 6 wells in about 3 days; keep the culture after 3 days For batch culture, adjust the cell density to 0.5×10 ⁶ cells/mL, 2.2 mL for batch culture, and 0.3×10 ⁶ cells/mL, 2 mL for seed preservation; 7-day 6-well batch culture supernatant was sent for testing and selection Cell lines with smaller antibody concentration and cell diameter were transferred to TPP for preservation and passage.

3 Recombinant Antibody Production

(1) Cell expansion

After recovery, the cells were first cultured in a 125mL shake flask with an inoculation volume of 30mL, the medium was 100% Dynamis medium, and placed in a shaker with a rotation speed of 120r/min, a temperature of 37°C, and 8% carbon dioxide. Cultivate for 72 hours, inoculate expansion culture at a seeding density of 500,000 cells/mL, the expansion volume is calculated according to production requirements, and the medium is 100% Dynamis medium. Then every 72h expansion once. When the amount of cells meets the production requirements, strictly control the inoculation density to about 500,000 cells/mL for production.

(2) Shake flask production and purification

Shake flask parameters: rotational speed 120r/min, temperature 37°C, carbon dioxide 8%. Fed-batch feeding: Feed feeding starts every day when the shake flask is cultured for 72 hours. HyCloneTM Cell BoostTM Feed 7a feeds 3% of the initial culture volume every day, and Feed 7b feeds 1/1000 of the initial culture volume every day. Replenish until the 12th day (feeding on the 12th day). Glucose was supplemented with 3g/L on the sixth day. Receive samples on the 13th day. Affinity purification was performed using a protein A affinity column. Take 2 μg of the purified antibody for reducing SDS-PAGE, and 2 μg of foreign control antibody as a control. The electropherogram is shown in Figure 1 below. After reducing SDS-PAGE, two bands are displayed, one Mr is 50KD, and the other Mr is 28KD .

Example 2

Antibody performance testing

(1) Activity detection

Dilute 2019-nCoV N protein antigen to 3 μg/mL in coating solution (main component NaHCO ₃ ) for microplate coating, 100 μl per well, overnight at 4°C; the next day, wash twice with washing solution and pat dry; add blocking solution (20%BSA+80%PBS), 120μL per well, 37°C, 1h, pat dry; add the diluted monoclonal antibody in Example 1, 100μL/well, 37°C, 30min-60min; wash with washing solution for 5 time, pat dry; add goat anti-mouse IgG-HRP, 100 μL per well, 37°C, 30 min; wash with washing solution (PBS) 5 times, pat dry; add chromogenic solution A (50 μL/well, containing 2.1g/L citric acid, 12.25g/L citric acid, 0.07g/L acetanilide and 0.5g/L carbamide peroxide), add color development solution B solution (50μL/well, containing 1.05g/L citric acid, 0.186g/LEDTA· 2Na, 0.45g/L TMB and 0.2mL/L concentrated HCl), 10min; add stop solution (50μL/well, containing 0.75g/EDTA·2Na and 10.2mL/L concentrated H ₂ SO ₄ ); (refer to 630nm) to read the OD value. The results are shown in Table 2 below.

Table 2 activity data

Concentration (ng/mL)	12.5	6.25	3.125	1.5625	0.78125	0.000
control	2.196	2.151	1.687	0.980	0.557	0.034
9B54	2.296	2.337	1.708	1.009	0.688	0.038

(2) Affinity detection

Using the AMC sensor, the purified antibody was diluted to 10 μg/mL with PBST, and the 2019-nCoV N protein antigen was serially diluted with PBST.

Operation process: Equilibrate in buffer 1 (PBST) for 60s, immobilize antibody in antibody solution for 300s, incubate in buffer 2 (PBST) for 180s, bind in antigen solution for 420s, dissociate in buffer 2 for 1200s, use 10mM pH 1.69GLY solution And buffer 3 for sensor regeneration, output data. K _D represents the equilibrium dissociation constant or affinity; kon represents the association rate; kdis represents the dissociation rate. The results are shown in Table 3 below.

Table 3 Affinity detection data

sample name	KD(M)	kon(1/Ms)	kdis(1/s)
control	2.43E-08	4.52E+03	1.10E-04
9B54	1.02E-10	1.11E+06	1.13E-04

(3) Stability assessment

Place the above antibody at 4°C (refrigerator), -80°C (refrigerator), and 37°C (incubator) for 21 days, take 7-day, 14-day, and 21-day samples for state observation, and conduct activity detection for 21-day samples , the results showed that under the three assessment conditions, there was no obvious change in the protein state of the antibody after 21 days of storage, and the activity did not show a downward trend with the increase of the assessment temperature, indicating that the above antibodies were stable. Table 4 below shows the OD results of the enzyme immunoassay activity test for 21 days.

Table 4

Sample concentration (ng/mL)	12.5	1.5625	0
4°C, 21 days sample	2.122	0.691	0.047
-80℃, 21 days sample	2.154	0.647	0.051
37°C, 21 days sample	2.133	0.666	0.057

(4) Performance evaluation

The above-mentioned 9B54 was used as a labeled antibody and used together with another new coronavirus antibody (purchased from Feipeng) to detect the new coronavirus.

1. mark

(1) Colloidal gold preparation: using the traditional sodium citrate reduction method, first heat the chloroauric acid solution to boiling, quickly add a certain proportion of trisodium citrate solution, stir evenly, and wait until the color of the solution turns wine red and does not change Stop heating at the time, be cooled to room temperature, obtain the colloidal gold solution that concentration is 4/10,000;

(2) Marking: add 0.2M K ₂ CO ₃ solution to the colloidal gold solution to adjust the pH to 6.0-7.5;

(3) Centrifugation: Add labeled antibody to the pH-adjusted colloidal gold solution and mix well, then add blocking agent to stop labeling, centrifuge at 10000rpm/7min/4°C, and remove the supernatant;

(4) Reconstitution: resuspend to 100 μL, sonicate 2-3 times;

(5) Gold coating: Dilute the concentrated gold obtained from the resuspension and spread it on a glass cellulose membrane, then freeze-dry it in a freeze dryer (1-2 hours) or dry it overnight in a drying room at 37°C.

2. Coated

(1) Assemble the nitrocellulose membrane and the colloidal gold PVC base plate for later use;

(2) Dilute the coated antibody to 1.0-2.0mg/mL, draw a line evenly on the NC membrane with a gold sprayer, and then put it in a 37°C incubator for drying for at least 45 minutes. Assemble the cut strips and add samples for detection.

3. Detection

(1) Quality control products: vaccine, 2019-nCoV N antigen, negative nasal swab, throat swab and saliva.

The antibody is used as a labeled antibody and used together with another new crown antibody, and the performance difference of the antibody is compared on the colloidal gold platform, and the antibody can achieve a better performance level than the control. See Table 5 below for details:

table 5

The above descriptions are only preferred embodiments of the present disclosure, and are not intended to limit the present disclosure. For those skilled in the art, the present disclosure may have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included within the protection scope of the present disclosure.

The partial amino acid sequences involved in this application are shown in Table 6 below:

Table 6

serial number	sequence fragment
SEQ ID NO: 1	DYNMG
SEQ ID NO: 2	AIIPNNGGTIYNQKFKG
SEQ ID NO: 3	EAYRDYDVKTWF
SEQ ID NO: 4	SASQGIRNYLN
SEQ ID NO: 5	YTSTLHS
SEQ ID NO: 6	QQYSKFP
SEQ ID NO: 7	EVLLQQSGPELVNPGASVKIPCKASGYTFT
SEQ ID NO: 8	WVKQSHGKSLEWIG
SEQ ID NO: 9	KATLTVDESSSTAYMELRRSLTSEDTAVYYCAR
SEQ ID NO: 10	AYWGQGTLVTVSA
SEQ ID NO: 11	DIQMTQTTSSLSASLGDRVTISC
SEQ ID NO: 12	WYQQKPDGTVKLLIY
SEQ ID NO: 13	GVPSRFSGSGSGTDYSLTISNLEPEDIATYFC
SEQ ID NO: 14	YTFGGGTKLEIK

Industrial Applicability

The anti-new coronavirus antibody provided in the present disclosure includes a heavy chain complementarity determining region and a light chain complementarity determining region. The antibody has a good affinity for the N protein of the new coronavirus, and the use of the antibody to detect the new coronavirus has sensitivity and specificity. The present disclosure provides a more excellent selection of antibodies for the detection of novel coronavirus, and therefore has excellent practical performance.

Claims (21)

1. An anti-new coronavirus or its N protein antibody or functional fragment thereof, including HCDR1, HCDR2, HCDR3 and LCDR1, LCDR2, LCDR3, characterized in that, the HCDR1, HCDR2, HCDR3 include the same as SEQ ID NO: 15 The amino acid sequence consistent with HCDR1, HCDR2 and HCDR3 of the heavy chain variable region is shown; the LCDR1, LCDR2 and LCDR3 include amino acid sequences consistent with the LCDR1, LCDR2 and LCDR3 of the light chain variable region shown in SEQ ID NO:16.
2. The antibody or its functional fragment according to claim 1, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined by Kabat, Chothia, IMGT, AbM or Contact system.
3. The antibody or its functional fragment according to claim 1, wherein the amino acid sequences of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 include or are sequentially shown in SEQ ID NO: 1-6.
4. An anti-new coronavirus or its N protein antibody or functional fragment thereof, characterized in that the antibody or its functional fragment comprises amino acid sequences such as HCDR1, HCDR2, and HCDR3 shown in SEQ ID NO: 1-3, And amino acid sequence such as LCDR1, LCDR2 and LCDR3 shown in SEQ ID NO:4-6.
5. The antibody or its functional fragment according to any one of claims 1-4, wherein the antibody or its functional fragment also has HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 at least one of;

   Optionally, the amino acid sequences of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3 and LFR4 sequentially include SEQ ID NO: 7-14 or a sequence having at least 80% homology with SEQ ID NO: 7-14 Amino acid sequence, or sequentially as shown in SEQ ID NO: 7-14 or an amino acid sequence having at least 80% homology to SEQ ID NO: 7-14;

   Optionally, the antibody or its functional fragments also have amino acid sequences such as HFR1, HFR2, HFR3, HFR4 shown in SEQ ID NO: 7-10, and amino acid sequences such as LFR1 shown in SEQ ID NO: 11-14 , LFR2, LFR3 and LFR4, or an amino acid sequence having at least 80% homology to each of said sequences;

   Optionally, the antibody or its functional fragment binds the novel coronavirus or its N protein with an affinity of K _D ≤ 10 ^-9 M;

   Optionally, the antibody or a functional fragment thereof comprises a heavy chain variable region shown in SEQ ID NO: 15, and a light chain variable region shown in SEQ ID NO: 16.
6. An antibody or functional fragment thereof against novel coronavirus or its N protein, characterized in that the antibody or functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain can be The amino acid sequence of the variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.
7. An antibody or functional fragment thereof against novel coronavirus or its N protein, characterized in that the antibody or functional fragment thereof comprises a heavy chain variable region and/or a light chain variable region, and the heavy chain can be The variable region contains the sequence structure of HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4, the light chain variable region contains the sequence structure of LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4, the HCDR1, HCDR2 The amino acid sequence of , HCDR3, LCDR1, LCDR2, LCDR3 is the amino acid sequence of HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, LCDR3 described in any one of claims 1-4, and said HFR1, HFR2, HFR3, HFR4, LFR1, The amino acid sequence of LFR2, LFR3, LFR4 is the amino acid sequence of HFR1, HFR2, HFR3, HFR4, LFR1, LFR2, LFR3, LFR4 described in claim 5;

   Optionally, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:15; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:16.
8. The antibody or functional fragment thereof according to any one of claims 1-7, wherein the antibody or functional fragment thereof further comprises a constant region;

   Optionally, said constant region comprises a heavy chain constant region and/or a light chain constant region;

   Optionally, the heavy chain constant region is selected from the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD; the light chain constant region is selected from the κ-type or λ-type light chain constant region ;

   Optionally, the species source of the constant region is bovine, horse, pig, sheep, goat, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose or human;

   Optionally, the species source of the constant region is mouse;

   Optionally, the sequence of the heavy chain constant region is shown in SEQ ID NO: 17 or has at least 80% homology with it, and the sequence of the light chain constant region is shown in SEQ ID NO: 18 or has at least 80% homology with it 80% homology;

   Optionally, the antibody or a functional fragment thereof comprises a heavy chain shown in SEQ ID NO: 19 and a light chain shown in SEQ ID NO: 20.
9. The antibody or functional fragment thereof according to any one of claims 1-8, wherein the functional fragment is selected from F(ab')2, Fab', Fab, Fv and scFv of the antibody any kind.
10. An antibody against novel coronavirus or its N protein, comprising heavy chain and/or light chain, characterized in that the heavy chain comprises the heavy chain variable region described in any one of claims 5-7 and the claim The heavy chain constant region described in 8; the light chain comprises the light chain variable region described in any one of claims 5-7 and the light chain constant region described in claim 8;

    Optionally, 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.
11. An antibody against novel coronavirus or its N protein, comprising heavy chain and/or light chain, characterized in that the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 19; the amino acid sequence of the light chain is as shown in Shown in SEQ ID NO:20.
12. An antibody conjugate, characterized in that the antibody conjugate comprises the antibody or a functional fragment thereof according to any one of claims 1-11;

    Optionally, the antibody or functional fragment thereof is labeled with a label;

    Optionally, the label is selected from fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent reagents and nanoparticle labels;

    Optionally, the fluorescent dye is selected from the group consisting of fluorescein dyes and derivatives thereof, rhodamine dyes and derivatives thereof, Cy series dyes and derivatives thereof, Alexa series dyes and derivatives thereof, protein dyes and derivatives thereof thing;

    Optionally, the enzyme is selected from horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase and glucose-6-phosphate deoxygenase;

    Optionally, the radioactive isotope is selected from ²¹² Bi, ¹³¹ I, ¹¹¹ In, ⁹⁰ Y, ¹⁸⁶ Re, ²¹¹ At, ¹²⁵ I, ¹⁸⁸ Re, ¹⁵³ Sm, ²¹³ Bi, ³² P, ⁹⁴ mTc, ⁹⁹ mTc, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁷ Sc, ¹¹⁰ mIn, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ¹⁷² Lu and ¹⁸ F;

    Optionally, the chemiluminescence reagent is selected from the group consisting of luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridyl ruthenium and its derivatives, acridinium esters and its derivatives, dioxane Ethane and its derivatives, lophine and its derivatives, and peroxalate and its derivatives;

    Optionally, the nanoparticle marker is selected from nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles and rare earth complex nanoparticles;

    Optionally, the colloid is selected from colloidal metals, disperse dyes, dye-labeled microspheres and latex;

    Optionally, the colloidal metal is selected from colloidal gold, colloidal silver and colloidal selenium;

    Optionally, the antibody or a functional fragment thereof is coated onto a solid phase;

    Optionally, the solid phase is selected from microspheres, plates and membranes;

    Optionally, the solid phase is selected from magnetic microspheres, plastic microspheres, plastic particles, microwell plates, glass, capillary tubes, nylon and nitrocellulose membranes;

    Optionally, the solid phase is selected from magnetic microspheres.
13. A reagent or kit, characterized in that the reagent or kit comprises the antibody or functional fragment thereof according to any one of claims 1-11 or the antibody conjugate according to claim 12;

    Optionally, the reagent or kit is a reagent or kit for detecting novel coronavirus or its N protein.
14. A nucleic acid, characterized in that it encodes the antibody or functional fragment thereof according to any one of claims 1-11.
15. A vector, characterized in that it contains a nucleic acid fragment encoding the antibody or a functional fragment thereof according to any one of claims 1-11.
16. A recombinant cell, characterized in that it contains the vector according to claim 15.
17. A method for preparing the antibody or functional fragment thereof according to any one of claims 1-11, characterized in that it comprises: culturing the recombinant cell according to claim 16.
18. The antibody or functional fragment thereof according to any one of claims 1-11, the antibody conjugate according to claim 12, or the reagent or kit according to claim 13, for detecting novel coronavirus or its N Use in the diagnosis of novel coronavirus infection or diseases related to novel coronavirus infection.
19. A method for diagnosing a subject's infection with novel coronavirus or a disease associated with novel coronavirus infection, comprising:

    A) under conditions sufficient for a binding reaction to occur, the antibody or functional fragment thereof according to any one of claims 1-11, the antibody conjugate according to claim 12, or the reagent according to claim 13 or The kit is contacted with a sample from said subject to perform a binding reaction; and

    B) Detection of immune complexes generated by the binding reaction.
20. A method for detecting novel coronavirus or its N protein in a test sample, comprising:

    a) Under conditions sufficient for an antibody/antigen binding reaction to occur, the novel coronavirus or its N protein antigen in the test sample is combined with the antibody or its functional fragment according to any one of claims 1-11, claim The antibody conjugate described in 12, or the reagent or kit described in claim 13 is contacted to form an immune complex; and

    b) detecting the presence of said immune complex indicating the presence of said antigen in said test sample;

    Optionally, in step a), the immune complex further includes a second antibody that binds to the antibody or antigen-binding fragment;

    Optionally, in step a), the immune complex further includes a second antibody that binds to the novel coronavirus or its N protein antigen.
21. According to the use according to claim 18 or the method according to claim 19, the diseases associated with novel coronavirus infection include respiratory symptoms, fever, cough, hypoxemia, pneumonia, acute respiratory distress syndrome, severe acute Respiratory syndrome, renal failure, or septic shock.

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