An antibody against the novel coronavirus and its application in detection

Abstract

The present invention discloses an antibody against novel coronavirus and its application in detection, relating to the technical field of antibodies. The antibody provided by the present invention comprises LCDR1, LCDR2 and LCDR3 with amino acid sequences shown in SEQ ID No.1 to SEQ ID No.3 in sequence, and HCDR1, HCDR2 and HCDR3 with amino acid sequences shown in SEQ ID No.4 to SEQ ID No.6 in sequence. This antibody has high specificity and sensitivity and can be used for the detection of novel coronavirus. The novel coronavirus antigen detection reagent developed based on the antibody can specifically detect novel coronavirus without being affected by the antigen protein mutation of virus mutant strains, and can reduce the detection cost, shorten the detection time and improve the detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of antibodies, and in particular, to an antibody against the novel coronavirus and its application in detection. Background Art

[0002] Coronaviruses are a large family of viruses that are known to cause colds and more severe diseases such as Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS).

[0003] The novel coronavirus (2019-nCoV, SARS-CoV-2) is the seventh type of coronavirus isolated from humans. This virus belongs to the genus β, has an envelope, and the particles are round or oval, often polymorphic, with a diameter of 60-140 nm. Its genetic characteristics are significantly different from those of SARS-CoV and MERS-CoV. The basic structure of SARS-CoV-2 is an envelope structure composed of genomic RNA and phosphorylated nucleocapsid protein (N protein). The envelope structure incorporates four proteins: spike glycoprotein (S protein), envelope glycoprotein (E protein), membrane glycoprotein (M protein), and hemagglutinin glycoprotein (HE protein). The N protein is buried in the phospholipid bilayer and covered by two different types of spike proteins. The membrane protein (M protein, a type III transmembrane glycoprotein) responsible for nutrient transport and the envelope protein (E protein) are located between the S proteins on the virus envelope.

[0004] Novel coronavirus variant strains generally refer to the situation where during the replication of the novel coronavirus, one or some gene sequences in individual viruses may undergo slight changes. The change in the gene sequence allows the virus to survive and further reproduce, thus forming a new novel coronavirus variant strain. The formation of a new novel coronavirus variant strain may be enhanced or weakened based on the original virus strain, and the clinical manifestations, transmission methods, transmission speed, etc. of the variant strain may all change. The novel coronavirus belongs to RNA viruses and is more prone to mutation than DNA viruses. As of September 26, 2021, the Delta variant strain has been detected in 39 countries, the Alpha variant strain has been detected in 45 countries, and the Beta variant strain has been detected in 40 countries. The Omicron mutant strain of the novel coronavirus that emerged at the end of 2021 has significantly more mutation sites than all other prevalent novel coronavirus variant strains, especially with more mutations in the virus spike (Spike) protein, resulting in enhanced transmission and immune escape capabilities of this virus. These novel coronavirus variant strains constantly emerging around the world have made the prevention and control of the epidemic and the detection difficulty continuously increase. Therefore, the prevention and control of the COVID-19 pandemic is a difficult problem that countries and regions around the world are jointly concerned about and facing.

[0005] The detection methods of the novel coronavirus mainly include the following.

[0006] a. Etiological detection: The etiological detection methods of viruses include cell culture, serological detection, nucleic acid detection, electron microscopy detection, etc. Compared with other methods, the detection of nucleic acid molecules has the characteristics of rapidity, high sensitivity, and strong specificity, and has become the mainstream method for the detection of coronaviruses. The existing nucleic acid detection methods for coronaviruses include whole genome sequencing, RT-PCR method, CRISPR, reverse transcription loop-mediated isothermal amplification method (RT-LAMP), and real-time RT-LAMP, etc. For the detection of SARS-CoV-2 nucleic acid, the most commonly applicable method currently is the real-time fluorescence quantitative PCR method, that is, after extracting the viral RNA in the specimen, reverse transcription, and PCR amplification of specific conserved sequences, it is displayed by conventional methods such as fluorescence. In addition, antigen detection targets the detection of the pathogen itself, and can detect whether there is a virus in the human body in the early stage of the disease, thus providing direct evidence of virus infection. The antigen detection of the novel coronavirus detects the viral antigen in the oral or nasopharyngeal swabs of patients on the test strip through the antigen-antibody binding reaction, which is convenient and fast, and generally can produce results in 15-20 minutes. The novel coronavirus antigen detection is relatively simple and fast.

[0007] b. Serological examination: Serological antibody detection has been included in one of the diagnostic criteria for novel coronavirus infection. Compared with nucleic acid detection, antibody detection has the characteristics of simple sampling and high detection efficiency. IgM antibody is the first antibody secreted in the immune response and is the earliest antibody to appear in the autoimmune process of virus infection, and can be used as a marker of recent acute infection. Under normal circumstances, IgM antibody is produced early, once infected, it is rapidly produced, usually starts to show positive after 3-5 days, has a short maintenance time, disappears quickly, and the positive detection of IgM in the blood can be used as an indicator of early infection. IgG antibody is produced late, has a long maintenance time, disappears slowly, and the titer in the recovery period is 4 times or more higher than that in the acute phase. The positive detection in the blood can be used as an indicator of current and past infections. Therefore, by detecting the positive conditions of IgM and IgG in the patient's serum, it will help to judge the different stages of the patient's SARS-CoV-2 infection.

[0008] Rapid immunoassay generally uses test strips for detection (1 red line for negative, 1 red line for positive), and most are based on the colloidal gold principle of antigen-antibody Figure 1)。The antibody 1 that can specifically bind to the antigen and is labeled with colloidal gold is fixed on the conjugate pad. Antibody 2 that specifically binds to the antigen is fixed at the test line on the NC membrane, and the secondary antibody that specifically recognizes the labeled antibody is fixed at the control line. When the test sample is added dropwise to the sample pad loading area, the sample moves towards the absorbent pad of the test strip. If it contains the antigen to be detected, the antigen binds to the gold-labeled antibody 1 on the conjugate pad to form an antigen-antibody complex, which is then captured by the antibody 2 on the test line and shows color. If the test sample does not contain the antigen to be detected, the test line does not show color. At the same time, the antibody on the control line specifically binds to the gold-labeled antibody and shows color to verify that the test strip is normal. The antibody detection principle is similar to that of antigen detection. If a "labeled antigen-antibody-captured antibody" complex is formed on the test line, it will show color, indicating that the test sample is positive.

[0009] However, considering the advantages and disadvantages of existing COVID-19 detection methods, nucleic acid detection has relatively high requirements for detection equipment or platforms. High-sensitivity RT-PCR instruments are expensive, and there are also relatively high requirements for the cleanliness of the laboratory and operators. In addition, nucleic acid detection takes a relatively long time. Considering the situation of sample transportation and sample backlog, it usually takes 24 hours to report the results. The antibodies stimulated in the blood samples for antibody detection are indirect evidence, which has a suggestive effect on clinical practice and there is a possibility of missed detection. In addition, the mutations in the main antigen proteins of COVID-19 variant strains significantly reduce the detection ability of antigen detection reagents, which is prone to causing false negative results in clinical practice.

[0010] Therefore, there is still a strong motivation to develop new COVID-19 detection antibodies to ensure that they can be used in the development of COVID-19 antigen detection reagents, are not affected by the mutations of the antigen proteins of virus mutant strains, reduce the detection cost, shorten the detection time, and improve the detection efficiency.

[0011] In view of this, the present invention is specifically proposed. Summary of the Invention

[0012] The object of the present invention is to provide an antibody against SARS-CoV-2 and its application in detection.

[0013] The present invention is implemented as follows:

[0014] In the first aspect, an embodiment of the present invention provides an antibody or its antigen-binding fragment that specifically targets the N protein of SARS-CoV-2. The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the light chain variable region includes LCDR1, LCDR2, and LCDR3 with amino acid sequences shown in SEQ ID No.1 to 3 in sequence; the heavy chain variable region includes HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID No.4 to 6 in sequence.

[0015] In a second aspect, embodiments of the present invention provide a nucleic acid encoding the antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0016] In a third aspect, embodiments of the present invention provide an antibody conjugate, which comprises: the antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0017] In a fourth aspect, embodiments of the present invention provide a reagent, kit or composition for detecting novel coronavirus, which comprises the antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0018] In a fifth aspect, embodiments of the present invention provide the use of the antibody or antigen-binding fragment thereof as described in the foregoing embodiments in the preparation of an immuno-diagnostic product.

[0019] In a sixth aspect, embodiments of the present invention provide an immunochromatographic test strip or kit, which comprises the antibody or antigen-binding fragment thereof as described in the foregoing embodiments.

[0020] The present invention has the following beneficial effects:

[0021] The innovation of the present invention lies in providing a monoclonal antibody that specifically targets novel coronavirus. The antibody can be used for the detection of novel coronavirus with high specificity and high sensitivity. The novel coronavirus antigen detection reagent developed based on the antibody can specifically detect novel coronavirus without being affected by the antigen protein mutation of virus mutant strains, and can reduce the detection cost, shorten the detection time, and improve the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for use in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0023] Figure 1 is the schematic diagram of colloidal gold immunoassay;

[0024] Figure 2 is the specific detection result of monoclonal antibody H2B4;

[0025] Figure 3 is the sensitivity test result of the lateral flow immunoassay reagent for inactivated novel coronavirus culture;

[0026] Figure 4 is the sensitivity test result of the lateral flow immunoassay reagent for standardized recombinant novel coronavirus N protein;

[0027] Figure 5Detection results of different SARS-CoV-2 mutant strains by lateral flow immunoassay reagents;

[0028] Figure 6 Clinical sample test results of lateral flow immunoassay reagents. Detailed implementation manners

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained by purchasing in the market.

[0030] First, the embodiments of the present invention provide an antibody or an antigen-binding fragment thereof that specifically targets the N protein of the novel coronavirus. The antibody or the antigen-binding fragment thereof includes a heavy-chain variable region and a light-chain variable region; the light-chain variable region includes LCDR1, LCDR2, and LCDR3 whose amino acid sequences are shown in SEQ ID No. 1 to 3 in sequence; the heavy-chain variable region includes HCDR1, HCDR2, and HCDR3 whose amino acid sequences are shown in SEQ ID No. 4 to 6 in sequence.

[0031] In the present invention, the term "antibody" encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (such as bispecific antibodies, trispecific antibodies, tetra-specific antibodies, etc.), murine antibodies, chimeric antibodies, full-length antibodies, etc., as long as they exhibit the desired antigen-binding activity.

[0032] In the present invention, the terms "complementary determining region", "CDR", or "CDRs" refer to the highly variable regions of the heavy and light chains of immunoglobulins, which are the regions within the variable domains of antibodies that mainly contribute to specific binding to antigens. In the specific implementation manners of the present invention, CDRs refer to two or more highly variable regions in the heavy and light chains of the antibody.

[0033] In the present invention, the heavy-chain complementary determining region is represented by HCDR, and the three CDR regions contained in the heavy-chain variable region are: HCDR1, HCDR2, and HCDR3; the light-chain complementary determining region is represented by LCDR, and the three CDR regions contained in the light-chain variable region are: LCDR1, LCDR2, and LCDR3.

[0034] In some embodiments, the amino acid sequence to which the antibody or the antigen-binding fragment thereof specifically targets and binds is shown in SEQ ID No. 10.

[0035] In some embodiments, the light chain variable region and the heavy chain variable region further include framework regions. In the present invention, the "framework region" or "FR" region refers to the regions other than CDRs in the heavy chain variable region and the light chain variable region of an antibody; the heavy chain framework region can be further subdivided into adjacent regions (FR1, FR2, FR3, and FR4) separated by CDRs, wherein the heavy chain framework region can be further subdivided into adjacent regions separated by CDRs, including HFR1, HFR2, HFR3, and HFR4 framework regions; the light chain framework region can be further subdivided into adjacent regions separated by LCDRs, including LFR1, LFR2, LFR3, and LFR4 framework regions. The heavy chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (ranging from the amino terminus to the carboxyl terminus): HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4; the light chain variable region is obtained by arranging and connecting the following numbered CDRs and FRs (ranging from the amino terminus to the carboxyl terminus): LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4.

[0036] In some embodiments, the antibody or its antigen-binding fragment further includes a constant region. Optionally, the constant region includes a heavy chain constant region and / or a light chain constant region. The light chain of a full-length antibody includes a light chain variable domain VL and a constant domain CL, VL is at the amino terminus of the light chain, and the CL domain is at the carboxyl terminus. The light chain includes a κ chain and a λ chain; the heavy chain of a full-length antibody includes a heavy variable domain VH and a constant region CH, VH is at the amino terminus of the heavy chain, and the CH domain is at the carboxyl terminus.

[0037] In some embodiments, the heavy chain constant region is selected from the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. The light chain constant region is a κ chain or a λ chain.

[0038] In some embodiments, the heavy chain constant region is IgG1; the light chain constant region is a κ chain.

[0039] In some embodiments, the species origin of the constant region is bovine, equine, porcine, ovine, rat, mouse, dog, cat, rabbit, donkey, deer, mink, chicken, duck, goose, or human.

[0040] In some embodiments, the amino acid sequence of the light chain is as shown in SEQ ID No.7, and the amino acid sequence of the heavy chain is as shown in SEQ ID No.8.

[0041] "Antigen-binding fragment" is a part of a whole antibody, and this part specifically binds to the antigen that the whole antibody binds to. Those skilled in the art can easily understand from the content described in the present invention that antigen-binding fragments can be prepared by methods known in the art, for example, by enzymatic digestion methods (including pepsin or papain) and / or by chemical reduction to cleave disulfide bonds, and can also be obtained by recombinant genetic techniques or by synthesis using an automated peptide synthesizer (such as an automated peptide synthesizer from Applied BioSystems).

[0042] In some embodiments, the antigen-binding fragment is selected from any one of F(ab’) 2 、Fab’, Fab, Fv, and scFv of an antibody.

[0043] On the other hand, embodiments of the present invention also provide a nucleic acid encoding the antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0044] On the other hand, embodiments of the present invention provide an antibody conjugate, which comprises: the antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0045] In some embodiments, the antibody conjugate further comprises a solid-phase carrier conjugated to the antibody or its antigen-binding fragment.

[0046] Optionally, the solid-phase carrier includes but is not limited to at least one of magnetic microspheres, plastic microspheres, plastic particles, microtiter plates, glass, capillary tubes, nylon, and nitrocellulose membranes.

[0047] In some embodiments, the antibody conjugate further comprises a detectable label conjugated to the antibody or its antigen-binding fragment.

[0048] In some embodiments, the label is selected from at least one of fluorescent dyes, enzymes, radioisotopes, chemiluminescent reagents, and nanoparticle-based labels.

[0049] In actual use, those skilled in the art can select other suitable labels according to the detection conditions or actual needs. No matter what label is used, it falls within the protection scope of the present invention.

[0050] In some embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (such as, but not limited to, fluorescein isothiocyanate (FITC), hydroxy fluorescein (FAM), tetrachloro fluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (such as, but not limited to, rhodamine B isothiocyanate (RBITC), tetramethyl rhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (such as, but not limited to, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, etc. or their analogs), Alexa series dyes and their derivatives (such as, but not limited to, Alexa Fluor 350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogs), and protein dyes and their derivatives (such as, but not limited to, phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), peridinin-chlorophyll protein (PerCP), etc.).

[0051] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate dehydrogenase.

[0052] In some embodiments, the radioisotopes 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 Rh177 Lu, 172 Lu and 18 F.

[0053] In some embodiments, the chemiluminescent reagent includes, but is not limited to, luminol and its derivatives, lucigenin, crustacean luciferin and its derivatives, ruthenium bipyridine and its derivatives, acridinium ester and its derivatives, dioxetane and its derivatives, rosalic acid and its derivatives, and peroxyoxalate and its derivatives.

[0054] In some embodiments, the nanoparticle-based labels include, but are not limited to, nanoparticles and colloids, and the nanoparticles include, but are not limited to, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0055] In some embodiments, the colloids include, but are not limited to, colloidal metals, colloidal selenium, disperse dyes, dye-labeled microspheres, and latex.

[0056] In some embodiments, the colloidal metals include, but are not limited to, colloidal gold or colloidal silver.

[0057] On the other hand, the embodiments of the present invention also provide the use of the antibody or its antigen-binding fragment as described in any of the foregoing embodiments in the preparation of a novel coronavirus detection product.

[0058] In some embodiments, the product includes any one of test strips, reagents, and reagent kits.

[0059] In some embodiments, the detection method is selected from any one of ELISA, immunofluorescence, chemiluminescent immunoassay, Western blot, immunochromatography, electrochemiluminescence immunoassay, and magnetic bead method.

[0060] In some embodiments, the detection product is an immunochromatographic test strip or a reagent kit.

[0061] In some embodiments, the capture antibody used on the conjugate pad of the immunochromatographic test strip is the antibody or its antigen-binding fragment.

[0062] In some embodiments, the detection antibody on the test line of the immunochromatographic test strip is the antibody or its antigen-binding fragment.

[0063] On the other hand, the embodiments of the present invention provide a lateral flow immunochromatographic reagent or a reagent kit, which includes the antibody or its antigen-binding fragment as described in any of the foregoing embodiments.

[0064] Based on the amino acid sequence of the antibody disclosed in the present invention, those skilled in the art can prepare the antibody by using genetic engineering techniques or other techniques (chemical synthesis, recombinant expression). For example, the antibody can be isolated and purified from the culture product of recombinant cells capable of recombinantly expressing the antibody described in any one of the above, which is easily achievable for those skilled in the art. Based on this, no matter which technique is used to prepare the antibody of the present invention, it falls within the protection scope of the present invention.

[0065] The features and properties of the present invention will be further described in detail below in conjunction with the examples.

[0066] Example 1

[0067] 1. Preparation of hybridoma monoclonal antibody:

[0068] (1) Design, preparation and carrier coupling of SARS-CoV-2 N antigen

[0069] The SARS-CoV-2 N protein sequence was obtained according to the SARS-CoV-2 protein sequence (SEQ ID No.9) on GenBank. After immunogenicity, hydrophilicity and surface accessibility analysis, the N-terminal 1-55 amino acid sequence of the N protein region was finally selected as the antigen (SEQ ID No.10). The synthetic peptide (COVID-KLH) antigen of this peptide and the C-terminal coupled keyhole limpet hemocyanin carrier protein KLH was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0070] (2) Immunization of mice

[0071] The synthetic polypeptide protein (COVID-KLH) was mixed with Freund's complete adjuvant at a ratio of 1:1 and emulsified to 500 μl, and then subcutaneously injected at multiple points into 3 female Balb / c mice aged 6-8 weeks. The antigen inoculation dose for each mouse was 50 μg. Three weeks later, the antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1 and emulsified to 500 μl, and then subcutaneously injected at multiple points. The antigen inoculation dose for each mouse was 50 μg. After an interval of three weeks, 50 μg was intraperitoneally injected without adjuvant, and the immunization was carried out 4 times in total.

[0072] (3) Determination of immune serum titer

[0073] The indirect ELISA method was used to determine the titer of immune serum. 50 μg of synthetic peptide COVID-KLH was dissolved in 10 mL of 0.05 M phosphate buffer with pH 9.6 to coat a 96-well polystyrene plate, 100 μl per well, overnight at 4 °C. The plate was washed three times with PBS (containing 0.05% (V / V) Tween-20), blocked with 100 μl of 10 mM PBS containing 1% BSA per well at 37 °C for 2 h, and then washed three times with PBS (containing 0.05% (V / V) Tween-20). Blood was collected from the tail vein of mice 10 days after the third immunization. The mouse immune serum was diluted 10 -2 to 10 -8 times with 10 mM PBS containing 1% BSA, added to the 96-well plate, 100 μl per well, incubated at 37 °C for 1 h. After washing the plate three times with PBS (containing 0.05% (V / V) Tween-20), 1:10000 diluted horseradish peroxidase-labeled goat anti-mouse IgG (Sigma, INC.) was added, 100 μl per well, incubated at 37 °C for 30 min. After washing the plate as above, TMB was used for color development, 100 μl per well, protected from light at room temperature for 10 min, then 50 μl of 2 M H 2 SO 4 was added to terminate the reaction, and the absorbance at 450 nm was measured. The serum of mice before immunization was used as a negative control, and the immune serum titer was determined with the ratio of the measured value to the control value ≥ 2.1 as the positive judgment value. The results are shown in Table 1.

[0074] Table 1 Immune serum titer

[0075] Dilution factor Immunized mouse 1 Immunized mouse 2 Immunized mouse 3 Immunized mouse 4 10000 2.54 2.65 2.53 2.58 50000 1.66 1.70 1.68 1.71 100000 1.01 1.08 1.05 1.10 500000 0.53 0.55 0.57 0.62 1000000 0.13 0.25 0.18 0.23

[0076] (4) Preparation of hybridoma

[0077] Mice with a serum titer greater than 1:10 5For BALB / c mice to be fused, 3 days before cell fusion, the synthetic peptide COVID-KLH was mixed with an equal volume of PBS and then intraperitoneally injected into the BALB / c mice to be fused at a dose of 50 μg / 500 μL per mouse for booster immunization. The spleens of the mice were aseptically removed and made into spleen cell suspensions, which were then mixed with the mouse myeloma cell line SP2 / 0 in the logarithmic growth phase at a ratio of 1:1. After centrifugation at 1000 g for 5 min at room temperature, the supernatant was discarded. The bottom of the centrifuge tube was gently flicked with a finger to loosen the precipitate. The centrifuge tube was placed in a 37°C water bath, and 50% polyethylene glycol (PEG, MW4000, Sigma), pre-warmed in a 37°C water bath, was added drop by drop to the centrifuge tube with a dropper while gently shaking the centrifuge tube. The addition was completed within 1 min. After dropping, it was left standing for 2 min, and then 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, and 10 mL of serum-free 1640 medium pre-warmed at 37°C were added every 1 min to terminate the action of polyethylene glycol. The cell mixture was centrifuged at 1000 g for 5 min at room temperature, the supernatant was discarded, and the cells were gently resuspended in HAT culture medium (hypoxanthine (H), aminopterin (A), and thymidine (T) (HAT, Sigma)). The cells were then dispensed into 96-well plates, 200 μL per well. After culturing for three days, the cell fusion was observed, and half of the HAT culture medium was replaced. This was continued for several days until clones formed. Seven days after fusion, the culture medium was replaced with HT culture medium (hypoxanthine (H) and thymidine (T) (HT, Sigma)) for culture.

[0078] (5) Screening of hybridoma cells secreting anti-SARS-CoV-2 N protein monoclonal antibodies

[0079] The cell culture supernatants were screened by indirect ELISA, and the positive clone hybridoma cells with higher titers were selected for subcloning and continuously cloned 2 - 3 times by the limiting dilution method until a 100% cell positive rate was achieved. The culture supernatants of the finally obtained 5 hybridoma cell lines with higher titers were detected by the indirect ELISA method, and at the same time, they were diluted with 0.02 M PBS for detection. The test results are shown in Table 2.

[0080] Table 2 Dilution test results

[0081]

[0082] Finally, a stable cell line secreting anti-SARS-CoV-2 N protein monoclonal antibody with the highest titer was obtained and labeled as H2B4. The cells with a 100% positive rate after cloning were amplified and cultured and then cryopreserved in liquid nitrogen.

[0083] (6) Preparation and purification of ascites

[0084] The hybridoma cell line H2B4 was injected intraperitoneally into BALB / c mice at a density of 1×10 6Inject an appropriate amount of liquid paraffin into the peritoneal cavity of 8-10-week-old female BALB / c mice pretreated, and observe the mice for 10-14 days. When the abdomen of the mice becomes enlarged, draw ascites. Purify the monoclonal antibody by affinity chromatography using Protein GSepharose Fast Flow, and determine the purity of the monoclonal antibody by SDS-PAGE. The purity reaches over 90%.

[0085] Example 2: Characterization of the monoclonal antibody of the present invention

[0086] (1) Determination of antibody concentration: The ascites prepared from the hybridoma cell line H2B4 was purified to obtain the monoclonal antibody H2B4 against the N protein of the new coronavirus, and its concentration was measured using a Nanodrop nucleic acid and protein analyzer produced by Thermofisher, and the concentration was 3.70 mg / ml.

[0087] (2) Antibody subtype identification: Use the mouse monoclonal antibody subtype identification kit of Thermofisher to identify the subtype of the hybridoma cell line. The subtype of the antibody secreted by H2B4 is IgG1, and the light chain is κ chain.

[0088] (3) Titer identification of the purified antibody: 50 μg of synthetic new coronavirus N protein peptide was dissolved in 10 ml of 0.05 M carbonate coating buffer at pH 9.6, added to a 96-well plate, 100 μL per well, and incubated overnight at 4°C. Wash the plate three times with PBS (containing 0.05% (v / v) Tween-20), block with 150 μL / well of 10 mM PBS containing 1% BSA for 2 h at 37°C, wash the plate three times with PBS (containing 0.05% (v / v) Tween-20), add 100 μL of the purified antibody to each well, incubate at 37°C for 1 h, wash the plate three times with PBS (containing 0.05% (v / v) Tween-20), add horseradish peroxidase-labeled goat anti-mouse IgG polyclonal antibody as the secondary antibody, incubate at 37°C for 30 min, wash the plate three times with PBS (containing 0.05% (v / v) Tween-20), add 100 μl to each well, develop color with TMB, incubate at 37°C for 15 min, and then add 2M H 2 SO 4 solution to terminate the reaction, and detect with an enzyme-linked immunosorbent assay (ELISA) reader at an absorbance value of 450 nm. Multiple results show that the titer of the antibody is above 1×10 6 .

[0089] (4) Affinity test:

[0090] Dilute the recombinant expressed N antigen protein of the novel coronavirus with 1×CB to 0.5 μg / ml and 1 μg / ml respectively, add 100 μl per well to the wells of the enzyme-linked immunosorbent assay (ELISA) plate, and make duplicate wells. Incubate overnight at 4°C or adsorb for 2 hours at 37°C. Spin-dry the coated microplate, wash it once according to the operating procedure (AFP procedure) set by the plate washer, add the blocking solution at 200 μl per well, place it in an incubator at 37°C for 2 hours, and then place it overnight at 4°C. Before use, take out the blocked microplate from 4°C, spin-dry it, and add the washing solution (1×PBS-T) to moisten the ELISA plate. Pre-dilute the monoclonal antibody H2B4 of the present invention to 30 μg / ml with 1×PBS, and record the pre-dilution multiple m. Dilute it 10 times to 3 μg / ml as the (S1) highest concentration, and then perform a 1:3 gradient dilution (dilute in a 96-deep well plate), with a total of 8 dilution gradients (S1 - S8).

[0091] Add 100 μl of the diluted antibody to the 96-well microplate patted dry on the absorbent paper, and incubate at 37°C for 30 min;

[0092] After incubation, spin-dry the ELISA plate, pat it dry on the absorbent paper, wash the ELISA plate 3 times with the plate washer. Add 100 μl of 1×PBS to each well in columns 1 - 4; add 200 μl of the urea treatment solution to each well in columns 5 and 6, and incubate at 37°C for 30 min. After incubation, spin-dry the ELISA plate, pat it dry on the absorbent paper, wash the ELISA plate 3 times with the plate washer, and add 100 μl of the GAM-HRP enzyme-labeled secondary antibody diluted 10,000 times with the secondary antibody dilution solution to each well, and incubate at 37°C for 30 min. After incubation, spin-dry the ELISA plate, pat it dry on the absorbent paper, wash the ELISA plate 3 times with the plate washer, take the TMB chromogenic solution, add 100 μl of the chromogenic solution to each well, and incubate at 37°C for 5 - 10 min. After color development, add 50 μl of the stop solution to each well. Set the ELISA reader to read at 450 nm / 630 nm. According to the measured values, calculate the concentration value K of the H2B4 antibody when the concentration of the antigen-antibody complex accounts for half of the total concentration at the two antigen concentrations of 0.5 μg / ml and 1 μg / ml 0.5 and K 1 , and calculate the affinity constant of the antibody H2B4 as: 1.35×10 10 .

[0093] Table 3 Affinity Results

[0094]

[0095] (5) Specificity Detection

[0096] The specificity of the monoclonal antibody H2B4 was detected by Western blot. The antigens used were the NP recombinant protein of influenza virus A, the NP recombinant protein of influenza virus B, the F protein of RSV virus, and the N protein of novel coronavirus. After the above proteins were diluted with loading buffer, they were transferred to a PVDF membrane using a Bio-Rad electrotransfer device after SDS-PAGE. The membrane was blocked with 5% skim milk for 1 h, washed three times with Tris-HCl buffer (pH 7.4) containing 0.1% (V / V) Tween-20 for 5 min each time. The purified anti-SARS-CoV-2 monoclonal antibody H2B4 was added at a ratio of 1:1000 and incubated overnight at 4°C. The membrane was washed three times with Tris-HCl buffer (pH 7.4) containing 0.1% (V / V) Tween-20 for 5 min each time. A goat anti-mouse IgG polyclonal antibody (Sigma) diluted 1:10000 was added as the secondary antibody and incubated at room temperature for 2 h. The membrane was washed three times with TBST, excess solution was blotted off with filter paper, laid flat on a clean piece of plastic wrap, 1.4 ml of Pierce-Thermo Scientific ECL Western Chemiluminescent Substrate Reagent (A:B = 1:1) was added to completely immerse the membrane in the reagent, quickly removed, excess liquid was blotted off with filter paper, laid on another piece of plastic wrap, wrapped with plastic wrap, placed in an X-ray film cassette, and developed in the darkroom. The anti-SARS-CoV-2 monoclonal antibody H2B4 reacted specifically with the N protein of novel coronavirus, showing a single specific band. The results showed that the detection results of the other antigens were negative except for the N protein of novel coronavirus. The results are as Figure 2 shown.

[0097] Example 3: Sequencing of monoclonal antibody H2B4

[0098] A total of 5 mg of the purified anti-SARS-CoV-2 N protein monoclonal antibody H2B4 was taken, and the light chain variable region and heavy chain variable region of the antibody were sequenced by Aibosen Biotechnology Co., Ltd. The specific sequences are shown in Table 4.

[0099] Table 4 Sequence information of the antibody

[0100]

[0101]

[0102] Example 4: Preparation of lateral flow immunoassay reagent

[0103] It should be noted that in other embodiments, the antibodies provided by the present invention can also be used for the preparation of immunoassay reagents such as enzyme-linked immunosorbent assay, chemiluminescence, and immunofluorescence detection. In this example, the preparation of a lateral flow chromatography reagent is taken as an example:

[0104] (1) Preparation of colloidal gold:

[0105] It is prepared by the reduction method, and the gold preparation conditions are 100 ml of chloroauric acid at 1 / 10,000 + 1.8 ml of 1% trisodium citrate.

[0106] (2) Optimization experiment of antibody pairing: The selected antibodies H2B4, F7A1, G4D8, E9C2, D7F1, F5H2, E7G6, C8A6, and C2F3 were respectively enzyme-labeled. In addition, the recombinant novel coronavirus N antigen was diluted to four concentrations of 0.1 μg / mL, 0.2 μg / mL, 0.5 μg / mL, and 1 μg / mL with 1×CB, and each sample was detected in duplicate wells, and 100 μL of the sample was added to each well. The results are shown in the following table.

[0107]

[0108] According to the above results, the antibody D7F1 with the best linear relationship was selected as the labeled monoclonal antibody for use. The sequence of monoclonal antibody D7F1 is as follows.

[0109] Light chain (the underlined part is the light chain variable region, and the bold part corresponds to CDR1 - CDR3 of the light chain):

[0110] CDR-L1 EASENIYSNLA CDR-L2 TATRLAD CDR-L3 QEFWGTPYT

[0111]

[0112] Heavy chain (the underlined part is the light chain variable region, and the bold part corresponds to CDR1 - CDR3 of the heavy chain):

[0113]

[0114]

[0115]

[0116] (3) Colloidal gold-labeled mouse anti-novel coronavirus N protein monoclonal antibody

[0117] By the physical adsorption method, the colloidal gold is combined with the antibody by adjusting the pH value. The specific labeling conditions are: under the condition of pH 8.3, 20 μL of 0.1 M potassium carbonate solution is added to each milliliter of colloidal gold, and the labeling concentration of mouse anti-novel coronavirus N protein monoclonal antibody (D7F1) is 16 μg / mL.

[0118] (4) Test line

[0119] Coat the mouse anti - SARS - CoV - 2 N protein monoclonal antibody H2B4 at an appropriate concentration of 1.0 mg / mL (in other embodiments, 0.1 - 2.8 mg / ml can be selected) on the nitrocellulose membrane to prepare the test line. Dry at 37°C. The spraying amount is 0.5 μL / mm (in other embodiments, 0.3 - 1.5 μL / mm can be selected).

[0120] d. Control line

[0121] Take goat anti - mouse IgG polyclonal antibody at 1.2 mg / mL (in other embodiments, selected from 0.5 - 2.5 mg / mL), and prepare the control line on the fiber membrane. Dry at 37°C. The spraying amount is 0.6 μL / mm (in other embodiments, 0.1 - 1.5 μL / mm can be selected).

[0122] (5) Test method

[0123] Restore the test card (test strip), sample diluent and sample to 18 - 30°C. The test method for the test card or test strip is as follows:

[0124] a. Take out the test card or test strip from the aluminum foil bag, mark the sample, and place it flat on the horizontal workbench;

[0125] b. Take 20 μL of the nasopharyngeal or oropharyngeal swab sample extract and directly add it to the sample addition hole (for the test card) or the sample addition place at the lower end of the indicating arrow (for the test strip);

[0126] c. Then add 100 μL (2 - 3 drops) of the sample diluent;

[0127] d. Interpret the result within 15 - 20 minutes. The test result is invalid after 20 minutes.

[0128] (6) Interpretation of test results

[0129] a. Positive test line: The test line and the control line are colored. It indicates that the sample detects the antigen of SARS - CoV - 2 (2019 - nCoV), and it may be in the early stage of infection or current infection. Final confirmation is required in combination with clinical symptoms.

[0130] b. Negative: Only one red control line appears in the test window. It means that the sample does not detect the antigen of SARS - CoV - 2 (2019 - nCoV).

[0131] c. Invalid: No red control line appears in the test window.

[0132] Example 5: Performance test of the COVID - 19 antigen lateral flow immunoassay reagent

[0133] 1) Sensitivity test

[0134] a. Inactivated COVID - 19 culture test

[0135] When the lateral flow immunoassay reagent prepared with the antibody of the embodiment of the present invention is used to detect the inactivated COVID-19 culture, the virus culture as low as 10 TCID50 / mL can be detected at the lowest. The experimental results are as follows Figure 3 as shown

[0136] b. Standardized recombinant COVID-19 N protein test

[0137] The lateral flow immunoassay rapid test reagent developed with the antibody described in Example 1 was detected using the reference material of the nucleocapsid protein solution of the novel coronavirus prepared by the National Institute of Metrology, China (Reference Material No.: GBW(E)091097). As Figure 4 shown, the standard recombinant COVID-19 N protein as low as 10 pg / mL can be detected

[0138] 2) Specificity test

[0139] a. Recombinant COVID-19 variant strain N protein test

[0140] A series of recombinant COVID-19 variant strain N proteins were used to detect the detection ability of the lateral flow immunoassay rapid test reagent developed with the antibody described in this patent for the recombinant COVID-19 variant strain N protein. The results showed that the detection reagent could detect the recombinant N proteins of different mutant strains, and the mutant strains were the novel coronavirus beta strain, the novel coronavirus delta strain, the novel coronavirus Gamma strain, and the novel coronavirus Omicron strain respectively. The specific results are as Figure 5 shown

[0141] Example 6: Clinical sample test of the lateral flow immunoassay reagent

[0142] The nasal swab samples of 100 normal people and the nasal swab samples of COVID-19 infected people were detected using the lateral flow immunoassay reagent provided in the embodiment of the present invention. The detection results are as Figure 6 shown (only some results are shown for the sake of space). It can be seen from the figure that the detection results are clearly visible and the background is clean, indicating that the product has good specificity and can be used for the rapid detection of clinical samples

[0143] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention

Claims

1. An antibody or its antigen-binding fragment that specifically targets the N protein of the novel coronavirus, wherein, the antibody or its antigen-binding fragment comprises a heavy-chain variable region and a light-chain variable region; the light-chain variable region comprises LCDR1, LCDR2, and LCDR3 with amino acid sequences shown in SEQ ID No. 1 to 3 in sequence; the heavy-chain variable region comprises HCDR1, HCDR2, and HCDR3 with amino acid sequences shown in SEQ ID No. 4 to 6 in sequence.

2. The antibody or its antigen-binding fragment according to claim 1, wherein, the amino acid sequence specifically targeted and bound by the antibody or its antigen-binding fragment is shown in SEQ ID No.

10.

3. The antibody or its antigen-binding fragment according to claim 1, wherein, the antibody is selected from: monoclonal antibodies.

4. The antibody or its antigen-binding fragment according to claim 1, wherein, the antibody is selected from any one of murine antibodies and chimeric antibodies.

5. The antibody or its antigen-binding fragment according to claim 1, wherein, The antigen-binding fragment is selected from any one of F(ab’) 2 , Fab’, Fab, Fv, and scFv of an antibody.

6. The antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein, the antibody or its antigen-binding fragment further comprises a constant region.

7. The antibody or its antigen-binding fragment according to claim 6, wherein, the constant region comprises a heavy-chain constant region and a light-chain constant region, the heavy-chain constant region is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM; the light-chain constant region is a κ chain or a λ chain.

8. The antibody or its antigen-binding fragment according to claim 7, wherein, the heavy-chain constant region is IgG1; the light-chain constant region is a κ chain.

9. The antibody or its antigen-binding fragment according to claim 1, wherein, the antibody or its antigen-binding fragment comprises a heavy chain and a light chain, the amino acid sequence of the light chain is shown in SEQ ID No. 7, and the amino acid sequence of the heavy chain is shown in SEQ ID No.

8.

10. A nucleic acid encoding the antibody or its antigen-binding fragment according to any one of claims 1 to 9.

11. An antibody conjugate, wherein, it comprises: the antibody or its antigen-binding fragment according to any one of claims 1 to 9 and a solid-phase carrier and / or a detectable label conjugated to the antibody or its antigen-binding fragment.

12. A reagent or kit for detecting the novel coronavirus, wherein, it comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 9.

13. The reagent or kit according to claim 12, wherein, the reagent, kit, or composition further comprises a reagent for an immunoassay method.

14. The reagent or kit for detecting the novel coronavirus according to claim 13, wherein, the immunoassay method comprises any one of enzyme-linked immunosorbent assay, chemiluminescence detection, lateral flow immunoassay, and immunofluorescence detection.

15. Use of the antibody or its antigen-binding fragment according to any one of claims 1 to 9 in the preparation of a product for detecting novel coronavirus.

16. According to the use described in claim 15, wherein, the method for detecting novel coronavirus includes any one of enzyme-linked immunosorbent assay, chemiluminescence detection, lateral flow immunoassay, and immunofluorescence detection.

17. According to the use described in claim 15, wherein, the product is selected from any one of reagents, test strips, and reagent kits.

18. A lateral flow immunoassay reagent or reagent kit, wherein, it comprises the antibody or its antigen-binding fragment according to any one of claims 1 to 9.

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