Monoclonal antibodies against novel coronavirus nucleoprotein and their applications

Abstract

The present invention discloses a monoclonal antibody against a novel coronavirus nucleoprotein and its application. In the monoclonal antibody, the amino acid sequence of the light chain CDR1 is shown in SEQ ID NO.2, the amino acid sequence of the light chain CDR2 is RTS, and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO.3; the amino acid sequences of the heavy chain CDR1, CDR2, and CDR3 are shown in SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7, respectively. The above-mentioned monoclonal antibody against the novel coronavirus nucleoprotein can cover multiple novel coronavirus variants, and has good specificity and affinity, and can be used to prepare products for detecting novel coronavirus products and preparing products for treating novel coronavirus patients.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a monoclonal antibody against the novel coronavirus nucleoprotein and its application. Background Art

[0002] After the novel coronavirus infects the body, the first thing that can be detected is viral nucleic acid, followed by viral proteins synthesized by the virus, namely antigens, and finally the body produces specific antibodies against the various antigenic components of the virus. Therefore, laboratory testing for novel coronavirus infection includes nucleic acid testing for viral genes, antigen testing for viral proteins, and antibody testing for specific antibodies. Nucleic acid testing and antigen testing are both pathogenic tests and provide direct evidence of infection. Compared with antibody testing, antigen testing can achieve early diagnosis 2-3 weeks earlier.

[0003] Currently, the novel coronavirus antigen detection is mainly based on the double-antibody sandwich principle, which requires high-affinity and high-specificity monoclonal antibodies. However, due to the rapid mutation of the novel coronavirus, approximately 4,000 novel coronavirus variants have been discovered worldwide, of which five variants are more harmful to humans and have higher infectivity, namely Alpha, Beta, Gamma, Delta, and Omicron. If the monoclonal antibodies used cannot recognize all strains, it will result in missed detection, posing a great risk to epidemic prevention and control. Therefore, the technology of screening and preparing monoclonal antibodies that target conserved epitopes between different strains and have high affinity and high specificity is extremely difficult and is the key to the development of novel coronavirus antigen detection reagents.

[0004] Furthermore, targeted disease therapy involves the process by which therapeutic drugs, after entering the body, specifically bind to target organs, cells, or pathogens through specific targeting mechanisms, allowing the drugs to exert their effects more directly and achieve the desired therapeutic effect. Targeted drug delivery can significantly increase drug concentration at the lesion site, improving efficacy while minimizing toxic side effects. Targeted therapies primarily include biological targeting and physicochemical targeting. Biological targeted therapy involves conjugating targeting molecules to drugs, allowing them to be administered to specific targets. In recent years, the use of monoclonal antibodies with high affinity and specificity conjugated to therapeutic drugs has become a hot topic and a major area of biological targeted drug therapy, demonstrating promising therapeutic effects. Monoclonal antibodies with high affinity and specificity that cover all viral strains are crucial to the success of this type of biological targeted therapy. Therefore, obtaining monoclonal antibodies with high coverage, affinity, and specificity is crucial for biological targeted therapy.

[0005] At present, research on the treatment of novel coronavirus infection mainly focuses on oral drug therapy and neutralizing antibody therapy. There is still a lack of research on biological targeted drug therapy using high-coverage, high-affinity and specific monoclonal antibodies targeting novel coronavirus pathogens. Summary of the Invention

[0006] To this end, the purpose of the present invention is to provide a monoclonal antibody against the nucleoprotein of the new coronavirus prepared using a fused hybridoma cell line. The monoclonal antibody obtained through the experiment can recognize five major new coronavirus strains and has good specificity and affinity. It can be used to detect the new coronavirus or its nucleoprotein or to prepare products for treating new coronavirus infection.

[0007] Therefore, one aspect of the present invention relates to a monoclonal antibody or an antigen-binding fragment thereof against the novel coronavirus nucleoprotein, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises CDR1, CDR2 and CDR3, and the heavy chain variable region comprises CDR1, CDR2 and CDR3, wherein:

[0008] The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO. 2, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO. 2, or an amino acid sequence comprising the above sequence;

[0009] The amino acid sequence of the light chain CDR2 is RTS or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence RTS, or an amino acid sequence comprising the above sequence;

[0010] The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO. 3, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO. 3, or an amino acid sequence comprising the above sequence;

[0011] The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO. 5, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO. 5, or an amino acid sequence comprising the above sequence;

[0012] The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO. 6, or an amino acid sequence having one or two conservative amino acid substitutions compared to the sequence shown in SEQ ID NO. 6, or an amino acid sequence comprising the above sequence;

[0013] The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.7 or an amino acid sequence having one or two conservative amino acid substitutions compared with the sequence shown in SEQ ID NO.7, or an amino acid sequence comprising the above sequence.

[0014] In a further aspect, the present invention also relates to a monoclonal antibody or an antigen-binding fragment thereof, wherein the light chain variable region sequence is the sequence shown in SEQ ID NO. 1, and the heavy chain amino acid sequence is the sequence shown in SEQ ID NO. 4. In a further aspect, the present invention also relates to a monoclonal antibody secreted by the mouse hybridoma cell line TCE1103, deposited with CGMCC No. 45247.

[0015] The present invention also relates to the above-mentioned monoclonal antibody or its antigen-binding fragment, which is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody. These antibodies or antigen-binding fragments retain the variable regions of the light chain and the heavy chain and are therefore able to recognize and bind to the novel coronavirus nucleoprotein.

[0016] The present invention also relates to a nucleic acid molecule comprising a nucleic acid encoding the aforementioned antibody or antigen-binding fragment thereof, and an expression vector comprising the aforementioned nucleic acid molecule, wherein the expression vector is capable of expressing the aforementioned antibody or antigen-binding fragment thereof. The present invention also relates to a recombinant comprising the aforementioned nucleic acid molecule or expression vector, wherein the recombinant is capable of producing the aforementioned antibody or antigen-binding fragment thereof.

[0017] On the other hand, the present invention relates to a monoclonal antibody hybridoma cell line against the new coronavirus nucleoprotein, wherein the monoclonal antibody hybridoma cell line secretes the above-mentioned monoclonal antibody. Further, the present invention relates to a monoclonal antibody hybridoma cell line against the new coronavirus nucleoprotein, wherein the monoclonal antibody hybridoma cell line is a mouse hybridoma cell line TCE1103, with a deposit number of CGMCC No. 45247.

[0018] In another aspect, the present invention relates to the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in the preparation of a product for detecting the novel coronavirus or treating novel coronavirus infection. Furthermore, the present invention relates to a kit for detecting the novel coronavirus, the kit comprising the above-mentioned monoclonal antibody or its antigen-binding fragment for recognizing and binding to the novel coronavirus nucleoprotein.

[0019] Description of biological material deposit

[0020] The monoclonal antibody hybridoma cell line of the present invention, mouse hybridoma cell line TCE1103, was deposited with the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) under the registration number CGMCC No. 45247 and the deposit date of August 17, 2022. The address of the General Microbiology Center of the China Culture Collection Administration of Microorganisms is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is an SDS-PAGE electrophoresis diagram showing the prokaryotic expression of the new coronavirus nucleoprotein, where the labels are: M is Marker; 1 is the whole bacterial lysate; 2 is the bacterial ultrasonic supernatant; 3 is the purified NP fusion protein.

[0022] Figure 2 This is a graph showing the subclass identification results of monoclonal antibodies against the new coronavirus nucleoprotein. The antibody subtype was identified as IgG1.

[0023] Figure 3 This is a schematic diagram showing the results of the colloidal gold immunochromatographic assay for detecting nucleoprotein using anti-novel coronavirus nucleoprotein monoclonal antibodies. A positive result is indicated by the appearance of two purple-red bands. One band is located in the test area (T line) and the other is located in the quality control area (C line). The color of the band in the test area (T line) can be dark or light, indicating a positive result. A negative result is indicated by the appearance of only one purple-red band in the quality control area (C line) and no band in the test area (T line). An invalid result is indicated by the absence of a purple-red band in the quality control area (C line). Regardless of whether a band appears in the test area (T line), the result is invalid. DETAILED DESCRIPTION

[0024] The purpose of the present invention is to provide an anti-novel coronavirus nucleoprotein monoclonal antibody prepared by using a fused hybridoma cell line, which can recognize the nucleoproteins of five major pathogenic virus strains.

[0025] Monoclonal antibodies were prepared by immunizing mice with a synthetic peptide (conjugated with KLH) that is a conserved epitope among the nucleoproteins of five major pathogenic virus strains. Eukaryotic expression of the novel coronavirus nucleoprotein was used to screen for monoclonal antibody hybridoma cell lines. A mouse hybridoma cell line expressing a monoclonal antibody with high coverage, high affinity, and high specificity was obtained and named TCE1103. This cell line was deposited with the General Microbiology Center of the China General Microbiology Culture Collection (CGMCC) under the accession number CGMCC No. 45247 on August 17, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China.

[0026] The inventors sequenced the monoclonal antibody produced by the mouse hybridoma cell line. The amino acid sequence of the light chain variable region of the monoclonal antibody is 111 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISCRAS ESVDNYG NSF MLWYQQKPGQPPKLLIY RTS NLDSGVP ARFSGSGSRTDFTLTINPVEAEDVATYYC QQSYDDPYT FGGGTKLEIK (SEQ ID NO. 1), where the underlined sequences are CDR1, CDR2, and CDR3, respectively. CDR1 is located at 27-36aa, with an amino acid sequence of ESVDNYGNSF (SEQ ID NO. 2); CDR2 is located at 54-56aa, with an amino acid sequence of RTS; and CDR3 is located at 93-101aa, with an amino acid sequence of QQSYDDPYT (SEQ ID NO. 3). The amino acid sequence of the heavy chain variable region is 110 amino acids, and its sequence is as follows: VQLQQSGPELVKPGASVKISCKTS GYTFTEYT MHWVKQSHGKSLEWIGG VNPKNGGT SYNQKFKGKATLTVDKSSSTAYMELRRSLTSEDSAVYYC TRWDY WGQGTTLTVS (SEQ ID NO. 4), where the underlined sequences are CDR1, CDR2, and CDR3, respectively. CDR1 is located at 25-32 aa, with an amino acid sequence of GYTFTEYT (SEQ ID NO. 5); CDR2 is located at 50-57 aa, with an amino acid sequence of VNPKNGGT (SEQ ID NO. 6); and CDR3 is located at 96-100 aa, with an amino acid sequence of TRWDY (SEQ ID NO. 7). The above monoclonal antibody has been determined to have high coverage, high affinity, and specificity for the 2019-nCoV nucleoprotein.

[0027] It is well known that the heavy and light chain CDR regions of antibodies are important sequences for recognizing and binding to corresponding antigens, and that one or two conservative amino acid substitutions in the amino acid sequence generally do not alter or only slightly alter the properties of the protein. Therefore, monoclonal antibodies or antigen-binding fragments thereof obtained by making one or two conservative amino acid substitutions in the light chain CDR1 and / or light chain CDR2 and / or light chain CDR3 and / or heavy chain CDR1 and / or heavy chain CDR2 and / or heavy chain CDR3 can still recognize and bind to the 2019-nCoV nucleoprotein. A conservative amino acid substitution refers to the replacement of an amino acid in a protein with another chemically similar amino acid. Examples include substitutions between aromatic amino acids Phe, Trp, and Tyr; substitutions between aliphatic amino acids Ala, Gly, Leu, Ile, and Val; substitutions between polar amino acids Gln and Asn; substitutions between basic amino acids Lys, Arg, and His; substitutions between acidic amino acids Asp and Glu; and substitutions between hydroxy amino acids Ser and Thr.

[0028] Furthermore, it is well known in the art that, in antibodies or antigen-binding fragments thereof, the framework region is located outside the light or heavy chain CDRs. Adding a small number of amino acids, such as one or two amino acids, between the CDR sequence and the framework region sequence has minimal effect on the three-dimensional structure of the antibody or antigen-binding fragment thereof, thereby still allowing recognition and binding to the corresponding antigen. Therefore, the light chain CDR1 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be a sequence comprising the aforementioned sequence, in addition to the sequence shown in SEQ ID NO. 2 or a sequence having one or two conservative amino acid substitutions therefrom. Similarly, the light chain CDR2 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be, in addition to being RTS or a sequence having one or two conservative amino acid substitutions compared to the sequence RTS, a sequence comprising the above sequence; the light chain CDR3 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be, in addition to being SEQ ID NO. 3 or a sequence having one or two conservative amino acid substitutions compared thereto, a sequence comprising the above sequence; the heavy chain CDR1 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be, in addition to being SEQ ID NO. 5 or a sequence having one or two conservative amino acid substitutions compared thereto, a sequence comprising the above sequence; the heavy chain CDR2 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be, in addition to being SEQ ID NO. 6 or a sequence having one or two conservative amino acid substitutions compared thereto, a sequence comprising the above sequence; the heavy chain CDR3 sequence of the monoclonal antibody or antigen-binding fragment thereof of the present invention may be, in addition to being SEQ ID NO. 7 or a sequence having one or two conservative amino acid substitutions compared thereto, a sequence comprising the above sequence.

[0029] Various antibody fragments capable of binding to the novel coronavirus nucleoprotein, i.e., antigen-binding fragments, such as but not limited to Fab, Fab', and F(ab')2, can also be prepared from the monoclonal antibodies of the present invention using existing techniques in the art. The Fab fragment is the region in the antibody structure that can bind to the antigen, consisting of a complete light chain and the variable region VH and constant region CH1 domain (Fd segment) of the heavy chain. Both the light chain and the heavy chain have a constant region and a variable region, and there is a disulfide bond link between the light and heavy chains. The antigen-binding fragment can be prepared as follows, for example, after enzymatic hydrolysis using papain, the antibody IgG is degraded into two Fab fragments and an Fc fragment (crystalline fragment). Under the action of pepsin, the antibody IgG is degraded into an F(ab')2 fragment and a pFc' fragment, and the F(ab')2 fragment is further reduced to form two Fab' fragments. The above-mentioned antigen-binding fragments can be used to prepare products for detecting the novel coronavirus or its nucleoprotein and therapeutic products for treating novel coronavirus patients.

[0030] Single-chain antibodies (scFv) can also be prepared from the monoclonal antibodies of the present invention using existing technologies in the art. A single-chain antibody is an antibody formed by connecting the heavy chain variable region and the light chain variable region of an antibody through a short peptide linker of several amino acids. It has only one chain and is an artificially synthesized antibody. The length and amino acid composition of the short peptide linker are well known in the art, and the short peptide linker that can be used for the monoclonal antibody of the present invention can be determined by simple repeated experiments. Single-chain antibodies can be expressed in, for example, Escherichia coli by genetic engineering technology. Single-chain antibodies have the advantages of small molecular weight, strong penetrability and weak antigenicity, and can be applied to the detection of the new coronavirus or its nucleoprotein, the diagnosis and treatment of new coronavirus patients.

[0031] The light chain constant region and heavy chain constant region of the monoclonal antibody of the present invention can be replaced with the amino acid sequence of an adult antibody by existing technologies in the art, thereby humanizing the mouse monoclonal antibody of the present invention to obtain a humanized antibody, so as to be used for antibody treatment of human novel coronavirus patients to reduce the immune side effects of mouse antibodies on the human body. Therefore, the humanized monoclonal antibody of the present invention can be applied to the detection of novel coronavirus or its nucleoprotein, the diagnosis and treatment of novel coronavirus patients.

[0032] Those skilled in the art can design and synthesize nucleic acid molecules encoding the above-mentioned anti-novel coronavirus nucleoprotein monoclonal antibody variable region amino acid sequence, and can also insert the synthesized nucleic acid molecules into nucleic acid vectors to construct expression vectors, which can express monoclonal antibodies or antigen-binding fragments thereof against novel coronavirus nucleoprotein. Those skilled in the art can also introduce the synthesized nucleic acid molecules or constructed expression vectors into organisms such as cells, bacteria, yeast, etc. to obtain recombinants, and express and produce antibodies or antigen-binding fragments thereof of the present invention via the above-mentioned recombinant, so that the antibodies or antigen-binding fragments thereof expressed can bind to and recognize novel coronavirus nucleoprotein, so the above-mentioned nucleic acid molecules, expression vectors and recombinants are within the scope of the claims of the present invention. And the above-mentioned technologies are all well-known technologies in the art, and those skilled in the art can carry out without creative work.

[0033] As mentioned above, the antibody of the present invention or its antigen-binding fragment can identify and bind to the different strains of novel coronavirus nucleoprotein, therefore can be used for preparing a test kit for detecting novel coronavirus or its nucleoprotein, the test kit can be any test kit utilizing the binding reaction of the antibody of the present invention or its antigen-binding fragment with the novel coronavirus nucleoprotein, such as but not limited to colloidal gold immunochromatography, fluorescent immunochromatography, enzyme-linked immunosorbent assay, chemiluminescence, immunohistochemical type test kit. For the test kit using colloidal gold immunochromatography to detect novel coronavirus or novel coronavirus nuclear antigen, the affinity and specificity of the colloidal gold labeled antibody are crucial for the detection method, which determines the sensitivity and specific performance of the test kit. Due to the anti-novel coronavirus nucleoprotein monoclonal antibody of the present invention having high coverage, high affinity and specificity, it is suitable as colloidal gold labeled antibody, the anti-novel coronavirus nucleoprotein polyclonal antibody of the present invention or monoclonal or polyclonal antibodies to any other anti-novel coronavirus nucleoprotein for other epitope segments can be used as detection line (T line) coated antibody. As quality control, the antibody pairs conventionally used in this area can be used, for example, colloidal gold labeled chicken IgY antibodies can be used, and the quality control line (C line) is coated with sheep anti-chicken IgY antibodies.

[0034] Because the monoclonal antibodies or antigen-binding fragments thereof of the present invention can specifically bind to the 2019-nCoV nucleoprotein, the antibodies or antigen-binding fragments thereof or humanized antibodies can be used to prepare products, such as drugs, for treating 2019-nCoV infection. Treatment can be, for example, but not limited to, targeted therapy, in which the monoclonal antibodies or antigen-binding fragments thereof or humanized antibodies of the present invention are conjugated to drugs for treating 2019-nCoV, and the conjugated drugs are targeted to 2019-nCoV by the antibodies or antigen-binding fragments thereof or humanized antibodies of the present invention, thereby improving the therapeutic effect.

[0035] In order to explain the technical content, achieved objectives and effects of the technical solution in detail, the following is an explanation in conjunction with specific embodiments.

[0036] Example 1: Screening and synthesis of novel coronavirus nucleoprotein dominant epitope peptides

[0037] According to the full-length protein sequences of the nucleoproteins of different novel coronavirus strains published in Genebank, multiple sequence alignments were performed in BLAST to determine the conserved amino acid segments between different strains. Then, Goldkey bioinformatics software was used for epitope analysis and prediction, and the dominant epitope segments were screened. The final screening determined that the highest score was the dominant epitope segment that was highly conserved among the main variant strains found so far, which was the amino acid sequence YKTFPPTEPKKDKKKKADETQ (SEQ ID NO.8) at positions 360 to 380, with a maximum score of 5.14558. The dominant epitope peptide of the novel coronavirus nucleoprotein was used for subsequent immunization of mice to screen hybridomas and prepare monoclonal antibodies. The synthesis of the dominant epitope peptide and KLH coupling were entrusted to Shanghai Dechi Biotechnology Co., Ltd.

[0038] Example 2: Prokaryotic expression and purification of the full-length 2019-nCoV nucleoprotein

[0039] Based on the full-length gene sequence of the original strain of the novel coronavirus nucleoprotein (Nuclear Protein) published in GeneBank, Beijing Qingke Biotechnology Co., Ltd. synthesized the full-length gene and ligated it into the pET-32a plasmid to generate the recombinant plasmid pET-32a-NP. The recombinant plasmid was transformed into BL21 competent cells, and a single colony was picked and incubated in 2 mL of LB liquid medium containing ampicillin sodium and shaken at 37°C overnight. The next day, the colony was inoculated into 200 mL of fresh LB liquid medium and incubated at 37°C and 160 rpm for 4 hours until the logarithmic growth phase. 120 μl of 1 mol / L IPTG induction solution was added and induced at 15°C for 12-14 hours. The induced cells were collected by centrifugation at 6000 rpm for 10 minutes at 4°C, resuspended in 25 mmol / L Tris-HCl (pH 8.5), sonicated on ice, and centrifuged at 12000 rpm for 10 minutes at 4°C. The supernatant was collected for later use. SDS-PAGE gel electrophoresis revealed that the nucleoprotein was primarily expressed in a soluble form in the supernatant. The antigen expressed by the pET-32a-NP recombinant plasmid was purified by Ni column, and the protein peaks were collected respectively. The purified products were analyzed by SDS-PAGE electrophoresis. The molecular weight of the NP fusion protein was about 63.8kDa, and the purity met the requirements of the immunogen purity. The results are as follows Figure 1 shown.

[0040] Example 3: Preparation of polyclonal antibodies against novel coronavirus nucleoprotein

[0041] Healthy male white rabbits were selected. 1 mg of NP fusion protein was mixed with 1 ml of complete Freund's adjuvant and thoroughly emulsified using a blender. The mixture was then injected subcutaneously at multiple sites along the spine, with at least 0.1 ml injected at each site. Four weeks later, 1 mg of protein was mixed with 1 ml of incomplete Freund's adjuvant and thoroughly emulsified using a blender for a second immunization at different sites. Four weeks later, a third booster immunization was performed to prepare polyclonal antibody serum. One week later, blood was collected from the heart. After clot formation and clot retraction, the blood was centrifuged at 5000 rpm for 15 minutes. The serum was aliquoted and stored at -20°C until further use. Polyclonal antibodies were purified according to the Montage antibody purification Prosp-G kit instructions, adjusting the antibody concentration to 1.0 mg / mL using antibody diluent.

[0042] The titer of the purified rabbit anti-novel coronavirus nucleoprotein polyclonal antibody was detected by indirect ELISA. The eukaryotic cell-expressed novel coronavirus N protein (product number DFHY-Ag-1301) purchased from Oriental Ocean (Beijing) Medical Research Institute Co., Ltd. was diluted to a concentration of 1.0 μg / mL with carbonate coating buffer, 100 μl was coated per well, and incubated at 4°C overnight; the plate was washed twice with washing solution, 200 μl per well; 100 μl of blocking solution was added to each well and blocked at room temperature for 6 hours; the plate was washed 5 times with washing solution, 200 μl per well; the rabbit anti-novel coronavirus nucleoprotein polyclonal antibody at a concentration of 1.0 mg / mL was diluted with antibody diluent at a dilution ratio of 1:2. 000, 1:8000, 1:32000, 1:128000, 1:512000, 1:2048000 and 1:8192000, add 100 μl to each well and set up a blank well (100 μl antibody diluent); incubate at 37°C for 30 min; wash the plate 5 times with washing solution, 200 μl per well; incubate HRP-labeled goat anti-rabbit secondary antibody at 37°C for 20 min; wash the plate 5 times with washing solution, 200 μl per well; add freshly prepared substrate solution, 100 μl per well, incubate at 37°C for 10 minutes; add 50 μl 2M H2SO4 to each well to terminate the reaction, measure the absorbance of each well at a wavelength of 450 nm using a microplate reader, and read the value within 10 minutes after termination. The results are shown in Table 1. The titer of the prepared rabbit anti-novel coronavirus nucleoprotein polyclonal antibody was 1:2048000 (OD value = 0.285).

[0043] Table 1 Titer determination of rabbit anti-novel coronavirus nucleoprotein polyclonal antibodies

[0044] Dilution ratio 1:2000 1:8000 1:32000 1:128000 1:512000 1:2048000 1:8192000 OD value 3.478 3.140 2.909 1.328 0.663 0.285 0.096

[0045] Example 4: Establishment of hybridoma cell lines

[0046] A KLH-coupled novel coronavirus nucleoprotein dominant epitope peptide was used as the immunogen. Eight-week-old BALB / c female mice were injected with the antigen plus an equal volume of Freund's complete adjuvant (50 μg / mouse) via the back and intraperitoneal route. A second and third immunization with the same dose was performed in the fourth and eighth weeks using Freund's incomplete adjuvant. Three days later, spleen cells were harvested for fusion. SP20 myeloma cells were resuscitated and cultured until they reached the logarithmic growth phase. Immunized BALB / c mice were ocularly removed and blood was collected to provide positive control serum. The mice were then sacrificed by cervical dislocation. The body surface was disinfected with 75% alcohol for 3-5 minutes, and the spleens were harvested for preparation of splenocyte suspensions.

[0047] The spleen cells and myeloma cells were mixed in a 5:1 ratio in serum-free DMEM medium and centrifuged at 1500 rpm for 5 minutes. The supernatant was thoroughly aspirated and the bottom of the centrifuge tube was gently shaken to disperse the cells. 1 mL of preheated 50% PEG was added to the fused cells within 45-60 seconds, gently shaking while adding. After addition, the tube was allowed to stand for 90 seconds. Serum-free DMEM medium was added to terminate the fusion (1 mL in the first minute, 2 mL in the second minute, and 8 mL in the third minute). The tube was allowed to stand at 37°C for 10 minutes and centrifuged at 1500 rpm for 5 minutes. The pellet was suspended in HAT medium and aliquoted into 96-well cell plates containing feeder cells. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 5 days. The medium was changed with HAT medium once, and on the 10th day, the medium was changed with HAT medium. When the fused cells covered 10%-50% of the bottom of the well, positive clones were screened using the same indirect ELISA method.

[0048] The eukaryotic cell-expressed novel coronavirus N protein (Cat. No. DFHY-Ag-1301) purchased from Oriental Marine (Beijing) Medical Research Institute Co., Ltd. was diluted with carbonate coating buffer to a concentration of 1.0 μg / ml. 100 μl was coated per well and incubated overnight at 4°C. The plate was washed twice with washing buffer (200 μl / well). 110 μl / well blocking buffer was added and blocked at room temperature for 6 hours. The plate was washed five times with washing buffer (200 μl / well). After adding 200 μl of sample diluent to each well, 10 μl of cell culture supernatant was added, incubated at room temperature for 30 minutes, and the solution was discarded. The plate was washed with 1× washing buffer (200 μL per well) and washed five times. The washed plate was inverted on absorbent paper and tapped vigorously to remove excess washing buffer. 100 μl / well of HRP-labeled anti-mouse IgG antibody was added and incubated at room temperature for 20 minutes. The plate was washed five times. Add 100 μl / well of freshly prepared substrate solution and incubate at room temperature in the dark for 10 minutes. Terminate the reaction by adding 50 μl / well of 2M H₂SO₄ stop solution. Measure the OD value of each well on a microplate reader at a wavelength of 450 nm, reading within 10 minutes after termination.

[0049] A total of 11 positive clones were obtained, and the clone with the highest detection value (OD450nm=3.327) was selected for subsequent experiments and named mouse hybridoma cell line TCE1103.

[0050] Example 5: Preparation of monoclonal antibodies against the novel coronavirus nucleoprotein and analysis of their subtypes

[0051] The TCE1103 hybridoma cell line was cultured in 1640 medium containing 10% fetal bovine serum. Each BALB / c male mouse was intraperitoneally injected with 0.5 mL of liquid paraffin. After 10 days, the cells were harvested, resuspended in 10 mL of saline, and injected intraperitoneally with 0.5 mL of paraffin per mouse (cell density approximately 1×10 7 After 2 weeks, ascites was collected. Antibodies were purified using the Melon Gel Monoclonal IgG Purification Kit from Thermo Fisher Scientific. The purified antibodies were aliquoted and stored at -20°C.

[0052] Antibody subtype identification was performed using the Pierce Papid Isotyping Kit-Mouse. First, the antibody was diluted to 100 ng / mL with sample diluent. Then, 150 μl of the diluted antibody was added to each well. After 5-10 minutes, the results were observed and recorded. The results showed that this monoclonal antibody was of mouse IgG1 subtype. Figure 2 shown.

[0053] Example 6: Determination of variable region sequences of monoclonal antibodies against the novel coronavirus nucleoprotein

[0054] Mouse hybridoma cell line TCE1103 was cultured, and total RNA was extracted from the hybridoma cells using the Trizol method. cDNA was reverse-transcribed using the Thermo Fisher High Capacity cDNA Rever Transcription Kit. Primers for the heavy and light chains of the antibody were designed based on the mouse monoclonal antibody primer sequences in Recombinant Antibodies (Science Press, edited by Shen Beifen, 2005). These primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. PCR amplification was performed using the following protocol: preheating at 95°C for 1 minute, 30 cycles of 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 45 seconds, followed by a final extension at 72°C for 5 minutes. The clones were ligated into the pMD18-T vector and expressed in Escherichia coli HB109. Positive clones were selected for sequencing. The determined sequences were compared with the mouse monoclonal antibody CDR region sequences using the BLAST website (https: / / www.ncbi.nlm.nih.gov / igblast / ).

[0055] After sequence analysis, it was found that the amino acid sequence of the light chain variable region is 111 amino acids, and its sequence is as follows: DIVLTQSPASLAVSLGQRATISCRAS ESVDNYGNSF MLWYQQKPGQPPKLLIY RTS NLDSGVP ARFSGSGSRTDFTLTINPVEAEDVATYYC QQSYDDPYT FGGGTKLEIK (SEQ ID NO.1), in which the underlined sequences are CDR1, CDR2 and CDR3, wherein CDR1 is located at 27-36aa, and the amino acid sequence is ESVDNYGNSF (SEQ ID NO.2); CDR2 is located at 54-56aa, and the amino acid sequence is RTS (SEQ ID NO.3); CDR3 is located at 93-101aa, and the amino acid sequence is QQSYDDPYT (SEQ ID NO.3).

[0056] The amino acid sequence of the heavy chain variable region is 110 amino acids and is as follows: VQLQQSGPELVKPGASVKISCKTS GYTFTEYT MHWVKQSHGKSLEWIGG VNPKNGGT SYN QKFKGKATLTVDKSSSTAYMELRRSLTSEDSAVYYC TRW DY WGQGTTLTVS (SEQ ID NO.4), in which the underlined sequences are CDR1, CDR2 and CDR3, wherein CDR1 is located at 25-32aa, and the amino acid sequence is GYTFTEYT (SEQ ID NO.5); CDR2 is located at 50-57aa, and the amino acid sequence is VNPKNGGT (SEQ ID NO.6); CDR3 is located at 96-100aa, and the amino acid sequence is TRWDY (SEQ ID NO.7).

[0057] Example 7: Activity determination of monoclonal antibodies against novel coronavirus nucleoprotein

[0058] The immunodetection performance of the prepared monoclonal antibody against the novel coronavirus nucleoprotein was tested by indirect enzyme-linked immunosorbent assay. In order to examine the specificity and affinity of the monoclonal antibody detection of the present invention, the novel coronavirus N protein (item number DFHY-Ag-1301), SARS virus N protein (item number DFHY-Ag-SARS), MERS virus N protein (item number DFHY-Ag-MERS), and respiratory syncytial virus protein (item number DFHY-Ag-RSV) purchased from Oriental Ocean (Beijing) Medical Research Institute Co., Ltd. were simultaneously tested during the experiment.

[0059] Each protein was diluted to a concentration of 1.0 μg / mL with carbonate coating buffer, 100 μl was coated in each well, and the plate was incubated at 4°C overnight; the plate was washed twice with washing solution, 200 μl per well; 100 μl of blocking solution was added to each well and blocked at room temperature for 6 hours; the plate was washed 5 times with washing solution, 200 μl per well; the mouse anti-novel coronavirus nucleoprotein monoclonal antibody with a concentration of 1.0 mg / mL was serially diluted with antibody diluent, and the dilution ratio was 1:2000, 1:8000, 1:32000, 1:10 ... :128000, 1:512000, 1:2048000 and 1:8192000, 100 μl of sample was added to each well and a blank well (100 μl of antibody diluent) was set; incubated at 37°C for 30 minutes; the plate was washed 5 times with washing solution, 200 μl per well; HRP-labeled goat anti-mouse secondary antibody was incubated at 37°C for 20 minutes; the plate was washed 5 times with washing solution, 200 μl per well; freshly prepared substrate solution was added, 100 μl per well, and incubated at 37°C for 10 minutes; 50 μl of 2M H2SO4 was added to each well to terminate the reaction, and the absorbance of each well was measured at a wavelength of 450 nm using a microplate reader. The results are shown in Table 2. The prepared monoclonal antibody can specifically recognize the new coronavirus nucleoprotein, and has no nonspecific reaction to the N protein of SARS virus and MERS virus, which belong to the same coronavirus, and the respiratory syncytial virus protein, indicating that the prepared monoclonal antibody has very high specificity. The titer of the prepared monoclonal antibody reached 1:2048000 (OD value = 0.251), indicating that the prepared monoclonal antibody has a very high affinity for the new coronavirus nucleoprotein.

[0060] Table 2 Titer determination of mouse anti-novel coronavirus nucleoprotein monoclonal antibodies (OD value)

[0061]

[0062] Example 8: Minimum detection line for novel coronavirus culture detection

[0063] Colloidal gold immunochromatography technology is used, using the high-affinity, high-specificity monoclonal antibody of the present invention as the labeling antibody and the polyclonal antibody of the present invention as the coating antibody for the T-line, to detect the novel coronavirus (2019-nCoV) N protein in novel coronavirus cultures. The test card contains: 1) colloidal gold-labeled mouse anti-novel coronavirus N protein monoclonal antibody and colloidal gold-labeled chicken IgY antibody; 2) a nitrocellulose membrane immobilized with a test line (T-line) and a quality control line (C-line). The T-line is immobilized with a rabbit anti-novel coronavirus N protein polyclonal antibody for coating; the C-line is immobilized with a goat anti-chicken IgY antibody for coating. The preparation process of the test card is as follows: 1) Coating buffer: PBS (pH 7.4) is used as the coating buffer; 2) Coating concentration: The coating concentration of the anti-novel coronavirus N protein polyclonal antibody is 2.0 mg / mL, and the coating concentration of the goat anti-chicken IgY antibody is 1.0 mg / mL; 3) NC membrane drying time: Dry for more than 24 hours after coating the NC membrane; 4) Optimal labeling amount: The labeling concentration of the anti-novel coronavirus N protein monoclonal antibody is 20 μg / mL, and the labeling concentration of the chicken IgY antibody is 20 μg / mL; 5) Colloidal gold label mixing ratio: The mixing ratio of the anti-novel coronavirus N protein monoclonal antibody-labeling colloidal gold label and the chicken IgY antibody colloidal gold label solution is 8:1; 6) Gold spraying amount: The gold label pad is sprayed with gold at 4 μL / cm; 7) Gold label pad drying time: 6 to 8 hours; 8) Membrane strip width: The width of the cut membrane strip should be no less than 3.0 mm.

[0064] When the sample to be tested is added to the sample hole of the test card, the sample will move forward along the test card under capillary action. If the sample contains N antigen, it will bind to the colloidal gold-labeled mouse anti-novel coronavirus N protein monoclonal antibody, and the complex will be captured by the coated rabbit anti-novel coronavirus N protein polyclonal antibody fixed on the nitrocellulose membrane, forming a purple-red T line. Regardless of whether the sample contains N antigen, a purple-red strip will form in the C line area. The result judgment criteria are as follows: Figure 3 Positive: Two purple-red bands appear, one in the test area (T line) and the other in the quality control area (C line). The color of the band in the test area (T line) can be dark or light, indicating a positive result. Negative: Only one purple-red band appears in the quality control area (C line), and no band appears in the test area (T). Invalid: No purple-red band appears in the quality control area (C line). Regardless of whether a band appears in the test area (T line), the result is invalid and a new test card must be taken for retesting.

[0065] The novel coronavirus culture was diluted to 1.9×10 4TCID50 / mL, the sample was tested using the new coronavirus 2019-nCoV nucleic acid detection kit (fluorescence PCR method) of Shengxiang Biotechnology Co., Ltd., and the test result was positive, with a Ct value (N gene) of 28.09 and a Ct value (ORF1ab) of 26.56. The sample was gradiently diluted into 8 dilution concentrations with a negative matrix according to the ratio of 1 / 2, 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, 1 / 128 and 1 / 256, and the colloidal gold immunochromatography method of the present invention was used for detection, and each sample was tested 20 times. During the detection, the test card was placed flat on the table, and 80μl of the gradient diluted virus culture was added to the sample well of the test card. Wait for the band to appear in the test card window, read the test at 15min, and the result was invalid after 20min. The results are shown in Table 3. Taking the virus level with a positive detection rate of 95% as the minimum detection limit, the low detection line of the novel coronavirus culture detected by colloidal gold immunochromatography technology using the high-affinity and high-specificity monoclonal antibodies and polyclonal antibodies of the present invention is 2.969×10 2 TCID50 / mL.

[0066] Table 3 Minimum detection limit for novel coronavirus culture detection

[0067]

[0068]

[0069] Example 9: Detection of clinical samples from patients infected with the new coronavirus

[0070] As described in Example 8, a colloidal gold immunochromatographic assay for the novel coronavirus nucleoprotein, developed using the high-coverage, high-affinity, and high-specificity monoclonal and polyclonal antibodies of the present invention, was used to test clinical samples. A total of 848 clinical trial samples were tested, of which 238 tested positive for novel coronavirus nucleic acid. These samples were collected during the acute infection period, within 0 to 7 days of symptom onset, and included the Alpha, Beta, Gamma, Delta, and Omicron variants of the novel coronavirus. Six hundred and ten tested negative for novel coronavirus nucleic acid. These samples were primarily from subjects with clinical manifestations of novel coronavirus pneumonia, such as fever or other respiratory symptoms, and who tested negative for novel coronavirus nucleic acid within 0 to 7 days of symptom onset. The test results showed that the detection technique of the present invention had a sensitivity of 98.28% with a 95% CI of [99.36%, 95.28%] and a specificity of 99.51% with a 95% CI of [98.56%, 99.83%]. In addition, 50 samples containing other common respiratory cross pathogens were tested, and the test results were all negative, indicating that common respiratory pathogens have no interference or cross-influence on the test results of the detection technology of the present invention.

[0071] In summary, the novel coronavirus nucleoprotein colloidal gold immunochromatographic detection technology established based on the high-coverage, high-affinity and specific monoclonal antibodies and polyclonal antibodies of the present invention is suitable for the early detection of novel coronavirus infection, covering a variety of common novel coronavirus strains, and has very high sensitivity and specificity.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against the novel coronavirus nucleoprotein, comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region comprises a light chain CDR1, a light chain CDR2, and a light chain CDR3, and the heavy chain variable region comprises a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3, characterized in that: The amino acid sequence of the light chain CDR1 is the sequence shown in SEQ ID NO.2; The amino acid sequence of the light chain CDR2 is RTS; The amino acid sequence of the light chain CDR3 is the sequence shown in SEQ ID NO.3; The amino acid sequence of the heavy chain CDR1 is the sequence shown in SEQ ID NO.5; The amino acid sequence of the heavy chain CDR2 is the sequence shown in SEQ ID NO.6; The amino acid sequence of the heavy chain CDR3 is the sequence shown in SEQ ID NO.

7.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The light chain variable region sequence is the sequence shown in SEQ ID NO.1, and the heavy chain variable region sequence is the sequence shown in SEQ ID NO.

4.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, characterized in that The antibody or antigen-binding fragment is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a single-chain antibody or a humanized antibody.

4. A nucleic acid molecule, characterized in that The nucleic acid comprises a nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

5. An expression vector, characterized in that The expression vector comprises the nucleic acid molecule of claim 4.

6. A recombinant, characterized in that The recombinant comprises the nucleic acid molecule according to claim 4 or the expression vector according to claim 5.

7. A monoclonal antibody hybridoma cell line against the novel coronavirus nucleoprotein, characterized in that: The monoclonal antibody hybridoma cell line is a mouse hybridoma cell line TCE1103, and its deposit number is CGMCC No.45247.

8. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 in the preparation of a product for detecting or treating novel coronavirus infection.

9. A kit for detecting a novel coronavirus, characterized in that: Comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3.

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