WO2023036153A1 - 一种新冠抗体及其应用 - Google Patents

一种新冠抗体及其应用 Download PDF

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Publication number
WO2023036153A1
WO2023036153A1 PCT/CN2022/117382 CN2022117382W WO2023036153A1 WO 2023036153 A1 WO2023036153 A1 WO 2023036153A1 CN 2022117382 W CN2022117382 W CN 2022117382W WO 2023036153 A1 WO2023036153 A1 WO 2023036153A1
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Prior art keywords
antibody
seq
cov
sars
amino acid
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PCT/CN2022/117382
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English (en)
French (fr)
Inventor
刘春艳
张翼
易嘉乐
罗沛
钟振宇
李瑞净
刘旭霞
秦汤
吴仁贞
阳馨滢
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广东菲鹏生物有限公司
菲鹏生物股份有限公司
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Publication of WO2023036153A1 publication Critical patent/WO2023036153A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present disclosure is in the field of protein detection. More specifically, it relates to a novel coronavirus antibody and its application.
  • Coronavirus is an enveloped single-stranded positive-sense RNA virus. According to the genetic evolution, serology and host-specific differences of coronaviruses, coronaviruses are divided into four genera: alpha-coronavirus, beta-coronavirus, gamma-coronavirus and delta-coronavirus. Betacoronaviruses can be divided into four groups A, B, C, and D. Alpha and beta coronaviruses mainly infect mammals including humans, livestock, and pets, and gamma and delta coronaviruses mainly infect birds and mammals. The new coronavirus belongs to the genus of ⁇ -coronavirus.
  • 2019-nCoV that is, 2019 new coronavirus, also known as "SARS-CoV-2"
  • SARS-CoV-2 2019 new coronavirus
  • the evolutionarily common outgroup is an HKU9-1 coronavirus that parasitizes fruit bats.
  • 2019-nCoV has an envelope, and the particles are round or oval, often pleomorphic, with a diameter of 50-200nm.
  • the present disclosure provides an antibody that binds to the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein.
  • the antibody does not bind to the 44-84 amino acid fragment, the 65-103 amino acid fragment, the 96-136 amino acid fragment, and the 130-180 amino acid fragment of the SARS-CoV-2 nucleocapsid protein .
  • the antibody binds to the same epitope as an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2 binds, Or the same epitope to which an antibody comprising the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:6 binds.
  • the present disclosure provides a combination of antibodies comprising Antibody 1 and Antibody 2, said Antibody 1 binding to an epitope with a heavy chain variable region comprising SEQ ID NO: 1 and a light chain of SEQ ID NO: 2
  • the antibodies of the variable regions bind to the same epitope
  • said Antibody 2 binds to an epitope that comprises a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO: 6
  • Antibodies from different regions bind to the same epitope.
  • the present disclosure further provides an antibody combination, comprising antibody 3 and antibody 4, the antibody 3 is a fragment that binds to the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein but does not bind to the SARS-CoV-2 nucleocapsid protein Antibodies for the 44th-84th amino acid fragment, the 65th-103rd amino acid fragment, the 96th-136th amino acid fragment, and the 130th-180th amino acid fragment, the antibody 4 binds to the SARS-CoV-2 nucleocapsid protein Antibodies to amino acid fragments 65-103.
  • Antibody 3 binds to the same epitope as an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2 binds, or Same epitope as bound by an antibody comprising the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:6.
  • Antibody 4 binds to the same epitope as an antibody comprising the heavy chain variable region of SEQ ID NO:9 and the light chain variable region of SEQ ID NO:10 binds.
  • the present disclosure also provides a kit comprising the antibody, or a combination of the antibodies.
  • the present disclosure also provides the use of the antibody, or the antibody combination, in the preparation of a kit.
  • the present disclosure also provides a method for preparing the antibody, the method comprising using the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein as an antigen, and preparing the antibody by hybridoma technology or phage display technology.
  • the present disclosure also provides a method for preparing the antibody, the method comprising: 1) immunizing an animal with an antigen or hapten comprising an amino acid fragment at positions 44-180 of the SARS-CoV-2 nucleocapsid protein; and 2) immunizing an animal from The ascites of the animal obtained an antibody binding to the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein.
  • the method for preparing the antibody also includes, through the 44th-84th amino acid fragment, the 65th-103rd amino acid fragment, the 96th-136th amino acid fragment, and the 130th amino acid fragment of the SARS-CoV-2 nucleocapsid protein -180 amino acid fragments, screening antibodies that do not bind to the above amino acid fragments.
  • the present disclosure also provides the use of the antibody, or the antibody combination, or the kit for detecting SARS-CoV-2.
  • the present disclosure also provides a method for detecting SARS-Cov-2, comprising:
  • the present disclosure also provides a method for diagnosing a subject infected with SARS-Cov-2 or a disease related to SARS-Cov-2 infection, comprising:
  • the diseases associated with SARS-Cov-2 infection include respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, and renal failure.
  • SARS-CoV-2 belongs to the Coronaviridae family and is a single-stranded positive-sense RNA virus without segments. It encodes two large overlapping open reading frames (ORF1a and ORF1b), four structural proteins (S, E, M and N proteins), and nine accessory proteins.
  • ORF1a and ORF1b two large overlapping open reading frames
  • S, E, M and N proteins four structural proteins
  • the N protein is the core component of the virion, which combines with the viral genome RNA and packages the RNA into a ribonucleoprotein (RNP) complex.
  • RNP ribonucleoprotein
  • the N protein plays an important role in viral mRNA transcription and replication, and is involved in immune regulation. It has been reported that N protein can combine with double-stranded RNA to have RNAi inhibitory activity, which can resist the antiviral response mediated by host RNAi. At the same time, N protein can also induce humoral and cellular immune responses after infection, making it an early and rapid diagnosis and
  • the SARS-CoV-2N protein has a full length of 419 amino acids and a size of 43-50kDa.
  • the N protein has three relatively conserved structural domains, one of which can intertwine with the viral genome RNA to form a viral nucleocapsid, which plays an important role in the synthesis of viral RNA and is related to viral genome replication and regulation of cell signaling pathways.
  • N protein is a phosphorylated protein. Phosphorylation can regulate the conformation of N protein, enhance the conformation with viral proteins, and enhance the affinity with viral RNA.
  • the N protein can interact with the M protein to promote the packaging of the nucleocapsid into virions.
  • N protein is often used as a diagnostic and detection tool for coronaviruses, and is the core raw material for rapid immunological diagnostic reagents.
  • 2019-nCoV N protein plays an important role in the diagnosis of novel coronavirus, and the development of monoclonal antibodies for the detection of 2019-nCoV N protein is of great significance.
  • the full length of the SARS-CoV-2 nucleocapsid protein is 419 amino acids, and its sequence is known, for example, see GenBank: UGZ64655.1.
  • Some embodiments of the present disclosure provide an antibody, wherein the antibody binds to a fragment of amino acids 44-180 of the nucleocapsid protein of SARS-CoV-2.
  • the antibody may also not bind to the 44-84 amino acid fragment, the 65-103 amino acid fragment, the 96-136 amino acid fragment, and the 130-180 amino acid fragment of the SARS-CoV-2 nucleocapsid protein amino acid fragments.
  • antibody is used in the broadest sense, which may include full-length antibodies and antigen-binding fragments thereof, and may include monospecific, bispecific, or multispecific antibodies, as well as chimeric antibodies, so long as they exhibit desired biological activity.
  • antigen-binding fragment is a substance comprising part or all of an antibody CDR (complementarity determining region), which lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to the antigen. Such fragments are biologically active in that they bind to the antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope.
  • Such fragments are selected from Fab (antigen-binding fragment) (composed of complete light chain and Fd), Fv (composed of VH and VL), scFv (single-chain antibody, VH and VL are connected by a linker peptide) or single domain antibodies (consisting of VH only).
  • Fab antigen-binding fragment
  • Fv fragment of VH and VL
  • scFv single-chain antibody, VH and VL are connected by a linker peptide
  • single domain antibodies consisting of VH only.
  • the antibody binds to an epitope to which an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2 binds The same, or the same epitope as that bound by an antibody comprising the heavy chain variable region of SEQ ID NO:5 and the light chain variable region of SEQ ID NO:6. In some embodiments, the antibody binds to the same epitope as an antibody comprising the heavy chain of SEQ ID NO: 3 and the light chain of SEQ ID NO: 4 binds, or to an antibody comprising the heavy chain of SEQ ID NO: 4. The heavy chain of ID NO: 7 and the light chain of SEQ ID NO: 8 bind to the same epitope.
  • an antibody recognizes the same epitope as other antibodies can be confirmed by competition between the two for the epitope. Competition between antibodies can be assessed by competition binding assays such as: ELISA, fluorescence energy transfer assay, or fluorescence microassay techniques.
  • the antibody binding to the amino acid fragment corresponding to the SARS-CoV-2 nucleocapsid protein means that the antibody can bind to the amino acid fragment, but the amino acid fragment is not necessarily the smallest binding fragment.
  • the present disclosure provides a combination of antibodies comprising Antibody 1 and Antibody 2, said Antibody 1 binding to an epitope with a heavy chain variable region comprising SEQ ID NO: 1 and SEQ ID NO: 1
  • the antibody of the light chain variable region of NO: 2 binds to the same epitope, and said antibody 2 binds to an epitope that is identical to the heavy chain variable region comprising SEQ ID NO: 5 and SEQ ID NO: 6 antibodies to the light chain variable region bound to the same epitope.
  • the present disclosure provides an antibody combination comprising Antibody 1 and Antibody 2, said Antibody 1 binding to an epitope with a heavy chain comprising SEQ ID NO: 3 and SEQ ID NO: 4
  • Antibody 2 binds to the same epitope as the antibody for the light chain of SEQ ID NO: 7 and the light chain of SEQ ID NO: 8 binds to the epitope
  • the epitope is the same.
  • the present disclosure also provides an antibody combination comprising Antibody 3 and Antibody 4.
  • the Antibody 3 binds to the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein. In some embodiments, Antibody 3 may also not bind to the 44-84 amino acid fragment, the 65-103 amino acid fragment, the 96-136 amino acid fragment, and the 130-180 amino acid fragment of the SARS-CoV-2 nucleocapsid protein amino acid fragments. In some embodiments, Antibody 3 binds to an epitope to which an antibody comprising the heavy chain variable region of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2 binds.
  • an antibody comprising a heavy chain variable region of SEQ ID NO: 5 and a light chain variable region of SEQ ID NO: 6; said antibody 4 binds to the SARS-CoV-2 nucleocoat A fragment of amino acids 65-103 of the shell protein.
  • Antibody 4 binds to an epitope to which an antibody comprising the heavy chain variable region of SEQ ID NO:9 and the light chain variable region of SEQ ID NO:10 binds. Bits are the same. The antibody 4 binds to the same epitope as the antibody comprising the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 12 binds.
  • the present disclosure also provides a kit, wherein the kit comprises the antibody, the antibody combination.
  • the effects of the disclosed antibodies can be tested using any suitable in vitro assay, cell-based assay, in vivo assay, animal model, etc.
  • the assay may include, for example, ELISA (enzyme-linked immunosorbent assay), FACS (fluorescent flow cytometry) binding assay, Biacore, competitive binding assay, and the like.
  • the reactivity of the antibody of the present disclosure combined with the antigen (antigenic peptide) is characterized, for example, in ELISA, for example, a reaction value > 0.5 at 405 nm read by a peroxidase-labeled ELISA method is determined to be better. Reactivity, can be used in immunoassays.
  • detectable labels such as colloidal gold, radiolabels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels, electron-dense labels, such as radioisotopes, fluorophores, rhodamine and derivatives thereof luciferase, luciferin, horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, carbohydrate oxidase, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, biotin/avidin, spin-labeled.
  • detectable labels such as colloidal gold, radiolabels, luminescent substances, colored substances, enzymes, such as fluorescent labels, chromophore labels, electron-dense labels, such as radioisotopes, fluorophores, rhodamine and derivatives thereof luciferase,
  • the present disclosure also provides the use of the antibody, or the antibody combination, in the preparation of a kit.
  • the present disclosure also provides methods of making the antibodies.
  • the antibody is prepared by using the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein as an antigen by hybridoma technology or phage display technology.
  • Some embodiments of the present disclosure provide a method for preparing the antibody, the antibody is a monoclonal antibody, and the method includes using the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein as an antigen to prepare the Monoclonal antibodies.
  • Some embodiments of the present disclosure also provide a method for preparing the antibody, the method comprising: 1) immunizing an animal with an antigen or hapten comprising the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein; and 2) obtaining an antibody binding to the 44th-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein from the ascites of the animal.
  • Some embodiments of the present disclosure also provide the use of the antibody, or the antibody combination, or the kit for detecting SARS-CoV-2.
  • Some embodiments of the present disclosure also provide a method for detecting SARS-Cov-2, including:
  • Some embodiments of the present disclosure also provide a method for diagnosing whether a subject is infected with SARS-Cov-2 or whether there is a disease related to SARS-Cov-2 infection, including:
  • the diseases associated with SARS-Cov-2 infection include at least one of respiratory symptoms, fever, cough, shortness of breath, dyspnea, pneumonia, severe acute respiratory syndrome, and renal failure.
  • the antibody provided by the disclosure has high sensitivity, good specificity, and no cross-reaction, and plays an important role in the diagnosis of novel coronavirus.
  • mice of the same strain as myeloma cells Take BALB/c mice of the same strain as myeloma cells at the age of 8-12 weeks, fully mix the 2019-nCoV N protein recombinant antigen containing 100 ⁇ g of protein per mouse and the same amount of Freund's complete adjuvant, and inject into the peritoneal cavity of the mice Within two weeks, 100 ⁇ g/mouse of 2019-nCoV N protein recombinant antigen was thoroughly mixed with an equal amount of Freund's incomplete adjuvant (FIA), and injected into the abdominal cavity of mice several times for booster immunization.
  • FIA Freund's incomplete adjuvant
  • mice After the detection of mouse serum (indirect ELISA method), those whose titer is above 1:2000 can be used for fusion, and 3 days before fusion, the mice are given intraperitoneal booster immunization again, with a dose of 50 ⁇ g/mouse.
  • BALB/c mouse peritoneal macrophages were used as feeder cells.
  • the BALB/c mice were killed by pulling the neck, soaked in 75% alcohol, and cut the abdominal skin with scissors under aseptic operation in an ultra-clean bench to expose the peritoneum. Wash repeatedly, recover the washing solution, centrifuge at 1000rpm for 5 minutes, save the precipitate, resuspend in RPMI 1640 screening culture medium (in RPMI 1640 complete culture medium containing HAT), adjust the cell concentration to 1 ⁇ 105 cells/mL, add 96 Orifice plate, 150 ⁇ L/well, 37 ° C, 5% CO 2 culture overnight.
  • the spleen was taken out under aseptic conditions, placed in a plate, washed once with RPMI 1640 basal culture medium, placed on a nylon mesh in a small beaker, ground and filtered to make a cell suspension. Centrifuge, discard the supernatant, resuspend in RPMI 1640 basal culture medium, repeat this three times, and count.
  • Immunized mouse splenocytes and mouse myeloma cells after PEG treatment, form a mixture of various cell components, including unfused myeloma cells and immune splenocytes; myeloma cell synkaryocytes and immune splenocytes symkaryon, and heterokaryon in myeloma cells and immune spleen cells. Only the latter form hybridoma cells. For this reason, unfused cells and allofused synkaryons must be removed from the various cell mixtures, and true hybrid cells must be selected. Therefore, on the 1st, 3rd, 5th, and 7th day after cell fusion, the above-mentioned HAT medium was used to replace the culture medium.
  • the reactivity in Table 2 is the OD 405 response value.
  • BSA bovine serum albumin
  • New crown N antibody coating Assemble the nitrocellulose membrane and the PVC bottom plate for use; dilute the new crown N coating antibody to 1.0-1.5mg/ml, and use a gold sprayer to draw a line on the NC membrane evenly. Put in a 37°C incubator for 60 minutes.
  • Quality control product 2019-nCoV N protein recombinant antigen, diluted to 1ng/ml with PBS for preliminary screening of antibody pairing;
  • the letter B represents no color development (not detected), and the number after the letter C represents color development. The larger the number, the better the color development. Weak (less active).
  • the 44-180aa fragment of the 2019-nCoV N protein recombinant antigen was expressed in small fragments, and different fragments of the 2019-nCoV N protein recombinant antigen were used to coat the microwells, and PBS+20% NBS was used as the diluent, and the monoclonal antibody was diluted to one Antibody concentration to 1 ⁇ g/ml, goat anti-mouse IgG-HRP was used as the secondary antibody, and the monoclonal antibody fragment was determined according to the reaction of each monoclonal antibody to different antigens.
  • the specific data are as follows:
  • the recombinant antigen expressed by Escherichia coli is obviously detected at 100pg/ml (C7+ color development), and can be detected at 25pg/ml (C8 or C8+ color development), and the minimum detection limit can reach 10pg /ml.
  • Adenovirus cytomegalovirus, Epstein-Barr virus, measles virus, mumps virus, mycoplasma pneumoniae, parainfluenza virus, respiratory and cellular virus, rotavirus, varicella-zoster virus, influenza A/B virus, etc.
  • pathogens are diluted in different concentrations, and the above three examples are paired for testing, and there is no cross-reaction.
  • 5C3, 1E5, and 7R1 were sequenced as follows.
  • a single-point mutation was performed on the variable region of the heavy chain of 5C3 to obtain a variant 5C3-1 that binds to the same epitope as 5C3; a single-point mutation was performed on the variable region of the heavy chain and the variable region of the light chain of 1E5 to obtain the variant 5C3-1 that binds to the same epitope as 1E5 Variant 1E5-1 of the epitope; a single point mutation was performed on the heavy chain variable region and the light chain variable region of 7R1 to obtain the variant 7R1-1 that binds to the same epitope as 7R1; 5C3-1, 1E5-1 Or 7R1-1 is used to replace 5C3, 1E5 or 7R1 in the original pairing accordingly, and the detection effect on the new crown recombinant antigen is equivalent. After the epitope experiment, it was confirmed that the replaced antibody had the same epitope as the original antibody.
  • the variant sequences are as follows.
  • the present disclosure also adopts the above-mentioned antibody preparation scheme, and uses the 44-180th amino acid fragment of the SARS-CoV-2 nucleocapsid protein as an immunogen to immunize and prepare the antibody that binds to the 44-180aa fragment, but does not bind to the 44-84aa fragment, 65 -Antibodies of 103aa fragment, 96-136aa fragment, and 130-180aa fragment, such as 1COV19-8, 3COV19-17, etc., which were evaluated by ELISA for OD 405 ⁇ 0.5 after reactivity screening, also meet the requirements of high sensitivity and good specificity. No cross-reactivity.
  • the present disclosure provides a novel coronavirus antibody and its application.
  • the disclosed antibody has high sensitivity, good specificity, and no cross-reaction, and plays an important role in the diagnosis of novel coronavirus, so it has excellent industrial applicability.

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Abstract

一种新冠抗体及其应用，该抗体结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段，不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段。

Description

一种新冠抗体及其应用
相关申请的交叉引用
本公开要求于2021年09月10日提交中国专利局的申请号为CN202111063235.1、名称为“一种新冠抗体或其抗原结合片段及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开属于蛋白检测领域。更具体地，涉及一种新冠抗体及其应用。
背景技术
冠状病毒是一种有囊膜的单股正链RNA病毒。根据冠状病毒的遗传学进化、血清学和宿主特异性差异，冠状病毒分为四个属：α-冠状病毒属、β-冠状病毒属、γ冠状病毒属和δ冠状病毒属。β冠状病毒属的病毒又可以分为A、B、C、D四个组群。α和β冠状病毒主要感染包括人类、家畜、宠物在内的哺乳动物，γ和δ冠状病毒主要感染鸟类及哺乳动物。新型冠状病毒属于β-冠状病毒属，从进化树位置上看，2019-nCoV(即2019新型冠状病毒，也称为“SARS-CoV-2”)与SARS和类SARS病毒的类群相邻，它们在进化上共同的外类群是一个寄生于果蝠的HKU9-1冠状病毒。2019-nCoV有包膜，颗粒呈圆形或椭圆形，常为多形性，直径50-200nm。
目前，全球对筛查、检测、诊断新型冠状病毒的试剂的灵敏度、特异性、是否有无交叉反应提出了更高的需求。
发明内容
本公开提供一种抗体，所述抗体结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段。
可选地，所述抗体不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段。
可选地，所述抗体与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，或者与包含SEQ ID  NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。
本公开提供一种抗体组合，包含抗体1和抗体2，所述抗体1与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，并且所述抗体2与表位结合，所述表位与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。
本公开又提供一种抗体组合，包含抗体3和抗体4，所述抗体3为结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段但不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段的抗体，所述抗体4为结合SARS-CoV-2核衣壳蛋白第65-103位氨基酸片段的抗体。
可选地，抗体3与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，或者与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。
可选地，抗体4与表位结合，所述表位与包含SEQ ID NO：9的重链可变区和SEQ ID NO：10的轻链可变区的抗体所结合的表位相同。
本公开还提供一种试剂盒，所述试剂盒包含所述抗体，或所述抗体组合。
本公开还提供所述抗体，或者所述抗体组合在制备试剂盒中的应用。
本公开还提供一种制备所述抗体的方法，所述方法包括以SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段作为抗原，通过杂交瘤技术或噬菌体展示技术制备所述抗体。
本公开还提供一种制备所述抗体的方法，所述方法包括：1)使用包含SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗原或半抗原免疫动物；和2)从所述动物腹水获得结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗体。
可选地，制备所述抗体的方法还包括，通过SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段，筛选不结合以上氨基酸片段的抗体。
本公开还提供所述抗体，或所述抗体组合，或所述试剂盒，用于检测SARS-CoV-2的用途。
本公开还提供一种检测SARS-Cov-2的方法，包括：
A)在足以发生结合反应的条件下，使所述抗体，或所述抗体组合，或所述试剂盒，与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开还提供一种诊断受试者在感染SARS-Cov-2或与SARS-Cov-2感染相关疾病中的方法，包括：
A)在足以发生结合反应的条件下，使所述抗体，或所述抗体组合，或所述试剂盒与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
可选地，所述与SARS-Cov-2感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭。
具体实施方式
SARS-CoV-2属于冠状病毒科，为不分节段的单股正链RNA病毒。它编码两个大的重叠开放阅读框(ORF1a和ORF1b)，4种结构蛋白(S、E、M和N蛋白)，以及9种辅助蛋白。其中，N蛋白是病毒粒子的核心成分，它与病毒基因组RNA结合，将RNA包装成核糖核蛋白(RNP)复合体。除了组装外，N蛋白还在病毒mRNA转录和复制中发挥重要作用，并参与免疫调节。有研究报道N蛋白可以与双链RNA结合而具有RNAi抑制活性，可以对抗宿主RNAi介导的抗病毒反应，同时，N蛋白还可诱导感染后的体液和细胞免疫应答，使其成为早期快速诊断和疫苗研发的关键靶标。
SARS-CoV-2N蛋白全长419个氨基酸，大小为43-50kDa。N蛋白有三个相对保守的结构域，其中一域可与病毒基因组RNA相互缠绕形成病毒核衣壳，在病毒RNA的合成过程中发挥着重要的作用，与病毒基因组复制和调节细胞信号通路有关。N蛋白是一个磷酸化蛋白，磷酸化能够调节N蛋白的构象，增强与病毒蛋白的构象，增强与病毒RNA的亲和力。在核衣壳包装过程中，N蛋白可与M蛋白相互作用，促进核衣壳包装成病毒粒子。感染早期机体就能产生抗N蛋白的高水平抗体，可以利用N蛋白建立快速检测2019-nCoV血清抗体方法。因此N蛋白常作为冠状病毒诊断检测工具，是免疫学快速诊断试剂的核心原料。
综上所述，2019-nCoV N蛋白检测对新型冠状病毒的诊断具有重要作用，开 发用于2019-nCoV N蛋白检测的单克隆抗体具有重要意义。
SARS-CoV-2核衣壳蛋白全长419个氨基酸，其序列公知，例如见GenBank:UGZ64655.1。本公开的一些实施方式提供一种抗体，其中，抗体结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段。在一些实施方式中，抗体还可以不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段。
如本文所用，术语“抗体”在最广义上使用，其可以包括全长抗体和其抗原结合片段，可以包括单特异性、双特异性或多特异性抗体，以及嵌合抗体，只要它们展示所需的生物学活性。术语“抗原结合片段”是包含抗体CDR(互补决定区)的一部分或全部的物质，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性，因为其结合至抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。此类片段选自Fab(抗原结合片段)(由完整的轻链和Fd构成)，Fv(由VH和VL构成)，scFv(单链抗体，VH和VL之间由一连接肽连接而成)或单域抗体(仅由VH组成)。此类片段可通过重组核酸技术产生，或可通过抗原结合分子(包括完整抗体)的酶裂解或化学裂解产生。
在一些实施方式中，所述抗体与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，或者与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。在一些实施方式中，所述抗体与表位结合，所述表位与包含SEQ ID NO：3的重链和SEQ ID NO：4的轻链的抗体所结合的表位相同，或者与包含SEQ ID NO：7的重链和SEQ ID NO：8的轻链的抗体所结合的表位相同。
本公开中，某抗体是否与其他抗体识别相同的表位，这可以通过两者对表位的竞争来确认。抗体间的竞争可以通过竞争结合测定来评价，其方法有：ELISA、荧光能量转移测定法或荧光微量测定技术等。
在一些实施方式中，抗体结合SARS-CoV-2核衣壳蛋白对应的氨基酸片段是指抗体能够结合所述氨基酸片段，但该氨基酸片段不一定是最小结合片段。
在一些实施方式中，本公开提供一种抗体组合，包含抗体1和抗体2，所述抗体1与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，并且所述抗体2与表位结合，所述表 位与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。
在一些实施方式中，本公开提供一种抗体组合，包含抗体1和抗体2，所述抗体1与表位结合，所述表位与包含SEQ ID NO：3的重链和SEQ ID NO：4的轻链的抗体所结合的表位相同，并且所述抗体2与表位结合，所述表位与包含SEQ ID NO：7的重链和SEQ ID NO：8的轻链的抗体所结合的表位相同。
在一些实施方式中，本公开还提供一种抗体组合，包含抗体3和抗体4。
在一些实施方式中，所述抗体3结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段。在一些实施方式中，抗体3还可以不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段。在一些实施方式中，所述抗体3与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，或者与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同；所述抗体4结合SARS-CoV-2核衣壳蛋白第65-103位氨基酸片段。
在一些实施方式中，所述抗体4与表位结合，所述表位与包含SEQ ID NO：9的重链可变区和SEQ ID NO：10的轻链可变区的抗体所结合的表位相同。所述抗体4与表位结合，所述表位与包含SEQ ID NO：11的重链和SEQ ID NO：12的轻链的抗体所结合的表位相同。
在一些实施方式中，本公开还提供一种试剂盒，其中，所述试剂盒包含所述抗体，所述抗体组合。
在一些实施方式中，可以使用任何适当的体外测定，基于细胞的测定，体内测定，动物模型等检测本公开抗体的效果如结合活性和/交叉反应性。在一些实施方式中，所述测定可以包括例如ELISA(酶联免疫吸附测定法)，FACS(流式细胞荧光分选技术)结合测定，Biacore，竞争性结合测定等。在一些实施方式中，例如在ELISA中表征本公开的抗体与抗原(抗原肽)结合的反应性，例如通过过氧化物酶标记的ELISA法读取405nm处的反应值≧0.5确定为有较好反应性，可用于免疫测定。
在一些实施方式中，其中所述抗体任选地用可检测的标记物标记。在一些实施方式中，可检测的标记例如胶体金、放射性标记、发光物质、有色物质、酶， 例如荧光标记、发色团标记、电子致密标记，例如放射性同位素、荧光团、罗丹明及其衍生物、荧光素酶、荧光素、辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖类氧化酶、葡萄糖氧化酶、半乳糖氧化酶，和葡萄糖-6-磷酸脱氢酶，生物素/抗生物素蛋白，自旋标记。
在一些实施方式中，本公开还提供所述抗体，或者所述抗体组合在制备试剂盒中的应用。
在一些实施方式中，本公开还提供制备所述抗体的方法。
通过杂交瘤技术或噬菌体展示技术，用SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段作为抗原制备所述抗体。
本公开一些实施方式提供了一种制备所述抗体的方法，所述抗体为单克隆抗体，所述方法包括以SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段作为抗原制备所述单克隆抗体。
本公开一些实施方式还提供了一种制备所述抗体的方法，所述方法包括：1)使用包含SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗原或半抗原免疫动物；和2)从所述动物腹水获得结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗体。
本公开一些实施方式还提供了所述抗体，或所述抗体组合，或所述试剂盒，用于检测SARS-CoV-2的用途。
本公开一些实施方式还提供一种检测SARS-Cov-2的方法，包括：
A)在足以发生结合反应的条件下，使所述抗体，或所述抗体组合，或所述试剂盒，与样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
本公开一些实施方式还提供一种诊断受试者是否感染SARS-Cov-2或是否有与SARS-Cov-2感染相关疾病的方法，包括：
A)在足以发生结合反应的条件下，使所述抗体，或所述抗体组合，或所述试剂盒与来自所述受试者的样品接触以进行结合反应；以及
B)检测结合反应产生的免疫复合物。
在一些实施方式中，所述与SARS-Cov-2感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭中的至少一种。
本公开提供的抗体灵敏度高、特异性好，无交叉反应，对新型冠状病毒的诊断具有重要作用。
实施例
下面将结合实施例对本公开的实施方式进行详细描述。
实施例1 新冠N蛋白单克隆抗体的制备
1、免疫动物
取8～12周龄与骨髓瘤细胞同种系的BALB/c小鼠，以含蛋白质100μg/只的2019-nCoV N蛋白重组抗原与等量福氏完全佐剂充分混匀，注入小鼠腹腔内，每隔2周100μg/只的2019-nCoV N蛋白重组抗原与等量福氏不完全佐剂(FIA)充分混匀，多次注入小鼠腹腔内加强免疫。经检测小鼠血清(间接ELISA法)，滴度在1∶2000以上者可用于融合，融合前3天经小鼠腹腔内再次加强免疫，剂量为50μg/只。
2、饲养细胞的制备
以BALB/c鼠腹腔巨噬细胞作饲养细胞。在融合前1天，BALB/c鼠拉颈处死，75％酒精全身浸泡，超净台内，无菌操作下用剪刀剪开腹部皮肤，暴露腹膜，用注射器腹腔注入RPMI 1640基础培养液5mL，反复冲洗，回收冲洗液，1000rpm，离心5分钟，留沉淀，用RPMI 1640筛选培养液(含HAT的RPMI 1640完全培养液中)重悬，调整细胞浓度1×10 5个细胞/mL，加入96孔板，150μL/孔，37℃，5％CO 2培养过夜。
3、免疫脾细胞的制备
小鼠末次免疫后三天，在无菌条件下取出脾脏，置于平皿中，RPMI 1640基础培养液冲洗一次，放于小烧杯的尼龙网上磨碎过滤，制成细胞悬液。离心，弃上清，RPMI 1640基础培养液重悬，如此重复三次，计数。
4、细胞融合
(1)取40mL HAT培养液，15mL DMEM无血清培养液和1mL50％PEG(M12000)分别置于37℃水浴中预温；
(2)分别取小鼠骨髓瘤细胞Sp2/0(菲鹏生物股份有限公司保存)(2～5×10 7)、上述免疫脾细胞(10 8)悬液加入50mL离心管中混匀，并加DMEM无血清培养液至40mL。离心10分钟，倒尽上清液，混匀；
(3)将离心管置于37℃预温的水中，取0.7mL预温的50％PEG溶液，静置90秒钟。立即滴加37℃15mL预温的无血清培养液；
(4)补加DMEM无血清培养液至40mL，离心10分钟，倒尽上清液。加入40mL含15％～20％(v)胎牛血清的HAT培养液。用吸管混匀，滴加到已含有饲养细胞的4块96孔细胞培养板的小孔中，每孔2滴，置37℃、7％CO 2的培养箱中培养。
5、杂交瘤细胞的选择培养
免疫小鼠脾细胞与小鼠骨髓瘤细胞，经PEG处理后，形成多种细胞成分的混合体，其中包括未融合的骨髓瘤细胞和免疫脾细胞；骨髓瘤细胞的共核体和免疫脾细胞的共核体，以及骨髓瘤细胞和免疫脾细胞的异核体。仅后者才能形成杂交瘤细胞。为此，在这多种细胞混合体中必须除去未融合的细胞和同种融合的共核体，并选择出真正的杂交细胞。因此，在细胞融合后第1、3、5、7天用前述的HAT培养液换液培养。
6、特异性抗体的检测及杂交瘤细胞克隆化
吸取每个培养孔的上清液，用间接ELISA法检测出培养液中含特异性识别2019-nCoV N蛋白重组抗原的抗体的培养孔。采用间接ELISA法鉴定细胞培养上清的交叉反应性，用2019-nCoV N蛋白重组抗原包被96孔板，封闭，加入杂交瘤细胞培养液上清孵育，加二抗，测在405nm处的反应值，挑选反应值(0.5以上)比较高的细胞株制备抗体进行下一轮筛选实验。筛选得到以下有较好反应性的抗体。
实施例2 抗体结合片段的鉴定
采用不同片段的2019-nCoV N蛋白重组抗原分别包被微孔，以PBS+20％NBS(N-溴代琥珀酰亚胺)为稀释液，稀释单抗为一抗浓度到1μg/ml，以羊抗鼠IgG-HRP(辣根过氧化物酶)为二抗，依据各单抗对不同抗原的反应情况来确定单抗片段。经统计，筛选得到的抗体分别是针对如下片段：
表1
Figure PCTCN2022117382-appb-000001
Figure PCTCN2022117382-appb-000002
其中结合44-180aa片段的十一株抗体对片段的反应情况摘取如表2：
表2
抗体 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1 5S7
反应性 2.362 2.616 2.491 2.493 2.486 2.528 2.941 2.949 2.444 2.552 2.778
表2中反应性即OD 405反应值。
实施例3 配对筛选
将以上抗体分别用于包被以及标记，实验过程如下：
1、新冠N抗体标记：取5ml浓度为4/万的胶体金，加入30-40μl 0.2M K 2CO 3，搅拌5min，加入新冠N标记抗体(所加抗体体积＝50μg/抗体浓度)，搅拌5min，再加入50μl 10％BSA(牛血清白蛋白)封闭终止标记；离心10000rpm，7min，去上清，沉淀用金子复溶液复溶，最后用金子复溶液定容到0.5ml(即1/10胶体金溶液体积)。
2、配制金子工作液：用金子复溶液将新冠N标记抗体浓缩金以20％的稀释比例配制成金子工作液，铺于玻璃纤维上。
3、制备干燥好的金子：将铺好的金子放入冻干机冻干(1-2h)或者放37℃干燥房干燥过夜。
4、新冠N抗体包被：将硝酸纤维素膜与PVC底板组装好备用；将新冠N包被抗体稀释至1.0-1.5mg/ml，用喷金画膜仪在NC膜上均匀地划线，放37℃恒温箱60min。
5、制备金标条：用切条机将金标条按需要的宽度切条，组装后加样进行检测。
6、检测
(1)质控品：2019-nCoV N蛋白重组抗原，使用PBS稀释到1ng/ml进行抗体配对初步筛选；
(2)检测方法：根据色卡比对，肉眼判读显色读值。
7、结果(每个片段展示一个抗体的检测结果)：
表3-1 3B8作为包被抗体的检测结果
标记抗体 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
质控品 C8 C8 C7 C8 C8 C8 C8 C8 C8 C7
PBS C9 B C8 C9 C9 B C9 C9 B B
标记抗体 5S7 6D9 4B7 1B5 2F4 7C5 5D9 4D1 6D5 4C6
质控品 C7 C8 C8 C8 C8 C8 C8 C8 C7 C7
PBS B C9 C9 B C9 B C9 C9 C8 C8
表3-2 5C3作为包被抗体的检测结果
标记抗体 6I3 9D2 4C9 3B8 1A6 1E5 8E4 6K1 6A5 7R1
质控品 C4 C4 C4 C8 C4 C3 C5 C5 C4 C3
PBS B B B C9 B B B B B B
标记抗体 5S7 6D9 4B7 1B5 2F4 7C5 5D9 4D1 6D5 4C6
质控品 C3 C7 C7 C8 C8 C8 C7 C7 C8 C8
PBS B B B B C9 B B B C9 B
表3-3 4B7作为包被抗体的检测结果
标记抗体 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
质控品 C7 C7 C7 C7 C8 C7 C7 C7 C7 C7
PBS C9 C9 C9 B C9 C9 C9 C9 C9 B
标记抗体 5S7 6D9 3B8 1B5 2F4 7C5 5D9 4D1 6D5 4C6
质控品 C7 C8 C9 C9 C9 C9 C8 C9 C9 C9
PBS B B C9 B C9 B B C9 C9 B
表3-4 1B5作为包被抗体的检测结果
标记抗体 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
质控品 C8 C6 C7 C6 C8 C6 C7 C9 C9 C6
PBS C9 B C9 B C9 B C9 C9 C9 B
标记抗体 5S7 6D9 4B7 3B8 2F4 7C5 5D9 4D1 6D5 4C6
质控品 C6 C7 C7 C7 C7 C8 C7 C7 C8 C8
PBS B C8 C9 C7 C8 C9 B C9 B B
表3-5 4C6作为包被抗体的检测结果
标记抗体 6I3 9D2 4C9 5C3 1A6 1E5 8E4 6K1 6A5 7R1
质控品 C8 C7 C7 C7 C8 C9 C7 C9 C9 C8
PBS B C9 C9 B B C9 B C9 B B
标记抗体 5S7 6D9 4B7 1B5 2F4 7C5 5D9 4D1 6D5 3B8
质控品 C9 C7 C8 C8 C7 C8 C7 C7 C8 C8
PBS B C8 C9 C9 C8 C9 B C9 B B
表3-1、3-2、3-3、3-4、3-5中，字母B代表不显色(未检出)，字母C后的数字代表显色，数字越大，显色越弱(活性越低)。
从检测结果来看，结合44-180aa区段的抗体在检测性能上表现出了较高的活性，故对该区段内的抗体做进一步的片段鉴定。
实施例4 优势抗体进一步片段鉴定
将2019-nCoV N蛋白重组抗原的44-180aa片段进行小片段表达，采用不同片段的2019-nCoV N蛋白重组抗原分别包被微孔，以PBS+20％NBS为稀释液，稀释单抗为一抗浓度到1μg/ml，以羊抗鼠IgG-HRP为二抗，依据各单抗对不同抗原的反应情况来确定单抗片段，具体数据如下：
表4-1
抗体编号 6I3 9D2 4C9 5C3 1A6 1E5
44-180 2.362 2.616 2.491 2.493 2.486 2.528
44-84 0.034 0.032 0.070 0.059 0.020 0.018
65-103 0.067 2.819 0.037 0.035 0.017 0.019
96-136 0.012 0.015 0.017 0.016 0.008 0.014
130-180 0.028 0.012 0.064 0.044 0.013 0.015
抗体识别区段 44-180 65-103 44-180 44-180 44-180 44-180
表4-2
抗体编号 8E4 6K1 6A5 7R1 5S7
44-180 2.941 2.949 2.444 2.552 2.778
44-84 0.018 0.059 0.014 0.017 0.016
65-103 0.025 0.058 0.013 2.056 2.366
96-136 0.013 0.036 0.014 0.010 0.017
130-180 0.016 0.053 0.011 0.014 0.015
抗体识别区段 44-180 44-180 44-180 65-103 65-103
从以上结果来看，9D2、7R1、5S7三株抗体进一步识别结合65-103aa，其余抗体均只识别44-180aa。
实施例5 优势抗体配对性能进一步验证
抗体标记、包被及组装等制备过程参见实施例3步骤1-5。
检测
(1)质控品：2019-nCoV N蛋白重组抗原，使用PBS稀释到25ng/ml、2ng/ml、500pg/ml、100pg/ml、25pg/ml、10pg/ml；
(2)检测方法：根据色卡比对，肉眼判读显色读值。
结果
1、活性：以下示例配对对大肠杆菌表达的重组抗原在100pg/ml时有明显检出(C7+显色)，对25pg/ml能检出(C8或C8+显色)，最低检测限可达10pg/ml。
表5
Figure PCTCN2022117382-appb-000003
2、特异性：
上述三种示例配对分别对采集的300例正常人鼻拭子、300例正常人咽拭子、300例正常人唾液标本进行胶体金显色测试，特异性均为100％。
3、交叉反应：
分别将腺病毒、巨细胞病毒、EB病毒、麻疹病毒、流行性腮腺炎病毒、肺炎支原体、副流感病毒、呼吸道和胞病毒、轮状病毒、水痘-带状疱疹病毒、流感A/B病毒等多种病原体进行不同浓度的稀释，以上述三种示例配对测试，不存在交叉反应。
4、正反配对：
将上述三种示例配对进行标记抗体与包被抗体的互换，配对性能保持相当。其中，对5C3、1E5、7R1进行测序，如下。
5C3
重链可变区SEQ NO.1：
Figure PCTCN2022117382-appb-000004
轻链可变区SEQ NO.2：
Figure PCTCN2022117382-appb-000005
重链SEQ NO.3：
Figure PCTCN2022117382-appb-000006
轻链SEQ NO.4：
Figure PCTCN2022117382-appb-000007
1E5
重链可变区SEQ NO.5：
Figure PCTCN2022117382-appb-000008
Figure PCTCN2022117382-appb-000009
轻链可变区SEQ NO.6：
Figure PCTCN2022117382-appb-000010
重链SEQ NO.7：
Figure PCTCN2022117382-appb-000011
轻链SEQ NO.8：
Figure PCTCN2022117382-appb-000012
7R1
重链可变区SEQ NO.9：
Figure PCTCN2022117382-appb-000013
轻链可变区SEQ NO.10：
Figure PCTCN2022117382-appb-000014
Figure PCTCN2022117382-appb-000015
重链SEQ NO.11：
Figure PCTCN2022117382-appb-000016
轻链SEQ NO.12：
Figure PCTCN2022117382-appb-000017
5、与5C3、1E5或7R1结合相同表位的抗体检测性能验证
对5C3重链可变区进行单点突变，得到与5C3结合相同表位的变体5C3-1；对1E5的重链可变区和轻链可变区进行单点突变，得到与1E5结合相同表位的变体1E5-1；对7R1的重链可变区和轻链可变区进行单点突变，得到与7R1结合相同表位的变体7R1-1；将5C3-1、1E5-1或7R1-1用以相应替换原配对中的5C3、1E5或7R1，对新冠重组抗原检出效果相当，经过表位实验确定替换的抗体与原抗体表位相同。变体序列如下。
5C3-1
重链可变区SEQ NO.13：
Figure PCTCN2022117382-appb-000018
重链SEQ NO.14：
Figure PCTCN2022117382-appb-000019
1E5-1
重链可变区SEQ NO.15：
Figure PCTCN2022117382-appb-000020
轻链可变区SEQ NO.16：
Figure PCTCN2022117382-appb-000021
重链SEQ NO.17：
Figure PCTCN2022117382-appb-000022
轻链SEQ NO.18：
Figure PCTCN2022117382-appb-000023
7R1-1
重链可变区SEQ NO.19：
Figure PCTCN2022117382-appb-000024
轻链可变区SEQ NO.20：
Figure PCTCN2022117382-appb-000025
重链SEQ NO.21：
Figure PCTCN2022117382-appb-000026
轻链SEQ NO.22：
Figure PCTCN2022117382-appb-000027
Figure PCTCN2022117382-appb-000028
实施例6 优势抗体再制备
本公开还通过以上所述的抗体制备方案，以SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段作为免疫原免疫制备得到结合44-180aa片段，但不结合44-84aa片段、65-103aa片段、96-136aa片段、及130-180aa片段的抗体，例如经反应性筛选为ELISA评价OD 405≧0.5的抗体1COV19-8、3COV19-17等，性能同样满足灵敏度高、特异性好，无交叉反应。
上述实施例为本公开可选的实施方式，但本公开的实施方式并不受上述实施例的限制，其他的任何未背离本公开的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本公开的保护范围之内。
工业实用性
本公开提供了一种新冠抗体及其应用，本公开的抗体具有灵敏度高、特异性好，无交叉反应，对新型冠状病毒的诊断具有重要作用，因此具有优异的工业实用性。

Claims (14)

  1. 一种抗体，其特征在于，所述抗体结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段。
  2. 如权利要求1所述的抗体，其特征在于，所述抗体不结合SARS-CoV-2核衣壳蛋白第44-84位氨基酸片段、第65-103位氨基酸片段、第96-136位氨基酸片段、及第130-180位氨基酸片段。
  3. 如权利要求1或2所述的抗体，其特征在于，所述抗体与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，或者
    与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。
  4. 一种抗体组合，其特征在于，包含抗体1和抗体2，所述抗体1与表位结合，所述表位与包含SEQ ID NO：1的重链可变区和SEQ ID NO：2的轻链可变区的抗体所结合的表位相同，并且
    所述抗体2与表位结合，所述表位与包含SEQ ID NO：5的重链可变区和SEQ ID NO：6的轻链可变区的抗体所结合的表位相同。
  5. 一种抗体组合，其特征在于，包含抗体3和抗体4，所述抗体3为权利要求2-3中任一项所述的抗体，所述抗体4为结合SARS-CoV-2核衣壳蛋白第65-103位氨基酸片段的抗体。
  6. 根据权利要求5所述的抗体组合，其特征在于，所述抗体4与表位结合，所述表位与包含SEQ ID NO：9的重链可变区和SEQ ID NO：10的轻链可变区的抗体所结合的表位相同。
  7. 一种试剂盒，其特征在于，所述试剂盒包含如权利要求1-3任一项所述的抗体，或如权利要求4-6任一项所述的抗体组合。
  8. 如权利要求1-3任一项所述的抗体，或者权利要求4-6任一项所述的抗体组合在制备试剂盒中的应用。
  9. 一种制备权利要求1-3任一项所述的抗体的方法，其特征在于，所述方法包括：1)使用包含SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗原或半抗原免疫动物；和2)从所述动物腹水获得结合SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段的抗体。
  10. 一种制备权利要求1-3任一项所述的抗体的方法，其特征在于，包括以SARS-CoV-2核衣壳蛋白第44-180位氨基酸片段作为抗原，通过杂交瘤技术或噬菌体展示技术制备所述抗体。
  11. 如权利要求1-3任一项所述的抗体，或权利要求4-6任一项所述的抗体组合，或权利要求7所述的试剂盒，用于检测SARS-CoV-2的用途。
  12. 一种检测SARS-Cov-2的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-3任一项所述的抗体，或权利要求4-6任一项所述的抗体组合，或权利要求7所述的试剂盒，与样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  13. 一种诊断受试者是否感染SARS-Cov-2或是否有与SARS-Cov-2感染相关疾病的方法，包括：
    A)在足以发生结合反应的条件下，使权利要求1-3任一项所述的抗体，或权利要求4-6任一项所述的抗体组合，或权利要求7所述的试剂盒与来自所述受试者的样品接触以进行结合反应；以及
    B)检测结合反应产生的免疫复合物。
  14. 根据权利要求13所述的方法，其中，所述与SARS-Cov-2感染相关的疾病包括呼吸道症状、发热、咳嗽、气促、呼吸困难、肺炎、严重急性呼吸综合征、肾衰竭中的至少一种。
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CN113773382A (zh) * 2021-09-10 2021-12-10 广东唯实生物技术有限公司 一种SARS-Cov-2检测方法和试剂盒

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