Monoclonal antibody Fab5 identifying the EB virus gB protein and its use

Abstract

The application belongs to the technical field of antibodies, and discloses a monoclonal antibody Fab5 for recognizing EB virus gB protein and application thereof. The monoclonal antibody or antigen-binding fragment thereof has high affinity to the gB protein and can significantly inhibit the infection of EBV on epithelial cells and B cells, and the epitope of the Fab5 binding to the gB is determined through structural analysis. The antibody can be used for detecting the presence or level of the gB protein in a sample, detecting the EB virus, diagnosing diseases caused by the EB virus infection, preventing the EB virus infection and / or treating and / or preventing diseases caused by the EB virus infection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of antibodies, and particularly relates to a monoclonal antibody Fab5 recognizing EB virus gB protein and application thereof. BACKGROUND

[0002] EB virus (EBV) is also known as human herpesvirus 4 type, and was first discovered by Epstein and Barr from Burkitt lymphoma cells through in vitro suspension culture and strain establishment in 1964. It is a DNA oncogenic virus of the lymphotropic virus genus of the gamma subfamily. EB virus particles are composed of four structural components, i.e. core protein, capsid, shell, and envelope, and the core is the core protein with DNA wound around it.

[0003] EBV is most commonly found in latent infection, and more than 90% of EBV infected persons are lifelong latent infection. The virus is activated and started under certain conditions to cause cancer. Current research has found that human EBV is closely related to the occurrence of nasopharyngeal carcinoma, infectious mononucleosis, Hodgkin's and non-Hodgkin's lymphoma, Burkitt's lymphoma, and epithelial cell carcinoma including gastric cancer, and other malignant tumors and autoimmune diseases such as multiple sclerosis. The initial replication site of EB virus is the oropharynx, which grows and reproduces in B lymphocytes and oral epithelial cells, and then infects B lymphocytes, which enter the blood circulation in large quantities to cause systemic infection. When the immune function of the body is low, the latent EBV is activated to form recurrent infection.

[0004] At present, there is no effective vaccine for EBV, and there is also a lack of specific treatment for diseases caused by EBV infection. Anti-viral drugs such as acyclovir are mostly used for the treatment of infectious mononucleosis, and these drugs can relieve symptoms to a certain extent, but cannot eliminate EB virus in B lymphocytes and throat epithelium. The treatment of EBV-related tumors is mainly chemotherapy and radiotherapy, but the effect is poor for patients with metastasis or recurrence.

[0005] Monoclonal antibodies can be produced in large quantities, and their high affinity and high specificity in binding with antigens greatly reduce adverse reactions in clinical application. These antibody molecules can also be modified to increase their antiviral efficacy. Antibodies are very promising means in the treatment of infectious diseases due to their specificity and flexibility of use, but so far no monoclonal antibody against EBV envelope glycoprotein gB has been marketed. Therefore, the development of monoclonal antibodies against EBV will provide more effective prevention and treatment means for EBV infection related diseases. SUMMARY

[0006] The first aspect of the present application aims to provide a monoclonal antibody or an antigen-binding fragment thereof.

[0007] The second aspect of the present application aims to provide a recombinant protein.

[0008] The third aspect of the present application aims to provide a biological material related to the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0009] The fourth aspect of the present application aims to provide a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application.

[0010] The fifth aspect of the present application aims to provide a conjugate.

[0011] The sixth aspect of the present application aims to provide use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application, the recombinant protein of the second aspect of the present application, the biological material of the third aspect of the present application, and / or the conjugate of the fifth aspect of the present application in the preparation of a product.

[0012] The seventh aspect of the present application aims to provide a medicament.

[0013] The eighth aspect of the present application aims to provide a vaccine.

[0014] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0015] The first aspect of the present application provides a monoclonal antibody or antigen-binding fragment thereof against Epstein-Barr virus gB, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region.

[0016] The heavy chain variable region comprises CDR-H1, CDR-H2 and CDR-H3.

[0017] The CDR-H1, CDR-H2 and CDR-H3 are CDR1, CDR2 and CDR3 with an amino acid sequence as shown in SEQ ID NO: 4.

[0018] The light chain variable region comprises CDR-L1, CDR-L2 and CDR-L3.

[0019] The CDR-L1, CDR-L2 and CDR-L3 are CDR1, CDR2 and CDR3 with an amino acid sequence as shown in SEQ ID NO: 17.

[0020] Preferably, the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L3 are in the order of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 18, SEQ ID NO: 19, respectively, and the amino acid sequence of the CDR-L2 is: DTS, and the CDRs are defined according to the IMGT definition scheme.

[0021] Preferably, the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 are in the order of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 19, respectively, and the CDRs are defined according to the Kabat definition scheme.

[0022] Preferably, the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 are in the order of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 19, respectively, and the CDRs are defined according to the Chothia definition scheme.

[0023] Preferably, the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 are in the order of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, respectively, and the CDRs are defined according to the Contact definition scheme.

[0024] Preferably, the amino acid sequence of the heavy chain variable region comprises:

[0025] a1 ) SEQ ID NO: 4; or

[0026] a2) an amino acid sequence which is obtained by substitution and / or deletion and / or addition of one or several amino acids of SEQ ID NO: 4 and has the same function as the protein shown in SEQ ID NO: 4; or

[0027] a3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology to SEQ ID NO: 4 and having the same function as the protein shown in SEQ ID NO: 4;

[0028] the amino acid sequence of the heavy chain variable region comprises:

[0029] b1) SEQ ID NO: 17; or

[0030] b2) an amino acid sequence having one or several amino acid substitutions and / or deletions and / or additions to SEQ ID NO: 17 and having the same function as the protein shown in SEQ ID NO: 17; or

[0031] b3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% homology to SEQ ID NO: 17 and having the same function as the protein shown in SEQ ID NO: 17.

[0032] Preferably, the monoclonal antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, a Fab, a Fab', a F(ab')2, a Fv, a scFv, a bispecific antibody, a multispecific antibody.

[0033] Preferably, the heavy chain further comprises a heavy chain constant region; and / or

[0034] the light chain further comprises a light chain constant region.

[0035] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0036] c1) an amino acid sequence consisting of amino acids 138-467 of SEQ ID NO: 3; or

[0037] c2) an amino acid sequence having one or several amino acid substitutions and / or deletions and / or additions to the amino acid sequence of c1) and having the same function as the protein of the amino acid sequence of c1); or

[0038] c3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of c1) and having the same function as the protein of the amino acid sequence of c1).

[0039] Preferably, the amino acid sequence of the heavy chain constant region comprises:

[0040] d1 ) an amino acid sequence consisting of amino acids at positions 127 to 232 of SEQ ID NO: 16; or

[0041] d2) an amino acid sequence having one or several amino acid substitutions and / or deletions and / or additions compared to the amino acid sequence of d1 ) and having the same function as the protein of the amino acid sequence of d1 ); or

[0042] d3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of d1 ) and having the same function as the protein of the amino acid sequence of d1 ).

[0043] Preferably, the heavy chain further comprises a heavy chain signal peptide; and / or

[0044] the light chain further comprises a light chain signal peptide.

[0045] Preferably, the amino acid sequence of the heavy chain signal peptide comprises:

[0046] e1 ) an amino acid sequence consisting of amino acids at positions 1 to 19 of SEQ ID NO: 3; or

[0047] e2) an amino acid sequence having one or several amino acid substitutions and / or deletions and / or additions compared to the amino acid sequence of e1 ) and having the same function as the protein of the amino acid sequence of e1 ); or

[0048] e3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of e1 ) and having the same function as the protein of the amino acid sequence of e1 ).

[0049] Preferably, the amino acid sequence of the light chain signal peptide comprises:

[0050] f1 ) an amino acid sequence consisting of amino acids at positions 1 to 19 of SEQ ID NO: 16; or

[0051] f2) an amino acid sequence having one or several amino acid substitutions and / or deletions and / or additions compared to the amino acid sequence of f1 ) and having the same function as the protein of the amino acid sequence of f1 ); or

[0052] f3) an amino acid sequence having 99%, 98%, 97%, 96%, 95%, 94%, or 93% homology to the amino acid sequence of f1 ) and having the same function as the protein of the amino acid sequence of f1 ).

[0053] Preferably, the amino acid sequence of the EB protein gB comprises:

[0054] g1 ) an amino acid sequence consisting of amino acids 25 to 685 of SEQ ID NO: 1 ; or

[0055] g2) an amino acid sequence of g1 ) which is subjected to substitution and / or deletion and / or addition of one or several amino acids and which has the same function as the protein of the amino acid sequence of g1 ); or

[0056] g3) an amino acid sequence which has 99%, 98%, 97%, 96%, 95%, 94% or 93% homology to the amino acid sequence of g1 ) and which has the same function as the protein of the amino acid sequence of g1 ).

[0057] In a second aspect of the present application, a recombinant protein is provided, comprising: the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application; and

[0058] an optional tag sequence which facilitates expression and / or purification.

[0059] Preferably, the tag sequence is selected from at least one of the group consisting of: His tag, GGGS sequence, FLAG tag; further His tag; and more further 6xHis tag.

[0060] In a third aspect of the present application, a biological material related to the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application is provided, the biological material comprising at least one of h1 ) to h16):

[0061] h1 ) a nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application, or the recombinant protein of the second aspect of the present application;

[0062] h2) an expression cassette comprising the nucleic acid molecule of h1 );

[0063] h3) a vector comprising the nucleic acid molecule of h1 );

[0064] h4) a vector comprising the expression cassette of h2);

[0065] h5) a transgenic cell line comprising the nucleic acid molecule of h1 );

[0066] h6) a transgenic cell line comprising the expression cassette of h2);

[0067] h7) a transgenic cell line comprising the vector of h3);

[0068] h8) a transgenic cell line comprising the vector of h4);

[0069] h9) a microorganism comprising the nucleic acid molecule of h1);

[0070] h10) a microorganism comprising the expression cassette of h2);

[0071] h11) a microorganism comprising the vector of h3);

[0072] h12) a microorganism comprising the vector of h4);

[0073] h13) a virus comprising the nucleic acid molecule of h1);

[0074] h14) a virus comprising the expression cassette of h2);

[0075] h15) a virus comprising the vector of h3);

[0076] h16) a virus comprising the vector of h4).

[0077] Preferably, the transgenic cell line does not comprise reproductive material.

[0078] Preferably, the nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application comprises a nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application and a nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application.

[0079] In a fourth aspect of the present application, there is provided a method for preparing the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application or the recombinant protein of the second aspect of the present application, using the biological material of the third aspect of the present application for expression and purification.

[0080] In a fifth aspect of the present application, there is provided a conjugate comprising: at least one of the monoclonal antibody or antigen binding fragment thereof of the first aspect of the present application and the recombinant protein of the second aspect of the present application;

[0081] and a conjugating moiety, the conjugating moiety comprising at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme.

[0082] Preferably, the detectable label is selected from a radioisotope, a fluorescent substance, a chemiluminescent substance, a colored substance, or any combination thereof.

[0083] Preferably, the conjugate is selected from the group consisting of: a fluorescent substance, a chemiluminescent label, a colored substance, a radioisotope, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme capable of generating a detectable product, a radionuclide, a biological toxin, a cytokine (such as IL-2, etc.), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle / nanorod, a viral particle, a liposome, a nanomagnetic particle, a prodrug-activating enzyme, a chemotherapeutic agent (e.g., cisplatin), or any form of nanoparticle, etc.

[0084] In a sixth aspect of the present application, the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect, the recombinant protein of the second aspect, the biomaterial of the third aspect, and / or the conjugate of the fifth aspect in the preparation of a product is provided.

[0085] The product comprises at least one of a drug, a reagent, a detection plate, a kit, a detection chip.

[0086] Preferably, the drug has at least one of the following functions i1)~i2):

[0087] i1) preventing EB virus infection;

[0088] i2) treating and / or preventing diseases caused by EB virus infection.

[0089] Preferably, the reagent, detection plate, detection chip or kit has at least one of the following functions j1)~j3):

[0090] j1) detecting the presence or level of gB protein in a sample;

[0091] j2) detecting EB virus;

[0092] j3) diagnosing diseases caused by EB virus infection.

[0093] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T cell lymphoma, lymphoproliferative disease, infectious mononucleosis.

[0094] Preferably, the drug comprises a pharmaceutically acceptable excipient.

[0095] Preferably, the drug comprises a vaccine.

[0096] Preferably, the vaccine comprises a pharmaceutically acceptable adjuvant.

[0097] In a seventh aspect of the present application, a product comprising at least one of k1)~k3) is provided:

[0098] k1) the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the application;

[0099] k2) the recombinant protein of the second aspect of the application;

[0100] k3) the conjugate of the fifth aspect of the application;

[0101] The product comprises at least one of a reagent, a detection plate, a kit, a detection chip.

[0102] Preferably, the product has at least one of the functions j1) to j3):

[0103] j1) detecting the presence or level of gB protein in a sample;

[0104] j2) detecting Epstein-Barr virus;

[0105] j3) diagnosing a disease caused by Epstein-Barr virus infection.

[0106] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disease, infectious mononucleosis.

[0107] In an eighth aspect of the application, a medicament is provided, the medicament comprising at least one of l1) to l4):

[0108] l1) the monoclonal antibody or antigen-binding fragment thereof of the first aspect of the application;

[0109] l2) the recombinant protein of the second aspect of the application;

[0110] l3) the biomaterial of the third aspect of the application;

[0111] l4) the conjugate of the fifth aspect of the application.

[0112] Preferably, the medicament further comprises a pharmaceutically acceptable carrier.

[0113] Preferably, the medicament has at least one of the functions i1) to i2):

[0114] i1) preventing Epstein-Barr virus infection;

[0115] i2) treating and / or preventing a disease caused by Epstein-Barr virus infection.

[0116] Preferably, the disease comprises at least one of nasopharyngeal carcinoma, gastric cancer, Hodgkin's lymphoma, Burkitt's lymphoma, NK / T-cell lymphoma, lymphoproliferative disease, infectious mononucleosis.

[0117] Preferably, the medicament comprises a vaccine.

[0118] Preferably, a vaccine comprises at least one of l1)~l4) and an adjuvant:

[0119] l1) a monoclonal antibody or antigen-binding fragment thereof of the first aspect of the present application;

[0120] l2) a recombinant protein of the second aspect of the present application;

[0121] l3) a biomaterial of the third aspect of the present application;

[0122] l4) a conjugate of the fifth aspect of the present application.

[0123] The beneficial effects of the present application are:

[0124] The present application provides a monoclonal antibody or antigen-binding fragment thereof against EBV gB, which has high affinity to EBV gB protein, can significantly inhibit the infection of EBV to epithelial cells and B cells, and can be used for detecting the presence or level of gB protein in a sample, detecting EB virus, diagnosing diseases caused by EB virus infection, preventing EB virus infection, and / or treating and / or preventing diseases caused by EB virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0125] Figure 1 is a graph of the affinity detection results of monoclonal antibody Fab5 and gB protein.

[0126] Figure 2 is a graph of the results of monoclonal antibody Fab5 blocking EBV infection of epithelial cells.

[0127] Figure 3 is a graph of the results of monoclonal antibody Fab5 blocking EBV infection of B cells.

[0128] Figure 4 is a graph of the results of cryo-EM complex structure of monoclonal antibody Fab5 and gB protein. DETAILED DESCRIPTION

[0129] The concept and technical effects of the present application will be described below in conjunction with the embodiments to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0130] The experimental methods in the following examples, unless otherwise specified, are generally performed according to routine conditions, or according to the conditions recommended by the manufacturer. The materials, reagents, etc. used in the present examples, unless otherwise specified, are commercially available reagents and materials.

[0131] Example 1 Preparation of anti-EB virus gB protein (EBV gB), animal immunization and antibody screening

[0132] 1.1 Expression and purification of EBV gB antigen protein

[0133] 1.1.1 Experimental materials

[0134] (1) Expression vector: eukaryotic expression vector: pcDNA3.1 (+) (ThermoFisher).

[0135] (2) Expression system: eukaryotic expression system cell HEK293F (ATCC).

[0136] (3) Reagents and consumables: Plasmid extraction kit (MN) cell transfection reagent PEI (Polyscience), 293F culture medium (Union), histidine-tagged protein purification agarose beads (Roche) and other conventional reagents and consumables are commercially available.

[0137] (4) Gene: gB gene of EB virus (M81 strain) is optimized and synthesized by Nanjing Kingsrui Biotechnology Co., Ltd. and inserted into pcDNA3.1 vector, hereinafter referred to as pcDNA3.1-gB.

[0138] The full-length gB extracellular segment recombinant protein has a total of 661 amino acid residues (not counting *), among which the bold annotations are signal peptides, which are as follows:

[0139] MPMGSLQPLATLYLLGMLVASCLGQTPEQPAPPATTVQPTATRQQTSFPFRVCELSSHGDLFRFSSDIQCPSFGTRENHTEGLLMVFKDNIIPYSFKVRSYTKIVTNILIYNGHRADSVTNRHEEKFSVESYETDQMDTIYQCYNAVKMTKDGLTRVYVDRDGVNITVNLKPTGGLANGVRRYASQTELYDAPGRVEATYRTRTTVNCLITDMMAKSNSPFDFFVTTTGQTVEMSPFYDGKNTETFHERADSFHVRTNYKIVDYDNRGTNPQGERRAFLDKGTYTLSWKLENRTAYCPLQHWQTFDSTIATETGKSIHFVTDEGTSSFVTNTTVGIELPDAFKCIEEQVNKTMHEKYEAVQDRYTKGQEAITYFITSGGLLLAWLPLTPRSLATVKNLTELTTPTSSPPSSPSPPAPPAARGSTSAAVLRRRRRNAGNATTPVPPAAPGKSLGTLNNPATVQIQFAYDSLRRQINRMLGDLARAWCLEQKRQNMVLRELTKINPTTVMSSIYGKAVAAKRLGDVISVSQCVPVNQATVTLRKSMRVPGSETMCYSRPLVSFSFINDTKTYEGQLGTDNEIFLTKKMTEVCQATSQYYFQSGNEIHVYNDYHHFKTIELDGIATLQTFISLNTSLIENIDFASLELYSRDEQRASNVFDLEGIFREYNFQAQNIAGLRKDLDNAVSHHHHHH*(SEQ ID NO: 1).

[0140] The DNA expression sequence thereof is 2071 base pairs, wherein the bold is the signal peptide, and the specific is:

[0141]

[0142] 1.1.2 Protein expression

[0143] First step: Transform and pick colonies

[0144] (1) Add the synthesized plasmid to the thawed DH5a bacteria and let it sit on ice for 5 minutes.

[0145] (2) Place the bacteria with the added plasmid in a shaking metal bath and heat shock at 42°C for 60-90 seconds at 120 rpm.

[0146] (3) Let the bacteria cool on ice for 5 minutes.

[0147] (4) Use a spreader to spread the bacteria onto a TB plate with ampicillin resistance and incubate overnight.

[0148] (5) The next day, use a sterile needle to pick a single colony and place it into 500 mL of medium with 0.1 mg / mL ampicillin and incubate at 37°C for 12-16 hours at 220 rpm.

[0149] Second step: Plasmid extraction

[0150] (1) Centrifuge the incubated bacteria at 4000 rpm and 4°C to discard the medium and keep the bacterial precipitate. Then, follow the kit instructions to extract the plasmid. The following is a brief plasmid extraction process.

[0151] (2) Resuspend the precipitate with RES resuspension buffer (containing RNAase A) in the NM extraction kit.

[0152] (3) Add LYS lysis buffer and let it stand for 5 minutes.

[0153] (4) Add NEU neutralization buffer. At this time, a precipitate should be produced and the solution should be clear and yellow.

[0154] (5) Use EQU equilibration buffer to equilibrate the hollow fiber column for extraction. Note that the solution should be poured from the top of the fiber column inlet so that the entire fiber sleeve is completely wet.

[0155] (6) Add the neutralized solution and precipitate to the equilibrated hollow fiber column.

[0156] (7) Add EQU equilibration buffer to equilibrate the hollow fiber column again so that the lower solution is clear and colorless and transparent.

[0157] (8) Discard the middle fiber sleeve and keep the outer adsorption column.

[0158] (9) Add Wash wash buffer to wash the adsorption membrane below the adsorption column.

[0159] (10) Place a centrifuge tube below the adsorption column for collection, then add an appropriate amount of Elu elution buffer for plasmid elution, and finally add isopropanol for plasmid precipitation.

[0160] (11) The precipitated plasmid solution is centrifuged at 4000 rpm for 30 minutes, the supernatant is discarded, and 70% ethanol is added for resuspension before centrifugation again.

[0161] (12) Place the centrifuged solution in a clean bench, discard the supernatant, resuspend the plasmid with an appropriate amount of sterile double distilled water, and store the resuspended plasmid at -20°C.

[0162] Step 3: Transfection of 293F cells with plasmid

[0163] (1) 293F cells should be cultured at 37°C, 120 rpm, 5% CO2 using culture medium, and the density should be maintained at 0.5-5*10 ^6 6 / mL (0.5-5X density), and the density should be maintained at 0.5-5*10

[0164] (2) Take out the plasmid and dilute it with 293 culture medium, generally 1 mg of plasmid is used for transfection per 1 L of 1X cells.

[0165] (3) Take out the dissolved PEI and dilute it with 293 culture medium, generally 5 mg of PEI is added for transfection system configuration per 1 mg of plasmid.

[0166] (4) Mix the plasmid and PEI diluted with 293 culture medium and stand for 10-15 minutes.

[0167] (5) Dilute the 293F cells to 1X density, then add the transfection mixture, and then return to 37°C, 120 rpm, 5% CO2 shaking culture for 5-7 days.

[0168] Step 4: Purification of secreted protein from eukaryotic culture supernatant

[0169] (1) Collect the supernatant of the cultured 293F cells using 8000 rpm, 4°C high-speed centrifugation for 1 hour, as the signal peptide sequence is added in the plasmid, the protein will be expressed in the supernatant, and the supernatant is used for protein purification.

[0170] (2) Collect the cell supernatant and filter twice with 0.22um filter to remove impurities.

[0171] (3) Take out the gravity purification column, add 1 mL His-tagged purification agarose beads into it, and add 0.22-μm-filtered double-distilled water (the liquid added to the purification column in the purification stage needs to be filtered through a 0.22-μm filter membrane), wash the beads, and then use PBS to equilibrate the purification column.

[0172] (4) Use the siphon method to transfer the supernatant to the purification column, so that the supernatant passes through the purification column completely. If repeated recovery is needed, the supernatant can be recovered at this step and purified again.

[0173] (5) Use PBS to equilibrate the purification column for three column volumes.

[0174] (6) Use PBS containing 300 mM imidazole to elute the purification column for two column volumes, and obtain the eluate.

[0175] (7) Take samples of the supernatant, the equilibration liquid, and the eluate for SDS-PAGE to verify the protein expression.

[0176] (8) Use an ultrafiltration tube of the corresponding size to concentrate the eluate, so that the volume after concentration is less than 1 mL. Centrifuge the concentrated eluate at 12,000 g and 4°C for 5 minutes to remove the precipitate.

[0177] (9) Subject the concentrated eluate to molecular sieve purification through an Akta purifier configured with a Superdex 200 Increase 10 / 300 GL molecular sieve column, and collect the protein sample at the molecular sieve peak position for SDS-PAGE verification with the previous sample.

[0178] 1.2 EBV gB mouse immunization experiment

[0179] 1.2.1 Vaccine configuration

[0180] (1) Take out the gB protein after thawing, where the gB is 5 μg per mouse, and the antigen uses sterile PBS as the solvent.

[0181] (2) Mix and emulsify 100 μg of the antigen solution with 100 uL of complete Freund's adjuvant to form a dose of 200 μL per mouse, and place it on a rolling incubator at 4°C overnight.

[0182] 1.2.2 Mouse spleen collection

[0183] (1) The mice are fed with conventional feed and conventional drinking water, and the age of the mice at the time of immunization is 6 weeks, and the mice are of the Balb / C strain.

[0184] (2) Fix the mouse, and using a 1-mL syringe, shake the vaccine manually again, and then inject it into the mouse through the abdominal subcutaneous immunization method.

[0185] (3) Observe the mice for 3-5 minutes and release them after they have returned to normal activity.

[0186] (4) The vaccine immunization process is 0 weeks-3 weeks-6 weeks-8 weeks, and the spleen collection time is 10 weeks.

[0187] (5) When collecting the mouse spleen, the spinal dislocation method is used according to the animal welfare and standard animal experiment process, the blood is collected, and the spleen is obtained by dissection.

[0188] 1.3 EBV gB mouse B cell gB-specific cell isolation and cloning

[0189] 1.3.1 Spleen lymphocyte isolation steps:

[0190] (1) The mice were sacrificed by cervical dislocation 10 weeks after immunization, and their peripheral lymph nodes and spleens were placed in EP tubes.

[0191] (2) Add an appropriate amount of DNAse and type IV collagenase to the 1640 medium to prepare a tissue digestion solution, and add an appropriate amount of digestion solution according to the size of the lymph nodes and spleen.

[0192] (3) Use the pressure plug in a sterile syringe to crush the tissue on a 70-μm cell screen until the tissue is almost crushed and minced, then rinse the cell screen with an appropriate amount of fresh culture medium to wash the remaining cells into the 50-mL centrifuge tube below.

[0193] (4) Add red blood cell lysis solution and incubate at room temperature for 10 minutes.

[0194] (5) Centrifuge at 2000g, 18°C for 5 min.

[0195] (6) Wash twice with fresh culture medium.

[0196] 1.3.2 B lymphocyte antigen staining and sorting

[0197] (1) Wash the cells to 1*10^8 cells per flow tube with PBS, centrifuge at 3000g, 18°C for 5 minutes, and discard the supernatant.

[0198] (2) Add 5 μL of Fc receptor blocking antibody solution to 100 μL of PBS, resuspend the cells and incubate on ice for 30 minutes.

[0199] (3) According to the cell grouping, PBS was used to prepare the flow cytometry antibody diluent. Different dilution gradients were set for the first use of the antibody. The most suitable gradient was selected according to the ratio of 1:20, 1:50, 1:100, 1:200, and 1:500 of the antibody to PBS. 100 μL of the diluted flow cytometry antibody cells and cell dead and live dyes were added to resuspend the cells for staining, and the cells were incubated at 4°C in the dark for 30 minutes. Mouse IgG and B220 antibodies were used as B cell separation reagents, and gB was used as a gB-specific B cell separation reagent.

[0200] (4) The cells were washed with PBS, centrifuged at 3000g and 4°C for 5 minutes, washed for 1 to 2 times, and the supernatant was discarded.

[0201] (5) The cells were resuspended in a flow tube with PBS, and the cells were flow sorted using a BD Rhaspody single cell sorter.

[0202] (6) IgG + B220 + The strongest gB positive signal in the cells was 0.2% of the cells sorted into a 96-well plate.

[0203] 1.3.3 B cell sequence cloning and expansion

[0204] (1) PCR was performed on the B cells in the 96-well plate by using mouse IgG heavy chain and light chain universal cloning primers to obtain the heavy chain and light chain sequences corresponding to a single B cell.

[0205] (2) The antibody heavy chain variable region was connected to the CMV fragment upstream and the constant region of mouse IgG1 downstream to express the complete heavy chain fragment. The antibody light chain variable region was connected to the CMV fragment upstream and the constant region of light chain κ / λ downstream to express the complete light chain fragment. The plasmid with the above-mentioned full-length sequence of the antibody heavy chain and light chain was co-transfected into 293F cells to realize the expression of the antibody. Protein A beads were used to purify the antibody. Among them, a total of 8 antibodies of Fab1-Fab8 were obtained by this method.

[0206] (3) ELISA was used to determine the gB affinity of the obtained antibodies, and the Fab5 antibody with the highest affinity was selected as the subsequent further verification.

[0207] Example 2 Preparation of monoclonal antibody (mAb) against Epstein-Barr virus gB protein (EBV gB)

[0208] By linking the variable region of the antibody heavy chain upstream to the CMV fragment and downstream to the constant region of human IgG1, the complete heavy chain fragment can be expressed; similarly, by linking the variable region of the antibody light chain upstream to the CMV fragment and downstream to the constant region of the light chain κ / λ, the complete light chain fragment can be expressed. Co-transfection of plasmids containing the full-length sequences of the antibody heavy and light chains into 293F cells achieves antibody expression, and protein A beads can be used for antibody purification.

[0209] The full-length heavy chain of Fab5 consists of 467 amino acid residues (excluding *), specifically:

[0210] QVQLQQPGAELVRPGASVKLSCKAS INWVKQRPGQGLEWIGN NYNQKFKDKATLTVDKSSSTAYMLLSSPTSEDSAVYYC WGQGTTLTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*(SEQ ID NO: 3);

[0211] The underlined portion of the sequence represents the amino acid sequence of the heavy chain variable region (SEQ ID NO: 4). The italicized portions represent the amino acid sequences (IMGT definition scheme) of the three complementary regions CDR-H1 (SEQ ID NO: 5), CDR-H2 (SEQ ID NO: 6), and CDR-H3 (SEQ ID NO: 7) within the heavy chain variable region. The bolded portion represents the signal peptide. Amino acids 138–467 constitute the heavy chain constant region. * indicates a stop codon.

[0212] Table 11 shows CDR-H1, CDR-H2, and CDR-H3 in the variable region of the heavy chain, which are defined by other CDR schemes.

[0213] Table 11 CDR-H1, CDR-H2, CDR-H3 in the heavy chain variable region in other defined CDR schemes

[0214]

[0215] Fab5 full-length light chain has 232 amino acid residues (not counting*), specifically:

[0216] QIVLTQSPAIMSASPGEKVTMTCSAS MYWYQQKPGSSPRLLIY NLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYC FGGGTK LEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*(SEQ ID NO: 16);

[0217] In the sequence, the underlined part is the amino acid sequence of the light chain variable region (SEQ ID NO: 17). The underlined and bolded part is the amino acid sequence of the three complementarity determining regions CDR-L1 (SEQ ID NO: 18), CDR-L2 (DTS) and CDR-L3 (SEQ ID NO: 19) in the light chain variable region (IMGT definition scheme) in order. The black bolded part is the signal peptide. The 127th to 232nd amino acids are the light chain constant region. * represents the stop codon.

[0218] The CDR-H1, CDR-H2, CDR-H3 in the light chain variable region in other defined CDR schemes are shown in Table 12.

[0219] Table 12 CDR-H1, CDR-H2, CDR-H3 in the light chain variable region in other defined CDR schemes

[0220]

[0221] Affinity determination of Fab5

[0222] The affinity of antibody Fab5 was determined by using biofilm interference technology (BLI).

[0223] BLI can be performed according to conventional methods in the art, and in this embodiment, the specific operation is as follows: a biosensor (Germany Sartorius The SA probe was immersed in a buffer (KB buffer, 0.1 wt% BSA and 0.02 v / v% Tween 20) for equilibration. Then it was taken out and immersed in a solution containing 5 μg / mL gB-Biotin (gB protein labeled with biotin) and the gB antigen in the solution was bound to the surface of the SA (streptavidin) biosensor, increasing the thickness of the surface film. Then the biosensor with the immobilized known concentration of antigen was immersed in the buffer as a baseline. By immersing the biosensor with the immobilized known concentration of antigen in a sample solution containing 31.3-500 nM Fab5 antibody for about 120 seconds, the specific binding between the antigen and the antibody caused an increase in the thickness of the film, and the biosensor with the bound Fab5 antibody was immersed in the buffer for dissociation for about 180 seconds, and the tested antibody (Fab5 antibody) was detached from the surface of the biosensor, causing a decrease in the thickness of the film. By monitoring the thickness of the biosensor biofilm in real time during the experiment, the kinetic constant of the tested sample (Fab5 antibody) was obtained. The results are shown in Figure 1: the KD (M) of the Fab5 antibody is less than 10 Figure 1 -12 M; it is shown that the Fab5 antibody has extremely high affinity with the gB antigen.

[0224] Example 4: Determination of the neutralization activity of the Fab5 antibody

[0225] (1) Preparation of EBV virus:

[0226] 1) CNE2 cells infected with EBV-GFP (the virus has been disclosed in the literature: An Antibody Targeting the Fusion Machinery Neutralizes Dual-Tropic Infection and Defines a Site of Vulnerability on Epstein-Barr Virus) were cultured in RPMI1640 + 5 v / v% FBS in a 37-degree incubator (5 v / v% CO2), and when the cells grew to 90% density (10 cm dish), TPA was added to a final concentration of 20 ng / mL and NaB (sodium butyrate) was added to a final concentration of 2.5 mM for induction, and the medium was changed after 12 hours.

[0227] 2) The culture supernatant was collected 48-72 hours after the medium change, and the virus was isolated and purified, and the supernatant was directly aspirated and centrifuged, and then filtered with a 0.45 μm filter. After concentration, resuspend in serum-free RPMI1640, and immediately use for infection or store at -80 degrees.

[0228] (2) Detection of the epithelial cell neutralization activity of the Fab5 monoclonal antibody

[0229] ​1) In each well of a 96-well plate, 1 x 10 6 epithelial cells were plated, and 100 μL of DMEM medium containing 10% FBS was added to each well.

[0230] 2) The next day, the Fab5 monoclonal antibody in the above example was adjusted to a concentration of 2 mg / mL. In a new 96-well plate, 60 uL of DMEM medium was added to each well, and 120 uL of the Fab5 antibody diluted with DMEM at 12.5 μg / mL was added to the first well (RPMI1640 medium was not included in the first well).

[0231] 3) After 2-fold gradient dilution (gradient dilution will draw 60 uL from the first well into the second well, and so on, and the last well will be discarded after 60 uL is drawn, and the final volume in each well is 60 uL); 60 uL of virus diluent (virus diluted with DMEM medium, with a titer of about 4*10 6 / mL) was added to each well, and after 2 hours of incubation at 37°C, the 293T cells plated the previous day were added. After 48 hours of culture in a 37°C incubator, detection was performed.

[0232] 4) After trypsin digestion of the 293T cells, a cell suspension was prepared, and the infection rate was detected by flow cytometry. The reduction ratio of the number of GFP-positive cells in the antibody treatment group compared with the infection control group (the same volume of DMEM was added) was detected, and the inhibition rate (neutralization efficiency, %) of the antibody in the 293T epithelial cell infection model was calculated. Prism was used to calculate and plot the IC 50 of the Fab5 monoclonal antibody.

[0233] The results are shown in Figure 2 : The IC 50 of the Fab5 monoclonal antibody in the epithelial cell infection model was 0.30 μg / mL; the Fab5 monoclonal antibody can significantly inhibit the infection of EBV on epithelial cells.

[0234] Example 5: Detection of the B cell neutralization activity of the Fab5 monoclonal antibody

[0235] 1) The Fab5 monoclonal antibody in the above example was adjusted to a concentration of 2 mg / mL. In a new 96-well plate, 60 uL of RPMI1640 medium was added to each well, and 90 uL of the Fab5 antibody diluted with RPMI1640 at 100 μg / mL was added to the first well (RPMI1640 medium was not included in the first well).

[0236] 2) After 3-fold gradient dilution (gradient dilution will draw 30 uL from the first well into the second well, and so on, and the last well will be discarded after 30 uL is drawn, and the final volume in each well is 60 uL); 60 uL of virus diluent (virus diluted with DMEM medium, with a titer of about 4*10 6After incubating at 37°C for 2 hours, add 1*10 mL to each well. 6 Raji cells were incubated at 37 degrees Celsius for 48 hours before being analyzed.

[0237] 3) Raji cells were aspirated to prepare a cell suspension. The infection rate was detected by flow cytometry. The inhibition rate (neutralization efficiency, %) of the antibody in the Raji B cell infection model was calculated by detecting the reduction in the number of GFP-positive cells in the antibody-treated group compared with the infection control group (which had an equal volume of RPMI 1640 added). The IC50 of the Fab5 monoclonal antibody was calculated using Prism. 50 .

[0238] The results are as follows Figure 3 As shown: IC50 of monoclonal antibody Fab5 in a B cell infection model 50 At a concentration of 0.9 μg / mL, the monoclonal antibody Fab5 can significantly inhibit EBV infection of B cells.

[0239] Example 6: Cryo-electron microscopy structures of monoclonal antibody Fab5 and gB protein

[0240] Brief steps for cryo-electron microscopy imaging:

[0241] (1) 1 mg / mL of Fab5 / gB was dropped into a glow-treated Quantifoil Cu R1.2 / 1.3 300 mesh copper mesh at a molar ratio of 1:1 (M / M) composite particle sample.

[0242] (2) The Vitrobot Mark IV automated freezing robot was used for freezing samples. The freezing time was 7 seconds and the humidity was 100%.

[0243] (3) The sample was observed using a 300kV Titan Krios transmission electron microscope. If the sample preparation was not up to standard, the sample was frozen again.

[0244] (4) Qualified samples are photographed using K2 Summit direct detection and their original images are recorded.

[0245] Brief steps for cryo-electron microscopy single-particle analysis:

[0246] (1) Use motionCor2 to align the photo data.

[0247] (2) Use PatchCTF to calculate the CTF value of the frozen data.

[0248] (3) Use Blob Picker to automatically pick particles without parameters to generate 2D classification control phases.

[0249] (4) Further selection of reference density for particle picking with reference and 2D classification based on best set in micrographs.

[0250] (5) Homogeneous reconstruction of the best 2D Class set of particles and resolution determination using gsFSC criteria.

[0251] Results are shown in Figure Figure 4 QVQLQQPGAELVRPGASVKLSCKAS INWVKQRPGQGLEWIGN NYNQKFKDKATLTVDKSSSTAYMLLSSPTSEDSAVYYC WGQGTTLTVSS QIVLTQSPAIMSASPGEKVTMTCSAS MYWYQQKPGSSPRLLIY NLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYC FGGGTK The structure shows that Fab5 binds to the DI domain of gB protein, where the Fab5 heavy chain is pink and the light chain is gray. Further analysis of the binding interface provides precise information on the key interaction regions between the light and heavy chains of Fab5 and gB. Meanwhile, the binding of Fab5 to the DI region is a significant feature that distinguishes it from previously reported antibodies 3A3 and 3A5 (Zhang et al. PNAS, 2023), highlighting that Fab5 is a completely new EB virus neutralizing antibody. The structure of the Fab5-gB protein complex provides important molecular biological evidence for the significant gB affinity and high EB virus neutralization of antibody Fab5.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against an Epstein-Barr virus gB protein, the monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region; the heavy chain variable region comprising CDR-H1, CDR-H2 and CDR-H3; the CDR-H1, CDR-H2 and CDR-H3 being CDR1, CDR2, CDR3 with amino acid sequences as shown in SEQ ID NO: 4; the light chain variable region comprising CDR-L1, CDR-L2 and CDR-L3; the CDR-L1, CDR-L2 and CDR-L3 being CDR1, CDR2, CDR3 with amino acid sequences as shown in SEQ ID NO:

17. 2.The monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein: the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L3 are in sequence as shown in SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 18, SEQ ID NO: 19, respectively, the sequence of the CDR-L2 is DTS, and the CDRs are defined according to the IMGT definition scheme; or the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 are in sequence as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 19, respectively, and the CDRs are defined according to the Kabat definition scheme; or the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 are in sequence as shown in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 10, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 19, respectively, and the CDRs are defined according to the Chothia definition scheme; or the amino acid sequences of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, CDR-L3 are in sequence as shown in SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, respectively, and the CDRs are defined according to the Contact definition scheme. 3.The monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, wherein: the monoclonal antibody or antigen-binding fragment thereof comprises at least one of a full-length antibody, Fab, Fab', F(ab') 2, Fv, scFv. ​ ​ ​ 4. A recombinant protein consisting of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3 and a tag sequence assisting expression and / or purification.

5. A biological material associated with the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4, the biological material comprising at least one of h1) to h16): h1) a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4; h2) an expression cassette comprising the nucleic acid molecule of h1); h3) a vector comprising the nucleic acid molecule of h1); h4) a vector comprising the expression cassette of h2); h5) a transgenic cell line comprising the nucleic acid molecule of h1); h6) a transgenic cell line comprising the expression cassette of h2); h7) a transgenic cell line comprising the vector of h3); h8) a transgenic cell line comprising the vector of h4); h9) a microorganism comprising the nucleic acid molecule of h1); h10) a microorganism comprising the expression cassette of h2); h11) a microorganism comprising the vector of h3); h12) a microorganism comprising the vector of h4); h13) a virus comprising the nucleic acid molecule of h1); h14) a virus comprising the expression cassette of h2); h15) a virus comprising the vector of h3); and h16) a virus comprising the vector of h4).

6. A method for preparing the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, or the recombinant protein according to claim 4, comprising the following steps: expressing and purifying using the biological material according to claim 5.

7. A conjugate consisting of at least one of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, and the recombinant protein according to claim 4; and a conjugating moiety; wherein the conjugating moiety is a detectable label.

8. The conjugate according to claim 7, wherein the detectable label comprises a radionuclide.

9. Use of at least one of (1) to (4) in the manufacture of a medicament: (1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (2) the recombinant protein according to claim 4; (3) the biological material according to claim 5; and (4) the conjugate according to claim 7 or 8; wherein the medicament has at least one of i1) to i2): i1) preventing Epstein-Barr virus infection; and i2) treating and / or preventing a disease caused by Epstein-Barr virus infection.

10. Use of at least one of (1) to (4) in the manufacture of a reagent, assay plate, assay chip, or kit: (1) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (2) the recombinant protein according to claim 4; (3) the biological material according to claim 5; and (4) the conjugate according to claim 7 or 8; wherein the reagent, assay plate, assay chip, or kit has at least one of j1) to j3): j1) detecting the presence or level of gB protein in a sample; j2) detecting the presence or level of an antigen-binding fragment of gB protein in a sample; and j3) detecting the presence or level of a gB protein in a sample. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ j2) detecting Epstein-Barr virus; j3) diagnosing a disease caused by Epstein-Barr virus infection.

11. A medicament comprising at least one of (11) to (14): (11) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (12) the recombinant protein according to claim 4; (13) the biomaterial according to claim 5; (14) the conjugate according to claim 7 or 8.

12. The medicament according to claim 10, further comprising a pharmaceutically acceptable carrier.

13. A kit for detecting Epstein-Barr virus, the kit comprising at least one of (11) to (14) and an adjuvant: (11) the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3; (12) the recombinant protein according to claim 4; (13) the biomaterial according to claim 5; (14) the conjugate according to claim 7 or 8. ​

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