EV-D68 Antibody and Its Application

Abstract

The present invention relates to the field of biomedicine technology, and provides EV-D68 antibodies and applications thereof. The present invention discloses two monoclonal antibody sequences of enterovirus D group 68 type VP1 and their applications in virus detection, reagent preparation, etc. Epitope identification shows that both monoclonal antibodies can recognize the conserved linear epitope P24 of EV-D68 embedded in the virus structure. Further experiments have shown that these two antibodies can efficiently identify all existing EV-D68 virus strains by Western Blot and ELISA methods. It can be seen that the present invention discloses two antibodies that can recognize highly conserved epitopes of EV-D68 virus, and based on these antibodies, a broad-spectrum and efficient Western Blot and ELISA detection method for detecting EV-D68 virus is established.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to EV-D68 antibodies and applications thereof. Background Art

[0002] Enterovirus D68 (EV-D68) belongs to the Picornaviridae family. It is a non-enveloped virus with an icosahedral capsid of approximately 30 nm. It is composed of 60 protomers consisting of four subunit proteins: VP1, VP2, VP3, and VP4. Among them, VP1 contains the main antigenic epitope of EV-D68 and the gene is 927 bp long.

[0003] EV-D68 was first isolated from a clinically collected throat swab sample from a patient with respiratory tract infection in the United States in 1962. EV-D68 usually presents as severe respiratory diseases and can also cause central nervous system symptoms, such as acute flaccid paralysis (AFP) in children, thus posing a threat to global public health security.

[0004] There are currently no antiviral drugs or commercial vaccines for EV-D68 infection, and there is also a lack of broad-spectrum detection reagents that can accurately and efficiently identify various EV-D68 virus strains. Summary of the invention

[0005] The present invention provides EV-D68 antibodies and applications thereof. The antibodies can recognize and bind to a highly conserved linear epitope P24 of EV-D68 embedded in the virus structure, and can broadly recognize various existing EV-D68 virus strains.

[0006] In a first aspect of the present invention, there is provided an EV-D68 antibody or an antigen-binding fragment thereof, comprising: VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6; or,

[0007] The amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 17-19, and the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 are shown in SEQ ID NOs: 20-22.

[0008] Optionally, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; or, a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 23 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 24.

[0009] Optionally, the antibody or antigen-binding fragment thereof comprises a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 33, and a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 34 or 35.

[0010] Optionally, the antibody or its antigen-binding fragment can recognize and / or specifically bind to the epitope VP1-P24 of EV-D68, and the amino acid sequence of the epitope VP1-P24 is shown in SEQ ID NO: 39, which is the amino acid sequence of positions 70-84 of VP1, and the specific sequence is LVENFLGRAALVSK.

[0011] Optionally, the antibody is a monoclonal antibody, a fully human antibody, a bispecific antibody or a multispecific antibody.

[0012] Optionally, the antigen-binding fragment of an antibody is selected from F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, scFv or any other antibody fragment that binds to an antigen but does not contain a complete antibody structure.

[0013] The second aspect of the present invention provides a nucleic acid encoding the above-mentioned EV-D68 antibody or an antigen-binding fragment thereof.

[0014] Optionally, the nucleic acid comprises the nucleotide sequences encoding VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NOs: 9-11 and the nucleotide sequences encoding VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NOs: 12-14, or,

[0015] The nucleotide sequences encoding VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 25-27 and the nucleotide sequences encoding VLCDR1, VLCDR2 and VLCDR3 are shown in SEQ ID NOs: 28-30.

[0016] Optionally, the nucleic acid comprises a nucleotide sequence encoding a heavy chain variable region and a light chain variable region as shown in SEQ ID NOs: 15-16, or a nucleotide sequence encoding a heavy chain variable region and a light chain variable region as shown in SEQ ID NOs: 31-32.

[0017] The third aspect of the present invention provides an expression vector comprising the above-mentioned nucleic acid.

[0018] The fourth aspect of the present invention provides a transgenic cell line or a recombinant bacterium comprising the above-mentioned nucleic acid or the above-mentioned expression vector.

[0019] The fifth aspect of the present invention provides a composition comprising the above-mentioned antibody or antigen-binding fragment thereof, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned transgenic cell line or recombinant bacteria.

[0020] In a sixth aspect of the present invention, an immunoconjugate is provided, comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the above-mentioned antibody or an antigen-binding fragment thereof, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme, a small molecule drug or a combination thereof.

[0021] In a seventh aspect, the present invention provides a detection reagent or kit for detecting EV-D68 virus, wherein the detection reagent or kit comprises the above-mentioned antibody or antigen-binding fragment thereof.

[0022] The eighth aspect of the present invention provides the use of the above-mentioned composition or the above-mentioned immunoconjugate in the preparation of a detection reagent for detecting EV-D68 or diagnosing a disease caused by EV-D68 infection; or, the use of the above-mentioned composition or the above-mentioned immunoconjugate in the preparation of a medicament for preventing or treating EV-D68 infection or a disease caused by infection.

[0023] Optionally, for the above application, the method for detecting EV-D68 or diagnosing a disease caused by EV-D68 infection comprises Western Blot or ELISA.

[0024] It can be seen from the above scheme that the present invention discloses two antibodies to enterovirus D group 68 type VP1, and also discloses the sequences of each region thereof and its application in virus detection and the like. The related scheme confirms that the two antibodies of the present invention can specifically recognize the highly conserved epitopes of EV-D68 virus and can efficiently recognize all existing EV-D68 virus strains. Based on the above antibodies, the present invention establishes a broad-spectrum and efficient detection method for detecting EV-D68 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figure shows the SDS-PAGE analysis of purified D1 and D2 antibodies. Lane M: protein marker; Lane 1: monoclonal antibody D1; Lane 2: monoclonal antibody D2.

[0026] Figure 2The binding characteristics of EV-D68 monoclonal antibody. ELISA and Western Blot test the ability of monoclonal antibody to recognize different antigens inactivated Fermon virus, inactivated MO-18947 virus, inactivated MO-18953 virus, and immunogen EV-D68 VLP (MO-18950). Figure A is ELISA test, and Figure B is Western Blot test.

[0027] Figure 3 Identification of linear B cell epitopes within the EV-D68 capsid protein. The entire sequence of VP1 (88 peptides) spanning EV-D68 was tested for reactivity with D1 and D2 by peptide ELISA. A in the figure is D1, and B in the figure is D2.

[0028] Figure 4 Alignment of the P24 peptide sequence with protein sequences of different strains of enterovirus EV-D68 and other enteroviruses such as CVA6. The P24 peptide sequence is at the top, and dots indicate residues identical to those of EV-D68.

[0029] Figure 5 The location of EV-D68 antigenic epitope VP1-P24 on the surface of the virus. In Figure A, VP1-P24 is located at the edge of the canyon area, and the structure is conserved. VP1-P24 is represented by blue. In Figure B, VP1 of EV-D68 (PDB: 4WM8; blue) overlaps with VP1 of EV71 (PDB: 3VBS; green), and the VP1 structures of EV-D68 and CVA10 (PDB: 6IIJ; pink) are similar. The arrow points to the antigenic epitope VP1-P24 of EV-D68. All figures were generated by PyMOL3.0.3. DETAILED DESCRIPTION

[0030] The present invention provides antibodies and / or antigen-binding fragments thereof that can specifically bind to enterovirus EV-D68.

[0031] The antibodies of the present invention may be polyclonal, monoclonal, genetically engineered and / or otherwise modified in nature, including monoclonal antibodies, fully human antibodies, bispecific antibodies or multispecific antibodies, etc. In various embodiments, the antibody comprises all or a portion of the constant region of the antibody. In some embodiments, the constant region is selected from the following types, including: IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 or IgG4) and IgM, etc. The present invention also discloses an antigen-binding fragment of an antibody that can specifically bind to EV-D68. Examples of antigen-binding fragments include F(ab')2, F(ab)2, Fab', Fab, Fv, Fd, scFv, etc.

[0032] For monoclonal antibodies of the present invention, any available or known method in the art can be used to obtain monoclonal antibodies, including expression in any eukaryotic, prokaryotic or phage system. Monoclonal antibodies for the present invention can be prepared using a wide variety of techniques known in the art, including hybridoma technology, recombinant technology or phage display technology, etc.

[0033] The embodiment of the present invention discloses the sequences of two monoclonal antibodies against enterovirus group D type 68 VP1, and the two monoclonal antibodies are represented by D1 and D2.

[0034] The sequence of monoclonal antibody D1 includes:

[0035] SEQ ID NOs: 1-3 are the amino acid sequences of heavy chain complementary determining regions CDR1, CDR2, and CDR3;

[0036] SEQ ID NOs: 4-6 are the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3;

[0037] SEQ ID NO: 7 is the amino acid sequence of the heavy chain variable region VH;

[0038] SEQ ID NO: 8 is the amino acid sequence of the light chain variable region VL;

[0039] SEQ ID NO: 33 is the amino acid sequence of the light chain constant region CL;

[0040] SEQ ID NO: 34 is the amino acid sequence of the heavy chain constant region CH;

[0041] SEQ ID NOs: 9-11 are the nucleotide sequences of heavy chain complementary determining regions CDR1, CDR2, and CDR3;

[0042] SEQ ID NOs: 12-14 are the nucleotide sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3;

[0043] SEQ ID NO: 15 is the nucleotide sequence of the heavy chain variable region VH;

[0044] SEQ ID NO: 16 is the nucleotide sequence of the light chain variable region VL;

[0045] SEQ ID NO: 36 is the nucleotide sequence of the light chain constant region CL;

[0046] SEQ ID NO: 37 is the nucleotide sequence of the heavy chain constant region CH.

[0047] The sequence of monoclonal antibody D2 includes:

[0048] SEQ ID NOs: 17-19 are the amino acid sequences of the heavy chain complementary determining regions CDR1, CDR2, and CDR3;

[0049] SEQ ID NOs: 20-22 are the amino acid sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3;

[0050] SEQ ID NO: 23 is the amino acid sequence of the heavy chain variable region VH;

[0051] SEQ ID NO: 24 is the amino acid sequence of the light chain variable region VL;

[0052] SEQ ID NO: 33 is the amino acid sequence of the light chain constant region CL;

[0053] SEQ ID NO: 35 is the amino acid sequence of the heavy chain constant region CH;

[0054] SEQ ID NOs: 25-27 are the nucleotide sequences of heavy chain complementary determining regions CDR1, CDR2, and CDR3;

[0055] SEQ ID NOs: 28-30 are the nucleotide sequences of the light chain complementary determining regions CDR1, CDR2, and CDR3;

[0056] SEQ ID NO: 31 is the nucleotide sequence of the heavy chain variable region VH;

[0057] SEQ ID NO: 32 is the nucleotide sequence of the light chain variable region VL;

[0058] SEQ ID NO: 36 is the nucleotide sequence of the light chain constant region CL;

[0059] SEQ ID NO: 38 is the nucleotide sequence of the heavy chain constant region CH.

[0060] For SEQ ID NOs: 33-35 in the sequence listing, the stop codon at the end of the sequence is not included.

[0061] Products in other aspects of the present invention also include intermediates in the process of preparing antibodies, including expression vectors, transgenic cell lines or recombinant bacteria, or compositions containing the above substances. Specifically, for expression vectors, they can be recombinant expression vectors such as eukaryotic expression vectors, and for transgenic cell lines or recombinant bacteria, they also include eukaryotic cells or prokaryotic cells, such as Escherichia coli, yeast or animal cells (such as mammalian cells, such as mouse cells, human cells, etc.), and cell lines such as 293T cell lines.

[0062] The present invention further provides the use of the above-mentioned antibodies or antigen-binding fragments, intermediates, etc. in the preparation of detection reagents for detecting EV-D68 or diagnosing diseases caused by EV-D68 infection.

[0063] Specifically, the present invention provides an immunoconjugate, comprising an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the antibody of the present invention or an antigen-binding fragment thereof, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme, a small molecule drug or a combination thereof.

[0064] Specifically, the present invention provides a kit or detection reagent for detecting EV-D68 virus, which kit or detection reagent comprises the antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the kit further comprises a solid phase substrate for the antibody or antigen-binding fragment thereof to attach, the solid phase substrate including but not limited to microplates, magnetic particles, filter paper for immunochromatography, polymers such as polystyrene, glass filters and other insoluble carriers. In some embodiments, the kit further comprises some other components, including but not limited to enzymes for labeling, corresponding substrates, radioactive isotopes, fluorescent substances, colored substances, buffers, etc.

[0065] The EV-D68 virus is detected using the above-mentioned kit or detection reagent. In some embodiments, the detection method includes: combining the antibody or antigen-binding fragment thereof of the present invention with the virus in the sample to be tested to form an antibody-virus or antibody fragment-virus complex; then, detecting the sample complex to determine whether the virus is present in the sample.

[0066] More specifically, in some embodiments, the detection reagent or kit comprises the antibody or antigen-binding fragment thereof of the present invention (as a first antibody), and a detectable, labeled second antibody. The specific detection method comprises: adsorbing the first antibody onto a solid support; then adding a sample to be tested that may contain EV-D68 virus to the support; adding a second antibody with a label to the support; and detecting the presence of the label to determine whether EV-D68 virus exists.

[0067] In addition, the specific detection method may also be known detection methods such as enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competitive method or sandwich method. Preferably, enzyme-linked immunosorbent assay (ELISA) and protein immunoblotting (Western Blot) can be used.

[0068] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0069] The embodiments of the present invention disclose a preparation and application scheme of a broad-spectrum EV-D68 virus detection antibody. By immunizing Balb / C mice with EV-D68 VLP antigens and using hybridoma technology, two EV-D68 binding-positive hybridoma cell lines D1 and D2 were screened, and the antibody types were IgG1 and IgG2a, respectively. And the nucleotide and amino acid sequences of the H chain and L chain variable regions of the two monoclonal antibodies were obtained by nested PCR. Epitope identification showed that both monoclonal antibodies could recognize the conserved linear epitope P24 embedded in the viral structure of EV-D68. Further experiments proved that these two antibodies can efficiently identify all existing EV-D68 virus strains by Western Blot and ELISA. Through comparison with the NCBI database, it was found that the epitope sequences recognized by these two antibodies were 98.5% conservative in all EV-D68 virus strains uploaded to Genebank. It can be seen that the present invention discloses two antibodies that can recognize highly conserved epitopes of EV-D68 virus, and based on these antibodies, establishes broad-spectrum and efficient Western Blot and ELISA detection methods for detecting EV-D68 virus.

[0070] Unless otherwise specified, the experimental materials used in the examples of the present invention can be obtained from commercial channels.

[0071] 1. Materials and Methods

[0072] 1.1 Cells and viruses

[0073] After 7-week-old female Balb / C mice were immunized with EV-D68 VLP (virus-like particle), positive hybridoma cells were screened. Hybridoma cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), and EV-D68 strains included Fermon strain, MO-18947 strain and MO-18953 strain. All viruses were amplified in RD cells (human embryonic kidney cells) and titrated by the 50% tissue culture infectious dose (TCID50) method.

[0074] 1.2 Monoclonal antibody preparation

[0075] Animal studies were approved by the Animal Welfare and Use Committee of Nanjing Medical University. All mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (SLAC, China).

[0076] First, 1 ml of paraffin oil was injected intraperitoneally into female Balb / C mice aged 6-8 weeks to reduce the mice's rejection of the inoculated hybridoma cells. At the same time, the selected hybridoma monoclonal clones were expanded and injected intraperitoneally into liquid paraffin-induced BAL B / c mice one week later. Then, ascites was collected and mAb was purified using Smart-Lifesciences rProtein A / G affinity chromatography columns.

[0077] 1.3 Antibody sequences

[0078] The cells were counted and collected at about 1 × 10 6 The hybridoma cell precipitates were used, RNA of the hybridoma cell samples was extracted by Trizol method, cDNA was obtained after reverse transcription, heavy chain and light chain variable regions were amplified and obtained, the amplified products were used for library construction and quality QC, sequenced using 3730xl, and compared with the database through bioinformatics analysis. The sequencing results showed that the hybridoma cell line antibody variable domain gene was successfully obtained.

[0079] 1.4 Western Blot

[0080] 1) Sample preparation: Add 60ul of purified EV-D68 virus to 12ul of 5× loading buffer, mix well and boil at 100℃ for 10 minutes.

[0081] 2) Gel electrophoresis: Perform SDS-PAGE gel protein electrophoresis of EV-D68.

[0082] 3) Transfer: After electrophoresis, remove the gel and cut off the concentrated gel, soak the separation gel in transfer buffer. Soak the prepared PVDF membrane in formaldehyde for activation, soak two filter papers in transfer buffer, stack them neatly on the positive electrode in the order of filter paper, PVDF membrane, gel, and filter paper, and cover the negative electrode for transfer.

[0083] 4) Blocking: After the transfer is completed, take out the PVDF membrane, put it in a blocking dish, add 30 ml of 5% skim milk, and block at RT for 2 hours.

[0084] 5) Primary antibody incubation: discard the blocking solution, wash with PBST 5 times, 5 min each time, add primary antibody at a concentration of 1:2000, and incubate at 4°C overnight.

[0085] 6) Secondary antibody incubation: discard the primary antibody, wash with PBST 5 times, 5 minutes each time, add goat anti-mouse HRP secondary antibody at a concentration of 1:10000, and incubate at RT for 2 hours.

[0086] 7) ECL chemiluminescence color development: Wash with PBST 5 times, 5 min each time, evenly cover the prepared ECL reagent on the PVDF membrane, develop by luminescence, press and expose in dark room conditions, exposure time 8 s, develop, and fix.

[0087] 1.5 Enzyme-linked immunosorbent assay (ELISA)

[0088] For epitope identification of monoclonal antibodies, ELISA plates were coated with 1 μg / well of individual peptides and incubated at 37°C for 2 h. After blocking in 5% milk in PBS-Tween-20 (PBST), purified monoclonal antibodies were added at a 1:100 dilution. After washing with PBST, the plates were incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG (1:5000 dilution; Sigma), and the absorbance of each well was measured at 450 nm using a microplate reader after color development.

[0089] Similarly, in order to observe the reactivity of monoclonal antibodies to different virus strains, 20 ng / well of inactivated virus or immunogen was coated, and then the same concentration of monoclonal antibody was used to bind to the antigen, and the other steps were similar to the above.

[0090] 1.6 Sequence alignment

[0091] The enterovirus strains used for sequence alignment included coxsackievirus D68 / Fermon (GenBank ID: AAR98503.1), coxsackievirus D68 / JPOC10-378 (BAK08578.1), coxsackievirus D68 NYC403 (AFM73548.1), coxsackievirus D68 / US / CO / 14-93 (AIX11070.1), coxsackievirus D68 / US / MO / 14-18947 (AIS73051.1), coxsackievirus D68 / US / KY / 14-18953 (AIS73057.1), coxsackievirus D68 / US / MO / 14-18950 (KM851228.1), and coxsackievirus D68 / US / CO / 14-93 (AIX11070.1). Gainesville (AMB37294.1), coxsackievirus D68 / VR1197 (ALG02131.1), coxsackievirus D68 / DZH-ZJUC (QCE20634.1), and coxsackievirus D68 / Ontario / PHL0008 (WXG26576.1).

[0092] In addition, this study also included rhinovirus strains Rhinovirus A1-ATCC VR-1559 (YP_009505608.1), Coxsackievirus A6 / Gdula (AAR38844.1), Coxsackievirus A9 / A242 / YN / CHN (AJD80555.1), Coxsackievirus A10 / S0273b (AOH73257.1), Coxsackievirus A16 / YY157 (AGK85257.1) and enterovirus 71 EV71 / HZ08 (AEB71507.1).

[0093] All these sequences were obtained from the National Center for Biotechnology Information (NCBI) database. Figures were generated by PyMOL3.0.3.

[0094] 2. Results

[0095] 2.1 Preparation of EV-D68 monoclonal antibody

[0096] In order to prepare EV-D68 monoclonal antibodies, EV-D68 VLP (MO-18950) was used to immunize 7-week-old female Balb / C mice, and positive hybridoma cells were screened. The positive hybridoma cells were subcloned by limiting dilution, and two stable clones were obtained, named D1 and D2. The monoclonal antibodies were typed, and the heavy chain of monoclonal antibody D1 belonged to the IgG1 subtype, while the heavy chain of monoclonal antibody D2 belonged to the IgG2a subtype, and the light chains were all kappa, as shown in Table 1.

[0097] Table 1 Characteristics of EV-D68 mAbs

[0098]

[0099] The monoclonal antibodies obtained by purifying mouse ascites were analyzed by SDS-PAGE, showing that the heavy and light chains of D1 and D2 antibodies were at 45 kDa and 23 kDa, respectively ( Figure 1 ), which indicates that the prepared monoclonal antibody contains one heavy chain and one light chain, which conforms to the basic structural characteristics of antibodies.

[0100] 2.2 Binding properties of EV-D68 mAbs

[0101] The ability of monoclonal antibodies to recognize different antigens was tested by ELISA, including inactivated EV-D68 Fermon, EV-D68 MO-18947, and EV-D68 VLP (MO-18950). The results showed that both monoclonal antibodies D1 and D2 could react with EV-D68 Fermon, EV-D68 MO-18947, and EV-D68 VLP (MO-18950), indicating that both monoclonal antibodies were specific for EV-D68. In addition, monoclonal antibody D1 showed stronger binding activity with EV-D68 MO-18947 than monoclonal antibody D2, while D2 showed stronger binding ability with Fermon and EV-D68 VLP (MO-18950) ( Figure 2 A in the figure).

[0102] The ability of the monoclonal antibodies to recognize different antigens was also tested by Western Blot, including the Fermon strain, MO-18947 strain, and MO-18953 strain of EV-D68. The results showed that both D1 and D2 could recognize the VP1 proteins of the three different strains, indicating that all three antibodies were specific for EV-D68 VP1 ( Figure 2 B in the figure).

[0103] 2.3 Identification of linear B cell epitopes of EV-D68 mAb

[0104] Identification of linear B cell epitopes of EV-D68 by ELISA. We previously screened a synthetic peptide library covering the entire VP1 sequence using sera from mice immunized with inactivated EV-D68. Figure 3 As shown in A and B in Figure 1, both D1 and D2 mAbs reacted strongly with VP1-derived peptide #24. These results indicate that the ELISA-positive peptide contains the B cell epitope recognized by mAbs D1 and D2, and also indicate that mAbs D1 and D2 contain the same linear B cell epitope recognized by the anti-EV-D68 antibody.

[0105] 2.4 Sequence analysis and localization of VP1-P24

[0106] Comparison of the VP1 protein sequences of 11 representative strains of EV-D68 showed that the VP1-P24 region (amino acid sequence from 70 to 84 of VP1, as shown in SEQ ID NO: 39) was highly conserved among various EV-D68 isolates ( Figure 4). In addition, a total of 67 sequences containing the VP1-P24 region were retrieved from the NCBI EV-D68 database through BLAST analysis. The VP1-P24 antigenic epitope was completely conserved in 66 EV-D68 strains and relatively conserved in 1 EV-D68 strain, in which the VP1-P24 antigenic epitope of ALG02131.1 underwent a single amino acid residue replacement. However, compared with other enteroviruses that cause hand, foot and mouth disease (EV71, CVA 10 and CVA 6, etc.), there are significant differences in the amino acid sequences between the VP1-P24 regions of EV-D68 and other enteroviruses ( Figure 4 ).

[0107] The VP1-P24 region is highly exposed on the surface of the virion and is located at the southern edge of the canyon ( Figure 5 In addition, if Figure 5 As shown in Figure B, the antigenic epitope VP1-P24 of EV-D68, the VP1 of EV-D68 overlaps with the VP1 of EV71, and the VP1 structures of EV-D68 and CVA10 are similar. In summary, these results show that VP1-P24 is highly conserved. Therefore, it can be considered that the monoclonal antibodies D1 and D2 of the present invention are a broad-spectrum monoclonal antibody that can be used for EV-D68 virus detection.

[0108] 2.5 Sequences of anti-EV-D68 mAbs

[0109] In order to determine the antibody sequence, RNA was extracted from the hybridoma cells, and then RT-PCR was performed, and the obtained cDNA was subjected to 5' end rapid amplification (5' RACE). The sequencing results showed that the monoclonal antibodies D1 and D2 of the present invention were related. The amino acid sequences of the CDR regions of the heavy and light chains of the above monoclonal antibodies are shown in Table 2.

[0110] Table 2 Amino acid sequence and nucleotide sequence of each CDR region

[0111]

[0112] The present invention has been described in detail above in conjunction with specific implementations and exemplary examples, but these descriptions cannot be understood as limiting the present invention. Those skilled in the art understand that, without departing from the spirit and scope of the present invention, a variety of equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its implementation methods, all of which fall within the scope of the present invention. The scope of protection of the present invention shall be subject to the attached claims.

Claims

1. An EV-D68 antibody or an antigen-binding fragment thereof, characterized in that: Include: VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6; or, The amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 17-19, and the amino acid sequences of VLCDR1, VLCDR2 and VLCDR3 are shown in SEQ ID NOs: 20-22.

2. The EV-D68 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: It comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 8; or, a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 23 and a light chain variable region with an amino acid sequence as shown in SEQ ID NO:

24.

3. The EV-D68 antibody or antigen-binding fragment thereof according to claim 2, characterized in that: It comprises a heavy chain constant region with an amino acid sequence as shown in SEQ ID NO: 33, and a light chain constant region with an amino acid sequence as shown in SEQ ID NO: 34 or 35.

4. The EV-D68 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody or antigen-binding fragment thereof can recognize and / or specifically bind to the epitope VP1-P24 of EV-D68, and the amino acid sequence of the epitope VP1-P24 is shown in SEQ ID NO:

39.

5. The EV-D68 antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The antibody is a monoclonal antibody or a fully human antibody; the antigen-binding fragment of the antibody is selected from F(ab')2, F(ab)2, Fab', Fab, Fv or scFv.

6. A nucleic acid encoding the EV-D68 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

7. The nucleic acid according to claim 6, characterized in that comprising the nucleotide sequences encoding VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NOs: 9-11 and the nucleotide sequences encoding VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NOs: 12-14, or, The nucleotide sequences encoding VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 25-27 and the nucleotide sequences encoding VLCDR1, VLCDR2 and VLCDR3 are shown in SEQ ID NOs: 28-30.

8. The nucleic acid according to claim 6, characterized in that The nucleotide sequence encoding the heavy chain variable region and the light chain variable region as shown in SEQ ID NO: 15-16, or the nucleotide sequence encoding the heavy chain variable region and the light chain variable region as shown in SEQ ID NO: 31-32.

9. An expression vector comprising the nucleic acid of claim 6.

10. A transgenic cell line or recombinant bacterium comprising the nucleic acid of claim 6 or the expression vector of claim 9.

11. A composition, characterized in that The method comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, the nucleic acid according to any one of claims 6 to 8, the expression vector according to claim 9, or the transgenic cell line or recombinant bacterium according to claim 10.

12. An immunoconjugate, characterized in that: The method comprises an antibody portion and a coupling portion coupled to the antibody portion, wherein the antibody portion comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, and the coupling portion is selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme or a combination thereof.

13. A detection reagent or kit for detecting EV-D68 virus, characterized in that: The detection reagent or kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.

14. Use of the composition of claim 11 or the immunoconjugate of claim 12 in the preparation of a detection reagent for detecting EV-D68 or diagnosing EV-D68 infection, or in the preparation of a medicament for preventing or treating EV-D68 infection.

15. The use according to claim 14, characterized in that: The method for detecting EV-D68 or diagnosing EV-D68 infection includes Western Blot or ELISA.

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