CN118994382B

CN118994382B - EV-D68 antibody and its application

Info

Publication number: CN118994382B
Application number: CN202411499151.6A
Authority: CN
Inventors: 代文龙; 戎震宇
Original assignee: Nanjing Medical University
Current assignee: Nanjing Medical University
Priority date: 2024-10-25
Filing date: 2024-10-25
Publication date: 2025-02-28
Anticipated expiration: 2044-10-25
Also published as: CN118994382A

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

EV-D68 antibody and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an EV-D68 antibody and application thereof.

Background

Enterovirus D68 (Enterovirus D, EV-D68) belongs to picornaviridae, is a non-enveloped virus, has twenty-face capsids of about 30nm, and consists of 60 protomers consisting of VP1, VP2, VP3 and VP4 subunit proteins, wherein VP1 contains EV-D68 main epitope and has a gene length of 927bp.

EV-D68 was first isolated in 1962 from a clinically collected pharyngeal swab sample from a patient with respiratory tract infection in the United states. EV-D68 is often manifested as a severe respiratory illness and also causes central nervous system symptoms such as Acute tardive paralysis (Acute FLACCID PARALYSIS, AFP) in children, thus posing a threat to global public health safety.

At present, no antiviral drugs and commercial vaccines for EV-D68 infection exist, and a broad-spectrum detection reagent capable of accurately and efficiently identifying each EV-D68 virus strain is also lacking.

Disclosure of Invention

The invention provides an EV-D68 antibody and application thereof, wherein the antibody can recognize and bind with a highly conserved linear epitope P24 embedded in a virus structure of EV-D68, and can recognize various existing EV-D68 virus strains in a broad spectrum.

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

VHCDR1, VHCDR2 and VHCDR3, shown in amino acid sequences SEQ ID NO 17-19, and VLCDR1, VLCDR2 and VLCDR3, shown in amino acid sequences SEQ ID NO 20-22.

Alternatively, the antibody or antigen binding fragment thereof comprises a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 7 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 8, or a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO. 23 and a light chain variable region having an amino acid sequence as shown in SEQ ID NO. 24.

Alternatively, the antibody or antigen binding fragment thereof comprises a light chain constant region having an amino acid sequence as set forth in SEQ ID NO. 33, and a heavy chain constant region having an amino acid sequence as set forth in SEQ ID NO. 34 or 35.

Optionally, the antibody or antigen binding fragment thereof can recognize and/or specifically bind to an epitope VP1-P24 of EV-D68, wherein the amino acid sequence of the epitope VP1-P24 is shown as SEQ ID NO. 39, and is the amino acid sequence of VP1 at positions 70-84, and the specific sequence is LVENFLGRAALVSK.

Alternatively, the antibody is a monoclonal antibody, a fully human antibody, a bispecific antibody, or a multispecific antibody.

Alternatively, the antigen binding fragment of the antibody is selected from the group consisting of F (ab ') 2, F (ab) 2, fab', fab, fv, fd, scFv, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure.

In a second aspect of the invention, there is provided a nucleic acid encoding the EV-D68 antibody or antigen-binding fragment thereof described above.

Alternatively, the nucleic acid comprises the nucleotide sequences encoding VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NO 9-11, or the nucleotide sequences encoding VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NO 12-14, or

The nucleotide sequences encoding VHCDR1, VHCDR2 and VHCDR3 as shown in SEQ ID NOS 25-27 and the nucleotide sequences encoding VLCDR1, VLCDR2 and VLCDR3 as shown in SEQ ID NOS 28-30.

Alternatively, the nucleic acid comprises the nucleotide sequences encoding the heavy and light chain variable regions as set forth in SEQ ID NOS.15-16 or the nucleotide sequences encoding the heavy and light chain variable regions as set forth in SEQ ID NOS.31-32.

In a third aspect of the invention, there is provided an expression vector comprising a nucleic acid as described above.

In a fourth aspect of the invention, there is provided a transgenic cell line or recombinant bacterium comprising a nucleic acid as described above or an expression vector as described above.

In a fifth aspect of the invention, there is provided a composition comprising an antibody or antigen binding fragment thereof as described above, a nucleic acid as described above, an expression vector as described above or a transgenic cell line or recombinant bacterium as described above.

In a sixth aspect of the invention there is provided an immunoconjugate comprising an antibody moiety comprising an antibody as described above or an antigen binding fragment thereof and a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from a fluorescent substance, a chemiluminescent substance, a coloured substance, an enzyme, a small molecule drug or a combination thereof.

In a seventh aspect of the invention there is provided a detection reagent or kit for detecting an EV-D68 virus, the detection reagent or kit comprising an antibody or antigen-binding fragment thereof as described above.

In an eighth aspect, the invention provides the composition, the application of the immunoconjugate in preparing a detection reagent for detecting EV-D68 or diagnosing diseases caused by EV-D68 infection, or the application of the composition and the immunoconjugate in preparing a medicament for preventing or treating EV-D68 infection or diseases caused by EV-D68 infection.

Alternatively, for the above-mentioned applications, the method of detecting EV-D68 or diagnosing a disease caused by EV-D68 infection includes Western Blot or ELISA method.

According to the scheme, the invention discloses two enterovirus D group 68 VP1 antibodies, and also discloses sequences of all regions and application of the sequences in virus detection and the like. Related schemes prove that the two antibodies can specifically recognize highly conserved epitopes of EV-D68 viruses and can efficiently recognize all the existing EV-D68 virus strains. Based on the antibody, the invention establishes a broad-spectrum efficient detection method for detecting EV-D68 virus.

Drawings

FIG. 1 is SDS-PAGE analysis of purified D1 and D2 antibodies. Lane M protein marker, lane 1 monoclonal antibody D1, lane 2 monoclonal antibody D2.

FIG. 2 shows binding characteristics of EV-D68 mab. ELISA and Western Blot detection of the ability of monoclonal antibodies to recognize different antigen-inactivated Fermon virus, inactivated MO-18947 virus, inactivated MO-18953 virus, and the immunogen EV-D68 VLP (MO-18950). In the figure, A is ELISA detection, and in the figure, B is Western Blot detection.

FIG. 3 is an identification of linear B cell epitopes within EV-D68 capsid proteins. The reactivity of the entire sequence of VP1 (88 peptides) spanning EV-D68 with D1, D2 was tested by peptide ELISA. In the figure, A is D1, and in the figure, B is D2.

FIG. 4 shows the alignment of P24 peptide sequences with protein sequences of enterovirus EV-D68, CVA6, etc. The P24 peptide sequence is at the top and the dots represent the same residues as EV-D68.

FIG. 5 shows the localization of the EV-D68 epitope VP1-P24 on the surface of the viral pathogen. VP1-P24 is shown at the edge of the canyon region, where structure conservation is shown with VP1-P24 in blue. In the figure, VP1 of EV-D68 (PDB: 4WM8; blue) overlaps VP1 of EV71 (PDB: 3VBS; green), VP1 of EV-D68 and CVA10 (PDB: 6IIJ; pink) are similar in structure, and the arrow indicates epitope VP1-P24 of EV-D68. The figures are each generated by pymol3.0.3.

Detailed Description

The present invention provides antibodies and/or antigen binding fragments thereof capable of specifically binding enterovirus EV-D68.

The antibodies of the invention may be polyclonal, monoclonal, genetically engineered, and/or otherwise modified in nature, including monoclonal antibodies, fully human antibodies, bispecific antibodies, or multispecific antibodies, and the like. 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 various types including IgA (e.g., igA1 or IgA 2), igD, igE, igG (e.g., igG1, igG2, igG3, or IgG 4), igM, and the like. The invention also discloses antigen binding fragments of antibodies capable of specifically binding EV-D68. Examples of antigen binding fragments include F (ab ') 2, F (ab) 2, fab', fab, fv, fd, scFv, and the like.

For monoclonal antibodies of the invention, they may be obtained from a single clone by any means available or known in the art, including expression in any eukaryotic, prokaryotic, or phage system. Monoclonal antibodies for use in the present invention can be prepared using a wide variety of techniques known in the art, including hybridoma techniques, recombinant techniques, phage display techniques, or the like.

The embodiment of the invention discloses sequences of monoclonal antibodies of two enterovirus D group 68 VP1, wherein the two monoclonal antibodies are expressed by D1 and D2.

The sequence of monoclonal antibody D1 includes:

1-3 are amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2, CDR 3;

SEQ ID NO. 4-6 shows the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, CDR 3;

SEQ ID NO.7 is the amino acid sequence of the heavy chain variable region VH;

SEQ ID NO. 8 is the amino acid sequence of the light chain variable region VL;

SEQ ID NO. 33 is the amino acid sequence of the light chain constant region CL;

SEQ ID NO. 34 shows the amino acid sequence of the heavy chain constant region CH;

SEQ ID NOS 9-11 shows the nucleotide sequences of the heavy chain complementarity determining regions CDR1, CDR2, CDR 3;

12-14 are the nucleotide sequences of the light chain complementarity determining regions CDR1, CDR2, CDR 3;

SEQ ID NO. 15 is the nucleotide sequence of the heavy chain variable region VH;

SEQ ID NO. 16 is the nucleotide sequence of the light chain variable region VL;

SEQ ID NO. 36 is the nucleotide sequence of the light chain constant region CL;

SEQ ID NO. 37 shows the nucleotide sequence of the heavy chain constant region CH.

The sequence of monoclonal antibody D2 includes:

17-19 are amino acid sequences of heavy chain complementarity determining regions CDR1, CDR2, CDR 3;

20-22 are the amino acid sequences of the light chain complementarity determining regions CDR1, CDR2, CDR 3;

SEQ ID NO. 23 is the amino acid sequence of the heavy chain variable region VH;

SEQ ID NO. 24 is the amino acid sequence of the light chain variable region VL;

SEQ ID NO. 33 is the amino acid sequence of the light chain constant region CL;

SEQ ID NO. 35 is the amino acid sequence of the heavy chain constant region CH;

25-27 are the nucleotide sequences of the heavy chain complementarity determining regions CDR1, CDR2, CDR 3;

28-30 are the nucleotide sequences of the light chain complementarity determining regions CDR1, CDR2, CDR 3;

SEQ ID NO. 31 is a nucleotide sequence of the heavy chain variable region VH;

SEQ ID NO. 32 is the nucleotide sequence of the light chain variable region VL;

SEQ ID NO. 36 is the nucleotide sequence of the light chain constant region CL;

SEQ ID NO. 38 shows the nucleotide sequence of the heavy chain constant region CH.

For SEQ ID NOS.33-35 of the sequence Listing, the stop codon at the end of the sequence is not entered.

The products of other aspects of the invention also include intermediates in the preparation of antibodies, including expression vectors, transgenic cell lines or recombinant bacteria, or compositions comprising the same. In particular, for expression vectors, recombinant expression vectors such as eukaryotic expression vectors, and for transgenic cell lines or recombinant bacteria, eukaryotic or prokaryotic cells such as E.coli, yeast or animal cells (e.g.mammalian cells such as mouse cells, human cells, etc.), cell lines such as 293T cell lines are also included.

The invention further provides the use of the above antibodies or antigen binding fragments, intermediates, etc. for the preparation of a detection reagent for detecting EV-D68 or diagnosing a disease caused by EV-D68 infection.

Specifically, the present invention provides an immunoconjugate comprising an antibody moiety comprising an antibody of the invention or an antigen binding fragment thereof, and a coupling moiety coupled to the antibody moiety, the coupling moiety being selected from a fluorescent substance, a chemiluminescent substance, a colored substance, an enzyme, a small molecule drug, or a combination thereof.

In particular, the invention provides a kit or detection reagent for detecting EV-D68 virus, which comprises the antibody or antigen-binding fragment thereof of the invention. In some embodiments, the kit further comprises a solid substrate for attachment of antibodies or antigen binding fragments thereof, including, but not limited to, microwell plates, magnetic microparticles, filter papers for immunochromatography, polymers such as polystyrene, glass filters, and other insoluble carriers, and the like. In some embodiments, the kit further comprises a number of other components including, but not limited to, enzymes for labeling, corresponding substrates, radioisotopes, fluorescent substances, colored substances, buffers, and the like.

Detection of EV-D68 virus is performed using the above-described kit or detection reagent, and in some embodiments, the detection method comprises binding the antibody or antigen-binding fragment thereof of the present invention to a virus in a sample to be tested to form an antibody-virus or antibody fragment-virus complex, and then detecting the sample complex to determine whether the virus is present in the sample.

More specifically, in some embodiments, the detection reagent or kit comprises an antibody of the invention or an antigen-binding fragment thereof (as a primary antibody), and a detectable, labeled secondary antibody. The specific detection method comprises adsorbing a first antibody to a solid support, adding a sample to be tested possibly containing 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 is present.

The specific detection method may be any known detection method such as enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescent immunoassay, immunochromatography, competition method or sandwich method. Preferably, enzyme-linked immunosorbent assay (ELISA), western Blot (Western Blot) can be used.

The technical scheme of the invention is described in detail below with reference to examples.

The embodiment of the invention discloses a preparation and application scheme of a broad-spectrum EV-D68 virus detection antibody. Two EV-D68 binding positive hybridoma cell lines D1 and D2 are screened by immunizing Balb/C mouse EV-D68 VLP antigen and utilizing a hybridoma technology, and the antibody types are IgG1 and IgG2a respectively. And the nucleotide and amino acid sequences of the variable regions of the H chain and the L chain of the two monoclonal antibodies are obtained by nested PCR. Epitope identification shows that both monoclonal antibodies can recognize the conserved linear epitope P24 of EV-D68 embedded in the virus structure. Further experiments prove that the two antibodies can efficiently identify all the existing EV-D68 virus strains by using a Western Blot and ELISA method. The epitope sequences recognized by the two antibodies are found to be 98.5% conserved in all the uploaded EV-D68 virus strains of Genebank through NCBI database comparison. It can be seen that the invention discloses two antibodies capable of recognizing highly conserved epitopes of EV-D68 virus, and based on the antibodies, a broad-spectrum and high-efficiency Western Blot and ELISA detection method for detecting EV-D68 virus is established.

Experimental materials used in the examples of the present invention are available from commercial sources unless otherwise specified.

1. Materials and methods

1.1 Cells and viruses

Positive hybridoma cells were selected after immunization of 7 week old female Balb/C mice with EV-D68 VLP (virus-LIKE PARTICLE). The hybridoma cells were cultured in RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS), and the EV-D68 strain included Fermon strain, MO-18947 strain and MO-18953 strain. All viruses were expanded in RD cells (human embryonic kidney cells) and titrated by the 50% tissue culture infection dose (TCID 50) method.

1.2 Preparation of monoclonal antibody

Animal studies were approved by the animal welfare and use committee of the university of south Beijing medical science. All mice were purchased from Shanghai Laek laboratory animal Limited (SLAC, china).

First, 1ml of paraffin oil was intraperitoneally injected into 6-8 week old female Balb/C mice, thereby reducing rejection of the mice to vaccinated hybridoma cells. Meanwhile, the selected hybridoma monoclonal is amplified, and after one week, the hybridoma monoclonal is injected into a liquid paraffin induced BAL B/c mouse. The ascites fluid was then collected and the mAb purified using a Smart-LIFESCIENCES RPROTEIN A/G affinity chromatography column.

1.3 Antibody sequences

Cell count was collected from about 1×10 ⁶ hybridoma cell pellets, RNA from hybridoma cell samples was extracted by Trizol method, cDNA was obtained after reverse transcription, heavy and light chain variable regions were amplified and obtained, amplified products were library constructed and quality QC was performed, sequencing was performed using 3730xl, and alignment was performed with databases by bioinformatics analysis. Sequencing results show that the hybridoma cell strain antibody variable domain gene is successfully obtained.

1.4 Western immunoblotting experiments (Western Blot)

1) Sample preparation 60ul of purified EV-D68 virus was added to 12ul of 5 Xloading buffer, mixed well and cooked at 100℃for 10 minutes.

2) Gel electrophoresis SDS-PAGE gel protein electrophoresis of EV-D68 was performed.

3) Transferring membrane, taking out gel after electrophoresis, cutting off concentrated gel, and soaking the separating gel in transfer buffer solution. Soaking the prepared PVDF film in formaldehyde for activation, soaking 2 sheets of filter paper in a transfer buffer solution, stacking the filter paper, the PVDF film, the gel and the filter paper on the positive electrode plate in order, and covering the negative electrode plate for film transferring.

4) After the membrane transfer is completed, the PVDF membrane is taken out, put into a sealing dish, 30ml of 5% skimmed milk is added, and RT sealing is carried out for 2 hours.

5) Primary antibody incubation, removing the blocking solution, washing with PBST 5 times, 5min each time, adding primary antibody according to the concentration of 1:2000, and standing at 4 ℃ overnight.

6) Secondary antibody incubation, namely discarding the primary antibody, washing with PBST for 5 times, 5min each time, adding goat anti-mouse HRP secondary antibody according to the concentration of 1:10000, and incubating for 2 hours at RT.

7) And (3) ECL chemiluminescence and color development, namely PBST washing for 5 times, each time for 5min, uniformly covering the prepared ECL reagent on the PVDF film, carrying out luminous development, tabletting and exposing under the condition of a darkroom for 8s, developing and fixing.

1.5 Enzyme-Linked adsorption assay (ELISA)

For epitope identification of monoclonal antibodies, the elisa plate was coated with 1 μg/Kong Shange peptide and incubated at 37 ℃ for 2 hours, after blocking in 5% milk in PBS-Tween-20 (PBST), purified monoclonal antibody was added at 1:100 dilution, after washing with PBST, plate was conjugated with horseradish peroxidase (HRP) anti-mouse IgG (1:5000 dilution; sigma), absorbance of each well was measured at 450: 450 nm using an elisa after development.

Similarly, to observe the reactivity of monoclonal antibodies to different virus strains, inactivated virus or immunogen was coated at 20 ng/well, followed by binding of the antigen with the same concentration of monoclonal antibody, and the other steps were similar to those described above.

1.6 Alignment of sequences

Enterovirus strains used for sequence alignment include Coxsackie D68/Fermon (GenBank ID: AAR 98503.1), coxsackie D68/JPOC-378 (BAK 08578.1), coxsackie D68 NYC403 (AFM 73548.1), coxsackie D68/US/CO/14-93 (AIX 11070.1), coxsackie D68/US/MO/14-18947 (AIS 73051.1), coxsackie D68/US/KY/14-18953 (AIS 73057.1), coxsackie D68/US/MO/14-18950 (KM 851228.1), coxsackie D68 GAINESVILLE (AMB 37294.1), coxsackie D68/VR 7 (ALG 02131.1), coxsackie D68/DZH-ZJUC (QCE 20634.1) and Coxsackie D68/Ontario/PHL0008 (WXG 26576.1).

In addition, the study included rhinovirus strain Rhinovirus A-ATCC VR-1559 (YP_ 009505608.1), coxsackie virus A6/Gdula (AAR 38844.1), coxsackie virus A9/A242/YN/CHN (AJD 80555.1), coxsackie virus A10/S0273b (AOH 73257.1), coxsackie virus A16/YY157 (AGK 85257.1) and enterovirus 71 type EV71/HZ08 (AEB 71507.1).

All of these sequences are from the National Center for Biotechnology Information (NCBI) database. The figures are each generated by pymol3.0.3.

2. Results

2.1 Preparation of EV-D68 monoclonal antibody

To prepare EV-D68 monoclonal antibodies, positive hybridoma cells were selected after immunization of EV-D68 VLP (MO-18950) with 7-week-old female Balb/C mice. Positive hybridoma cells were subcloned by limiting dilution and 2 stable clones were obtained, named D1 and D2, respectively. Monoclonal antibodies were typed, with the monoclonal antibody D1 heavy chain belonging to the IgG1 subtype, the monoclonal antibody D2 heavy chain belonging to the IgG2a subtype, and the light chain all belonging to kappa, as shown in table 1.

TABLE 1 EV characterization of the D68 mab

Analysis of SDS-PAGE of monoclonal antibodies obtained by purifying mouse ascites shows that the heavy chain and the light chain of the D1 and the D2 antibodies are respectively positioned at 45kDa and 23kDa (figure 1), which shows that the prepared monoclonal antibodies respectively contain one heavy chain and one light chain, and conform to the basic structural characteristics of the antibodies.

2.2 Binding Properties of EV-D68 monoclonal antibody

Monoclonal antibodies were tested for their ability to recognize different antigens, including inactivated EV-D68 Fermon, EV-D68 MO-18947 and EV-D68 VLP (MO-18950) by ELISA. The results showed that both mAbs D1 and D2 were able to react with EV-D68 Fermon, EV-D68 MO-18947 and EV-D68 VLP (MO-18950), indicating that both of these 2 mAbs were EV-D68 specific. In addition, mab D1 showed stronger binding activity to EV-D68 MO-18947 than mab D2, whereas D2 showed stronger binding ability to Fermon and EV-D68 VLP (MO-18950) (A in FIG. 2).

The ability of monoclonal antibodies to recognize different antigens, including strain Fermon of EV-D68, strain MO-18947, and strain MO-18953, was also detected by Western Blot. The results show that both D1 and D2 recognize VP1 proteins of three different strains, indicating that all three antibodies are EV-D68 VP1 specific (B in FIG. 2).

2.3 Identification of linear B cell epitope of EV-D68 monoclonal antibody

Linear B cell epitopes of EV-D68 were identified by ELISA. Previously we screened a synthetic peptide library encompassing the entire VP1 sequence using serum from inactivated EV-D68 immunized mice. As shown in FIG. 3A, B, both D1 and D2 mab reacted strongly with VP 1-derived peptide # 24. These results indicate that the ELISA positive peptide contains B cell epitopes recognized by monoclonal antibodies D1 and D2, and also indicate that the D1 and D2 monoclonal antibodies contain the same linear B cell epitope recognized by 1 anti-EV-D68 antibody.

2.4 Sequence analysis and positioning of VP1-P24

Alignment of VP1 protein sequences of 11 representative strains of EV-D68 revealed that the VP1-P24 region (amino acid sequence at positions 70 to 84 of VP1, as shown in SEQ ID NO: 39) was highly conserved among various EV-D68 isolates (FIG. 4). In addition, a total of 67 sequences containing VP1-P24 regions were retrieved from NCBI EV-D68 database by BLAST analysis. The VP1-P24 epitope is completely conserved in 66 EV-D68 strains, and is relatively conserved in 1 EV-D68 strain, wherein the VP1-P24 epitope of ALG02131.1 is replaced by a single amino acid residue. However, there was a significant difference in amino acid sequence between the EV-D68 and other enterovirus VP1-P24 regions compared to other enteroviruses causing hand-foot-and-mouth disease (EV 71, CVA 10, CVA 6, etc.) (fig. 4).

The VP1-P24 region is highly exposed on the surface of the virion, at the southern isthmus (A in FIG. 5). Furthermore, as shown in FIG. 5B, the epitope VP1-P24 of EV-D68, VP1 of EV-D68 overlaps with VP1 of EV71, and VP1 structures of EV-D68 and CVA10 are similar. Taken together, these results indicate that VP1-P24 is highly conserved. Thus, the monoclonal antibodies D1 and D2 of the invention are considered to be a broad-spectrum monoclonal antibody useful for EV-D68 virus detection.

2.5 Sequences of anti-EV-D68 monoclonal antibodies

To determine the antibody sequences, RNA was extracted from the hybridoma cells, followed by RT-PCR, and the resulting cDNA was subjected to rapid amplification of the 5 'end (5' RACE). Sequencing results showed that the monoclonal antibodies D1 and D2 of the invention are related. The amino acid sequences of the CDR regions of the above monoclonal antibody heavy and light chains are shown in Table 2.

TABLE 2 amino acid and nucleotide sequences of the CDR regions

The invention has been described in detail in connection with the specific embodiments and exemplary examples thereof, but such description is not to be construed as limiting the invention. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, and these fall within the scope of the present invention. The scope of the invention is defined by the appended claims.

Claims

1. 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,

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 its antigen-binding fragment according to claim 1, characterized in that 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.

5. The EV-D68 antibody or its antigen-binding fragment 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 it 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,

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 it 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.

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 it 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 it 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 comprises Western Blot or ELISA method.