Antibodies targeting chicken egg drop syndrome virus and kits for detecting chicken egg drop syndrome

Abstract

The present application provides an antibody targeting chicken egg drop syndrome virus and a kit for detecting chicken egg drop syndrome. The antibody of the present application can recognize different strains of chicken egg drop syndrome virus, and does not cross-react with viruses such as fowl adenovirus I group type 4 (FAdV) and chicken infectious bronchitis virus (IBV). Therefore, it can be used to detect EDSV virus without cross-reacting with other viruses, and has high specificity and sensitivity.

Description

Technical Field

[0001] The present application belongs to the field of veterinary biotechnology detection technology, and specifically, relates to an antibody against chicken egg drop syndrome virus and a kit for detecting chicken egg drop syndrome. Background Art

[0002] Egg Drop Syndrome (EDS) is caused by the Egg Drop Syndrome virus (EDSV), which mainly causes a sudden drop in egg production in laying hens, accompanied by a decrease in egg quality, such as the appearance of thin-shelled eggs, sandy-shelled eggs, and shellless eggs. Since the disease was first reported in the Netherlands in 1967, EDS has been reported in many countries, including France, Italy, Brazil, India, Japan, Singapore, and North Korea. In 1991, the virus was isolated from a chicken farm in Nanjing, my country. At present, EDSV infection has been detected in chicken farms in many provinces and regions in my country, including Beijing, Shandong, Jiangsu, and Taiwan. In just a few years, it has become prevalent throughout the country, causing huge economic losses to the poultry industry. The natural hosts of EDSV are usually ducks and geese, but chickens of different ages and breeds can also be infected with EDSV. Most poultry, such as turkeys, sparrows, and quails, can also be infected with EDSV and can carry the virus. EDS can be transmitted vertically and through contact. It is usually not pathogenic before animals reach sexual maturity and is difficult to detect. After sexual maturity, the virus activates and causes the disease in laying birds. During an outbreak, the disease can also cause horizontal transmission of EDS through fecal-oral transmission.

[0003] EDSV is a non-enveloped linear double-stranded DNA virus belonging to the Adenoviridae family, genus avian adenovirus, group III. The EDSV strains isolated so far all belong to the same serotype, but there are different genotypes. There are many studies on the detection methods for EDSV, from etiological methods to immunological methods to molecular biological detection methods, and the sensitivity of the detection methods also varies. The detection methods included in national regulations include the chicken inspection method of the 2020 edition of the National Standard of the People's Republic of China, the Pharmacopoeia of the People's Republic of China (Part III). In the inspection of finished poultry-derived live vaccines, the chicken inspection method of the 2020 edition of the Pharmacopoeia of the People's Republic of China (Part III) is the hemagglutination inhibition method to detect viral antibodies. However, the above-mentioned statutory methods all have shortcomings. The EDSV hemagglutination inhibition test antibody detection must be obtained through the chicken inspection method to obtain chicken serum for detection, which is time-consuming and costly. EDSV contamination in vaccines is a potential risk for the spread and epidemic of EDS. Therefore, it is urgent to establish a stable and efficient method for detecting EDSV for quality inspection of live poultry vaccines and diagnosis of EDS diseases. Summary of the invention

[0004] In view of the shortcomings of the prior art in detecting EDSV, the purpose of the present application is to provide an antibody targeting chicken egg drop syndrome virus and a kit for detecting chicken egg drop syndrome virus.

[0005] Specifically, this application involves the following aspects:

[0006] 1. An antibody targeting chicken egg drop syndrome virus, wherein the antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein:

[0007] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6; or

[0008] The amino acid sequence of CDR-H1 is shown in SEQ ID NO:7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO:8, the amino acid sequence of CDR-H3 is shown in SEQ ID NO:9, the amino acid sequence of CDR-L1 is shown in SEQ ID NO:10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO:11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:12.

[0009] 2. The antibody targeting chicken egg drop syndrome virus according to item 1, wherein the antibody comprises a heavy chain variable region, wherein:

[0010] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO:13 or SEQ ID NO:14, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13 or SEQ ID NO:14.

[0011] 3. An antibody targeting chicken egg drop syndrome virus according to item 1 or 2, wherein the antibody comprises a light chain variable region, wherein:

[0012] The amino acid sequence of the light chain variable region is as shown in SEQ ID NO:15 or SEQ ID NO:16, or is an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15 or SEQ ID NO:16.

[0013] 4. An antibody targeting chicken egg drop syndrome virus according to any one of items 1 to 3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, wherein:

[0014] The amino acid sequence of the heavy chain variable region is such as SEQ ID NO:13, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:14, and the amino acid sequence of the light chain variable region is such as SEQ ID NO:15, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO:15; or

[0015] The amino acid sequence of the heavy chain variable region is such as SEQ ID NO:14, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:14; the amino acid sequence of the light chain variable region is such as SEQ ID NO:16, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:16.

[0016] 5. The antibody targeting chicken egg drop syndrome virus according to any one of items 1 to 4, wherein the antibody is a chimeric antibody, a humanized antibody or a fully human antibody.

[0017] 6. An isolated nucleic acid comprising a polynucleotide sequence encoding the antibody of any one of items 1-5.

[0018] 7. A vector comprising the nucleic acid described in item 6.

[0019] 8. A host cell comprising the nucleic acid described in item 6 or the vector described in item 7.

[0020] 9. A kit for detecting egg drop syndrome, wherein the kit comprises:

[0021] The antibody of any one of items 1-5.

[0022] 10. The kit according to item 9, wherein the kit further comprises fluorescently labeled goat anti-mouse IgG that recognizes the antibody according to any one of items 1 to 5.

[0023] 11. Use of the antibody described in any one of items 1 to 5 in the preparation of a kit for detecting chicken egg drop syndrome.

[0024] 12. Use of the antibody described in any one of items 1 to 5 in the preparation of a kit for detecting exogenous viruses in live avian virus vaccines.

[0025] The antibody of the present application can recognize different strains of egg drop syndrome virus, and does not cross-react with viruses such as fowl adenovirus group I type 4 (FAdV) and infectious bronchitis virus (IBV), so it can be used to detect EDSV virus without cross-reacting with other viruses, and has high specificity and sensitivity. The kit of the present application can be used for the detection of exogenous viruses in avian virus live vaccines, which is conducive to further ensuring the purity and safety of avian virus live vaccines in my country, thereby improving the quality of vaccines, and can also be used for clinical detection of EDSV, determination of virus content and epidemiological surveys. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph of small-scale protein expression, where M: Marker; 1: uninduced control (Codon plus); 2-3: IPTG-induced (Codon plus).

[0027] Figure 2 This is a diagram of protein mass expression, where M: Marker; 1: whole bacteria after ultrasound; 2: supernatant after ultrasound; 3: precipitate after ultrasound.

[0028] Figure 3 This is a protein purification diagram, where M: Marker; 1: 5-fold dilution of the purified protein; 2: 10-fold dilution of the purified protein.

[0029] Figure 4 shows the results of the indirect immunofluorescence kit test, where Figure 4A and Figure 4B Infected with different EDSV viruses: A is the Beijing 911 strain; B is the 127 strain. DETAILED DESCRIPTION

[0030] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0031] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0032] The term "antibody" herein is used in a broad sense, covering various antibody structural molecules that bind to EDSV and contain one or more CDR domains disclosed herein, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) and antibody fragments (e.g., Fv, Fab, Fab', Fab'-SH, F(ab')2), linear antibodies and single-chain antibody molecules (e.g., scFv), etc., as long as they exhibit the desired binding activity to EDSV.

[0033] The modifier "monoclonal" in the term "monoclonal antibody" as used herein means that the antibody is obtained from a substantially homogeneous antibody population and contains only trace amounts of naturally occurring mutations or mutations that occur during the preparation of the monoclonal antibody. Compared to polyclonal antibody preparations that typically include different antibodies against different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on the antigen. The monoclonal antibodies of the present application can be manufactured by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods, and methods using transgenic animals containing all or part of the human immunoglobulin loci.

[0034] A "chimeric antibody" is an antibody having at least a portion of a heavy chain variable region and at least a portion of a light chain variable region derived from one species and at least a portion of a constant region derived from another species. For example, in one embodiment, a chimeric antibody may comprise a murine variable region and a human constant region.

[0035] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise at least one, typically two substantially entire variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to those of non-human antibodies, and all or substantially all FRs correspond to those of human antibodies. Optionally, a humanized antibody may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody (e.g., a non-human antibody) refers to an antibody that has undergone humanization.

[0036] "Human antibodies" may also be referred to as "human antibodies", "fully human antibodies" or "fully human antibodies" and are antibodies whose amino acid sequences correspond to those produced by humans or human cells. This definition of human antibodies specifically excludes humanized antibodies comprising non-human antigen-binding residues. Human antibodies can be prepared using various techniques known in the art, including phage display library technology, such as those described in Hoogenboom and Winter, J. Mol. Biol., 227: 381 (1991); Marks et al., J. Mol. Biol., 222: 581 (1991). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol., 147 (1): 86-95 (1991). Human antibodies can be prepared by administering antigens to transgenic animals (e.g., immune xenogeneic mice) that have been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been disabled (for XENOMOUSE™ technology, see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584). For human antibodies produced via human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006).

[0037] In some embodiments, the antibody of the present application is a full-length antibody, which typically refers to an antibody composed of two "heavy chains" and two "light chains". A "heavy chain" is generally a polypeptide that consists of a heavy chain variable region (VH), a heavy chain constant region 1 (CH1), a hinge region (HR), an antibody heavy chain constant region 2 (CH2), and a heavy chain constant region 3 (CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH3; in some embodiments, a "full-length antibody heavy chain" is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. A "full-length antibody light chain" is generally a polypeptide consisting of a light chain variable region (VL) and a light chain constant region (CL) in the N-terminal to C-terminal direction, abbreviated as VL-CL.

[0038] In the present application, the light chain constant region and the heavy chain constant region can be any light chain constant region and heavy chain constant region. The light chain constant region (CL) can be κ (kappa) or λ (lambda). The heavy chain constant region can be any one of the heavy chain constant regions of IgG, IgM, IgA, IgE, and IgD.

[0039] It is well known to those skilled in the art that each heavy chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs), and each light chain variable region can be composed of three complementary determining regions (CDRs) and four framework regions (FRs). The complementary determining regions (CDRs, usually CDR1, CDR2, and CDR3) are the regions in the variable region that have the greatest impact on the affinity and specificity of the antibody. In some embodiments, from the N-terminus to the C-terminus, the heavy chain variable region and the light chain variable region both include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0040] The CDR sequences of the heavy chain variable region or the light chain variable region can be identified by reference to any numbering system known in the art, such as the Kabat system (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991); the Chothia system (Chothia and Lesk, "Canonical Structures for the Hypervariable Regions of Immunoglobulins", J. Mol. Biol. 196, 901-917 (1987)); or the IMGT system (Lefranc et al., "IMGT Unique Numbering for Immunoglobulin and Cell Receptor Variable Domains and Ig Superfamily V-like Domains"). s and Ig superfamily V-likedomains), "Dev. Comp. Immunol.", 27, 55-77 (2003). In the embodiments of the present application, the CDR sequence is defined and determined using the IMGT system.

[0041] As used herein, the term "identity" may be defined as the percentage of identical residues in amino acid or nucleotide sequence variants after alignment and introduction of gaps. Methods and computer programs for alignment are well known in the art. As used herein, an amino acid sequence that is referenced to have a percentage identity with an amino acid sequence refers to a sequence that has that percentage identity over the entire length of the referenced amino acid sequence.

[0042] In this article, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host, which transfers the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used to express the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell that contains the vector.

[0043] In this article, the term "nucleic acid" or "polynucleotide" or "nucleic acid molecule" generally refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers in single-stranded or double-stranded form thereof. Unless otherwise specified, the term may include nucleic acids containing analogs of natural nucleotides, which have similar binding properties to reference nucleic acids (e.g., sequence information is shown) and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specified, the sequence of a nucleic acid may include variants modified in a conservative manner thereof, such as degenerate codon substitutions, alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0044] As used herein, "affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, "binding affinity" as used in this specification refers to the intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant (K D Affinity can be determined by common methods known in the art.

[0045] Herein, the term "antibody targeting EDSV" refers to an antibody that can bind to EDSV with sufficient affinity and can be used as a diagnostic agent and / or a therapeutic agent targeting EDSV.

[0046] The antibody targeting EDSV of the present application does not bind to proteins unrelated to the target. Here, "unrelated protein" refers to other proteins other than the EDSV protein as the target; Here, "unrelated" means: when the binding ability of the antibody targeting EDSV protein of the present application to the EDSV protein as its target is taken as 100%, the binding ability of the antibody targeting EDSV protein of the present application to the unrelated protein is less than 10%, for example, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.

[0047] In this article, the indirect immunofluorescence test is to use a specific antibody to react with the corresponding antigen in the specimen, then use a fluorescein-labeled second antibody (anti-antibody) to bind to the first antibody in the antigen-antibody complex, and after washing, observe the specific fluorescence under a fluorescence microscope to detect unknown antigens or antibodies.

[0048] The present application provides an antibody targeting EDSV, wherein the antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3).

[0049] In some embodiments, the amino acid sequence of CDR-H1 is as shown in SEQ ID NO:1 (GYTFTEYT), the amino acid sequence of CDR-H2 is as shown in SEQ ID NO:2 (FNPKNGVT), the amino acid sequence of CDR-H3 is as shown in SEQ ID NO:3 (VRLEVRVYAMDY), the amino acid sequence of CDR-L1 is as shown in SEQ ID NO:4 (QSLLYSNIQKNY), the amino acid sequence of CDR-L2 is as shown in SEQ ID NO:5 (WAS), and the amino acid sequence of CDR-L3 is as shown in SEQ ID NO:6 (QQYYSYPFT).

[0050] In some embodiments, the amino acid sequence of CDR-H1 is as shown in SEQ ID NO:7 (GFTFSGSA), the amino acid sequence of CDR-H2 is as shown in SEQ ID NO:8 (INTGGQLT), the amino acid sequence of CDR-H3 is as shown in SEQ ID NO:9 (AKTYYGNYDY), the amino acid sequence of CDR-L1 is as shown in SEQ ID NO:10 (QRIVHNNGNTY), the amino acid sequence of CDR-L2 is as shown in SEQ ID NO:11 (KVS), and the amino acid sequence of CDR-L3 is as shown in SEQ ID NO:12 (FQGSHVPLT).

[0051] In some embodiments, the antibody comprises a heavy chain variable region, wherein: the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 13

[0052] (EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKTLE WI),

[0053] or SEQ ID NO:14

[0054] (EVQLVESGGGLVRPGGSLKLSCVASGFTFSGSAMSWVRQTPEKRLEW VATINTGGQLTYYPDSVKGRFTISRDNAKNTLYLQMSRLRSEDTAMYYCAKT YYGNYDYWGQGTTLTVSS),

[0055] Or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13 or SEQ ID NO:14.

[0056] In some embodiments, the antibody comprises a light chain variable region, wherein: the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 15

[0057] (DIVMSQSPSSLDVSVGEKVTLSCKSSQSLLYSNIQKNYLAWYQQKPG QSPKLLIYWASVRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYS YPFTFGSGTKLEIK),

[0058] or SEQ ID NO:16

[0059] (DVLMTQTPLSLPVSLGDQASISCRSSQRIVHNNGNTYLEWYLQKPGQ SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP LTFGSGTKLELK),

[0060] Or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15 or SEQ ID NO:16.

[0061] In some embodiments, the amino acid sequence of the heavy chain variable region is such as SEQ ID NO:13, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13, and the amino acid sequence of the light chain variable region is such as SEQ ID NO:15, or an amino acid sequence that is 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:15.

[0062] In some embodiments, the amino acid sequence of the heavy chain variable region is such as SEQ ID NO:14, or an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:14, and the amino acid sequence of the light chain variable region is such as SEQ ID NO:16, or an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:16.

[0063] The present application also provides an isolated nucleic acid comprising a polynucleotide sequence encoding any of the aforementioned antibodies.

[0064] The present application also provides a vector comprising the above-mentioned nucleic acid.

[0065] The present application also provides a host cell, which comprises the above-mentioned nucleic acid or vector.

[0066] The present application also provides a kit for detecting egg drop syndrome, which can detect the presence and / or amount of EDSV, wherein the kit comprises any one of the above antibodies.

[0067] In some embodiments, the kit further comprises a fluorescently labeled antibody that recognizes any one of the above antibodies, such as goat anti-mouse IgG.

[0068] In some embodiments, the kit may further include a sample diluent and a washing solution.

[0069] In some embodiments, the kit may further include, for example, a buffer, a preservative, or a protein stabilizer.

[0070] In some embodiments, the kit may further comprise components necessary for detecting the detectable label.

[0071] In some embodiments, the kit may further include a container, instructions for use, and the like.

[0072] In some embodiments, the kit may also contain a control sample or a series of control samples that can be assayed and compared to the test sample. Each component of the kit may be contained in a separate container, and all multiple containers may be placed in a single package, with instructions on how to use the kit, e.g., instructions for collecting samples and / or performing tests and / or analyzing results, written on the package insert.

[0073] In some embodiments, the kit includes any one of the above-mentioned antibodies targeting EDSV, fluorescently labeled goat anti-mouse IgG that recognizes the antibody targeting EDSV, a sample diluent, and a washing solution.

[0074] The present application also provides the use of any one of the above antibodies in preparing a kit for detecting chicken egg drop syndrome.

[0075] The present application also provides the use of any one of the above antibodies in the preparation of a kit for detecting exogenous viruses in live avian virus vaccines.

[0076] The avian virus live vaccine can be various types of live vaccines available in the art, such as fowlpox live vaccine, Marek's disease turkey herpes live vaccine, Muscovy duck parvovirus, gosling plague bivalent live vaccine, Marek's disease type I, III bivalent live vaccine, Newcastle disease live vaccine, avian encephalomyelitis, fowlpox bivalent live vaccine, infectious bronchitis live vaccine, and infectious laryngotracheitis recombinant fowlpox virus genetic engineering vaccine.

[0077] Example

[0078] Example 1 - EDSV Hexon protein sequence analysis

[0079] Different EDSV strains have only one serotype. The reference sequences of different EDSV strains were downloaded from NCBI. Through analysis, it was found that Hexon protein has high homology in different strains. DNAStar software was used to analyze the secondary structure, hydrophilicity, antigenicity and functional region of Hexon protein. The results showed that Hexon protein is highly conserved, with very low antigenicity differences, and can be used as an alternative fragment for expressing protein. Based on the secondary structure analysis, hydrophilicity, antigenicity, etc. of the protein sequence, Hexon protein was selected as the immunogen. According to the protein structure analysis, the nucleotide fragment (1707bp) of the full-length Hexon protein was selected for sequence synthesis (sequence 1), and EcoRⅠ and Xho I restriction sites were added at the 5' and 3' ends for vector cloning. Sequence synthesis was carried out at Beijing Liuhe BGI Protein Research and Development Center Co., Ltd.

[0080] Example 2 - Construction and purification of recombinant expression plasmid

[0081] 1. Construction of recombinant expression plasmid

[0082] The synthetic sequence and plasmid pET30a were double-digested with restriction endonucleases EcoRⅠ and Xho I, respectively. The purified and recovered fragments and the digested products of the expression vector were connected with a DNA Ligation Kit and transformed into competent cells (Codonplus).

[0083] 2. Small-scale expression of recombinant proteins

[0084] The clones identified as positive by PCR were picked and placed in 1.5 mL of LB liquid medium containing the corresponding resistance, and cultured at 37°C, 200 r / min; cultured until OD = 0.6-0.8, induced by IPTG (0.5 mM), cultured at 37°C, 200 r / min for 2 h; 1 mL of induced bacterial solution was taken, centrifuged at 12000 r / min for 1 min, the supernatant was discarded, and the precipitate was blown off with 50-100 μL Tris-HCl (pH 8.0) solution (the amount of buffer added depends on the amount of bacteria), and 2× loading buffer with the same volume as the buffer was added, and electrophoresis was performed at 100°C for 5 min. The results showed that a specific target band of the recombinant Hexon protein appeared at a size of about 69 KD, which was consistent with the expected size (such as Figure 1 ), indicating that Hexon protein was successfully expressed.

[0085] 3. Large-scale expression of recombinant proteins

[0086] The cultured bacterial suspension was transferred to 250 mL of the corresponding resistance LB liquid medium at a ratio of 1:50, and cultured at 37°C with a shake of 200 r / min until OD = 0.6-0.8, and induced with IPTG (0.5 mM) at 16°C overnight; harvesting bacteria: centrifugation at 8000 r / min for 6 min. Discard the supernatant; ultrasonic bacterial disruption: the bacterial bodies were blown away with 20-30 ml of 10 mM Tris-HCl (pH 8.0) solution, and ultrasonically broken (500 W, 180 times, 5 s each time, 5 s interval); electrophoresis was used to determine the expression form: 100 μL of the ultrasonic bacterial suspension was taken, centrifuged at 12000 r / min for 10 min, 50 μL of the supernatant was taken to another EP tube, the supernatant was discarded, and the precipitate was blown away with 50 μL of 10 mM Tris-HCl (pH 8.0) solution. The supernatant and precipitate were taken for SDS-PAGE detection, and a large amount of target protein (such as Figure 2 As shown), indicating that the expression form of the recombinant bacteria is inclusion body expression. The expressed protein was purified as follows: 20-30 ml 10mM Tris-HCl (pH8.0) solution was used to resuspend the precipitate obtained by ultrasonic centrifugation, and the mixture was allowed to stand for 10 minutes; 12000 r / min, centrifuged for 10 minutes, and the supernatant was transferred to another tube for storage; 20-30 ml 10mM Tris-HCl (pH8.0) solution was used to resuspend the precipitate, and the mixture was allowed to stand for 10 minutes; 12000 r / min, centrifuged for 10 minutes, and the supernatant was discarded. Repeat the above resuspension and centrifugation steps once; first add a small amount of 10mM Tris-HCl (pH8.0) solution to resuspend the precipitate, and then add 5-10 mL of 10mM Tris-HCl (pH8.0) solution containing 8M urea to dissolve the protein; 12000 r / min, centrifuged for 10 minutes, collected the supernatant, and took 50 μL of the sample for SDS-PAGE electrophoresis detection (the results are shown in Figure 3 As shown). Using BSA as the standard, the purified protein concentration was estimated to be >1.0 mg / mL and the purity was >85% by SDS-PAGE gel scanning analysis, and was named EDSV-Hexon.

[0087] Example 3 - Preparation of monoclonal antibodies

[0088] 1. Immunization of Mice

[0089] After emulsifying the purified protein of EDSV-Hexon with Freund's complete adjuvant in a ratio of 1:1, 4 SPF BALB / c female mice were subcutaneously injected at a dose of 30 μg protein / mouse (primary immunization). Subcutaneous booster immunization was performed 2 weeks, 4 weeks, and 6 weeks after the initial immunization, with an immunization dose of 30 μg / mouse. Blood was collected from the eye sockets 10 days after the third booster immunization, and the serum titer was measured by indirect ELISA. Mice with high serum titers were selected and immunized once with 50 μg of the immunogen by intraperitoneal injection. Cell fusion was performed 3 days after immunization.

[0090] 2. Cell fusion assay

[0091] The spleen of the immunized mouse was aseptically obtained and a spleen cell suspension was prepared. The immune spleen cells and SP2 / 0 cells (stored in this laboratory) were fused using the conventional 50% PEG method, and the fused cells were placed in 5 96-well plates and selectively cultured with HAT culture medium (purchased from Sigma).

[0092] 3. Cloning and screening of hybridoma cells

[0093] The culture in the above 96-well plate was screened by ELISA plate coated with EDSV-Hexon protein and tag protein. The screening method is as follows:

[0094] (1) Coating ELISA plate

[0095] The purified EDSV-Hexon protein was diluted with sodium carbonate-sodium bicarbonate buffer at pH 9.6 to a final concentration of 2 μg / mL, 100 μL / well, 4°C, overnight; then washed three times with PBST (PBS containing 0.05% Tween).

[0096] (2) Closed

[0097] The cells were blocked with PBS containing 2% milk, 200 μL / well, incubated at 37°C for 2 h, and then washed three times with PBST (PBS containing 0.05% Tween).

[0098] (3) Incubation with primary antibody

[0099] Hybridoma cell culture supernatant, negative control (SP2 / 0 culture supernatant), blank control (PBS), and positive control (positive serum diluted 1000 times with PBS) were added as primary antibodies, all at 100 μL / well, and incubated at 37°C for 1 h.

[0100] (4) Washing

[0101] The cells were washed three times with PBST (PBS containing 0.05% Tween).

[0102] (5) Incubation with secondary antibody

[0103] Goat anti-mouse IgG / HRP diluted 20,000-fold with PBS was added as secondary antibody, 100 μL / well, and incubated at 37°C for 1 h.

[0104] (6) Washing

[0105] The cells were washed three times with PBST (PBS containing 0.05% Tween).

[0106] (7) Color rendering

[0107] Add 100ul / well of color developing solution (citrate buffer containing 1% solution A and 10% solution B, solution A: 1% TMB in DMSO; solution B: 0.1% H2O2) and the color developing time is about 5 minutes.

[0108] (8) Add 50 μL of stop solution (containing 2 M sulfuric acid) to each well to stop the reaction.

[0109] (9) Reading

[0110] Measure the absorbance at dual wavelengths (450, 630nm) and record and save the data.

[0111] After testing, two ELISA-positive hybridoma cell lines, 7# and 9#, were screened out from 328 hybridoma cell lines.

[0112] The screening results are shown in Table 1.

[0113] Table 1 ELISA screening results of hybridoma cell lines

[0114]

[0115] 4. Indirect Immunofluorescence Detection

[0116] The supernatants of the two positive hybridoma cell lines (No. 7 and No. 9) obtained by ELISA screening were used as primary antibodies, and a cell line with good reactivity to the whole virus of different strains of EDSV was screened by indirect immunofluorescence. The cell line was named EDSV-Hexon protein hybridoma cell Mab-Hexon-9. The cell lines that were positive in immunofluorescence detection were selected for subcloning by limiting dilution method until the cell clone antibody positive rate reached 100%. The positive cell lines with a positive rate of 100% after subcloning were expanded and cultured and stored in liquid nitrogen. The indirect immunofluorescence detection method is as follows:

[0117] (1) Preparation of positive virus plates

[0118] The virus solutions of different EDSV strains (the information of different strains is shown in Table 2) were diluted to 100 TCID 50 / 0.1ml, inoculate 96-well plates with well-grown chicken embryo liver cells (prepared in our laboratory), inoculate 4 wells of each strain, 100μL / well, and set up blank controls. Then, culture in an incubator at 37°C with 5% CO2 for 5 days and perform fluorescence staining.

[0119] Table 2 Information of different EDSV strains and control strains

[0120]

[0121] The above virus strains are from the Veterinary Microbiology Center of the China National Microbiological Culture Collection Administration (see China Veterinary Drug Administration, China National Veterinary Culture Collection Administration Veterinary Microbiology Center, Catalogue of Chinese Veterinary Species, First Edition, Chinese-English Combined Volume 1992, Science and Technology Press of China, pages 125-127, 132, 137 and 151); avian leukosis virus (ALV) RAV-1 strain is from the China National Veterinary Drug Administration (Mao Yaqing, Wang Jia, Wu Tao, Wang Zhe, Jiang Taozhen, Expression of avian leukosis virus p27 protein in Escherichia coli and preparation of polyclonal antibodies, Chinese Journal of Veterinary Drugs, Issue 11, 2013, pp. 61-66).

[0122] (2) Dyeing

[0123] 1) Fixed

[0124] Discard the cell culture medium in the 96-well plate, add about 250 μL of cell culture medium to each well, gently wash the cell surface once, discard PBS as much as possible, then add 100 μL of cold methanol to each well, fix at room temperature for 10 to 15 minutes, discard the methanol, and dry naturally for 2 to 5 minutes.

[0125] 2) Add primary antibody (monoclonal antibody) and wash the cell surface once with PBS (pH 7.2). Dilute the supernatant of the monoclonal cell line to be tested 10 times with PBS and add it to the EDSV virus-positive cell plate (washed once with PBS before use), 50 μL per well, and incubate at 37°C in the dark for 1 hour.

[0126] 3) Washing

[0127] The monoclonal antibodies in the wells were discarded, and the wells were washed 5 times with PBS, adding 0.3 mL of washing solution to each well each time, and washing was performed with slight shaking.

[0128] 4) Fluorescent secondary antibody staining

[0129] Discard the washing solution as much as possible, add 50 μL of fluorescently labeled goat anti-mouse IgG appropriately diluted with PBS (1:100-1:200) to each well, and incubate at 37°C in the dark for 1 hour.

[0130] 5) Washing

[0131] Same method as 3).

[0132] 6) Observation and result determination

[0133] Under a fluorescent inverted microscope, the cells were observed with blue excitation light (wavelength 490nm), and the cells had complete cell morphology. When specific green fluorescence appeared in the field of view of the inoculated well, and when it was magnified to 200-400 times, the nucleus and cytoplasm of the infected cells were both stained, the well was determined to be positive for EDSV detection. When the inoculated well did not show specific green fluorescence, the field of view was dark, proving that the cells were not infected, and the well was determined to be negative for EDSV detection.

[0134] 5. Identification of hybridoma cells

[0135] (1) Culture characteristics

[0136] Use DMEM culture medium containing 10% to 15% fetal bovine serum, culture in a 37°C, 5% CO2 incubator, and examine the cell morphology of the hybridoma cell line under a microscope. The cell morphology should be consistent.

[0137] (2) Purity test

[0138] Sterility test, mycoplasma test and exogenous virus test are carried out according to the appendix methods of the current "Chinese Pharmacopoeia of Veterinary Medicine" (compiled by the Chinese Pharmacopoeia Committee, Pharmacopoeia of Veterinary Medicine of the People's Republic of China, 2020 edition, China Agriculture Press, 2020, referred to as "Chinese Pharmacopoeia" in this invention).

[0139] (3) Cytological examination

[0140] The hybridoma cells cultured for 24 hours were examined for chromosome number using the colchicine method to observe whether the chromosome characteristics were consistent with the staining characteristics of the hybridoma cells.

[0141] (4) Preparation of ascites

[0142] 8-10 week old BALB / C mice were intraperitoneally injected with 0.5 ml of pristane per mouse. 7-10 days later, the mice were intraperitoneally injected with 10 hybridoma cells. 6 ~10 7 / 0.5ml / mouse. After 7-10 days, observe the state of the mice. When the abdomen of the mice is obviously swollen and the mice are inconvenient to move, extract the mouse ascites, centrifuge at 3000r / min for 10min, take the supernatant, and store at -40℃. After an interval of 2-3 days, if ascites is produced again, it can be collected again. This ascites is the mouse anti-EDSV Hexon monoclonal antibody. Two monoclonal antibodies were obtained, of which the first monoclonal antibody corresponds to the 7# hybridoma cell line, and the second monoclonal antibody corresponds to the 9# hybridoma cell line.

[0143] The first monoclonal antibody:

[0144] The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1 (GYTFTEYT), the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2 (FNPKNGVT), the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3 (VRLEVRVYAMDY), the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4 (QSLLYSNIQKNY), the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5 (WAS), and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6 (QQYYSYPFT);

[0145] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13 (EVQLQQSGPELVKPGASVKISCKTSGYTFTEYTMHWVKQSHGKTLEWI), and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 15 (DIVMSQSPSSLDVSVGEKVTLSCKSSQSLLYSNIQKNYLAWYQQKPGQSP KLLIYWASVRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYSYPFT FGSGTKLEIK).

[0146] Second monoclonal antibody:

[0147] The amino acid sequence of CDR-H1 is shown in SEQ ID NO:7 (GFTFSGSA), the amino acid sequence of CDR-H2 is shown in SEQ ID NO:8 (INTGGQLT), the amino acid sequence of CDR-H3 is shown in SEQ ID NO:9 (AKTYYGNYDY), the amino acid sequence of CDR-L1 is shown in SEQ ID NO:10 (QRIVHNNGNTY), the amino acid sequence of CDR-L2 is shown in SEQ ID NO:11 (KVS), and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:12 (FQGSHVPLT).

[0148] The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 14

[0149] The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 16.

[0150] (DVLMTQTPLSLPVSLGDQASISCRSSQRIVHNNGNTYLEWYLQKPGQ SPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP LTFGSGTKLELK).

[0151] (5) Ascites titer determination

[0152] The ascites of each monoclonal antibody was diluted 2-fold from 1:100 to 1:51200, and the fluorescent antibody titer of the ascites was determined by indirect immunofluorescence detection method according to 4. in Example 3, which was 1:2000.

[0153] Example 4 - Assembly and application of indirect immunofluorescence kit for detecting EDSV

[0154] In this example, the two monoclonal antibodies obtained in Example 3 were assembled into kits and cited.

[0155] The kit components include anti-EDSV monoclonal antibody, commercial FITC-labeled goat anti-mouse antibody (purchased from Sigma), sample diluent and washing solution.

[0156] The steps and criteria for detecting the egg drop syndrome virus in the kit are as follows:

[0157] 1. Sample Inoculation

[0158] 100 μL of the treated sample was inoculated into a chicken embryo liver cell monolayer covering a 96-well plate, incubated at 37°C for 1 hour, and then 100 μL of M199 culture medium containing 2% bovine serum was added to each well and cultured at 37°C for 5-7 days.

[0159] 2. Fluorescence staining and result determination

[0160] (1) Fixed

[0161] Discard the cell culture medium in the 96-well plate, add about 0.3 mL PBS (pH 7.2) to each well to gently wash the cell surface once, discard the PBS as much as possible, then add 0.2 mL cold methanol to each well, fix at room temperature for 15 minutes, discard the methanol, and dry naturally (5 minutes).

[0162] (2) Add primary antibody

[0163] After natural drying, the cell surface was washed once with PBS (pH 7.2), and then 50 μL of EDSV monoclonal antibody appropriately diluted with PBS (pH 7.2-7.4) was added to each well and incubated at 37° C. for 1 h.

[0164] (3) Washing

[0165] The EDSV monoclonal antibody was discarded, and the wells were washed five times with PBS (pH 7.2), adding 0.3 mL of washing solution to each well each time, and washing was performed with slight shaking.

[0166] (4) Fluorescent secondary antibody staining

[0167] Discard the washing solution as much as possible, add 50 μL of FITC-labeled goat anti-mouse IgG appropriately diluted with PBS (pH 7.2-7.4) to each well, and incubate at 37°C for 1 hour.

[0168] (5) Washing

[0169] The method is the same as (3).

[0170] (6) Observation and judgment

[0171] Under a fluorescent inverted microscope, the cells were observed with blue excitation light (wavelength 490nm), and the cells had complete cell morphology. When specific green fluorescence appeared in the field of view of the inoculated well, and when it was magnified to 200-400 times, the nucleus and cytoplasm of the infected cells were both stained, the well was determined to be positive for EDSV detection. When the inoculated well did not show specific green fluorescence, the field of view was dark, proving that the cells were not infected, and the well was determined to be negative for EDSV detection.

[0172] 3. Test kit specificity

[0173] According to the established indirect immunofluorescence operation method, the indirect immunofluorescence kit was used to detect different EDSV (Beijing 911 strain, 127 strain), fowl adenovirus I group type 4 (FAdV-4), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), avian leukosis virus (ALV), avian reticuloendotheliosis virus (REV), infectious laryngotracheitis virus (ILTV), infectious bursal disease virus (IBDV) and chicken embryo liver cells without virus inoculation as a control, and the coloring of the virus-infected cells was observed to determine the specificity of the indirect immunofluorescence method.

[0174] The test results using the first monoclonal antibody are shown in Figure 4, and the results show that the kit can specifically identify and detect different EDSV strains (see AB in Figure 4, where: A is Beijing 911 strain; B is 127 strain, and the reaction results are all positive), while the reactions with FAdV-4, NDV, IBV, ALV, REV, ILTV, IBDV and chicken embryo liver cells that are not inoculated with the virus are all negative, proving that the established method has good specificity, and the indirect immunofluorescence kit can be applied to the specific detection of EDSV. The test results using the second monoclonal antibody are similar, and different EDSV strains (Beijing 911 strain, 127 strain) can also be specifically identified and detected, while FAdV-4, NDV, IBV, ALV, REV, ILTV, IBDV and chicken embryo liver cells that are not inoculated with the virus do not react.

[0175] 4. Sensitivity test of the kit

[0176] EDSV (Beijing 911 strain) was diluted to 10 7.0 TCID 50 / 100μL, and based on this, 10-fold serial dilutions were performed to 10 0.001 TCID 50 / 100 μL, the above 8 dilution samples were inoculated into chicken embryo liver cells that covered the monolayer, 100 μL / well, and 6 replicates for each sample. At the same time, an uninoculated chicken embryo liver cell group was set as a negative control. Detection was performed according to the steps and criteria for detecting EDSV in Example 4.

[0177] Results The EDSV test results at each dose gradient were as follows: when the infection dose was greater than or equal to 10 0.01 TCID 50 The above results show that the minimum detection limit of EDSV contamination of this kit is 10 0.01 TCID 50 .

[0178] 5. Initial application of the kit

[0179] This kit was applied to the detection of exogenous viruses in live vaccines of poultry viruses. In this experiment, key varieties of live vaccines for poultry produced by 10 domestic manufacturers were selected, and EDSV detection was performed according to the kit of this patent, and serological tests for EDSV contamination were performed on the selected live vaccines for poultry diseases according to the third edition of the 2020 edition of the Chinese Pharmacopoeia of Veterinary Medicine. At the same time, the PBS group was set as a negative control, and the group infected with EDSV (Beijing 911 strain) was set as a positive control. The results showed that the results of the serological test were consistent with those of the test using this kit. The selected live vaccines for poultry were not contaminated with EDSV, the negative control was negative for EDSV detection, and the positive control was positive for EDSV detection. The vaccine information and test results are shown in Table 3, among which the test results of the two monoclonal antibodies were the same, as shown in Table 3.

[0180] Table 3 Test results of vaccines from domestic and foreign companies using the kit of this application

[0181]

[0182]

Claims

1. An antibody targeting egg drop syndrome virus (EDSV), wherein: The antibody comprises three heavy chain complementary determining regions (CDR-H1, CDR-H2, CDR-H3) and three light chain complementary determining regions (CDR-L1, CDR-L2, CDR-L3), wherein: The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 1, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 2, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 3, the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 4, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 5, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO: 6; or The amino acid sequence of CDR-H1 is shown in SEQ ID NO: 7, the amino acid sequence of CDR-H2 is shown in SEQ ID NO: 8, the amino acid sequence of CDR-H3 is shown in SEQ ID NO: 9, the amino acid sequence of CDR-L1 is shown in SEQ ID NO: 10, the amino acid sequence of CDR-L2 is shown in SEQ ID NO: 11, and the amino acid sequence of CDR-L3 is shown in SEQ ID NO:

12.

2. The antibody targeting chicken egg drop syndrome virus according to claim 1, wherein The antibody comprises a heavy chain variable region, wherein: The amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 13 or SEQ ID NO: 14, or is an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 13 or SEQ ID NO:

14.

3. The antibody targeting chicken egg drop syndrome virus according to claim 1 or 2, wherein: The antibody comprises a light chain variable region, wherein: The amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 15 or SEQ ID NO: 16, or is an amino acid sequence with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 15 or SEQ ID NO:

16.

4. The antibody targeting chicken egg drop syndrome virus according to claim 1 or 2, wherein: The antibody comprises a heavy chain variable region and a light chain variable region, wherein: The amino acid sequence of the heavy chain variable region is such as SEQ ID NO: 13, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is such as SEQ ID NO: 15, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with SEQ ID NO: 15; or The amino acid sequence of the heavy chain variable region is such as SEQ ID NO: 14, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 14, and the amino acid sequence of the light chain variable region is such as SEQ ID NO: 16, or an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:

16.

5. The antibody targeting chicken egg drop syndrome virus according to claim 1 or 2, wherein: The antibody is a chimeric antibody.

6. An isolated nucleic acid comprising a polynucleotide sequence encoding the antibody of any one of claims 1-5. A vector comprising the nucleic acid according to claim 6.

8. A host cell comprising the nucleic acid of claim 6 or the vector of claim 7.

9. A kit for detecting egg drop syndrome, wherein: The kit comprises: The antibody according to any one of claims 1 to 5.

10. The kit according to claim 9, wherein The kit further comprises fluorescently labeled goat anti-mouse IgG that recognizes the antibody of any one of claims 1-5.

11. Use of the antibody according to any one of claims 1 to 5 in preparing a kit for detecting chicken egg drop syndrome.

12. Use of the antibody according to any one of claims 1 to 5 in the preparation of a kit for detecting foreign viruses in live avian virus vaccines.

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