Preparation and application of monoclonal antibody 2B3-H4-G8 against 100K protein of serotype 4 fowl adenovirus

Abstract

The application discloses preparation and application of a 100K protein monoclonal antibody 2B3-H4-G8 of a serum 4 type avian adenovirus. The application provides a hybridoma cell strain 2B3-H4-G8 secreting FADV4 antibody, and the preservation number is CCTCC NO: C2022177. The application also provides the antibody secreted by the hybridoma cell strain 2B3-H4-G8 and application of the antibody. The application obtains the hybridoma cell strain 2B3-H4-G8 secreting the monoclonal antibody. The monoclonal antibody secreted by the hybridoma cell strain 2B3-H4-G8 has positive reaction with GX2019-010 and 18 FAdV-4 isolates, but has negative reaction with FAdV-4 CACC AV211. The prepared 100K recombinant protein monoclonal antibody has good reaction originality and specificity, and lays a foundation for research and development of FAdV-4 antigen and antibody detection reagents.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to the preparation and application of a monoclonal antibody 2B3-H4-G8 against avian adenovirus type 4 100K protein. Background Technology

[0002] Fowl adenovirus serotype 4 (FAdV-4) is the pathogen causing highly pathogenic and infectious pericardial effusion hepatitis syndrome. Infected poultry experience acute death, with a mortality rate of 20%-80%. FAdV-4 infection can lead to host immunosuppression, and the resulting immune failure, secondary infections, and mixed infections further exacerbate the harm of pericardial effusion hepatitis syndrome. Before 2015, the disease was sporadically found in many parts of my country, but since June 2015, it has experienced an explosive epidemic in multiple provinces including Henan, Hebei, Anhui, Shandong, Guangdong, and Jiangsu, as well as the Guangxi Zhuang Autonomous Region.

[0003] According to reports, the virulence of the FAdV-4 strain circulating in my country after 2015 has significantly increased, and it has been defined as a novel FAdV-4. Genetic evolution analysis shows that it is not in the same branch as the FAdV-4 strains abroad (including non-pathogenic strains ON1 and KR5, and pathogenic strain MX-SHP95). The right end of its genome has a deletion of 1966bp (including deletions of ORF19, ORF48, and ORF27 genes). There are multiple mutations at amino acid sites of the major structural proteins Hexon, Penton, and Fiber (related to the virulence of the strain), but there is no related analysis or report on the genetic variation of the 100K gene.

[0004] Current research on FAdV-4 monoclonal antibodies has focused on developing immunogens based on the three main structural proteins: penton, Fiber 2, and Hexon. Wang Ping et al. (2018) immunized mice with whole FAdV-4 virus and then developed a Fiber-2 monoclonal antibody using the prokaryotic expression product of Fiber-2 as a screening antigen. This monoclonal antibody was positive for FAdV-4 and negative for FAdV-8; the reactivity with other serotypes of FAdV was not determined. Liu Na et al. (2018) screened monoclonal antibodies using Fiber-2 and Hexon proteins and developed colloidal gold test strips. These monoclonal antibodies were positive for FAdV-4, but the reactivity with other serotypes of FAdV was not determined. Hou Lidan et al. (2022) immunized mice with the prokaryotic expression protein of the penton gene as an antigen and screened and prepared a penton monoclonal antibody. This monoclonal antibody could recognize all representative serotypes of group I avian adenovirus.

[0005] The 100K protein is a non-structural protein of FAdV-4, highly expressed in the later stages of viral infection. It promotes the proper folding of Hexon proteins and plays a crucial role in viral infection and replication. Serological detection methods established using recombinant 100K protein or monoclonal antibodies can distinguish between antibodies produced by FAdV-4 viral infection and those produced by inactivated vaccine immunization, providing technical support for FAdV-4 eradication. Currently, no research reports on 100K protein monoclonal antibodies have been found. Summary of the Invention

[0006] The purpose of this invention is to provide the preparation and application of monoclonal antibody 2B3-H4-G8 against avian adenovirus type 4 100K protein.

[0007] In a first aspect, the present invention provides a hybridoma cell line 2B3-H4-G8 that secretes FADV4 antibody, the accession number of which is CCTCC NO:C2022177.

[0008] In a second aspect, the present invention provides an antibody secreted by the hybridoma cell line 2B3-H4-G8 described in the first aspect.

[0009] The antibody mentioned above is a monoclonal antibody.

[0010] Thirdly, the present invention provides for the use of the antibody described in the second aspect in any of the following:

[0011] 1) Prepare products for the detection or auxiliary detection of avian adenovirus type 4 in serum;

[0012] 2) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains avian adenovirus type 4;

[0013] 3) Prepare products that bind to FAdV4-100K protein;

[0014] 4) Prepare products that bind to avian adenovirus type 4 in serum;

[0015] 5) Prepare products that can differentiate or assist in differentiating between the GX2019-010 isolate and the FAdV-4CACC AV211 strain.

[0016] In the above-described applications, the serotype 4 avian adenovirus does not include the FAdV-4CACC AV211 strain, but specifically GX2019-010 or other serotype 4 avian adenovirus isolates.

[0017] The above products are indirect immunofluorescence assay kits or Western blot detection kits.

[0018] The detection or combination in the above applications can be achieved by ELISA, indirect immunofluorescence assay or Western blot.

[0019] Fourthly, the present invention provides a kit comprising the antibody described in the second aspect.

[0020] The reagent kit described above is any one of the following 1) to 2):

[0021] 1) A kit comprising the antibody described in the second aspect and an antibody labeled with a fluorescent group (such as FITC-labeled goat anti-mouse IgG antibody); used in embodiments of the present invention for indirect immunofluorescence assays;

[0022] 2) A kit comprising the antibody described in the second aspect and an enzyme-labeled antibody (such as HRP-labeled goat anti-mouse IgG antibody). Used in embodiments of the invention for Western blot detection.

[0023] The reagent kit described above has any of the following functions:

[0024] 1) Detection or auxiliary detection of avian adenovirus type 4 in serum;

[0025] 2) To detect or assist in the detection of whether the sample to be tested contains avian adenovirus type 4;

[0026] 3) Binds to FAdV4-100K protein;

[0027] 4) Combined with serum type 4 avian adenovirus;

[0028] 5) Differentiate between the GX2019-010 isolate and the FAdV-4CACC AV211 strain.

[0029] Fifthly, the present invention provides the kit described in the fourth aspect, or, the antibody described in the second aspect and the antibody labeled with a fluorescent group, or the antibody described in the second aspect and the antibody labeled with an enzyme, in any of the following applications:

[0030] 1) Prepare products for the detection or auxiliary detection of avian adenovirus type 4 in serum;

[0031] 2) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains avian adenovirus type 4;

[0032] 3) Prepare products that bind to FAdV4-100K protein;

[0033] 4) Prepare products that bind to avian adenovirus type 4 in serum;

[0034] 5) Prepare products that can differentiate or assist in differentiating between the GX2019-010 isolate and the FAdV-4CACC AV211 strain.

[0035] In the above text, the serotype 4 avian adenovirus does not include the FAdV-4CACC AV211 strain, but specifically the GX2019-010 or other serotype 4 avian adenovirus isolates.

[0036] The other serotype 4 avian adenovirus isolates mentioned above are those isolated after 2015, specifically GX2017-001, GX2017-002, GX2017-003, GX2017-004, GX2018-005, GX2018-006, GX2017-007, GX2018-008, GX2019-009, GX2019-011, GX2019-012, GX2019-013, GX2019-014, GX2019-015, GX2019-016, GX2019-017, GX2019-018, and GX2020-019.

[0037] The experiments of this invention demonstrate that by immunizing mice with the 100K recombinant protein of GX2019-010 (FAdV-4) as an immunogen and screening them by ELISA, a hybridoma cell line 2B3-H4-G8 secreting monoclonal antibodies was successfully developed. The monoclonal antibody secreted by this hybridoma cell line 2B3-H4-G8 showed positive reactions only with GX2019-010 and 18 FADV-4 isolates (isolated after 2017), but negative reactions with FADV-4 CACC AV211 purchased by our laboratory from the China Institute of Veterinary Drug Control in 2009. The prepared 100K recombinant protein monoclonal antibody exhibits good reactivity and specificity, laying the foundation for the development of FADV-4 antigen and antibody detection reagents. Attached Figure Description

[0038] Figure 1 The results of the IFA assay for the FADV4-100K monoclonal antibody.

[0039] Figure 2 The results are from a Western blot experiment of the FADV4-100K monoclonal antibody.

[0040] Instructions for the Preservation of Biological Materials

[0041] Bacterial strain name: Hybridoma cell line

[0042] Strain number: 2B3-H4-G8

[0043] Preservation Institution: China Center for Type Culture Collection

[0044] Abbreviation for depository institution: CCTCC

[0045] Address: Wuhan University, Wuhan, China

[0046] Deposit date: August 25, 2022

[0047] Collection Center Registration Number: CCTCC No.: C2022177 Detailed Implementation

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0050] The materials used in some of the embodiments are as follows:

[0051] FAdV-4CACC AV211 was purchased from the China Institute of Veterinary Drug Control in 2009. The website address is http: / / cvcc.ivdc.org.cn / , and the public can order the strain directly online.

[0052] FAdV-4 Guangxi isolate (GX2019-010) 100K recombinant protein (hereinafter also referred to as FADV4-100K protein, amino acid sequence is sequence 1);

[0053] Information on 19 strains of FAdV-4 is as follows:

[0054] GX2017-01, GX2017-02, GX2017-03, GX2017-04, GX2017-05, GX2017-06, GX2018-07, GX2018-08, and GX2019-09 are recorded in the following non-patent literature: “Rashid F, Xie Z, Zhang L, et al. Genetic characterization of fowl aviadenovirus 4 isolates from Guangxi, China, during 2017–2019[J]. Poultry Science, 2020, 99(9):4166-4173.”;

[0055] GX2019-010, GX2019-011, GX2019-012, GX2019-013, GX2019-014, GX2019-015, and GX2019-016 are recorded in the following non-patent literature: "Luan Yongjiao. Whole genome sequencing analysis, detection method and pathogenicity study of Guangxi isolates of avian adenovirus type 4 [D]. Guangxi University, 2021.", and are preserved by the Veterinary Biotechnology Laboratory of the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

[0056] Information for GX2019-017, GX2019-018, and GX2019-019 is shown in Table 1 and is preserved by the Veterinary Biotechnology Laboratory of the Guangxi Zhuang Autonomous Region Veterinary Research Institute.

[0057] LMH cells and myeloma SP2 / 0 cells are described in the following non-patent literature: Wei You, Xie Zhixun, Deng Xianwen, Li Xiaofeng, Xie Zhiqin, Fan Qing, Zhang Yanfang, Huang Jiaoling, Wang Sheng. Truncated expression of Fiber-2 protein of avian adenovirus type 4 and preparation of monoclonal antibody [J]. Journal of Southern Agriculture, 2022, 53(08):2341-2349. Preserved by the Veterinary Biotechnology Laboratory of the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

[0058] Avian adenovirus types 1–11 (FADV-1–FADV-11) and avian reovirus (ARV) were purchased from the China Institute of Veterinary Drug Control. The website is http: / / cvcc.ivdc.org.cn / , and the public can order the strains online.

[0059] Newcastle disease virus (NDV) is described in the following non-patent literature: “Zeng Tingting, Xie Zhixun, Hua Jun, Xie Liji, Li Meng, Huang Jiaoling, Zhang Minxiu, Zhang Yanfang, Yuan Yadong, Sa Wei. Etiological analysis of two strains of genotype XII Newcastle disease virus from geese in Guangxi[J]. Journal of Southern Agriculture, 2022, 53(07):2007-2014.”, preserved by the Veterinary Biotechnology Laboratory of the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

[0060] Egg Drop Syndrome Virus (EDSV) is described in the following non-patent literature: “Xie Zhixun, Xie Zhiqin, Liu Jiabo, Pang Yaoshan, Tang Xuping, Liang Shaofeng, Lan Fanshun. Serological survey of egg drop syndrome [J]. Chinese Poultry, 1994(01):28-29.”, preserved by the Veterinary Biotechnology Laboratory of the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

[0061] Infectious bronchitis virus (IBV) is described in the following non-patent literature: “Xie Zhiqin, Xie Zhixun, Lü Huagang. Cloning and sequence analysis of S1 gene of 30 standard strains and isolates of chicken infectious bronchitis virus [J]. Southwest China Journal of Agricultural Sciences, 2008, 21(06):1733-1736.”, preserved by the Veterinary Biotechnology Laboratory of the Veterinary Research Institute of Guangxi Zhuang Autonomous Region.

[0062] SPF-grade female BALB / c mice were purchased from the Experimental Animal Center of Guangxi Medical University; Freund's complete adjuvant, Freund's incomplete adjuvant, β-propiolactone inactivation agent, HAT selective medium, HT medium, PEG fusion reagent, and mouse MAb subclass identification kit were all purchased from Sigma-Aldrich; DMEM medium was purchased from Hycon; fetal bovine serum was purchased from Invitrogen; BW-V1160Vira Trap™ Adenovirus Purification Miniprep Kit was purchased from Biomiga; 96-well microplates, BSA, tetramethylbenzidine (TMB), HRP, and FITC-labeled goat anti-mouse IgG antibodies were purchased from Shenzhen Jingmei Biotechnology Co., Ltd.

[0063] Table 1 contains information for GX2019-017, GX2019-018, and GX2019-019.

[0064] GX2019-017 MW439044 January 4, 2021 2019.4 Rong County, Guangxi GX2019-018 MW439045 January 4, 2021 2018.8 Nanning, Guangxi GX2020-019 OP378126 September 7, 2022 2020.9 Yulin, Guangxi

[0065] Example 1: Preparation and Identification of FAdV-4 Monoclonal Antibody

[0066] I. Preparation of FAdV-4 Monoclonal Antibody

[0067] 1. Cultivation, purification, and concentration of FAdV-4 isolates from Guangxi

[0068] GX2019-010 was cultured using SPF chicken embryo hepatocytes. The cell culture medium was harvested, and the GX2019-010 virus was purified and concentrated according to the instructions of the BW-V1160ViraTrap™ Adenovirus Purification Miniprep Kit.

[0069] 2. Obtaining hybridoma cells

[0070] 1) Animal immunity and cell fusion

[0071] FADV4-100K protein (amino acid sequence 1) was diluted with PBS to 120 μg / ml and emulsified with an equal volume of Freund's complete adjuvant at a 1:1 ratio. This emulsified protein was used as an immunogen and administered subcutaneously to multiple sites in female BALB / c mice 6-8 weeks post-immunization (1 ml / mouse). Booster immunizations were performed 21 and 35 days post-immunization using FADV4-100K protein emulsified with Freund's incomplete adjuvant. Blood samples were collected 49 days after the initial immunization to measure antibody titers. BALB / c mice with the highest antibody titers were selected for booster immunization with FADV4-100K protein 3 days before cell fusion. Spleens from immunized mice were aseptically harvested and placed in a cell strainer to prepare a spleen cell suspension. Cell counts were calculated, and spleen cells were fused with myeloma cells (Sp2 / 0) at a 5:1 ratio for a cell fusion assay.

[0072] 2) Hybridoma cell screening

[0073] Fusion cells were cultured in HAT medium containing hypoxanthine, aminopterin, and thymidine deoxyribonucleoside. Cell culture supernatants were collected at 9, 12, and 15 days post-fusion. The fusion cell supernatants were analyzed using FADV4-100K-ELISA (an ELISA method established using FADV4-100K protein as the antigen). Cells from wells showing positive ELISA results were subcloned three times consecutively using limiting dilution. After 10 days of culture, the cell culture supernatants from the subclones were collected for FADV4-100K-ELISA analysis. The fusion cell supernatants from the selected positive wells were further screened using FADV4-ELISA (an ELISA method established using GX2019-010 virus as the antigen).

[0074] Cell lines with stable growth, high and stable antibody titers were selected for preservation.

[0075] The supernatant of fusion cells at days 9, 12, and 15 was screened using FAdV4-100K-ELISA, yielding 172 positive hybridoma cell lines. Further analysis of the supernatant using FAdV4-ELISA revealed 39 positive hybridoma cells with high OD values. After three rounds of subcloning and screening, a single positive hybridoma cell line, 2B3-H4-G8, was obtained.

[0076] Hybridoma cells 2B3-H4-G8 were deposited on August 25, 2022, at the China Center for Type Culture Collection (CCTCC, Wuhan University, Wuhan, China), with accession number CCTCC NO:C2022177.

[0077] 3) Cell cryopreservation and thawing

[0078] The hybridoma cells 2B3-H4-G8 selected above were prepared into 10 using cryopreservation solution. 4 Cell suspensions of cells / ml were stored long-term in liquid nitrogen. Upon thawing, the cryovials were removed and immediately placed in a 37°C water bath for rapid thawing. After centrifugation to remove the cryopreservation solution, the cells were transferred to culture flasks for incubation.

[0079] 3. Preparation and purification of FADV4-100K monoclonal antibody

[0080] 1) Obtain monoclonal antibodies from culture supernatant

[0081] Hybridoma cells 2B3-H4-G8, which were frozen for different times according to 2) above, were placed in cell culture medium (using Gibco brand DMEM / F12 medium containing 10% fetal bovine serum), cultured at 37°C for 5 days, centrifuged at 400g for 10 hours, and the supernatant was collected and recorded as FADV4-100K monoclonal antibody from the supernatant.

[0082] 2) Preparation of antibodies from ascites fluid

[0083] The hybridoma cells 2B3-H4-G8, frozen after step 2) above, were cultured in DMEM / F12 medium (containing 10% fetal bovine serum, Gibco brand) at 37°C for 72-96 hours until the cell density reached approximately 70%. The cells were digested with trypsin EDTA solution, resuspended in PBS solution to form a cell suspension, and counted.

[0084] 8-week-old BALB / c mice were injected intraperitoneally with 1 mL of sterile injection-grade white oil, followed by an intraperitoneal injection of 2 × 10⁻⁶ g of the same oil 14 days later. 6 The above-mentioned positive hybridoma cells 2B3-H4-G8, frozen for 3 months and then thawed during the logarithmic growth phase, were used to closely monitor the health status and ascites signs in BALB / c mice. Ascites was produced 7-10 days after cell inoculation. The ascites was collected, purified according to the Protein G column instructions, and then subjected to titer determination and subsequent experiments. This was designated as the FADV4-100K monoclonal antibody derived from the ascites.

[0085] II. Biological Characteristics Analysis of Monoclonal Antibodies

[0086] 1. Analysis of antibody secretion stability:

[0087] The FADV4-100K monoclonal antibody secreted from the supernatant of the positive hybridoma cells 2B3-H4-G8 obtained in step 3 of the above method (1) after being frozen in liquid nitrogen for 3 months and 6 months was used in the following experiments:

[0088] Following the FADV4-100K-ELISA detection method established by Wu Xiaoqian, the cells were coated with FADV4-100K protein (6 μg / mL) and incubated overnight. After washing three times, different dilutions of FADV4-100K monoclonal antibody from the supernatant were added, and the cells were incubated at 37°C for 1 h. After washing three times, goat anti-mouse enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG antibody) was added at a ratio of 1:3000, and the cells were incubated at 37°C for 1 h. After washing three times, the cells were developed with TMB chromogenic buffer, and the OD450 value was read to determine the ability of positive hybridoma cells 2B3-H4-G8 to secrete antibodies.

[0089] The results showed that after thawing and passage of hybridoma cells 2B3-H4-G8 for 3 and 6 months, the ELISA titers of FADV4-100K monoclonal antibody in the hybridoma cell culture supernatant were greater than 2. 7 The antibody secretion stability is good.

[0090] 2. Ascites antibody titer test

[0091] The same method was used to detect the titer of FADV4-100K monoclonal antibody secreted from ascites by positive hybridoma cells 2B3-H4-G8 that had been frozen in liquid nitrogen for 3 months.

[0092] Ascites antibody titer assay results showed that hybridoma cells could induce mice to produce high-titer antibodies, with the monoclonal antibody FADV4-100K derived from ascites fluid reaching a titer of 2. 19 .

[0093] 3. Identification of monoclonal antibody subclasses

[0094] The FADV4-100K monoclonal antibody secreted from the supernatant of positive hybridoma cells 2B3-H4-G8 obtained in step 3 of the above was collected and frozen in liquid nitrogen for 3 months. Subclass identification was performed according to the instructions of the mouse MAb subclass identification kit.

[0095] The identification results of the monoclonal antibody subclasses from the supernatant are shown in Table 2. The heavy chain of the antibody secreted by the 2B3-H4-G8 (accession number: CCTCCNO: C2022177) hybridoma cells is the IgG2b subclass, and the light chain is the K chain.

[0096] Table 2 shows the ELISA identification results of monoclonal antibody subclasses.

[0097]

[0098] 4. Monoclonal antibody specificity detection

[0099] Other serotypes of avian adenovirus (FADV-1 to FADV-11), NDV, ARV, EDSV, and IBV were used as coating antigens. The primary antibody was the FADV4-100K monoclonal antibody secreted from ascites fluid by positive hybridoma cells 2B3-H4-G8, obtained from step 3 of the above method and cryopreserved in liquid nitrogen for 3 months. Details are as follows:

[0100] The virus was coated with GX2019-010, FADV-4CACC AV211 strain, and other serotypes of FADV, as well as whole viruses of NDV, ARV, EDSV, and IBV, and incubated overnight. After washing three times, different dilutions of FADV4-100K monoclonal antibody from ascites fluid were added, and the mixture was incubated at 37°C for 1 hour. After washing three times, goat anti-mouse enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG antibody) was added at a ratio of 1:3000, and the mixture was incubated at 37°C for 1 hour. After washing three times, the mixture was developed with TMB chromogenic solution, and the OD450 value was read to evaluate the specificity of the FADV4-100K monoclonal antibody from ascites fluid.

[0101] The results are shown in Table 3. The monoclonal antibody secreted by 2B3-H4-G8 hybridoma cells only reacted well with the GX2019-010 isolate and showed no cross-reactivity with FADV-4CACC AV211 strain or other serotypes of FADV, NDV, ARV, EDSV, and IBV, indicating that the antibody secreted by the positive hybridoma cells 2B3-H4-G8 has good specificity.

[0102] Table 3 shows the specificity detection of monoclonal antibodies.

[0103]

[0104]

[0105] Eighteen FADV-4 isolates (viruses isolated at different times and from different farms) were used as coating antigens. The FADV4-100K monoclonal antibody secreted by positive hybridoma cells 2B3-H4-G8 from ascites fluid after being frozen in liquid nitrogen for 3 months was used as the primary antibody and incubated at 37°C for 1 hour. After washing 3 times, goat anti-mouse enzyme-labeled secondary antibody (HRP-labeled goat anti-mouse IgG antibody) was added at a ratio of 1:3000 and incubated at 37°C for 1 hour. After washing 3 times, TMB chromogenic solution was used for color development, and the OD450 value was read to evaluate the broad spectrum of the FADV4-100K monoclonal antibody from ascites fluid.

[0106] The results are shown in Table 4. The FADV4-100K monoclonal antibody secreted by 2B3-H4-G8 hybridoma cells in ascites reacted well with 18 FADV-4 isolates, indicating that the antibody secreted by positive hybridoma cells has a good broad-spectrum reaction with FADV-4 isolates.

[0107] Table 4 shows the detection results of monoclonal antibodies against 18 FADV-4 isolates.

[0108]

[0109]

[0110] Example 2: Application of FADV4-100K monoclonal antibody

[0111] I. FADV4-100K monoclonal antibody for indirect immunofluorescence assay (IFA) detection of FADV-4 virus

[0112] Experimental group: SPF chicken embryo hepatocytes were cultured in 12-well cell culture plates to 90% confluence, inoculated with GX2019-010, infected for 1.5 h, washed twice with PBS, and cultured for another 48 h in DMEM / F12 medium containing 2.5% bovine serum. The culture medium was discarded, and the cells were fixed with ice-cold methanol. The primary antibody was FADV4-100K monoclonal antibody secreted from ascites fluid by positive hybridoma cells 2B3-H4-G8 obtained in Example 1 and cryopreserved in liquid nitrogen for 3 months. The cells were incubated at 37°C for 1 h, and the secondary antibody was FITC-labeled goat anti-mouse IgG antibody. A negative control group was also set up. The cells were incubated at 37°C for 1 h, washed 3 times, air-dried, and observed and photographed under a fluorescence microscope.

[0113] The negative control group consisted of SPF chicken embryo hepatocytes that were not inoculated with GX2019-010 and were directly subjected to IFA assays using the FADV4-100K monoclonal antibody obtained from ascites fluid in Example 1.

[0114] The results were observed under a fluorescence microscope as follows: Figure 1 As shown in the figure, the upper figure is the experimental group and the lower figure is the negative control group. The cells infected with GX2019-010 have bright green fluorescence, while no green fluorescence is observed in the negative control cells. This indicates that the FADV4-100K monoclonal antibody can be used as an antibody to detect FADV-4 by indirect immunofluorescence assay (IFA).

[0115] Additionally, the same test was conducted using the FAdV-4CACC AV211 strain (purchased from the China Institute of Veterinary Drug Inspection), and the result for 2B3-H4-G8 was negative.

[0116] The above results indicate that the FADV4-100K monoclonal antibody can distinguish between GX2019-010 and FADV-4CACCAV211.

[0117] II. FADV4-100K monoclonal antibody used for Western blot identification and detection of FADV-4 virus

[0118] Experimental group: LMH cells were infected with GX2019-010 at 0.5 MOI. Cell proteins were collected 48 h after infection, transferred to PVDF membranes after SDS-PAGE electrophoresis, blocked with 5% skim milk for 2 h, washed three times with PBST, and then incubated overnight at 4°C with HRP-labeled goat anti-mouse IgG antibody (HRP-labeled goat anti-mouse IgG antibody) after being cryopreserved in liquid nitrogen for 3 months as described in Example 1. After washing three times with PBST, the cells were incubated at room temperature for 1 h, washed three times with PBST, and then developed and exposed. The control group was not treated with GX2019-010.

[0119] The results are as follows Figure 2 As shown, + represents the experimental group infected with GX2019-010, and - represents the control group without GX2019-010. The FADV4-100K monoclonal antibody of 2B3-H4-G8 can specifically detect FADV-4 in the GX2019-010 strain and has good Western blot reactivity.

[0120] Additionally, the same test was conducted using the FAdV-4CACC AV211 strain, and the test for 2B3-H4-G8 was negative.

Claims

1. The hybridoma cell line 2B3-H4-G8 that secretes FADV4 antibody, with accession number CCTCC NO: C2022177.

2. A monoclonal antibody secreted by the hybridoma cell line 2B3-H4-G8 as described in claim 1.

3. The use of the monoclonal antibody of claim 2 in any of the following: 1) Prepare products for detection or auxiliary detection of GX2019-010 isolates; 2) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains the GX2019-010 isolate.

4. A kit comprising the monoclonal antibody of claim 2.

5. The reagent kit according to claim 4, characterized in that: The kit is any one of the following 1) to 2): 1) A kit comprising the monoclonal antibody as described in claim 2 and a FITC-labeled goat anti-mouse IgG antibody; 2) A kit comprising the monoclonal antibody as described in claim 2 and HRP-labeled goat anti-mouse IgG antibody.

6. The use of the kit according to any one of claims 4-5 in any of the following: 1) Prepare products for detection or auxiliary detection of GX2019-010 isolates; 2) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains the GX2019-010 isolate.

7. The use of the monoclonal antibody of claim 2 and the FITC-labeled goat anti-mouse IgG antibody in any of the following: 1) Prepare products for detection or auxiliary detection of isolate GX2019-010; 2) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains the GX2019-010 isolate.

8. The use of the monoclonal antibody of claim 2 and the HRP-labeled goat anti-mouse IgG antibody in any of the following: 1) Prepare products for detection or auxiliary detection of GX2019-010 isolates; 2) Prepare products for detecting or assisting in the detection of whether the sample to be tested contains the GX2019-010 isolate.

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