Chicken parvovirus vp2 monoclonal antibody and application thereof

Abstract

The application provides a chicken parvovirus VP2 monoclonal antibody and application thereof, relates to the field of biotechnology and virology, and 1 strain of chicken parvovirus VP2 protein monoclonal antibody screened by the application can specifically combine chicken parvovirus and VP2 protein expressed in vitro, and the light chain of the 1 strain of monoclonal antibody is kappa chain, and the heavy chain is IgG2a. A double antibody sandwich ELISA detection platform of ChPV VP2 protein is established by taking monoclonal antibody 3F8 as a coating antibody, the minimum detection line of the chicken parvovirus reaches 15.6 ng / mL, the specificity is strong, the sensitivity is high, the stability is good, and parallel detection of 192 clinical samples by the double antibody sandwich ELISA detection platform and nested PCR shows that the total coincidence rate reaches 81.3%. The chicken parvovirus monoclonal antibody provided by the application can be used for preparing chicken parvovirus related detection reagents, and also provides raw materials for basic researches on functions of the chicken parvovirus VP2 protein, is helpful for prevention and control of the chicken parvovirus in the poultry industry, and is also helpful for basic researches on the chicken parvovirus related proteins.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biotechnology and virology, and particularly relates to a monoclonal antibody of chicken parvovirus and application of the monoclonal antibody, in particular, application to detection of chicken parvovirus VP2 protein, and the monoclonal antibody can also be applied to related basic research of chicken parvovirus VP2 protein. BACKGROUND

[0002] Avian parvoviruses belong to the Parvoviridae family and the Parvovirinae subfamily, and the Parvovirinae subfamily has eight genera. Among them, avian adeno-associated virus (AAAV), goose parvovirus (GPV), muscovy duck parvovirus (MDPV) and other avian parvoviruses belong to the Dependovirus genus, and chicken parvovirus (ChPV) belongs to the Aveparvovirus genus. ChPV is one of the main pathogens causing the running-tunting syndrome (RSS), also known as malabsorption syndrome (MAS), which mainly leads to diarrhea, growth retardation and increased mortality of chicks. In 1984, ChPV was first discovered in the United States. In 1987, WO Diting discovered ChPV in chicken flocks in China, and chicks infected with ChPV showed severe growth arrest. In recent years, ChPV infection has been detected in Shandong, Henan, Hebei and Guangxi, China. From 2014 to 2019, ChPV was detected in broilers in Guangxi, and the detection rate of ChPV in 1-7 week-old broilers was as high as 74.46%, and the detection rate of ChPV in 8-20 week-old broilers was 53.56%, indicating that ChPV is a widespread infection in chicken flocks, and the virus can also be detected in healthy chickens. Due to the lack of commercial ChPV vaccines, it will cause significant or potential economic losses to the poultry industry, and it is urgent to establish ChPV rapid screening and related safety prevention measures.

[0003] The ChPV genome is about 5kb in size, and contains three open reading frames (ORF) that encode four proteins. ORF1 and ORF3 encode non-structural proteins NS1 and NP1, respectively, and ORF2 encodes structural proteins VP1 and VP2. In mouse parvovirus, goose parvovirus and porcine parvovirus, the VP2 protein, which is composed of 537 amino acids, is the main capsid protein of ChPV and can self-assemble into a natural-like capsid in the absence of VP1. The VP2 protein is related to the assembly of virus particles, the pathogenicity of the virus and the tropism of the host cell. In addition, the VP2 protein is the main protective antigen of the virus and has strong immunogenicity. The body stimulated by the protein will produce a strong immune response, and the VP2 protein of porcine parvovirus and canine parvovirus is often used for serological diagnosis and vaccine preparation.

[0004] Due to the unique replication mode of ChPV, it is difficult to normally proliferate in chicken embryos and cells, further increasing the difficulty of immunodiagnosis of the virus. Therefore, at present, molecular biology methods are often used for detection, including PCR, nested PCR and dye method quantitative PCR detection and other related nucleic acid detection methods. However, nucleic acid extraction and PCR require special instruments and higher requirements for detection personnel, and are not suitable for on-site rapid detection. At present, there is still a lack of serological detection methods for ChPV antigens, and the functional research on the interaction between ChPV VP2 protein and host cells is also relatively insufficient, which is mainly due to the lack of specific antibody detection tools for VP2 protein.

[0005] Due to the difficulty in isolating ChPV, foreign studies have shown that virus isolation can be carried out by inoculating chicken embryos. The inventors have tried many times using the isolation method reported abroad, but still have not isolated the virus. In order to strengthen the research on the pathogenic mechanism of ChPV and the related functions of viral proteins, the inventors have previously established an infectious clone of ChPV, which serves as an important means for studying the pathogenic mechanism of the virus and the function of the protein, and provides favorable conditions for screening viral protein monoclonal antibodies in the present application. The inventors have also failed in using a polyclonal antibody as a capture antibody and a monoclonal antibody as a detection antibody. On this basis, the present application unexpectedly screens a ChPV VP2 protein monoclonal antibody using monoclonal antibody preparation technology, and establishes a double-antibody sandwich ELISA detection platform with relatively higher sensitivity and specificity. The detection platform not only provides a material basis for serological detection of ChPV, specific detection of VP2 protein, and functional research on VP2 protein, but also provides related technical support for the prevention and control of ChPV in the chicken industry. SUMMARY

[0006] The purpose of the present application is to at least partially overcome the deficiencies of the prior art, and to provide a monoclonal antibody for recognizing chicken parvovirus.

[0007] To achieve the above-mentioned purpose or one of the purposes, the present application provides the following technical solutions:

[0008] In a first aspect, the present application provides a monoclonal antibody or an antigen binding fragment thereof, which is monoclonal antibody 3F8 or an antigen binding fragment thereof against chicken parvovirus VP2 protein; a hybridoma cell line 3F8 capable of secreting monoclonal antibody 3F8, which has been preserved in China Center for Type Culture Collection on September 14, 2023, with a preservation number of CCTCC NO: C2023272 and a preservation address of China. Wuhan. Wuhan University.

[0009] Preferably, in the monoclonal antibody or the antigen binding fragment thereof, the light chain of the monoclonal antibody 3F8 or the antigen binding fragment thereof is a kappa chain; and / or the heavy chain of the monoclonal antibody 3F8 or the antigen binding fragment thereof is an IgG2a subtype.

[0010] Preferably, in the monoclonal antibody or the antigen binding fragment thereof, the monoclonal antibody 3F8 or the antigen binding fragment thereof can specifically bind to chicken parvovirus, an infectious clone strain containing chicken parvovirus, or an antigen binding site thereof.

[0011] In a second aspect, the present application provides a host cell capable of producing monoclonal antibody 3F8 or an antigen binding fragment thereof.

[0012] In a third aspect, the present application provides a preparation method for preparing monoclonal antibody 3F8 or an antigen binding fragment thereof, and / or for preparing monoclonal antibody 3F8 or an antigen binding fragment thereof, comprising the following steps: 1) immunizing a mouse with purified chicken parvovirus VP2 protein under suitable conditions; 2) preparing and screening a positive hybridoma cell line of monoclonal cells by using a cell fusion method of spleen cells and myeloma cells; 3) preparing a corresponding monoclonal antibody from the positive hybridoma cell line; and 4) verifying the specificity of the monoclonal antibody by an indirect ELISA method.

[0013] In a fourth aspect, the present application provides a kit for detecting chicken parvovirus, comprising: monoclonal antibody 3F8 or an antigen binding fragment thereof, and / or monoclonal antibody 3F8 or an antigen binding fragment thereof.

[0014] Preferably, in the kit for detecting chicken parvovirus, a double antibody sandwich ELISA method is used, monoclonal antibody 3F8 or an antigen binding fragment thereof, or monoclonal antibody 3F8 or an antigen binding fragment thereof is used as a capture antibody, a chicken anti-chicken parvovirus VP2 protein polyclonal antibody is used as a detection antibody, and a HRP-labeled goat anti-chicken IgG is used as an enzyme-labeled antibody.

[0015] In a fifth aspect, the present application provides a method for detecting chicken parvovirus, comprising using monoclonal antibody 3F8 or an antigen-binding fragment thereof, or monoclonal antibody 3F8 or an antigen-binding fragment thereof; the method does not include disease diagnosis and treatment methods.

[0016] Preferably, the method for detecting chicken parvovirus is a double antibody sandwich ELISA method, monoclonal antibody 3F8 or an antigen-binding fragment thereof, or monoclonal antibody 3F8 or an antigen-binding fragment thereof is used as a capture antibody, a polyclonal antibody of chicken anti-chicken parvovirus VP2 protein is used as a detection antibody, and HRP-labeled goat anti-chicken IgG is used as an enzyme-labeled antibody.

[0017] In a sixth aspect, the present application provides a use, comprising monoclonal antibody 3F8 or an antigen-binding fragment thereof, and / or monoclonal antibody 3F8 or an antigen-binding fragment thereof, a host cell, a kit for detecting chicken parvovirus, or a method for detecting chicken parvovirus, in any one of the following: 1) preparation of chicken parvovirus-related products; 2) chicken parvovirus detection, the detection including: epidemiological analysis, detection of ex vivo, non-living samples, import and export quarantine detection; 3) functional verification of chicken parvovirus VP2 protein; the use does not include disease diagnosis and treatment methods.

[0018] Advantages of the present application:

[0019] The present application uses a purified ChPV VP2 protein as an immunogen to screen a cell strain 3F8 capable of secreting a monoclonal antibody through a monoclonal antibody preparation method, injects the hybridoma cell strain into a mouse body, obtains a corresponding ascites, and the ascites titer is 8.6x10 6 Thus, a corresponding monoclonal antibody 3F8 is obtained. Through subtype identification of the monoclonal antibody, the light chain of the monoclonal antibody 3F8 is a kappa chain, and the heavy chain is IgG2a; through indirect immunofluorescence identification and Western-blot identification, the monoclonal antibody 3F8 can specifically bind to chicken parvovirus and in-vitro expressed VP2 protein, and is a specific monoclonal antibody against chicken parvovirus structural protein VP2 protein.

[0020] In specific identification of the monoclonal antibody, the Western-blot identification result shows that the monoclonal antibody 3F8 can specifically react with prokaryotic expressed VP2 recombinant protein and VP2 protein expressed by LMH cells transfected by an infectious clone plasmid pBluescript II SK(+)-ChPV. The IFA result shows that the monoclonal antibody 3F8 can specifically react with the protein expressed by LMH cells transfected by the infectious clone plasmid pBluescript II SK(+)-ChPV, and the LMH cells all appear specific green fluorescence. It can be seen that the monoclonal antibody 3F8 has high specificity.

[0021] The monoclonal antibody 3F8 described in the present application is produced by the hybridoma cell line 3F8 classified as secreting, which has been deposited with the China Center for Type Culture Collection on September 14, 2023, with the accession number CCTCC NO: C2023272, and the address is Wuhan, Wuhan University, China. The monoclonal antibody 3F8 described in the present application is a specific monoclonal antibody for parvovirus VP2 protein. The successful preparation of this strain of monoclonal antibody provides a material basis for the serological detection method of the pathogen. The preparation of this strain of monoclonal antibody into a detection reagent related to chicken parvovirus can be applied to the rapid detection of ChPV on the spot in the chicken breeding industry; it also provides specific detection materials and tools for in-depth study of the related functions of the VP2 protein of the virus and the replication mechanism of the virus in cells.

[0022] Currently, the detection of ChPV etiology mainly detects pathogen nucleic acid, but nucleic acid extraction and PCR require special instruments and have high requirements for detection personnel, which is not suitable for rapid detection on the spot. The double antibody sandwich ELISA method for detecting antigens is quick and convenient to operate, does not require complex processing of the detection sample, does not require the use of special experimental instruments, saves experimental costs, and is more easily applied to the detection of antigens on the spot in the breeding farm. Among them, the double antibody sandwich ELISA method is often used to establish detection of the corresponding pathogen. However, before the establishment of the double antibody sandwich ELISA detection platform for ChPV VP2 protein described in the present application, attempts were made to use chicken polyclonal antibodies as capture antibodies and monoclonal antibodies as detection antibodies. The results showed that the OD 450nm value of the positive control was less than 0.2, which was presumably that the specific binding site of the target protein to the monoclonal antibody was preferentially bound by the polyclonal antibody, resulting in the inability of the monoclonal antibody to bind to the target protein, and in turn resulting in a low OD 450nm value. Replacing the capture antibody with the monoclonal antibody described in the present application and using polyclonal antibodies as detection antibodies, the experimental results unexpectedly showed that the OD 450nm value and P / N value were significantly improved.

[0023] Based on the monoclonal antibody provided in the application, a double antibody sandwich ELISA (VP2-DAS-ELISA) detection platform for the ChPV VP2 protein is established: the monoclonal antibody 3F8 is used as a capture antibody (or a coating antibody), a polyclonal antibody against the ChPV VP2 protein of a chicken is used as a detection antibody, and a HRP-labeled goat anti-chicken IgG is used as an enzyme-labeled antibody to detect the chicken parvovirus antigen. Further, a series of condition optimization tests are conducted, mainly including that the optimal coating concentration of the capture antibody is 1:4000, the optimal concentration of the detection antibody is 1:8000, the optimal coating condition is 4 DEG C coating for 18 hours, the optimal blocking incubation time is 45 min, the optimal incubation concentration of the enzyme-labeled antibody is 1:4000, the optimal color development time is 8 min, and the positive and negative critical value is 0.09. The optimized ChPV VP2-DAS-ELISA detection platform has a minimum detection line of 15.6 ng / mL for the chicken parvovirus, and has high specificity and sensitivity. The enzyme-labeled plate prepared from the monoclonal antibody 3F8 has a batch and batch variation coefficient of less than 5% in repeated detection, and has good reagent kit performance stability. The ChPV VP2-DAS-ELISA detection platform provided in the application is compared with a third-party reagent nested PCR for parallel detection, and the parallel detection of 192 clinical samples shows that the total coincidence rate reaches 81.3%.

[0024] The ChPV VP2-DAS-ELISA detection platform constructed in the application is a serological detection method for the ChPV antigen, and provides convenience for the field detection of the ChPV. This is also the first double antibody sandwich ELISA method for detecting the ChPV antigen based on the ChPV VP2 protein, and can provide technical support for the field rapid detection of the ChPV in the chicken industry, and is helpful for the prevention and control of the chicken parvovirus in the chicken industry.

[0025] The ChPV VP2-DAS-ELISA detection platform for the ChPV VP2 protein is established by using the monoclonal antibody 3F8 against the ChPV VP2 protein, and can be used for the chicken parvovirus antigen detection platform and the development and research of the ChPV VP2 protein structure and function. Since there is also a lack of ChPV commercial vaccines at present, the ChPV is mainly prevented and controlled through rapid screening means and biological safety prevention measures. The double antibody sandwich ELISA antigen detection method provided in the application is fast and convenient to operate, does not need to perform complex treatment on the detection sample, does not need to use special experimental instruments, saves experimental cost, is used for detecting the corresponding ChPV pathogen, and is more easily applied to the field detection of the antigen in the breeding farm.

[0026] In conclusion, the monoclonal antibody 3F8 provided by the application has good specificity, provides raw materials for further study of ChPV VP2 protein function, and helps basic research on chicken parvovirus related proteins. The VP2-DAS-ELISA detection platform provided by the application has strong specificity, high sensitivity, good repeatability, and a total coincidence rate of 81.3%, which helps to improve the serological detection of ChPV, realizes rapid detection of ChPV antigens, provides a simple and rapid detection method for the epidemiological investigation of chicken parvovirus, and is conducive to the prevention and control of ChPV. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Figure 1 is an indirect immunofluorescence (IFA) detection diagram of chicken parvovirus VP2 monoclonal antibody, wherein A is chicken parvovirus VP2 monoclonal antibody 3F8, and B is negative serum;

[0028] Figure 2 Figure 1 is an indirect immunofluorescence (IFA) detection diagram of chicken parvovirus VP2 monoclonal antibody, wherein A is chicken parvovirus VP2 monoclonal antibody 3F8, and B is negative serum;

[0029] Figure 3 Figure 1 is an indirect immunofluorescence (IFA) detection diagram of chicken parvovirus VP2 monoclonal antibody, wherein A is chicken parvovirus VP2 monoclonal antibody 3F8, and B is negative serum;

[0030] Figure 4 Figure 1 is an indirect immunofluorescence (IFA) detection diagram of chicken parvovirus VP2 monoclonal antibody, wherein A is chicken parvovirus VP2 monoclonal antibody 3F8, and B is negative serum; 450nm

[0031] Figure 5 Figure 1 is an indirect immunofluorescence (IFA) detection diagram of chicken parvovirus VP2 monoclonal antibody, wherein A is chicken parvovirus VP2 monoclonal antibody 3F8, and B is negative serum; 450nm DETAILED DESCRIPTION

[0032] ​​Exemplary embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein like reference numerals identify like elements in the figures, and the same or similar concepts are referred to using the same or similar reference numerals, such as chicken parvovirus = ChPV, Newcastle disease virus = NDV, Mycoplasma synoviae = MS, Mycoplasma synoviae = MS, serotype 4 fowl adenovirus = FadV-4, chicken infectious anemia virus = CIAV, chicken infectious bronchitis virus = IBV, avian metapneumovirus = amPV, H9N2 subtype avian influenza virus = AIV, egg drop syndrome virus = EDSV, avian circovirus type 2 = AGV2, monoclonal antibody = mAb, polyclonal antibody = pAb, etc.

[0033] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the present disclosure. The following examples are merely illustrative of the present application and should not be construed as limiting the scope of the present application.

[0034] The experimental methods used in the following examples are conventional methods, and the materials, reagents, etc. used are commercially available unless otherwise specified. Among them, ChPV VP2 protein (i.e. prokaryotic expression of chicken parvovirus VP2 protein) and chicken anti-ChPV VP2 protein polyclonal antibody are prepared and purified according to the literature (“Prokaryotic expression of chicken parvovirus NS1 and VP2 protein and preparation of polyclonal antibody” Liao Jianqi et al., Southwestern Agricultural Sciences, 2023, Vol. 36, No. 02, pp. 419-426); ChPV infectious clone strain is the cell supernatant after repeated freeze-thaw of the cell suspension harvested after transfection of ChPV infectious clone plasmid pBluescript II SK(+)-ChPV into LMH cells, prepared and preserved by the Veterinary Biological Technology Room of Guangxi Zhuang Autonomous Region Animal Husbandry Institute; mouse myeloma cells SP2 / 0 and chicken hepatoma cells (LMH) are preserved by the Veterinary Biological Technology Room of Guangxi Zhuang Autonomous Region Animal Husbandry Institute; 8-week-old SPF BALB / c mice are purchased from Changsha Tianqing Biotechnology Co., Ltd., production license number Scxk (Xiang) 2019-0013; 50 ChPV-negative clinical samples are derived from SPF chickens; 192 clinical samples are taken from anal swabs in Nanning market, numbered after repeated freeze-thaw and stored at -80°C; Newcastle disease virus (NDV) GX6 / 02 strain, chicken mycoplasma (MS) PMS156 strain, serotype 4 avian adenovirus (FadV-4) FAdV-4-GX2018001 strain, chicken infectious anemia virus (CIAV) GXC060821 strain, chicken infectious bronchitis virus (IBV) GXIB / 02 strain, avian metapneumovirus (amPV) APV / MN-10 strain, H9N2 subtype avian influenza virus (AIV) 066C strain and avian egg drop syndrome virus (EDSV) GEV strain are isolated and preserved by the Veterinary Biological Technology Room of Guangxi Zhuang Autonomous Region Animal Husbandry Institute; avian circovirus (AGV2) is derived from clinical samples and identified as AGV2 positive by the Veterinary Biological Technology Room of Guangxi Zhuang Autonomous Region Animal Husbandry Institute; 180 kDa protein marker is purchased from Beijing Solabio Biotechnology Co., Ltd.; BCA protein quantification kit, BCIP / NBT alkaline phosphatase color reagent kit, alkaline phosphatase (AP) labeled goat anti-mouse IgG (H+L), HRP labeled goat anti-chicken IgG, HRP labeled goat anti-mouse IgG (H+L), Western-blot blocking solution and FITC labeled goat anti-mouse IgG (H+L) are purchased from Shanghai Biyun Tian Biotechnology Co., Ltd.; HT, HAT culture medium additives, 50% PEG solution and mouse monoclonal antibody subtype identification kit are purchased from Proteintech company; DMEM / F-12 cell culture medium is purchased from Gibco company;Nikon Eclipse Ti2 inverted microscope system was purchased from Nikon Precision (Shanghai) Co., Ltd., The Trans-Blot Turbo transfer system was purchased from Bio-Rad Laboratories (Shanghai) Co., Ltd.; EasyPure Viral DNA / RNA Kit was purchased from Beijing Zixingjin Biotechnology Co., Ltd.; Reverse transcription kit, Premix Taq; TM , Reverse transcription kit and cloning vector pMD-18T were purchased from Baorai Biotechnology (Beijing) Co., Ltd.; Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from SIGMA-Aldrich, USA. However, it is obvious that one or more embodiments can be implemented without these specific details, and the specific conditions not specified in the embodiments are carried out according to the conventional conditions or the conditions recommended by the manufacturer.

[0035] The molecular biology experimental methods not specifically described in the following examples were carried out according to the specific methods listed in the book "Molecular Cloning Experiment Guide" (third edition) J. Sambrook or according to the kit and product instructions. The application object of the implementation of the present application is derived from the biological product to be detected, which belongs to inanimate samples; the direct purpose of the detection is to ensure the safety of the biological product to be detected, thereby facilitating the improvement of the quality control standards of biological products, and there is no process to obtain the diagnostic results of diseases or health conditions. Therefore, the present application does not belong to the diagnostic method of diseases, which meets the basic requirements of the patent protection object of the "Patent Law".

[0036] The present application provides a strain of monoclonal antibody of chicken parvovirus, a preparation method and application thereof. First, by monoclonal antibody preparation technology, the purified ChPV VP2 protein is used as an immunogen, and a hybridoma cell strain 3F8 capable of secreting ChPV VP2 protein monoclonal antibody is unexpectedly screened, the hybridoma cell strain is injected into the mouse body, and the corresponding ascites is obtained; by immunological method, the types of light chain and heavy chain of monoclonal antibody 3F8 are identified, and the double antibody sandwich ELISA (VP2-DAS-ELISA) detection platform of ChPV based on monoclonal antibody 3F8 is obtained, and the reaction conditions of ChPV VP2-DAS-ELISA detection are further optimized. The specific embodiments of the present application are described in detail as follows:

[0037] Example 1, preparation and verification of chicken anti-ChPV VP2 protein monoclonal antibody

[0038] The application uses purified ChPV VP2 protein to immunize 8-week-old BALB / c mice, and after four immunizations, the mouse spleen cells are fused with myeloma cells, positive hybridoma cell lines are screened using an indirect ELISA method, and they are subcloned three times by the limited dilution method to obtain a positive hybridoma cell line 3F8 Hybridoma cell line 3F8 that can stably secrete antibodies and can secrete monoclonal antibody 3F8. The hybridoma cell line 3F8 has been preserved in the China Center for Type Culture Collection on September 14, 2023, with the preservation number being CCTCC NO: C2023272 and the preservation address being China. Wuhan. Wuhan University.

[0039] The obtained hybridoma cell line is injected intraperitoneally into Balb / c mice to prepare ascites; an indirect ELISA is used to determine the ascites titer, a mouse MAb subclass identification kit is used to detect the monoclonal antibody ascites, Western-blot and IFA are used to identify the specificity of the monoclonal antibody. The specific preparation and identification process is as follows:

[0040] 1) Animal immunization

[0041] The concentration of the purified ChPV VP2 protein is determined using a BCA protein quantification kit, 8-week-old BALB / c mice are immunized, a total of four times, with an interval of 21 days between the two immunizations: for the first immunization, complete Freund's adjuvant and an equal volume of ChPV VP2 protein are mixed and emulsified, 1 mL of the emulsified suspension is used for each mouse, each 1 mL contains 100 μg of VP2 recombinant protein, 0.5 mL is injected intraperitoneally and 0.5 mL is injected subcutaneously in multiple points for each mouse; for the second and third immunizations, incomplete Freund's adjuvant and an equal volume of ChPV VP2 protein are mixed and emulsified, 1 mL of the emulsified suspension is used for each mouse, each 1 mL contains 100 μg of VP2 recombinant protein, 0.5 mL is injected intraperitoneally and 0.5 mL is injected subcutaneously in multiple points for each mouse; for the fourth immunization, 400 μg / mouse of ChPV VP2 protein without adjuvant is used for intraperitoneal injection.

[0042] 2) Isolation of spleen cells, cell fusion and monoclonalization

[0043] After immunization, the BALB / c mice are anesthetized, exsanguinated from the eye orbit, and sacrificed, the sacrificed mice are dissected under sterile conditions, the mouse spleen is removed, the surrounding connective tissue is peeled off, and after sterile homogenization, serum-free DMEM medium is added for grinding, mixed, and then centrifuged to discard the supernatant, and the precipitate is resuspended in serum-free DMEM medium to obtain mouse spleen cells.

[0044] Mix the mouse spleen cells and myeloma cells well, centrifuge at 1600 rpm for 10 minutes, discard the supernatant, and obtain the mixed cells. Place the centrifuge tube containing the mixed cells in a 37°C water bath environment, slowly add 1 mL of preheated 50% PEG, mix well while adding, then slowly add 10 mL of preheated DMEM medium within 5 minutes, stand at 37°C for 10 minutes, then centrifuge at 1000 rpm for 8 minutes, discard the supernatant. Resuspend the centrifuge tube containing the mixed cells with 50 mL of HAT medium, evenly spread the mixed cell suspension in a 96-well cell plate, add 100 μL of cell suspension to each well, and after limited dilution, each well of the 96-well cell plate is in a single cell state, place it in a 37°C, 5% CO2 cell incubator for 7-10 days. When the area of the fused cell colony exceeds 1 / 10 of the culture well, take the fused cell supernatant in the culture well for indirect ELISA method screening of positive hybridoma cell strains.

[0045] Indirect ELISA method: use purified ChPV VP2 protein as detection antigen, first dilute the purified ChPV VP2 protein to 5 μg / mL with carbonate buffer (PBS) as coating antigen, add 50 μL / well to the 96-well plate, and coat at 4°C overnight; add the fused cell supernatant to be detected, add enzyme-labeled antibody (HRP-labeled goat anti-mouse IgG (H+L)); after washing, add substrate for color development, and use an enzyme label instrument to detect the indirect ELISA result.

[0046] Through the indirect ELISA method, the positive well is subjected to 3 times of monoclonalization by limited dilution method, and 1 strain of ChPV VP2 protein positive hybridoma cell strain is obtained, named cell strain 3F8.

[0047] 3) Ascites preparation and titer determination

[0048] The 1 strain of ChPV VP2 protein positive hybridoma cell strain 3F8 obtained by screening is injected into the abdominal cavity of a mouse to obtain the corresponding mouse ascites. The prepared mouse ascites is diluted by doubling dilution to 1:16000, 1:32000, 1:64000, 1:128000, 1:256000, 1:4096000, 1:8192000, 1:16384000, 1:32768000, and the same dilution factor is used as a negative control. The above indirect ELISA method is used to determine the OD 450 value to determine the titer of the ascites. When the OD value (P value) of the test sample is greater than 2.1 than the OD value (N value) of the negative control serum, it is judged to be positive. The results show that the ascites titer of cell strain 3F8 is 8.6 x 10 6 The monoclonal antibody is named monoclonal antibody 3F8.

[0049] 4) Subclass identification of monoclonal antibody

[0050] After diluting the corresponding ascites of the cell strain 3F8 to 1:100000, the monoclonal antibody obtained from 3F8 was identified according to the instruction of the mouse monoclonal antibody subtype identification kit, and the IgGl, IgG2a, IgG2b, IgG2c, IgG3, IgM, Kappa, Lambda, total 8 subtypes of the monoclonal antibody were identified, and the identification results are shown in Table 1, that is, the heavy chain of the monoclonal antibody 3F8 is IgG2a, and the light chain is kappa chain.

[0051] Table 1 Subtype identification results of monoclonal antibody 3F8

[0052]

[0053] 5) Specific identification of monoclonal antibody

[0054] The specific identification of the monoclonal antibody includes indirect immunofluorescence (Immunofluorescence assay, IFA) identification and Western-blot identification.

[0055] Preparation of LMH positive infected cells: culture LMH cells, when the cells grow to about 80% confluence, transfect ChPV infectious clone plasmid pBluescript II SK(+)-ChPV into LMH cells, and normal cells are used as negative control, and then incubate in a 5% CO2, 37°C incubator for 3 days, then collect the LMH cells to obtain ChPV infectious clone plasmid pBluescript II SK(+)-ChPV transfected LMH positive infected cells, wherein the VP2 protein expressed by the LMH positive infected cells is about 61 kD.

[0056] The specific operation of IFA identification is as follows: use 6-well plates to prepare LMH positive infected cells according to the above method, use cell fixing solution (50% methanol + 50% acetone) to fix the LMH positive infected cells at room temperature for 20 min; after blocking at 37°C with 5% skimmed milk powder, use monoclonal antibody 3F8 diluted to 1:500 as the primary antibody, and use mouse negative serum as the primary antibody for the negative control, and incubate with the fixed LMH positive infected cells at 37°C for 1 h; wash with phosphate buffered saline (PBST) for 3 times, add FITC-goat anti-mouse IgG (H+L) antibody (1:500) and incubate at 37°C for 1 h; after washing with PBST, observe the fluorescence under an inverted microscope and take pictures. The results are shown in Figure 1 The results show that the LMH positive infected cells incubated with monoclonal antibody 3F8 appear green fluorescence, and no green fluorescence is detected in the negative control, that is, the monoclonal antibody 3F8 specifically binds to the protein produced by the LMH cells infected with ChPV infectious clone plasmid pBluescript II SK(+)-ChPV.

[0057] Western-blot identification: to verify the purified ChPV VP2 protein (VP2 protein is about 87 kD) and LMH positive infected cells, specific reaction with monoclonal antibody 3F8. The specific operation of Western-blot identification is as follows: LMH positive infected cells were prepared by cell culture bottle according to the above method, and then the transfected LMH positive infected cells were lysed by cell lysis solution to obtain cell lysate solution. After SDS-PAGE electrophoresis of the purified ChPV VP2 protein and the cell lysate solution, the proteins in the gel were transferred to the PVDF membrane using a transmembrane instrument, 5% skim milk was blocked overnight, monoclonal antibody 3F8 (1:1000) was used as the first antibody, and incubated at 37°C for 1h; AP-labeled goat anti-mouse IgG (1:2000 dilution) was used as the second antibody, and incubated at 37°C for 1h, and then developed according to the BCIP / NBT alkaline phosphatase color reagent kit instructions. The results are shown in Figure 2 Figure 2, monoclonal antibody 3F8 specifically binds to purified ChPV VP2 protein, and the protein size is about 87 kD; Figure 3 Figure 3 shows that monoclonal antibody 3F8 can specifically bind to VP2 protein expressed in positive cell lysate, and the protein size is about 61 kD.

[0058] Example 2, establishment of double antibody sandwich ELISA detection platform for monoclonal antibody application

[0059] 1) Optimization of antibody concentration

[0060] Specific detection verification was carried out with monoclonal antibody 3F8 as the capture antibody (or coating antibody) and ChPV VP2 protein, chicken anti-ChPV VP2 protein polyclonal antibody as the detection antibody, and HRP-labeled goat anti-chicken IgG as the enzyme-labeled antibody, to establish a double antibody sandwich ELISA (Double Antibody Sandwich ELISA, VP2-DAS-ELISA) detection platform for detecting ChPV VP2 protein, and the optimal use concentration of the capture antibody and the detection antibody was screened by square array titration method.

[0061] Coating antibody: monoclonal antibody 3F8 was diluted to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000 with coating buffer to prepare coating solution, and each dilution concentration of coating solution was added to 2 columns of enzyme-labeled holes (1-2, 3-4, 5-6, 7-8, 9-10, 11-12) for enzyme-labeled plate coating, 100 μL of coating solution with corresponding monoclonal antibody 3F8 concentration was added to each enzyme-labeled hole, 8 holes per column, a total of 12 columns, after coating at 4°C overnight, the liquid in the holes was discarded, each enzyme-labeled hole was washed with PBST for 3 times, and the liquid in the hole was patted dry on the absorbent paper after each washing.

[0062] Using PBST solution containing 1% bovine serum albumin (BSA) to prepare 5 μg / mL ChPV VP2 protein solution. Selecting 1 column of enzyme-labeled wells containing 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 and 1:32000 monoclonal antibody 3F8 (coating antibody concentration, 2 columns for each dilution, see coating antibody step), adding 100 μL of 5 μg / mL ChPV VP2 protein solution in turn, adding 100 μL of PBST solution containing 1% bovine serum albumin (BSA) to the other 6 columns as negative control, placing in a 37°C incubator for 1 h, then discarding the liquid in the wells, and washing 3 times with PBST.

[0063] Detection antibody: taking chicken anti-ChPV VP2 protein polyclonal antibody, using PBST containing 1% BSA to prepare detection antibody solution by diluting chicken anti-ChPV VP2 protein polyclonal antibody to 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, 1:128000 in turn, adding each concentration of detection antibody solution to each row (A-H) of the above-mentioned incubated enzyme-labeled plate, 12 wells in each row, adding 100 μL of detection antibody solution to each well, placing in a 37°C incubator for 45 min, then discarding the liquid in the wells, and washing 3 times with PBST.

[0064] Enzyme-labeled, color development: adding 100 μL of HRP-labeled goat anti-chicken IgG enzyme-labeled antibody (1:2000) to each enzyme-labeled well of the enzyme-labeled plate, placing in a 37°C incubator for 45 min, then adding 100 μL of single-component TMB color developing liquid to each enzyme-labeled well, developing for 10 min in the dark, and then adding 100 μL of stop solution to each enzyme-labeled well to terminate the reaction. Using an enzyme-labeled instrument to read the OD value of each well and recording, detection well OD value (P value): negative control well OD value (N value) = P / N value. 450nm

[0065] The test results of the VP2-DAS-ELISA detection platform are shown in Table 2.

[0066] Table 2 Concentration optimization results of capture antibody and detection antibody in VP2-DAS-ELISA detection

[0067]

[0068] ​The optimal dilution concentration of the capture antibody and the detection antibody in the VP2-DAS-ELISA detection results shown in Table 2 is indicated in bold. It can be seen that the optimal dilution concentration of the capture antibody (monoclonal antibody 3F8) is 1:4000, and the optimal dilution concentration of the detection antibody (polyclonal antibody of chicken anti-ChPV VP2 protein) is 1:8000.

[0069] Result determination: When the enzyme-labeled hole corresponding to the maximum P / N value of the capture antibody and the detection antibody concentration can be used as the optimal concentration. The detection result of OD 450nm value is: when P / N value > 2.1, the detection result is positive.

[0070] 2) Optimization of coating time and coating temperature

[0071] After the preliminary establishment of VP2-DAS-ELISA detection, the coating conditions of the capture antibody were further optimized, including further optimization of coating time and coating temperature. The monoclonal antibody 3F8 was prepared into a coating solution at a dilution of 1:4000 as described above, and the coating time and coating temperature were set to 4°C for 18h, 37°C for 1h, 37°C for 2h, and 37°C for 3h, respectively. The detection antibody was prepared into a detection antibody solution at a concentration of 1:8000, and the rest of the reaction conditions were performed according to the optimization steps of the above antibody concentration. The test results of the VP2-DAS-ELISA detection platform are shown in Table 3 as follows:

[0072] Table 3 Optimization results of coating conditions

[0073]

[0074] In the VP2-DAS-ELISA detection results shown in Table 3, the optimal coating conditions of VP2-DAS-ELISA detection are indicated in bold. It can be seen that the optimal coating time and temperature of the capture antibody are under the condition of 4°C for 18h, and the P / N value reaches the maximum. Therefore, the optimal coating conditions of the VP2-DAS-ELISA detection platform are preferably that the coating solution is placed in 4°C for 18h.

[0075] 3) Optimization of blocking time

[0076] According to the above-mentioned optimized VP2-DAS-ELISA detection platform: monoclonal antibody 3F8 is prepared as a coating solution at the above-mentioned dilution of 1:4000, the coating time and temperature are preferably 4°C for 18h, and the blocking is performed at 37°C using 5% skimmed milk. The blocking time is set at 30min, 45min, 60min and 90min for a total of four gradients, after blocking, 100μL of detection antibody (1:8000) is added to each well at 37°C for 45min; after washing with PBST solution for three times, 100μL of HRP-labeled goat anti-chicken IgG enzyme-labeled antibody (1:2000) is added to each well, and incubated at 37°C for 45min, then 100μL of single-component TMB color developing solution is added to each enzyme-labeled well, and the color is developed in the dark for 10min, and then 100μL of termination solution is added to each enzyme-labeled well to terminate the reaction. The OD 450nm value of each well is read using an enzyme-labeled instrument and recorded, the OD value of the detection well (P value) is calculated by the OD value of the negative control well (N value) = P / N value, and the test results of the VP2-DAS-ELISA detection platform are shown in Table 4 below:

[0077] Table 4 Optimization results of blocking time

[0078]

[0079] In the VP2-DAS-ELISA detection results shown in Table 4, the best time for monoclonal antibody 3F8 blocking is indicated in bold, and it can be seen that the P / N value is the highest when the blocking time is 45min, so the best blocking time in the VP2-DAS-ELISA detection platform is 45min.

[0080] 4) Optimization of enzyme-labeled antibody concentration

[0081] According to the above-mentioned optimized VP2-DAS-ELISA detection platform: monoclonal antibody 3F8 is prepared as a coating solution at the above-mentioned dilution of 1:4000, the coating time and temperature are preferably 4°C for 18h, and the blocking is performed at 37°C using 5% skimmed milk. The blocking time is set at 30min, 45min, 60min and 90min for a total of four gradients, after blocking, 100μL of detection antibody (1:8000) is added to each well at 37°C for 45min; after washing with PBST solution for three times, 100μL of HRP-labeled goat anti-chicken IgG enzyme-labeled antibody (1:2000) is added to each well, and incubated at 37°C for 45min, then 100μL of single-component TMB color developing solution is added to each enzyme-labeled well, and the color is developed in the dark for 10min, and then 100μL of termination solution is added to each enzyme-labeled well to terminate the reaction. The OD

[0082] Table 5 Optimization results of enzyme-labeled antibody concentration

[0083]

[0084] The optimal dilution concentration of the enzyme-labeled antibody in the VP2-DAS-ELISA detection results shown in Table 5 is indicated in bold. As can be seen, when the HRP-labeled goat anti-chicken IgG enzyme-labeled antibody is diluted at 1:4000, the P / N value is the highest, and therefore the optimal dilution of the HRP-labeled goat anti-chicken IgG enzyme-labeled antibody in the VP2-DAS-ELISA detection platform is 1:4000.

[0085] 5) Optimization of color development time

[0086] According to the above-optimized VP2-DAS-ELISA detection platform: the monoclonal antibody 3F8 is prepared as a coating solution at the above-mentioned dilution of 1:4000, the coating time and temperature are preferably 4°C for 18h, the blocking time after coating of the capture antibody (monoclonal antibody 3F8) is 45min, the detection antibody concentration is diluted at 1:8000 for incubation for 45min, and the HRP-labeled goat anti-chicken IgG enzyme-labeled antibody is diluted at 1:4000 to prepare an enzyme-labeled antibody solution. Single-component TMB color developing solution is used for color development, and the color development time is optimized and verified, with the color development time set at 5min, 8min, 11min, and 14min, respectively, and the remaining reaction conditions being according to the above-optimized steps. The test results of the VP2-DAS-ELISA detection platform are shown in Table 6 below:

[0087] Table 6: Optimization results of color development time

[0088]

[0089] The optimal color development time in the VP2-DAS-ELISA detection results shown in Table 6 is indicated in bold. As can be seen, when the color development time is 8min, the P / N value is the highest, and therefore the optimal color development time in the VP2-DAS-ELISA detection platform is preferably 8min.

[0090] 6) Determination of the critical value

[0091] According to the above-optimized VP2-DAS-ELISA detection platform: the monoclonal antibody 3F8 is prepared as a coating solution at the above-mentioned dilution of 1:4000, the coating time and temperature are preferably 4°C for 18h, the blocking time after coating of the capture antibody (monoclonal antibody 3F8) is 45min, the detection antibody concentration is diluted at 1:8000 for incubation for 45min, and the HRP-labeled goat anti-chicken IgG enzyme-labeled antibody is diluted at 1:4000 to prepare an enzyme-labeled antibody solution. Single-component TMB color developing solution is used for color development for 8min, and a total of 50 ChPV-negative clinical samples are detected. The VP2-DAS-ELISA detection platform obtains OD 450nm values for the 50 ChPV-negative clinical samples, and the OD 450nmThe average value (X) and the standard deviation (s) were calculated according to OD 450nm The positive and negative critical values of the sample were determined as X+3s. The average value (X) of the VP2-DAS-ELISA detection result was 0.066 and the standard deviation (s) was 0.008, so the critical value of the VP2-DAS-ELISA detection platform was X+3s=0.09, i.e. OD 450nm The detection result was determined as positive when the value was greater than or equal to 0.09, otherwise it was determined as negative.

[0092] Example 3, Performance verification of the double antibody sandwich ELISA (VP2-DAS-ELISA) detection platform using monoclonal antibodies

[0093] 1) Kit composition

[0094] According to the above-mentioned optimized VP2-DAS-ELISA detection platform, a ChPV double antibody sandwich ELISA (VP2-DAS-ELISA) detection kit was constructed, and the kit composition included: an enzyme-labeled plate coated with monoclonal antibody 3F8, a detection antibody solution, an enzyme-labeled antibody, a ChPV positive control, a ChPV negative control, a washing solution, a blocking solution, a color developing solution, and a termination solution.

[0095] Preferably, the enzyme-labeled plate was prepared by coating with the monoclonal antibody 3F8, wherein the coating concentration of the monoclonal antibody 3F8 was 1:4000, and the temperature and time of the coating reaction were 4°C for 18h;

[0096] Preferably, the detection antibody solution was prepared by the polyclonal antibody of the chicken anti-ChPV VP2 protein, wherein the concentration of the antibody was 1:8000;

[0097] Preferably, the enzyme-labeled antibody was HRP-labeled goat anti-chicken IgG, wherein the concentration of the antibody was 1:4000;

[0098] Preferably, the ChPV positive control was prepared by ChPV VP2 protein, wherein the concentration of the ChPV VP2 protein was 5μg / mL, and the negative control was a washing solution containing 1% bovine serum albumin (BSA);

[0099] Preferably, the blocking solution was a washing solution containing 5% skimmed milk powder; the washing solution was a PBST solution prepared by adding 5mL Tween-20 to 1L PBS solution and mixing; and the color developing solution was a single-component TMB color developing solution.

[0100] Preferably, the ChPV double antibody sandwich ELISA detection kit further included a sample diluent and a standard.

[0101] Further preferably, the standard was ChPV VP2 protein at a concentration of 5μg / mL.

[0102] The detection steps recorded in the ChPV double antibody sandwich ELISA test kit instruction book at least include:

[0103] 1) Prepare all reagents and samples to be tested, and equilibrate at room temperature for at least 30 min;

[0104] 2) Add 100 μL of samples to be tested, negative controls, positive controls, and / or standards to the enzyme-labeled wells, respectively, and incubate in a 37°C incubator for 1 h, then discard the liquid in the wells and wash with PBST for 3 times;

[0105] 3) Add 100 μL of detection antibody solution to the enzyme-labeled wells, respectively, and incubate at 37°C for 45 min, then discard the liquid in the wells and wash with PBST for 3 times;

[0106] 4) Add 100 μL of enzyme-labeled antibody to the enzyme-labeled wells, respectively, and incubate at 37°C for 45 min;

[0107] 5) Add color developing solution to the enzyme-labeled wells, avoid light, and incubate at room temperature for 8 min, then add 100 μL of stop solution to stop the reaction;

[0108] 7) Within 30 min, use an enzyme-labeled instrument to measure the optical density OD value at 450 nm wavelength. 450nm

[0109] 2) Specificity

[0110] The samples to be tested were selected from ChPV infectious clones, Newcastle disease virus (NDV), synovial bursa mycoplasma (MS), serum 4 type avian adenovirus (FadV-4), chicken infectious anemia virus (CIAV), chicken infectious bronchitis virus (IBV), avian metapneumovirus (amPV), H9N2 subtype avian influenza virus (AIV), egg drop syndrome virus (EDSV) isolate, and avian circovirus type 2 (AGV2) clinical positive samples, and the enzyme-labeled plate prepared from the monoclonal antibody 3F8 was detected by the above-mentioned optimized double antibody sandwich ELISA method for detecting ChPV VP2 protein, and the negative cloacal swab sample of chicken was used as a negative control, and the detection results of the VP2-DAS-ELISA detection method are shown in Figure 4 The detection results of the enzyme-labeled plate prepared from the monoclonal antibody 3F8 can be seen: only ChPV positive samples react, and no reaction occurs with other avian viruses (positive samples of NDV, MS, FadV-4, CIAV, IBV, amPV, H9N2 subtype AIV, EDSV, and AGV2), which proves that the detection specificity of the VP2-DAS-ELISA detection platform prepared from the monoclonal antibody 3F8 of the application is good.

[0111] 2) Sensitivity

[0112] ​ChPV VP2 protein was diluted with PBS to concentrations of 1000 ng / mL, 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.2 ng / mL, 15.6 ng / mL, 7.8 ng / mL, and 3.9 ng / mL as test samples. Sensitivity tests were performed using the optimized reaction conditions of the VP2-DAS-ELISA detection platform. The detection results of the VP2-DAS-ELISA method are as follows: Figure 5 As shown, in the test samples with different concentrations of ChPV VP2 protein, when the concentration is 15.6 ng / mL, the OD value of the detection results from the VP2-DAS-ELISA detection platform is [not specified]. 450nm The value is still greater than the critical value. Therefore, the lowest detection concentration of the VP2-DAS-ELISA method described in this invention can be identified as 15.6 ng / mL.

[0113] 3) Repeatability

[0114] ChPV VP2 protein was sequentially diluted to 250 ng / mL, 500 ng / mL, 750 ng / mL, and 1000 ng / mL. Clinical samples from SPF chickens with negative ChPV molecular detection results were used as negative controls. The reproducibility and stability of the VP2-DAS-ELISA detection platform were validated using the optimized reaction conditions. First, using the same batch of coated ELISA plates, three replicates of each ChPV VP2 protein dilution were performed for intra-batch reproducibility testing, and OD was measured. 450nm The intra-batch coefficient of variation was calculated. Then, at least three batches of coated ELISA plates were selected, and inter-batch reproducibility assays were performed on the four dilutions of ChPV VP2 protein. Three replicates were performed for each dilution of ChPV VP2 protein. The OD value of the VP2-DAS-ELISA detection method was... 450nm The mean, variance, and coefficient of variation of the three replicates are shown in Table 7. It can be seen that the intra-assay coefficient of variation and inter-assay coefficient of variation of the VP2-DAS-ELISA detection platform of the present invention are both less than 5%. Therefore, the repeatability of the VP2-DAS-ELISA method of the present invention is good.

[0115] Table 7. Repeatability of VP2-DAS-ELISA

[0116]

[0117] 4) Clinical sample validation

[0118] The 192 clinical samples were detected in parallel by referring to the optimal reaction conditions of the VP2-DAS-ELISA detection platform and by using the ChPV nested PCR method (refer to A novel tool for specific detection and quantification of chicken / turkey parvoviruses to trace poultry fecal contamination in the environment, Carratala A et al., Applied and environmental microbiology, 2012, vol. 78, No. 20, pp. 7496-7499) as a third-party reagent. The detection results of the VP2-DAS-ELISA method and the ChPV nested PCR method were compared and analyzed.

[0119] The results show that in the detection results of the VP2-DAS-ELISA method, 130 clinical samples are positive and 62 clinical samples are negative; in the detection results of the ChPV nested PCR method, 162 clinical samples are positive and 30 clinical samples are negative. The comparative analysis results are shown in Table 8. Through further calculation, compared with the ChPV nested PCR method, the positive coincidence rate of the VP2-DAS-ELISA method is 78.0%, the negative coincidence rate is 43.8%, and the total coincidence rate is 81.3%. Details are shown in Table 9.

[0120] Table 8 Comparison of positive rates of clinical sample detection results

[0121]

[0122] Table 9 Comparison of coincidence rates of clinical sample detection results

[0123]

[0124] The application screens 1 cell strain 3F8 capable of secreting monoclonal antibody by cell fusion technology of spleen cells and myeloma cells of immunized mice, and further prepares corresponding monoclonal antibody 3F8 by using purified ChPV VP2 protein as immunogen; the monoclonal antibody 3F8 can specifically combine chicken parvovirus and VP2 protein expressed in vitro by indirect immunofluorescence identification and Western-blot identification; the amino acid sequence of the monoclonal antibody 3F8 shows that the light chain of the monoclonal antibody is kappa chain and the heavy chain is IgG2a by mouse monoclonal antibody subtype identification kit and molecular biology sequence determination; the double antibody sandwich ELISA detection platform of the ChPV VP2 protein is established based on the monoclonal antibody 3F8, and the detection conditions are optimized, including that the optimal coating concentration of the capture antibody is 1:4000, the optimal concentration of the detection antibody is 1:8000, the optimal coating condition is 4 DEG C coating for 18h, the optimal incubation time of blocking is 45min, the optimal incubation concentration of the enzyme-labeled antibody is 1:4000, the optimal color development time is 8min, and the positive and negative critical value is 0.09. The optimized ChPV VP2-DAS-ELISA detection platform has a minimum detection line of 15.6ng / mL for chicken parvovirus, has strong specificity, high sensitivity and good stability, and has a total coincidence rate of 81.3% compared with 192 clinical samples by nest PCR method. The monoclonal antibody of the chicken parvovirus provided by the application can be used for preparing detection reagents related to chicken parvovirus, and provides raw materials for basic research of functions of the chicken parvovirus VP2 protein, and is helpful to basic research of proteins related to the chicken parvovirus. The ChPV VP2-DAS-ELISA detection platform constructed by the application can be used for rapid detection of ChPV in clinic, and is helpful to prevention and control of the chicken parvovirus in the poultry industry.

[0125] The specific embodiments of the application have been described in detail, so that those skilled in the art will easily understand. However, all the descriptions can be modified or replaced differently, and these changes are within the protection scope of the application. The whole scope of the application is given by the appended claims and any equivalents thereof.

Claims

1. A monoclonal antibody or its antigen-binding fragment thereof, specifically a monoclonal antibody 3F8 against chicken parvovirus VP2 protein or its antigen-binding fragment thereof; the hybridoma cell line 3F8 capable of secreting said monoclonal antibody 3F8 was deposited on September 14, 2023, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2023272, located at Wuhan University, Wuhan, China.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that: The light chain of the monoclonal antibody 3F8 or its antigen-binding fragment is a kappa chain; and / or, the heavy chain of the monoclonal antibody 3F8 or its antigen-binding fragment is an IgG2a subtype.

3. A host cell capable of producing the monoclonal antibody or antigen-binding fragment thereof as described in any one of claims 1-2.

4. A preparation method for preparing the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2, comprising the following steps: 1) The hybridoma cell line 3F8 with accession number CCTCC NO:C2023272 was used; 2) Culture the hybridoma cell line under suitable conditions to induce it to secrete monoclonal antibody 3F8; 3) The monoclonal antibody or its antigen-binding fragment is purified from the culture supernatant or ascites.

5. A kit for detecting chicken parvovirus, comprising: The monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-2.

6. The kit for detecting chicken parvovirus according to claim 5, characterized in that: The kit uses a double-antibody sandwich ELISA method, with the monoclonal antibody described in any one of claims 1-2 as the capture antibody, the polyclonal antibody against chicken parvovirus VP2 protein as the detection antibody, and HRP-labeled goat anti-chicken IgG as the enzyme-labeled antibody.

7. A method for detecting chicken parvovirus, comprising using a monoclonal antibody or an antigen-binding fragment thereof as described in any one of claims 1-2; the method does not include methods for diagnosing and treating the disease.

8. The method for detecting chicken parvovirus according to claim 7, characterized in that: The method is a double-antibody sandwich ELISA method, using the monoclonal antibody described in any one of claims 1-2 as the capture antibody, the polyclonal antibody against chicken parvovirus VP2 protein as the detection antibody, and HRP-labeled goat anti-chicken IgG as the enzyme-labeled antibody.

9. An use comprising the monoclonal antibody of any one of claims 1-2, and the host cell of claim 3, in any one of the following: 1) Prepare a kit for detecting chicken parvovirus; 2) Chicken parvovirus detection, the detection including: Epidemiological analysis, detection of isolated, non-living samples, and import / export quarantine inspection; The uses described do not include the diagnosis and treatment of diseases.

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