Rabbit monoclonal antibody against p27 protein of avian leukosis virus subgroup j, kit and application thereof in immunodetection

Abstract

The present application relates to rabbit monoclonal antibody against P27 protein of subgroup J of avian leukosis virus, kit and application thereof in immunodetection, and the present application has screened a pair of rabbit monoclonal antibody 13D11 and 6H9 against P27 protein of subgroup J of avian leukosis virus for the first time, the pair of antibody has the advantage of recognizing P27 protein of subgroup J, and the signal-to-noise ratio of CLIA pairing screening is as high as 43362.Based on the antibody, three detection systems of antibody sandwich ELISA, magnetic microsphere chemiluminescence immunoassay and color latex microsphere chromatography are established, the detection sensitivity is high, there is no cross reaction with common avian pathogens, and the present application has the characteristics of high sensitivity, strong specificity, multi-platform adaptation and the like, provides important technical means for clinical sample detection of subgroup J of avian leukosis and purification monitoring of breeding chicken group, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of animal disease immunology detection technology, specifically relating to monoclonal antibodies against avian leukosis virus subgroup J (ALV-J) P27 protein, kits, and their application in immunological detection. Background Technology

[0002] Avian leukosis (AL) is a neoplastic infectious disease in birds caused by avian leukosis virus (ALV). It can manifest in various pathological types, including lymphoid leukosis, myeloid leukosis, hemangioma, and renal tumor, and is often accompanied by decreased egg production, growth retardation, and increased mortality, resulting in performance losses. Based on the virus's host range, envelope antigenicity, and neutralizing properties, ALV can be divided into several subgroups, including A, B, C, D, E, and J. Subgroups A, B, C, D, and J are mostly exogenous pathogenic subgroups, while subgroup E is mostly endogenous non-pathogenic or low-pathogenic. Since its isolation and reporting in the late 1980s, subgroup J avian leukosis virus (ALV-J) has been persistently prevalent in chicken flocks in multiple countries, causing myeloma and immunosuppression in broilers, local breeds, and some laying hens, resulting in significant economic losses to large-scale poultry farming.

[0003] Because ALV is a retrovirus, there are currently no safe and effective therapeutic drugs or universally applicable preventive vaccines available in clinical practice. Control of avian leukosis mainly relies on the purification of breeder flocks: this involves regularly testing breeder serum, egg white, cloacal swabs, meconium, or cell cultures for viral antigens or specific antibodies, culling positive individuals, and cutting off vertical and horizontal transmission. Therefore, rapid, sensitive, specific, and standardized immunoassay reagents are crucial for ALV monitoring and purification. The ALV gag gene encodes a specific antigenic group, among which the P27 protein is the main structural protein of the viral nucleocapsid / capsid, with a molecular weight of approximately 27 kDa. It is a non-glycosylated or minimally modified conserved protein. P27 shows high homology among different exogenous ALV subgroups, has a high content in viral particles (accounting for more than 30% of the total viral protein), and contains multiple antigenic sites that can be recognized by antibodies. Therefore, it is widely used as a universal target for ALV antigen detection.

[0004] Existing ALV-P27 detection antibodies mainly fall into two categories. One category consists of polyclonal antibodies from rabbits, goats, etc., which have short preparation cycles and can recognize multiple epitopes, but suffer from large batch-to-batch variability and a high risk of cross-reactivity with endogenous ALV-E, making standardization difficult. The other category consists of murine hybridoma monoclonal antibodies, which can stably recognize a single epitope and are suitable for sandwich ELISA systems, but murine immune responses tend to favor a few linear epitopes, resulting in narrow epitope coverage of the monoclonal antibodies. Furthermore, long-term hybridoma culture is prone to chromosome loss and decreased antibody secretion, leading to limited batch-to-batch consistency and sensitivity. In addition, with the progress in ALV-J subset purification, an increasing number of clinically positive samples (such as cloacal swabs, meconium, and egg white) have low viral loads and complex matrices. Existing kits lack sufficient sensitivity when detecting such extremely low concentration positive samples, easily resulting in missed detections. This causes ALV evolution to stagnate after a certain stage, and the positive rate no longer continues to decline. Rabbit monoclonal antibodies are well-suited for developing standardized diagnostic reagents due to the high diversity of the rabbit immune system, the ability to produce high-affinity antibodies, the recognition of more conformations and weak immunogenic epitopes, and the stable expression through gene recombination, overcoming the instability of mouse hybridomas. However, rabbit monoclonal antibodies against ALV-J P27 and their application in immunoassay are currently lacking. Therefore, there is an urgent need to develop a high-affinity, high-specificity, and stably produced anti-ALV-J P27 rabbit monoclonal antibody to meet the requirements for sensitivity, specificity, and batch-to-batch consistency in avian leukosis eradication detection, reduce the false negative rate of low-load samples, and overcome the bottleneck in ALV-J subgroup virus eradication. Summary of the Invention

[0005] This invention addresses a gap in existing technologies by providing a monoclonal antibody against the P27 protein of avian leukosis virus subgroup J (ALV-J), a reagent kit, and its application in immunoassay. This invention is the first to screen and obtain a pair of high-affinity rabbit monoclonal antibodies against the P27 protein of avian leukosis virus. After CLIA pairing screening, the signal-to-noise ratio reached as high as 43362. Three detection systems were successfully established: antibody sandwich ELISA, magnetic microparticle chemiluminescent immunoassay, and color latex microsphere chromatography. These systems feature high sensitivity, strong specificity, and multi-platform compatibility, providing important technical means for clinical sample detection of avian leukosis subgroup J and monitoring of breeder flock purification, with broad application prospects.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a rabbit monoclonal antibody against avian leukosis virus subgroup J P27 protein, comprising: monoclonal antibody 13D11 and / or monoclonal antibody 6H9, wherein: the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 of monoclonal antibody 1 are shown in SEQ ID NO. 3-5, and the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 8-10, respectively; the amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 of monoclonal antibody 6H9 are shown in SEQ ID NO. 13-15, respectively, and the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 18-20, respectively.

[0007] Furthermore, the amino acid sequence of the light chain variable region of the monoclonal antibody 13D11 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.17.

[0008] Furthermore, monoclonal antibody 13D11 is used as the capture antibody, and monoclonal antibody 6H9 is used as the detection antibody. The capture and detection antibodies recognize different non-overlapping epitopes of the ALV P27 protein.

[0009] The present invention also provides a polynucleotide encoding the above-mentioned monoclonal antibody.

[0010] The present invention also provides a recombinant vector comprising the above-mentioned polynucleotides.

[0011] The present invention also provides antibody conjugates, namely, solid-phase antibody conjugates formed by binding the above-mentioned monoclonal antibodies with a solid-phase carrier, or labeled antibody conjugates formed by binding with a detection marker.

[0012] Furthermore, when the solid-phase antibody conjugate is formed by binding with a solid-phase carrier, the solid-phase carrier is selected from any one or more of the following: enzyme-labeled plate, magnetic beads, nitrocellulose membrane, polystyrene microspheres, agarose gel microspheres, glass fiber membrane, and nylon membrane; when the labeled antibody conjugate is formed by binding with a detection label, the detection label is selected from any one or more of the following: enzyme label, chemiluminescent label, fluorescent label, colored particle label, and radioactive label.

[0013] Further, the enzyme label is selected from any one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, and glucose oxidase; the chemiluminescent label is selected from any one or more of acridinium ester, luminol and its derivatives, isoluminol and its derivatives, and ruthenium tripyridine; the fluorescent label is selected from any one or more of fluorescein isothiocyanate, phycoerythrin, Cy3, Cy5, Alexa Fluor series fluorescent dyes, and quantum dots; the colored particle label is selected from any one or more of colored latex microspheres, colloidal gold particles, carbon nanoparticles, and selenium nanoparticles; the radioactive label is selected from: 125 I, 3 H, 14 C 32 Any one or more of P.

[0014] This invention also provides the application of the above-mentioned monoclonal antibodies, polynucleotides, recombinant vectors, and antibody conjugates in the preparation of avian leukosis virus detection kits.

[0015] Furthermore, the kit includes: a chromatography kit, a chemiluminescent immunoassay kit, or an enzyme-linked immunosorbent assay kit.

[0016] Furthermore, the kit includes: an antibody sandwich enzyme-linked immunosorbent assay kit, a magnetic microparticle chemiluminescence immunoassay kit, a colored latex microsphere immunochromatographic assay kit, a colloidal gold immunochromatographic assay kit, or a fluorescence immunochromatographic assay kit.

[0017] Furthermore, in the case of an antibody sandwich enzyme-linked immunosorbent assay kit, monoclonal antibody 13D11 is used as a capture antibody coated on the ELISA plate, and monoclonal antibody 6H9 is used as a detection antibody linked to horseradish peroxidase.

[0018] Furthermore, in the case of a magnetic microparticle chemiluminescent immunoassay kit, monoclonal antibody 13D11 is used as a capture antibody and coupled to the surface of carboxyl magnetic beads via EDC / NHS, while monoclonal antibody 6H9 is used as a detection antibody and linked to acridine ester, HRP, APC, or isoluminol.

[0019] Furthermore, when using a color latex microsphere immunochromatographic assay kit or a colloidal gold immunochromatographic assay kit, monoclonal antibody 6H9 is coated onto the detection line (T line) of a nitrocellulose membrane, and monoclonal antibody 13D11 is coupled to color latex microspheres or colloidal gold particles.

[0020] The present invention also provides a kit for detecting avian leukosis virus P27 antigen, the kit containing the above-mentioned monoclonal antibody or antibody conjugate.

[0021] Furthermore, monoclonal antibody 13D11 was used as the capture antibody, and monoclonal antibody 6H9 was used as the detection antibody.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The present invention successfully screened a pair of antibodies 13D11 (capture antibody) and 6H9 (detection antibody) from the spleen of New Zealand white rabbits immunized with ALV-J P27 protein. The two antibodies recognize different non-overlapping epitopes. After CLIA pairing screening, the signal-to-noise ratio reached as high as 43362, which laid a solid foundation for establishing a high-sensitivity detection method. Based on this antibody pair, three detection systems were simultaneously established: sandwich ELISA, magnetic microparticle chemiluminescence immunoassay, and color latex microsphere chromatography. These systems are suitable for high-throughput laboratory detection, precise quantification of trace antigens, and rapid on-site screening in poultry farms, respectively. The detection limit for recombinant ALV-J P27 protein can reach 0.5 ng / ml, and the detection sensitivity for ALV-J virus culture medium can reach 400-fold dilution, showing significant advantages in detecting samples with low viral load. All three detection methods show no cross-reactivity with various common avian pathogens such as avian influenza virus and Newcastle disease virus, and can specifically recognize ALV P27 antigen, ensuring accurate and reliable detection results. The antibody pair and three detection systems provided by this invention have the advantages of high affinity, multi-platform compatibility, high sensitivity, strong specificity, simple operation, and low cost. They can be used for clinical sample detection of avian leukosis and for the purification monitoring of avian leukosis in breeder flocks, and have important application value. Attached Figure Description

[0023] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The results of detecting different concentrations of ALV-JP27 protein standards using a color latex microsphere chromatography method based on rabbit monoclonal antibodies in Example 4 of this invention are shown. The concentrations from left to right are 16 ng / ml, 8 ng / ml, 4 ng / ml, 2 ng / ml, 1 ng / ml, and 0.5 ng / ml, with two replicates for each concentration.

[0025] Figure 2 The results of detecting ALV-J virus solution at different dilutions using a color latex microsphere chromatography method based on rabbit monoclonal antibody in Example 4 of this invention are shown. The dilutions from left to right are 100 times, 200 times, 400 times and 800 times, with two replicates for each dilution.

[0026] Figure 3The results of the specific detection of ALV using the color latex microsphere chromatography method based on rabbit monoclonal antibodies in Example 4 of this invention are shown from left to right as follows: avian influenza virus, Newcastle disease virus, infectious bronchitis virus, Salmonella pullorum, avian leukosis virus subgroup J positive, avian leukosis virus negative N1, avian leukosis virus subgroup K positive, avian leukosis virus negative N2, positive control, and negative control. Detailed Implementation

[0027] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning understood by those skilled in the art. Unless specifically stated, the reagents, methods, and equipment used in this invention are conventional reagents, methods, and equipment in this technical field. Unless specifically stated, the reagents and materials used in the following embodiments are commercially available.

[0028] Example 1: Preparation and screening of monoclonal antibodies against avian leukosis virus subgroup J p27 protein In this embodiment, New Zealand white rabbits were immunized with ALV-J P27 protein. Following this, B lymphocytes capable of recognizing the avian leukosis virus J subset P27 protein were enriched and screened from the immunized rabbit spleen using single-cell labeling and sorting techniques. The isolated B lymphocytes were cultured as single cells to obtain positive clones secreting monoclonal antibodies. Finally, using recombinant gene expression technology, naturally paired antibody light chain variable region (VL) and heavy chain variable region (VH) genes were obtained from the positive clone B lymphocytes via PCR amplification. These genes were then tandemly inserted into expression vectors along with the light chain constant region (CL) and heavy chain constant region (CH), respectively. These vectors were co-transfected into host cells, cultured, and purified to obtain rabbit-derived monoclonal antibodies against ALV-J P27 protein. Through cross-pairing screening, the optimal antibody pairs for the antibody sandwich assay were obtained. Specific experiments are as follows.

[0029] 1. Animal immunization Preparation of P27 protein: Based on the ALV-J P27 sequence (GeneBank accession number: OL799231.1, sites 1311~2051), the P27 gene was synthesized. Hind III, Xho I, and His tags were introduced upstream and downstream. The synthesized P27 gene fragment was then ligated into the vector pcDNA3.1. The ligation product was transformed into DH5α competent cells. After plasmid extraction and sequencing confirmation, the recombinant plasmid pcDNA3.1-JP27 was obtained. This recombinant plasmid was transfected into CHO cells. When the cell viability was ≤60%, the cell culture medium was harvested. After cell lysis, the cells were centrifuged at 12000×g for 20 min to remove cell debris. An appropriate amount of supernatant was taken for SDS-polyacrylamide gel electrophoresis. At the same time, the culture supernatant after centrifugation was filtered through a 0.45 μm filter and purified using a Ni column. After dialysis and medium replacement, the ALV-J P27 protein was obtained.

[0030] Animal Immunization: Two New Zealand white rabbits were immunized with purified ALV-J P27 protein. For the first immunization, each rabbit received 500 μg of protein, which was emulsified by mixing the P27 protein with Freund's complete adjuvant at a 1:1 ratio and then injected subcutaneously at multiple sites on the back and abdomen. Booster immunizations were then performed every two weeks, with each rabbit receiving 200 μg of protein, which was emulsified by mixing the protein with Freund's incomplete adjuvant at a 1:1 ratio and then injected subcutaneously at multiple sites on the back and abdomen. Three booster immunizations were performed. On the tenth day after the fourth immunization, blood was collected from the marginal ear vein to separate serum, and the antibody titer in the rabbit serum was detected using indirect enzyme-linked immunosorbent assay (ELISA). Rabbits with high antibody titers received a third booster immunization, and four days later, the spleen was sacrificed to separate spleen cells.

[0031] The indirect ELISA method for determining the titer of immune serum includes the following steps: (1) Coating: Prepare 0.5 μg / mL ALV-JP27 protein solution with carbonate buffer (pH 9.6), add 100 μL / well to the microplate, and coat overnight at 4℃; (2) Washing and blocking: Discard the liquid in the wells, add 300 μL of washing buffer PBST to each well, let stand for 30 s and then discard, repeat washing 3 times, pat dry the last time, then add 200 μL of blocking buffer to each well, and block at 37℃ for 1 h; (3) Gradual dilution and addition of serum to be tested: Discard the blocking solution, wash the plate 3 times and pat dry as described in step (2), then perform gradient dilution of the serum to be tested, starting with a 1:1000 dilution, and then perform a three-fold gradient dilution, adding the serum dilution solution at 100 μL / well. Add μL / well to the plate and incubate at room temperature for 1 h; (4) Secondary antibody incubation: Discard the liquid in the wells, wash the plate 3 times and pat dry as described in step (2), then add 100 μL / well of horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (purchased from ThermoFisher, catalog number 31460) diluted 1:5000 and incubate at room temperature in the dark for 1 h; (5) Color development and termination: Discard the secondary antibody, wash the plate 3 times and pat dry as described in step (2), then add 100 μL / well of TMB color development solution and react at 25℃ in the dark for 15 min. Finally, add 100 μL / well of termination buffer to terminate the reaction and measure the absorbance at 450 nm. Rabbit serum before immunization was used as a negative control, and the detection system without immune serum was used as a blank control (NC).

[0032] Table 1. Results of ALV-J P27 antibody titer in rabbit serum after quadruple immunization (OD) 450 )

[0033] The serum titer test results are shown in Table 1. It can be seen that both rabbits achieved the required titers after four immunizations, with titers exceeding 1:243000, indicating a strong immune response, suitable for subsequent monoclonal antibody isolation. Rabbit number 2, with the higher titer, was selected for a booster immunization. Four days later, the spleen was sacrificed to isolate spleen cells for subsequent B lymphocyte sorting.

[0034] 2. Isolation of B lymphocytes from the spleen and sorting and culture of antigen-specific B lymphocytes For relevant methods, please refer to the published patents “Method for efficiently isolating single antigen-specific B lymphocytes from spleen cells (Publication No.: CN110016462A, Publication Date: 2019-07-16)” and “An in vitro culture system for B lymphocytes and its application (Publication No.: CN111518765A, Publication Date: 2020-08-11)”.

[0035] The cultured B lymphocyte supernatant was used to identify positive clones that could recognize and bind to ALV-J P27 protein using an indirect ELISA method coated with P27 protein. A total of 1344 cell supernatants were tested, of which 91 were positive. Further testing and verification were performed on the 91 cell supernatants, and 6 clones with the highest values ​​were selected: 6C9, 2A5, 13D11, 4F9, 6H9 and 8B12. The results of the retesting of these 6 cell supernatants are shown in Table 2.

[0036] Table 2 Results of antigen-specific B lymphocyte screening

[0037] Table 2 shows that all six candidate B cell clones secreted antibodies that specifically bind to the ALV-J P27 protein. Among them, 6C9 and 2A5 showed the strongest binding activity; 13D11 and 4F9 were next; 6H9 and 8B12 had relatively lower activity but still maintained high levels. Overall, these six clones all possess good antigen recognition capabilities and can be used as candidates for subsequent rabbit monoclonal antibody pairing screening.

[0038] 3. Cloning of the rabbit monoclonal antibody gene B lymphocytes corresponding to positive clones were collected, and total RNA was extracted and reverse transcribed into cDNA. Using the cDNA as a template, the naturally paired rabbit antibody light chain variable region (VL) and heavy chain variable region (VH) genes were amplified by PCR. The PCR reaction system consisted of: 4 μL cDNA, 1 μL forward primer (10 mM), 1 μL reverse primer (10 mM), 12.5 μL 2×Gloria HiFi (from Ibotek), and 6.5 μL H2O. The PCR amplification program consisted of: 98℃ pre-denaturation for 30 s, followed by 40 cycles of 95℃ for 15 s, 60℃ for 15 s, and 72℃ for 40 s, and finally holding at 72℃ for 5 min.

[0039] The amplified DNA product was sequenced, and the sequence of the rabbit constant region was obtained by querying the IMGT online database, resulting in antibody gene sequences of the complete light chain (FL) and the complete heavy chain (FH).

[0040] 4. Expression and large-scale production of rabbit monoclonal antibodies Rabbit monoclonal antibodies recognizing the ALV-J P27 protein were produced on a large scale using recombinant expression technology. The heavy chain and light chain genes of the rabbit monoclonal antibody-positive clones selected in the above steps were loaded downstream of the signal peptide of the expression vector pcDNA3.1 via homologous recombination. Sequencing confirmed the successful construction of the recombinant plasmids. The aforementioned process was outsourced to Wuhan Aibote Biotechnology Co., Ltd.

[0041] The light and heavy chain recombinant expression vectors of each antibody were co-transfected into 293F cells. 72-96 hours after transfection, the cell supernatant was collected, containing the recombinant rabbit monoclonal antibody recognizing the ALV-J P27 protein. The target antibody was purified from the cultured cell supernatant using Protein G affinity gel resin (purchased from Tiandi Renhe; catalog number: SA020100), following the Protein G affinity gel resin manufacturer's instructions. The antibody was then collected by dialysis and its purity was verified by 12% polyacrylamide gel electrophoresis (SDS-PAGE) to be ≥ 95%. After successful verification, the antibody was aliquoted and stored at -20°C for later use.

[0042] 5. Rabbit monoclonal antibody pairing screening Since subsequent detection systems such as antibody sandwich ELISA need to be established, it is necessary to screen for paired antibody pairs that can recognize different non-overlapping epitopes on the ALV-J P27 protein.

[0043] Chemiluminescent immunoassay (CLIA) was used for pairing screening (see Example 3 for detailed operation). Six purified antibodies (6C9, 2A5, 13D11, 4F9, 6H9, and 8B12) were paired as capture and detection antibodies, respectively, forming a total of 36 pairing combinations. The signal-to-noise ratio (S / N = positive RLU / negative RLU) was calculated based on the detection results of positive and negative standards. The antibody pairing screening values ​​and S / N ratio data are shown in Table 3.

[0044] Table 3 Results of rabbit monoclonal antibody pairing screening

[0045] The results showed significant differences in signal-to-noise ratio (SNR) among different pairings. The 13D11 and 6H9 and 2A5 and 13D11 pairs exhibited relatively high SNRs, indicating that these antibodies recognize different epitopes on the ALV-J P27 protein and have no steric hindrance between them, making them suitable as antibody pairs for the antibody sandwich assay. The 13D11 and 6H9 pair had the highest SNR at 43362.25; therefore, subsequent experiments preferred 13D11 as the capture antibody and 6H9 as the detection antibody.

[0046] The sequence information of antibodies 13D11 and 6H9 is shown in Table 4. In the table, VL represents the light chain variable region, LCDR1-3 represent the light chain complementarity-determining regions CDR1-3 respectively, VH represents the heavy chain variable region, and HCDR1-3 represent the heavy chain complementarity-determining regions CDR1-3 respectively.

[0047] Table 4 Sequence information of rabbit monoclonal antibodies

[0048] Example 2: Analysis of the effectiveness of antibody sandwich enzyme-linked immunosorbent assay (ELISA) for detecting ALV-J based on rabbit monoclonal antibodies. This embodiment utilizes the two antibodies screened above to detect ALV-J P27 protein in test samples using antibody sandwich enzyme-linked immunosorbent assay (ELISA). The principle is as follows: a monoclonal antibody recognizing the ALV-J antigen (capture antibody, 13D11) is coated onto a solid-phase carrier (ELISA plate). After adding the test sample, the P27 antigen in the sample specifically binds to the capture antibody. After washing, another horseradish peroxidase-labeled monoclonal antibody recognizing a different epitope (detection antibody, 6H9) is added, forming a "capture antibody-P27 antigen-detection antibody-HRP" complex. The amount of labeled antibody is directly proportional to the content of P27 antigen in the sample. Finally, an enzyme-catalyzed substrate is added for color development, and the result is measured by optical density (OD) at 450 nm. 450 The P27 antigen concentration in a sample can be detected by measuring the P27 value.

[0049] The antibody sandwich ELISA system includes the following components: an enzyme-labeled plate coated with P27 antigen capture antibody (13D11), washing buffer, sample dilution buffer (for diluting meconium samples), enzyme-labeled detection antibody 6H9, substrate solution, stop solution, and negative and positive controls for quality control. The washing buffer is PBS buffer (PBST buffer) containing 1% Tween-20, usually prepared as a 10-fold concentrated form; the sample dilution buffer is phosphate buffer containing 0.5% (wt) casein (pH 7.4 ± 0.1, concentration 0.2M); the substrate solution is 0.3 g / L TMB chromogenic solution; the stop solution is 2 M hydrochloric acid solution; the negative control (NC) is normal DF1 cell culture supernatant with an OD value of approximately 0.037; the positive control (PC) is a solution containing avian leukosis virus P27 antigen diluted with DF1 cell culture supernatant.

[0050] The preparation method of the ELISA plate coated with P27 antigen capture antibody includes: diluting the purified capture antibody 13D11 to 0.5 μg / mL, adding 100 μL / well to a polystyrene microplate (96-well ELISA plate), and coating overnight at 4°C. The next day, discard the liquid in the wells, add 150 μL / well of PBST to wash once, and blot dry. Then add 200 μL / well of blocking buffer, incubate overnight at 4°C to block unbound sites, discard the blocking buffer, blot dry, dry in a desiccant chamber, seal in an aluminum foil bag containing desiccant, and store at 4°C for later use.

[0051] Preparation of enzyme-labeled antibody 6H9: The antibody was prepared using a horseradish peroxidase (HRP) rapid labeling kit (purchased from Huzhou Yingchuang Biotechnology Co., Ltd., catalog number HRP-L-100). In actual use, the antibody was appropriately diluted according to the titer of each batch. The diluent used was Tris-HCl buffer (pH 7.6) containing 1% (wt) bovine serum albumin (BSA).

[0052] The antibody sandwich ELISA detection includes the following steps: (1) Sample preparation: Dilute the sample to be tested with an equal volume of dilution buffer, i.e., add 50 μL of dilution buffer to 50 μL of sample to be tested and mix well; (2) Sample addition: Add 100 μL of diluted sample to the microplate coated with P27 protein capture antibody, and set up 2 wells for positive control and 2 wells for negative control, and incubate at 37℃ for 30 min; (3) Washing: Discard the liquid in the well, add 300 μL of washing buffer to each well, wash 4 times, and pat dry; (4) Add enzyme-labeled detection antibody: Add 100 μL of enzyme-labeled detection antibody to each well and incubate at 37℃ for 30 min; (5) Washing: Repeat step (3) to wash the plate; (6) Color development: Add 100 μL of TMB color development solution to each well and incubate at 37℃ in the dark for 10 min; (7) Termination: Add 50 μL of termination solution to each well and gently shake to mix; (8) Measurement: Measure 450 μL using a microplate reader. Optical density (OD) at nm 450 )value.

[0053] The criteria for determining the validity of the experiment are: the average value of the positive control (PC) ≥ 0.2 and the average value of the negative control (NC) ≤ 0.15. The S / P value is calculated as follows: S / P = (sample OD...) / (sample OD...) 450 Value - Negative control OD 450 (mean) / (positive control OD) 450 Mean - Negative Control OD 450 (mean); if the S / P ratio of the sample to be tested is ≥ 0.2, it is judged as positive for P27 antigen; if the S / P ratio is < 0.2, it is judged as negative for P27 antigen.

[0054] The sandwich ELISA method for detecting 13D11 capture-6H9, established as described above, was compared with the sensitivity of a commercially available kit of the same type. Recombinant ALV-J P27 protein standards were serially diluted to specified concentrations, and the isolated ALV-J subset virus culture medium (starting concentration 10) was used. 5.2 TCID 50The samples were diluted 100, 200, 400, 800, and 1600 times (dilution matrix: PBS) respectively. The above-mentioned ELISA detection kit and the IDVet avian leukosis virus ELISA group-specific antigen detection kit (veterinary drug production license number 154718819) were used for detection. The detection results are shown in Table 5.

[0055] Table 5. Comparison of analytical sensitivity for detecting ALV-J P27 antigen using sandwich ELISA based on rabbit monoclonal antibodies.

[0056] Table 5 shows that the antibody sandwich ELISA detection system established based on the two monoclonal antibodies 13D11 and 6H9 screened in this invention has comparable sensitivity to the detection of ALV-J virus culture medium by the IDVet commercial kit, both detecting positive results within 200-fold dilutions. This indicates that the antibodies of this invention have good recognition ability for the native conformation P27 antigen. In the detection of recombinant P27 protein, the limit of detection of the method of this invention is 1 ng / mL, which is comparable to the sensitivity of imported commercial kits.

[0057] The specificity of the 13D11-6H9 antibody sandwich ELISA established in this invention for detecting ALV was further validated. Positive samples P1 (Avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Salmonella pullorum, and ALV-J subgroup (ALV-J), positive sample P2 (ALV-K), and negative samples N1 and N2 (Avian leukosis virus) were detected. Positive and negative samples of Avian leukosis virus were confirmed using a commercially available IDVet ELISA kit. The detection results are shown in Table 6.

[0058] Table 6. Specificity of ALV Detection Based on Antibody Sandwich ELISA Established with Rabbit Monoclonal Antibody

[0059] The results showed that, except for the positive samples of avian leukosis virus with an S / P value ≥ 0.2 (classified as positive), the S / P values ​​of other common avian pathogens and negative samples were all < 0.2, and were all classified as negative. This indicates that the detection system established in this invention has good specificity for ALV P27 antigen and no cross-reaction with other common avian pathogens, and can be used for avian leukosis eradication.

[0060] Example 3: Analysis of the effectiveness of a chemiluminescent immunoassay based on rabbit monoclonal antibodies for detecting ALV-J P27 protein. This embodiment, based on the two antibodies screened above, further employs antibody sandwich magnetic microparticle chemiluminescent immunoassay (CLIA) technology to detect ALV-J P27 protein in the test sample. The principle is as follows: a monoclonal antibody recognizing ALV-J P27 protein (capture antibody 13D11) is coated onto the surface of magnetic microparticles, and another monoclonal antibody recognizing a different epitope (detection antibody, 6H9) is labeled with acridine ester. When the P27 antigen is present in the test sample, the antigen can simultaneously bind to both the capture antibody on the magnetic beads and the acridine ester-labeled detection antibody, forming a "magnetic bead-capture antibody-antigen-detection antibody-acridine ester" complex. After washing to remove unbound labeled antibodies, pre-activation and activation solutions are added. The acridine ester emits light under alkaline H2O2 conditions, and the luminescence intensity (RLU) is directly proportional to the P27 antigen content in the sample. The concentration of P27 antigen in the test sample is detected by the optical system of the chemiluminescent immunoassay analyzer.

[0061] The magnetic microparticle chemiluminescent immunoassay (CLIA) system comprises the following components: magnetic beads coated with P27 protein capture antibody (13D11), acrid ester-labeled detection antibody (6H9), wash buffer, pre-activation solution, activation solution, and positive and negative calibrators for calibration, and quality control solutions C1 and C2 for quality control. The wash buffer is an aqueous solution (pH 7.2) containing 25 mM Tris, 150 mM NaCl, and 0.1% Tween 20; the pre-activation solution is an aqueous solution containing 0.1% H2O2 and 0.1 M HCl; and the activation solution is an aqueous solution containing 0.2 M NaOH and 2% Triton X-100. The negative calibrator and quality control solution C1 are PBS, and the positive calibrator and quality control solution C2 are recombinant ALV P27 protein standards.

[0062] The preparation method of P27 protein capture antibody-coated magnetic beads includes: taking carboxyl magnetic beads (purchased from JSR, catalog number MS160) into a centrifuge tube, performing magnetic separation on a magnetic rack, discarding the supernatant, and washing the magnetic beads three times with 100 mM MES buffer (pH 5.0). Resuspending the magnetic beads in 100 mM MES buffer (pH 5.0), adding 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS) to a final concentration of 2.5 mg / mL, and reacting at room temperature for 30 min. Then, magnetic separation is performed, washing the magnetic beads three times, resuspending the magnetic beads in 100 mM MES buffer (pH 5.0) containing 0.05% Tween 20, adding the capture antibody 13D11 at an antibody-to-magnetic-bead mass ratio of 1:20, mixing well, and reacting at room temperature for 2 h. Finally, wash the magnetic beads three times, dilute them to 0.5 mg / mL, and store them at 4°C for later use.

[0063] The preparation method of acridine ester-labeled detection antibody includes: taking 50 μL of purified detection antibody 6H9 (concentration 2 mg / mL) solution into a brown centrifuge tube, adding 35 μL of 0.02 M PBS, mixing well, then adding 15 μL of 5 mM acridine ester solution dissolved in dimethyl sulfoxide (DMSO), mixing well, briefly centrifuging, and labeling at 1500 rpm at room temperature for 2 h in the dark. After briefly centrifuging the acridine ester-labeled antibody, it is placed in a dialysis bag, using 0.02 M PBS as the dialysis buffer, changing the dialysis buffer every 4 h, and after 3 changes of the dialysis buffer, the labeled substance is recovered, diluted to 0.2 μg / mL, and stored at -20℃ in the dark for later use.

[0064] The reaction procedure for magnetic microparticle chemiluminescence method is as follows: (1) Sample addition: Add 20 μL of the sample to be tested (meconium, egg white or cell culture supernatant), 30 μL of P27 protein capture antibody coated magnetic beads (13D11-MBs) and 30 μL of acridinium ester labeled detection antibody (6H9-AE); (2) Incubation: Incubate at 37℃ for 20 min; (3) Wash; (4) Add substrate: Add 100 μL of pre-activation solution and 100 μL of activation solution, react for 64 s; (5) Detection.

[0065] The criteria for determining the validity of the test are as follows: For control sample C1 (negative), the S / Co ratio must be between 0 and 0.04; for control sample C2, the S / Co ratio must be between 1.90 and 2.30. The S / Co value is calculated as: S / Co = Sample luminescence value / Cutoff value. A sample with an S / Co ratio ≥ 1.0 is considered positive for P27 antigen, and a sample with an S / Co ratio < 1.0 is considered negative for P27 antigen. The Cutoff value is determined by using ROC curves on a large number of clinical samples and is set to 50,000.

[0066] The 13D11-6H9 magnetic microparticle chemiluminescence method established above was compared with the sensitivity of a commercially available kit of the same type. Recombinant ALV-J P27 protein standards were serially diluted to specified concentrations, and the isolated ALV-J subpopulation virus culture medium (starting concentration 10) was used. 5.2 TCID 50 The samples were diluted 100, 200, 400, 800, and 1600 times (dilution matrix: PBS) respectively. The above detection system was used in conjunction with the IDVet avian leukosis virus ELISA group-specific antigen detection kit (veterinary drug production license number 154718819) for detection. The detection results are shown in Table 7.

[0067] Table 7. Analytical sensitivity of the chemiluminescent immunoassay based on rabbit monoclonal antibody for detecting ALV-J P27 antigen.

[0068] The results showed that the 13D11-6H9 magnetic microparticle chemiluminescence detection system established in this invention has higher detection sensitivity for ALV-J virus culture medium than the commercial IDVet kit, still detecting positive results at a 400-fold dilution, while the commercial IDVet kit showed negative results at the same dilution. In the detection of recombinant P27 protein, the method of this invention showed a higher signal response than the imported commercial kit, with a detection limit of 500 pg / ml (0.5 ng / mL), demonstrating higher detection sensitivity than traditional methods. In summary, the CLIA method based on 13D11 and 6H9 dual rabbit monoclonal antibodies established in this invention has higher detection sensitivity for P27 antigen in natural viral fluids and meets the high-sensitivity detection requirements for purified P27 protein, making it suitable for accurate screening of avian leukosis-positive samples.

[0069] The specificity of the 13D11-6H9 magnetic particle chemiluminescence method for detecting ALV established in this invention was further validated by detecting positive samples P1 for avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Salmonella pullorum, and avian leukosis virus subgroup J (ALV-J), positive sample P2 for ALV-K, and negative samples N1 and N2 for avian leukosis virus. The positive and negative samples for avian leukosis virus were confirmed by commercially available IDVet ELISA kits, and the results are shown in Table 8.

[0070] Table 8. Specificity of ALV detection using a magnetic microparticle chemiluminescent immunoassay based on monoclonal antibodies.

[0071] The results showed that, except for the positive sample of avian leukosis virus with S / Co > 1.0 (judged as positive), the S / Co values ​​of the other samples were all < 1.0, and all were judged as negative. This indicates that the detection system of this embodiment has good specificity for ALV P27 antigen and no cross-reaction with other common avian pathogens, and can be used for avian leukosis eradication.

[0072] Example 4: Analysis of the effectiveness of a color latex microsphere chromatography method for detecting ALV-J P27 protein based on rabbit monoclonal antibodies. This embodiment, based on the antibodies screened above, further employs colored latex microsphere immunochromatography to detect ALV-J P27 protein in the sample. The principle is as follows: a monoclonal antibody recognizing ALV-J P27 protein (corresponding to the detection antibody 6H9 mentioned above) is coated onto the detection line (T line) of a nitrocellulose membrane (NC membrane), and goat anti-rabbit is coated onto the control line (C line). Separately, colored latex microspheres are used to label monoclonal antibodies recognizing different epitopes (corresponding to the capture antibody 13D11 mentioned above), and these labeled antibodies are sprayed onto the conjugation pad. When the sample to be tested (meconium, egg white, or cell culture supernatant) is added to the sample pad, under chromatographic action, if the sample contains P27 antigen, it binds to the latex microsphere-labeled antibody to form a "labeled antibody-antigen" complex, which continues to move forward and is captured by the capture antibody on the T line, forming a visible colored band. Unbound latex microsphere-labeled antibodies continue to move to the C line and are captured by the control line, forming a colored band. Regardless of the presence of antigen in the sample, the C line should develop color; otherwise, the test strip is invalid. This method is suitable for rapid on-site screening of avian leukosis virus subgroup J. The "detection antibody" corresponds to the antibody that generates the signal; monoclonal antibody 6H9 is used as the detection line (T line) coated on a nitrocellulose membrane.

[0073] The colored latex microsphere immunochromatographic system includes the following components: substrate (PVC plate), sample pad (glass fiber), conjugate pad (sprayed with antibody 13D11 labeled with colored latex microspheres), nitrocellulose membrane (NC membrane): T line coated with antibody 6H9 (0.5 mg / mL), C line coated with goat anti-rabbit (1 mg / mL), and absorbent pad.

[0074] Methods for preparing colored latex microspheres labeled with monoclonal antibodies include: (1) Preparation of colored latex microspheres: Red carboxylated polystyrene latex microspheres were prepared by emulsion polymerization. Appropriate amounts of styrene monomer, divinylbenzene, and acrylic acid were weighed and added to deionized water containing surfactant. Nitrogen gas was introduced for protection, and the mixture was heated to 70°C. Potassium persulfate was added to initiate the polymerization reaction, and the reaction was continuously stirred for 8 h. After the reaction, the mixture was filtered through a 0.45 μm filter membrane and washed three times by centrifugation with ultrapure water to remove unreacted monomers and surfactants. The precipitate was resuspended in MES buffer (50 mM, pH 6.0) containing 0.1% SDS, and the microsphere concentration was adjusted to 2% (w / v). Dynamic light scattering analysis showed that the average particle size of the latex microspheres was 200-300 nm, and the dispersion index (PDI) was <0.1. Ultraviolet-visible spectrophotometry showed that the maximum absorption wavelength was 520 nm (for red microspheres), indicating that the microspheres passed the test.

[0075] (2) Determination of optimal labeling pH: 100 μL of colored latex microsphere solutions with a series of pH values ​​(5.5, 6.0, 6.5, 7.0, 7.5, 8.0) were taken, and 10 μg of antibody 13D11 was added to each solution. After mixing, the solutions were incubated at room temperature for 30 min. The pH corresponding to the highest antibody conjugation rate was determined by measuring the absorbance of the supernatant at 520 nm. The optimal labeling pH was found to be pH 6.0.

[0076] (3) Determination of optimal antibody dosage: Take 100 μL of colored latex microsphere solution (2%, w / v), add 2.5, 5, 10, 15, 20, and 25 μg of antibody 13D11 respectively, and make up to 200 μL with MES buffer (50 mM, pH 6.0). React at room temperature for 2 h. After the reaction, centrifuge and wash twice, resuspend in PBS containing 1% BSA, measure the absorbance at 520 nm, and calculate the labeling efficiency. The minimum antibody dosage at which the labeling efficiency reaches the plateau phase is the optimal labeling dosage. The result is 10 μg antibody / 100 μL microspheres.

[0077] (4) Colored latex microspheres labeled with antibody: Take 200 μL of the prepared colored latex microspheres (2%, w / v) into a centrifuge tube, add MES buffer (50 mM, pH 6.0) to a total volume of 500 μL. Add 20 μL each of EDC solution (10 mg / mL) and NHS solution (10 mg / mL), and activate at room temperature for 30 min. Centrifuge (12000 rpm, 15 min), discard the supernatant, and resuspend the precipitate with MES buffer. Add an appropriate amount of antibody 13D11 (20 μg), and make up to 500 μL with MES buffer. React at room temperature for 2 h. After the reaction, add 10% BSA to a final concentration of 1% to block unbound sites, and incubate at room temperature for 30 min. Finally, centrifuge at 12,000 rpm for 15 min at 4°C, discard the supernatant, and resuspend the precipitate in 200 μL of phosphate buffer (pH 7.4) containing 1% BSA, 2% sucrose, and 0.05% Tween-20. Store at 4°C for later use.

[0078] Assembly of colored latex microsphere test strips: (1) Coating the NC membrane: Using a membrane scribing instrument, coat the T-line position with antibody 6H9 (0.5 mg / mL) and the C-line position with goat anti-rabbit (1 mg / mL). Dry at 37℃. (2) Treating the conjugate pad: Immerse the glass fiber in the treatment solution (0.02 M Tris-HCl buffer containing 0.5% BSA, 0.5% PVP, 0.05% Tween-20, and 2% sucrose, pH 8.0), soak for 10 min, and then dry at 37℃. Spray the prepared colored latex microsphere labeled antibody onto the conjugate pad and dry at 37℃. (3) Treating the sample pad: Immerse the glass fiber in the treatment solution (0.02 M Tris-HCl buffer containing 1% BSA, 0.2% PEG20000, and 0.1% Triton 100, pH 8.0), soak for 10 min, and then dry at 37℃. (4) Assembly: The prepared sample pad, conjugate pad, nitrocellulose membrane with detection lines T and C, absorbent pad and other materials are pasted onto the base plate in sequence, with each component overlapping by 1 mm. Cut into 3.0 mm wide test strips with a strip cutter, put them into the cartridge, add desiccant and seal for storage.

[0079] Color latex microsphere chromatography detection procedure and criteria for test validity: Detection procedure: Add 50 μL of the sample to be tested (meconium, egg white, or cell culture supernatant) to the sample well of the test strip, incubate at room temperature for 15 min, and read the results. Result interpretation: Positive: Both the C line and T line show colored bands. Negative: Only the C line shows a colored band; the T line does not show color. Invalid: The C line does not show color, regardless of whether the T line shows color, the test strip is considered invalid.

[0080] The established color latex microsphere chromatography strip based on 13D11 and 6H9 was compared with commercially available kits for parallel sensitivity analysis. Recombinant ALV-J P27 protein standards (starting concentration 1 mg / ml) were diluted to concentrations of 16 ng / ml, 8 ng / ml, 4 ng / ml, 2 ng / ml, 1 ng / ml, 0.5 ng / ml, 0.25 ng / ml, 0.1 ng / ml, and 0.05 ng / ml, respectively. The isolated ALV-J subset virus culture medium (starting concentration 10) was also used. 5.2 TCID 50 The samples were diluted 100, 200, 400, 800, and 1600 times (dilution matrix: PBS) respectively. The above detection system was used in conjunction with the IDVet Avian Leukosis Virus P27 Protein Detection Kit (Veterinary Drug Approval Number 154718819) for detection. The results are shown in Table 9. Figure 1-2 As shown.

[0081] Table 9. Analytical sensitivity of ALV-J P27 protein detection using color latex microsphere chromatography based on rabbit monoclonal antibody.

[0082] From Table 9 and Figure 1-2 As can be seen, the limit of detection for recombinant ALV-J P27 protein using the color latex microsphere chromatography test strip developed in this invention can reach 2 ng / ml. In the detection of ALV-J virus culture medium, the test strip of this invention can still detect positive results at a 200-fold dilution, which is comparable to the detection sensitivity of the commercial IDVet kit. This proves that the test strip of this invention has good capture and detection capabilities for P27 antigen in natural virus particles, and has the advantages of simple operation, rapid detection, and no need for instruments. It is suitable for rapid on-site screening in avian leukosis eradication work.

[0083] The specificity of the 13D11-6H9 color latex microsphere chromatography method for detecting ALV established in this invention was further validated. Positive samples of avian influenza virus (AIV), Newcastle disease virus (NDV), infectious bronchitis virus (IBV), Salmonella pullorum, avian leukosis virus subgroup J (ALV-J), positive samples of avian leukosis virus subgroup K (ALV-K), and negative samples N1 and N2 of avian leukosis virus were detected. Positive and negative samples of avian leukosis virus were confirmed by commercially available IDVet ELISA kits. The detection results are shown in Table 10. Figure 3 As shown.

[0084] Table 10. Specificity of ALV-J Detection Based on Colored Latex Microsphere Chromatography Using Rabbit Monoclonal Antibodies

[0085] The results showed that only samples positive for avian leukosis virus subgroup J showed color development on both the C and T lines, and were therefore considered positive. The remaining samples showed color development on the C line but no color development on the T line, and were therefore considered negative. This indicates that the 13D11-6H9 color latex microsphere chromatography method established in this invention has good specificity for the ALVP27 antigen and shows no cross-reactivity with other common avian pathogens, making it suitable for rapid and specific on-site screening of avian leukosis virus.

[0086] In summary, this invention successfully screened a pair of rabbit monoclonal antibodies (13D11 and 6H9) with high affinity for recognizing the J subset from the spleen of New Zealand white rabbits immunized with ALV-J P27 protein. The signal-to-noise ratio reached 43362 after CLIA pairing screening. Based on this antibody pair, three detection kits were developed: sandwich ELISA, magnetic microparticle chemiluminescence immunoassay, and color latex microsphere chromatography. The sandwich ELISA and color latex microsphere chromatography methods showed a detection sensitivity of up to 200-fold dilution for ALV-J virus culture medium. The color latex microsphere chromatography method showed a sensitivity of up to 2 ng / ml for recombinant ALV-J P27 protein, and the sandwich ELISA method showed a sensitivity of up to 1 ng / ml for recombinant ALV-J P27 protein, comparable to commercially available imported kits. The magnetic microparticle chemiluminescence immunoassay method achieved a detection limit of 0.5 ng / ml for recombinant ALV-J P27 protein and improved the detection sensitivity of ALV-J virus culture medium to 400-fold dilution, significantly outperforming commercially available imported kits and demonstrating superior detection capability for low viral load samples. All three detection methods showed no cross-reactivity with common avian pathogens such as avian influenza virus, Newcastle disease virus, infectious bronchitis virus, and Salmonella pullorum, and specifically recognized ALV virus, exhibiting excellent specificity. The antibody pair and detection system of the present invention have the advantages of high affinity, multi-platform compatibility, high sensitivity and strong specificity, and can be used for clinical sample detection of avian leukosis J subgroup and monitoring of breeder flock purification.

[0087] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A rabbit monoclonal antibody against the P27 protein of avian leukosis virus subgroup J, characterized in that, include: Monoclonal antibody 13D11 and / or monoclonal antibody 6H9, wherein: The amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 of the monoclonal antibody 13D11 are shown in SEQ ID NO. 3-5, and the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 8-10, respectively. The amino acid sequences of the light chain variable regions LCDR1, LCDR2, and LCDR3 of the monoclonal antibody 6H9 are shown in SEQ ID NO. 13-15, and the amino acid sequences of the heavy chain variable regions HCDR1, HCDR2, and HCDR3 are shown in SEQ ID NO. 18-20, respectively.

2. The monoclonal antibody according to claim 1, characterized in that, The amino acid sequence of the light chain variable region of the monoclonal antibody 13D11 is shown in SEQ ID NO.2, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.7; the amino acid sequence of the light chain variable region of the monoclonal antibody 6H9 is shown in SEQ ID NO.12, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.

17.

3. A polynucleotide encoding the monoclonal antibody of any one of claims 1-2.

4. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide of claim 3.

5. An antibody conjugate, characterized in that, A solid-phase antibody conjugate formed by binding a monoclonal antibody as described in claim 1 or 2 to a solid-phase carrier, or a labeled antibody conjugate formed by binding a detection marker.

6. The antibody conjugate according to claim 5, characterized in that, When a solid-phase antibody conjugate is formed by binding with a solid-phase carrier, the solid-phase carrier is selected from any one or more of the following: ELISA plate, magnetic beads, nitrocellulose membrane, polystyrene microspheres, agarose gel microspheres, glass fiber membrane, and nylon membrane; When a labeled antibody conjugate is formed by binding with a detection marker, the detection marker is selected from any one or more of the following: enzyme labeling, chemiluminescent labeling, fluorescent labeling, colored particle labeling, and radioactive labeling.

7. The use of the monoclonal antibody of any one of claims 1-2, the polynucleotide of claim 3, the recombinant vector of claim 4, or the antibody conjugate of any one of claims 5-6 in the preparation of an avian leukosis virus detection kit.

8. The application according to claim 7, characterized in that, The kits include: chromatography kits, chemiluminescent immunoassay kits, or enzyme-linked immunosorbent assay kits.

9. A kit for detecting avian leukosis virus P27 antigen, characterized in that, The kit contains the monoclonal antibody of any one of claims 1-2 or the antibody conjugate of any one of claims 5-6.

10. The reagent kit according to claim 9, characterized in that, Monoclonal antibody 13D11 was used as the capture antibody, and monoclonal antibody 6H9 was used as the detection antibody.

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