Monoclonal antibody of chicken infectious bursal disease virus, detection test paper and application thereof

Abstract

This invention discloses a set of paired monoclonal antibodies against infectious bursal disease (IBDV) in chickens, quantum dot test strips, and their preparation methods. The invention obtains monoclonal antibodies 1D7 and 9G9 by immunizing mice with IBDV-VP2 recombinant protein; both antibodies can specifically bind to IBDV. The test strip of this invention uses the quantum dot-labeled monoclonal antibody 9G9 as the detection probe, utilizing the double-antibody sandwich principle, employing 1D7 as the capture antibody and 9G9 as the labeling antibody. This test strip can rapidly, specifically, and sensitively detect IBDV in chickens. The test strip of this invention is suitable for rapid on-site diagnosis, animal quarantine, and epidemiological investigation of IBDV in chickens, and has significant application value.

Description

Technical Field

[0001] This invention relates to the field of bioimmunology, specifically to monoclonal antibodies against infectious bursal disease virus in chickens, test strips, and their applications. Background Technology

[0002] Infectious bursal disease (IBD) is an acute, highly contagious, immunosuppressive infectious disease caused by the infectious bursal disease virus (IBDV). It primarily affects the bursa of Fabricius, the central immune organ of chickens, leading to weakened immune function and making the chicken highly susceptible to secondary infections or vaccine failure, causing huge economic losses to the global poultry industry.

[0003] IBDV belongs to the genus Avian diRNAvirus in the family DiRNAviridae. Its main structural proteins include VP2 and VP3. The VP2 protein, located on the outer surface of the virus particle, is the main protective antigen and contains multiple neutralizing epitopes. The VP2 protein's amino acid region from position 206 to 350 is a hypervariable region; significant differences in the amino acid sequence of this region exist between different strains and are closely related to the virus's antigenicity, virulence, and immune evasion.

[0004] Currently, the prevention and control of infectious bursal disease (IBD) in chickens mainly relies on vaccination. However, existing vaccines have limitations such as unsatisfactory immune protection and difficulty in responding to viral mutations. In terms of diagnosis, traditional laboratory testing methods, such as virus isolation and identification and serological testing, have limitations including complex procedures, long testing cycles, and the need for specialized equipment and technicians, failing to meet the needs for rapid on-site testing. Therefore, developing convenient, efficient, and highly specific on-site testing technologies to achieve early and accurate identification of the disease is of great significance for the effective prevention and control of IBD in chickens and for ensuring the healthy and sustainable development of the poultry industry.

[0005] Quantum dots (QDs), as a novel fluorescent labeling material, have advantages such as broad excitation spectrum, narrow emission spectrum, high fluorescence intensity, and good photochemical stability. When applied to immunochromatographic test strips, they can significantly improve the sensitivity and stability of detection, making them an ideal alternative to traditional colloidal gold labels. Summary of the Invention

[0006] The purpose of this invention is to provide a set of paired monoclonal antibodies (1D7 as a capture antibody and 9G9 as a labeling antibody) for detecting infectious bursal disease virus (IBDV) in chickens. These paired monoclonal antibodies can achieve specific recognition of IBDV in chickens.

[0007] This invention also provides a quantum dot test strip with high specificity and sensitivity for detecting infectious bursal virus (IBDV) in chickens, and its preparation method.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A group of paired monoclonal antibodies against infectious bursal disease virus (IBDV) in chickens, wherein the monoclonal antibodies can specifically bind to IBDV, including monoclonal antibody 1D7 and monoclonal antibody 9G9, wherein monoclonal antibody 1D7 is a capture antibody and monoclonal antibody 9G9 is a labeling antibody; both monoclonal antibody 1D7 and monoclonal antibody 9G9 include a light chain variable region and a heavy chain variable region.

[0010] The amino acid sequence of the light chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID No. 8, and the nucleotide sequence is shown in SEQ ID No. 9; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID No. 17, and the nucleotide sequence is shown in SEQ ID No. 18.

[0011] The amino acid sequence of the light chain variable region of the monoclonal antibody 9G9 is shown in SEQ ID No. 26, and the nucleotide sequence is shown in SEQ ID No. 27; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 9G9 is shown in SEQ ID No. 35, and the nucleotide sequence is shown in SEQ ID No. 36.

[0012] The light chain variable region of the monoclonal antibody 1D7 contains four light chain framework regions, namely 1D7-LFR-1, 1D7-LFR-2, 1D7-LFR-3 and 1D7-LFR-4, with amino acid sequences of SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, respectively.

[0013] The heavy chain variable region of the monoclonal antibody 1D7 contains four heavy chain framework regions, namely 1D7-HFR-1, 1D7-HFR-2, 1D7-HFR-3, and 1D7-HFR-4; their amino acid sequences are SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, respectively.

[0014] The light chain variable region of the monoclonal antibody 9G9 contains four light chain framework regions, namely 9G9-LFR-1, 9G9-LFR-2, 9G9-LFR-3 and 9G9-LFR-4, with amino acid sequences of SEQ ID No.22, SEQ ID No.23, SEQ ID No.24 and SEQ ID No.25, respectively.

[0015] The heavy chain variable region of the monoclonal antibody 9G9 contains four heavy chain framework regions, namely 9G9-HFR-1, 9G9-HFR-2, 9G9-HFR-3, and 9G9-HFR-4; their amino acid sequences are SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, and SEQ ID No. 34, respectively.

[0016] The light chain variable region of the monoclonal antibody 1D7 includes three complementarity-determining regions, namely 1D7-LCDR-1, 1D7-LCDR-2 and 1D7-LCDR-3, with amino acid sequences of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively.

[0017] The heavy chain variable region of monoclonal antibody 1D7 includes three complementarity-determining regions, namely 1D7-HCDR-1, 1D7-HCDR-2 and 1D7-HCDR-3, with amino acid sequences of SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, respectively.

[0018] The light chain variable region of monoclonal antibody 9G9 includes three complementarity-determining regions, namely 9G9-LCDR-1, 9G9-LCDR-2 and 9G9-LCDR-3, with amino acid sequences of SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21, respectively.

[0019] The heavy chain variable region of monoclonal antibody 9G9 includes three complementarity-determining regions, namely 9G9-HCDR-1, 9G9-HCDR-2 and 9G9-HCDR-3, with amino acid sequences of SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30, respectively.

[0020] A quantum dot-labeled chicken infectious bursal disease virus antigen test strip includes a base plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are arranged sequentially along the sample flow direction; the nitrocellulose membrane has a detection line and a control line, the detection line is coated with the monoclonal antibody 1D7 as described in claim 1, the control line is coated with coated SPA, and the conjugate pad is coated with the quantum dot-labeled monoclonal antibody 9G9.

[0021] The test strips contain 1 μL of quantum dot-labeled monoclonal antibody 9G9 sprayed onto the pad; the concentration of monoclonal antibody 1D7 sprayed onto the detection line is 0.8 mg / mL; and the concentration of SPA coating is 1 mg / mL.

[0022] The method for preparing the test strip includes the following steps:

[0023] (1) Preparation of quantum dot-labeled monoclonal antibody: 2.5 μL of 8 μM QDs and 11.5 μL of EDC solution were added to a centrifuge tube, mixed well, and then placed in a shaker at 25 °C with a rotation speed of 220 r / min for 30 min in the dark. After the reaction, 7.28 μL of monoclonal antibody 9G9 and 18.72 μL of BBS buffer were added, and the mixture was shaken at 25 °C in the dark for 2.5 h to obtain quantum dot-labeled monoclonal antibody 9G9.

[0024] (2) Preparation of nitrocellulose membrane: The detection line of the nitrocellulose membrane was sprayed with the capture antibody 1D7 and the control line was sprayed with SPA. The membrane was then dried at 50°C for 3 h.

[0025] (3) Assembly of test strips: Fix the conjugate pad, sample pad, absorbent pad and nitrocellulose membrane to the base plate in sequence, with each part overlapping by 2 mm at the edges; finally cut into test strips of a certain size.

[0026] The method for interpreting the test results of the test strips is as follows: when fluorescent bands appear on both the test line and the control line, the result is positive; when only the control line shows a fluorescent band and the test line does not show a fluorescent band, the result is negative, indicating that the sample does not contain infectious bursal disease virus; when no fluorescent band appears on the control line, the result is invalid regardless of whether a band appears on the test line.

[0027] The application of the monoclonal antibody in reagents or kits for preparing infectious bursal virus in chickens.

[0028] Beneficial technical effects of the present invention:

[0029] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0030] (1) This invention is the first to screen out paired monoclonal antibodies 1D7 and 9G9 that can specifically recognize IBDV VP2 protein. The titers of these antibodies were determined to be no less than 1:1.024×10⁻⁶. 6 The affinity constants are as high as 3.51 × 10⁻⁶. 9 L / mol and 2.5×10 9 With a concentration of L / mol, these two antibodies exhibit extremely high antigen-binding activity and affinity. They target different epitopes of the IBDV VP2 protein and can form stable sandwich complexes, providing an important foundation for highly sensitive immunological detection.

[0031] (2) The quantum dot-labeled immunochromatographic test strip constructed in this invention combines the advantages of high fluorescence intensity of quantum dots and the specificity of double antibody sandwich. Experiments have shown that the test strip can complete the detection within 15 minutes and shows extremely high specificity when detecting IBDV. It has no cross-reaction with other common avian disease viruses and can effectively distinguish between negative and positive samples, greatly reducing the risk of false detection and false negative detection.

[0032] (3) The test strip of the present invention does not require special instruments and equipment. The operator only needs to drop the sample to be tested onto the sample pad, and the result can be read by the naked eye under ultraviolet light within 15 minutes. It greatly simplifies the detection process, lowers the technical threshold, and is very suitable for rapid on-site screening and epidemiological investigation in scenarios such as farms, grassroots veterinary stations, and port quarantine. Attached Figure Description

[0033] Figure 1 The agarose gel electrophoresis pattern of this invention is used to identify positive expression bacteria;

[0034] In this context, lane M represents the DNA Marker; lanes 1-5 contain bacterial PCR products.

[0035] Figure 2 SDS-PAGE was used to identify the VP2 recombinant protein expression product for this invention;

[0036] Lane M is the protein marker; lanes 1-2 are the supernatant and precipitate of the induced product, respectively.

[0037] Figure 3 This is a Western Blot identification result diagram of the present invention;

[0038] In this context, lane M is the marker; lane 1 is the reaction between the target protein and positive serum.

[0039] Figure 4 The serum titer of mice immunized with the VP2 recombinant protein of this invention before fusion;

[0040] Figure 5 The SDS-PAGE identification results of the monoclonal antibodies 1D7 and 9G9 of this invention;

[0041] Where M: protein marker; 1: ascites fluid before purification; 2: monoclonal antibody IgG after ascites purification.

[0042] Figure 6 The results of titer determination of the monoclonal antibodies 1D7 and 9G9 of this invention;

[0043] Figure 7 The graph shows the affinity assay results for the monoclonal antibodies 1D7 and 9G9 of this invention. Detailed Implementation

[0044] The specific embodiments of the present invention will be further described in detail below with reference to examples.

[0045] Unless otherwise specified, the instruments and equipment involved in the following examples are all conventional instruments and equipment; the biochemical reagents involved are all commercially available; and the experimental methods involved are all conventional methods.

[0046] Biomaterials:

[0047] The VP2 gene was extracted from fragment A of IBDV HLJ0504 strain (GenBank: GQ166972.1) and synthesized by Zhengzhou Qingke Biotechnology; the pET-28a-SUMO vector, Escherichia coli DH5α clones, and BL21(DE3) competent cells (pTf16 molecular chaperone) were preserved in our laboratory.

[0048] Main reagents and consumables:

[0049] BamHI, XhoI, Prime STAR® HS DNA Polymerase, 5× Prime STAR Buffer, dNTPs, rTaq enzyme, and T4 DNA ligase were all purchased from TakaRa; PVDF membranes and 0.22 μm sterile filter heads were purchased from Millipore; DL2000 Marker and 6× DNA loading buffer were purchased from Tiangen Biotech; positive serum reference was purchased from the China Institute of Veterinary Drug Control; ECL chemiluminescence kit was purchased from Kangwei Century; HAT medium, HT medium, PEG-1500, RPM1-1640 cell culture medium and fetal bovine serum were purchased from Gibco; 0.25% trypsin cell digestion solution was purchased from Beyotime; Freund's complete adjuvant, Freund's incomplete adjuvant, HRP-labeled goat anti-mouse IgG and HRP-labeled goat anti-chicken IgG were purchased from Sigma; 96-well cell culture plates and T25 / T75 cell culture flasks were purchased from Corning; isopropanol and chloroform were purchased from Sinopharm Group; DEPC water was purchased from Biosharp.

[0050] Example 1: Preparation of recombinant protein of chicken infectious bursal disease virus VP2

[0051] 1.1 Amplification of the IBDV-VP2 encoding gene:

[0052] The nucleotide sequence of the VP2 protein of IBDV strain HLJ0504 was extracted from fragment A of IBDV strain HLJ0504 (GenBank: GQ166972.1), as shown in SEQ ID NO.37, and its amino acid sequence is shown in SEQ ID NO.38.

[0053] Using the cDNA of IBDV HLJ0504 strain as a template, primers were designed using SnapGene software, and two restriction enzyme sites, BamHI and XhoI, and protective bases were introduced into the forward and reverse primers, respectively.

[0054] Its forward primer is shown in SEQ ID NO.39, and its reverse primer is shown in SEQ ID NO.40.

[0055] 1.2 Construction of pET-28a-SUMO-VP2 plasmid: Using the VP2 gene of IBDV HLJ0504 strain as a template, PCR amplification was performed using the forward and reverse primers in 1.1. After purification and recovery, the target gene was obtained. The amplification reaction system of the target fragment of the VP2 gene is shown in Table 1, with a total of 50 μL.

[0056] Table 1 PCR reaction system

[0057]

[0058] The target gene and pET-28a-SUMO vector were double-digested with BamHI and XhoI, and then recovered via gel extraction. The double-digested prokaryotic expression vector pET-28a-SUMO and the target gene were ligated using T4 DNA ligase to obtain the recombinant plasmid pET-28a-SUMO-VP2. The recombinant plasmid pET-28a-SUMO-VP2 was transformed into BL21(DE3) (pTf16 molecular chaperone) competent cells and plated on LB agar plates containing kanamycin and chloramphenicol. Single colonies were picked for colony PCR identification. The colony PCR reaction volume is shown in Table 2, with a total volume of 10 μL.

[0059] Table 2. Bacterial PCR Reaction System

[0060]

[0061] The target gene for the VP2 protein is 1350 bp in size. The experimental results of bacterial PCR amplification are as follows: Figure 1 As shown, a bright band can be clearly seen at approximately 1350 bp in lanes 1-5, indicating that the length of the obtained product is consistent with the length of the target gene, and the band is single and bright. The positive recombinant plasmid was sent for sequencing, and the sequencing was correct, indicating that the recombinant plasmid pET-28a-SUMO-VP2 was successfully constructed.

[0062] 1.3 VP2 recombinant protein induced expression:

[0063] Positive strains were induced to express recombinant VP2 protein using IPTG and L-arabinose. The bacterial sludge was then collected and ultrasonically disrupted. The supernatant and precipitate after induction and disruption were analyzed by SDS-PAGE. Results are shown below. Figure 2 The recombinant VP2 protein expressed was approximately 60 kDa, which is consistent with expectations.

[0064] The purified VP2 recombinant protein was identified using positive serum, and the results are shown below. Figure 3 The obtained recombinant VP2 protein can be recognized by positive serum, indicating that the protein expression was successful and the protein maintained good immunoreactivity.

[0065] Example 2. Preparation of hybridoma cell lines for monoclonal antibodies against VP2 protein of infectious bursal disease virus in chickens.

[0066] 2.1 Immunization of BALB / c mice

[0067] (1) The dose of VP2 recombinant protein for the first immunization was 50 μg. After mixing the protein with an equal volume of Freund's complete adjuvant, the mixture was emulsified into a water-in-oil state using an emulsifier and then transferred to a 1 mL syringe. BALB / c mice were immunized by subcutaneous injection at multiple points on the back.

[0068] (2) The mice were immunized a second time 14 days after the first immunization. The immunization dose and method were the same as the first immunization, except that Freund's complete adjuvant was replaced with Freund's incomplete adjuvant;

[0069] (3) The mice were immunized for the third time 28 days after the first immunization, and blood was collected from the tail of the mice on day 35. The serum was collected after centrifugation at 3000 r / min for 10 min, and the titer of the polyclonal antibody serum of the mice was determined by ELISA.

[0070] (4) If the measured serum titer of mice is not lower than 1:1×10 4 Mice with high antibody titers and good growth status were selected for pulse immunization on day 42. No adjuvants were used during the pulse immunization. The immunogen protein was diluted to 0.5 μg / μL with sterile PBS, and the mice were immunized intraperitoneally at a dose of 50 μg / mouse. The growth status of the mice was observed daily. Spleen cells were harvested 3-5 days after the pulse immunization for cell fusion experiments using traditional hybridoma techniques.

[0071] The measurement results are as follows Figure 4 As shown, mice 1 and 2 represent the corresponding polyclonal antibody sera, and NC represents the polyclonal antibody sera from mice that were not immunized. ELISA results showed that the serum from mouse 2 had the highest titer, reaching 1:102400. Therefore, mouse 2 was selected for cell fusion to prepare monoclonal antibodies.

[0072] 2.2 Cell Fusion

[0073] (1) Preparation of spleen cell suspension: The fur and peritoneum of the mouse abdomen were cut open with sterile scissors and tweezers, the spleen was removed and rinsed with GNK washing solution. The spleen was placed on a sterile 200-mesh nylon mesh and ground into a spleen cell homogenate with scissors. GNK washing solution was added continuously during the process to prevent damage to the spleen cells. After grinding, a spleen cell suspension was obtained.

[0074] (2) Cell counting: Transfer the spleen cell suspension to a centrifuge tube and centrifuge at 1200 r / min for 10 min. Discard the supernatant, resuspend the cells with fresh GNK washing buffer, mix gently, and take out a small amount of cell suspension for counting. Centrifuge the remaining cell suspension at 1200 r / min for 10 min to ensure that a large number of spleen cells are centrifuged. Then place the cells in a cell culture incubator for later use.

[0075] (3) Cell fusion: Cell fusion was performed at a ratio of 8:1 for spleen cells to SP2 / 0 cells. After centrifugation, the supernatant was discarded, and the cells at the bottom of the centrifuge tube were gently tapped with a fingertip and placed in a 37 ℃ water bath. 1 mL of preheated PEG1500 fusion agent was slowly added to the cells at the bottom of the centrifuge tube within 90 s, strictly controlled. After completion, the tube was allowed to stand for 90 s. Then, within 90 s, GNK washing buffer was added at the following rate: 1 mL in the first 30 s, 3 mL in the 30-60 s, and the remaining 11 mL in the 60-90 s to terminate the effect of PEG 1500. After standing at 37 ℃ for 5 min, GNK was added to a final volume of 40 mL, and the tube was centrifuged at 1000 r / min for 10 min.

[0076] (4) Plating: After discarding the supernatant, resuspend the cells in an appropriate amount of HAT medium, dilute the cells to a suitable density, and add 100 μl / well to a 96-well plate that has been pre-coated with feeder cells one day in advance. Incubate statically in a 37 ℃ 5% CO2 cell culture incubator. About 5 days after confluence, obvious cell clusters can be observed under a microscope.

[0077] 2.3 ELISA screening for positive hybridoma cell lines

[0078] (1) About 7 days after cell fusion, observe the cells at the bottom of the cell culture plate. When the cell cluster occupies about 1 / 3 of the bottom of the plate under a 10x microscope, use ELISA to detect the antibody titer secreted in the cell culture supernatant. Use blood from the eyeballs of fused mice diluted at 1:1000 as a positive control.

[0079] (2) Coat VP2 recombinant protein in the enzyme label strip at a ratio of 50 ng / well, add cell supernatant at a ratio of 50 μl / well under sterile conditions, incubate at 37 ℃ for 30 min, dilute HRP-Goat AntiMouse IgG with 5% skim milk at a ratio of 1:5000, add 50 μL / well to the enzyme label strip, incubate at 37 ℃ for 30 min, and develop TMB color for 10 min.

[0080] (3) Selecting at OD 450 The hybridoma cell clusters with the highest readings were transferred to 48-well cell culture plates and cultured in HT medium when their confluence at the bottom of the plate reached approximately 90%. The assay was repeated three times. Cell clusters showing a positive reaction with VP2 recombinant protein in all three assays were selected.

[0081] 2.4 Subcloning of positive hybridoma cells

[0082] The selected positive wells were subjected to subcloning screening using the limiting dilution method. After 3-4 rounds of subcloning, until the positive rate reached 100%, two hybridoma cell lines that could stably secrete specific monoclonal antibodies were finally obtained and named 1D7 and 9G9.

[0083] 2.5 Extraction of total RNA from hybridoma cell lines

[0084] (1) Culture the monoclonal hybridoma cell line in a T25 cell culture flask. When the cell density reaches about 80-90%, discard the cell culture supernatant and wash twice with sterile PBS. On the second wash, gently blow the cells down and transfer them to an enzyme-free centrifuge tube. Centrifuge at 1000 r / min for 5 min, discard the supernatant and add 1 mL of Trizol to vigorously blow the cells to completely rupture them.

[0085] (2) Add chloroform to the lysed cell solution at a rate of 200 μL / tube, and vortex vigorously until the upper layer of the solution in the tube is milky white. After standing at room temperature for 10 min, centrifuge at 12000 r / min at 4 ℃ for 10 min.

[0086] (3) After centrifugation, the solution in the tube is divided into 3 layers. Carefully transfer the upper transparent aqueous phase to a new enzyme-free centrifuge tube and add an equal volume of isopropanol. Gently invert and mix about 10 times. Let stand at room temperature for 15 min, and centrifuge at 4 ℃ and 12000 r / min for 10 min.

[0087] (4) After centrifugation, discard the upper liquid and add 1 mL of 75% ethanol to the precipitate at the bottom of the tube for resuspension. Gently shake to mix, and centrifuge at 12000 r / min for 10 min at 4 ℃. Repeat this step twice.

[0088] (5) After centrifugation, discard the supernatant, open the centrifuge tube cap in a clean bench, and after the ethanol at the bottom of the tube has dried completely, add 20 μL of DEPC water to the precipitate at the bottom of the tube. Use NanoDrop™ to determine the RNA concentration and store at -80 ℃.

[0089] 2.6 Total RNA reverse transcription and amplification of antibody variable region sequences

[0090] Using the Takara PrimeScript™ RT Reagent Kit, the total RNA extracted in the previous step was reverse transcribed into cDNA. Using this cDNA as a template, the variable region sequences of the antibody heavy and light chains were amplified separately using antibody variable region-specific primer pairs. The PCR amplification products were identified by 1.5% agarose gel electrophoresis, and the target band of the expected size was recovered using a DNA gel recovery kit. The purified target DNA fragment was sent to Zhengzhou Qingke Biotechnology Co., Ltd. for Sanger sequencing to obtain the nucleotide sequence of the antibody variable region.

[0091] 2.7 Variable region sequence of monoclonal antibodies

[0092] The variable region sequences of the heavy and light chains after sequencing were input into NCBI's IgBlast module for sequence alignment, and then aligned and labeled on the Weseq online server.

[0093] a. The sequence of monoclonal antibody 1D7 is as follows:

[0094] The amino acid and nucleotide sequences of the light chain variable region are shown in SEQ ID No. 8 and SEQ ID No. 9, respectively; the three complementarity-determining regions of the light chain variable region are 1D7-LCDR-1, 1D7-LCDR-2 and 1D7-LCDR-3, and their amino acid sequences are SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, respectively.

[0095] The light chain variable region contains four light chain framework regions, namely 1D7-LFR-1, 1D7-LFR-2, 1D7-LFR-3 and 1D7-LFR-4, with amino acid sequences of SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, respectively; see Table 3 for details.

[0096] Table 3. Amino acid sequence of the variable region of the light chain of monoclonal antibody 1D7

[0097]

[0098] The amino acid and nucleic acid sequences of the heavy chain variable region are shown in SEQ ID No. 17 and SEQ ID No. 18, respectively; the three complementarity-determining regions of the heavy chain variable region are 1D7-HCDR-1, 1D7-HCDR-2 and 1D7-HCDR-3, and their amino acid sequences are SEQ ID No. 10, SEQ ID No. 11 and SEQ ID No. 12, respectively.

[0099] The heavy chain variable region contains four heavy chain framework regions, namely 1D7-HFR-1, 1D7-HFR-2, 1D7-HFR-3, and 1D7-HFR-4; their amino acid sequences are SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, respectively; the specific sequences are shown in Table 4.

[0100] Table 4. Amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1D7

[0101]

[0102] b. Sequence of monoclonal antibody 9G9

[0103] The amino acid and nucleic acid sequences of the light chain variable region of monoclonal antibody 9G9 are shown in SEQ ID No. 26 and SEQ ID No. 27, respectively. The three complementarity-determining regions of the light chain variable region are 9G9-LCDR-1, 9G9-LCDR-2 and 9G9-LCDR-3, and their amino acid sequences are SEQ ID No. 19, SEQ ID No. 20 and SEQ ID No. 21, respectively.

[0104] The four light chain framework regions of the light chain variable region are 9G9-LFR-1, 9G9-LFR-2, 9G9-LFR-3 and 9G9-LFR-4, and their amino acid sequences are SEQ ID No.22, SEQ ID No.23, SEQ ID No.24 and SEQ ID No.25, respectively; see Table 5 for the specific sequences.

[0105] Table 5. Amino acid sequence of the variable region of the light chain of monoclonal antibody 9G9

[0106]

[0107] The amino acid and nucleic acid sequences of the heavy chain variable region of monoclonal antibody 9G9 are SEQ ID No. 35 and SEQ ID No. 36, respectively; the three complementarity-determining regions of the heavy chain variable region are 9G9-HCDR-1, 9G9-HCDR-2, and 9G9-HCDR-3, with amino acid sequences of SEQ ID No. 28, SEQ ID No. 29, and SEQ ID No. 30, respectively; the four heavy chain framework regions of the heavy chain variable region are 9G9-HFR-1, 9G9-HFR-2, 9G9-HFR-3, and 9G9-HFR-4, with amino acid sequences of SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, and SEQ ID No. 34, respectively; the specific sequences are shown in Table 6.

[0108] Table 6. Amino acid sequence of the variable region of the heavy chain of monoclonal antibody 9G9

[0109]

[0110] Example 3: Preparation of monoclonal antibody against VP2 protein of infectious bursal disease virus in chickens

[0111] 3.1 Preparation of monoclonal antibody ascites

[0112] Multiparous BALB / c female mice were intraperitoneally injected with 500 μL of Freund's incomplete adjuvant. Seven days after injection, slight abdominal distension was observed. Hybridoma cells were resuspended in sterile PBS and counted, then divided into groups at a ratio of 1 × 10⁻⁶. 6 Cells were injected into the peritoneal cavity of mice via intraperitoneal injection at a dose of 500 μL. The collected ascites fluid was centrifuged at 6000 r / min for 15 min, and the clear, pale yellow intermediate layer was collected and stored at -20 ℃ for later use.

[0113] 3.2 Purification of Monoclonal Antibodies

[0114] Ascites fluid was purified using a Protein A affinity chromatography column, following the manufacturer's instructions. The purified antibody was analyzed by SDS-PAGE, and the purified product was identified by electrophoresis. Results are shown below. Figure 5 As shown, the monoclonal antibodies purified by Protein A chromatography exhibited specific bands in the approximately 50 kDa (heavy chain) and 25 kDa (light chain) regions, with a significant reduction in background contaminants, indicating that the final monoclonal antibodies 1D7 and 9G9 were of high purity.

[0115] 3.3 Monoclonal antibody titer

[0116] The titer of monoclonal antibodies was determined by indirect ELISA. Results are shown below. Figure 6The titers of both monoclonal antibodies 1D7 and 9G9 are not less than 1:1.024×10. 6 .

[0117] 3.4 Affinity Measurement

[0118] CBS was used as the coating buffer. The VP2 recombinant protein was diluted to 2 μg / mL and 1 μg / mL, respectively. 100 μL of the diluted protein was added to each well, and the mixture was incubated overnight at 4°C. The remaining steps were as described in 3.3. Finally, the OD values ​​were read. 450 Values. Based on the concentration of each monoclonal antibody and OD. 450 A standard curve was established based on the proportional relationship, and the 50% OD was calculated. 450 The reciprocal of.

[0119] A linear equation is obtained by constructing a curve based on the reciprocals of the two equations, and then applying 50% OD. 450 Substituting the reciprocal of the equation into the linear equation, the corresponding antibody concentration is calculated. The units are then converted to molar concentrations and substituted into formulas (1) and (2) to calculate the affinity constant Kaff of the monoclonal antibody ([Ag]t represents the original coating concentration, and [Ab]t represents the corresponding monoclonal antibody molar concentration):

[0120] Kaff=(n-1) / 2(n[Ab']t-[Ab]t) (1)

[0121] n=[Ag]t / [Ag']t (2)

[0122] See results Figure 7 The affinity constant of monoclonal antibody 1D7 was calculated to be 3.51 × 10⁻⁶. 9 The affinity constant of monoclonal antibody 9G9 is 2.5 × 10 L / mol. 9 L / mol.

[0123] Example 4: Preparation of a quantum dot-labeled test strip for infectious bursal disease in chickens

[0124] 1. Preparation of quantum dot-labeled monoclonal antibodies

[0125] Immunofluorescent probes were prepared by labeling monoclonal antibodies with carboxylated water-soluble quantum dots using the EDC activation method. The specific steps are as follows:

[0126] Add 2.5 μL of QDs (8 μM) to a 1.5 mL centrifuge tube, followed by 11.5 μL of EDC solution. Mix well by pipetting and place the centrifuge tube in a shaker at 25°C for 30 min in the dark at 220 rpm to activate the carboxyl groups on the quantum dot surface. Then add 7.28 μL of monoclonal antibody 9G9 and 18.72 μL of BBS buffer, and react at 25°C with shaking for 2.5 h in the dark to obtain quantum dot-labeled monoclonal antibody 9G9.

[0127] 2. Preparation of nitrocellulose membrane: The capture antibody 1D7 (detection T line) and SPA (quality control C line) were sprayed onto nitrocellulose using a BioDot sprayer. The concentration of the capture antibody 1D7 was 0.8 mg / mL and the concentration of the coating SPA was 1 mg / mL. The membrane was then dried at 50℃ for 3 h.

[0128] 3. Assembly of the test strips

[0129] Spray quantum dot-labeled monoclonal antibody 9G9, 1 μL / strip, onto the conjugate pad. Fix the conjugate pad, sample pad, absorbent pad, and nitrocellulose membrane onto a PVC substrate, with each part overlapping by about 2 mm at the edges. Use a BioDot cutter to cut the chromatography card into 3 mm × 75 mm test strips, place them in an aluminum foil bag (containing desiccant), and seal at room temperature.

[0130] Method for determining test results:

[0131] Place 100 μL of virus sample onto the test strip and observe the results within 15 minutes.

[0132] If fluorescent bands appear on both the control line and the test line, the result is positive, meaning the sample contains infectious bursal disease virus (IBDV). If a fluorescent band appears on the control line but not on the test line, the result is negative, meaning the sample does not contain IBDV. If no fluorescent band appears on the control line, the result is invalid regardless of whether a band appears on the test line.

[0133] Partial sequence from the instruction manual:

[0134] The amino acid sequence of the variable region of the light chain of monoclonal antibody 1D7 is SEQ ID No. 8:

[0135] DIVLTQSPASLAVSLGQRATISYRAS KSVSTSGYSY MHWNQQKPGQPPRLLIY LVS NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC QHIRELTR SEGGPSWK

[0136] The nucleotide sequence of the variable region of the light chain of monoclonal antibody 1D7 is SEQ ID No. 9:

[0137] ATGGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTT GTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAA

[0138] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1D7, SEQ ID No. 17:

[0139] GASVKISCKTS GYTFTEYI MHWVKQSHGKSLEWIGG INPNNGGT SYNQKFKGKATLTVDKSSSTAYMELRRSLTSEDSAVYYC ARSITTPFAY WGQGTTVTVSS

[0140] The nucleotide sequence of the variable region of the heavy chain of monoclonal antibody 1D7 is SEQ ID No. 18:

[0141] ATGGGGGCTTCAGTGAAGATATCCTGCAAGACTTCTGGATACACATTCACTGAATATATCATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGGTATTAATCCTAACAATGGTGGTACTAGCTACAATCAGAAGTTCAAGG GCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAAGATTCTGCAGTCTATTACTGTGCAAGATCGATTACTACGCCTTTTGCTTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCATAA

[0142] The amino acid sequence of the variable region of the light chain of monoclonal antibody 9G9, SEQ ID No. 26:

[0143] QIVLTQSPAIMSASPGEKVTITCSAS SSVSY IHWFQQKPGTSPKLWIY STS NLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYC QQRSRFPWT FGGGTKLEIK

[0144] The nucleotide sequence of the variable region of the light chain of monoclonal antibody 9G9, SEQ ID No. 27:

[0145] ATGCAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAGTTACATTCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAAACTCTGGATTTATAGCACATCCAACCTG GCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGCCGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAAAGGAGTCGTTTCCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAATAA

[0146] The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 9G9, SEQ ID No. 35:

[0147] GASVKISCKAS GYTFTDYN MDWVRQSHGKSLEWIGT INPNYDST FYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYC AREGGNYRMGYAMDY WGQGTTVTVSS

[0148] The nucleotide sequence of the variable region of the heavy chain of monoclonal antibody 9G9, SEQ ID No. 36:

[0149] ATGGGGGCTTCAGTGAAGATATCCTGCAAGGCTTCTGGCTACACATTCACTGACTACAACATGGACTGGGTTAGGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAACTATTAATCCTAACTATGATAGTACTTTCTACAACCAGAAGTTCAAGGGAAAGGCCACATTGACTGTAGACAAGTCCTCCAGTACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACACTGCAGTCTATTACTGTGCAAGAGAGGGGGGTAACTACCGTATGGGCTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCATAA

[0150] The nucleotide sequence of the VP2 protein coding gene of IBDV HLJ0504 strain SEQ ID NO.37:

[0151]

[0152] The amino acid sequence of the VP2 protein of IBDV strain HLJ0504, SEQ ID NO.38:

[0153] QQIVPFIRSLLMPTTGPASIPDDTLEKHTLRSETSTYNLTVGDTGSGLIVFFPGFPGSIBGAHYTLQGNGNYKFDQMLLTAQNLPASYNYCRLVSRSLTVRSSTLPGGVYA LNGTINAVTFQGSLSELTDVSYNGLMSATANINDKIGNVLVGEGVTVLSLPTSYDLGYVRLGDPIPAIGLDPKMVATCDSSDRPRVYTITAADDYQFSSQYQAGGVTITLF SANIDAITSLSIGGELVFQTSVQGLILGATIYLIGFDGTAVITRAVAADNGLTAGTDNLMPFNIVIPTSEITQPITSIKLEIVTSKSSGGQAGDQMSWSASGSLAVTIHGG NYPGALRPVTLVAYERVATGSVVTVAGVSNFELIPNPELAKNLVTEYGRFDPGAMNYTKLILSERDRLGIKTVWPTREYTDFREYFMEVADLNSPLKIAGAFGFKDIIRALR

[0154] IBDV VP2 target gene PCR forward primers are shown in SEQ ID NO. 39:

[0155] CGGGATCCATGCAGCAGATCGTACCGTT

[0156] The reverse primer for IBDV VP2 target gene PCR is shown in SEQ ID NO.40:

[0157] CCGCTCGAGTTAACGGAGCGCACGGATGA

Claims

1. A group of paired monoclonal antibodies against infectious bursal disease virus in chickens, characterized in that: The monoclonal antibodies can specifically bind to infectious bursal virus in chickens, including monoclonal antibody 1D7 and monoclonal antibody 9G9, wherein monoclonal antibody 1D7 is a capture antibody and monoclonal antibody 9G9 is a labeling antibody; both monoclonal antibody 1D7 and monoclonal antibody 9G9 include a light chain variable region and a heavy chain variable region. The amino acid sequence of the light chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID No. 8, and the nucleotide sequence is shown in SEQ ID No. 9; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 1D7 is shown in SEQ ID No. 17, and the nucleotide sequence is shown in SEQ ID No.

18. The amino acid sequence of the light chain variable region of the monoclonal antibody 9G9 is shown in SEQ ID No. 26, and the nucleotide sequence is shown in SEQ ID No. 27; the amino acid sequence of the heavy chain variable region of the monoclonal antibody 9G9 is shown in SEQ ID No. 35, and the nucleotide sequence is shown in SEQ ID No.

36.

2. The monoclonal antibody according to claim 1, characterized in that: The light chain variable region of the monoclonal antibody 1D7 contains four light chain framework regions, namely 1D7-LFR-1, 1D7-LFR-2, 1D7-LFR-3 and 1D7-LFR-4, with amino acid sequences of SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7, respectively. The heavy chain variable region of the monoclonal antibody 1D7 contains four heavy chain framework regions, namely 1D7-HFR-1, 1D7-HFR-2, 1D7-HFR-3, and 1D7-HFR-4; their amino acid sequences are SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, and SEQ ID No. 16, respectively. The light chain variable region of the monoclonal antibody 9G9 contains four light chain framework regions, namely 9G9-LFR-1, 9G9-LFR-2, 9G9-LFR-3 and 9G9-LFR-4, with amino acid sequences of SEQ ID No.22, SEQ ID No.23, SEQ ID No.24 and SEQ ID No.25, respectively. The heavy chain variable region of the monoclonal antibody 9G9 contains four heavy chain framework regions, namely 9G9-HFR-1, 9G9-HFR-2, 9G9-HFR-3, and 9G9-HFR-4; their amino acid sequences are SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 33, and SEQ ID No. 34, respectively.

3. The monoclonal antibody according to claim 2, characterized in that: The light chain variable region of the monoclonal antibody 1D7 includes three complementarity-determining regions, namely 1D7-LCDR-1, 1D7-LCDR-2 and 1D7-LCDR-3, with amino acid sequences of SEQ ID No.1, SEQ ID No.2 and SEQ ID No.3, respectively. The heavy chain variable region of monoclonal antibody 1D7 includes three complementarity-determining regions, namely 1D7-HCDR-1, 1D7-HCDR-2 and 1D7-HCDR-3, with amino acid sequences of SEQ ID No.10, SEQ ID No.11 and SEQ ID No.12, respectively. The light chain variable region of monoclonal antibody 9G9 includes three complementarity-determining regions, namely 9G9-LCDR-1, 9G9-LCDR-2 and 9G9-LCDR-3, with amino acid sequences of SEQ ID No.19, SEQ ID No.20 and SEQ ID No.21, respectively. The heavy chain variable region of monoclonal antibody 9G9 includes three complementarity-determining regions, namely 9G9-HCDR-1, 9G9-HCDR-2 and 9G9-HCDR-3, with amino acid sequences of SEQ ID No. 28, SEQ ID No. 29 and SEQ ID No. 30, respectively.

4. A quantum dot-labeled antigen test strip for infectious bursal disease virus in chickens, characterized in that: The test strip includes a base plate, on which a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad are sequentially arranged along the sample flow direction. The nitrocellulose membrane has a detection line and a control line. The detection line is coated with the monoclonal antibody 1D7 as described in claim 1, the control line is coated with coated SPA, and the conjugate pad is coated with quantum dot-labeled monoclonal antibody 9G9.

5. The test strip according to claim 4, characterized in that: The amount of quantum dot-labeled monoclonal antibody 9G9 sprayed on the pad was 1 μL / strip; the concentration of monoclonal antibody 1D7 sprayed on the detection line was 0.8 mg / mL; and the concentration of SPA coating was 1 mg / mL.

6. A method for preparing the test strip according to claim 5, characterized in that: The preparation method includes the following steps: (1) Preparation of quantum dot-labeled monoclonal antibody: 2.5 μL of 8 μM QDs and 11.5 μL of EDC solution were added to a centrifuge tube, mixed well, and then placed in a shaker at 25 °C with a rotation speed of 220 r / min for 30 min in the dark. After the reaction, 7.28 μL of monoclonal antibody 9G9 and 18.72 μL of BBS buffer were added, and the mixture was shaken at 25 °C in the dark for 2.5 h to obtain quantum dot-labeled monoclonal antibody 9G9. (2) Preparation of nitrocellulose membrane: The detection line of the nitrocellulose membrane was sprayed with the capture antibody 1D7 and the control line was sprayed with SPA. The membrane was then dried at 50°C for 3 h. (3) Assembly of test strips: Fix the conjugate pad, sample pad, absorbent pad and nitrocellulose membrane to the base plate in sequence, with each part overlapping by 2 mm; finally cut into test strips of a certain size.

7. The method for preparing the test strip according to claim 6, characterized in that: The interpretation method for the test results is as follows: when fluorescent bands appear on both the test line and the control line, the result is positive; when only the control line shows a fluorescent band and the test line does not show a fluorescent band, the result is negative, indicating that the sample does not contain infectious bursal disease virus; when no fluorescent band appears on the control line, the result is invalid regardless of whether a band appears on the test line.

8. The use of a monoclonal antibody according to any one of claims 1-3 in a reagent or kit for preparing infectious bursal virus in chickens.

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