Hybridoma cell line secreting GCRV VP4 protein monoclonal antibody, monoclonal antibody and application thereof

Abstract

The present invention provides a hybridoma cell line that secretes a monoclonal antibody against the VP4 protein of grass carp reovirus, its monoclonal antibody and its application, which belong to the technical field of aquatic reovirus detection. The hybridoma cell line is named GCRV‑VP4‑6E7B12 and has been deposited in the China Center for Type Culture Collection on December 10, 2024, with a deposit number of CCTCC NO: C2024160. The monoclonal antibody 6E7B12 against the VP4 protein of grass carp reovirus secreted by the hybridoma cell line GCRV‑VP4‑6E7B12 has the advantage of specific binding to the antigen. The enzyme-labeled monoclonal antibody is used as the detection antibody, and the VP4 protein polyclonal antibody is used as the capture antibody. The double-antibody sandwich ELISA method can quantitatively detect the content of the VP4 protein of grass carp reovirus in the sample to be tested, with a detection range of 1~32ng / mL and a lower limit of detection of 1ng / mL. This detection method has the advantages of high sensitivity and a wide detection range.

Description

Technical Field

[0001] The present invention belongs to the technical field of aquatic reovirus detection, and particularly relates to a hybridoma cell line secreting a monoclonal antibody against the VP4 protein of grass carp reovirus (GCRV), the monoclonal antibody secreted therefrom, and applications thereof. Background Art

[0002] Grass carp reovirus (GCRV) is a double-stranded RNA virus belonging to the genus Aquatic Reovirus, family Reoviridae. Currently prevalent in China, GCRV strains are classified into three genotypes: GCRV-873 and GCRV-GZ1208 represent genotype I, GCRV-104 represents genotype III, and the majority of strains are genotype II. GCRV can cause severe hemorrhagic disease in grass carp during the summer, with mortality rates reaching 100% when water temperatures range from 24°C to 30°C. In addition to grass carp (Ctenopharyngodon idellus), GCRV has also been found to infect black carp (Mylopharyngodon piceus), broadhead carp (Pseudorasbora parva), and the rare minnow (Gobiocypris rarus), causing significant economic losses to my country's freshwater fish aquaculture industry.

[0003] Existing methods for detecting grass carp reovirus include PCR-based nucleic acid detection (e.g., methods disclosed in CN106811550B, CN109971890A, CN108504783A, etc.), RT-RPA-based detection (e.g., method disclosed in CN112680544B), loop-mediated isothermal nucleic acid amplification (e.g., methods disclosed in CN104673935A and CN104561379A), and RT-LAMP-based visualization detection (e.g., methods disclosed in CN104450961A, CN104450962A, and CN102703608A). In addition, there is an ELISA-based detection method (CN102876809B) based on IgM antibodies, which detects the presence of GCRV-II antibodies in grass carp serum samples. Currently, there are no ELISA detection methods or kits that directly detect genotype II grass carp reovirus. Summary of the Invention

[0004] The first object of the present invention is to provide an immunogenic grass carp reovirus VP4 protein. The nucleotide sequence encoding the grass carp reovirus VP4 protein is shown in SEQ ID No. 1.

[0005] A second objective of the present invention is to provide a hybridoma cell line that secretes a monoclonal antibody against the VP4 protein of grass carp reovirus. This hybridoma cell line, designated GCRV-VP4-6E7B12, was deposited with the China Center for Type Culture Collection at Wuhan University, Wuhan, Hubei Province, China, on December 10, 2024, with the deposit number CCTCC NO: C2024160. This hybridoma cell line secretes a monoclonal antibody against the VP4 protein of grass carp reovirus, encoded by the sequence set forth in SEQ ID No. 1.

[0006] The third object of the present invention is to provide the use of the hybridoma cell line with the deposit number CCTCC NO: C2024160 in preparing monoclonal antibodies against the VP4 protein of grass carp reovirus.

[0007] The fourth object of the present invention is to provide a monoclonal antibody 6E7B12 against the VP4 protein of grass carp reovirus secreted by the hybridoma cell line with the deposit number CCTCC NO: C2024160. The monoclonal antibody 6E7B12 is identified as an IgG1 antibody, and its light chain is of the κ subtype.

[0008] The fifth object of the present invention is to provide the use of the grass carp reovirus VP4 protein encoded by the sequence shown in SEQ ID No. 1 or the hybridoma cell line with the deposit number CCTCC NO: C2024160 or the monoclonal antibody 6E7B12 in the preparation of products for detecting grass carp reovirus.

[0009] In a preferred embodiment, the product for detecting grass carp reovirus is a kit.

[0010] In a further preferred embodiment, the kit is a double-antibody sandwich ELISA kit.

[0011] A sixth object of the present invention is to provide an ELISA kit for detecting grass carp reovirus, wherein the ELISA kit comprises the monoclonal antibody 6E7B12.

[0012] In a preferred embodiment, the ELISA kit further comprises a polyclonal antibody against the VP4 protein of grass carp reovirus as a capture antibody; and the monoclonal antibody 6E7B12 is enzyme-labeled and used as a detection antibody.

[0013] In a further preferred embodiment, the monoclonal antibody 6E7B12 is labeled with HRP (horseradish peroxidase) and used as a detection antibody.

[0014] In a further preferred embodiment, the ELISA kit further comprises a blocking solution, which is a 1×PBS buffer solution containing 0.1% (v / v) Tween-20, 0.5 wt % BSA and 0.04 wt % Block ACE.

[0015] In a further preferred embodiment, the ELISA kit further comprises grass carp reovirus VP4 protein as an antigen, and the coating concentration of the antigen is 1-32 ng / mL.

[0016] The technical solution of the present invention has the following beneficial effects:

[0017] (1) The monoclonal antibody 6E7B12 against the VP4 protein of grass carp reovirus secreted by the hybridoma cell line GCRV-VP4-6E7B12 in the present invention has the advantages of strong specificity and high antigen binding ability.

[0018] (2) An ELISA kit constructed using the enzyme-labeled grass carp reovirus VP4 protein monoclonal antibody provided by the present invention as the detection antibody and the grass carp reovirus VP4 protein polyclonal antibody as the capture antibody can quantitatively detect the content of grass carp reovirus VP4 protein in the test sample. The detection range is 1-32 ng / mL, and the detection limit is 1 ng / mL. The kit has the advantages of high sensitivity and a wide detection range. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a diagram showing the SDS-PAGE electrophoresis results of various solutions during the purification of the VP4 protein when preparing the VP4 protein in the present invention;

[0020] Figure 2 This is a diagram showing the SDS-PAGE electrophoresis result of the target protein collected after the VP4 protein is purified when preparing the VP4 protein in the present invention;

[0021] Figure 3 This is a diagram showing the SDS-PAGE electrophoresis results of the purified VP4 protein monoclonal antibody prepared in the present invention;

[0022] Figure 4 This is a diagram showing the results of Western-Blot identification of the VP4 protein polyclonal antibody prepared in the present invention;

[0023] Figure 5 The figure is a standard curve diagram of double antibody sandwich ELISA detection of VP4 protein using the VP4 protein monoclonal antibody and VP4 protein polyclonal antibody prepared in the present invention.

[0024] Deposit description: The hybridoma cell line in this application that secretes the grass carp reovirus VP4 protein monoclonal antibody encoded by the nucleotide sequence shown in SEQ ID No. 1 is named GCRV-VP4-6E7B12 (Hybridoma cell line GCRV-VP4-6E7B12), which was deposited in the China Center for Type Culture Collection on December 10, 2024, and the deposit address is Wuhan University, Wuhan City, Hubei Province, China, with the deposit number CCTCC NO: C2024160. DETAILED DESCRIPTION

[0025] The following content clearly and completely describes the technical solution of the present application in conjunction with the embodiments so that those skilled in the art can fully understand the present application. Obviously, the embodiments described are only some preferred embodiments of the present application, rather than all embodiments. Any equivalent transformation or substitution made by those of ordinary skill in the art to the following embodiments without creative work falls within the scope of protection of the present application.

[0026] The sources or components of the raw materials such as culture media, kits, plasmids, and competent cells used in the following examples are as follows:

[0027] LB liquid medium main components: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride;

[0028] LB solid medium main components: 10g / L tryptone, 5g / L yeast extract, 10g / L sodium chloride, 7.5g / L agar powder;

[0029] 5× reducing loading buffer was SDS-PAGE protein loading buffer (5×) purchased from Shanghai Biyuntian Biotechnology Co., Ltd., product number P0015;

[0030] The main components of incomplete 1640 medium are: 80% 1640 basal medium, 20% fetal bovine serum;

[0031] The main components of HAT complete medium are: 80% 1640 basal medium, 20% fetal bovine serum, and 1×HAT (Gibco™ 50×HAT, supplemented by a liquid mixture of sodium hypoxanthine (5mM), aminopterin (20µM), and thymidine (0.8mM)).

[0032] The pET-28a vector was purchased from Wuhan Tiande Biotechnology Co., Ltd., catalog number TD-M028. Competent BL21(DE3) cells were purchased from Wuhan Tiande Biotechnology Co., Ltd. BALB / c mice were purchased from Albirex Biotechnology Co., Ltd. SP2 / 0 cells were purchased from Wuhan Tiande Biotechnology Co., Ltd. HRP-conjugated goat anti-mouse IgG was purchased from Wuhan Tiande Biotechnology Co., Ltd. New Zealand white rabbits were purchased from Albirex Biotechnology Co., Ltd. Type II GCRV virus was isolated from diseased grass carp in the wild. Viral RNA was amplified by RT-PCR (forward primer: 5'-ATTCCACCACCTCCTGTGC-3'; reverse primer: 5'-CCACACCTCATCTGGCCATT-3'), Sanger sequenced, and sequence alignment was performed (reference sequence: Grass carpreovirus isolate 109 segment 11, complete sequence, NCBI accession number: KF712485.1). The alignment results were verified to be 100% identical. HRP-conjugated goat anti-rabbit IgG was purchased from Wuhan Tiande Biotechnology Co., Ltd. Block ACE was purchased from Bio-RAD with the product number BUF029.

[0033] Example

[0034] 1. Preparation of grass carp reovirus VP4 protein antigen

[0035] 1.1. Construction of a recombinant expression vector for GCRV VP4 protein: Based on the gene sequence of GCRV VP4 protein (Gene ID: AND67146.1), a full-length fragment was selected as the antigen. This fragment was optimized according to the codon preference of E. coli, and the nucleotide sequence shown in SEQ ID No. 1 was artificially synthesized (by Wuhan Tiande Biotechnology Co., Ltd.). The nucleotide sequence shown in SEQ ID No. 1 was ligated into the pET-28a vector via the NdeI and XhoI restriction sites to generate the recombinant expression vector pET-28a-VP4.

[0036] 1.2 Transformation with the recombinant expression vector: Transform 1-2 μL of the recombinant expression vector into competent BL21(DE3) cells. Place on ice for 30 minutes, then heat shock in a 42°C water bath for 90 seconds. Add an appropriate amount of antibiotic-free LB liquid medium and incubate at 37°C and 150 rpm for 40 minutes. Spread the mixture onto LB solid medium containing the appropriate resistance (e.g., kanamycin) and incubate overnight in a 37°C incubator.

[0037] 1.3. Testing the inducible expression conditions of the recombinant expression vector: Pick a single colony from the culture dish and transfer it to 3 mL of LB medium with the appropriate resistance (e.g., containing kanamycin). Cultivate at 37°C, 200 rpm for 3-4 hours. Transfer 1.5 mL of the bacterial culture to a new 15 mL centrifuge tube and add 1.5 μL of IPTG (final concentration 1 mM). Induce the culture at 37°C, 200 rpm for 5 hours. Add 1.5 μL of IPTG to the remaining 1.5 mL of bacterial culture and induce the culture overnight at 16°C, 200 rpm. After induction, transfer the culture to a separate 1.5 mL centrifuge tube, centrifuge at 12,000 g for 1 minute, and discard the supernatant. Add 1 mL of 1× PBS buffer to each pellet and sonicate to lyse the cells. Centrifuge at 12,000 g for 1 minute. Remove 100 μL of the supernatant, label it "NPE," discard the excess supernatant, and resuspend the pellet in 100 μL of 1× PBS (containing 8 M urea) and label it "DPE." Add 25 μL of 5× reducing loading buffer to the resuspended suspension, boil for 10 minutes, and analyze by 12% SDS-PAGE.

[0038] Based on the results of the above expression tests, the optimal expression conditions of VP4 protein were determined (see Table 1), and then amplified expression was performed.

[0039] Table 1 Optimal expression conditions of VP4 protein

[0040]

[0041] 1.4. Amplify expression: Take the correctly constructed recombinant expression vector pET28a-VP4 and induce expression according to the optimal expression conditions obtained in step 1.3. Then, centrifuge at 4°C, 10,000 rpm for 5 minutes to collect the cells.

[0042] 1.5. Purification of VP4 protein: Use an appropriate amount of lysis buffer (PBS with a pH of 7.5, 10% glycerol) to lyse the bacterial cells collected in step 1.4. After thorough mixing, use an ultrasonic disruptor to lyse for 20 minutes, then centrifuge at 12,000 g for 10 minutes, separate the supernatant (the supernatant protein mixture to be purified), and retain the precipitate. Add Ni resin carrying a His protein tag to the supernatant mixture, incubate in a shaker at 4°C for 30 minutes, collect the resin, temporarily retain the flow-through (FT), and then purify according to the procedure in Table 2 (the elution time for each step is 5 minutes). The supernatant IN, flow-through FT, wash buffer W1, wash buffer W2, and eluents E1-E6 after the purification process were sampled for 12% SDS-PAGE detection. The results are as follows. Figure 1According to the test results, the target protein was collected and dialyzed into 1× PBS buffer (pH=7.4). The protein concentration was detected by ultra-micro spectrophotometer and 12% SDS-PAGE. The results are shown in Figure 2 shown.

[0043] Table 2 VP4 protein purification procedure

[0044]

[0045] Figure 1 IN indicates the supernatant after disruption, FT indicates the flowthrough, W1 indicates the wash buffer 1, W2 indicates the wash buffer 2, E1~E6 indicate the eluents E1-E6, and M indicates the protein marker. Figure 1 As can be seen from the above, the eluates E1-E6 all contain the target protein. Figure 2 The upper M indicates protein marker, Figure 2 As can be seen from the above, the protein is of high purity and can be used for subsequent experiments.

[0046] From the test results, it can be seen that most of the VP4 proteins finally obtained exist in the supernatant in a soluble form. The sample information of the VP4 protein is shown in Table 3.

[0047] Table 3 Information of the prepared VP4 protein samples

[0048]

[0049] 2. Preparation of VP4 protein monoclonal antibodies

[0050] 2.1. Mouse immunization: 0.8 mg of the prepared VP4 protein antigen was mixed with an equal amount of adjuvant and emulsified. Four female BALB / c mice aged 4 to 6 weeks were immunized with a dose of 0.2 mg per mouse per time. The immunization method was subcutaneous injection in the abdomen. The specific operation steps are shown in Table 4 below. The serum collected before immunization was used as a negative serum control. On the tenth day after each immunization, blood was collected from the tail vein of the immunized mice. The collected serum was tested for antibody titer by indirect ELISA. When the mouse serum titer reached 10 5 At the above time, the antigen without adjuvant was used for booster immunization, and cell fusion was performed on the 3rd day. The results of mouse serum titer test are shown in Table 5.

[0051] Table 4 Immunization schedule for BALB / c mice

[0052]

[0053] Table 5 BALB / c mouse serum titer test results

[0054]

[0055] In Table 5, VP4-1 to VP4-4 represent the corresponding immunized mouse numbers. As can be seen from the table, VP4-4 and VP4-1 had the highest titers, so mice numbered VP4-4 and VP4-1 were selected for the subsequent cell fusion step.

[0056] 2.2 Cell Fusion

[0057] 2.2.1. One week before cell fusion, resuscitate SP2 / 0 cells and passage them normally so that the number of SP2 / 0 cells in the cell suspension is 1×10 7 At the same time, on the 3rd day after the booster immunization with the antigen without adjuvant, the mice were killed by removing the eyeballs and bleeding, and the collected serum was used as the positive serum control for the post-fusion test. The mice were soaked in a sterile beaker containing 75% ethanol for 5 minutes for disinfection. After absorbing the liquid on the surface of the mouse with absorbent paper, the spleen of the mouse was removed with a sterile scalpel, and the spleen was placed on the cell filter. The spleen was gently ground with the needle core of a sterile syringe. During the grinding process, the culture medium (incomplete 1640 culture medium) was continuously added to keep the spleen cells moistened. The filtered cells were collected in a sterile tube with a disposable sterile Pasteur pipette, and the spleen cells were repeatedly and gently blown to form a single-cell suspension. The suspension was centrifuged at room temperature and 1500r / min for 10 minutes, the supernatant was discarded, and the precipitate was suspended with incomplete 1640 culture medium so that the number of spleen cells in the suspension was 5×10 7 , spare.

[0058] 2.2.2 Perform cell fusion as follows: Thoroughly mix 15 mL of the spleen cell suspension prepared above using a sterile disposable Pasteur pipette and slowly add it along the tube wall to 15 mL of pre-prepared SP2 / 0 cells. Centrifuge at 1500 rpm for 8 minutes at room temperature. Discard the supernatant and gently tap the bottom of the tube to loosen the pellet. Equilibrate PEG 1450 (polyethylene glycol 1450) to room temperature in the dark. Place the tube in a 37°C water bath and add 1 mL of PEG 1450 at a constant rate over 1 minute, gently shaking the tube. Add 1 mL of incomplete 1640 medium preheated at 37°C at a constant rate over 1 minute. Then, add 3 mL of incomplete 1640 medium preheated at 37°C at a constant rate over 3 minutes. Finally, add 10 mL of incomplete 1640 medium preheated at 37°C at a constant rate over 10 minutes. Gently shake the centrifuge tube during addition. After completion of addition, incubate the tube in a 37°C water bath for 10 minutes. Centrifuge at 800 rpm for 10 minutes at room temperature. Discard the supernatant and resuspend the cells in 5 mL of complete HAT medium supplemented with 10% FBS (fetal bovine serum). Add the remaining HAT medium to a total of 50 mL. Use a pipette to add the cell suspension to a 96-well cell culture plate at a volume of 100 μL / well. Add 100 μL / well of complete HAT medium and incubate the plate in a 37°C cell culture incubator with 5% CO2. On day 4 after fusion, observe the cells and perform a half-well medium exchange: discard 100 μL of culture medium from each well and replace with 100 μL of fresh complete HAT medium containing 10% FBS. On day 8-10 after fusion, observe the cells under a microscope. Mark wells containing cell clusters and perform indirect ELISA to detect the presence of specific antibodies in the cell culture supernatant.

[0059] 2.2.3. The operating steps of the indirect ELISA detection method are as follows: (1) Antigen coating: The prepared C-terminal antigen immune complex of VP4 protein was diluted with coating solution (50mM CB buffer, pH 9.6) at 10μg / mL, 100μL per well was added to the well, and the plate was refrigerated at 4℃ overnight. The liquid in the plate was discarded, the water was shaken off, and 200μL PBST (PBS buffer containing 0.1% (v / v) Tween-20, pH 7.4) was added to each well and placed on a micro-oscillator for 60s, the water was shaken off, and the plate was washed 3 to 5 times with PBST. (2) Blocking: The blocking solution (1× PBS buffer containing 1wt% BSA and 0.1% (v / v) Tween-20) was added to the well at 100μL / well and blocked at 4℃ overnight. Discard the liquid in the plate, spin dry the water, add PBST at 200 μL / well, spin dry the water, and wash the plate 3 to 5 times with PBST. (3) Primary antibody incubation: Add 100 μL of the supernatant of the fusion well cells prepared in step 2.2.2 and the positive serum control (diluted 32,000 times with blocking solution), the negative serum control (diluted 100 times with blocking solution), and the blank control (no serum added, only blocking solution added) to the enzyme labeling plate per well and incubate at 37°C for 70 minutes. Discard the supernatant of the fusion well cells in the plate, spin dry the water, add PBST at 200 μL / well, spin dry the liquid, and wash with PBST 3 to 5 times. (4) Secondary antibody incubation: Dilute HRP-goat anti-mouse IgG with PBS buffer at 1:10,000 (volume ratio), add 100 μL of the diluted secondary antibody to each well, and incubate at 37°C for 60 minutes. Discard the secondary antibody in the plate, shake dry, add PBST at 200 μL / well, shake dry, and wash the plate 3-5 times with PBST. (5) Color development: Add 100 μL of 0.32 μmol / L TMB (3,3',5,5'-tetramethylbenzidine) color development solution to each well and allow to react at 37°C for 15 minutes (protect from light). (6) Stop the reaction: Add 50 μL of 2 mol / L sulfuric acid to each well to stop the reaction. Read the OD value using a microplate reader as soon as possible. 450nm Value. OD of fusion cell supernatant sample 450nm The OD values ​​of negative serum control samples were compared 450nm A ratio greater than 2.1 was considered positive.

[0060] 2.3 Monoclonal Antibody Screening and Monoclonal Hybridoma Cell Line Construction

[0061] Select the fused positive cell clone wells in the 96-well cell culture plate in step 2.2, wait until all spleen cells die, the fused positive cell clone colonies grow to 1 / 3 to 1 / 2 of the bottom of the well, and the cell culture medium is changed 3 times to make the antibodies secreted by the original spleen cells completely disappear, then aspirate the hybridoma cell culture supernatant 3 to 4 days after the medium change or when the cell culture medium turns yellow, and use indirect ELISA to detect positive hybridoma cell colonies. After the limiting dilution method is used for subcloning more than 3 times, when all clone cell colony clone wells are detected as positive, it indicates that this clone is a monoclonal clone and the antibody secreted by this cell line is a monoclonal antibody. Finally, 5 monoclonal cell lines that can stably secrete antibodies were obtained, namely 6E7B3, 6E7B12, 6H5, 6A5B1, and 6A5C4. Among them, 6E7B12 has the highest titer. The OD value of ELISA detection after the monoclonal cell line is stable 450nm The values ​​are shown in Table 6.

[0062] Table 6 ELISA detection values ​​of cell culture supernatants of 5 monoclonal cell lines after stabilization

[0063]

[0064] 2.4 Monoclonal Antibody Purification and Subtype Identification

[0065] 2.4.1. Preparation of ascites: 8-week-old female BALB / c mice were selected and intraperitoneally injected with 0.5 mL of sterile liquid paraffin. Seven days later, 6 × 10 6 The hybridoma cells generated in step 2.3 were in the logarithmic growth phase. Ascites was collected when the mouse abdomen was noticeably enlarged and felt ripple upon pressing. The ascites was centrifuged at 6000 rpm for 10 minutes to remove the liquid paraffin and fat. The supernatant was aspirated to obtain the mouse ascites antibody.

[0066] 2.4.2. Purify mouse ascites using a Hitrap Protein G HP immunoaffinity chromatography column as follows: (a) Dilute the ascites 8-10-fold with binding buffer (0.01 M PBS) and place on the upper shelf of a refrigerator at 4°C until ready for use. (b) Add 60-80 μL of Tris-HCl (1 mol / L, pH 9.0) to the collection tube to prevent any effects on the proteins in the ascites. (c) Rinse the purification column with ultrapure water to remove any ethanol from the column, maintaining a flow rate of 0.5-1 mL / min. (d) Equilibrate the purification column by flushing with 10 mL of binding buffer, maintaining a flow rate of 0.4-0.7 mL / min. (e) Use a syringe to add 10 mL of the ascites dilution prepared in step (a), maintaining a flow rate of 0.2-0.4 mL / min. (f) Rinse the purification column with 10 mL of binding buffer to remove any unbound proteins. Repeat this step once. (g) Elute with 5 mL of elution buffer (0.1 M Gly-HCl solution, pH 2.5-3.0) at a flow rate of 0.2 mL / min. Collect the eluate in a collection tube to obtain the purified monoclonal antibody.

[0067] 2.4.3 Monoclonal Antibody Detection and Subtype Identification

[0068] (A) The purified monoclonal antibody was detected using 12% SDS-PAGE. Figure 3 As shown. Figure 3 It can be seen from the above that Figure 3 It can be seen from the above that the purity of the purified antibody is above 85%, and the purification effect is good.

[0069] (B) Monoclonal antibody subtype identification: The mouse IgG antibody subtype identification kit (Detai Biotechnology, DTT03) was used for analysis. The purified monoclonal antibody was diluted 8000-fold with the diluent provided in the kit and then tested. The operation was performed strictly according to the kit instructions. The results showed that 6E7B12 was an IgG1 antibody with a kappa light chain.

[0070] 3. Preparation, screening and validation of VP4 protein polyclonal antibodies

[0071] 3.1. Preparation of VP4 protein polyclonal antibodies

[0072] Two New Zealand white rabbits were immunized with 1.6 mg / 1 mL of the VP4 protein prepared in step 1.5 as an immunogen. For the initial immunization, an equal volume of complete Freund's adjuvant was added for emulsification. For the subsequent three immunizations, an equal volume of incomplete Freund's adjuvant was added. The emulsified immune complex was administered subcutaneously to the neck and back of four-month-old male New Zealand white rabbits at a dose of 800 μL per rabbit. A second immunization was performed on day 14, a third on day 28, and a fourth on day 42. Blood was collected from the jugular vein on day 45 and stored overnight at 4°C. The overnight stored blood was centrifuged at 5,000 rpm for 10 minutes, and serum was collected. Polyclonal antibodies were purified from a portion of the serum using the caprylic acid-ammonium sulfate salting-out method as follows: 5 mL of serum was centrifuged at 10,000 rpm and 4°C for 10 minutes to remove impurities. To the pretreated serum, add 10 mL of 0.06 M acetic acid (pH 5.0) and adjust the pH to 4.8 with 1 M HCl. Caprylic acid was added dropwise at a ratio of 11 μL per mL of diluted serum, with stirring at room temperature, over 30 minutes. The mixture was allowed to stand at 4°C for 2 hours. The mixture was then centrifuged at 13,000 rpm for 30 minutes, and the pellet was discarded. The supernatant was passed through a 125 μm nylon sieve, and 0.01 M PBS buffer (1 / 10 volume) was added. The pH was adjusted to 7.4 with 1 M NaOH. An equal volume of saturated ammonium sulfate solution was slowly added to the serum supernatant with stirring to a final concentration of 1:1 (v / v). The mixture was incubated at 4°C for 3 hours to fully precipitate the protein. The mixture was centrifuged at 13,000 rpm at 4°C for 10 minutes, and the supernatant was discarded. The pellet was resuspended in 12 mL of 0.01 M PBS buffer, and 8 mL of saturated ammonium sulfate solution was slowly added with stirring at 4°C for 1 hour. Centrifuge at 13,000 r / min and 4°C for 10 minutes, discard the supernatant, and resuspend the precipitate in 13.3 mL of 0.01 M PBS buffer. Slowly add 6.7 mL of saturated ammonium sulfate solution while stirring, and incubate at 4°C for 1 hour. Centrifuge at 13,000 r / min and 4°C for 10 minutes, discard the supernatant, and resuspend the precipitate in an appropriate amount of 0.01 M PBS buffer and place it in a dialysis bag with a permeability of 5 kDa. Place the dialysis bag in 50 volumes of 0.01 M PBS buffer and stir on a magnetic stirrer for 6 hours. During this period, change the dialysate three times to fully remove the ammonium sulfate. Recover the dialyzed product and centrifuge to obtain the purified polyclonal antibody.

[0073] 3.2 Screening and validation of VP4 protein polyclonal antibodies

[0074] Use indirect ELISA (refer to the indirect ELISA detection method in step 2.2.3, replace the primary antibody with rabbit antiserum) to detect the titer of polyclonal antibodies in the serum collected in step 3.1. 450nm Absorbance value / OD of negative serum control 450nmAbsorbance values ​​> 2.1 were considered positive. The titer test results of the polyclonal antibodies are shown in Table 7.

[0075] Table 7 Results of indirect ELISA testing of polyclonal antibody titers

[0076]

[0077] From the results in the table, it can be seen that the titer of the obtained serum can reach 1:512000, indicating that high-titer specific polyclonal antibodies were prepared using VP4 protein as the immunogen.

[0078] 3.3. Polyclonal Antibody Western Blot Verification

[0079] The type II GCRV virus isolated from the infected wild-type grass carp was added with 2 times the volume of RIPA lysis buffer (strong) (Shanghai Biyuntian Biotechnology Co., Ltd., product number P0013B) to obtain the virus lysate. 1μL, 2μL, 4μL, 8μL, and 16μL of the virus lysate were taken for detection, respectively. The primary antibodies were antiserum P4-1# and antiserum P4-2# in Table 7, with a dilution concentration of 1:1000 (volume ratio); the secondary antibody was HRP-labeled goat anti-rabbit IgG (H+L), with a dilution concentration of 1:10000 (volume ratio). Chemiluminescence (ECL) was used for color development and photography was taken. The results are shown in the figure. Figure 4 As shown. Figure 4 As can be seen from the figure, there is a specific band between 60 and 75 kDa, and the size of this band is equivalent to the theoretical molecular weight of VP4 protein, 68.3 kDa.

[0080] 4. Establishment of an antibody sandwich ELISA assay for GCRV VP4 protein

[0081] 4.1. The antibody sandwich ELISA reaction steps are as follows: (I) Coat the deep-well plate with the purified polyclonal antibody prepared above, 100 μL per well, at 4°C overnight. (II) Add blocking solution, 250 μL per well, and incubate at 37°C for 2 hours. (III) Add VP4 protein to the deep wells, 50 μL per well. (IV) Continue to add enzyme-labeled monoclonal antibody and secondary antibody (enzyme-labeled monoclonal antibody is 6E7B3-HRP or 6E7B12-HRP, secondary antibody is HRP-labeled goat anti-mouse IgG (H+L)) to the deep wells, 100 μL of each antibody per well, and incubate at 37°C for 1 hour. (V) Add 0.32 μmol / L TMB color development solution to the deep wells, 100 μL per well, and develop for 10 minutes. (VI) Then add 2 mol / L sulfuric acid to each well at a volume of 50 μL / well to terminate the reaction, and measure the OD 450nmReaction value. Wash the plate 3-5 times with PBST between each step (I)-(V), each time for 90 seconds. Process the data from each test and calculate the P / N value of the reaction. The reaction condition with the highest P / N value is used as the basis for determining the optimal ELISA reaction conditions. The P / N value is calculated as follows: P / N value = OD value of positive control well. 450nm Mean / negative control well OD 450nm Mean.

[0082] 4.2 Screening of optimal working concentrations of monoclonal and polyclonal antibodies

[0083] Dilute the polyclonal antibody obtained in step 3.2 from 1:200 to 1:25,600 by volume with coating buffer (50 mM CB buffer, pH 9.6). Select the two most potent purified, labeled, enzyme-labeled monoclonal antibodies from Table 6 and dilute them from 1:200 to 1:6,400 by volume with blocking buffer. Detection was performed using the checkerboard titration method as in step 4.1. OD 450 nm values ​​were read using a microplate reader to determine the optimal binding dilution. The results are shown in Table 8.

[0084] Table 8 Results of ELISA array titration of polyclonal and monoclonal antibodies at different concentrations

[0085]

[0086] The data in the table show that when the polyclonal antibody dilution factor was 1:3200 and the 6E7B3-HRP monoclonal antibody dilution factor was 1:800, the curvature of the P / N value of the reaction was higher than that of other dilutions. Therefore, a dilution factor of 1:3200 for the polyclonal antibody and a dilution factor of 1:800 for the 6E7B3-HRP monoclonal antibody were determined to be the optimal reaction dilutions. When the polyclonal antibody dilution factor was 1:3200 and the 6E7B12-HRP monoclonal antibody dilution factor was 1:1600, the curvature of the P / N value of the reaction was higher than that of other dilutions. Therefore, a dilution factor of 1:3200 for the polyclonal antibody and a dilution factor of 1:1600 for the 6E7B12-HRP monoclonal antibody were determined to be the optimal reaction dilutions.

[0087] 4.3. Screening for the best detection antibody

[0088] According to the method in step 4.1, ELISA detection was performed using 6E7B3-HRP and 6E7B12-HRP as detection antibodies and antiserum P4-2# as capture antibody. The experimental results are shown in Table 9.

[0089] Table 9 Detection antibody screening results

[0090]

[0091] As can be seen from the data in the table, the background value of 6E7B12-HRP monoclonal antibody is lower, so 6E7B12-HRP monoclonal antibody is selected as the best detection antibody for preparing the standard curve.

[0092] 4.4. Screening of blocking solution

[0093] Following the method in step 4.1, 6E7B12-HRP at a dilution factor of 1:1600 was selected as the detection antibody, and antiserum P4-2# at a dilution factor of 1:3200 was selected as the capture antibody. PBST buffer (1× PBS buffer containing 0.1% (v / v) Tween-20) containing different concentrations of BSA, Block ACE, and gelatin was used as the blocking buffer for ELISA detection. The results are shown in Table 10.

[0094] Table 10 Blocking solution screening results

[0095]

[0096] The percentages in Table 10 are all weight percentages. From the data in the table, it can be seen that 1 wt% BSA + 0.04 wt% Block ACE has the best blocking effect. Considering the N value, 1× PBS buffer containing 0.1% (v / v) Tween-20, 0.5 wt% BSA, and 0.04 wt% Block ACE was selected as the blocking solution.

[0097] 4.5. Establishing a standard curve for double antibody sandwich ELISA

[0098] Select 6E7B12-HRP with a dilution ratio of 1:1600 as the detection antibody, antiserum P4-2# with a dilution ratio of 1:3200 as the capture antibody, and the blocking solution selected in step 4.4 as the blocking solution. Dilute the VP4 protein prepared in step 1.5 to 32ng / mL, 16ng / mL, 8ng / mL, 4ng / mL, 2ng / mL, and 1ng / mL, respectively, and make two parallels for each concentration. Perform ELISA detection according to the method in step 4.1, and the standard curve results are shown in Tables 11 and Figure 5 .

[0099] Table 11 Detection results of VP4 protein at different concentrations

[0100]

[0101] from Figure 5 As can be seen from the figure, when the VP4 protein concentration is 1~32ng / mL, the VP4 protein concentration is closely related to OD 450nm The values ​​have a good linear relationship, and the linear regression equation is y=0.034x+0.0428 (R2 =0.9912). The horizontal axis x is the VP4 protein concentration in ng / mL; the horizontal axis y is the OD 450nm Therefore, the detection range of VP4 protein by double antibody sandwich ELISA is 1-32 ng / mL, and the detection limit is 1 ng / mL.

[0102] In summary, the method for double antibody sandwich ELISA detection of VP4 protein using the VP4 protein monoclonal antibody in this application as the detection antibody and the VP4 protein rabbit polyclonal antibody as the capture antibody is as follows:

[0103] S1. Polyclonal antibody coating: Dilute the VP4 protein polyclonal antibody at a volume ratio of 1:3200 with coating solution (50 mM CB buffer, pH 9.6), then add 100 μL / well to the deep-well plate and place in a 4°C refrigerator overnight.

[0104] S2. Blocking: A mixture of 0.5% BSA and 0.04% Block ACE was added to the deep-well plate at a volume of 250 μL / well as blocking solution and incubated at 37°C for 2 h.

[0105] S3. Add antigen: Add VP4 protein to the deep-well plate at a volume of 50 μL / well. The final concentration of VP4 protein is 1-32 ng / mL.

[0106] S4. Add enzyme-labeled secondary antibody: Dilute HRP-labeled 6E7B12 monoclonal antibody (6E7B12-HRP) to 1:1600 (volume ratio) with blocking buffer (0.5% BSA + 0.04% Block ACE). Add 100 μL / well to the deep-well plate and incubate at 37°C for 2 h.

[0107] S5. Substrate color development: Add 0.32 μmol / L TMB color development solution to the deep-well plate at a volume of 100 μL / well and develop color for 10 min.

[0108] S6. Stop the reaction: Add 2 mol / L sulfuric acid to the deep-well plate at a volume of 50 μL / well to stop the reaction. Measure the absorbance of the reaction system at 450 nm (OD 450nm ).

[0109] Between each step of steps S1 to S5, the plate was washed 3 to 5 times with PBST for 90 seconds each time.

[0110] The embodiments described above are merely preferred embodiments of the present application and are not intended to limit the scope of protection of the present application. For any person skilled in the art, the present application may have various modifications and variations. Any simple equivalent changes and modifications made based on the scope of protection of the present application and the contents of the specification should be included in the scope of protection of the present application.

Claims

1. A hybridoma cell line secreting a monoclonal antibody against the VP4 protein of grass carp reovirus, characterized in that: The hybridoma cell line is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: C2024160.

2. Use of the hybridoma cell line according to claim 1 in preparing monoclonal antibodies against the VP4 protein of grass carp reovirus.

3. A monoclonal antibody against grass carp reovirus VP4 protein, characterized in that: The monoclonal antibody is secreted by the hybridoma cell line or its successive cell line according to claim 1.

4. The monoclonal antibody according to claim 3, characterized in that The monoclonal antibody is an IgG1 antibody, and its light chain is of the κ subtype.

5. Use of the hybridoma cell line according to claim 1, the monoclonal antibody according to claim 3, or the monoclonal antibody according to claim 4 in the preparation of a product for detecting grass carp reovirus.

6. The use according to claim 5, characterized in that The product for detecting grass carp reovirus is a kit.

7. An ELISA kit for detecting grass carp reovirus, characterized in that: The ELISA kit comprises the monoclonal antibody according to claim 3 or 4.

8. The ELISA kit according to claim 7, wherein The ELISA kit comprises a polyclonal antibody against the VP4 protein of grass carp reovirus as a capture antibody; and the monoclonal antibody described in claim 3 or 4 is used as a detection antibody after being enzyme-labeled.

9. The ELISA kit according to claim 8, wherein The ELISA kit includes a blocking solution, which is a 1×PBS buffer containing 0.1% (v / v) Tween-20, 0.5wt% BSA, and 0.04wt% Block ACE; or / and, the ELISA kit contains a grass carp reovirus VP4 protein encoded by the nucleotide sequence shown in SEQ ID No. 1 as an antigen, and the coating concentration of the antigen is 1-32 ng / mL.

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