A monoclonal antibody against gE protein of pseudorabies virus of swine, a hybridoma cell strain and application thereof

Abstract

This invention discloses a monoclonal antibody against porcine pseudorabies virus gE protein, a hybridoma cell line, and their applications, belonging to the field of biotechnology. The monoclonal antibody 3G10 comprises a light chain and a heavy chain; the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO.1, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO.8. This invention obtains a hybridoma cell line 3G10 that secretes a specific monoclonal antibody against porcine pseudorabies virus gE protein, and establishes a gE antibody blocking ELISA method and a gE antibody blocking ELISA kit based on the monoclonal antibody 3G10 secreted by this cell line. This method exhibits high sensitivity and specificity for detecting porcine pseudorabies virus gE antibodies, providing a new approach for the monitoring and control of porcine pseudorabies.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a monoclonal antibody against the gE protein of porcine pseudorabies virus, a hybridoma cell line, and their applications. Background Technology

[0002] Porcine pseudorabies (PR) is an acute infectious disease caused by the porcine pseudorabies virus (PRV), posing a significant threat to the pig farming industry. It primarily causes abortion, stillbirth, and mummified fetuses in sows, while newborn piglets exhibit fever and neurological symptoms, with a mortality rate approaching 100%.

[0003] In 2011, a PRV variant emerged in my country. Epidemiological surveillance revealed that the PRV circulating in large-scale pig farms in my country since 2011 is still predominantly a variant. Whole-genome genetic evolution analysis of domestic and international PRV strains revealed that they belong to different branches: the foreign strain is genotype I, while the domestic strain is genotype II. These two genotypes differ significantly in sequence and antigenicity. Furthermore, the newly circulating PRV variant also differs from strains prevalent in China before 2011. Control of porcine pseudorabies primarily relies on vaccines and corresponding diagnostic kits. Currently, both live and inactivated vaccines against the newly circulating PRV variant have been developed and are on the market. However, domestic PRV antibody detection kits are mainly imported (with foreign PRV strains as the coating antigen), and there are almost no domestically available kits that can compete with them, especially those using circulating strains as antigens.

[0004] gE protein is one of the main glycoproteins on the surface of PRV virus particles. The absence of this protein can significantly reduce viral virulence without affecting viral replication. Therefore, almost all porcine pseudorabies vaccines, whether live or inactivated, lack the main virulence gene gE. Detecting PRV gE protein antibodies in porcine serum can monitor wild-type virus infection status and is of great significance for the prevention and control of the disease. Summary of the Invention

[0005] The purpose of this invention is to provide a monoclonal antibody against porcine pseudorabies virus gE protein, a hybridoma cell line, and their applications, to address the problems existing in the prior art. This invention obtains a hybridoma cell line 3G10 that secretes a specific monoclonal antibody against porcine pseudorabies virus (PRV) gE protein. Based on the monoclonal antibody 3G10 secreted by this cell line, a gE antibody blocking ELISA method and a gE antibody blocking ELISA kit were established. This method exhibits high sensitivity and specificity for detecting porcine pseudorabies virus gE antibodies, providing a new approach for the monitoring and control of porcine pseudorabies.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a monoclonal antibody 3G10 against porcine pseudorabies virus gE protein, wherein the monoclonal antibody 3G10 comprises a light chain and a heavy chain; The amino acid sequence of the CDR1 region of the light chain is shown in SEQ ID NO.2, the amino acid sequence of the CDR2 region is LVS, and the amino acid sequence of the CDR3 region is shown in SEQ ID NO.3. The amino acid sequence of the CDR1 region of the heavy chain is shown in SEQ ID NO.9, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.10, and the amino acid sequence of the CDR3 region is shown in SEQ ID NO.11.

[0007] Further, the amino acid sequence of the FR1 region of the light chain is shown in SEQ ID NO.4, the amino acid sequence of the FR2 region is shown in SEQ ID NO.5, the amino acid sequence of the FR3 region is shown in SEQ ID NO.6, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.7; The amino acid sequence of the FR1 region of the heavy chain is shown in SEQ ID NO.12, the amino acid sequence of the FR2 region is shown in SEQ ID NO.13, the amino acid sequence of the FR3 region is shown in SEQ ID NO.14, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.15.

[0008] The present invention also provides a hybridoma cell line that secretes the above-mentioned monoclonal antibody 3G10. The hybridoma cell line is named hybridoma cell line 3G10 against porcine pseudorabies virus gE protein. It has been deposited at the China General Microbiological Culture Collection Center on March 26, 2026, with accession number CGMCC No. 46794.

[0009] The present invention also provides the use of the above-mentioned hybridoma cell line in the production of monoclonal antibodies against porcine pseudorabies virus gE protein.

[0010] The present invention also provides the use of the above-mentioned monoclonal antibody 3G10 or the above-mentioned hybridoma cell line in the preparation of products for detecting porcine pseudorabies virus.

[0011] The present invention also provides the use of the above-mentioned monoclonal antibody 3G10 or the above-mentioned hybridoma cell line in the preparation of products for detecting porcine pseudorabies virus gE protein antibodies.

[0012] Optionally, the product includes a reagent kit.

[0013] The present invention also provides a kit for detecting porcine pseudorabies virus gE antibody, the kit comprising an enzyme-labeled plate and an enzyme-labeled antibody, the enzyme-labeled antibody being made from the above-mentioned monoclonal antibody 3G10.

[0014] Furthermore, the ELISA plate is coated with inactivated porcine pseudorabies virus.

[0015] Optionally, the kit may also include a washing solution, a sample diluent, a substrate solution, and a stop solution.

[0016] The present invention discloses the following technical effects: This invention obtained a hybridoma cell line 3G10 that secretes a specific monoclonal antibody against porcine pseudorabies virus (PRV) gE protein, and established a gE antibody blocking ELISA method and a gE antibody blocking ELISA kit based on the monoclonal antibody 3G10 secreted by this cell line.

[0017] The hybridoma cell line 3G10 of this invention can stably secrete monoclonal antibodies that specifically recognize PRV gE protein. The antigenic epitope recognized by this monoclonal antibody is a conformational epitope, which is more stable and specific than linear epitopes, and can be used for specific recognition and detection of PRV with strong reactivity. The hybridoma cell line 3G10 and its secreted monoclonal antibody can also be used to prepare a blocking antibody detection kit. This kit is easy to operate, has good specificity, and exhibits superior sensitivity in detecting porcine pseudorabies virus gE antibodies compared to imported kits and other domestic kits. It can better monitor the infection status of pig herds, and the optimal concentration of the enzyme-labeled monoclonal antibody can reach 1:5000, greatly saving raw materials. This invention provides a new solution for the monitoring and control of porcine pseudorabies. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 To determine the reactivity of different strains of gE monoclonal antibodies against porcine pseudorabies virus using ELISA assays; Figure 2 To determine the blocking efficiency of the two monoclonal antibodies using an ELISA assay; Figure 3 The critical value for detecting porcine pseudorabies virus gE blocking ELISA antibodies; Figure 4 The results of a specificity test for the ELISA antibody detection method for porcine pseudorabies virus gE blocking; Figure 5 The results of sensitivity comparison of five serum samples tested using different kits. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0026] The porcine pseudorabies virus (HeN1 strain, TP strain) involved in the following examples was preserved and provided by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences, and is available to the public from there.

[0027] Example 1: Preparation and identification of monoclonal antibodies that specifically recognize PRV gE protein (1) Preparation of immunogen Porcine pseudorabies virus (HeN1 strain, TP strain) was inoculated at a rate of 0.1% onto Vero cells (purchased from ATCC) that had grown into a monolayer and cultured at 37°C for 24 hours. The virus solution was harvested when more than 90% of the cells showed cytopathic effects. Since the gE protein is located on the surface of the virus particle and is a lipid bilayer envelope protein, it is easily destroyed. Therefore, this invention selected β-propiolactone as the inactivating agent, which only inactivates viral nucleic acid without destroying the antigen structure. The virus solution and the inactivating agent were mixed at a ratio of 2000:1 (v / v), and the mixture was inactivated at 4°C for 24 hours, followed by a 1-hour water bath at 37°C to terminate the inactivation reaction. The inactivated virus solution was centrifuged at 4000 rpm for 20 minutes, and the supernatant was collected. The supernatant was then ultracentrifuged at 16500 rpm for 2 hours, the supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS to obtain the purified immunogen.

[0028] (2) Mouse immunity and cell fusion In mouse immunization, to maintain the native structure of the antigen, this embodiment uses whole PRV virus to immunize mice. Because PRV encodes numerous proteins, the monoclonal antibodies produced after direct immunization of mice with whole virus are diverse, making it difficult to prepare monoclonal antibodies specifically targeting the gE protein. Therefore, this invention employs an immune tolerance strategy to immunize mice. First, mice are immunized with inactivated and purified whole PRV TP strain (deleted gI, gE, US9, US2, and TK genes) as a tolerogen. This is followed by cyclophosphamide (CY) treatment to induce tolerance in mice to all PRV-encoded proteins except gI, gE, US9, US2, and TK. Subsequently, mice are immunized with inactivated and purified whole PRV HeN1 strain (wild-type) to induce a response against gI, gE, US9, US2, and TK proteins. The main immunization procedure is as follows: First, 6-7 week old BALB / c mice are intraperitoneally injected with 50 mg of PRV TP strain (tolerogen). Then, mice were administered 100 mg / kg cyclophosphamide (CY) at 15 minutes, 12 hours, 24 hours, and 48 hours after tolerogen injection. Two weeks later, the tolerogen injection and CY treatment regimen were repeated. Two weeks after cyclophosphamide treatment, mice were immunized with 50 mg of wild-type PRV (HeN1 strain) as an immunogen. The first immunization was emulsified with Freund's complete adjuvant, followed by a second immunization two weeks later using Freund's incomplete adjuvant emulsification. A third immunization was performed two weeks later, following the same procedure as the second immunization. Three days before cell fusion, 50 mg of PRV HeN1 strain was injected intraperitoneally. Spleen cells from immunized mice were then fused with myeloma cells SP2 / 0 (purchased from ATCC) under PEG conditions. After culturing in HAT selective medium for 10 days, indirect immunofluorescence was performed.

[0029] (3) Screening and cloning purification of positive hybridoma cells First, Vero cells infected with the HeN1 variant of porcine pseudorabies virus (PRV) in 96-well plates were used as antigens. The supernatant from the fusion cell culture was used for indirect immunofluorescence assays to screen hybridoma cells that secreted antibodies capable of reacting with PRV. Then, 293T cells expressing gE protein were used as antigens, and the supernatant from the selected hybridoma cells was further identified using indirect immunofluorescence assays to determine whether the monoclonal antibody targeted the gE protein. Positive hybridoma cells were cloned and purified using a limiting dilution method. After three limiting dilutions, purified hybridoma cells capable of stably secreting antibodies were obtained. After multiple experiments, three hybridoma cell lines capable of secreting antibodies against the PRV HeN1 strain gE protein were screened and named 1E4, 3G10, and 4F3, respectively.

[0030] (4) Identification of the reactivity of monoclonal antibodies The reactivity of the monoclonal antibodies was identified using an indirect ELISA method. The specific method was as follows: Inactivated and purified porcine pseudorabies virus (HeN1 strain) antigen was coated in the wells of an ELISA plate. Cell culture supernatant from three monoclonal antibody strains was added to each well at 100 μL / well. DMEM was used as a negative control. The plate was incubated at 37°C for 30 min. The liquid in the wells was discarded. 300 μL of PBST was added to each well, and the plate was washed four times. The plate was then dried. 100 μL of goat anti-mouse IgG enzyme-labeled antibody (1:5000 dilution) was added to each well, and the plate was incubated at 37°C for 30 min. The liquid in the wells was discarded. 300 μL of PBST was added to each well, and the plate was washed four times. 100 μL of TMB substrate solution was added to each well, and the plate was incubated at 37°C (in the dark) for 10 min. 50 μL of stop solution was added to each well, and the mixture was gently vortexed to mix thoroughly. The OD was read at 450 nm using an ELISA reader. 450nm Value (the reading should be completed within 15 minutes after the addition of the stop solution).

[0031] The results showed that two of the three monoclonal antibodies could produce a good immune response to the PRV (HeN1 strain) viral antigen coated on the ELISA plate, while one antibody showed poor reactivity (see [link to ELISA plate]). Figure 1 ).

[0032] (5) Verification of whether the reaction between monoclonal antibodies and the virus can be blocked by PRV-positive serum. In order to screen for monoclonal antibodies that can establish blocking or competitive ELISA antibody detection methods, this invention further identified two monoclonal antibodies (1E4 and 3G10) that react well with the virus to determine whether their binding to the virus can be blocked by positive serum for porcine pseudorabies virus gE antibody.

[0033] The wells of the ELISA plate were coated with inactivated and purified porcine pseudorabies virus (HeN1 strain) antigen. Porcine pseudorabies virus positive serum was diluted 1:2 and added to each well (100 μL / well). Negative serum was included as a control. The plates were incubated at 37°C for 30 min. The liquid in the wells was discarded. 300 μL of PBST was added to each well, and the plates were washed four times and then patted dry. 100 μL of monoclonal antibody cell culture supernatant was added to each well, and the plates were incubated at 37°C for 30 min. The liquid in the wells was discarded. 300 μL of PBST was added to each well, and the plates were washed four times and then patted dry. 100 μL of goat anti-mouse IgG enzyme-labeled antibody (1:5000 dilution) was added to each well, and the plates were incubated at 37°C for 30 min. The liquid in the wells was discarded. 300 μL of PBST was added to each well, and the plates were washed four times and then patted dry. 100 μL of TMB substrate solution was added to each well, and the plates were incubated at 37°C (in the dark) for 10 min for color development. Add 50 μL of stop solution to each well, gently shake to mix thoroughly, and then read the OD value using a microplate reader at a wavelength of 450 nm. 450nm The results showed that the binding of the 1E4 strain monoclonal antibody to the virus could not be blocked by positive serum, while the blocking efficiency of the 3G10 strain monoclonal antibody reached over 90%. Figure 2 Therefore, hybridoma cell line 3G10 was selected for subsequent experiments.

[0034] The hybridoma cell line 3G10, named the hybridoma cell line 3G10 resistant to porcine pseudorabies virus gE protein, was deposited on March 26, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 46794.

[0035] (6) Sequencing of monoclonal antibody 3G10.

[0036] Sequencing of the monoclonal antibody secreted by the 3G10 hybridoma cell line yielded the following results: Variable region sequence of light chains: DIVMTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELFGQGTKLEIK, SEQ ID NO.1; Among them, CDR1: KSVSTSGYSY, SEQ ID NO.2; CDR2: LVS; CDR3: QHIREL, SEQ ID NO.3; FR1: DIVMTQSPASLAVSLGQRATISYRAS, SEQ ID NO.4; FR2: MHWNQQKPGQPPRLLIY, SEQ ID NO.5; FR3: NLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYC, SEQ ID NO.6; FR4: FGQGTKLEIK, SEQ ID NO.7; Variable region sequence of heavy chain: EVQLQQSGPELVKPGASVKISCKASGYPFTGYYMYWVTQSQVKSLEWIGRINPYNGVPTYNQNFKDKASLTVDRSSSTAYMELHSLTSEDSAVYYCGRIDKGATDYWGQGTSVTVSS, SEQ ID NO.8; Among them, CDR1: GYPFTGYY, SEQ ID NO.9; CDR2: INPYNGVP, SEQ ID NO.10; CDR3: GRIDKGATDY, SEQ ID NO.11; FR1: EVQLQQSGPELVKPGASVKISCKAS, SEQ ID NO.12; FR2:MYWVTQSQVKSLEWIGR, SEQ ID NO.13; FR3: TYNQNFKDKASLTVDRSSSTAYMELHSLTSEDSAVYYC, SEQ ID NO.14; FR4: WGQGTSVTVSS, SEQ ID NO.15.

[0037] Example 2: Purification and labeling of monoclonal antibody 3G10 Preparation of 3G10 monoclonal antibody ascites: 6-8 week old BALB / c mice were intraperitoneally injected with Freund's incomplete adjuvant, 0.5 mL / mouse. Seven days after inoculation, each mouse was intraperitoneally injected with 5 × 10⁻⁶ mg / mL of the adjuvant. 5 One well-grown 3G10 hybridoma cells were collected. One week after inoculation, when the peritoneum of the mice showed obvious distension, the peritoneal fluid was aspirated, centrifuged at 5000 r / min for 10 minutes, and the supernatant was collected and stored at -70℃ for later use.

[0038] Purification of 3G10 monoclonal antibody: Dissolve 1 mL of the prepared 3G10 monoclonal antibody in ascites fluid at room temperature (15-25℃), centrifuge at 10000 r / min for 10 minutes, discard the upper layer of oil and the precipitate at the bottom of the tube, and collect the supernatant. Mix the ascites supernatant with binding buffer 1:10 and filter through a 0.45 μm filter membrane. Connect a syringe containing 10 mL of binding buffer to a HiTrap protein G column, open the bottom cap of the column, and push the syringe to allow the binding buffer to flow through the column at a flow rate of 1 mL / min. Aspirate the sample into the syringe and allow it to flow through the column at a flow rate of 0.2-1 mL / min. Elute unbound sample with 10 mL of binding buffer. Add 60 μL of 1MTris-HCl to the collection tube. Elute the monoclonal antibody bound to the column with 1 mL of Elution Buffer, collect the filtrate into the collection tube, determine the protein concentration, and store at -70℃.

[0039] Horseradish peroxidase (HRP) labeling of monoclonal antibody 3G10: Weigh 5 mg of HRP and dissolve it in 0.5 mL of 0.06 M acetate buffer (pH 5.6). Add 0.5 mL of freshly prepared NaIO4 solution and mix well. Incubate at 4°C for 25 min. Add 0.5 mL of ethylene glycol (0.16 mol / L) and incubate at room temperature (25°C) in the dark for 30 min. Mix the activated enzyme solution with 1.5 mg of purified 3G10 monoclonal antibody, adjust the pH to approximately 9.0, transfer to a dialysis bag, and dialyze gently with freshly prepared carbonate buffer (0.05 mol / L, pH 9.5) for 16 hours. Change the medium at 2, 4, and 12 hours of dialysis. After removing the dialysate, add 0.2 mL of NaBH4 solution (5 mg / mL), mix well, and incubate at 4°C for 2 hours. Slowly add an equal volume of saturated ammonium sulfate solution to the above solution, mix well, and centrifuge at 3000 r / min for 30 min at 4℃ (centrifuge pre-cooled to 4℃). Discard the supernatant, dissolve the precipitate in 1 mL of PBS (0.02 mol / L, pH 7.4), place in a dialysis bag, and dialyze overnight at 4℃ with PBS (0.02 mol / L, pH 7.4) to remove salts. Change the medium at 2 hours, 4 hours, and 12 hours of dialysis. The next day, centrifuge at 10000 rpm for 30 minutes to remove insoluble matter to obtain the enzyme-antibody (HRP-IgG) conjugate. Transfer the labeled antibody to a new centrifuge tube and freeze with glycerol. Serially dilute the enzyme-labeled antibody and use an ELISA plate coated with porcine pseudorabies virus (HeN1 strain) to determine the titer of the labeled monoclonal antibody. The maximum dilution with a P / N ratio greater than 2.1 is its ELISA titer (P / N = OD of the sample).450nm Value / Negative Control OD 450nm The results showed that the labeled antibody ELISA titer could reach 32,000-fold (Table 1).

[0040] Table 1 Results of enzyme-labeled antibody titer assay Example 3: Establishment of a porcine pseudorabies gE antibody blocking ELISA method 1. Preparation of coating antigen (1) Purification of coating antigen Porcine pseudorabies virus strain HeN1 was inoculated into Vero E6 cells at a ratio of 0.1% and cultured for 24-36 hours to harvest the virus. The virus solution was then added to β-propiolactone solution at a ratio of 2000:1 (v / v) and inactivated at 2-8℃ for 24 hours. The inactivated virus solution was then transferred to a centrifuge tube and centrifuged at 4000 r / min for 20 minutes, collecting the supernatant. The supernatant was transferred to another centrifuge tube and centrifuged at 16500 r / min for 2 hours. The supernatant was discarded, and the precipitate was resuspended in an appropriate amount of PBS. Further purification was performed using density gradient centrifugation: 4 mL of 20% sucrose solution was added to a centrifuge tube. Then, using a long needle, 4 mL of 40% sucrose solution was added to the bottom of the centrifuge tube, followed by 4 mL of 60% sucrose solution. Finally, 1 mL of virus resuspension was slowly added to the top layer of the 20% sucrose solution. The mixture was centrifuged at 26000 r / min for 3 hours. The milky white virus band between the 40% and 60% sucrose solutions was aspirated and added to a centrifuge tube. The tube was then filled with PBS (0.01 mol / L, pH 7.4) and centrifuged at 16500 r / min for 2 hours. The supernatant was discarded, and the precipitate was resuspended in PBS (0.01 mol / L, pH 7.4) at 1 / 1000 of the original volume of the virus solution. The precipitate was then transferred to a 1.5 mL EP tube and stored at 2–8 °C. The protein concentration of the purified virus solution was determined using the BCA method according to the kit instructions and stored at -70 °C.

[0041] 2. Establishment of PRV gE antibody blocking ELISA method (1) Antigen coating: ELISA plates were coated with purified and inactivated porcine pseudorabies virus strain HeN1 at 1 µg / well and incubated overnight at 4°C. (2) Blocking: Take out the coated ELISA plate, wash 4 times with PBST, and pat dry; add 200 μL / well of 1% BSA blocking solution, and incubate at 37℃ for 1.5 h; wash 4 times with PBST, and pat dry; (3) Incubation of the serum to be tested: Mix the serum to be tested with the diluent at a ratio of 1:1 and add it to the ELISA plate. Add 100 μL to each well. At the same time, set up positive serum and negative serum as controls. Incubate at 37℃ for 30 min. Wash 4 times with PBST and pat dry. (4) Enzyme-labeled monoclonal antibody incubation: HRP-labeled 3G10 was diluted 5000 times with PBST, 100 μL was added to each well, and incubated in a constant temperature incubator at 37℃ for 30 min. The mixture was washed 4 times with PBST and then patted dry. (5) Substrate color development: Add substrate TMB for color development, add 100 μL to each well, and incubate in a constant temperature incubator at 37℃ for 10 min in the dark; (6) Termination of reaction: Add 50 μL HF to each well to terminate the reaction, and measure the absorbance A value at 650 nm using an ELISA reader; (7) Calculate the S / N value: S / N = OD of the serum to be tested 650nm Value / Negative Control OD 650nm value.

[0042] Following the optimized reaction conditions described above, 309 swine serum samples known to be PRV antibody negative and 113 swine serum samples known to be PRV antibody positive were tested and analyzed using Medcalcv 15.8 software. The results showed that when the cut-off value was 0.6 (see...), the... Figure 3 The sensitivity and specificity of the detection were both 100%. Therefore, the criteria for determining the result of the blocking ELISA test are as follows: when the S / N value is ≤0.6, it is determined to be gE antibody positive, and when the S / N value is >0.6, it is determined to be gE antibody negative.

[0043] 3. Specificity, sensitivity, repeatability, and comparison with commercially available kits of the ELISA antibody detection method for porcine pseudorabies virus gE blocking. 3.1 Specificity test Following an optimized blocking ELISA method, specific sera for porcine pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), African swine fever virus (ASFV), classical swine fever virus (CSFV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), and porcine circovirus (PCV2) were tested (all specific sera were prepared and preserved by the Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences). Positive and negative results were determined based on cutoff values ​​to evaluate the specificity of this method. Results showed that only PRV-specific sera were positive; the S / N values ​​of positive sera for other viruses were all higher than the cutoff value (see [link to ELISA method]). Figure 4 This indicates that the blocking ELISA method established in this invention has good specificity.

[0044] 3.2 Sensitivity Test Five PRV antibody-negative piglets were inoculated with a virulent variant of porcine pseudorabies virus (HeN1 strain). Serum was collected two weeks later and serially diluted 2-fold (1:2~1:2048) before detection using an optimized blocking ELISA method. Positive and negative results were determined based on cutoff values. The results were compared with those from two domestic and international companies (A: Porcine pseudorabies virus gE antibody detection kit (blocking ELISA), purchased from IDEXX; B: Porcine pseudorabies virus gE protein blocking ELISA antibody detection kit, purchased from Putai). The sensitivity of this invention was evaluated. Results showed that the detection sensitivity of this kit for these five serum samples was comparable to that of the international company A's kit and 0-4 times higher than that of the domestic company B's kit (see...). Figure 5 ).

[0045] 3.3 Repeatability Test Three batches of ELISA plates were coated, and three plates were randomly selected from each batch. Five serum samples were then tested, and the results were statistically analyzed to evaluate the intra- and inter-batch reproducibility of the method. The results showed that the coefficients of variation for both intra- and inter-batch reproducibility were less than 5%, indicating that the method established in this invention has good reproducibility (see Table 2).

[0046] Table 2 Results of intra-batch and inter-batch repeatability tests Example 4: Assembly and Application of a Swine Pseudorabies gE Antibody Blocking ELISA Detection Kit 1. Principle of the porcine pseudorabies gE antibody blocking ELISA detection kit The basic principle of the porcine pseudorabies gE antibody blocking ELISA kit is that the enzyme-labeled monoclonal antibody competitively binds to the PRV antigen in the microplate, and the presence of PRV gE antibody in the serum is determined by the binding rate of the monoclonal antibody to the antigen. The test serum is used after a two-fold dilution. The reaction of the serum and the enzyme-labeled monoclonal antibody with the antigen is carried out in two steps, and the entire test is completed in microplates coated with PRV antigen. In the first incubation phase, the PRV antibody in the test serum reacts with the antigen in the wells of the plastic plate. After washing, the enzyme-labeled monoclonal antibody is added to the microplate and competitively binds to the viral antigen in the second incubation. If there is no PRV gE antibody in the test serum, the enzyme-labeled antibody reacts freely with the antigen. Conversely, if PRV gE antibody is present in the test serum, the reaction between the enzyme-labeled monoclonal antibody and the antigen is blocked. After incubation, the unreacted enzyme-labeled antibody is washed away, and substrate / chromogenic reagent solution is added. In the presence of the enzyme, the substrate is converted into a substance that reacts with the chromogenic reagent to produce a blue color. The absorbance A(650) at 650 nm was measured using a spectrophotometer. The sample / negative ratio (S / N) was calculated by dividing the A(650) value of the tested sample by the average A(650) value of the negative control. The content of PRV antibodies in the sample is inversely proportional to its A(650) S / N value. The presence of PRV gE antibodies indicates previous infection with wild-type PRV.

[0047] 2. Main components and content of the reagent kit The main components and their contents of the kit are shown in Table 3.

[0048] Table 3 Main components and content 3. Storage All reagents should be stored at 2-8°C and have a shelf life of 12 months.

[0049] 4. Usage 4.1 How to use (1) Preparation of reagents: Before use, restore all reagents and samples to (20~25℃). The reagents should be gently rotated or shaken to mix.

[0050] (2) Preparation of washing solution: Take 1 part of 10 times concentrated washing solution and add it to 9 parts of double distilled water, and mix well.

[0051] The prepared detergent solution should be used within 3 days.

[0052] 4.2 ELISA Operation Steps (1) Serum preparation: Take a sample dilution plate, add 60 μL of sample diluent to each well, and then add 60 μL of serum to be tested. Mix well.

[0053] (2) Sample addition: According to the number of samples, take a removable coated plate and add 100 μL of diluted serum to be tested to each well. At the same time, set up 2 wells each of positive control serum and negative control serum (no dilution is required for positive and negative controls).

[0054] (3) Incubation: Incubate at 37℃ for 30 minutes.

[0055] (4) Washing: Discard the liquid in the well, add 300 μL of washing solution to each well, wash 4 times, and pat dry.

[0056] (5) Add enzyme-labeled monoclonal antibody: Add 100 μL of enzyme-labeled monoclonal antibody to each well.

[0057] (6) Incubation: Incubate at 37℃ for 30 minutes.

[0058] (7) Washing: Same as (4).

[0059] (8) Color development: Add substrate solution to each well, 100 μL / well, and incubate at 37°C in the dark for 10 minutes.

[0060] (9) Termination: Add 50 μL of stop solution (HF solution) to each well, gently shake to mix thoroughly, and read the OD at a wavelength of 650 nm. 650nm Value (the reading should be taken within 5 minutes after the stop solution is added).

[0061] (10) Calculation: Calculate the SN value according to the following formula.

[0062] S / N = OD of the serum to be tested 650nm Value / Negative control mean OD 650nm value 4.3 Judgment (1) Conditions for the experiment to be valid: the average OD of the negative control serum 650nm If the value is between 0.8 and 1.7, and the average S / N value of the positive control is ≤0.4, the test is considered valid.

[0063] (2) Judgment: When the S / N value of the serum sample to be tested is ≤0.6, it is judged as positive; when the S / N value is >0.6, it is judged as negative.

[0064] 4.4 Precautions (1) The kit should be transported and stored at 2~8℃.

[0065] (2) All the strips must be sealed with sealing film during storage to prevent moisture from damaging the lining board. Otherwise, it must not be used.

[0066] (3) Do not expose the substrate solution to strong light or oxides. Once all reagents have been removed, they must not be added back to the bottle.

[0067] (4) Read the instructions carefully and do not use expired components or mix reagents from different batches.

[0068] (5) When diluting the concentrated washing solution by 10 times, if crystals are found, place it at 37°C to dissolve them before use.

[0069] (6) Pay attention to the sample addition and washing process to ensure the accuracy of the test. Do not use your mouth to suck the liquid.

[0070] (7) Do not use serum that has become putrid.

[0071] (8) The testing utensils must be clean, and contact with metal objects should be avoided during the operation.

[0072] (9) The operation should be carried out strictly in accordance with the instructions of the reagent kit, and the time and temperature specified in each operation step should be strictly observed.

[0073] (10) Wear gloves during operation. The stop solution is hydrofluoric acid (HF), which is corrosive. Use with care.

[0074] All waste liquids and wastes must be treated to render them harmless.

[0075] 4.5 Detection of Clinical Samples Following the optimized blocking ELISA method, 290 serum samples were tested, and the results were compared with those from the IDEXX antibody detection kit. The results from this method showed 81 positive serum samples and 209 negative serum samples. The concordance rate between the blocking ELISA method established in this experiment and the IDEXX kit was 98.96% (see Table 4).

[0076] Table 4. Concordance rate between this method and the IDEXX kit for detecting clinical samples. The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody 3G10 against porcine pseudorabies virus gE protein, characterized in that, The monoclonal antibody 3G10 comprises a light chain and a heavy chain; The amino acid sequence of the CDR1 region of the light chain is shown in SEQ ID NO.2, the amino acid sequence of the CDR2 region is LVS, and the amino acid sequence of the CDR3 region is shown in SEQ ID NO.

3. The amino acid sequence of the CDR1 region of the heavy chain is shown in SEQ ID NO.9, the amino acid sequence of the CDR2 region is shown in SEQ ID NO.10, and the amino acid sequence of the CDR3 region is shown in SEQ ID NO.

11.

2. The monoclonal antibody 3G10 according to claim 1, characterized in that, The amino acid sequence of the FR1 region of the light chain is shown in SEQ ID NO.4, the amino acid sequence of the FR2 region is shown in SEQ ID NO.5, the amino acid sequence of the FR3 region is shown in SEQ ID NO.6, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.

7. The amino acid sequence of the FR1 region of the heavy chain is shown in SEQ ID NO.12, the amino acid sequence of the FR2 region is shown in SEQ ID NO.13, the amino acid sequence of the FR3 region is shown in SEQ ID NO.14, and the amino acid sequence of the FR4 region is shown in SEQ ID NO.

15.

3. A hybridoma cell line secreting the monoclonal antibody 3G10 of claim 1, characterized in that, The hybridoma cell line, named 3G10, is an anti-porcine pseudorabies virus gE protein hybridoma cell line. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC) on March 26, 2026, with accession number CGMCC No. 46794.

4. The use of the hybridoma cell line according to claim 3 in the production of monoclonal antibodies against porcine pseudorabies virus gE protein.

5. The use of the monoclonal antibody 3G10 according to claim 1 or 2 or the hybridoma cell line according to claim 3 in the preparation of products for detecting porcine pseudorabies virus.

6. The use of the monoclonal antibody 3G10 according to claim 1 or 2 or the hybridoma cell line according to claim 3 in the preparation of a product for detecting porcine pseudorabies virus gE protein antibodies.

7. The application according to claim 5 or 6, characterized in that, The product includes a reagent kit.

8. A kit for detecting porcine pseudorabies virus gE antibodies, characterized in that, The kit contains an enzyme-labeled plate and an enzyme-labeled antibody, wherein the enzyme-labeled antibody is made from the monoclonal antibody 3G10 as described in claim 1 or 2.

9. The reagent kit according to claim 8, characterized in that, The ELISA plate is coated with inactivated porcine pseudorabies virus.

10. The reagent kit according to claim 8, characterized in that, The kit also includes a washing solution, a sample diluent, a substrate solution, and a stop solution.

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