An indirect elisa kit for detecting duck plague virus antibody and application thereof

Abstract

The application discloses an indirect ELISA kit for detecting duck plague virus antibodies and application thereof, and belongs to the technical field of biological detection. The indirect ELISA kit for detecting duck plague virus antibodies disclosed by the application comprises an enzyme-labeled plate coated with an antigen; and the antigen is a gC recombinant protein. The indirect ELISA kit for detecting duck plague virus antibodies disclosed by the application has the advantages of high specificity, high sensitivity, simple operation, good safety and easiness in standardization.

Description

Technical Field

[0001] This invention relates to the field of biological detection technology, and more specifically to an indirect ELISA kit based on duck plague virus gC recombinant protein, its preparation method and uses. Background Technology

[0002] Duck plague (DPV) is an acute, septicemic infectious disease of ducks, geese, and other waterfowl caused by duck plague virus (DPV). It is characterized by high morbidity and mortality, posing a serious threat to waterfowl farming. Therefore, establishing rapid, accurate, and specific antibody detection methods for duck plague virus is of great significance for disease monitoring, immunization efficacy evaluation, and prevention and eradication.

[0003] Currently, the main methods for detecting duck plague virus antibodies include virus neutralization assay, agar diffusion assay, and indirect ELISA. Among these, indirect ELISA is widely used due to its simplicity, objective results, and suitability for large-scale screening. Existing technologies have reported methods for establishing indirect ELISA using whole virus antigens (such as patent CN103777011A), which uses the whole duck plague virus strain from Guizhou, propagated and purified in duck embryos, as the coating antigen.

[0004] However, the above whole virus antigen scheme still has the following technical problems: (1) The whole virus antigen is complex, containing a variety of viral structural proteins and miscellaneous proteins from duck embryos in addition to the target antigen, which can easily lead to non-specific reactions and affect the accuracy of detection; (2) The preparation of whole virus depends on duck embryo proliferation and live virus operation, which poses a biosafety risk, and there are large differences between batches, making it difficult to achieve standardized production; (3) It cannot distinguish between wild virus infection and gC gene deletion vaccine immunization, and is not suitable for the purification and control of duck plague.

[0005] Therefore, providing an indirect ELISA kit for detecting duck plague virus antibodies and its application is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides an indirect ELISA kit for duck plague virus antibodies that is highly specific, sensitive, easy to operate, safe, and easy to standardize, in order to overcome the shortcomings of existing whole virus antigen schemes.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] An indirect ELISA kit for detecting duck plague virus antibodies includes an enzyme-labeled plate coated with an antigen; the antigen is a gC recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Furthermore, the method for preparing the enzyme-labeled plate coated with the antigen includes the following steps: The recombinant gC protein was added to the wells at a concentration of 0.5 μg / mL and incubated at 37°C for 2 hours, then at 4°C for 14 hours. The supernatant was removed, and the plate was washed three times with PBST and the supernatant was discarded. 5% skim milk powder was added, and the plate was blocked at 37°C for 2 hours to obtain the enzyme-labeled plate coated with the antigen.

[0010] Furthermore, the indirect ELISA kit also includes enzyme-labeled secondary antibody, coating solution, washing solution, chromogenic solution, stop solution, negative standard (negative control serum: serum from healthy mice) and positive standard (positive control serum: serum from immunized mice).

[0011] Furthermore, the enzyme-labeled secondary antibody is HRP-labeled goat anti-mouse IgG.

[0012] Furthermore, the coating solution is a carbonate buffer solution with a pH of 9.6.

[0013] Furthermore, the washing solution is a PBS buffer containing 0.05% Tween-20.

[0014] Furthermore, the colorimetric solution is a TMB solution.

[0015] Furthermore, the terminating solution is 2M H2SO4.

[0016] Furthermore, the indirect ELISA kit described herein is used for detecting duck plague virus antibodies for non-disease diagnostic purposes.

[0017] Furthermore, a method for detecting duck plague virus antibodies for non-disease diagnostic purposes includes the step of detecting duck plague virus antibodies using the above-mentioned indirect ELISA kit: Add 100 µL of negative standard, positive standard, and test sample solution to the enzyme-labeled plate coated with antigen, respectively, and incubate at 37°C for 45 min. After incubation, rinse 3 times with washing solution. Add 1:5000 diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 45 min. After incubation, rinse 3 times with washing buffer. Add the colorimetric solution to each reaction well and incubate at 37°C in the dark for 15 minutes; Add stop solution to each reaction well and measure the absorbance at 450 nm using an ELISA reader. Result interpretation: When OD450nm≥0.193, it is judged as positive, that is, the test sample contains duck plague virus antibodies; when OD450nm≤0.173, it is judged as negative, that is, the test sample does not contain duck plague virus antibodies.

[0018] As can be seen from the above technical solution, compared with the prior art, the present invention discloses an indirect ELISA kit for detecting duck plague virus antibodies and its application, which has the following beneficial effects: (1) High specificity: Using a single gC recombinant protein as the coating antigen avoids the non-specific background caused by miscellaneous proteins in the whole virus antigen.

[0019] (2) Good safety: It does not involve live virus operations and has no biosafety risks.

[0020] (3) Quality controllable: Recombinant proteins can be produced in a standardized and large-scale manner with high batch-to-batch consistency.

[0021] (4) High sensitivity: The lowest detection limit for duck plague virus antibody can reach a dilution of 1:12800 (based on spiked experiment verification).

[0022] (5) Good repeatability: The coefficient of variation within the batch is less than 5%, and the results are stable and reliable. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 Analysis of hydrophilicity / hydrophobicity and antigenic epitope characteristics of DEV gC protein; where A: hydrophilicity / hydrophobicity analysis of gC protein; B: antigenic epitope characteristics analysis of gC protein.

[0025] Figure 2 The result represents the amplification of the target band; where M represents the protein molecular weight standard and 1 represents the target gene.

[0026] Figure 3 The image shows the enzyme digestion verification results of pET-28a-DPV-gC; where M: DL12000 DNA Maker; 1: pET-28a-DPV-gC; 2: BamH1 single enzyme digestion product of pET-28a-DPV-gC; 3: BamH1 and Xho1 double enzyme digestion product of pET-28a-DPV-gC.

[0027] Figure 4 To verify the sequencing results; the selected area is the first 20 bases containing ATG, including... Bam HI restriction site.

[0028] Figure 5SDS-PAGE results; where M: 15~130 kDa Protein Marker; Determination of induction concentrations in lanes 1~7: Lane 1: pET28a empty vector, Lanes 2~6: gC recombinant plasmid IPTG induction concentrations of 0.1, 0.3, 0.5, 0.7, 1.0 mmol / L respectively, Lane 7: gC recombinant plasmid not induced; Determination of induction time in lanes 8~11: 3h, 4h, 5h, 6h respectively; Identification of expression forms in lanes 12~13: 12: supernatant of bacterial lysate, 13: resuspension of precipitate after bacterial lysate.

[0029] Figure 6 To determine the appropriate imidazole washing and elution concentrations; where M: protein marker; 1: pET28a empty vector, uninduced; 2: flow-through buffer; 3-5: 20, 40, and 60 mM imidazole washing buffers; 6-9: 100, 200, 300, and 500 mM imidazole elution buffers.

[0030] Figure 7 The results show the large-scale purification of gC recombinant protein; where M: protein marker; 1: flow-through buffer; 2: 40 mM imidazole washing buffer; 3: 500 mM imidazole elution buffer.

[0031] Figure 8 For SDS-PAGE gel purity verification; where M: protein maker; 1: concentrated and purified gC recombinant protein; 2: purified gC recombinant protein; 3: unpurified gC recombinant protein.

[0032] Figure 9 To determine the titer of mouse anti-gC recombinant protein polyclonal antibody.

[0033] Figure 10 Western blot validation of mouse anti-gC recombinant protein polyclonal antibody.

[0034] Figure 11 To verify the expression of gC protein in duck plague virus vaccine; where M: protein Maker; 1: duck plague virus vaccine; 2: turkey herpesvirus vaccine; 3: gC recombinant protein; 4: pET-28a empty vector.

[0035] Figure 12 This is the result of an indirect ELISA specific assay.

[0036] Figure 13 These are the results of an indirect ELISA sensitivity test. Detailed Implementation

[0037] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0038] (1) Vaccine, plasmid and cell sources Duck plague live attenuated vaccine (CVCC AV1222) and MD turkey herpesvirus live vaccine (Fc-126 strain) were purchased from Harbin Pharmaceutical Group Biological Vaccine Co., Ltd. The prokaryotic expression vector plasmid pET-28a(+) was preserved by the Poultry Disease Research Laboratory of Guangxi University. Escherichia coli DH5α competent cells were purchased from Escherichia coli and BL21(DE3) competent cells were purchased from Beijing TransGen Biotech Co., Ltd.

[0039] (2) Laboratory animals Healthy Balb / c mice were purchased from Changsha Tianqin Biotechnology Co., Ltd. The animals were housed in a clean, pollution-free animal facility for one week for acclimatization, with a continuous supply of water and feed to ensure environmental adaptation and prevent stress. This study strictly adhered to the national "Regulations on the Management of Laboratory Animals," and all experimental protocols were reviewed by the Laboratory Animal Ethics Committee of Guangxi University (Ethics Review No.: GXU-2026-197, License No.: SYXK Gui 2025-0001) to ensure compliance with animal welfare and ethical requirements. Duck embryos were purchased from a poultry market in Guangxi.

[0040] (3) Main consumables and reagents DNA extraction kits were purchased from Tiangen Biotech (Beijing) Co., Ltd.; DNA purification and recovery kits were purchased from Sangon Biotech (Shanghai) Co., Ltd.; DL2000 and DL5000 DNA Markers, restriction endonucleases EcoRI and HindIII, and high-fidelity DNA polymerase were purchased from Nanjing Novizan Biotechnology Co., Ltd.; T4 DNA ligase was purchased from Beijing TransGen Biotech Co., Ltd.; isopropyl-β-D-thiogalactoside (IPTG), kanamycin (Kan), and filter paper were purchased from Beijing Lanjieke Technology Co., Ltd.; plasmid extraction kit and Ni-NTA agarose affinity chromatography pre-packed column were purchased from Comvita Biotechnology Co., Ltd.; Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA), BCA protein quantification kit, and MD25 dialysis bags (8000-14000D) were all purchased from Beijing Solarbio Technology Co., Ltd.; HRP-labeled goat anti-mouse IgG was purchased from Abmart Biopharmaceutical (Shanghai) Co., Ltd.; ultrasensitive electrochemiluminescence (ECL) kit was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., and PVDF membrane was purchased from Immobilon.

[0041] (4) Main instruments The 96-well VeritiPro thermal cycler was purchased from Thermo Fisher Scientific; the constant temperature water bath was purchased from Jiangsu Jinyi Instrument Technology Co., Ltd.; the gel imaging system was purchased from Suzhou Beirui Instrument Technology Co., Ltd.; the vertical electrophoresis apparatus, membrane transfer apparatus, and ChemiDoc Mp all-in-one imaging system were all purchased from Bio-Rad; and the microplate reader was purchased from TECAN.

[0042] Example 1 Using the ExPASy ProtParam and ExPASy ProtScale tools, the physicochemical properties of the gC protein amino acid sequence of duck plague virus (as shown in SEQ ID NO.1) were first analyzed, and then the hydrophilicity and hydrophobicity of the gC protein were analyzed in detail.

[0043] >AGW24811.1 gC [anatid alphaherpesvirus 1]CVCC AV1222: MGPLVMVAFSVSLLLTITITRSQNAAQMTCSSTKQLAYHGEKITFGCEPKGNATNAKNWTMVVTFYHKPTQAVDLLSPVEWNGMQYDPVGSKQAQPRIIYTAMPSNADSGLGEYDLHGLDEKHYGFEPTEDSSFPLTILKPTALDEGLYEWVLSTPFVESNTNKTKRRTEAIGAITLKIIKIPSGISVIAHPMVYGQRYRATCILENFFPPGSGRMTWVVDDSTNLPINSVRTYSTAKSEEDTVSLIGSLLLGSSIDSMPPEITCKAIWNGEGETRTFNASAVPVVYSKPNVILTFESGHAVCNARCVADTAAVGIKWMVGRELKENIWKDGMVEVVGQCIDHPGTVNIRSRYPLEYTGTVTQYTCRVEGYPEELPIFEDAALYDSSPYSEGKPMIMIIVTVLTAGILTGGIILIMAVCFYYSRDKADNII; SEQ ID NO.1。

[0044] gC protein gene sequence: ATGGGGCCATTAGTGAT GCGAGATAAAGCAGAC AATATTATT TGA; SEQ ID NO.2.

[0045] Physicochemical properties: Analysis showed that the protein consists of 431 amino acids, has a molecular weight of 47.35 kDa, a theoretical isoelectric point of 5.31, and is acidic. The instability index is 34.79, indicating a stable protein structure. The average hydrophilicity index (GRAVY) is -0.049, indicating an overall hydrophilic protein with good water solubility, which is beneficial for the expression and purification of recombinant proteins.

[0046] Results of hydrophilicity / hydrophobicity: Figure 1 Analysis A showed that most of the protein was hydrophilic, with only localized hydrophobic regions. Overall, it was highly hydrophilic, consistent with the structural characteristics of enveloped glycoproteins, making it suitable as a coating antigen for the establishment of subsequent ELISA methods.

[0047] Results of antigenic epitope feature analysis: The BepiPred 2.0 algorithm of the IEDB platform was used to predict B cell linear antigenic epitopes of the gC protein amino acid sequence of duck enteritis virus, with 0.5 as the antigen positivity threshold. Figure 1 Results B showed that the overall antigen score of the gC protein was 0.473. Multiple positive antigen regions exceeding the detection threshold were found within the protein sequence, with the highest peak antigen scores observed in amino acid segments 65-94, 156-171, 221-231, and 323-339, representing dominant linear B-cell epitopes. Combined with hydrophilicity analysis, these high antigenic index segments correspond to highly hydrophilic, extracellularly exposed regions of the protein, structurally providing a good basis for antibody recognition.

[0048] Example 2 Preparation of duck plague virus gC recombinant protein 1) Obtaining the template The commercially available duck plague attenuated live vaccine (CVCC AV1222) was dissolved and diluted with DMEM, and the genomic DNA of the DPV was extracted using the DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., following the instructions.

[0049] 2) Cloning and expression of the gC gene Primers (gC-F: 5'-CG) were designed based on the duck plague virus gC gene sequence (as shown in SEQ ID NO. 2) from GenBank accession number KF263690.1. GGATCC ATGGGGCCATTAGTGAT-3'; BamHI; SEQ ID NO.3; gC-R: 5'-CCG CTC GAGAATAATATTGTCTGCTTTATCTCGC-3'; XhoI; SEQ ID NO.4), using viral genomic DNA as a template for PCR amplification, the reaction system (50 μL) contained: 25 μL 2× Phanta Max Master Mix, 2.5 μL each of forward and reverse primers, 5 μL template DNA, and ddH2O to a final volume of 50 μL. The PCR reaction program was: 95 ℃ pre-denaturation for 3 min; 95 ℃ denaturation for 15 s, 58 ℃ annealing for 15 s, 72 ℃ extension for 1 min, for a total of 35 cycles; and a final extension at 72 ℃ for 5 min. The product was detected by 1% agarose gel electrophoresis (…). Figure 2 Afterwards, the target fragment was purified using a DNA gel extraction kit. The amplified product was then subjected to... Bam HⅠ and Xho I. After enzyme digestion, the cells were ligated into the enzyme-digested and recovered pET-28a linear vector and transformed into Escherichia coli BL21(DE3) competent cells.

[0050] Enzyme digestion system: vector plasmid / target fragment 25 µL, restriction enzyme Bam HI 1 µL, endonuclease Xho I 1 µL, CutSmart Buffer 5 µL, ddH2O 3 µL. Total volume 35 µL.

[0051] The enzyme digestion system was placed in a 37°C water bath for 8 h. After gel purification, the digestion products were ligated. The ligation system consisted of: 5 µL of the purified target fragment, 2 µL of the purified vector, 1 µL of 10×T4 DNA Ligase buffer, 1 µL of T4 DNA Ligase, and ddH2O to a final volume of 10 µL. The mixture was gently mixed, briefly centrifuged, and incubated overnight at 4°C. The ligation product was then transformed into... E. coli DH5α competent cells were plated on LB agar plates containing 50 μg / mL kanamycin and incubated overnight at 37 °C with the plates inverted. The next day, single colonies were picked and identified by PCR using gC-F / gC-R primers. Positive clones were expanded and plasmids were extracted, then cultured in 10 µL of water. Bam HI and 10 µL Xho The constructed recombinant plasmid (1 µg) was identified by double digestion with restriction enzyme I; or by digestion with 10 µL of restriction enzyme. Bam The constructed recombinant plasmid (1 µg) was identified by single-enzyme digestion with HI endonuclease; the digestion system was placed in a 37°C water bath for 1 h, and the results were analyzed by 1% agarose gel electrophoresis. Figure 3 The extracted positive plasmids were sent to BGI Genomics for bidirectional sequencing identification. Sequencing results ( Figure 4The results showed that all bases corresponded correctly. The recombinant plasmid with completely correct sequencing was named pET-28a-DPV-gC.

[0052] 3) Expression and purification of gC protein (1) Optimization of expression conditions and identification of expression form: The recombinant plasmid pET-28a-DPV-gC with correct sequencing results was transformed into the expression host bacteria. E. coli In BL21(DE3), positive single colonies were picked and inoculated into 5 mL LB liquid medium containing 50 μg / mL kanamycin, and cultured at 37 ℃ with shaking at 220 r / min for 12–16 h. The next day, 100 mL of the culture was expanded at a ratio of 1:100 to explore the induction conditions for gC recombinant protein expression. When the OD600nm of the bacterial culture reached 0.5, 5 mL of the bacterial culture was dispensed into 15 mL centrifuge tubes in a clean bench. Under induction conditions of 37 ℃, the IPTG induction concentration was set to 0.1, 0.3, 0.5, 0.7, and 1.0 mmol / L, and the induction time was 5 h. Alternatively, with IPTG at 0.5 mmol / L and an induction temperature of 37 ℃, the induction time was set to 3 h, 4 h, 5 h, and 6 h. Optimal induction conditions were determined using whole-cell loading. After induction, the supernatant was discarded by centrifugation. TBS and lysis buffer were added according to the instructions for the lysis buffer. After lysis, 5× loading buffer was added and mixed thoroughly. The samples were then boiled at 100°C for 10 min to complete sample preparation. Empty vector was used as a blank control, and the recombinant vector was used as an uninduced control. Samples were prepared using the same method, and the optimal expression conditions for the recombinant protein were determined by SDS-PAGE. Under optimal induction conditions, the supernatant and precipitate of the bacterial culture after lysis with the lysis buffer were separated. The precipitate was resuspended in PBS, and the expression form of the recombinant protein was determined by SDS-PAGE after sample preparation.

[0053] Induction concentration results: SDS-PAGE electrophoresis results showed that the gC recombinant plasmid could be expressed normally at 37℃. The IPTG induction concentration was explored, and the results of SDS-PAGE electrophoresis were used to determine the optimal concentration. Figure 5 As can be seen from lanes 1-7 in the diagram, the effect of different final concentrations of the inducer on the expression of gC protein is not significant. Considering the toxic effect of the inducer on the competent cells, the concentration of the inducer was set at 0.5 mmol / L.

[0054] Induction time results: Four time gradients were set at 3h, 4h, 5h, and 6h, as shown by SDS-PAGE electrophoresis results ( Figure 5 As can be seen from lanes 8-11 in the image, the bands are darker and wider after 5 hours of induction, therefore 5 hours is selected as the optimal induction time for gC recombinant plasmids.

[0055] Identification of expression form: The results were obtained by analyzing the supernatant and precipitate after bacterial cell disruption. Figure 5 Lanes 12-13 in the study show that the gC recombinant protein is mainly expressed in the form of inclusion bodies.

[0056] (2) Optimization of purification conditions and Western blot identification A. High expression of recombinant gC protein Bacterial culture was added to 500 mL of resistant LB medium at a ratio of 1:100 and cultured until the OD600nm value of the bacterial culture reached 0.5. Induction was then performed at 37°C for 5 hours with a final IPTG concentration of 0.5 mM. After induction, the bacterial cells were collected by centrifugation at 3000 rpm for 10 min at 4°C, washed with pre-cooled PBS buffer (pH 7.4), and resuspended. The cells were then sonicated on ice (3 s on, 5 s off, total power 300 W, total time 20 min) until the bacterial culture changed from turbid to clear. The lysate was centrifuged at 4000 rpm for 10 min at 4°C, and the inclusion body precipitate was collected. The precipitate was dissolved thoroughly in 8 mol / L urea, centrifuged again, and the supernatant was filtered through a 0.22 μm filter for protein purification.

[0057] B. Optimization of purification conditions and Western blotting identification of recombinant gC protein Proteins were purified using a His-tagged Ni-NTA affinity chromatography pre-packed column.

[0058] After equilibrating the chromatography column with binding buffer, the supernatant from partial inclusion body lysis was slowly passed through the column to ensure thorough binding of the target protein to the Ni column. The supernatant flowing through the Ni column was collected as the flow-through buffer, and then washed sequentially with wash buffers containing 20 mmol / L, 40 mmol / L, and 60 mmol / L imidazole to remove impurities. Elution buffers containing 100 mmol / L, 200 mmol / L, 300 mmol / L, and 500 mmol / L imidazole were prepared for staged elution of the target protein. The eluent was collected in separate tubes and identified by Western blotting. The purified recombinant gC protein was mixed with 5×SDS loading buffer and boiled at 100 °C for 10 min for denaturation. 12% SDS-PAGE electrophoresis was performed, and the electrophoretically separated proteins were transferred to a PVDF membrane using a semi-dry transfer method. Block the membrane with PBST containing 5% skim milk powder at room temperature for 2 h; after washing, add mouse anti-His-Tag monoclonal antibody and incubate overnight at 4 ℃; wash the membrane 3 times the next day, add HRP-labeled goat anti-mouse IgG (1:5000 dilution) and incubate at room temperature for 1 h; after washing the membrane 3 times, add ECL ultrasensitive luminescent solution and expose and develop in a chemiluminescent gel imaging system to verify the reactivity of the recombinant protein.

[0059] Determination of imidazole concentration in gC recombinant protein: After large-scale induction of gC protein expression, a portion of the inclusion body lysis supernatant was used to determine the imidazole concentration in the washing and elution buffers. The results were obtained from Western blotting (WB). Figure 6 It can be seen that when the imidazole concentration is 40 mmol / L, the target protein will not be washed off. When the imidazole concentration is 500 mmol / L, the protein band is wider and darker. Therefore, the washing solution with an imidazole concentration of 40 mmol / L is selected to wash away impurities, and the elution solution with an imidazole concentration of 500 mmol / L is selected to elute the target protein.

[0060] C. Large-scale purification and concentration of recombinant gC protein All inclusion body lysate supernatants were purified according to the optimized purification steps and conditions described above to obtain a large amount of purified gC recombinant protein. Figure 7 The collected purified protein was placed in dialysis bags (molecular weight cutoff 8-14 kDa) and dialyzed at 4 °C in dialysate for 48 h. The dialysate was dialyzed using a urea gradient of 6, 5, 4, 3, 2, 1, and 0 mol / L to remove urea and imidazole, with each concentration dialyzed for 4-6 h. After dialysis, the protein was concentrated using PEG-2000. The concentration of the concentrated protein was determined using a BCA kit, aliquoted, and stored at -80 °C for later use.

[0061] Samples prepared with flow-through buffer, washing buffer, and elution buffer were subjected to Western blotting for identification. Unpurified protein, purified protein, and concentrated protein were stained with Coomassie Brilliant Blue R-250 and then destained to observe the purity and molecular weight of the protein bands.

[0062] The results of extensive purification and testing show that a recombinant gC protein with good antigenicity was obtained.

[0063] gC recombinant protein concentration verification: After purification, the protein was concentrated by... Figure 8 The results showed that the concentrated and purified protein was consistent with the estimated protein size, with a single band and no other contaminating proteins, indicating that the protein purity was high.

[0064] Protein concentration determination: Protein concentration was determined using a BCA protein quantification kit, and a BSA standard curve was plotted using standards. The standard curve equation is y = 2.4279x - 0.4717, R0. 2 = 0.9925; where y is the value of the sample measured using an ELISA reader. D The 562 nm value, where x is the concentration (µg / mL) of the protein sample being tested. The protein sample measured... D The 562 nm value was substituted into the y value of the standard curve equation obtained above to calculate the x value, and the concentration of the concentrated gC recombinant protein was found to be 1.9 mg / mL.

[0065] Example 3 Preparation of polyclonal antibodies Balb / c mice were immunized with purified gC recombinant protein at a dose of 50 μg per mouse, three times, followed by a powdered immunization. Serum was collected three days after the last immunization. Indirect ELISA showed a polyclonal antibody titer of 1:1638400, and Western blotting confirmed that the antibody specifically recognized the gC recombinant protein.

[0066] Purified recombinant gC protein was used as an immunogen to immunize BALB / c mice to prepare polyclonal antibodies. The specific immunization procedure is as follows: (1) Primary immunization (Day 0): Take an appropriate amount of purified gC recombinant protein (50 μg per mouse, 200 μL in total), add an equal volume of Freund's complete adjuvant (FCA), and shake for 5 min until it reaches a water-in-oil state (does not spread when dropped onto pre-cooled water). Immunize 4 BALB / c mice by multiple subcutaneous and intraperitoneal injections. Two mice injected with an equal volume of PBS and FCA emulsion serve as negative controls.

[0067] (2) Secondary immunization (Day 14): Equal volumes of gC recombinant protein and Freund's incomplete adjuvant (FIA) were fully emulsified and the mice were boosted with the same dose and route as described above.

[0068] (3) Third immunization (Day 28): Repeat the procedure of the second immunization (protein + FIA) for a second booster immunization. One week after immunization, serum was collected from the tail tip and the antibody titer was measured by indirect ELISA. Mice with titers higher than 1:102400 were selected for shock immunization.

[0069] (4) Four immunizations (shock immunization, Day 35): Depending on the previous immunization, 50 μg of purified gC recombinant protein was diluted with PBS and injected intraperitoneally for adjuvant-free shock immunization.

[0070] (5) Serum collection (Day 38): Three days after the shock immunization, blood was collected from the eyes of all mice. The collected whole blood was allowed to stand at room temperature for 30 to 60 minutes to coagulate. Then, the blood clot was gently peeled off along the tube wall with a sterile pipette tip and allowed to stand for another 10 to 15 minutes to allow the blood clot to shrink and sink. The centrifuge was set to 4 ℃ and 3000 r / min for 10 minutes. The upper light yellow transparent serum was collected and stored at -80 ℃ for later use, which yielded the mouse anti-gC recombinant protein polyclonal antibody.

[0071] The antibody titer of immune serum was determined using an indirect ELISA method. Recombinant gC protein was diluted to 0.5 μg / mL with coating buffer and coated onto an ELISA plate. The plate was incubated at 37°C for 2 hours, followed by overnight incubation at 4°C. After washing, 5% skim milk blocking buffer was added, and the plate was blocked at 37°C for 2 hours. The blocking buffer was discarded, and serially diluted mouse anti-gC recombinant protein polyclonal antibody was added as the primary antibody, and the plate was incubated at 37°C for 1 hour. After washing, horseradish peroxidase-labeled goat anti-mouse IgG secondary antibody (1:3000) was added, and the plate was incubated at 37°C for 1 hour. After thorough washing, TMB chromogenic buffer was added, and the plate was incubated at room temperature in the dark for 15 minutes. The reaction was terminated by adding 2 mol / L H₂SO₄, and the absorbance at 450 nm was measured using an ELISA reader. The highest serum dilution with a positive serum OD450 nm / negative serum OD450 nm (P / N) ratio ≥ 2.1 was determined as the titer of the polyclonal antibody. Results are shown below. Figure 9 The titer of the mouse anti-gC recombinant protein polyclonal antibody was 1:1638400.

[0072] The purified protein was used as the antigen for polyclonal antibody validation. The steps were as follows: 40 μL of sample was added to 8 μL of 5× Loading buffer and boiled in a metal bath at 100℃ for 10 min. The sample was subjected to SDS-PAGE electrophoresis and transferred to a PVDF membrane. After transfer, the membrane was washed three times with washing buffer, blocked with 5% skim milk powder at 37℃ for 2 h, washed three times with washing buffer, and polyclonal antibody diluted 1:1000 was incubated at 4℃ for 12-16 h, washed three times for 10 min each time, and HRP-goat anti-mouse IgG diluted 1:5000 was incubated at 37℃ for 1 h, washed three times, ECL chromogenic solution was added, and the image was captured and saved using a Western blot imaging system. The results are shown below. Figure 10 The mouse anti-polyclonal antibodies were able to specifically bind to the corresponding structural proteins, showing a specific reaction band at 60.9 kDa, consistent with the expected band size, indicating that they all had good reactivity.

[0073] Verification of gC protein expression in duck plague virus vaccine The lyophilized powder of DEV vaccine CVCC AV1222 was reconstituted with DMEM, and then lysed with 1 mM PMSF and RIPA lysis buffer on ice for 30 min. After centrifugation at 12000 rpm for 10 min, the supernatant was collected, loaded with loading buffer, and denatured in a metal bath at 100°C for 10 min for subsequent testing. The pET-28a empty vector was selected as a blank control. The Herpesvirus of Turkey (HVT) vaccine was prepared using the same sample treatment as the DEV vaccine and used as a specific control sample. The gC recombinant protein was used as a positive control. Both samples were loaded for Western blotting (WB) detection.

[0074] See results Figure 11 The polyclonal antibody binds normally to the gC antigen, but the duck plague virus vaccine does not have a gC recombinant protein band, indicating that the gC recombinant protein does not exist in the duck plague virus vaccine in the form of a structural protein, or that the abundance of gC protein in the vaccine is insufficient, so that it cannot be detected by Western blotting.

[0075] Example 4: Establishment of the ELISA method Experimental materials The purified duck plague virus gC recombinant protein prepared in Example 2; mouse anti-gC recombinant protein polyclonal antibody (as standard positive serum, prepared by immunizing BALB / c mice with purified gC recombinant protein in Example 3); and serum from healthy, unimmunized BALB / c mice (as negative control serum). Since no duck-derived standard positive serum was available at the stage of establishing the detection method in this study, a basic indirect ELISA reaction system was established using a laboratory-prepared mouse polyclonal antibody with a defined titer. The relevant reaction conditions were optimized to lay the foundation for subsequent detection of actual samples.

[0076] Main consumables and reagents The ELISA plate was purchased from BIOFIL Biotechnology Co., Ltd., the skim milk powder and TMB chromogenic solution were purchased from Lanjieke Technology Co., Ltd., the PBST powder was purchased from Servicebio Biotechnology Co., Ltd., and the HRP-labeled goat anti-mouse IgG (ELISA secondary antibody) was purchased from AbMART.

[0077] Preparation of main reagents (1) Carbonate buffer (pH 9.6): Weigh 1.5g Na2CO3 and 2.9g NaHCO3, shake well and then dilute to 1 L with pure water and store at 4℃.

[0078] (2) PBST washing solution: 1 L of 1×PBS diluent and 0.5 mL of Tween-20 are added. After shaking and mixing, it is used.

[0079] (3) 1% BSA: Weigh 0.15 g of BSA powder, add 15 mL of washing solution, shake to mix, and store at 4℃.

[0080] (4) 5% skim milk powder: Weigh 0.75 g of skim milk powder and add 15 mL of washing solution, shake to mix well and store at 4℃.

[0081] (5) Termination solution: Slowly add 21.7 mL of concentrated sulfuric acid to 178.3 mL of pure water, stir gently to mix well, and use after cooling.

[0082] Determination of optimal antigen coating concentration and primary antibody dilution The optimal coating concentration of the gC recombinant protein and the optimal dilution of the test serum (primary antibody) were determined using checkerboard titration.

[0083] (1) Antigen coating: The purified gC recombinant protein was horizontally diluted with carbonate buffer (pH 9.6) at different concentration gradients (5, 2.5, 2, 1, 0.5, 0.25, 0.125 μg / mL), and 100 μL was added to each well. The protein was then coated overnight at 4 °C.

[0084] (2) Washing and blocking: The next day, discard the liquid in the wells, wash 3 times with PBST, add 200 μL of 5% skim milk powder to each well as blocking solution, and incubate at 37 ℃ for 2 h. Wash the plate 3 times.

[0085] (3) Primary antibody incubation: Mouse anti-gC recombinant protein polyclonal antibody (positive serum) and healthy mouse serum (negative serum) were serially diluted longitudinally at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400 and 1:12800, respectively. 100 μL was added to each well and incubated at 37 ℃ for 1 h. The plate was washed 3 times.

[0086] (4) Secondary antibody incubation and color development: Add 100 μL of HRP-goat anti-mouse IgG at a fixed dilution (e.g., 1:3000) to each well and incubate at 37 °C for 1 h.

[0087] (5) Color development and termination: After washing the plate, add 100 μL of TMB color development solution to each well, develop color in the dark for 15 min, and add 50 μL of 2M H2SO4 (termination solution) to terminate the reaction.

[0088] (6) Data Analysis: The OD value of each well was measured at 450 nm using an ELISA reader. The ratio of OD values ​​of positive serum to negative serum (P / N value) was calculated. Three replicate wells were set up for each different reaction condition, and the average OD450 nm value of the three wells was calculated. The antigen concentration and serum dilution corresponding to the well with an OD450 nm value of around 1.0 and the largest P / N value were selected as the optimal working conditions.

[0089] The results of optimized reaction conditions (coating concentration, serum dilution matrix) are shown in Table 1. The antigen coating concentration was 0.5 μg / mL; the optimal dilution of the serum to be tested was 1:6400.

[0090] Table 1

[0091] Determination of optimal antigen coating conditions After determining the optimal antigen coating concentration, different coating temperatures and times were set to optimize the coating conditions, aiming to reduce non-specific binding, lower background interference, and improve the sensitivity and specificity of the detection. Different coating conditions were set: overnight at 4°C, 1 h at 37°C followed by 14 h at 4°C, 2 h at 37°C followed by 14 h at 4°C, 3 h at 37°C followed by 14 h at 4°C, and 14 h at room temperature (25°C) for the same steps. The optimal coating conditions were determined by reading the microplate reader and selecting the coating condition corresponding to the highest P / N ratio. The results are shown in Table 2. The optimal antigen coating conditions were 2 hours at 37°C followed by 14 hours at 4°C.

[0092] Table 2

[0093] Optimization of sealing fluid type and time After determining the optimal antigen coating concentration and coating conditions, the blocking solution was optimized to reduce nonspecific background. 1% BSA and 5% skim milk powder were prepared as blocking solutions. After coating ELISA plates with the determined antigen concentrations, different blocking solutions were added and the plates were blocked at 37 ℃ for 1, 1.5, 2, and 2.5 h. Positive and negative sera were then added at the optimal dilution, and the remaining steps were the same. By comparing the P / N values ​​and the background OD values ​​of the negative wells under different blocking conditions, the optimal blocking solution type and concentration were determined. The results are shown in Table 3. The optimal blocking solution conditions were 5% skim milk powder and blocking for 2 h.

[0094] Table 3

[0095] Determination of incubation time for the serum to be tested The optimal antigen coating concentration and conditions were determined for coating, and the optimal blocking solution and blocking time were optimized for blocking. The optimal dilution of the test serum was explored for dilution, and incubation times of 30 min, 45 min, 60 min, and 90 min were set to determine the optimal incubation time for the test serum. The results are shown in Table 4. The optimal reaction time for the test serum was 45 minutes.

[0096] Table 4

[0097] Determination of the optimal working concentration of enzyme-labeled secondary antibody Under the determined antigen coating, primary antibody dilution, and blocking conditions, HRP-goat anti-mouse IgG was serially diluted at the following ratios: 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:7500, and 1:10000, and added to the corresponding wells. Finally, by measuring the OD450 value and calculating the P / N value, the secondary antibody dilution corresponding to moderate color development, lowest background, and highest P / N value was selected. The results are shown in Table 5. The optimal dilution of the secondary antibody was 1:5000.

[0098] Table 5

[0099] Determination of the optimal reaction time for enzyme-labeled secondary antibodies Based on the established antigen coating conditions, primary antibody dilution, blocking system, and secondary antibody dilution, the incubation time for the secondary antibody was explored according to the ELISA procedure. Incubation was set at three conditions: 30 min, 45 min, and 60 min. The results are shown in Table 6. The optimal reaction time for the secondary antibody was 45 minutes.

[0100] Table 6

[0101] Determination of negative and positive cut-off values Twenty serum samples from healthy Balb / c mice that were confirmed to be free of duck plague virus and had not undergone related immunization were collected and tested using an optimized indirect ELISA reaction system. The OD450 values ​​of all negative serum samples were recorded, and their average values ​​were calculated. X and standard deviation (SD). According to statistical principles, when the OD450 value of the sample to be tested is greater than or equal to... When X+3SD, it is considered positive; when the OD450 value of the sample is less than or equal to When the result is X+2SD, it is considered negative; if it falls between the two, it is considered suspicious and requires retesting.

[0102] The cut-off values ​​are shown in Table 7. The distribution of OD450 values ​​and the threshold values ​​for 20 negative serum samples are displayed. Based on the experimental results, the mean OD450 nm value for negative serum samples detected by the established indirect ELISA method was 0.133, and the standard deviation was 0.02. Therefore, when the OD450 nm value of the tested sample is ≥ [value missing], the cut-off value is determined. A positive result is defined as X+3SD=0.193; the tested sample OD450nm ≤ When X+2SD=0.173, it is judged as negative; when the OD450nm of the tested sample is between 0.173 and 0.193, it is judged as suspicious.

[0103] Table 7

[0104] Indirect ELISA specific assay To verify the specificity of this system, an optimized ELISA method was used to simultaneously detect mouse anti-gC recombinant protein polyclonal antibody, as well as mouse antisera for infectious bursal virus (IBV) and Newcastle disease virus (NDV). If only the anti-gC serum well showed a positive high OD value, and the OD values ​​of the other serum wells were all below the cut-off value, it indicated that the method did not cross-react with other pathogens and had good specificity. Results are shown below. Figure 12 This indicates that the established ELISA method has good specificity.

[0105] Indirect ELISA Sensitivity Test Mouse anti-gC recombinant protein polyclonal antibody-positive serum with known titers was serially diluted 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, for a total of seven gradients. The established ELISA method was used for detection, and the average OD450nm of each gradient was recorded in three replicates. The cutoff value was used to determine the detectable positive dilution, and the sensitivity was evaluated. Results are shown below. Figure 13 This indicates that the established ELISA method has a detection sensitivity of 1:12800.

[0106] Repeatability test (1) Intra-batch reproducibility: On the same batch of coated ELISA plates, 3 positive serum samples and 3 negative serum samples were selected respectively, and 3 replicate wells were set for each sample for detection. The average OD450 value was calculated. X), standard deviation (SD), and coefficient of variation (CV%).

[0107] (2) Inter-batch repeatability: Using three ELISA plates coated under the same conditions but from different batches, the same six positive serum samples were tested. The mean OD450nm value and the inter-batch coefficient of variation were calculated.

[0108] If the coefficient of variation (CV) within and between batches is less than 10%, it indicates that the indirect ELISA method has good stability and reproducibility.

[0109] The intra-batch repeatability results are shown in Table 8, indicating good intra-batch repeatability.

[0110] Table 8

[0111] The results of batch-to-batch repeatability are shown in Table 9, indicating good batch-to-batch repeatability.

[0112] Table 9

[0113] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An indirect ELISA kit for detecting duck plague virus antibodies, characterized in that, An enzyme-labeled plate containing an antigen; the antigen is a gC recombinant protein, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 1, characterized in that, The method for preparing the enzyme-labeled plate coated with the antigen includes the following steps: The recombinant gC protein was added to the wells at a concentration of 0.5 μg / mL and incubated at 37°C for 2 hours, then at 4°C for 14 hours. The supernatant was removed, and the plate was washed three times with PBST and the supernatant was discarded. 5% skim milk powder was added, and the plate was blocked at 37°C for 2 hours to obtain the enzyme-labeled plate coated with the antigen.

3. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 1, characterized in that, The indirect ELISA kit also includes enzyme-labeled secondary antibody, coating solution, washing solution, chromogenic solution, stop solution, negative standard and positive standard.

4. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 3, characterized in that, The enzyme-labeled secondary antibody is HRP-labeled goat anti-mouse IgG.

5. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 3, characterized in that, The coating solution is a carbonate buffer solution with a pH of 9.

6.

6. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 3, characterized in that, The washing solution is a PBS buffer containing 0.05% Tween-20.

7. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 3, characterized in that, The colorimetric solution is a TMB solution.

8. The indirect ELISA kit for detecting duck plague virus antibodies according to claim 3, characterized in that, The terminating solution is 2M H2SO4.

9. The use of the indirect ELISA kit according to any one of claims 1-8 in detecting duck plague virus antibodies for non-disease diagnostic purposes.

10. A method for detecting duck plague virus antibodies for non-disease diagnostic purposes, characterized in that, Includes the step of detecting duck plague virus antibodies using the indirect ELISA kit according to any one of claims 1-9: Add 100 µL of negative standard, positive standard, and test sample solution to the enzyme-labeled plate coated with antigen, respectively, and incubate at 37°C for 45 min. After incubation, rinse 3 times with washing solution. Add 1:5000 diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 45 min. After incubation, rinse 3 times with washing buffer. Add the colorimetric solution to each reaction well and incubate at 37°C in the dark for 15 minutes; Add stop solution to each reaction well and measure the absorbance at 450 nm using an ELISA reader. Result interpretation: When OD450nm≥0.193, it is judged as positive, that is, the test sample contains duck plague virus antibodies; when OD450nm≤0.173, it is judged as negative, that is, the test sample does not contain duck plague virus antibodies.

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