Double-antibody sandwich detection kit for duck tembusu virus as well as detection method and application of double-antibody sandwich detection kit

Abstract

The invention is applicable to the field of immunodetection, and provides a double-antibody sandwich detection kit for duck tembusu virus as well as a detection method and application thereof, and the double-antibody sandwich detection kit comprises the steps of preparation of a mouse monoclonal antibody, preparation of a duck polyclonal antibody, enzyme-linked immunosorbent assay (ELISA) detection procedure and the like. Wherein the capture antibody is a mouse monoclonal antibody of an anti-TMUV-prM protein, and is derived from a hybridoma cell strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.45754; the detection antibody is an anti-TMUV duck polyclonal antibody. The invention provides a kit for detecting TMUV (TetramethylUV) prM protein and / or TMUV and a detection method. The kit and the method have excellent specificity, sensitivity and repeatability, can be used for rapidly and accurately detecting TMUV, and are suitable for being popularized in clinical detection.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology and biological products, and particularly relates to a double-antibody sandwich detection kit for duck Tembusu virus, a detection method and an application thereof. Background Art

[0002] Tembusu virus disease (TMUV) is an infectious disease caused by the Tembusu virus (TMUV), characterized by viral encephalitis in ducklings and growing ducks and decreased egg production in laying ducks. In recent years, the disease has become increasingly pathogenic in chickens and geese. The disease spreads rapidly and widely, causing significant economic losses to the poultry industry.

[0003] TMUV, a member of the genus Orthoflavivirus, is a single-stranded, positive-sense RNA virus. Its genome contains an approximately 11 kb open reading frame (ORF). The virus encodes three structural proteins: capsid protein (C), pre-membrane protein (prM), and envelope protein (E), as well as seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). The prM protein is the precursor of the outer membrane protein (M) in mature viral particles and has a molecular weight of approximately 19 kDa. The prM protein acts as a bridge between the virus and the host cell, providing anchoring. Located on the surface of the viral particle, the prM protein is easily recognized by antibodies and is a key protein in detecting TMUV.

[0004] The prM protein has high homology among different TMUV strains and the antigenic epitope is stable. Using monoclonal antibodies against prM protein as capture antibodies can ensure specific recognition and efficient capture of TMUV; duck polyclonal antibodies against TMUV can recognize multiple epitopes on the TMUV surface with strong affinity.

[0005] Currently, TMUV diagnosis relies primarily on virus isolation and identification, RT-PCR, and virus neutralization assays. These tests rely on specialized laboratories and require lengthy experimental cycles. Therefore, a method that is simple to operate, low-cost, and suitable for large-scale pathogen detection is needed. The double-antibody sandwich ELISA method, which utilizes both capture and detection antibodies for dual screening, offers advantages such as high sensitivity and strong specificity and is commonly used for accurate viral serological testing. Summary of the Invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide a double antibody sandwich detection kit for duck Tembusu virus and its detection method and application. The detection method provided by the present invention can be used for rapid and accurate detection of TMUV and is suitable for promotion in clinical testing.

[0007] To achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides a double antibody sandwich kit for detecting duck Tembusu virus, comprising: an ELISA plate coated with anti-Tembusu virus prM protein monoclonal antibody, and anti-Tembusu virus duck positive serum.

[0008] The monoclonal antibody against TMUV-prM protein is obtained by secretion from a hybridoma cell line with a deposit number of CGMCC No. 45754. The hybridoma cell line is deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms.

[0009] The ELISA plate coated with anti-Tembusu virus prM protein monoclonal antibody is prepared by the following steps: (1) Coating antibody: The monoclonal antibody against Tembusu virus prM protein secreted by the hybridoma cell line with the deposit number CGMCC No. 45754 was used as the capture antibody to coat the plate in a 96-well ELISA plate, incubate overnight at 4°C, wash with PBST containing 0.05% Tween, and spin dry; (2) Blocking: Block with 5% skim milk powder diluted with PBST, incubate at 37°C, wash, and spin dry.

[0010] The method for preparing the anti-Tembusu virus duck positive serum comprises the following steps: The virus liquid inactivated by TMUV was prepared into inactivated vaccine to inoculate SPF ducks. After the booster immunization treatment, serum was collected and the serum titer was determined. The positive serum with a titer of not less than 1:3,200 was collected.

[0011] Preferably, the virus solution is 1*10 5 TCID 50 / mL inoculated SPF ducks, inoculated with 0.2mL.

[0012] The second aspect of the present invention provides the use of the double-antibody sandwich kit in conducting an epidemiological survey of duck Tembusu virus.

[0013] In a third aspect, the present invention provides a method for detecting duck Tembusu virus, which uses the above-mentioned double-antibody sandwich kit.

[0014] The coating dosage of the monoclonal antibody against Tembusu virus prM protein in the ELISA plate is 100 ng / well.

[0015] The detection method comprises the following steps: (1) Add Tembusu virus supernatant to the ELISA plate coated with anti-Tembusu virus prM protein monoclonal antibody, incubate, wash, and dry, add 1:200 diluted anti-Tembusu virus duck positive serum, incubate, wash, and dry; (2) Add 1:2000 diluted enzyme-labeled goat anti-duck antibody, incubate, wash, and dry; (3) Add TMB substrate colorimetric solution, incubate at 37°C in the dark, then add stop solution to terminate the colorimetric reaction. Measure the absorbance at 450nm. Determine the positive and negative critical values ​​based on the absorbance at 450nm and make judgments on the results.

[0016] In step (3), the positive / negative critical value = negative sample OD 450nm The mean value + standard deviation 3SD is used to obtain the positive and negative critical value. 450nm A value above 0.505 was considered positive.

[0017] Beneficial effects of the present invention: The present invention provides a kit and method for detecting TMUV prM protein and / or TMUV using a mouse monoclonal antibody against TMUV-prM protein as a capture antibody and a duck polyclonal antibody against TMUV as a detection antibody. The detection method provided by the present invention has excellent specificity, sensitivity, and repeatability for duck Tembusu virus detection, can be used for rapid and accurate TMUV detection, and is suitable for promotion in clinical testing. DETAILED DESCRIPTION

[0018] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0019] As mentioned above, duck Tembusu virus spreads rapidly and widely, causing huge economic losses to the poultry industry. It is of great significance to establish a simple, low-cost and large-scale detection method for Tembusu virus.

[0020] Based on this, the present invention provides a double-antibody sandwich detection kit for duck Tembusu virus, as well as its detection method and application. The method includes the preparation of mouse monoclonal antibodies, duck polyclonal antibodies, and an ELISA detection procedure. The capture antibody is a mouse monoclonal antibody against the TMUV-prM protein, derived from the hybridoma cell line with the deposit number CGMCC No. 45754; the detection antibody is a duck polyclonal antibody against TMUV. The kit and detection method for detecting TMUV prM protein and / or TMUV provided by the present invention have excellent specificity, sensitivity, and reproducibility, can be used for rapid and accurate TMUV detection, and are suitable for promotion in clinical testing.

[0021] The following detailed description is for illustrative purposes only and is intended to provide further explanation of the present invention, rather than to limit the scope of the present invention.

[0022] The Tembusu virus strain TMUV-SDDZ (MH764606) and the hybridoma cell line secreting monoclonal antibodies against duck Tembusu virus (deposit number CGMCC No. 45754) used in the following examples were all deposited by the Avian Disease Laboratory of Shandong Agricultural University.

[0023] The HRP-labeled goat anti-duck antibody used in the following examples was purchased from KPL Company; TMB colorimetric solution was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd. Six-week-old BALB / c female mice used in the following examples were purchased from Jinan Pengyue Animal Co., Ltd.

[0024] Example 1: Concentration and purification of TMUV C6 / 36 cells preserved in the laboratory were revived and subcultured. When the cells were cultured to a cell density of 90%, the TMUV-SDDZ strain was inoculated and the DMEM medium containing 2% FBS was replaced. The culture was continued for 5-7 days. When the cytopathic effect reached more than 70%, the virus solution was collected and repeatedly frozen and thawed three times at -80°C. The supernatant was centrifuged at 12,000 r / min for 30 min, and the supernatant was taken and filtered using a 0.22 μm filter to remove impurities. The virus solution was concentrated using a 100 KDa ultrafiltration tube. The concentrated virus solution was ultracentrifuged at 32,000 r / min for 2 h, the supernatant was discarded, and PBS was added to dissolve the precipitate to obtain purified Tembusu virus (TMUV). TMUV was used as the detection antigen and the TCID was determined. 50 Then, the virus solution was diluted to 10 9 TCID 50 / mL and store it in aliquots at -80℃ for future use.

[0025] Example 2: Preparation of monoclonal antibodies against duck Tembusu virus prM protein 1. Preparation of Ascites 6-week-old female BALB / c mice in good condition were intraperitoneally injected with 0.5 mL of sterilized liquid paraffin. 14 days later, 10 hybridoma cells with the deposit number of CGMCC No. 45754 were intraperitoneally injected. 6 / 0.5mL. After 7 to 10 days, the mouse abdomen was obviously swollen. Before the mouse was on the verge of death, ascites was slowly extracted using a syringe.

[0026] 2. Purification of Antibodies The collected ascites was centrifuged at 4°C and 12,000 rpm for 10 minutes. After carefully removing the supernatant, the supernatant was collected using a pipette, taking care not to aspirate the precipitate at the bottom. The ascites was filtered through a 0.45 μm filter and purified using Protein G affinity chromatography to obtain the monoclonal antibody TMUV-prM. The purification method is as follows: Load 1 mL of Protein G resin into the chromatography column. Add 5 mL of wash buffer to equilibrate the column and allow the wash buffer to flow out. Dilute the ascites fluid 1:1 with wash buffer and add it to the column. Allow the fluid to flow out slowly at a flow rate of 0.2-0.5 mL / min. Repeat loading 2-3 times. After loading, wash the column with 30 mL of wash buffer. After the wash buffer has drained away, elute the antibody with 10 mL of elution buffer at a flow rate of 0.5 mL / min and collect the eluted product. Add neutralization buffer to the eluate to adjust the pH of the eluate containing the eluted product to 7.4. Transfer the eluate to a dialysis bag and dialyze overnight at 4°C against 1 L of 0.01 M Tris-HCl buffer (pH 7.4). Dilute the purified mAb to 1 μg / mL in 1× carbonate buffer (pH 9.6).

[0027] Example 3: Preparation of anti-duck Tembusu virus polyclonal antibodies The TMUV obtained in Example 1 was inactivated using a 2.0‰ formaldehyde solution and mixed with Tween-80 at a volume ratio of 96:4 to prepare an aqueous phase. The oil phase and the aqueous phase were emulsified in proportion to prepare a TMUV inactivated vaccine. 200 μL of the TMUV inactivated vaccine (1*10 5 TCID 50 / mL) was inoculated into 10-day-old SPF ducks, and a booster immunization was given 7 days later with the same dose. Serum was subsequently collected every 3 days and the serum titer was determined by indirect ELISA. Positive sera with a titer of no less than 1:3,200 were collected.

[0028] Example 4: Establishment and optimization of TMUV double antibody sandwich ELISA method 1. Establish the optimal dilution concentration of capture antibody and detection antibody The optimal working concentrations of the capture and detection antibodies were determined using a checkerboard assay. A 1 mg / mL anti-TMUV-prM monoclonal antibody was diluted in carbonate buffer to 0.5, 1, 2, and 5 μg / mL, respectively. The ELISA plate was then loaded horizontally, with 100 μL added to each well, resulting in 50, 100, 200, and 500 ng of monoclonal antibody coated per well. Coating was incubated overnight at 4°C and washed three times with PBST. Each well was blocked with 200 μL of 5% skim milk, incubated at 37°C for 1 hour, and washed three times with PBST.

[0029] The virus solution prepared in Example 1 was added to an ELISA plate, and parallel wells were set up, with 100 μL per well, incubated at 37°C for 1 hour, and the plate was washed three times with PBST. The detection antibody, duck polyclonal antibody against Tembusu virus, was added to a 96-well plate at 100 μL / well after dilution, incubated at 37°C for 1 hour, and washed three times with PBST. TMB colorimetric solution was added and incubated at room temperature in the dark for 15 minutes. Finally, 50 μL of stop solution (2M H2SO4) was added to terminate the reaction, and the OD value was measured by a microplate reader. 450nm The test results are shown in Table 1 and Table 2.

[0030] Table 1: Matrix Titration OD 450nm Value test results Note: P indicates positive samples, N indicates negative samples Table 2: P / N values ​​of square array titration Negative sample OD 450 The value is lower than 0.3, the positive sample OD 450 Value / negative sample OD 450 The value corresponding to the well with the largest value (P / N value) determines the optimal coating concentration of the capture antibody and the optimal dilution concentration of the detection antibody.

[0031] As shown in Tables 1 and 2, the optimal coating concentration of the capture antibody is 1 μg / mL, and the optimal dilution concentration of the detection antibody is 1:200.

[0032] 2. Optimal coating temperature and time for capture antibody ELISA plates were coated with 1 μg / mL capture antibody diluted in carbonate buffer. Different coating conditions were set: 37°C for 1 hour, 2 hours, 3 hours, and 4°C overnight, while all other conditions remained the same. Testing revealed that the plate coated overnight at 4°C had the highest P / N value, indicating that overnight at 4°C was the optimal coating condition.

[0033] Table 3: Optimal coating time for capture antibodies 3. Determination of optimal sealing conditions According to the determined optimal conditions, anti-TMUV-prM monoclonal antibody at a concentration of 1 μg / mL was used as the capture antibody at 4°C overnight, and duck polyclonal antibody against Tembusu virus at a dilution concentration of 1:200 was used as the detection antibody. 1% skim milk, 1% BSA, 5% skim milk, and 5% BSA were used as blocking solutions, respectively. Each blocking solution was used for 60 minutes. Each group was repeated 3 times, and double antibody sandwich ELISA was performed to read the OD value. 450nm The results are shown in Table 4, and the optimal blocking solution was determined to be 5% skim milk.

[0034] Table 4: Optimal blocking conditions 4. Determination of the optimal antigen detection time 10 5 TCID 50 / mL TMUV was used as the detection antigen, and the antibody was incubated for 0.5h, 1h, 2h, and 3h, respectively. As shown in Table 5, the P / N ratios for 1h and 2h were not much different. Considering the detection speed, 1h was selected as the optimal antigen detection time.

[0035] Table 5: Optimal antigen detection time 5. Working concentration of enzyme-labeled antibody According to the optimal conditions determined above, the HRP-labeled goat anti-duck IgG (H+L) enzyme-labeled secondary antibody was set at three working concentrations of 1:1000, 1:2000, and 1:3000, respectively. As shown in Table 6, the P / N of 1:1000 and 1:2000 is not much different. Considering the cost, the optimal working concentration of the enzyme-labeled antibody is 1:2000.

[0036] Table 6 Optimal working concentration of enzyme-labeled antibodies 6. Determination of critical value The double antibody sandwich ELISA method of Example 4 was used to detect 30 TMUV negative samples, and the OD values ​​of the 30 samples were calculated. 450nm The mean (Mean) and standard deviation (SD) are used to calculate the critical value of positive and negative samples according to the formula (positive and negative critical value = Mean + 3SD). The result is 0.3011 + 3 × 0.068 = 0.505 Example 5: Sensitivity testing The virus content of Example 1 was 10 9 TCID 50 The Tembusu virus solution was diluted 10 times with a dilution ratio of 10 / mL, and viruses of different dilutions were extracted. The virus was detected using the method in Example 4 and the optimized optimal detection conditions. The results are as follows: As shown in Table 7. As can be seen from Table 7, the minimum detectable virus content of this method is 10TCID 50 / mL.

[0037] Table 7: Sensitivity Tests The above description is only a preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification made within the spirit and principles of the present application shall be Equivalent replacements, improvements, etc. should all be included in the protection scope of this application.

Claims

1. A double antibody sandwich kit for detecting duck Tembusu virus, characterized in that: The double antibody sandwich test kit comprises: an enzyme labeling plate coated with anti-Tembusu virus prM protein monoclonal antibody and anti-Tembusu virus duck positive serum.

2. The double antibody sandwich kit according to claim 1, characterized in that The monoclonal antibody against TMUV-prM protein is obtained by secretion from a hybridoma cell line with a deposit number of CGMCC No.45754.

3. The double antibody sandwich kit according to claim 1 or 2, characterized in that The ELISA plate coated with anti-Tembusu virus prM protein monoclonal antibody is prepared by the following steps: (1) Coating antibody: The monoclonal antibody against Tembusu virus prM protein secreted by the hybridoma cell line with the deposit number CGMCC No. 45754 was used as the capture antibody to coat the plate in a 96-well ELISA plate, incubate overnight at 4°C, wash with PBST containing 0.05% Tween, and spin dry; (2) Blocking: Block with 5% skim milk powder diluted with PBST, incubate at 37°C, wash, and spin dry.

4. The double antibody sandwich kit according to claim 1, characterized in that The method for preparing the anti-Tembusu virus duck positive serum comprises the following steps: The inactivated virus liquid after TMUV inactivation was used to prepare inactivated vaccine and inoculated SPF ducks. After booster immunization, serum was collected and the serum titer was determined. The positive serum with a titer of not less than 1:3,200 was collected.

5. Use of the double antibody sandwich kit according to claim 1 in conducting an epidemiological survey of duck Tembusu virus.

6. A method for detecting duck Tembusu virus, characterized in that: The detection method uses the double antibody sandwich kit according to claim 1.

7. The detection method according to claim 6, characterized in that The coating dosage of the monoclonal antibody against Tembusu virus prM protein in the ELISA plate is 100 ng / well.

8. The detection method according to claim 6, characterized in that The detection method comprises the following steps: (1) Add Tembusu virus supernatant to the ELISA plate coated with anti-Tembusu virus prM protein monoclonal antibody, incubate, wash, and dry, add 1:200 diluted anti-Tembusu virus duck positive serum, incubate, wash, and dry; (2) Add 1:2000 diluted enzyme-labeled goat anti-duck antibody, incubate, wash, and dry; (3) Add TMB substrate colorimetric solution, incubate at 37°C in the dark, then add stop solution to terminate the colorimetric reaction. Measure the absorbance at 450nm. Determine the positive and negative critical values ​​based on the absorbance at 450nm and make judgments on the results.

9. The detection method according to claim 8, characterized in that In step (3), the positive / negative critical value = negative sample OD 450nm The mean value + standard deviation 3SD is used to obtain the positive and negative critical value. 450nm A value above 0.505 was considered positive.

Images