Anti-S11 type riemerella anatipestifer monoclonal antibody and blocking ELISA antibody detection kit and application thereof

Abstract

The invention discloses an anti-S11 type riemerella anatipestifer monoclonal antibody as well as a blocking ELISA antibody detection kit and application thereof, and belongs to the technical field of poultry immunology. The monoclonal antibody provided by the invention comprises a heavy chain and a light chain, the heavy chain comprises VHCDR1, VHCDR2 and VHCDR3 of which the amino acid sequences are as shown in SEQ ID NO: 1-3, and the light chain comprises VLCDR1 of which the amino acid sequence is as shown in SEQ ID NO: 4, VLCDR2 of which the amino acid sequence is LVS and VLCDR3 of which the amino acid sequence is as shown in SEQ ID NO: 5; the blocking ELISA antibody detection kit prepared from the monoclonal antibody can specifically detect the level of a riemerella anatipestifer serotype 11 (S11 type) antibody in a sample, has no cross reactivity with riemerella anatipestifer S1 type, riemerella anatipestifer S2 type, riemerella anatipestifer S5 type, riemerella anatipestifer S6 type and riemerella anatipestifer S14 type, avian pathogenic escherichia coli, avian pasteurella multocida and salmonella, and is high in sensitivity and specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of avian immunology, and particularly to a monoclonal antibody against Riemerella anatipestifer serotype S11, a blocking ELISA antibody detection kit therefor, and applications thereof. Background Art

[0002] The information disclosed in the background art of the present invention is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Riemerella anatipestifer (RA) is a Gram-negative bacterium that can infect domestic ducks, geese, turkeys and other poultry, causing infectious serositis. The disease is characterized by fibrinous pericarditis, perihepatitis, airsacculitis and meningitis, and can cause the death of poultry in severe cases. It is one of the important pathogens endangering the poultry industry worldwide. At present, it is known that there are at least 21 serotypes of Riemerella anatipestifer, and there is no cross-immune protection between different serotypes. The serotype classification of some strains is not clear. In recent years, the host range and epidemic characteristics of this pathogen have shown a complex trend, and there is an urgent need to monitor the disease dynamics through precise diagnostic techniques.

[0004] At present, bacterial isolation and identification are the most effective and reliable methods for diagnosing Riemerella anatipestifer, but it takes a long time and the procedure is cumbersome; the slide agglutination test is one of the classic methods for identifying this bacterium and is also the most effective means for determining the bacterial serotype. However, due to the complex serotype of this bacterium and the lack of commercialization of standard sera, the application and popularization of this method are severely restricted. The polymerase chain reaction (PCR) diagnostic technique has been widely used in the detection of Riemerella anatipestifer, but the current PCR detection methods cannot distinguish serotypes, and have the disadvantages of cumbersome process, long time consumption, and the need for special instruments. Therefore, there is an urgent need to establish a method with high speed, sensitivity and specificity to meet the needs of rapid clinical detection.

[0005] Enzyme-Linked Immunosorbent Assays (ELISA) is a mature detection technique that has been widely used in various fields. This method is fast, convenient and highly sensitive, and does not require special instrument equipment. However, there is no commercial kit for the detection of specific antibodies against Riemerella anatipestifer serotype 11 (S11). Existing methods have problems such as insufficient antigen purity, low detection sensitivity or complex operation steps, and it is difficult to meet the accurate diagnostic needs of clinical S11 infections.

[0006] Therefore, the development of an antibody detection technology for Riemerella anatipestifer serotype S11, especially a standardized kit based on the ELISA principle, is of great significance for the rapid screening of infections of this serotype, epidemiological monitoring, and evaluation of vaccine immunization effects. Summary of the Invention

[0007] In view of this, the present invention provides a monoclonal antibody against Riemerella anatipestifer serotype S11, a blocking ELISA antibody detection kit thereof, and applications. The ELISA antibody detection kit developed based on the monoclonal antibody against Riemerella anatipestifer serotype S11 has the advantages of good specificity, high sensitivity, and simple detection, and can be applied to the specific detection of Riemerella anatipestifer serotype S11.

[0008] In the first aspect, the present invention provides a monoclonal antibody against Riemerella anatipestifer serotype S11. The monoclonal antibody against Riemerella anatipestifer serotype S11 comprises a heavy chain and a light chain. The heavy chain contains VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences shown in SEQ ID NO: 1 - 3, and the light chain contains VLCDR1 with an amino acid sequence shown in SEQ ID NO: 4, VLCDR2 with an amino acid sequence of LVS, and VLCDR3 with an amino acid sequence shown in SEQ ID NO: 5.

[0009] Preferably, the amino acid sequence of the heavy chain of the monoclonal antibody against Riemerella anatipestifer serotype S11 is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain of the monoclonal antibody against Riemerella anatipestifer serotype S11 is shown in SEQ ID NO: 7.

[0010] In the second aspect, the present invention provides an application of the above - mentioned monoclonal antibody against Riemerella anatipestifer serotype S11, and the application is for the preparation of products for detecting Riemerella anatipestifer serotype S11.

[0011] Preferably, the products include ELISA detection kits, colloidal gold detection kits, or chemiluminescent immunoassay kits.

[0012] In the third aspect, the present invention provides a blocking ELISA antibody detection kit, which includes an enzyme - linked reaction plate and an enzyme - labeled antibody; wherein, the enzyme - linked reaction plate is coated with Riemerella anatipestifer serotype S11, and the enzyme - labeled antibody is the above - mentioned monoclonal antibody against Riemerella anatipestifer serotype S11 labeled with an enzyme.

[0013] Preferably, the blocking ELISA antibody detection kit further comprises substrate solution A, substrate solution B and termination solution; the substrate solution A is a tetramethylbenzidine solution at a concentration of 0.1 - 0.3 mg / mL, the substrate solution B is a citrate phosphate buffer solution containing urea peroxide, and the volume ratio of the substrate solution A to the substrate solution B is 1:(0.9 - 1.1); the termination solution is a hydrofluoric acid solution with a volume fraction of 0.2 - 0.4%.

[0014] Preferably, the blocking ELISA antibody detection kit further comprises sample diluent and 10-fold concentrated washing solution; the sample diluent is a phosphate buffer solution containing Tween-20 with a volume fraction of 0.04 - 0.06%; the 10-fold concentrated washing solution is a phosphate buffer solution containing Tween-20 with a volume fraction of 0.4 - 0.6%.

[0015] Preferably, the enzyme of the enzyme-labeled antibody includes any one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme or malate dehydrogenase.

[0016] Preferably, the blocking ELISA antibody detection kit further comprises positive control serum and negative control serum; the positive control serum is positive serum prepared from ducks immunized with inactivated vaccine of Riemerella anatipestifer serotype S11; the negative control serum is the serum of ducks without Riemerella anatipestifer serotype S11 pathogen and not immunized.

[0017] Fourthly, the present invention provides an application of the above-mentioned blocking ELISA antibody detection kit, and the application is for preparing a reagent for detecting a sample to be tested for Riemerella anatipestifer serotype S11 infection or vaccination.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects: The present invention immunizes mice with Riemerella anatipestifer serotype S11 as an antigen, prepares a monoclonal antibody against Riemerella anatipestifer serotype S11 with good specificity and high sensitivity. The developed blocking ELISA antibody detection reagent for Riemerella anatipestifer serotype S11 can specifically detect the level of antibodies against Riemerella anatipestifer serotype 11 in a sample, with high sensitivity and strong specificity, and has no cross-reactivity with Riemerella anatipestifer serotypes S1, S2, S5, S6 and S14, avian pathogenic Escherichia coli, avian Pasteurella and Salmonella, and has broad application prospects. Detailed Embodiments

[0019] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0020] The technical solution of the present invention will be further described below in conjunction with specific embodiments. There are no special restrictions on the sources of the reagents used in the following embodiments, and commercially available products well-known to those skilled in the art can be used.

[0021] Example 1 This example provides the preparation of a monoclonal antibody against Riemerella anatipestifer serotype S11.

[0022] 1. Antigen preparation: Take the glycerol bacteria of Riemerella anatipestifer serotype S11 stored at -70°C (self-preserved in the laboratory) and inoculate it into tryptone soy agar (TSA) medium (purchased from Haibo Biotechnology Co., Ltd., Qingdao High-tech Industrial Park), and culture it at 37°C for 24 h. Pick a single colony suspected of being Riemerella anatipestifer and perform three-zone streaking on the TSA plate, and culture it anaerobically at 37°C for 18 h. After stably passing three generations on the solid medium, use the self-made diagnostic serum of Riemerella anatipestifer serotype S11 to perform an agglutination test on the single colonies on the TSA plate to verify the serotype of the revived Riemerella anatipestifer. Pick a single colony and inoculate it into TSA medium for overnight culture for 15 h, add sterile PBS to scrape the bacteria, centrifuge the scraped bacterial liquid at 4000 rpm for 8 min, discard the supernatant, resuspend it with an appropriate amount of sterile PBS, and then centrifuge it at 4000 rpm for 8 min. After washing twice, resuspend it with an appropriate amount of sterile PBS, measure the concentration of the bacterial liquid, and adjust the concentration of the bacterial suspension to 1.0×10 9 CFU / mL.

[0023] 2. Animal immunization: Mix the bacterial suspension obtained in step 1 with Freund's complete adjuvant at a volume ratio of 1:1, fully emulsify it, and immunize six-week-old BALB / c mice subcutaneously at multiple points. The immunization dose is 1×10 8 CFU / mouse. Thereafter, immunize at the same dose every two weeks. After 3 immunizations, collect blood from the mouse tail vein and detect the antibody titer of the serum by indirect ELISA. Select the mouse with the highest serum titer and boost immunize it with the primary immunization dose without adjuvant.

[0024] 3. Cell fusion, screening and subcloning: On the 3rd day after boost immunization, harvest the immune spleen cells of the mouse. Fusion of SP2 / 0 cells and immune spleen cells is carried out at a ratio of 1:10 in terms of quantity. When the cell clones to be fused grow to cover 1 / 2 of the area of the cell well bottom, detect the cell culture supernatant by indirect ELISA, select the cell wells with strong positive results in the ELISA detection, and perform 3 subclonings by the limiting dilution method. Select the strong positive cell wells with only single clone growth and perform large-scale culture.

[0025] 4. Determination of the titer of the monoclonal antibody: Culture the monoclonal positive hybridoma cells and detect the titer of the monoclonal antibody to finally determine the positive hybridoma cell line used.

[0026] 5. Monoclonal Antibody Sequencing: Collect hybridoma cells (number greater than 10 6 ), and send them to Sangon Biotech (Shanghai) Co., Ltd. for subsequent construction and sequencing to obtain the gene sequencing results.

[0027] The heavy chain of the monoclonal antibody contains VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1 - 3; the light chain contains VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, VLCDR2 with an amino acid sequence of LVS, and VLCDR3 with an amino acid sequence as shown in SEQ ID NO: 5. The amino acid sequence of the heavy chain is as shown in SEQ ID NO: 6, and the amino acid sequence of the light chain is as shown in SEQ ID NO: 7.

[0028] VHCDR1 (SEQ ID NO: 1): GYTFTSYV.

[0029] VHCDR2 (SEQ ID NO: 2): INPYNDGT.

[0030] VHCDR3 (SEQ ID NO: 3): ARGGMIVWFAY.

[0031] VLCDR1 (SEQ ID NO: 4): QSLLYSNGKTY.

[0032] VLCDR2: LVS.

[0033] VLCDR3 (SEQ ID NO: 5): VQGTHFPYT.

[0034] Heavy chain (SEQ ID NO: 6): EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGMIVWFAYWGQGTLVTVSA.

[0035] Light chain (SEQ ID NO: 7): DIVITQSPLTLSVTIGQPASISCKSSQSLLYSNGKTYLTWLLQRPGQSPKRLIYLVSILDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPYTFGGGSKLEIK.

[0036] Example 2 This embodiment provides a method for preparing and using a blocking ELISA antibody detection kit.

[0037] 1. Components: (1) Sample diluent, which is phosphate buffer solution (0.01M, pH 7.4) containing 0.05% Tween-20. The preparation method is as follows: Take KH 2 PO 4 0.2 g, NaHPO 4 ·12H 2 O 2.9 g, NaCl 8 g, 0.5 mL Tween-20, mix and make up the volume to 1000 mL with double-distilled water.

[0038] (2) Concentrated washing solution (10×), which is phosphate buffer solution (0.1M, pH 7.4) containing 0.5% Tween-20. The preparation method is as follows: Take KH 2 PO 4 2 g, NaHPO 4 ·12H 2 O 29 g, NaCl 80 g, 5 mL Tween-20, mix and make up the volume to 1000 mL with double-distilled water.

[0039] (3) Chromogenic solution, which is tetramethylbenzidine (TMB)-hydrogen peroxide urea solution. The preparation method is as follows: Take 200 mg tetramethylbenzidine (TMB), dissolve it in 100 mL absolute ethanol, and make up the volume to 1000 mL with double-distilled water to obtain substrate solution A; Take citric acid (C 6 H 8 O 7 ·H 2 O) 21 g, disodium hydrogen phosphate anhydrous (Na 2 HPO 4 ) 28.2 g, 6.4 mL 0.75% hydrogen peroxide urea, make up the volume to 1000 mL with double-distilled water, and adjust the pH value to 5.0 to obtain substrate solution B; Mix equal volumes of substrate solution A and substrate solution B, which is the chromogenic solution.

[0040] (3) Stop solution, which is 0.31% hydrofluoric acid solution. The preparation method is as follows: Take 0.31 mL hydrofluoric acid and make up the volume to 100 mL with double-distilled water.

[0041] (4) Positive serum control: The positive serum of Riemerella anatipestifer serotype S11 obtained by screening is diluted 1:50 with the sample diluent (OD 450 ≥1.0), add penicillin and streptomycin with a final concentration of 1000 U / mL, and filter sterilize it to be used as a positive control.

[0042] (5)Negative serum control: The negative serum of Riemerella anatipestifer serotype S11 obtained by screening was diluted 1:50 with the sample diluent (OD 450 ≤0.20), added with penicillin and streptomycin at a final concentration of 1000 U / mL, and sterile filtered to serve as the negative control.

[0043] (6)Enzyme conjugate solution: Horseradish peroxidase-labeled goat anti-mouse secondary antibody (HRP Goat Anti-Mouse IgG(H+L), purchased from Abbkine Scientific Co., Ltd.) was diluted 4000-fold with 1× washing solution.

[0044] 2. Determination of the optimal working concentration of the antigen by indirect ELISA method using mouse positive serum Riemerella anatipestifer serotype S11 was serially diluted 10-fold with ELISA coating buffer (1×, pH 9.6, purchased from Solarbio Life Sciences Co., Ltd.) to 10 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 CFU / mL at 4 dilution ratios, and the ELISA reaction plate was coated at a dose of 100 μL / well.

[0045] Mouse positive serum control and negative serum control were serially diluted 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 with the sample diluent respectively; indirect ELISA was performed; chromogenic solution was added for color development, and the reaction was terminated with the termination solution; the OD value at a light wavelength of 450 nm was measured, and the results are shown in Table 1.

[0046] Select the antigen dilution concentration corresponding to the well with an OD 450 value close to 1.0 and the largest ratio of positive OD value / negative OD value as the optimal coating concentration of the antigen. The optimal coating concentration of Riemerella anatipestifer serotype S11 is 10 7 CFU / mL.

[0047] Table 1 OD 450 values at different coating concentrations and dilution ratios of bacterial solutions

[0048] 3. Usage method of the blocking ELISA antibody detection kit (1)All reagents should be equilibrated at room temperature for 20 min before use and stored for standby; liquid reagents should be gently shaken and mixed evenly before use; (2)Before use, the concentrated washing solution should be completely dissolved and mixed at room temperature. The concentrated washing solution (10×) was diluted 10-fold with distilled water to obtain the washing buffer (1×); (3) Dilute the serum to be tested 50-fold with the sample diluent in the dilution plate. Use the negative and positive control sera directly. (4) Take out the required strips, put the remaining strips into an aluminum foil bag, seal it, and store it at 4°C. Add the negative serum, positive serum, and diluted serum to be tested to the ELISA reaction plate coated with the antigen (Riemerella anatipestifer serotype S11), 100 μL per well. Set 1 well for each serum to be tested, and set 2 wells for both the negative control and the positive control. (5) Place it in an incubator at 37°C for 60 min; discard the reaction solution, add 300 μL of washing buffer (1×) to each well, wash the plate continuously 3 times and then pat it dry. Avoid drying the micro-wells during the intervals. (6) Add 100 μL of the anti-Riemerella anatipestifer serotype S11 monoclonal antibody of Example 1 to each well; place it in an incubator at 37°C for 60 min; discard the reaction solution, add 300 μL of washing buffer to each well, wash the plate continuously 3 times and then pat it dry. (7) Add 100 μL of the enzyme conjugate solution to each well; place it in an incubator at 37°C for 30 min. (8) Discard the enzyme conjugate solution, add 300 μL of washing buffer to each well, wash the plate continuously 3 times and then pat it dry. (9) Add 100 μL of the chromogenic solution to each well, mix well by shaking, and react in the dark at room temperature for 15 min. (10) Add 50 μL of the chromogenic stop solution to each well, mix well by shaking to terminate the reaction, and measure the results within 15 minutes. (11) Conditions for a valid test: The OD 450 values of the negative control should all be ≥ 1.0, and the blocking rate of the positive control wells should be ≥ 50%. (12) Judgment: Measure the OD values of each well on an ELISA reader. Blocking rate = 100% × (1 - OD of the sample 450 value / mean OD of the negative control 450 ). Determine the presence or absence of antibodies by calculating the blocking rate of each sample. Negative: blocking rate ≤ 40%; Positive: blocking rate ≥ 60%; Suspected: 40% < blocking rate < 60%.

[0049] Example 3 This example provides a specific detection of the blocking ELISA antibody detection kit constructed in Example 2.

[0050] The blocking ELISA antibody detection kit constructed in Example 2 was used to detect S11 type Riemerella anatipestifer positive serum, S1 type Riemerella anatipestifer positive serum, S2 type Riemerella anatipestifer positive serum, S5 type Riemerella anatipestifer positive serum, S6 type Riemerella anatipestifer positive serum, S14 type Riemerella anatipestifer positive serum, duck Escherichia coli positive serum, duck Pasteurella positive serum, and duck Salmonella positive serum. The test results are shown in Table 2. The mean OD of the negative serum control 450 was 2.07, and the mean OD of the positive serum control 450 was 0.126. The results showed that except for the S11 type Riemerella anatipestifer positive serum which was positive, the rest were negative, indicating that the detection kit had no cross-reaction with S1 type Riemerella anatipestifer positive serum, S2 type Riemerella anatipestifer positive serum, S5 type Riemerella anatipestifer positive serum, S6 type Riemerella anatipestifer positive serum, duck Escherichia coli positive serum, duck Pasteurella positive serum, and duck Salmonella positive serum.

[0051] Table 2 Specificity test results

[0052] Example 4 This example provided a sensitivity test for the blocking ELISA antibody detection kit constructed in Example 2.

[0053] The S11 type Riemerella anatipestifer positive serum and negative serum were diluted at 1:200 - 1:3200 respectively, and the blocking ELISA detection was carried out under the optimal reaction conditions for the rest of the conditions. The test results are shown in Table 3.

[0054] Table 3 Sensitivity test results

[0055] The results showed that when the S11 type Riemerella anatipestifer positive serum was diluted to 1:800, its OD 450 was 0.27 and the blocking rate was 84.25%. It was proved that the blocking ELISA antibody detection kit of the present invention had high sensitivity.

[0056] Example 5 This example provided a repeatability test for the blocking ELISA antibody detection kit constructed in Example 2.

[0057] The blocking ELISA antibody detection kit constructed in Example 2 was used to detect 6 positive sera of Riemerella anatipestifer serotype S11 respectively. Each sample was detected 3 times, and its coefficient of variation CV% (CV = S.D. / X×100%, S.D.: standard deviation, X: arithmetic mean) was measured, as shown in Table 4. The results showed that the maximum coefficient of variation was 1.05% and the minimum was 0.23%. The coefficients of variation of the 6 sera were all small, indicating that the detection kit had good repeatability.

[0058] Table 4 Results of repeatability experiment

[0059] Example 6 This example provides the practical application of the blocking ELISA antibody detection kit constructed in Example 2.

[0060] The samples tested in this example were 42 duck sera at different stages after immunization with the S11 type Riemerella anatipestifer vaccine (16 samples at 2 weeks after the first immunization, 16 samples at 1 week after the second immunization, and 10 samples at 2 weeks after the second immunization). The blocking ELISA antibody detection kit constructed in Example 2 was used for detection, and 31 positive sera were detected. The results were in line with the antibody growth and decline law, indicating that the blocking ELISA antibody detection kit established in the present invention was applicable to the clinical detection of antibodies against Riemerella anatipestifer serotype S11.

[0061] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various modifications and changes. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An anti-S11 Riemerella anatipestifer monoclonal antibody, characterized in that: The anti-S11 type Riemerella anatipestifer monoclonal antibody includes a heavy chain and a light chain, wherein the heavy chain comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NOs: 1-3, and the light chain comprises VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, VLCDR2 with an amino acid sequence of LVS and VLCDR3 with an amino acid sequence as shown in SEQ ID NO:

5.

2. The anti-S11 Riemerella anatipestifer monoclonal antibody according to claim 1, characterized in that: The amino acid sequence of the heavy chain of the anti-S11 Riemerella anatipestifer monoclonal antibody is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain of the anti-S11 Riemerella anatipestifer monoclonal antibody is shown in SEQ ID NO:

7.

3. The use of the anti-S11 Riemerella anatipestifer monoclonal antibody according to claim 1 or 2, characterized in that: The application is the application in preparing a product for detecting Riemerella anatipestifer S11 type.

4. The use according to claim 3, characterized in that The products include ELISA detection kits, colloidal gold detection kits or chemiluminescent immunoassay kits.

5. A blocking ELISA antibody detection kit, characterized in that: It comprises an enzyme-linked reaction plate and an enzyme-labeled antibody; wherein the enzyme-linked reaction plate is coated with S11 Riemerella anatipestifer, and the enzyme-labeled antibody is the anti-S11 Riemerella anatipestifer monoclonal antibody according to claim 1 or 2 labeled with an enzyme.

6. The blocking ELISA antibody detection kit according to claim 5, characterized in that: The blocking ELISA antibody detection kit also includes substrate solution A, substrate solution B and stop solution; the substrate solution A is a 0.1-0.3 mg / mL tetramethylbenzidine solution, the substrate solution B is a citric acid phosphate buffer solution containing urea hydrogen peroxide, and the volume ratio of the substrate solution A to the substrate solution B is 1:(0.9-1.1); the stop solution is a hydrofluoric acid solution with a volume fraction of 0.2-0.4%.

7. The blocking ELISA antibody detection kit according to claim 5, characterized in that: The blocking ELISA antibody detection kit also includes a sample diluent and a 10-fold concentrated washing solution; the sample diluent is a phosphate buffer containing 0.04-0.06% Tween-20 by volume; the 10-fold concentrated washing solution is a phosphate buffer containing 0.4-0.6% Tween-20 by volume.

8. The blocking ELISA antibody detection kit according to claim 5, characterized in that: The enzyme of the enzyme-labeled antibody includes any one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme or malate dehydrogenase.

9. The blocking ELISA antibody detection kit according to claim 5, characterized in that: The blocking ELISA antibody detection kit also includes positive control serum and negative control serum; the positive control serum is the positive serum prepared by immunizing ducks with S11 Riemerella inactivated vaccine; the negative control serum is the serum of ducks without S11 Riemerella pathogens and without immunization.

10. Use of the blocking ELISA antibody detection kit according to any one of claims 5 to 9, characterized in that: The application is the application in preparing a reagent for detecting a sample to be tested for infection or vaccination with Riemerella anatipestifer S11.

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