Monoclonal antibody against Riemerella anatipestifer S11 and its blocking ELISA antibody detection kit and application

Abstract

The present invention discloses an anti-S11 Riemerella anatipestifer monoclonal antibody and a blocking ELISA antibody detection kit and application thereof, belonging to the technical field of avian immunology. The monoclonal antibody provided by the present invention comprises a heavy chain and a light chain, wherein the heavy chain comprises VHCDR1, VHCDR2 and VHCDR3 as shown in the amino acid sequence of SEQ ID NO:1-3, and the light chain comprises VLCDR1 as shown in the amino acid sequence of SEQ ID NO:4, VLCDR2 with the amino acid sequence of LVS and VLCDR3 as shown in the amino acid sequence of SEQ ID NO:5; the blocking ELISA antibody detection kit prepared from the monoclonal antibody can specifically detect the level of antibodies to Riemerella anatipestifer serotype 11 (S11 type) in a sample, and has no cross-reactivity with Riemerella anatipestifer S1, S2, S5, S6 and S14 types, avian pathogenic Escherichia coli, avian Pasteurella and Salmonella, with high sensitivity and strong specificity.

Description

Technical Field

[0001] The present invention relates to the technical field of poultry immunology, in particular to an anti-S11 Riemerella anatipestifer monoclonal antibody and a blocking ELISA antibody detection kit and application thereof. Background Art

[0002] The information disclosed in the background of the invention is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Riemerella anatipestifer (RA) is a Gram-negative bacillus that infects domestic ducks, geese, turkeys, and other poultry, causing an infectious serositis characterized by fibrinous pericarditis, perihepatitis, air sac inflammation, and meningitis. In severe cases, it can lead to death. It is a major pathogen endangering the poultry industry worldwide. Currently, at least 21 serotypes of R. anatipestifer are known, and there is a lack of cross-immunity between serotypes. Some strains have unclear serotype classification. In recent years, the host range and epidemiological characteristics of this pathogen have become increasingly complex, necessitating the use of precise diagnostic technologies to monitor disease dynamics.

[0004] Currently, bacterial isolation and identification is the most effective and reliable method for diagnosing Riemerella anatipestifer, but it is time-consuming and cumbersome. The slide agglutination test is a classic method for identifying the bacterium and the most effective means of determining bacterial serotypes. However, the complex serotypes of the bacterium and the lack of commercially available standard sera severely restrict its application and widespread adoption. Polymerase chain reaction (PCR) diagnostic technology has been widely used for the detection of Riemerella anatipestifer, but current PCR methods cannot distinguish between serotypes and are cumbersome, time-consuming, and require specialized equipment. Therefore, a rapid, sensitive, and specific method is urgently needed to meet the needs of rapid clinical testing.

[0005] Enzyme-linked immunosorbent assays (ELISAs) are a mature detection technology that has been widely used in various fields. This method is rapid, convenient, highly sensitive, and does not require special instruments or equipment. However, there are no commercially available kits 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, making it difficult to meet the clinical needs for accurate diagnosis of S11 infection.

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

[0007] In view of this, the present invention provides an anti-S11 Riemerella anatipestifer monoclonal antibody and its blocking ELISA antibody detection kit and application. The ELISA antibody detection kit developed based on the anti-S11 Riemerella anatipestifer monoclonal antibody has the advantages of good specificity, high sensitivity and simple detection, and can be used for the specific detection of S11 Riemerella anatipestifer.

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

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

[0010] In a second aspect, the present invention provides a use of the above-mentioned anti-S11 Riemerella anatipestifer monoclonal antibody, wherein the use is in preparing a product for detecting S11 Riemerella anatipestifer.

[0011] Preferably, the product comprises an ELISA detection kit, a colloidal gold detection kit or a chemiluminescence immunoassay kit.

[0012] In a third aspect, the present invention provides a blocking ELISA antibody detection kit, comprising 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 above-mentioned anti-S11 Riemerella anatipestifer monoclonal antibody labeled with an enzyme.

[0013] Preferably, the blocking ELISA antibody detection kit further comprises substrate solution A, substrate solution B and a 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%.

[0014] Preferably, the blocking ELISA antibody detection kit further comprises 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.

[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 a positive control serum and a negative control serum; the positive control serum is a positive serum prepared after immunizing ducks with an inactivated vaccine of Riemerella anatipestifer S11; and the negative control serum is a serum from ducks that are free of the Riemerella anatipestifer S11 pathogen and have not been immunized.

[0017] In a fourth aspect, the present invention provides a use of the above-mentioned blocking ELISA antibody detection kit, wherein the use is in preparing a reagent for detecting a sample to be tested for infection or vaccination with Riemerella anatipestifer S11.

[0018] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0019] The present invention uses S11 Riemerella anatipestifer as an antigen to immunize mice, and prepares an anti-S11 Riemerella anatipestifer monoclonal antibody with good specificity and high sensitivity. The S11 Riemerella anatipestifer blocking ELISA antibody detection reagent developed based on the antibody can specifically detect the level of antibodies to Riemerella anatipestifer serotype 11 in a sample with high sensitivity and strong specificity, and has no cross-reactivity with S1, S2, S5, S6 and S14 Riemerella anatipestifer, avian pathogenic Escherichia coli, avian Pasteurella and Salmonella, and has broad application prospects. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. 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 invention belongs.

[0021] The technical solution of the present invention is further described below with reference to specific examples. The present invention has no particular limitation on the sources of the reagents used in the following examples, and commercially available products known to those skilled in the art can be used.

[0022] Example 1

[0023] This example provides the preparation of monoclonal antibodies against Riemerella anatipestifer S11.

[0024] 1. Antigen Preparation: Inoculate tryptone soy agar (TSA) medium (purchased from Haibo Biotechnology Co., Ltd., Qingdao High-Tech Industrial Park) with glycerol culture of R. anatipestifer S11 stored at -70°C (laboratory stock) and incubate at 37°C for 24 hours. Pick a single colony suspected of R. anatipestifer and streak it three times onto a TSA plate. Incubate anaerobically at 37°C for 18 hours. After three stable passages on solid culture medium, perform agglutination tests on the single colony on the TSA plate using a homemade R. anatipestifer S11 diagnostic serum to verify the serotype of the recovered R. anatipestifer. A single colony was picked and inoculated into TSA medium for overnight culture for 15 h. Sterile PBS was added to scrape the bacteria. The scraped bacterial solution was centrifuged at 4000 rpm for 8 min, the supernatant was discarded, and the suspension was resuspended with an appropriate amount of sterile PBS. The suspension was centrifuged at 4000 rpm for 8 min. After washing twice, the suspension was resuspended with an appropriate amount of sterile PBS. The concentration of the bacterial suspension was measured and adjusted to 1.0 × 10 9 CFU / mL.

[0025] 2. Animal immunization: Take the bacterial suspension from step 1 and mix it with Freund's complete adjuvant in a volume ratio of 1:1, fully emulsify it, and subcutaneously immunize six-week-old BALB / c mice at multiple points. The immunization dose is 1×10 8 CFU / mouse. Immunize once every two weeks with the same dose. After three immunizations, collect blood from the tail vein of the mice and test the antibody titer of the serum using indirect ELISA. Select the mouse with the highest serum titer and boost it with the primary dose without adjuvant.

[0026] 3. Cell fusion, screening, and subcloning: On the third day after booster immunization, immune spleen cells of mice were harvested and SP2 / 0 cells were fused with immune spleen cells at a ratio of 1:10. When the fused cell clones grew to cover 1 / 2 of the bottom area of ​​the cell wells, the cell culture supernatant was detected by indirect ELISA. The cell wells with strong positive results by ELISA were selected and subcloned three times by limiting dilution. The strongly positive cell wells with only a single clone were selected for expansion culture.

[0027] 4. Monoclonal antibody titer determination: Culture monoclonal positive hybridoma cells, perform monoclonal antibody titer detection, and ultimately determine the positive hybridoma cell line used.

[0028] 5. Monoclonal antibody sequence determination:

[0029] Collect hybridoma cells (number greater than 10 6 ), and sent it to Shanghai Sangon Biotechnology Co., Ltd. for subsequent construction and sequencing to obtain the gene sequencing results.

[0030] The heavy chain of the monoclonal antibody comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences set forth in SEQ ID NOs: 1-3; the light chain comprises VLCDR1 with an amino acid sequence set forth in SEQ ID NO: 4, VLCDR2 with an amino acid sequence of LVS, and VLCDR3 with an amino acid sequence set forth in SEQ ID NO: 5. The amino acid sequence of the heavy chain is set forth in SEQ ID NO: 6, and the amino acid sequence of the light chain is set forth in SEQ ID NO: 7.

[0031] VHCDR1 (SEQ ID NO: 1): GYTFTSYV.

[0032] VHCDR2 (SEQ ID NO: 2): INPYNDGT.

[0033] VHCDR3 (SEQ ID NO: 3): ARGGMIVWFAY.

[0034] VLCDR1 (SEQ ID NO: 4): QSLLYSNGKTY.

[0035] VLCDR2:LVS.

[0036] VLCDR3 (SEQ ID NO: 5): VQGTHFPYT.

[0037] Heavy chain (SEQ ID NO: 6):

[0038] EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARGGMIVWFAYWGQGTLVTVSA.

[0039] Light chain (SEQ ID NO: 7):

[0040] DIVITQSPLTLSVTIGQPASISCKSSQSLLYSNGKTYLTWLLQRPGQSPKRLIYLVSILDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCVQGTHFPYTFGGGSKLEIK.

[0041] Example 2

[0042] This embodiment provides a method for preparing and using a blocking ELISA antibody detection kit.

[0043] 1. Components:

[0044] (1) Sample diluent: phosphate buffer (0.01 M, pH 7.4) containing 0.05% Tween-20. The preparation method is as follows: take 0.2 g of KH2PO4, 2.9 g of NaHPO4·12H2O, 8 g of NaCl, and 0.5 mL of Tween-20, mix them, and add double-distilled water to make up to 1000 mL.

[0045] (2) Concentrated washing solution (10×) is phosphate buffer (0.1 M, pH 7.4) containing 0.5% Tween-20. The preparation method is as follows: take 2 g of KH2PO4, 29 g of NaHPO4·12H2O, 80 g of NaCl, and 5 mL of Tween-20, mix them, and add double distilled water to make up to 1000 mL.

[0046] (3) The color developing solution is tetramethylbenzidine (TMB)-hydrogen peroxide urea solution, and the preparation method is as follows:

[0047] Dissolve 200 mg of tetramethylbenzidine (TMB) in 100 mL of anhydrous ethanol and dilute to 1000 mL with double-distilled water to obtain substrate solution A.

[0048] Take 21 g of citric acid (C6H8O7·H2O), 28.2 g of anhydrous disodium hydrogen phosphate (Na2HPO4), 6.4 mL of 0.75% urea hydrogen peroxide, dilute to 1000 mL with double distilled water, and adjust the pH to 5.0 to obtain substrate solution B;

[0049] Mix equal volumes of substrate solution A and substrate solution B to make the color developing solution.

[0050] (3) Stop solution: 0.31% hydrofluoric acid solution. The preparation method is as follows: take 0.31 mL of hydrofluoric acid and dilute to 100 mL with double distilled water.

[0051] (4) Positive serum control: The positive serum of Riemerella anatipestifer type S11 obtained by screening was diluted 1:50 with sample diluent (OD 450≥1.0), add penicillin-streptomycin to a final concentration of 1000 U / mL and sterile filter as a positive control.

[0052] (5) Negative serum control: The negative serum of Riemerella anatipestifer S11 obtained by screening was diluted 1:50 with the sample diluent (OD 450 ≤0.20), add penicillin-streptomycin to a final concentration of 1000 U / mL and sterile filter as a negative control.

[0053] (6) Enzyme conjugate solution: Horseradish peroxidase-labeled goat anti-mouse secondary antibody (HRP Goat Anti-Mouse IgG (H+L), purchased from Abotek Biotechnology Co., Ltd.), diluted 4000 times with 1× washing solution.

[0054] 2. Determine the optimal working concentration of the antigen using the mouse positive serum indirect ELISA method

[0055] The S11 Riemerella anatipestifer was diluted 10-fold to 10 using ELISA coating solution (1×, pH 9.6, purchased from Solaibao Biotechnology Co., Ltd.). 5 CFU / mL, 10 6 CFU / mL, 10 7 CFU / mL, 10 8 ELISA reaction plates were coated with 4 dilutions of CFU / mL at a dose of 100 μL / well.

[0056] Mouse positive serum control and negative serum control were serially diluted with sample diluent at 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800, respectively; indirect ELISA assay was performed; color development solution was added to develop the color, and the reaction was terminated with stop solution; the OD value at a wavelength of 450 nm was measured. The results are shown in Table 1.

[0057] Select OD 450 The antigen dilution concentration corresponding to the well with the value close to 1.0 and the largest ratio of positive OD value to negative OD value was taken as the optimal coating concentration of the antigen. The optimal coating concentration of Riemerella anatipestifer S11 was 10 7 CFU / mL.

[0058] Table 1 OD at different bacterial coating concentrations and dilution ratios 450 value

[0059]

[0060] 3. How to use the blocking ELISA antibody test kit

[0061] (1) All reagents should be equilibrated at room temperature for 20 minutes before use; liquid reagents should be gently shaken to mix thoroughly before use;

[0062] (2) The concentrated washing solution was completely dissolved and mixed at room temperature before use. The concentrated washing solution (10×) was diluted 10 times with distilled water to obtain the washing buffer solution (1×).

[0063] (3) Dilute the serum to be tested 50-fold with sample diluent in a dilution plate, and use the negative and positive control sera directly;

[0064] (4) Take out the required strips, seal the remaining strips in an aluminum foil bag, and store at 4°C. Add the negative serum, positive serum, and diluted test serum to the ELISA reaction plate coated with antigen (S11 Riemerella anatipestifer), 100 μL / well, set up 1 well for each test serum, and 2 wells each for negative control and positive control;

[0065] (5) Place in a 37°C incubator and react for 60 minutes; discard the reaction solution, add 300 μL of washing buffer (1×) to each well, wash the plate three times in succession, and pat dry. Avoid drying the microwells during the intervals.

[0066] (6) Add 100 μL of the monoclonal antibody against Riemerella anatipestifer S11 of Example 1 to each well; place in a 37°C incubator and react for 60 min; discard the reaction solution, add 300 μL of washing buffer to each well, wash the plate three times, and then pat dry;

[0067] (7) Add 100 μL of enzyme conjugate solution to each well; place in a 37°C incubator and react for 30 minutes;

[0068] (8) Discard the enzyme binding solution, add 300 μL of washing buffer to each well, wash the plate three times and pat dry;

[0069] (9) Add 100 μL of color development solution to each well, shake and mix, and react at room temperature in the dark for 15 minutes;

[0070] (10) Add 50 μL of color stop solution to each well, shake and mix to terminate the reaction, and measure the results within 15 minutes;

[0071] (11) Test conditions: Negative control OD 450 The values ​​should all be ≥1.0, and the blocking rate of the positive control wells should be ≥50%;

[0072] (12) Determination: Measure the OD value of each well on the microplate reader. Blocking rate = 100% × (1 - sample OD 450 Value / negative control OD 450 The presence of antibodies was determined by calculating the blocking rate of each sample. Negative: blocking rate ≤ 40%; Positive: blocking rate ≥ 60%; Suspicious: 40% < blocking rate < 60%.

[0073] Example 3

[0074] This example provides specific detection of the blocking ELISA antibody detection kit constructed in Example 2.

[0075] The blocking ELISA antibody detection kit constructed in Example 2 was used to detect S11 Riemerella anatipestifer positive serum, S1 Riemerella anatipestifer positive serum, S2 Riemerella anatipestifer positive serum, S5 Riemerella anatipestifer positive serum, S6 Riemerella anatipestifer positive serum, S14 Riemerella anatipestifer positive serum, duck Escherichia coli positive serum, duck Pasteurella positive serum, duck Salmonella positive serum, and the test results are shown in Table 2. The negative serum control OD 450 The mean value was 2.07, and the positive serum control OD 450 The mean value was 0.126. The results showed that except for the positive serum of Riemerella anatipestifer S11, the rest were negative, indicating that the detection kit had no cross reaction with the positive serum of Riemerella anatipestifer S1, Riemerella anatipestifer S2, Riemerella anatipestifer S5, Riemerella anatipestifer S6, duck Escherichia coli, duck Pasteurella, and duck Salmonella.

[0076] Table 2 Specificity test results

[0077]

[0078] Example 4

[0079] This example provides a sensitivity test of the blocking ELISA antibody detection kit constructed in Example 2.

[0080] The positive and negative sera of Riemerella anatipestifer S11 were diluted 1:200 to 1:3200, respectively, and the rest of the conditions were used for blocking ELISA detection according to the optimal reaction conditions. The test results are shown in Table 3.

[0081] Table 3 Sensitivity test results

[0082]

[0083] The results showed that when the positive serum of Riemerella anatipestifer S11 was diluted to 1:800, its OD 450 The blocking rate was 0.27, and the blocking rate was 84.25%, which proved that the blocking ELISA antibody detection kit of the present invention has high sensitivity.

[0084] Example 5

[0085] This example provides a repeatability test of the blocking ELISA antibody detection kit constructed in Example 2.

[0086] The blocking ELISA antibody detection kit constructed in Example 2 was used to test six Riemerella anatipestifer S11-positive sera. Each sample was tested three times, and the coefficient of variation (CV%) (CV = SD / X × 100%, where SD is standard deviation and X is the arithmetic mean) was determined, as shown in Table 4. The results showed that the maximum coefficient of variation was 1.05% and the minimum was 0.23%. The coefficient of variation for all six sera was small, demonstrating that the detection kit has excellent reproducibility.

[0087] Table 4 Repeatability test results

[0088]

[0089] Example 6

[0090] This example provides the practical application of the blocking ELISA antibody detection kit constructed in Example 2.

[0091] The samples tested in this example were 42 duck sera at different stages after immunization with the S11 Riemerella anatipestifer vaccine (16 samples 2 weeks after the first immunization, 16 samples 1 week after the second immunization, and 10 samples 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 consistent with the antibody growth and decline law, which shows that the blocking ELISA antibody detection kit established by the present invention is suitable for clinical application in the detection of S11 Riemerella anatipestifer antibodies.

[0092] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A monoclonal antibody against Riemerella anatipestifer S11, characterized in that: The anti-S11 type Riemerella anatipestifer monoclonal antibody includes a heavy chain and a light chain, the heavy chain comprises VHCDR1, VHCDR2 and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 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 use of the anti-S11 Riemerella anatipestifer monoclonal antibody according to claim 1, characterized in that: The application is the application in preparing a product for detecting Riemerella anatipestifer S11 type.

3. The use according to claim 2, characterized in that The products include ELISA detection kits, colloidal gold detection kits or chemiluminescence immunoassay kits.

4. 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 labeled with an enzyme.

5. The blocking ELISA antibody detection kit according to claim 4, wherein The blocking ELISA antibody detection kit also includes substrate solution A, substrate solution B and a 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%.

6. The blocking ELISA antibody detection kit according to claim 4, wherein 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.

7. The blocking ELISA antibody detection kit according to claim 4, wherein The enzyme of the enzyme-labeled antibody includes any one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme or malate dehydrogenase.

8. The blocking ELISA antibody detection kit according to claim 4, wherein The blocking ELISA antibody detection kit also includes a positive control serum and a negative control serum; the positive control serum is the positive serum prepared by immunizing ducks with an inactivated S11 Riemerella vaccine; the negative control serum is the serum of ducks without the S11 Riemerella pathogen and without immunization.

9. Use of the blocking ELISA antibody detection kit according to any one of claims 4 to 8, 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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