Universal duck rimer's bacillus monoclonal antibody, detection kit and application thereof

Abstract

This invention relates to the field of avian immunology, and more particularly to a universal monoclonal antibody against Riemerella anatipestifer, a detection kit, and their applications. Specifically, this invention successfully designed a universal monoclonal antibody against Riemerella anatipestifer, and the detection kit developed based on this monoclonal antibody can rapidly detect the level of Riemerella anatipestifer serum antibodies in samples with high sensitivity and strong specificity. Furthermore, it shows no cross-reactivity with Enterococcus faecalis, Salmonella typhi, Escherichia coli, and Salmonella enteritidis, thus it can be used for rapid screening, epidemiological monitoring, and evaluation of vaccine immunization efficacy against Riemerella anatipestifer infection, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of avian immunology technology, and in particular to a universal monoclonal antibody against Riemerella anatipestifer, a detection kit, and their applications. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Riemerella anatipestifer ( Riemerella anatipestifer Rasogenic serositis (RA) is a Gram-negative bacillus with a wide host spectrum, infecting various poultry such as ducks, geese, and turkeys, causing infectious serositis. The typical pathological features of this disease include fibrinous pericarditis, perihepatitis, air sacculitis, and meningitis. It often leads to outbreaks of contagious diseases in poultry, with infection and mortality rates exceeding 75%, making it a significant pathogenic factor hindering the development of the global poultry industry.

[0004] At least 21 serotypes of Riemerella anatipestifer have been identified, and there is no cross-immunoprotective effect between different serotypes. Furthermore, the serotype classification of some strains remains unclear. This complexity significantly increases the difficulty of treating and controlling RA infection. In recent years, cases of Riemerella anatipestifer infection in chickens have appeared in multiple locations, indicating that the host range of this pathogen is gradually expanding and its epidemiological characteristics are becoming increasingly complex. Therefore, it is urgent to rely on precise diagnostic technologies to conduct dynamic disease monitoring and provide technical support for prevention and control efforts.

[0005] In current RA detection technologies, bacterial isolation and identification is the gold standard for diagnosis, boasting high reliability. However, this method is cumbersome and time-consuming, making it difficult to meet the needs of rapid diagnosis. Slide agglutination tests, a classic method for identifying RA and determining its serotype, are limited by the complexity of serotypes, the lack of commercially available standard sera, and low detection sensitivity, significantly hindering their application and promotion. While polymerase chain reaction (PCR) technology is widely used in RA detection, existing PCR methods cannot effectively distinguish between different serotypes and also suffer from cumbersome procedures, long processing times, and reliance on specialized equipment. Furthermore, existing enzyme-linked immunosorbent assays (ELISA) technologies and products suffer from poor specificity, insufficient sensitivity, or difficulty in universally detecting different RA serotypes, failing to meet the precise detection needs of clinical scenarios such as rapid RA infection screening and vaccination efficacy evaluation. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a universal monoclonal antibody against Riemerella anatipestifer, a detection kit, and their applications. Specifically, the present invention successfully designed a universal monoclonal antibody against Riemerella anatipestifer, and the detection kit developed based on this monoclonal antibody can rapidly detect the level of Riemerella anatipestifer serum antibodies in samples, with high sensitivity and strong specificity. This invention is based on the above research results.

[0007] Specifically, the technical solution of the present invention is as follows:

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

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

[0010] A second aspect of the present invention provides the application of the above-mentioned universal Riemerella anatipestifer monoclonal antibody in the preparation of products for detecting Riemerella anatipestifer.

[0011] In the application described, the product can be a detection kit. The detection kit can be an ELISA detection kit, a colloidal gold detection kit, or a chemiluminescent immunoassay kit. In one specific embodiment of the present invention, the detection kit is an ELISA detection kit.

[0012] The serotypes of *Rimerella anatipestifer* include, but are not limited to, S1, S2, S5, S6, S7, S10, S11, and S14.

[0013] A third aspect of the present invention provides a detection kit comprising at least the above-mentioned universal Riemerella anatipestifer monoclonal antibody, wherein the universal Riemerella anatipestifer monoclonal antibody is used as a primary antibody.

[0014] Furthermore, the detection kit is an ELISA detection kit, specifically a blocking ELISA antibody detection kit. This detection kit uses the antibody in the sample to be tested to compete with the universal Riemerella anatipestifer monoclonal antibody in the kit to bind to the Riemerella anatipestifer antigen on the enzyme-linked reaction plate, and finally determines the content of the antibody to be tested based on the color intensity.

[0015] Therefore, the test kit also includes an enzyme-linked reaction plate and an enzyme-labeled antibody; wherein the enzyme-linked reaction plate is coated with Riemerella anatipestifer antigen, and the enzyme-labeled antibody is an enzyme-labeled goat anti-mouse secondary antibody.

[0016] The enzyme in the enzyme-labeled antibody is any one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, or malate dehydrogenase, preferably horseradish peroxidase.

[0017] The detection kit also includes a chromogenic solution and a stop solution; the chromogenic solution is 3,3',5,5'-tetramethylbenzidine (TMB) chromogenic solution; the stop solution is ELISA stop solution (2 mol / L sulfuric acid solution).

[0018] The test kit also includes a sample diluent and a 10-fold concentrated wash buffer; the sample diluent is a phosphate buffer containing 0.04-0.06% Tween-20 by volume; the 10-fold concentrated wash buffer is a phosphate buffer containing 0.4-0.6% Tween-20 by volume.

[0019] The test kit also includes a positive control serum and a negative control serum; the positive control serum is a universal positive serum for Riemerella anatipestifer, and the negative control serum is a negative serum for Riemerella anatipestifer.

[0020] A fourth aspect of the present invention provides the use of the above-described test kit in the preparation of reagents for detecting Riemerella anatipestifer infection or vaccination.

[0021] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0022] The above-mentioned technical solution uses *Riemerella anatipestifer* as an antigen to immunize mice, and prepares a universal monoclonal antibody against *Riemerella anatipestifer* with good specificity and high sensitivity. Based on this, a universal ELISA antibody detection reagent for blocking *Riemerella anatipestifer* can specifically detect the level of serum antibodies against *Riemerella anatipestifer* in samples. It has high sensitivity and strong specificity, and has no cross-reactivity with *Enterococcus faecalis*, *Salmonella typhi*, *Escherichia coli*, and *Salmonella enteritidis*. Therefore, it can be used for rapid screening, epidemiological monitoring, and evaluation of vaccine immunization efficacy against *Riemerella anatipestifer* infection, and has broad application prospects. Detailed Implementation

[0023] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0024] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0025] The following examples further illustrate the present invention, but do not constitute a limitation thereof. It should be understood that these examples are for illustrative purposes only and not for limiting the scope of the invention. Unless otherwise specified, the materials, reagents, instruments, and methods used in the following examples are all conventional materials, reagents, instruments, and methods in the art and are commercially available. The *Riemerella anatipestifer* SD22-32-D strain selected in the examples has been described in the paper "Emerging...". Riemerella anatipestifer infection in chickens: pathogenic characteristics and hostimmune response profiles” (doi.org / 10.1016 / j.psj.2025.105687).

[0026] Example 1

[0027] This embodiment provides the preparation of a universal monoclonal antibody against Riemerella anatipestifer.

[0028] 1. Antigen Preparation: Glycerol-containing *Riemerella anatipestifer* strain SD22-32-D, stored at -70℃, was inoculated onto tryptophan-soybean agar (TSA) medium (purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.) and incubated at 37℃ for 24 hours. Single colonies suspected to be *Riemerella anatipestifer* were picked and streaked in three zones on TSA plates, then anaerobically cultured at 37℃ for 18 hours. After three stable passages on solid medium, agglutination tests were performed on single colonies on TSA plates using self-prepared diagnostic sera containing all known serotypes of *Riemerella anatipestifer* to verify the agglutination reaction of the revived *Riemerella anatipestifer* with all known serotypes. Single colonies were picked and inoculated into TSA medium and cultured overnight for 15 h. Bacteria were scraped off with sterile PBS, centrifuged at 4000 rpm for 8 min, the supernatant was discarded, and the bacterial suspension was resuspended in sterile PBS. The suspension was then centrifuged again at 4000 rpm for 8 min, washed twice, and resuspended in sterile PBS. The bacterial concentration was measured, and the concentration of the bacterial suspension was adjusted to 1.0 × 10⁻⁶. 9 CFU / mL.

[0029] 2. Animal immunization: Mix the bacterial suspension from step 1 with Freund's complete adjuvant at a 1:1 volume ratio, emulsify thoroughly, and subcutaneously immunize six-week-old BALB / c mice at multiple sites. The immunization dose is 1×10⁻⁶. 8 CFU / mouse was administered, followed by immunization with the same dose every two weeks. After three immunizations, blood was collected from the tail vein of the mice, and the serum antibody titer was detected by indirect ELISA. Mice with the highest serum titer were selected and boosted with a primary dose without adjuvant.

[0030] 3. Cell fusion and screening, subcloning: On the 3rd day after booster immunization, mouse immune spleen cells were harvested. 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 well, the cell culture supernatant was detected by indirect ELISA. Cell wells with strong positive ELISA were selected, and subcloning was performed 3 times using the limiting dilution method. Cell wells with strong positive cell clones growing with only a single clone were selected for expansion culture.

[0031] 4. Monoclonal antibody titer determination: Culture monoclonal positive hybridoma cells, perform monoclonal antibody titer testing, and finally determine the positive hybridoma cell line used.

[0032] 5. Monoclonal antibody sequencing:

[0033] Collect hybridoma cells (number greater than 10) 6 The sample was sent to Shanghai Sangon Biotech Co., Ltd. for further construction and sequencing, and the gene sequencing results were obtained.

[0034] The heavy chain of the monoclonal antibody comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3; the light chain comprises VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, VLCDR2 with an amino acid sequence of DTS, and VLCDR3 with an amino acid sequence as shown in SEQ ID NO: 5. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain is shown in SEQ ID NO: 7.

[0035] VHCDR1 (SEQ ID NO: 1): GYAFTNYL

[0036] VHCDR2 (SEQ ID NO: 2): INPGTGGT

[0037] VHCDR3 (SEQ ID NO: 3): TKGTMINRFDF

[0038] VLCDR1 (SEQ ID NO: 4): QGISNY

[0039] VLCDR2: DTS

[0040] VLCDR3 (SEQ ID NO: 5): QQYSKIPPT

[0041] Heavy chain (SEQ ID NO: 6): QVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWIKQGPGQGLEWIGVINPGTGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSGDSAVFFCTKGTMINRFDFWGQGTTLTVSS

[0042] Light chain (SEQ ID NO: 7): DIQMTQTTSSLSASLGDRVTISCSASQGISNYLNWYQQKPDGTVKLLIYDTSSLQSGVPSRFSGSGSGTDYSLTITNLEPEDIATYYCQQYSKIPPTFGGGTKLEIK

[0043] Example 2

[0044] This embodiment provides a method for preparing and using a blocking ELISA antibody detection kit.

[0045] 1. Components:

[0046] (1) The sample dilution solution is a phosphate buffer (0.01M, pH 7.4) containing 0.05% Tween-20. The preparation method is as follows: take 0.2g of KH2PO4, 2.9g of NaHPO4·12H2O, 8g of NaCl, and 0.5mL of Tween-20, mix them, and add double-distilled water to make up to 1000mL.

[0047] (2) Concentrated washing solution (10×) is a phosphate buffer (0.1M, pH 7.4) containing 0.5% Tween-20. The preparation method is as follows: take 2g of KH2PO4, 29g of NaHPO4·12H2O, 80g of NaCl, 5 mL of Tween-20, mix them, and add double-distilled water to make up to 1000 mL.

[0048] (3) Universal monoclonal antibody against Riemerella anatipestifer, preparation is described in Example 1.

[0049] (4) Colorimetric solution: Commercially available TMB single-component colorimetric solution.

[0050] (5) Stop solution: Commercially available ELISA stop solution (2 mol / L sulfuric acid solution).

[0051] (6) Positive serum control: Serum prepared by immunizing SPF chickens with Riemerella anatipestifer (SD22-32-D strain) was used as positive serum. It was diluted 1:20 with sample diluent, and streptomycin with a final concentration of 1000 U / mL was added. The serum was then filtered aseptically and used as a positive control.

[0052] (7) Negative serum control: The negative serum of Riemerella anatipestifer obtained by screening was diluted 1:20 with sample diluent, and streptomycin with a final concentration of 1000 U / mL was added. The serum was then filtered aseptically and used as a negative control.

[0053] (8) Enzyme conjugate: Horseradish peroxidase-labeled goat anti-mouse secondary antibody (HRP Goat Anti-Mouse IgG (H+L), purchased from Aibote Biotechnology Co., Ltd.) was diluted 5000 times with 1× washing buffer.

[0054] 2. Instructions for use of the blocking ELISA antibody detection kit

[0055] (1) All reagents should be equilibrated at room temperature for 20 minutes before use; liquid reagents should be gently shaken and mixed before use.

[0056] (2) Before use, the concentrated washing solution should be completely dissolved and mixed at room temperature. The concentrated washing solution (10×) should be diluted 10 times with distilled water to obtain the washing buffer (1×).

[0057] (3) Dilute the serum to be tested 20 times with sample diluent in the dilution plate. The negative and positive control serums can be used directly.

[0058] (4) Take out the required strips, put the remaining strips into an aluminum foil bag, seal it, and store it at 4℃. Add negative serum, positive serum, and diluted test serum to the ELISA reaction plate coated with antigen (Riemerella anatipestifer SD22-32-D strain), 100 μL / well, one well for each test serum sample, and two wells each for negative control and positive control;

[0059] (5) Place in a 37℃ incubator and react for 60 min;

[0060] (6) Discard the reaction solution, add 300 μL of washing buffer (1×) to each well, wash the plate 3 times consecutively and then pat dry. Avoid drying the microwells during the interval.

[0061] (7) Add 100 μL of the universal duck plague Riemerella monoclonal antibody from Example 1 to each well;

[0062] (8) Place in a 37℃ incubator and react for 60 minutes;

[0063] (9) Discard the reaction solution, add 300 μL of washing buffer to each well, wash the plate 3 times and then pat dry;

[0064] (10) Add 100 μL of enzyme conjugate to each well;

[0065] (11) Place in a 37℃ incubator and react for 30 min;

[0066] (12) Discard the enzyme conjugate solution, add 300 μL of washing buffer to each well, wash the plate three times and then pat dry.

[0067] (13) Add 100 μL of colorimetric solution to each well, shake to mix, and react at room temperature in the dark for 15 min;

[0068] (14) Add 50 μL of colorimetric stop solution to each well, shake to mix and stop the reaction, and measure the results within 15 minutes;

[0069] (15) Conditions for the test to be valid: The OD450 value of the negative control should be ≥1.0 and the blocking rate of the positive control well should be ≥50%.

[0070] (16) Judgment: Measure the OD value of each well on an ELISA reader. Blocking rate = 100% × (1 - sample OD450 value / mean OD450 value of negative control). Determine the presence or absence of antibody by calculating the blocking rate of each sample. Negative: blocking rate ≤ 40%; Positive: blocking rate ≥ 60%; Suspicious: 40% < blocking rate < 60%.

[0071] Example 3

[0072] This embodiment provides a universality test for the blocking ELISA antibody detection kit constructed in Example 2.

[0073] The blocking ELISA antibody detection kit constructed in Example 2 was used to detect positive sera of R. anatipestifer type S1, S2, S5, S6, S7, S10, S11, and S14 of R. anatipestifer. The detection results are shown in Table 1. The mean OD450 value of the negative serum control was 2.278, and the mean OD450 value of the positive serum control was 0.108. The results showed that positive sera for Riemerella anatipestifer S1, S2, S5, S6, S7, S10, S11, and S14 were all positive, indicating that the kit can detect antibodies against all known serotypes of Riemerella anatipestifer.

[0074] Table 1 Results of General Purpose Tests

[0075] sample OD value Blocking rate (%) Mean of negative serum 2.278 0 Mean of positive serum 0.108 95.3 S1 0.179 92.4 S2 0.096 95.8 S5 0.212 90.7 S6 0.179 92.4 S7 0.225 94.6 S10 0.102 95.5 S11 0.306 86.6 S14 0.342 85.0

[0076] Example 4

[0077] This embodiment provides specific detection of the blocking ELISA antibody detection kit constructed in Example 2.

[0078] The blocking ELISA antibody detection kit constructed in Example 2 was used to detect positive sera of *R. anatipestifer* types S1, S2, S5, S6, S7, S10, S11, and S14, as well as positive sera of *Enterococcus faecalis*, *Salmonella typhi*, *Escherichia coli*, and *Salmonella enteritidis*. The detection results are shown in Table 2. The negative serum control OD... 450 The mean value was 2.246, and the OD value of the positive serum control was... 450 The mean value was 0.106. The results showed that all serotypes of *Rimeria anatidae* tested were positive, while the rest were negative, indicating that the test kit did not cross-react with serotypes of *Enterococcus faecalis*, *Salmonella typhi*, *Escherichia coli*, or *Salmonella enteritidis*.

[0079] Table 2 Specificity test results

[0080] Sample Name OD value Blocking rate (%) Mean of negative serum 2.246 0 Mean of positive serum 0.105 95.3 S1 0.202 91.0 S2 0.114 94.9 S5 0.278 87.6 S6 0.228 89.8 S7 0.290 87.1 S11 0.351 84.4 S14 0.249 88.9 S10 0.106 95.3 Enterococcus faecalis 2.093 6.8 Typhoid fever monk 2.102 6.4 E. coli 2.376 0 Salmonella enteritis 2.016 1.0 Pasteurella multocida 1.933 1.4

[0081] Example 5

[0082] This embodiment provides a sensitivity test for the blocking ELISA antibody detection kit constructed in Example 2.

[0083] Positive and negative sera of Riemerella anatipestifer were diluted 1:10 to 1:1600, respectively, and the blocking ELISA was performed under the optimal reaction conditions. The results are shown in Table 3.

[0084] Table 3 Sensitivity Test Results

[0085]

[0086] The results showed that when Riemerella anatipestifer positive serum was diluted to 1:400, the blocking rate was 69.2%. This demonstrates that the blocking ELISA antibody detection kit of the present invention has high sensitivity.

[0087] Example 6

[0088] This embodiment provides a repeatability test of the blocking ELISA antibody detection kit constructed in Example 2.

[0089] The blocking ELISA antibody detection kit established in Example 2 was used to detect eight positive sera for *R. anatidae* (one each of *R. anatidae* types S1, S2, S5, S6, S7, S10, S11, and S14). Each sample was tested three times, and the coefficient of variation (CV%) was determined (CV = SD / X × 100%, where SD is the standard deviation and X is the arithmetic mean), as shown in Table 4. The sample detection results showed that the maximum CV was 4.78% and the minimum was 0.57%. The CVs of all eight sera were relatively small, indicating that the detection kit has good reproducibility.

[0090] Table 4 Results of Repeatability Experiments

[0091] Sample Name OD value 1 OD value 2 OD value 3 Average blocking rate (%) CV (%) Mean of negative serum 2.312 2.290 2.246 0 0 Mean of positive serum 0.100 0.117 0.105 95.3 0.37 S1 0.137 0.149 0.202 92.9 1.77 S2 0.101 0.126 0.114 95.0 0.58 S5 0.178 0.220 0.278 90.1 2.62 S6 0.214 0.209 0.228 90.5 0.64 S7 0.217 0.347 0.290 87.5 3.33 S10 0.094 0.120 0.106 95.3 0.57 S11 0.300 0.341 0.351 85.5 1.57 S14 0.381 0.434 0.249 84.5 4.78

[0092] Example 7

[0093] This embodiment provides a practical application of the blocking ELISA antibody detection kit constructed in Example 2.

[0094] The 162 samples tested in this embodiment included 32 chicken serum samples (16 pre-immunization sera and 16 post-immunization sera), 86 duck serum samples (25 pre-immunization sera, 29 post-single immunization sera with the S6+S7+S10 triple vaccine, and 32 post-second immunization sera with the S1+S2+S7 triple vaccine), and 44 fresh-entry duck serum samples. The blocking ELISA antibody detection kit constructed in Example 2 was used for testing, and 70 positive sera were detected. The results were consistent with the antibody fluctuation pattern and the current immune status of *Riemerella anatipestifer*, indicating that the blocking ELISA antibody detection kit established in this invention is suitable for clinical detection of antibodies against all serotypes of *Riemerella anatipestifer*.

[0095] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A universal monoclonal antibody against Riemerella anatipestifer, characterized in that, The heavy chain of the universal Riemerella anatipestifer monoclonal antibody comprises VHCDR1, VHCDR2, and VHCDR3 with amino acid sequences as shown in SEQ ID NO: 1-3, and the light chain of the universal Riemerella anatipestifer monoclonal antibody comprises VLCDR1 with an amino acid sequence as shown in SEQ ID NO: 4, VLCDR2 with an amino acid sequence of DTS, and VLCDR3 with an amino acid sequence as shown in SEQ ID NO:

5.

2. The application of the universal Riemerella anatipestifer monoclonal antibody according to claim 1 in the preparation of products for detecting Riemerella anatipestifer, wherein the serotypes of Riemerella anatipestifer include S1, S2, S5, S6, S7, S10, S11 and S14.

3. The application as described in claim 2, characterized in that, The product in question is a test kit.

4. The application as described in claim 3, characterized in that, The detection kit is an ELISA detection kit, a colloidal gold detection kit, or a chemiluminescent immunoassay kit.

5. A test kit, characterized in that, The test kit contains at least the universal monoclonal antibody against Riemerella anatipestifer as described in claim 1.

6. The detection kit as described in claim 5, characterized in that, The test kit also includes an enzyme-linked reaction plate and an enzyme-labeled antibody; wherein, the enzyme-linked reaction plate is coated with Riemerella anatipestifer antigen, and the enzyme-labeled antibody is an enzyme-labeled goat anti-mouse secondary antibody; The enzyme in the enzyme-labeled antibody is any one of horseradish peroxidase, alkaline phosphatase, glucose oxidase, β-galactosidase, lysozyme, or malate dehydrogenase.

7. The detection kit as described in claim 5, characterized in that, The detection kit also includes a chromogenic solution and a stop solution; the chromogenic solution is 3,3',5,5'-tetramethylbenzidine chromogenic solution; the stop solution is an ELISA stop solution.

8. The use of the test kit according to any one of claims 5-7 in the preparation of a test sample for detecting Riemerella anatipestifer infection or vaccination, wherein the serotypes of Riemerella anatipestifer include S1, S2, S5, S6, S7, S10, S11 and S14.

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