ELISA kit for Actinobacillus pleuropneumoniae

Abstract

The present invention discloses an ELISA kit for Actinobacillus pleuropneumoniae, and relates to the field of immunoassay technology. The ELISA kit comprises the following components: an ELISA plate coated with an antigen, a washing and diluting solution, an enzyme-labeled secondary antibody, a color developing solution, and a stop solution; the antigen is a recombinant PET-32a-apfA fusion protein; the amino acid sequence of the recombinant PET-32a-apfA fusion protein is shown in SEQ ID NO.4. The ELISA kit has high specificity and sensitivity, provides a reliable technical means for the rapid and accurate diagnosis of Actinobacillus pleuropneumoniae, and contributes to the early detection and prevention and control of porcine contagious pleuropneumonia. The ELISA kit can be used for vaccine immune effect evaluation, can provide objective and quantitative data support, and is conducive to promoting the improvement of vaccine research and development and evaluation systems.

Description

Technical Field

[0001] The present invention relates to the technical field of immune detection, in particular to an ELISA kit for Actinobacillus pleuropneumoniae. Background Art

[0002] Swine Infectious Pleuropneumonia (SIPP) is a serious respiratory infection of pigs caused by Actinobacillus pleuropneumoniae (APP), causing significant economic losses to the global swine industry. The disease's main pathological features include fibrinous pleuritis and hemorrhagic and necrotizing pneumonia. However, diagnosis based solely on these pathological findings remains challenging.

[0003] Currently, inactivated vaccines are widely used clinically to prevent porcine contagious pleuropneumonia. However, to more effectively prevent and control the disease and evaluate the immune efficacy of vaccines, there is an urgent need to develop a convenient and efficient serological diagnostic method. This new method will not only significantly improve the accuracy of disease diagnosis but also provide a reliable basis for evaluating vaccine effectiveness. It is of vital importance to promote the prevention, control and ultimate elimination of porcine contagious pleuropneumonia. Summary of the Invention

[0004] The present invention aims to provide an ELISA kit for Actinobacillus pleuropneumoniae to solve the problems existing in the prior art. The ELISA kit has high specificity and sensitivity, and provides a reliable technical means for the rapid and accurate diagnosis of Actinobacillus pleuropneumoniae.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an ELISA detection kit for Actinobacillus pleuropneumoniae, comprising the following components:

[0007] Antigen-coated ELISA plates, washing and diluents, enzyme-labeled secondary antibodies, developing solution, and stop solution;

[0008] The antigen is a recombinant PET-32a-apfA fusion protein;

[0009] The amino acid sequence of the recombinant PET-32a-apfA fusion protein is shown in SEQ ID NO.4.

[0010] Furthermore, the preparation method of the antigen-coated ELISA plate includes the following steps: adding a carbonate buffer solution containing the recombinant PET-32a-apfA fusion protein to a multi-well plate, coating overnight at 4°C, then adding a blocking agent, and blocking at 37°C for 60 minutes to obtain the antigen-coated ELISA plate.

[0011] Furthermore, in the carbonate buffer solution, the concentration of the recombinant PET-32a-apfA fusion protein is 2 μg / mL.

[0012] Furthermore, the washing and diluting solution is PBST solution.

[0013] Furthermore, the enzyme-labeled secondary antibody is HRP-labeled rabbit anti-pig IgG.

[0014] Furthermore, the color developing solution is TMB solution.

[0015] Furthermore, the stop solution is 10% sulfuric acid solution.

[0016] The present invention also provides a method for detecting antibodies to Actinobacillus pleuropneumoniae for non-disease diagnosis purposes, comprising the step of detecting antibodies to Actinobacillus pleuropneumoniae using the above-mentioned ELISA detection kit.

[0017] Furthermore, the steps of using the ELISA detection kit to detect Actinobacillus pleuropneumoniae antibodies include:

[0018] Add 400-fold diluted standard Actinobacillus pleuropneumoniae positive serum, standard Actinobacillus pleuropneumoniae negative serum or the sample to be tested to the antigen-coated ELISA plate, incubate at 37° C. for 60 minutes, and wash the plate 5 times with PBST after incubation;

[0019] Add 100 μL of 2000-fold diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 45 min. After incubation, wash the plate five times with PBST.

[0020] Add 100 μL of the color development solution to each reaction well and react in the dark for 10 min;

[0021] After adding 50 μL of the stop solution to each reaction well to terminate the reaction, the OD value was read using a microplate reader. 450nm Value, determine the test result:

[0022] When the OD of the standard Actinobacillus pleuropneumoniae positive serum 450nm ≥1.0, and the OD of the standard A. pleuropneumoniae negative serum 450nm When ≤0.2, the test results are valid;

[0023] When the sample to be tested OD 450nm OD values ​​compared with negative control450nm The ratio of the values ​​is ≥2.0 and the OD 450nm When OD ≥ 0.4, it was judged as positive; 450nm When <0.4, it was judged as negative.

[0024] The present invention discloses the following technical effects:

[0025] The present invention uses a truncated apfA protein as the coating antigen of an ELISA test plate to develop an ELISA kit for Actinobacillus pleuropneumoniae, which significantly improves the specificity and sensitivity of detection.

[0026] The present invention provides a reliable technical means for the rapid and accurate diagnosis of Actinobacillus pleuropneumoniae, which is helpful for the early detection and prevention and control of porcine contagious pleuropneumoniae.

[0027] The kit of the present invention can be applied to the evaluation of vaccine immune effects, can provide objective and quantitative data support, and is conducive to promoting the improvement of vaccine research and development and evaluation systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0029] Figure 1 This is a diagram showing the verification results of the recombinant plasmid PET-32a-apfA;

[0030] Figure 2 Figure 2 is the SDS-PAGE test result of the recombinant PET-32a-apfA fusion protein; A is the solubility test result; B is the purity test result; in A, M is a marker, 1 is the recombinant induced bacteria, 2 is the protein induction supernatant, and 3 is the protein induction precipitate; in B, M is a marker, and 1 is the concentrated protein;

[0031] Figure 3 is the OD under different antigen coating conditions 450nm Statistical chart of value and P / N value;

[0032] Figure 4 is the OD under different blocking agent conditions 450nm Statistical chart of value and P / N value;

[0033] Figure 5 OD under different closure time conditions 450nm Statistical chart of value and P / N value;

[0034] Figure 6 is the OD under different serum action time conditions 450nm Statistical chart of value and P / N value;

[0035] Figure 7 OD under different enzyme-labeled secondary antibody dilution conditions 450nm Statistical chart of value and P / N value;

[0036] Figure 8 OD under different enzyme-labeled secondary antibody action time conditions 450nm Statistical chart of value and P / N value;

[0037] Figure 9 is the OD under different color development time conditions 450nm Statistical chart of value and P / N value;

[0038] Figure 10 This is the result diagram of the specificity detection experiment;

[0039] Figure 11 This is the result of the sensitivity detection experiment. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1

[0046] 1. Obtain the target gene

[0047] According to the apfA gene sequence (MT740280.1) of Actinobacillus pleuropneumoniae on GenBank, it was truncated and expressed in the prokaryotic expression system Escherichia coli BL21 (DE3), and then purified by affinity chromatography according to the treatment method of soluble protein to obtain the truncated apfA protein.

[0048] Primers were designed for amplification of the gene encoding the truncated apfA protein. To facilitate cloning of the PCR product, BamHI and XhoI restriction sites were added to the upstream and downstream primers, respectively. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. and the primer sequences are as follows:

[0049] F:5'-cgc ggatcc attcgaccgcttactaacgcg-3' (SEQ ID NO. 1);

[0050] R:5'-ccg ctcgag ttaagtcctcccctttagattt-3' (SEQ ID NO. 2).

[0051] Nucleotide sequence of the gene encoding the truncated protein of apfA (SEQ ID NO.3):

[0052] ATTCGACCGCTTACTAACGCGTTTACTTTAATTGAATTGATGATCGTGATTGCGATTATTGCCATTTTAGCTACGGTTGCAATTCCGTCATATAACAGTTATACCCAAAAAGCGGCGCTTTCGGAGCTATTGGCGGCATCGGCTTCTTATAAAACGGATGTCGAGATCTGCATATATAACACCGGAGATTCTAAAAA CTGTAGCGGCGGTCAAAACGGTGTCAGAAAAATGACGGAGCTTAGACAGGCTAAATATTTAAATGCCATTACGGTGGAAGGCGGAACGATTACAGTAACGGGGAAAGGGAATCTACAGGAATACGGTTATACGATGACACCGATTCATAACGGTAGCACTATTTCTTGGGAAACGAAATGTAAAGGGGAGGACTTAA.

[0053] Amino acid sequence of apfA truncated protein (SEQ ID NO.4):

[0054] IRPLTNAFTLIELMIVIAIIAILATVAIPSYNSYTQKAALSELLAASASYKTDVEICIYNTG DSKNCSGGQNGVRKMTELRQAKYLNAITVEGGTITVTGKGNLQEYGYTMTPIHNGSTISW ETKCKGEDL.

[0055] Genomic DNA from Actinobacillus pleuropneumoniae was extracted and used as a template for PCR amplification to obtain the truncated apfA gene. The PCR amplification reaction system and procedure are as follows:

[0056] 50 μL PCR reaction system: 2 μL template DNA, 5 μL 10× PCR buffer, 4 μL 25 mmol / L MgCl2, 2 μL 2 μmol / L dNTPs, 5 μL DMSO, 2 μL each of 10 μmol / L primers F and R, 0.25 μL 5 U / μL ExTaq DNA Polymerase, and 27.75 μL sterile deionized water.

[0057] The PCR reaction procedure was as follows: pre-denaturation at 95°C for 5 min, followed by 35 cycles (denaturation at 95°C for 30 s, annealing at 54°C for 90 s, and extension at 72°C for 3 min), and finally extension at 72°C for 10 min. The PCR product was recovered using a DNA recovery kit.

[0058] 2. Prokaryotic expression of truncation of apfA protein

[0059] The recovered apfA truncated gene was connected to the pET-32a(+) expression vector to construct the recombinant plasmid PET-32a-apfA and verified ( Figure 1 ).

[0060] The recombinant plasmid PET-32a-apfA was transformed into BL21 (D3) competent cells; the single colony with the correct bacterial solution PCR and sequencing was selected for expansion culture, and the bacterial solution OD 600nm When the p-terminal pH value was 0.6-1.0, isopropyl-β-D-thiogalactoside (IPTG, 1 mM) was added and expressed at 37°C for 6 h. The bacteria were collected and ultrasonically disrupted, and the protein was purified using a nickel column and verified by SDS-PAGE; and the recombinant PET-32a-apfA fusion protein was obtained by concentration.

[0061] SDS-PAGE analysis showed that the recombinant PET-32a-apfA fusion protein was soluble and consistent with the expected size ( Figure 2 After protein concentration, SDS-PAGE showed that the protein was highly pure ( Figure 2 Middle B).

[0062] Example 2 Optimization of ELISA reaction conditions

[0063] An ELISA detection method for Actinobacillus pleuropneumoniae was constructed using the recombinant PET-32a-apfA fusion protein prepared in Example 1.

[0064] 1. Optimization of optimal antigen coating concentration and serum dilution

[0065] The optimal coating concentration and serum dilution of the recombinant PET-32a-apfA fusion protein antigen were determined by the checkerboard method. The recombinant PET-32a-apfA fusion protein was diluted in carbonate buffer to a coating concentration of 2, 2 -1 , 2 -2 , 2 -3 , 2 -4 , 2 -5 , 2 -6 , 2 -7 , 2 -8 , 2 -9 , 2 -10 , 2 -11μg / mL, 100 μL per well (coated in a horizontal row), and the serum was titrated in the order of 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600 (coated in a vertical row) to determine the optimal coating concentration and serum dilution concentration;

[0066] The results showed that the optimal antigen coating concentration was 2 μg / mL and the optimal positive serum dilution was 1:400 (Table 1).

[0067] Table 1 Determination of optimal antigen coating concentration and optimal serum dilution

[0068]

[0069] 2. Determination of antigen coating conditions

[0070] Based on the optimal antigen coating concentration and serum dilution, the antigen coating conditions were optimized and the antigen was coated at 4°C overnight, 37°C for 2 hours, and 37°C for 2 hours followed by 4°C overnight (each with 3 replicates). 450nm The values ​​and P / N values ​​were calculated to determine the optimal antigen coating conditions (three replicates were performed and the average value was calculated).

[0071] P / N = OD of the sample to be tested 450nm Value / negative control OD 450nm value.

[0072] The results showed that the optimal antigen coating condition was 4°C overnight ( Figure 3 ).

[0073] 3. Determination of optimal sealing conditions

[0074] Based on the optimal antigen coating concentration, serum dilution, and antigen coating conditions, the blocking conditions were optimized. The ELISA plate wells were grouped and blocked with 5% skim milk, 1% BSA, and 10% fetal bovine serum at 37°C for 60 min, 90 min, and 120 min (each with 3 replicates). The OD values ​​of positive and negative sera for Actinobacillus pleuropneumoniae were compared. 450nm The values ​​of P / N were used to determine the optimal blocking conditions (three replicates were set and the average value was calculated).

[0075] The results showed that the best blocking agent was 1% BSA ( Figure 4 ), the optimal blocking time is 37℃60min( Figure 5 ).

[0076] 4. Determination of the optimal serum action time

[0077] The optimal serum reaction time was optimized based on the optimal antigen coating concentration, serum dilution, antigen coating conditions, and blocking conditions. The ELISA plates were coated and divided into groups. The serum was diluted in proportion and incubated at 37°C for 60 min, 90 min, and 120 min, respectively. The OD values ​​of positive and negative serum samples for Actinobacillus pleuropneumoniae were compared. 450nm The value and P / N value were used to determine the optimal action time of the serum (three replicates were set and the average value was calculated).

[0078] The results showed that the optimal action time of serum was 60 min ( Figure 6 ).

[0079] 5. Determination of the optimal enzyme-labeled secondary antibody dilution and reaction time

[0080] Based on the optimal antigen coating concentration, serum dilution, antigen coating conditions, blocking conditions and serum reaction time, the dilution and effect of enzyme-labeled secondary antibody were optimized. The ELISA plates were coated and grouped, and rabbit anti-swine IgG-HRP antibodies were diluted at dilutions of 1:10000, 1:20000, 1:40000, 1:80000, 1:160000, 1:320000, 1:640000, and 1:1280000, respectively. Each dilution was incubated at 37°C for 30 min, 45 min, and 60 min, respectively. The OD values ​​of positive and negative sera for Actinobacillus pleuropneumoniae were compared. 450nm The optimal enzyme-labeled secondary antibody dilution and action time were determined by the values ​​of PCR and P / N (three replicates were set and the average value was calculated).

[0081] The results showed that the optimal dilution of enzyme-labeled secondary antibody was 1:10000 ( Figure 7 ), the optimal enzyme-labeled secondary antibody reaction time is 45 min ( Figure 8 ).

[0082] 6. Determination of color development time

[0083] The color development time was optimized based on the optimal antigen coating concentration, serum dilution, antigen coating conditions, blocking conditions, serum reaction time, and enzyme-labeled secondary antibody dilution and reaction time. The coated ELISA plates were divided into groups and the TMB substrate reaction time was incubated at 37°C for 5 minutes, 10 minutes, and 15 minutes, respectively. The OD values ​​of positive and negative sera for Actinobacillus pleuropneumoniae were compared. 450nm The optimal color development time was determined by the value and P / N value (three replicates were set and the average value was calculated).

[0084] The results showed that the optimal color development time was 10 min ( Figure 9 ).

[0085] 7. Determination of critical value

[0086] Based on the above-mentioned optimal antigen coating concentration, serum dilution, antigen coating conditions, blocking conditions, serum action time, and enzyme-labeled secondary antibody dilution, action time, and display time, 41 known standard positive sera (sera collected from healthy pigs immunized with porcine Actinobacillus pleuropneumoniae vaccine or diagnosed with porcine Actinobacillus pleuropneumoniae disease) and 43 negative sera (sera collected from healthy pigs not immunized with porcine Actinobacillus pleuropneumoniae vaccine) were tested, and the critical value was determined by ROC curve analysis.

[0087] The results showed that when OD 450nm When OD < 0.4, the sensitivity (100%) and specificity (97.8%) were high, and this OD value was selected as the critical value; when OD 450nm When OD ≥ 0.4, it was judged as positive; 450nm When <0.4, it was judged as negative.

[0088] Example 3

[0089] The ELISA kit for the detection of Actinobacillus pleuropneumoniae includes the following components:

[0090] ELISA plate: ELISA plate coated with antigen (recombinant PET-32a-apfA fusion protein prepared in Example 1);

[0091] Washing and dilution solution: PBST solution;

[0092] Enzyme-labeled secondary antibody: HRP-labeled rabbit anti-pig IgG;

[0093] Color development solution: TMB solution;

[0094] Stop solution: 10% (V / V) sulfuric acid solution;

[0095] Standard Actinobacillus pleuropneumoniae-positive serum;

[0096] Standard Actinobacillus pleuropneumoniae negative serum.

[0097] The preparation method of the ELISA plate coated with the antigen is as follows:

[0098] 100 μL of carbonate buffer solution containing 2 μg / mL recombinant PET-32a-apfA fusion protein was added to a 96-well microplate, placed in a 37°C incubator for 2 hours and then incubated at 4°C overnight for coating. Then, 200 μL of 1% BSA was added and blocked at 37°C for 60 minutes to obtain an antigen-coated ELISA plate.

[0099] The detection method of this ELISA detection kit is as follows:

[0100] (1) Add 400-fold diluted standard A. pleuropneumoniae-positive serum, standard A. pleuropneumoniae-negative serum, or serum to be tested to the antigen-coated ELISA plate, incubate at 37°C for 60 min, and wash the plate five times with PBST.

[0101] (2) Add 100 μL of 10,000-fold diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 45 min. After incubation, wash the plate five times with PBST.

[0102] (3) Add 100 μL of color development solution (TMB solution) to each reaction well and react for 10 min in the dark;

[0103] (4) Add 50 μL of stop solution (10% sulfuric acid solution) to each reaction well to terminate the reaction and read the OD value using a microplate reader. 450nm value.

[0104] (5) Criteria for determining positive results of samples to be tested:

[0105] The average value of negative control = (well A1 + well A2) / 2; the average value of positive control = (well A3 + well A4) / 2.

[0106] When the OD of the standard Actinobacillus pleuropneumoniae positive serum 450nm ≥1.0, and the OD of the standard A. pleuropneumoniae negative serum 450nm When ≤0.2, the test results are valid;

[0107] If the sample to be tested OD 450nm OD values ​​compared with negative control 450nm The ratio of values ​​(P / N) is ≥2.0, when the OD of the sample to be tested is 450nm When the OD value is ≥0.4, it is considered positive; when the OD 450nm When <0.4, it was judged as negative.

[0108] Example 4 Specificity Detection

[0109] The ELISA test kit of Example 3 was used to test the standard positive serum of Streptococcus suis (S.suis), Pasteurella multocida (P.multocida), Escherichia coli (E.coli), Staphylococcus aureus (S.aureus), and Haemophilus parasuis (HPS) with known antibody positive results at a dilution of 1:400. At the same time, the positive and negative serum of Actinobacillus pleuropneumoniae were used as controls. Finally, the results were analyzed based on the OD value. 450nm The specificity of the ELISA detection method was verified.

[0110] The results showed that after the PET-32a-apfA fusion protein antigen reacted with different pathogen positive sera, OD 450nmThe values ​​were all less than 0.4, indicating that the PET-32a-apfA fusion protein antigen did not non-specifically bind to the positive serum of the above pathogens, indicating that the ELISA method established in the present invention has good specificity ( Figure 10 ).

[0111] Example 5 Sensitivity Detection

[0112] The positive and negative sera of porcine Actinobacillus pleuropneumoniae were diluted with PBS at 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 and 1:25600 times, respectively. Then, the ELISA detection kit of Example 3 was used to perform ELISA detection on the positive sera of different dilutions.

[0113] The results showed that when the serum dilution ratio was 1:6400, the test result was still positive (OD 450nm >0.4), indicating that the method has high sensitivity ( Figure 11 ).

[0114] Example 6 Clinical Application

[0115] The ELISA test kit of Example 3 was used to test 88 pig serum samples submitted for clinical testing (32 of which were positive samples, as confirmed by nucleic acid testing). The results are shown in Table 2.

[0116] Table 2 Sample test results

[0117] Total number of samples / pieces Positive number / sample Negative number / copy Positive rate 88 32 56 36.36％

[0118] The results showed that the positive detection rate of samples was 36.36%.

[0119] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. An ELISA detection kit for Actinobacillus pleuropneumoniae, characterized in that: Includes the following components: Antigen-coated ELISA plates, washing and diluents, enzyme-labeled secondary antibodies, developing solution, and stop solution; The antigen is a recombinant PET-32a-apfA fusion protein; The amino acid sequence of the recombinant PET-32a-apfA fusion protein is shown in SEQ ID NO.4; The preparation method of the antigen-coated ELISA plate comprises the following steps: adding a carbonate buffer solution containing the recombinant PET-32a-apfA fusion protein to a multi-well plate, coating at 4°C overnight, then adding a blocking agent, and blocking at 37°C for 60 minutes to obtain the antigen-coated ELISA plate.

2. The ELISA detection kit according to claim 1, wherein The blocking agent was 1% BSA.

3. The ELISA test kit according to claim 1, wherein In the carbonate buffer solution, the concentration of the recombinant PET-32a-apfA fusion protein was 2 μg / mL.

4. The ELISA detection kit according to claim 1, wherein The washing and diluting solution is PBST solution.

5. The ELISA detection kit according to claim 1, wherein The enzyme-labeled secondary antibody is HRP-labeled rabbit anti-pig IgG.

6. The ELISA detection kit according to claim 1, wherein The color developing solution is TMB solution.

7. The ELISA detection kit according to claim 1, wherein The stop solution is 10% sulfuric acid solution.

8. A method for detecting antibodies to Actinobacillus pleuropneumoniae for non-disease diagnosis purposes, characterized in that: The method comprises the step of using the ELISA detection kit according to any one of claims 1 to 7 to detect antibodies against Actinobacillus pleuropneumoniae.

9. The method according to claim 8, characterized in that The steps of using the ELISA detection kit to detect antibodies to Actinobacillus pleuropneumoniae include: Add 400-fold diluted standard Actinobacillus pleuropneumoniae positive serum, standard Actinobacillus pleuropneumoniae negative serum or the sample to be tested to the antigen-coated ELISA plate, incubate at 37° C. for 60 minutes, and wash the plate 5 times with PBST after incubation; Add 100 μL of 2000-fold diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 45 min. After incubation, wash the plate five times with PBST. Add 100 μL of the color development solution to each reaction well and react in the dark for 10 minutes; After adding 50µL of the stop solution to each reaction well to terminate the reaction, the OD value was read using a microplate reader. 450nm Value, determine the test result: When the OD of the standard Actinobacillus pleuropneumoniae positive serum 450nm ≥1.0, and the OD of the standard A. pleuropneumoniae negative serum 450nm When ≤0.2, the test results are valid; When the sample to be tested OD 450nm OD values ​​compared with negative control 450nm The ratio of the values ​​is ≥2.0 and the OD of the sample to be tested is 450nm When the value is ≥0.4, it is judged as positive; when the OD 450nm When the value is <0.4, it is judged as negative.

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