ELISA kit for actinobacillus pleuropneumoniae

Abstract

The invention discloses an enzyme-linked immunosorbent assay (ELISA) kit for actinobacillus pleuropneumoniae, and relates to the technical field of immunodetection. The ELISA detection kit comprises the following components: an elisa plate coated with an antigen, a washing and diluting solution, an elisa secondary antibody, a developing solution and a stop solution, the antigen is a recombinant PET-32a-apfA (Polyethylene Terephthalate-32a-apfA) fusion protein; and the amino acid sequence of the recombinant PET-32a-apfA fusion protein is as shown in SEQ ID NO. 4. The ELISA kit has high specificity and sensitivity, provides a reliable technical means for rapid and accurate diagnosis of actinobacillus pleuropneumoniae, and is helpful for early discovery and prevention and control of porcine contagious pleuropneumonia. The ELISA kit can be applied to vaccine immune effect evaluation, can provide objective and quantitative data support, and is beneficial to promoting vaccine research and development and improving an evaluation system.

Description

Technical Field

[0001] The 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 infectious disease of pigs caused by Actinobacillus pleuropneumoniae (APP), which has caused huge economic losses to the global pig industry. The main pathological characteristics of the disease include fibrinous pleurisy, hemorrhagic and necrotizing pneumonia, but it is still quite challenging to make a diagnosis based on these pathological manifestations alone.

[0003] At present, inactivated vaccines are widely used in clinical practice to prevent porcine contagious pleuropneumonia, but in order to more effectively prevent and control the disease and evaluate the immune effect of the vaccine, it is urgent to develop a convenient and efficient serological diagnosis method. This new method can not only significantly improve the accuracy of disease diagnosis, but also provide a reliable evaluation basis for the effectiveness of the vaccine, which is of vital significance for promoting the prevention and control of porcine contagious pleuropneumonia and its ultimate purification. Summary of the invention

[0004] The purpose of the present invention is to provide an ELISA kit for Actinobacillus pleuropneumoniae to solve the problems existing in the above-mentioned 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] ELISA plates coated with antigens, washing and diluents, enzyme-labeled secondary antibodies, color development solutions, and stop solutions;

[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 comprises the following steps: adding a carbonate buffer solution containing the recombinant PET-32a-apfA fusion protein into 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 a 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 using the above-mentioned ELISA detection kit to detect antibodies to Actinobacillus pleuropneumoniae.

[0017] Furthermore, the steps of using the ELISA detection kit to detect antibodies to Actinobacillus pleuropneumoniae 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 min, and wash the plate with PBST for 5 times 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 5 times with PBST;

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

[0021] After adding 50 μL of the stop solution to each reaction well to terminate the reaction, the OD was read using an ELISA 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 result is valid;

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

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

[0025] The present invention uses truncated apfA protein as the coating antigen of an ELISA detection 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, and can provide objective and quantitative data support, which 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

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

[0030] Figure 2 The figure 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 a recombinant induced bacteria, 2 is a protein induced supernatant, and 3 is a protein induced precipitate; in B, M is a marker, and 1 is a 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 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] Fig. 9 is the OD under different color development time conditions 450nm Statistical chart of value and P / N value;

[0038] Fig.10 This is the result diagram of the specific detection experiment;

[0039] Fig.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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are 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 by the prokaryotic expression system Escherichia coli BL21 (DE3), and then affinity chromatography purification was performed according to the treatment method of soluble protein to obtain the truncated apfA protein.

[0048] Primers were designed for amplification of the truncated protein encoding gene of apfA. To facilitate cloning of PCR products, BamH I and Xho I restriction sites were added to the upstream and downstream primers, respectively. The primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd. 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] The genomic DNA of Actinobacillus pleuropneumoniae was extracted and used as a template to obtain the truncated gene of apfA by PCR amplification. The reaction system and reaction procedure of PCR amplification are as follows:

[0056] 50μL PCR reaction system: 2μL template DNA, 5μL 10×PCR buffer, 4μL 25mmol / L MgCl2, 2μL 2μmol / L dNTPs, 5μL DMSO, 2μL each of 10μmol / L primers F and R, 0.25μL 5U / μL ExTaq DNA Polymerase, and 27.75μL 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 truncated 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 transferred into BL21(D3) competent cells; single colonies with correct bacterial solution PCR and sequencing were selected for expansion culture, and the bacterial solution OD 600nm When the p-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. The protein was purified using a nickel column and verified by SDS-PAGE. The recombinant PET-32a-apfA fusion protein was obtained by concentration.

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

[0062] Example 2 Optimization of ELISA reaction conditions

[0063] The recombinant PET-32a-apfA fusion protein prepared in Example 1 was used to construct an ELISA detection method for Actinobacillus pleuropneumoniae.

[0064] 1. Optimization of the 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 chessboard method. The recombinant PET-32a-apfA fusion protein was diluted in carbonate buffer to a coating concentration of 2 and 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 horizontal rows in sequence), and the serum was titrated in square arrays at 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800, and 1:25600 (coated in vertical rows in sequence) 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. The antigen was coated at 4°C overnight, 37°C for 2h, and 37°C for 2h followed by 4°C overnight (three replicates were set for each). Comparison of OD values ​​of positive and negative sera of Actinobacillus pleuropneumoniae 450nm The values ​​and P / N values ​​were calculated to determine the optimal antigen coating conditions (three replicates were set and the average value was calculated).

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

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

[0073] 3. Determination of the Optimal Closure 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 respectively (three replicates were set for each). Comparison of OD values ​​of positive and negative sera of Actinobacillus pleuropneumoniae 450nm The values ​​of P / N were measured to determine the best blocking conditions (three replicates were performed and the average value was calculated).

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

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

[0077] The optimal serum action time was optimized based on the optimal antigen coating concentration, serum dilution, antigen coating conditions and blocking conditions. The ELISA plates were coated and grouped, and 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 of 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 best action time of serum was 60min ( Figure 6 ).

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

[0080] Based on the optimal antigen coating concentration, serum dilution, antigen coating conditions, blocking conditions and serum action time, the enzyme-labeled secondary antibody dilution and action were optimized. The ELISA plates were coated and grouped, and the rabbit anti-swine IgG-HRP antibody was diluted at 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 of Actinobacillus pleuropneumoniae were compared. 450nm The optimal enzyme-labeled secondary antibody dilution and action time were determined by the value and P / N value (three replicates were set and the average value was calculated).

[0081] The results showed that the optimal enzyme-labeled secondary antibody dilution was 1:10000 ( Figure 7 ), the optimal enzyme-labeled secondary antibody action time is 45min ( 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 action time, and enzyme-labeled secondary antibody dilution and action time. The coated ELISA plates were grouped and the TMB substrate action time was incubated at 37°C for 5 min, 10 min, and 15 min, respectively. The OD values ​​of positive and negative sera of 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 ( Fig. 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 respectively, 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 ≥0.4, it was judged as positive; when OD 450nm When <0.4, it was judged as negative.

[0088] Example 3

[0089] ELISA test kit for Actinobacillus pleuropneumoniae, comprising 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 developing 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 antigen is as follows:

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

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

[0100] (1) Add 400-fold diluted standard Actinobacillus pleuropneumoniae positive serum, standard Actinobacillus 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 5 times with PBST after incubation;

[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 colorimetric 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 an ELISA reader. 450nm value.

[0104] (5) Criteria for positive determination 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 result is valid;

[0107] If the sample to be tested OD 450nm OD value and negative control 450nm The ratio of values ​​(P / N) is ≥2.0. 450nm When the value is ≥0.4, it is judged as positive; when 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 at a dilution of 1:400, and the positive and negative serum of Actinobacillus pleuropneumoniae were used as controls. Finally, the OD values ​​were calculated. 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 ( Fig.10 ).

[0111] Example 5 Sensitivity Detection

[0112] The positive serum and negative serum of porcine Actinobacillus pleuropneumoniae were diluted 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, 1:12800 and 1:25600 times with PBS, and 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 ( Fig.11 ).

[0114] Example 6 Clinical Application

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

[0116] Table 2 Sample test results

[0117] Total number of samples / pieces Positive number / copy 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 only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An ELISA detection kit for Actinobacillus pleuropneumoniae, characterized in that: Includes the following components: ELISA plates coated with antigens, washing and diluents, enzyme-labeled secondary antibodies, color development solutions, and stop solutions; 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.

2. The ELISA test kit according to claim 1, characterized in that 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 into 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.

3. The ELISA test kit according to claim 2, characterized in that The blocking agent was 1% BSA.

4. The ELISA test kit according to claim 2, characterized in that: In the carbonate buffer solution, the concentration of the recombinant PET-32a-apfA fusion protein was 2 μg / mL.

5. The ELISA detection kit according to claim 1, characterized in that The washing and diluting solution is PBST solution.

6. The ELISA detection kit according to claim 1, characterized in that The enzyme-labeled secondary antibody is HRP-labeled rabbit anti-pig IgG.

7. The ELISA test kit according to claim 1, characterized in that: The color developing solution is TMB solution.

8. The ELISA test kit according to claim 1, characterized in that The stop solution is 10% sulfuric acid solution.

9. 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 8 to detect antibodies against Actinobacillus pleuropneumoniae.

10. The method according to claim 9, characterized in that The steps of using the ELISA test 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 min, and wash the plate with PBST for 5 times 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 5 times with PBST; Add 100 μL of the color developing solution to each reaction well and react for 10 min in the dark; After adding 50 μL of the stop solution to each reaction well to terminate the reaction, the OD was read using an ELISA 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 result is valid; When the sample OD 450nm OD value and negative control 450nm The ratio of the values ​​is ≥2.0 and the OD 450nm ≥0.4, it was judged as positive; when OD 450nm When <0.4, it was judged as negative.

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