Indirect ELISA kit for rapidly detecting porcine delta coronavirus IgA antibody

Abstract

The invention discloses an indirect ELISA (Enzyme-Linked Immunosorbent Assay) kit for rapidly detecting a porcine delta coronavirus IgA antibody, and relates to the field of immunological detection. The indirect ELISA kit comprises an ELISA plate coated with an antigen, the antigen is a recombinant PDCoV S2 protein, and the amino acid sequence of the antigen is as shown in SEQ ID NO. 2. According to the indirect ELISA kit, a recombinant PDCoV S2 protein is used as a coating antigen to capture a PDCoV specific IgA antibody, a goat anti-pig IgA antibody labeled by horse radish peroxidase is used as a detection antibody, a 'PDCoV-S2 protein-PDCoV-S2 specific IgA antibody-enzyme labeled antibody' trimer compound can be formed on an ELISA plate, and the PDCoV specific IgA antibody-enzyme labeled antibody can be used for detecting the PDCoV specific IgA antibody. And the result can be directly judged by measuring the color change of the substrate and the absorbance value, so that the detection efficiency can be obviously improved. The indirect ELISA kit can be used for clinical detection and immune evaluation of the PDCoV.

Description

Technical Field

[0001] The invention relates to the field of immunological detection, and in particular to an indirect ELISA kit for rapidly detecting porcine delta coronavirus IgA antibodies. Background Art

[0002] Porcine deltacoronavirus (PDCoV) is a newly discovered novel porcine enteric coronavirus that can infect pigs of all ages, mainly causing acute diarrhea, vomiting and death in piglets under 10 days old. In December 2021, the journal Nature reported that porcine deltacoronavirus was detected and isolated from serum samples of three febrile children in Haiti, indicating that the virus has the risk of infecting humans, and rapid and accurate disease diagnosis is crucial for the prevention and control of the disease.

[0003] As an intestinal pathogen, PDCoV's replication characteristics on the mucosal surface make it particularly important to pay attention to IgA antibodies. The immune system of newborn piglets is not yet fully mature, and piglets mainly obtain maternal anti-PDCoV specific IgA by ingesting colostrum. After the IgA in the colostrum is absorbed by the piglets through the digestive tract, it can provide passive immune protection for the piglets. By detecting the concentration of PDCoV-specific IgA in breast milk or serum, the immune status of the sow and the level of passive immunity obtained by the piglets can be evaluated, which is of great significance for formulating effective vaccination strategies and disease prevention and control measures. The present invention intends to develop an indirect ELISA detection kit for detecting porcine delta coronavirus IgA antibodies, in order to provide technical support for the detection of PDCoV-specific IgA. Summary of the invention

[0004] The purpose of the present invention is to provide an indirect ELISA kit for rapid detection of porcine delta coronavirus IgA antibodies to solve the problems existing in the above-mentioned prior art.

[0005] In order to solve the above technical problems, the present invention provides the following solutions:

[0006] The present invention provides an indirect ELISA kit for rapid detection of porcine delta coronavirus IgA antibodies, comprising an ELISA plate coated with an antigen;

[0007] The antigen is a recombinant PDCoV S2 protein, and its amino acid sequence is shown in SEQ ID NO.2.

[0008] Furthermore, the method for preparing the antigen-coated ELISA plate comprises the following steps:

[0009] The suspension of the recombinant PDCoV-S2 protein was added to the ELISA well, incubated in a 37°C incubator for 30 min, the supernatant was removed, washed 3 times with PBST and the supernatant was discarded, a blocking solution was added, and the plate was blocked at 37°C in a shaker for 2 h to obtain the antigen-coated ELISA plate.

[0010] Furthermore, the concentration of the recombinant PDCoV-S2 protein in the suspension is 0.125-2.0 μg / mL.

[0011] Preferably, the concentration of the recombinant PDCoV-S2 protein in the suspension is 1 μg / mL.

[0012] Furthermore, the blocking solution is a 1-5 g / L skim milk powder PBS solution.

[0013] Preferably, the blocking solution is a 5 g / L skim milk powder PBS solution.

[0014] Furthermore, the indirect ELISA kit also includes an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a stop solution, a negative standard substance and a positive standard substance.

[0015] Furthermore, the enzyme-labeled secondary antibody is goat anti-pig IgA labeled with HRP.

[0016] Furthermore, the diluent is a 0.01M PBS buffer with a pH of 7.4 and containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300; and the washing solution is a 0.01M PBST buffer with a pH of 7.4 and containing 0.5% Tween 20.

[0017] Furthermore, the color developing solution is TMB solution.

[0018] Furthermore, the stop solution is a 2 mol / L H2SO4 solution.

[0019] The present invention also provides a method for detecting porcine delta coronavirus IgA antibodies for non-disease diagnosis purposes, comprising the steps of using the above-mentioned indirect ELISA kit to detect porcine delta coronavirus IgA antibodies:

[0020] Add 100 μL of diluent, negative standard, positive standard, and sample solution to the antigen-coated ELISA plate, incubate at 37°C for 30 min, and rinse with washing solution three times after incubation.

[0021] Add diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 30 min. After incubation, rinse three times with washing solution.

[0022] Add colorimetric solution to each reaction well and incubate at 37°C with the light off for 10 min;

[0023] Stop solution was added to each reaction well, and the absorbance was measured at a wavelength of 450 nm using an enzyme reader;

[0024] Result judgment: When OD 450nm When OD is ≥0.273, it is judged as positive, that is, the sample solution contains porcine delta coronavirus IgA antibodies; 450nm When <0.273, it is judged as negative, that is, the sample fluid to be tested does not contain porcine delta coronavirus IgA antibodies.

[0025] The present invention discloses the following technical effects:

[0026] The invention designs primers according to the S2 gene sequence of the epidemic strain PDCoV HNZK-04 strain (GenBank accession number: MH708124), amplifies the PDCoV S2 gene, constructs a recombinant expression plasmid pET-32a(+), transforms Escherichia coli BL21, optimizes the expression conditions, obtains the recombinant PDCoV S2 protein, and purifies it by Ni-NTA affinity chromatography. The ELISA plate made with the recombinant PDCoV S2 protein as the coating antigen captures the PDCoV-specific IgA antibody, and the goat anti-swine IgA labeled with horseradish peroxidase is used as the detection antibody, and a "PDCoV-S2 protein-PDCoV-S2 specific IgA antibody-enzyme labeled antibody" trimer complex can be formed on the ELISA plate, and the result can be directly determined by the determination of the substrate color change and the absorbance value.

[0027] The kit constructed based on the indirect ELISA detection technology of the present invention can strictly control the overall reaction time within 1.5-2h under the condition of ensuring high sensitivity and strong specificity, greatly shortening the operation time and improving the detection efficiency. The present invention optimizes the parameter conditions of different reagents in the kit and finally obtains the optimal detection conditions: the optimal coating antigen concentration is 1μg / mL, the optimal blocking solution is 5% skim milk powder, the blocking time is 2h, the optimal enzyme-labeled antibody (1mg / mL) dilution is 1:60000, and the optimal color development time is 10min. The results of detecting porcine delta coronavirus IgA antibodies under this optimal condition show that when OD 450 nm When OD is ≥0.273, it is judged as positive, that is, the sample to be tested contains porcine delta coronavirus IgA antibodies; when OD 450nm When the value is less than 0.273, it is judged as negative, that is, the sample to be tested does not contain porcine delta coronavirus IgA antibody. The invention is convenient and easy to operate, efficient and low in cost, and the detection specificity reaches 100%. 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 The results of PDCoV S2 gene electrophoresis are shown in Figure 1. Lane 1: PDCoV S2; Lane 2: negative control;

[0030] Figure 2 It is the double restriction enzyme digestion verification of the recombinant plasmid pET32-PDCoV-S2; among them, lane 1: undigested plasmid; lane 2: pET32-PDCoV-S2 plasmid identified by double restriction enzyme digestion of BamH I and EcoR I;

[0031] Figure 3 This is the SDS-PAGE analysis of PDCoV S2 protein; wherein, lane 1: uninduced whole bacteria; lane 2: induced whole bacteria; lane 3: supernatant after induction; lane 4: precipitate after induction;

[0032] Figure 4 This is the SDS-PAGE analysis of the purified PDCoV S2 protein; wherein, lane 1: purified S2 protein;

[0033] Figure 5 Western blotting identification of purified PDCoV S2 protein; Lane 1: PDCoV positive serum; Lane 2: PDCoV negative serum;

[0034] Figure 6 The results of PDCoV S2 protein dosage optimization were obtained;

[0035] Figure 7 Optimize the blocking solution conditions.

[0036] Figure 8 Optimize the results for enzyme-labeled antibody conditions;

[0037] Fig. 9 Optimize results for color development time;

[0038] Fig.10 It is the result of specific test;

[0039] Fig.11 This is the result of the sensitivity test. 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 those skilled in the art. 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. Preparation of PDCoV-S2 protein

[0047] a. According to the S2 gene sequence of PDCoV HNZK-04 strain (GenBank accession number: MH708124), a pair of primers for amplifying the gene were designed using the Primmer5.0 software. The primer information is shown in Table 1. The cDNA of the correctly sequenced PDCoV HNZK-04 strain preserved by the Animal Molecular Pathogens Team of Henan Agricultural University was used as a template, and F-BamH I and R-EcoR I were designed as primers to PCR amplify the S2 gene. The PCR reaction system is shown in Table 2. After the mixed system is centrifuged and mixed, it is placed in the PCR instrument for amplification. The reaction procedure is: 95°C pre-denaturation for 30s, 95°C denaturation for 15s, 57°C annealing for 15s, 72°C extension for 60s, a total of 32 cycles, 72°C thorough extension for 5min, and 4°C end.

[0048] After amplification, 6 μL of PCR product was detected by 1% agarose gel electrophoresis ( Figure 1 ) and the product was recovered by gel. The purified double-enzyme PCR product and the pET-32a empty vector were double-enzyme digested with restriction endonucleases EcoR I and Xho I, and the recombinant plasmid pET-32a-PDCoV-S2 was obtained by ligation with T4 ligase ((The double-enzyme digestion verification results of the recombinant plasmid pET32-PDCoV-S2 are shown in Figure 2 ), the recombinant plasmid pET-32a-PDCoV-S2 was transferred into DH5α, and the positive recombinant strain BL21-S2 was screened on LB medium agar plates containing ampicillin (50 μg / mL), and the bacteria were enriched in LB liquid medium. After the enrichment, the strains were lyophilized and stored at -80°C for later use.

[0049] Table 1 Primer information

[0050]

[0051] Table 2 PCR reaction system

[0052]

[0053]

[0054] b. Take the recombinant Escherichia coli BL21-S2 expressing PDCoV-S2 protein (the coding gene sequence of the protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2) and streak it on LB agar plate (containing 50 μg / mL ampicillin), culture it at 37°C for 16 hours, pick a single colony and inoculate it in LB liquid medium (containing 50 μg / mL ampicillin), and culture it at 37°C, 180 r / min until OD 600nmAfter reaching 0.7-0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.5mmol / L aseptically, shake and induce at 180r / min in a shaker at 37℃ for 8 hours, and then lyse by ultrasonic lysis in an ice bath to obtain PDCoV-S2 protein. SDS-PAGE detection should show a band of about 70KDa ( Figure 3 ).

[0055] SEQ ID NO.1:

[0056]

[0057] SEQ ID NO.2:

[0058] .

[0059] c. Dissolve the protein with 8M urea solution, and then replace the high concentration urea solution with 6M, 4M, 3M, 2M, and 1M urea solutions. Finally, use 2mM imidazole PBS buffer (8.0g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24gKH2PO4 are dissolved in 800mL distilled water, and the solution is adjusted to 7.4 with HCl, and finally distilled water is added to 1L) as the cleaning buffer for the impurity protein, and 100mM imidazole concentration PBS buffer is used as the elution buffer for the PDCoV recombinant S2 protein.

[0060] d. After purification, high-purity PDCoV S2 protein was obtained, and SDS-PAGE observation showed a single clear band ( Figure 4), the protein concentration was determined to be 1000 μg / mL by Bradford method. The results of Western blotting of purified PDCoV S2 protein are shown in Figure 5 .

[0061] 2. Preparation of ELISA Plate

[0062] Add the diluted 20-0.1μg / mL PDCoV-S2 protein suspension to the ELISA wells, incubate in a 37°C incubator for 30 minutes, remove the supernatant, wash 3 times with PBST and discard the supernatant. Add blocking solution (5g skim milk powder (SM) dissolved in 950mL PBS, dilute to 1000mL with purified water) and block in a shaker at 37°C for 2h to occupy the gaps on the surface of the ELISA wells that are not bound to the PDCoV-S2 protein, and prepare an ELISA plate coated with the PDCoV-S2 protein.

[0063] Example 2

[0064] An indirect ELISA kit for rapid detection of porcine delta coronavirus IgA antibodies, comprising the following components:

[0065] ELISA plate: ELISA plate coated with PDCoV-S2 protein prepared in Example 1, with a coating antigen concentration of 1 μg / mL;

[0066] Enzyme-labeled secondary antibody: HRP-labeled goat anti-swine IgA;

[0067] Diluent: 0.01M PBS buffer, pH 7.4, containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300;

[0068] Washing solution: 0.01 M PBST buffer, pH 7.4, containing 0.5% Tween 20;

[0069] Color developing solution: TMB solution;

[0070] Stop solution: 2mol / LH2SO4 solution;

[0071] Negative standard: containing negative control serum of uninfected PDCoV antibody;

[0072] Positive standard: PDCoV antibody positive quality control serum.

[0073] Example 3

[0074] The method for detecting PDCoV IgA antibodies using the indirect ELISA kit of Example 2 comprises the following steps:

[0075] (1) diluting the milk or serum sample 64 times with a diluent and mixing thoroughly to obtain a sample solution; the diluent is a 0.01 M pH 7.4 PBS buffer containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300;

[0076] (2) Add 100 μL of diluent, negative standard, positive standard, and sample solution to the ELISA wells as blank group, negative control group, positive control group, and experimental group, respectively, and perform duplicate well experiments, then incubate at 37°C for 30 min;

[0077] The above positive standard is the PDCoV antibody positive quality control serum, which is dissolved in diluent before use;

[0078] The above negative standard is a negative control serum containing antibodies to uninfected PDCoV, which is dissolved in diluent before use;

[0079] (3) Discard the unbound sample solution and rinse three times with 200 μL of washing solution; the washing solution is 0.01 M PBST buffer with pH 7.4 containing 0.5% Tween 20;

[0080] (4) diluting the enzyme-labeled secondary antibody (HRP-labeled goat anti-swine IgA) with an original concentration of 1 mg / mL by 6000 times with a diluent, adding 100 μL of the diluted enzyme-labeled secondary antibody to each reaction well, and repeating step (3) for washing;

[0081] (5) Add 200 μL of TMB substrate solution (Shanghai Sangon Biotechnology Co., Ltd.) to each reaction well and incubate at 37°C in the dark for 10 min;

[0082] (6) Add 50 μL of stop solution (2 mol / L H2SO4 solution) to each reaction well, and measure the absorbance of each reaction well at a wavelength of 450 nm using an ELISA reader within 30 min;

[0083] (7) Result determination: 20 PDCoV-negative sera were tested, and the average value (X) and standard deviation (SD) of the absorbance at 450 nm were calculated. According to statistical data, when OD 450nm When ≥X+3SD, the serum sample is judged as positive, otherwise OD 450nm <X+3SD means negative.

[0084] Optimize different conditions in the above detection methods:

[0085] 1. Determination of the optimal antigen coating amount

[0086] Use coating solution to dilute PDCoV-S2 protein to the following concentrations: 0.125μg / mL, 0.25μg / mL, 0.5μg / mL, 1.0μg / mL, 2.0μg / mL. Add the diluted protein solution of different concentrations to the enzyme-labeled wells, 100μL per well, and then place in a 4℃ refrigerator for coating overnight. Follow the routine steps and select the best protein coating concentration by P / N value after color development. The results are as follows: Figure 6 As shown. According to the highest P / N value, the concentration of PDCoV-S2 protein is determined to be 1 μg / mL, so the concentration of the desired coating antigen selected by the present invention is 1 μg / mL.

[0087] 2. Determination of the Optimal Blocking Solution

[0088] Since different blocking solutions have different effects and the blocking effects of different blocking solution concentrations vary, the ELISA plate coated with the optimal protein concentration will be used for blocking. The type of blocking solution selected is the antigen coating concentration determined in step 1. After coating overnight, select the blocking solution: 1% BSA, 2.5% BSA, 5% BSA, 1% skim milk, 2.5% skim milk, 5% skim milk, 1% trehalose, 2.5% trehalose, 5% trehalose, and add 200 μL to each well. Place in a 37°C incubator for blocking for 2 hours. After the blocking is completed, perform subsequent routine operations, and finally select the best blocking solution based on the P / N value. The results are as follows Figure 7 As shown, from the calculated signal-to-noise ratio, it was found that the P / N value of 5% skim milk was the highest, so 5% skim milk was selected as the optimal blocking condition.

[0089] 3. Determination of the optimal dilution of the best secondary antibody

[0090] The enzyme-labeled antibody binds to the enzyme-labeled wells through the antigen-antibody binding reaction, which affects the test results. Therefore, the dilution of the enzyme-labeled antibody is optimized. The secondary antibody is diluted at a ratio of 1:10000, 1:20000, 1:40000, 1:60000, and 1:80000, respectively. Each dilution is repeated for 8 wells, and then the conventional steps are performed. OD is measured with an enzyme-labeled instrument 450nm The optimal dilution of the secondary antibody is selected with the maximum P / N value. Figure 8 As shown, the P / N value is the highest when the antibody is diluted 60,000 times, so the enzyme-labeled antibody dilution of 1:60,000 is selected as the best reaction condition.

[0091] 4. Determination of the optimal color development time

[0092] Horseradish peroxidase (HRP) can catalyze the oxidation of substrate TMB to oxTMB, which shows a blue reaction. The reaction is terminated by adding a stop solution. Therefore, the color development time is selected to be 5, 10, 20, and 30 minutes, and the OD is measured. 450nmTo calculate the P / N value, select the best color development time. Fig. 9 As shown, the optimal color development time of the present invention is 10 minutes.

[0093] 5. Determination of critical value

[0094] According to the method established under the optimized optimal reaction conditions, 20 PDCoV negative sera were tested. The results were calculated as the mean (X) and standard deviation (SD) of the absorbance value at 450nm. According to data statistics, when OD 450nm ≥0.273, the sample to be tested is judged to be positive, that is, the sample to be tested contains porcine delta coronavirus IgA antibodies; when OD 450nm When the value is less than 0.273, it is judged as negative, that is, the sample to be tested does not contain porcine delta coronavirus IgA antibodies.

[0095] Example 4

[0096] Specificity experiments:

[0097] The method established in Example 3 was used to detect positive sera for classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine circovirus type 2 (PCV2). At the same time, a standard positive control group and a standard negative control group were set up, and the specificity of the method was determined by measuring the absorbance value.

[0098] The results are as follows Fig.10 As shown, the antigen of the present invention specifically binds to the PDCoV-S2 IgA antibody and does not bind to other antibodies, so the detection method of the present invention has good specificity.

[0099] Example 5

[0100] Sensitivity test:

[0101] The PDCoV standard positive sample was diluted in multiples (from 1:2, 1:4, ..., until the reaction was negative, and each dilution was repeated 3 times), and then tested using the method established in Example 3. The results are shown in Figure 3. Fig.11 As shown, when the PDCoV positive samples were diluted to 1:2048 (V / V), the results were all positive, while when the positive sample dilution was 1:4096 (V / V), the detection result was negative. This shows that the detection method established by the present invention has high sensitivity.

[0102] Example 6

[0103] Intra-batch and inter-batch repeatability tests:

[0104] Randomly select 10 samples to be tested (5 positive samples and 5 negative samples), use the same batch of purified recombinant PDCoV-S2 protein to coat the plate, and use the indirect ELISA method established in Example 3 to test the 10 sera for 3 consecutive days for intra-batch repeatability test; use 3 batches of purified recombinant protein to coat the plate, and use the established indirect ELISA method to test the 10 sera for 3 consecutive days, and repeat 3 wells for each serum to do intra-batch repeatability test. Calculate OD 450nm The mean value, standard deviation and coefficient of variation were used to verify the repeatability of the method. The results are shown in Table 3. The coefficients of variation of intra-batch and inter-batch repeated tests were 1.538%-4.951% and 1.510%-7.367%, respectively, both less than 10%, indicating that the method has good repeatability and stability.

[0105] Table 3 Intra-batch and inter-batch repeatability results

[0106]

[0107]

[0108] 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 indirect ELISA kit for rapid detection of porcine delta coronavirus IgA antibodies, characterized in that: Including an ELISA plate coated with an antigen; The antigen is a recombinant PDCoV S2 protein, and its amino acid sequence is shown in SEQ ID NO.

2.

2. The indirect ELISA kit according to claim 1, characterized in that The method for preparing the antigen-coated ELISA plate comprises the following steps: The suspension of the recombinant PDCoV-S2 protein was added to the ELISA well, incubated in a 37°C incubator for 30 min, the supernatant was removed, washed 3 times with PBST and the supernatant was discarded, a blocking solution was added, and the plate was blocked at 37°C in a shaker for 2 h to obtain the antigen-coated ELISA plate.

3. The indirect ELISA kit according to claim 2, characterized in that The concentration of the recombinant PDCoV-S2 protein in the suspension was 1 μg / mL.

4. The indirect ELISA kit according to claim 2, characterized in that The blocking solution is a 5 g / L skim milk powder PBS solution.

5. The indirect ELISA kit according to claim 1, characterized in that The indirect ELISA kit also includes an enzyme-labeled secondary antibody, a diluent, a washing solution, a color developing solution, a stop solution, a negative standard substance and a positive standard substance.

6. The indirect ELISA kit according to claim 5, characterized in that The enzyme-labeled secondary antibody is a goat anti-swine IgA antibody labeled with HRP.

7. The indirect ELISA kit according to claim 5, characterized in that The diluent is a 0.01M PBS buffer with a pH of 7.4 and containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300; the washing solution is a 0.01M PBST buffer with a pH of 7.4 and containing 0.5% Tween 20.

8. The indirect ELISA kit according to claim 5, characterized in that The color developing solution is TMB solution.

9. The indirect ELISA kit according to claim 5, characterized in that The stop solution is 2 mol / L H2SO4 solution.

10. A method for detecting porcine delta coronavirus IgA antibodies for non-disease diagnosis purposes, characterized in that: The method comprises the steps of detecting porcine delta coronavirus IgA antibodies using the indirect ELISA kit according to any one of claims 1 to 9: Add 100 μL of diluent, negative standard, positive standard, and sample solution to the antigen-coated ELISA plate, incubate at 37°C for 30 min, and rinse with washing solution three times after incubation. Add diluted enzyme-labeled secondary antibody to each reaction well and incubate at 37°C for 30 min. After incubation, rinse with washing solution three times; Add colorimetric solution to each reaction well and incubate at 37°C with the light off for 10 min; Stop solution was added to each reaction well, and the absorbance was measured at a wavelength of 450 nm using an enzyme reader; Result judgment: When OD 450nm When OD is ≥0.273, it is judged as positive, that is, the sample solution contains porcine delta coronavirus IgA antibodies; 450nm When <0.273, it is judged as negative, that is, the sample fluid to be tested does not contain porcine delta coronavirus IgA antibodies.

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