An elisa method for detecting porcine epidemic diarrhea virus antibody sIgA

Abstract

The application discloses an ELISA method for detecting porcine epidemic diarrhea virus antibody SIgA and belongs to the technical field of biology. The method comprises the steps of taking S1 protein as a coating antigen and detecting porcine epidemic diarrhea virus antibody SIgA in a sample to be detected, wherein the amino acid sequence of the S1 protein is a truncated sequence from the 19th to the 719th. The application preliminarily establishes an indirect ELISA method for detecting porcine epidemic diarrhea virus specific SIgA antibodies in serum, intestinal tissue and milk by optimizing conditions, and determines that the optimal antigen coating concentration and coating time are 0.25 mu g / ml and 4 DEG C overnight 12h-16h respectively, and the optimal detection conditions are determined. The method for detecting PEDV specific SIgA in serum, intestinal tissue and milk established in the research is simple and rapid, has good sensitivity and specificity, and provides a basis and a detection standard for evaluating the mucosal immune level of PEDV.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an ELISA method for detecting porcine epidemic diarrhea virus antibody SIgA. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV). It is characterized by severe watery diarrhea, vomiting, and dehydration in piglets, and is most prevalent in autumn and winter, often causing high mortality rates in piglets. Clinically, PEDV frequently co-infects with other viruses such as transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus (PDCoV), causing significant economic losses to the global pig farming industry.

[0003] PEDV is a type of coronavirus with four structural proteins (spiking (S) protein, membrane (M) protein, envelope (E) protein, and nucleocapsid (N) protein). The S protein is the main antigenic determinant of PEDV, composed of two subunits: S1 (1–789 aa) and S2 (790–1383 aa). This protein can induce the production of neutralizing antibodies and plays an important role in the body's immune protection. The S1 subunit contains multiple antigenic epitopes and receptor-binding domains, enabling it to recognize and bind to specific receptors on the host cell surface; it is the main functional region of the S protein. The S2 subunit mainly mediates the fusion of the virus with the host cell membrane. Therefore, the S1 region can serve as a good candidate protein for the preparation of antibody detection ELISA kits.

[0004] PEDV is primarily transmitted through the respiratory and digestive tracts, causing lesions in mucosal epithelial cells. Secretory immunoglobulin A (SIgA) is a major effector factor of mucosal immunity, mainly distributed in the mucus of the digestive, respiratory, and reproductive tracts of animals. Currently, there are many studies on prevention and control through mucosal immunity. For example, the inactivated vaccine for TGEV, administered orally, expresses dendritic cell-targeting probiotic peptides and porcine epidemic diarrhea virus (COE) antigen, producing good immunoprotective effects. Therefore, SIgA has become an important indicator for detecting and assessing the level of mucosal immune antiviral activity. However, current methods and criteria for PEDV SIgA detection mostly target specific samples such as serum, intestinal tissue, or milk, and suffer from low sensitivity, making widespread application difficult. Therefore, establishing a safe, stable, broad-spectrum, and highly sensitive ELISA detection method is of significant clinical importance for assessing the level of PEDV mucosal immunity. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention proposes an ELISA method for detecting porcine epidemic diarrhea virus (PEDV) antibody SIgA. Using S1 protein as the coating antigen, an indirect ELISA method is established that can specifically detect PEDV SIgA. This method can be used with high sensitivity for the detection of porcine epidemic diarrhea virus.

[0006] To achieve the above objectives, the present invention provides an ELISA method for detecting porcine epidemic diarrhea virus antibody SIgA, comprising the steps of using S1 protein as a coating antigen and detecting porcine epidemic diarrhea virus antibody SIgA in the sample to be tested, wherein the amino acid sequence of the S1 protein is a truncated sequence from position 19 to 719.

[0007] Preferably, the ELISA method specifically includes the following steps:

[0008] (1) Coating antigen: Add the coating antigen S1 protein and coating solution to the ELISA plate and coat overnight;

[0009] (2) Sealing: Use 5% skim milk powder as the sealing solution and seal at 37℃ for 1-3 hours;

[0010] (3) Sample addition: Add diluted serum, feces or milk sample and incubate;

[0011] (4) Add primary antibody: Dilute the primary antibody at a volume ratio of 1:(20-200) and add it, then incubate for 30-120 min;

[0012] (5) Add enzyme-labeled antibody: Dilute the secondary antibody at a volume ratio of 1:(10000-25000) and add it, then incubate for 30-90 min;

[0013] (6) TMD color rendering;

[0014] (7) Terminate the reaction.

[0015] After each of the first five steps, the plate should be washed with PBST.

[0016] Preferably, in step (1), the coating concentration of the coated antigen S1 protein is 0.0625-2 μg / mL, more preferably 0.25 μg / mL; the overnight coating is performed at 4°C for 12-16 h.

[0017] Preferably, in step (1), the S1 protein is fused with a trimerizing tag Fd, and the amino acid sequence of Fd is shown in SEQ ID NO: 1.

[0018] Preferably, in step (2), the sealing conditions are: sealing at 37°C for 1 hour.

[0019] Preferably, in step (3), serum and milk samples are diluted at a volume ratio of 1:(100-400), and fecal samples are diluted at a volume ratio of 1:(5-100). The incubation conditions are: 37°C for 1 hour.

[0020] Preferably, in step (4), the dilution ratio of the primary antibody is 1:200 or 1:20, and the incubation conditions are: incubation at 37°C for 0.5 h;

[0021] In step (5), the enzyme-labeled antibody is diluted at a ratio of 1:20000 and incubated at 37°C for 0.5 hours.

[0022] The present invention also provides an ELISA kit for detecting porcine epidemic diarrhea virus antibody SIgA, comprising an enzyme-labeled plate coated with S1 protein, wherein the amino acid sequence of the S1 protein is a truncated sequence from position 19 to 719, and the S1 protein is a trimerized protein fused with a trimerizing tag.

[0023] Preferably, the kit further includes washing solution, blocking solution, TMD chromogenic solution, stop solution, positive control, and negative control.

[0024] Preferably, the blocking solution is 5% skim milk powder; the positive control is porcine epidemic diarrhea virus (PEDV) SIgA antibody-positive porcine serum, feces, and milk samples, and the negative control is porcine epidemic diarrhea virus (PEDV) SIgA antibody-negative porcine serum, feces, and milk samples.

[0025] Compared with the prior art, the present invention has the following advantages and technical effects:

[0026] This invention clones the PEDV S1 gene (19-719aa) into the pcDNA3.1 vector and expresses the PEDV S1 protein in the mammalian cell expression system 293F. Expressing the S1 protein using the mammalian cell expression system maintains the spatial conformation of the antigen. After purification, the protein, analyzed by SDS-PAGE and Western blot, can be used to coat antigens in an indirect ELISA method. Specifically, this invention uses S1 protein as the coating antigen and preliminarily establishes an indirect ELISA for detecting PEDV-specific SIgA antibodies in serum and intestinal tissue through optimized conditions. The optimal antigen coating concentration and coating time were determined to be 0.25 μg / ml and overnight (12h-16h) at 4°C, respectively; the optimal dilution for serum and milk was 1:200; the optimal dilution for intestinal tissue was 1:20; the optimal blocking solution and optimal blocking time were 5% skim milk powder and 0.5h at 37°C, respectively; the optimal incubation time for serum, intestinal tissue, and milk was 0.5h at 37°C; and the optimal dilution and optimal incubation time for enzyme-labeled secondary antibody were 1:20000 and 0.5h at 37°C. Finally, this method was used to detect serum, intestinal tissue, and milk samples from pigs, and the concordance rates with the neutralization experiment results were 94.4%, 97.8%, and 93.5%, respectively. The method for detecting PEDV-specific SIgA in serum, intestinal tissue, and breast milk established in this invention is simple and rapid, with good sensitivity and specificity. This provides a basis and detection standard for evaluating the mucosal immune level of PEDV, offers new ideas for PEDV immunological diagnosis, and provides a scientific tool for the prevention and control of PEDV. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0028] Figure 1 SDS-PAGE analysis and Western blot identification results for PEDV S1 protein; M: marker, PEDV-S1: PEDV-S1 protein;

[0029] Figure 2 For the definition of the critical value for the indirect ELISA method;

[0030] Figure 3 This is a specific detection result from indirect ELISA;

[0031] Figure 4This is the result of sensitivity testing using indirect ELISA. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0034] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0035] The following examples involve the following materials: 293F cells, recombinant expression plasmid pcDNA3.1-S1, PEDV negative serum, PEDV positive serum, PEDV negative intestinal sample, PEDV, TGEV, PDCoV, and rotavirus (RV) positive intestinal sample; antisera against atypical pneumonia virus (APPV), TGEV, PDCoV, RV, and porcine reproductive and respiratory syndrome virus (PRRSV) were all prepared and preserved in our laboratory, and the clinical serum to be tested was collected by our laboratory from multiple pig farms in Jiangsu Province.

[0036] Some reagents and equipment: Tans5α competent cells were purchased from Beijing TransGen Biotech Co., Ltd.; endotoxin-free plasmid extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd.; Opti-MEM was purchased from Gibco, USA; Sinofection Transfection Reagent and SMM 293-TII Expression Medium were both purchased from Beijing Sinobiological Co., Ltd.; trypan blue staining agent (0.4%) was purchased from Invitrogen, USA; HRP-labeled goat anti-pig IgA was purchased from Bethyl Laboratories, USA, and TMB substrate chromogenic solution was purchased from Huzhou Yingchuang Biotechnology Co., Ltd.; cell shake flasks were purchased from Guangzhou Jet Biofil Co., Ltd.; CO2-tolerant level shaker (CO-06U) was purchased from Seiki Co., Ltd., USA; HisTrap HP protein purification column was purchased from Cytiva, USA; BCA protein concentration assay kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0037] Example 1: Establishment of PEDV SIgA Detection Method

[0038] 1. Eukaryotic expression and purification of S1 protein from PEDV mutant strain

[0039] The recombinant eukaryotic expression plasmid pcDNA3.1-S1 was synthesized by GenScript and used the pcDNA3.1 vector. The expressed protein is amino acids 19-719 of the AH2012 / 12 strain (GeneBank accession number: KU646831) S1 protein. A trimerizing tag Fd (amino acid sequence — GYIPEAPRDGQAYVRKDGEWVLLSTFL, SEQ ID NO: 1) was fused to the protein to trimerize it, mimicking the native conformation of the viral S protein. Finally, a His tag was fused to the carboxyl terminus to facilitate protein purification.

[0040] pcDNA3.1-S1 was transfected into 293F cells using a transient transfection method. One day prior to transfection, cell density was sampled and counted, cell viability was calculated, and cells were diluted to 2 × 10⁶ cells / mL with fresh SMM 293-TII Expression Medium. 6 Cells / mL were cultured in a constant temperature shaker at 37℃, 5% CO2, and 150 rpm. On the day of transfection, samples were taken to count cell density and viability. When the cell density was 3-5 × 10⁶ cells / mL, the cell density was determined. 6 When the cell density is 3 × 10⁶ cells / mL and the viability is higher than 90%, adjust the cell density to 3 × 10⁶ cells / mL. 6 After reaching a cell / mL concentration, plasmid transfection was performed. 24 hours after transfection, the bottle cap was loosened to meet the dissolved oxygen and CO2 emission requirements for subsequent high-density cell growth. 96 hours post-transfection, the supernatant from the 293F-PEDV-S1 suspension cells was collected and processed using Ni... + After purification by column chromatography, protein concentration was determined using a BCA kit. The purified protein was desalted, aliquoted, and stored at -80°C. Western blotting was performed to identify the purified PEDV-S1 protein using His-tagged antibody (1:10000) as the primary antibody and goat anti-mouse HRP-IgG (1:10000) as the secondary antibody.

[0041] 2. Establishment and optimization of the indirect ELISA method

[0042] The purified S1 protein was diluted with PBS solution to a series of coating concentrations (2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.0625 μg / mL) using a matrix method. The dilution factors for PEDV-positive and PEDV-negative swine serum, positive and negative milk (1:100, 1:200, 1:400), PEDV-positive and PEDV-negative swine intestinal samples (1:5, 1:20, 1:50, 1:100), coating times (4℃ overnight, 37℃ 2h, 4℃ overnight + 37℃ 2h), and blocking solutions (1% gelatin, 1% BSA, 3% BSA, 5% BSA) were also determined. The optimal reaction conditions for this method were determined by optimizing the following parameters: BSA, 5% skim milk powder and non-blocked conditions (37℃ for 1h, 37℃ for 2h, 37℃ for 3h, room temperature for 1h, room temperature for 2h and 3h), serum and intestinal incubation time (30min, 45min, 60min, 90min, 120min), goat anti-porcine HRP-IgA (1:10000, 1:15000, 1:20000, 1:25000), secondary antibody incubation time (30min, 60min, 90min), and TMB color development time (5min, 10min, 15min, 20min).

[0043] 3. Determination of the critical value for negative samples

[0044] Forty PEDV-negative porcine serum samples, 15 negative intestinal samples, and 13 negative milk samples were tested under determined optimal reaction conditions, with each sample performed in triplicate. The OD values ​​of the negative samples were calculated. 450nm average and standard deviation (S), when At a statistically significant level, a result of 99.9% is considered positive; otherwise, it is considered negative.

[0045] 4. Specificity test

[0046] The indirect ELISA method established in this invention was used to detect TGEV, PDCoV, RV, APPV, and PRRSV positive sera, as well as TGEV, PDCoV, and RV positive intestinal samples and breast milk samples. Each positive serum and intestinal sample was tested in triplicate, and the average value was taken. PEDV positive and negative serum controls were included. OD was measured. 450nm The specificity of the method is determined by the value.

[0047] 5. Sensitivity test

[0048] Three PEDV-positive swine serum samples, three PEDV-positive swine intestinal samples, and three PEDV-positive swine milk samples preserved in this experiment were taken and serially diluted 2-fold (1:100 to 1:12800). The indirect ELISA method established in this invention was used for detection to determine the sensitivity of the method.

[0049] 6. Repeatability test

[0050] Six porcine serum samples (three PEDV-positive and three PEDV-negative sera), six porcine intestinal samples (three PEDV-positive and three PEDV-negative samples), and six porcine milk samples (three PEDV-positive and three PEDV-negative samples) were tested using ELISA reaction plates coated with the same batch. Each sample was tested three times. The above experiments were repeated using ELISA reaction plates coated with three different batches. The coefficients of variation for the intra-batch and inter-batch repeated experiments were calculated based on the test results to evaluate the repeatability of the method.

[0051] 7. Compliance Rate Test

[0052] The established ELISA method was used to detect 54 serum samples with different antibody titers, 46 intestinal samples with different antibody titers, and 31 breast milk samples with different antibody titers. The results were compared with those of the neutralization experiment to analyze the concordance rate of the method.

[0053] 8. Results and Analysis

[0054] 8.1 Expression and purification of pcDNA3.1-S1 protein

[0055] Sample collection via Ni + After column purification, the purified protein was identified by SDS-PAGE. The results showed a protein band at approximately 100 kDa, consistent with the Western blotting results (see [link to SDS-PAGE]). Figure 1 The protein concentration obtained by BCA method was 0.8 mg / mL.

[0056] 8.2 Determination of optimal reaction conditions for indirect ELISA

[0057] The optimal antigen coating concentration for the established ELISA method was 0.25 mg / L. Serum, intestinal and milk samples were diluted 1:200 (Table 1), 1:20 (Table 2) and 1:200 (Table 3), respectively. The optimal antigen coating time was overnight at 4°C (Table 4).

[0058] Table 1. Determination of optimal antigen coating concentration and optimal serum dilution.

[0059]

[0060] Table 2 Determination of optimal antigen coating concentration and optimal dilution of intestinal samples

[0061]

[0062] Table 3. Determination of Optimal Antigen Coating Concentration and Optimal Dilution of Milk Samples

[0063]

[0064]

[0065] Table 4 Determination of Optimal Antigen Coating Time

[0066]

[0067] The optimal sealing temperature and time are 37℃ for 30 min (Table 5, Table 6).

[0068] Table 5. Determination of the optimal blocking agent (serum)

[0069]

[0070] Table 6. Determination of Optimal Blocking Time (Serum)

[0071]

[0072]

[0073] The optimal action time for the primary antibody is 30 min (Table 7).

[0074] Table 7 Determination of Optimal Primary Antibody Incubation Time

[0075]

[0076] The optimal dilution concentration of the enzyme-labeled secondary antibody is 1:10000 (Table 8), and the optimal incubation time of the secondary antibody is 30 min (Table 9).

[0077] Table 8. Determination of Optimal Secondary Antibody Dilution Ratio (Serium)

[0078]

[0079]

[0080] Table 9. Determination of Optimal Secondary Antibody Reaction Time (Serium)

[0081]

[0082] The optimal color development time is 15 min (Table 10).

[0083] Table 10 Determination of Optimal Color Development Time

[0084]

[0085] In summary, based on the data obtained from Tables 1-10 above, the basic procedure for ELISA testing is shown in Table 11.

[0086] Table 11 Indirect ELISA Operation Procedure

[0087]

[0088]

[0089] 8.3 Determination of Critical Values

[0090] Using the optimized indirect ELISA method described above, 40 serum samples, 15 intestinal samples, and 13 breast milk samples were analyzed. The results are as follows: Figure 2 As shown, the statistical results can be calculated as follows:

[0091] The positive cutoff value for serum sample detection was 0.163; that is, the criterion for interpreting indirect ELISA detection results was: the average OD value of the two wells in the negative control was [value missing]. 450nm Value <0.14, average OD of the two wells in the positive control 450nm The detection result is valid when OD is ≥0.28. 450nm <0.14 is considered negative, 0.14≤OD 450nm <0.163 is considered suspicious, OD 450nm A value ≥0.163 is considered positive. If a suspected sample is retested and the value is still less than 0.163, it is considered negative.

[0092] The positive cutoff value for intestinal sample detection was 0.346; therefore, the criterion for interpreting indirect ELISA results was set as follows: the average OD value of the two negative control wells was [value missing]. 450nm Value <0.293, average OD of the two wells in the positive control 450nm The detection result is valid when the OD value is ≥0.586. 450nm <0.293 is considered negative, 0.293≤OD 450nm <0.346 is considered suspicious, OD 450nm A value ≥0.346 is considered positive. If a suspected sample is retested, and the value is still less than 0.346, it is considered negative.

[0093] The positive cutoff value for detecting milk samples was 0.24; therefore, the criterion for interpreting indirect ELISA results was set as follows: the average OD value of the two negative control wells was [value missing]. 450nm Value <0.19, average OD of the two wells in the positive control 450nm The detection result is valid when OD is ≥0.38. 450nm <0.19 is considered negative, 0.19≤OD 450nm<0.24 is considered suspicious, OD 450nm A value ≥0.24 is considered positive. If a suspected sample is retested and the value is still less than 0.24, it is considered negative.

[0094] 8.4 Analytical Specificity of Indirect ELISA Method

[0095] The indirect ELISA method established in this invention was used to detect positive serum samples and positive intestinal tissue samples for five swine pathogens (APPV, PRRSV, TGEV, RV, and PDCoV); positive milk samples for TGEV, RV, and PDCoV; and positive serum, intestinal, and milk samples for PEDV antibodies. The results are as follows: Figure 3 As shown, this method can specifically detect PEDV antibody in serum, intestinal tissue, and milk samples, and has good specificity.

[0096] 8.5 Analytical Sensitivity of Indirect ELISA Method

[0097] The established indirect ELISA method was used to detect serially diluted PEDV-positive serum, intestinal tissue, and breast milk samples (dilutions of 1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800). The results showed that this method could detect positive serum and breast milk samples at a dilution as low as 1:6400, and positive intestinal samples at a dilution as low as 1:800 (see [link to ELISA method]). Figure 4 ).

[0098] 8.6 Analytical repeatability of indirect ELISA method

[0099] The established indirect ELISA method was used to perform intra-assay and inter-assay repeatability tests on 6 serum samples, 6 intestinal samples, and 6 milk samples. The results (Tables 12-14) showed that the intra-assay coefficients of variation for serum, intestinal, and milk samples were all between 0.52% and 7.60%, and the inter-assay coefficients were all between 1.57% and 8.31%, indicating that the method has good repeatability.

[0100] Table 12. Repeatability of Indirect ELISA (Serium)

[0101]

[0102] Table 13. Repeatable experiments of indirect ELISA (intestinal tract)

[0103]

[0104] Table 14. Repeatable experiments of indirect ELISA (breast milk)

[0105]

[0106] 8.7 Compliance Rate Experiment

[0107] Using the indirect ELISA method established in this invention and a neutralization experiment, 54 porcine serum samples, 46 porcine intestinal samples, and 31 milk samples were simultaneously tested. The results showed that the overall concordance rate of serum was 94.4% (Table 15), the overall concordance rate of intestinal tissue was 97.8% (Table 16), and the overall concordance rate of milk was 93.5%.

[0108] Table 15 Concordance Rate Test for Indirect ELISA (Serium)

[0109]

[0110] Table 16 Concordance Rate Tests for Indirect ELISA (Intestinal)

[0111]

[0112] Table 17 Concordance Rate Test for Indirect ELISA (Breast Milk)

[0113]

[0114] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. An ELISA method for detecting porcine epidemic diarrhea virus antibody SIgA, characterized by, The method comprises the step of detecting the porcine epidemic diarrhea virus SIgA antibody in the sample to be tested by taking the S1 protein as a coated antigen, wherein the amino acid sequence of the S1 protein is a truncated sequence of 19-719 of the S1 protein of the AH2012 / 12 strain with the GeneBank accession number KU646831.

2. The ELISA method of claim 1, wherein, The ELISA method specifically comprises the following steps: (1) coating antigen: adding the coating antigen S1 protein and coating solution to an enzyme-labeled plate for coating overnight; (2) blocking: using 5% skimmed milk powder as a blocking solution, and blocking at 37°C for 1-3 hours; (3) sample addition: adding diluted serum, fecal or milk samples, and incubating; (4) first antibody addition: adding the diluted first antibody at a volume ratio of 1:(20-200), and incubating for 30-120 minutes; (5) enzyme-labeled antibody addition: adding the diluted second antibody at a volume ratio of 1:(10000-25000), and incubating for 30-90 minutes; (6) TMD color development; (7) reaction termination.

3. The ELISA method of claim 2, wherein, In step (1), the coating concentration of the coating antigen S1 protein is 0.0625-2 μg / mL; and the overnight coating is 12-16 hours at 4°C.

4. The ELISA method of claim 3, wherein, In step (1), the S1 protein is fused with a trimerization tag Fd, and the amino acid sequence of the Fd is shown in SEQ ID NO:

1.

5. The ELISA method of claim 2, wherein, In step (2), the blocking condition is 37°C for 1 hour.

6. The ELISA method of claim 2, wherein, In step (3), the serum and milk samples are diluted at a volume ratio of 1:(100-400), the fecal sample is diluted at a volume ratio of 1:(5-100), and the incubation condition is 37°C for 1 hour.

7. The ELISA method of claim 2, wherein, In step (4), the dilution ratio of the first antibody is 1:200 or 1:20, and the incubation condition is 37°C for 0.5 hours; In step (5), the dilution ratio of the enzyme-labeled antibody is 1:20000, and the incubation condition is 37°C for 0.5 hours.

8. An ELISA kit for detecting porcine epidemic diarrhea virus antibody SIgA, characterized by, The kit further comprises a washing solution, a blocking solution, a TMD color developing solution, a terminating solution, a positive control and a negative control.

9. The ELISA kit of claim 8, wherein The blocking solution is 5% skimmed milk powder; the positive control is a pig serum, fecal and milk sample positive for the porcine epidemic diarrhea virus SIgA antibody; and the negative control is a pig serum, fecal and milk sample negative for the porcine epidemic diarrhea virus SIgA antibody.

10. The ELISA kit of claim 9, wherein ​

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