An indirect elisa kit for rapid detection of porcine epidemic diarrhea virus IgA antibody and application thereof

Abstract

The application discloses an indirect ELISA kit for rapidly detecting porcine epidemic diarrhea virus IgA antibody and application thereof and belongs to the technical field of immunology. The application provides a recombinant PEDV-RBD protein, and an amino acid sequence of the recombinant PEDV-RBD protein is shown as SEQ ID NO. 3. The application constructs an indirect ELISA kit for detecting porcine epidemic diarrhea virus IgA antibody based on the recombinant PEDV-RBD protein, can accurately capture PEDV IgA antibody in sow milk or serum to realize detection, has the advantages of high sensitivity and strong specificity, and provides a technical means for prevention and control of porcine epidemic diarrhea.

Description

Technical Field

[0001] This invention relates to the field of immunology, and in particular to an indirect ELISA kit for rapid detection of porcine epidemic diarrhea virus IgA antibodies and its application. Background Technology

[0002] Porcine epidemic diarrhea (PED) is a highly contagious intestinal disease caused by porcine epidemic diarrhea virus (PEDV), posing a serious threat to the global pig industry. PEDV is primarily transmitted via the fecal-oral route. Infection in piglets can cause severe watery diarrhea, vomiting, and dehydration, with extremely high mortality rates. Particularly during the cold winter and spring seasons, PEDV spreads rapidly, and pigs of all ages are susceptible to infection, especially young suckling piglets, where morbidity and mortality rates can reach 100%. Therefore, rapid and accurate detection of PEDV IgA antibodies in pigs is of great significance for disease control, immunization efficacy evaluation, and vaccine development.

[0003] Currently, the main methods for controlling PED include vaccination and improved husbandry practices. However, the effectiveness of vaccines largely depends on antibody levels in the pig herd, particularly IgA antibodies in colostrum, which play a crucial protective role for newborn piglets. Therefore, developing a rapid and accurate method for detecting IgA antibodies in PED is of great significance for evaluating vaccine efficacy, developing immunization programs, and conducting epidemiological surveys. Summary of the Invention

[0004] The purpose of this invention is to provide an indirect ELISA kit for rapid detection of porcine epidemic diarrhea virus (PEDV) IgA antibodies and its application, thereby addressing the problems existing in the prior art. This invention constructs an indirect ELISA kit for detecting PEDV IgA antibodies based on recombinant PEDV-RBD protein. This kit accurately captures PEDV IgA antibodies in porcine sow's milk or serum for detection, exhibiting high sensitivity and strong specificity, thus providing a technical means for the prevention and control of porcine epidemic diarrhea.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a recombinant PEDV-RBD protein, the amino acid sequence of which is shown in SEQ ID NO.3.

[0007] The present invention also provides the encoding gene of the above-mentioned recombinant PEDV-RBD protein, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0008] In this invention, the gene encoding the recombinant PEDV-RBD protein is obtained by PCR amplification using the cDNA of PEDV HN2021 strain as a template.

[0009] The primer pairs for the PCR amplification are shown in SEQ ID NO.1-2.

[0010] The PCR amplification reaction system is as follows: KOD One™ PCR Master Mix 25 μL, template DNA 2 μL, primer F 1 μL, primer R 1 μL, ddH2O 21 μL.

[0011] The PCR amplification reaction program is as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s, for a total of 32 cycles, 72℃ complete extension for 5 min, and 4℃ end.

[0012] The present invention also provides the use of the above-mentioned recombinant PEDV-RBD protein in the preparation of reagents or kits for detecting porcine epidemic diarrhea virus IgA antibodies.

[0013] Preferably, the reagent or kit is used to detect porcine epidemic diarrhea virus IgA antibodies in breast milk or serum.

[0014] The present invention also provides an indirect ELISA kit for detecting porcine epidemic diarrhea virus IgA antibodies, wherein the kit contains an enzyme-labeled plate coated with the above-mentioned recombinant PEDV-RBD protein.

[0015] Preferably, the coating concentration of the recombinant PEDV-RBD protein is 0.5-2 μg / mL.

[0016] The coating time for the recombinant PEDV-RBD protein was 2 hours, and the coating temperature was 37°C.

[0017] Preferably, the blocking solution used for coating the recombinant PEDV-RBD protein is 2.5% BSA.

[0018] The sealing liquid is applied at a temperature of 4°C for 14-16 hours.

[0019] Preferably, the kit further includes an HRP-labeled goat anti-porcine IgA solution, a diluent, a washing solution, a TMB substrate solution, and a stop solution.

[0020] The diluent is a 0.01M pH 7.4 PBS buffer containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300.

[0021] The washing solution is a 0.01M pH 7.4 PBST buffer containing 0.5% Tween 20.

[0022] The terminating solution is a 2 mol / L H2SO4 solution.

[0023] Preferably, the concentration of the HRP-labeled goat anti-porcine IgA solution is 12.5-50 ng / mL.

[0024] Preferably, when using the kit, after adding the TMB substrate solution, the reaction is allowed to proceed for 15-30 minutes, and then the stop solution is added to terminate the reaction.

[0025] The detection principle of the kit is as follows: the sample to be tested is co-incubated with an ELISA plate coated with recombinant PEDV-RBD protein in the kit. The specific antibody in the serum is accurately captured by the PEDV-RBD protein. The HRP-labeled goat anti-pig IgA antibody is used as the detection antibody and binds to the specific antibody captured by the PEDV-RBD protein in the ELISA plate to form a "PEDV-RBD protein-PEDV-RBD specific IgA antibody-enzyme-labeled antibody" trimer complex. After the TMB substrate solution is added, a color reaction occurs, and the result can be directly determined by measuring the absorbance value.

[0026] The present invention discloses the following technical effects:

[0027] This invention utilizes porcine epidemic diarrhea virus (PEDV) strain HN2021 to prepare recombinant PEDV-RBD protein. Based on this recombinant protein, an indirect ELISA kit for detecting PEDV IgA antibodies is established. While ensuring high sensitivity and strong specificity, the overall reaction time is strictly controlled within 1.5-2 hours, significantly shortening the operation time and improving detection efficiency. This invention further improves the sensitivity of the kit by optimizing the parameters of different reagents. 450nm When ≥0.3195, the sample is considered positive, meaning it contains porcine epidemic diarrhea virus IgA antibody; when OD... 450nm A value <0.3195 is considered negative, meaning the sample does not contain porcine epidemic diarrhea virus IgA antibodies. This invention is convenient, easy to operate, efficient, and low-cost, with 100% detection specificity, providing a technical means for the prevention and control of porcine epidemic diarrhea. Attached Figure Description

[0028] 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.

[0029] Figure 1 The results of double enzyme digestion of recombinant plasmid pET-32a-PEDV-RBD are shown in the following: lane M: marker; lane 1: undigested plasmid; lane 2: pET32-PEDV-RBD plasmid identified by double enzyme digestion with BamHI and EcoRI.

[0030] Figure 2 SDS-PAGE analysis results of purified PEDV-RBD protein, where lane M: marker; lane 1: purified RBD protein;

[0031] Figure 3 The results of Western blotting identification of purified PEDV-RBD protein are shown. Lane M: Marker; Lane 1: Purified RBD protein.

[0032] Figure 4 The results show the optimized coating concentration of recombinant PEDV RBD protein;

[0033] Figure 5 The results show the optimization of the type of sealing liquid;

[0034] Figure 6 The results show the optimized dilution of the enzyme-labeled secondary antibody;

[0035] Figure 7 The result is the optimized color development time;

[0036] Figure 8 ROC curve plot for determining the critical value;

[0037] Figure 9 This represents the specific detection results of the indirect ELISA detection method;

[0038] Figure 10 This represents the sensitivity test results for the indirect ELISA detection method. Detailed Implementation

[0039] 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.

[0040] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0041] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0042] 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 readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0043] 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.

[0044] The technical solution of the present invention is as follows:

[0045] Primers were designed based on the RBD gene sequence of PEDV HN2021 strain published in GenBank, and the RBD gene was amplified. The RBD gene was ligated to a vector to obtain a recombinant vector. The recombinant vector was then transformed into Escherichia coli to construct a recombinant bacterium. After the recombinant bacterium was propagated, it was induced to express by IPTG. The recombinant bacterium after induction was then broken up and purified to isolate the recombinant PEDV-RBD protein.

[0046] An indirect ELISA detection method was established using recombinant PEDV-RBD protein as the antigen. The method was validated by optimizing the antigen coating amount, blocking buffer type, secondary antibody dilution, and color development time. Finally, the established indirect ELISA detection method was validated in terms of specificity, sensitivity, and repeatability.

[0047] A primer is a macromolecule with a specific nucleotide sequence that is stimulated to synthesize at the initiation of nucleotide polymerization, and is usually linked to the reactant by hydrogen bonds. Primers are typically two artificially synthesized oligonucleotide sequences. In some embodiments of this invention, the primers are designed using Primmer 5.0 software.

[0048] "Vector" refers to a self-replicating DNA molecule used in recombinant DNA technology to transfer a DNA fragment (target gene) into a recipient cell. In some embodiments of the present invention, the vector may be selected from bacterial plasmids, bacteriophages, and animal and plant viruses; in other embodiments of the present invention, the vector is selected from bacterial plasmids, and further selected from pET-32a vector.

[0049] A "recombinant vector" refers to a DNA sequence capable of inserting a foreign DNA sequence and undergoing autonomous replication, typically composed of DNA fragments from two or more different sources. In some embodiments of the present invention, the recombinant vector is obtained by recombination and ligation of an RBD gene with a vector. The recombination and ligation method can be selected from enzyme digestion ligation, homologous recombination, and overlap extension PCR. In other embodiments of the present invention, the recombination and ligation method is selected from enzyme digestion ligation, and further selected from the method using EcoR1 and HindIII as restriction enzyme sites and employing T4 ligase for ligation.

[0050] "Transduction" refers to the process of transferring DNA or RNA from one cell to another. It is one of the ways in which bacteria transfer genetic material. In some embodiments of the present invention, the transduction method may be selected from heat shock, electroporation, electroporation, microinjection, gene gun, and virus-mediated transduction; in other embodiments of the present invention, the transduction method is selected from heat shock.

[0051] "Induced expression" refers to the process by which a foreign gene is activated or enhanced under certain conditions. This expression mode usually depends on a specific inducer, such as a metabolite, chemical substance, or other environmental signal. In some embodiments of the present invention, the inducer may be selected from a chemical substance; in other embodiments of the present invention, the inducer is selected from IPTG (isopropyl-β-D-thiogalactopyranoside).

[0052] The "indirect ELISA detection method" works by attaching an antigen to a solid-phase carrier. The antibody to be tested in the sample binds to this carrier, forming a solid-phase antigen-antibody complex. Then, an enzyme-labeled secondary antibody (an antibody targeting the antibody to be tested) binds to the antibody in the solid-phase immune complex, forming a solid-phase antigen-antibody-enzyme-labeled secondary antibody complex. The degree of color development after substrate addition can be visually inspected or quantitatively measured using a spectrophotometer to determine the content of the antibody to be tested. In some embodiments of this invention, the enzyme-labeled secondary antibody may be selected from enzyme-labeled goat anti-porcine IgA antibody, and further selected from HRP-labeled goat anti-porcine IgA antibody.

[0053] The sources of some reagents and biological materials involved in the embodiments of this invention have been specified, but this is not intended to limit the technical solution of this invention, and they can also be purchased through conventional channels; the sources of reagents and biological materials not specified are all commercially available products and can be purchased through conventional channels.

[0054] Example 1: Preparation of recombinant PEDV-RBD protein

[0055] 1. Preparation of PEDV-RBD protein

[0056] a. Based on the RBD gene sequence of PEDV HN2021 strain (GenBank accession number: OR707084.1), a pair of primers was designed using Primmer 5.0 software. Primer information is shown in Table 1. Using the correctly sequenced cDNA of PEDV HN2021 strain preserved by the Animal Molecular Pathogen Team of Henan Agricultural University as a template, the RBD gene was amplified by PCR using primers PEDV RBD F and PEDV RBD R. The PCR reaction system is shown in Table 2. After centrifugation, the mixture was placed in a PCR instrument for amplification. The reaction program was as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 15 s, for a total of 32 cycles, with a final extension at 72℃ for 5 min, and a final step at 4℃. After amplification, 6 μL of PCR product was detected by 1% agarose gel electrophoresis and the product was recovered from the gel. The purified double-digested PCR product and the empty pET-32a vector were double-digested using restriction endonucleases Ecor I and Hind III. The recombinant plasmid pET-32a-PEDV-RBD was then ligated using T4 ligase to obtain the recombinant plasmid. The resulting recombinant plasmid pET-32a-PEDV-RBD was then double-digested with BamHI and EcoRI. The digestion products were verified by gel electrophoresis, and the verification results are shown below. Figure 1 As shown, the recombinant plasmid was successfully constructed.

[0057] The recombinant plasmid pET-32a-PEDV-RBD was transformed into the DH5α strain. The recombinant strain BL21-N was screened using LB agar plates containing ampicillin (50 μg / mL). The strain was then enriched in LB liquid medium. After enrichment, the strain was freeze-dried and stored at -80℃ for later use.

[0058] Table 1 Primer Information

[0059]

[0060]

[0061] Table 2 PCR reaction system

[0062] KODOne™ PCRMasterMix 25μL Template DNA 2μL Primer F 1μL Primer R 1μL <![CDATA[ddH2O]]> 21μL Total volume 50μL

[0063] b. Recombinant Escherichia coli BL21-N expressing PEDV-RBD protein was streaked onto LB agar plates (containing 50 μg / mL ampicillin) and incubated at 37°C for 16–20 h. Single colonies were picked and inoculated into LB liquid medium (containing 50 μg / mL ampicillin) and incubated at 37°C and 180 rpm until OD500. 600nm After reaching a concentration of 0.7–0.8, IPTG was aseptically added to a final concentration of 0.1 mmol / L. After inducing the protein expression by shaking at 180 r / min in a shaker at 37°C for 6–8 hours, the protein was lysed by sonication in an ice bath to obtain PEDV-RBD protein. The appearance of a band of approximately 55–70 kDa by SDS-PAGE indicated successful induction of expression.

[0064] c. Collect the successfully induced bacterial culture by centrifugation, collect the bacterial cells, wash three times with pre-cooled PBS or Buffer A solution, and then rinse with an appropriate amount of PBS or Buffer A solution (50 mmol / L). -1 Tris, 0.5 mmol / L -1 EDTA, 50 mmol / L -1 Resuspend the bacterial cells in NaCl. Disrupt the cells 4-5 times using a pressure disruptor, centrifuge, and remove the supernatant. In an ice-water bath, wash with an appropriate amount of inclusion body washing buffer (1 mol / L). -1 Tris-HCL, 1% Triton X-100, 1mmol L -1Wash the precipitate with EDTA (adjust volume to 500 mL), stir thoroughly, centrifuge, and discard the supernatant. Repeat 3 times. Wash the precipitate with 2 mol / L urea in an ice-water bath, stir thoroughly for 10 min, and discard the supernatant. Wash the precipitate with 1 mol / L sodium chloride solution in an ice-water bath, stir thoroughly for 10 min, centrifuge, and discard the supernatant. Denaturation treatment: Denature inclusion body proteins with 8 mol / L urea and 0.5 M DTT at 4°C for 2 h. After protein renaturation, centrifuge and retain the supernatant. Add 1 mL of oxidized glutathione to the denatured protein solution, followed by 1 mL of reduced glutathione, and renature in an ice bath for 2 h. Dialyze the renatured protein solution overnight at 4°C with 4 M urea. The next day, change the dialysate (4 M urea) and dialyze for 12 h, then change to 2 M urea and dialyze overnight.

[0065] d. After purification, high-purity recombinant PEDV-RBD protein was obtained. SDS-PAGE observation showed a single, clear band, as shown in the image. Figure 2 As shown, the Western blotting results are as follows: Figure 3 As shown, the concentration of this protein was determined to be 2 mg / mL using the Bradford method.

[0066] The amino acid sequence of the recombinant PEDV-RBD protein is shown in SEQ ID NO.3, and the gene sequence encoding the protein is shown in SEQ ID NO.4.

[0067] SEQ ID NO.3:

[0068] GISYQPCTANCIGYAANVFATEPNGHIPEGFSFNNWFLLSNDSTLVHGKVVSNQPLLVNCLLAIPKIYGLGQFFSFNQTIDGVCNGAAVQRAPEALRFNINDSSVILAEGSIVLHTALGTNFSFVCSNSSNPHLATFAIPLGATQVPYYCFFKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLDAVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQRTTIQRILYCDDPVSQLKCSQVAFDLDDGFYTISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANLIASATTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVTDVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDS。

[0069] SEQ ID NO.4:

[0070]

[0071] Example 2: Preparation of PEDV antibody-positive control serum

[0072] Healthy pigs aged 3-5 weeks that tested negative for both PEDV antigen and antibody were used to administer recombinant PEDV HN2021 inactivated virus (TCID). 50 =10 7 The vaccine was prepared by emulsifying a mixture of 0.1 mL of the positive serum and Sepik 206 adjuvant, followed by intramuscular immunization of pigs twice in the neck. Serum was collected after passing the initial test. The isolated positive serum was then used to prepare positive control serum. The prepared positive control serum was tested, and the results showed that all serum samples were sterile, and the PEDV neutralization test titer was ≥1:8 in five repeated tests, indicating that the prepared positive control serum was of acceptable quality and suitable for subsequent research.

[0073] Example 2: Establishment of an Indirect ELISA Detection Method

[0074] 1. Preliminary establishment of detection methods

[0075] (1) Add the diluted 0.25-4 μg / mL PEDV-RBD protein suspension to the wells of the enzyme labeling plate and incubate at 37℃ for 2 h. Remove the supernatant, wash three times with PBST and discard the supernatant. Add a certain amount of blocking buffer (2.5 g BSA dissolved in 95 mL PBS, and bring the volume to 100 mL with purified water) and block at 4℃ for 14-16 h. The purpose is to occupy the gaps on the surface of the wells that are not bound to PEDV-RBD protein to obtain the enzyme labeling plate.

[0076] (2) Dilute the milk or serum sample 64 times with diluent and mix thoroughly to obtain the sample solution; the above diluent is 0.01M pH7.4 PBS buffer containing 10% bovine serum albumin and 0.1% liquid biological preservative Proclin-300.

[0077] (3) Add 100 μL of diluent, standard solution, negative standard, positive standard and sample solution to the well containing enzyme label, respectively, as blank group, standard group, negative control group, positive control group and experimental group, and perform replicate experiment, and then incubate at 37℃ for 60 min.

[0078] The above positive standard is PEDV antibody positive control serum (obtained by infecting pigs with PEDV HN2021 strain, titer 1 / 400), which is dissolved in sample diluent before use.

[0079] The above negative standard is a negative control serum containing antibodies against uninfected PEDV. It should be dissolved in sample diluent before use.

[0080] (4) Discard the unbound sample solution and rinse 3 times with 200 μL of washing solution; the washing solution is 0.01 M pH 7.4 PBST buffer containing 0.5% Tween 20.

[0081] (5) Dilute the original concentration of 1 mg / mL enzyme-labeled secondary antibody (HRP-labeled goat anti-pig IgA antibody, purchased from abcam) by 10,000-80,000 times with diluent, add 100 μL of the diluted enzyme-labeled secondary antibody to each well, and repeat step (4) for washing.

[0082] (6) Add 100 μL of TMB substrate solution (Sangon Biotech (Shanghai) Co., Ltd.) to each well and incubate at 37°C in the dark for 15 min.

[0083] (7) Add 50 μL of stop solution (2 mol / L H2SO4 solution) to each well. Within 30 min, measure the absorbance of each centrifuge tube at a wavelength of 450 nm using an ELISA reader.

[0084] (8) Result Interpretation: 150 PEDV serum samples were tested, and ROC curves were plotted accordingly. The highest Youden index was selected as the criterion, with a corresponding sensitivity and specificity of 92.2% and 98.0%, respectively. The corresponding absorbance value was 0.3195. When the OD... 450nm A serum sample is considered positive if its OD value is ≥0.3195, otherwise it is considered positive. 450nm A value less than 0.3195 indicates a negative result.

[0085] 3. Optimize different conditions in the detection method.

[0086] (1) Determination of the optimal antigen coating amount

[0087] PEDV-RBD protein was diluted with coating buffer to concentrations of 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL. 100 μL of each diluted protein solution was added to each well and incubated at 37°C for 2 hours. Subsequent standard procedures were performed. After color development, the optimal protein coating concentration was determined using the P / N ratio. The results are shown below. Figure 4 As shown. Based on the highest P / N value, the concentration of PEDV-RBD protein was determined to be 2 μg / mL. Therefore, the required concentration of the coating antigen in this invention was selected as 2 μg / mL.

[0088] (2) Determination of the optimal sealing solution

[0089] Because different blocking solutions have different effects and their concentrations result in varying blocking efficiency, the optimal protein concentration for coating the microplate was determined before blocking. Using a predetermined antigen coating concentration, after overnight coating, the following blocking solutions were selected: 1% BSA, 2.5% BSA, 5% BSA, 1% skim milk, 2.5% skim milk, and 5% skim milk, with 200 μL added to each well. The plates were incubated at 4°C for 14-16 hours. After blocking, routine procedures were performed, and the optimal blocking solution was ultimately selected based on the P / N ratio. Results are as follows: Figure 5 As shown, the calculated P / N values ​​show that 2.5% BSA has the highest P / N value, therefore 2.5% BSA is selected as the optimal closure condition.

[0090] (3) Determination of the optimal dilution of the secondary antibody

[0091] Enzyme-labeled antibodies bind to the enzyme-labeled wells through an antigen-antibody binding reaction, thus affecting the detection results. Therefore, the dilution of the enzyme-labeled antibodies was optimized. Secondary antibodies were diluted at ratios of 1:10000, 1:20000, 1:40000, 1:60000, and 1:80000, with each dilution replicated in eight wells. After completing subsequent experiments, the OD was measured using an enzyme-labeled immunosorbent assay (ELISA) reader. 450nm The optimal dilution for the secondary antibody was selected based on the highest P / N value. The results are as follows: Figure 6 As shown, the P / N value is highest when the antibody is diluted 40,000 times. Therefore, the optimal reaction conditions are selected when the enzyme-labeled antibody is diluted 1 / 40,000.

[0092] (4) Determining the optimal color development time

[0093] Horseradish peroxidase (HRP) catalyzes the oxidation of the substrate TMB to oxTMB, resulting in a blue reaction. This reaction is terminated by adding a stop solution. Therefore, the color development time was selected as 5, 10, 15, 20, and 30 minutes, and the reaction was observed by measuring the OD. 450nm The P / N value is calculated to select the optimal color development time. The results are as follows: Figure 7 As shown, the optimal color development time of the present invention is 15 min.

[0094] (5) Determination of critical values

[0095] Based on the optimized reaction conditions (optimal coating antigen concentration of 2 μg / mL, optimal blocking buffer of 2.5% BSA, blocking time of 2 h, optimal enzyme-labeled antibody dilution of 1 mg / mL of 1 / 40000, and optimal color development time of 15 min), an indirect ELISA method was established. 150 clinical serum samples with PEDV were tested, and ROC curves were plotted accordingly. Figure 8As shown, the largest Youden index was selected as the basis, with a corresponding sensitivity of 92.2% and a specificity of 98.0%, while the corresponding absorbance value was 0.3195, the area under the curve was 0.9845, and the 95% confidence interval was 0.9684 to 1.000. Therefore, when OD... 450nm A value greater than 0.3195 is considered positive, while a value less than 0.3195 is considered negative.

[0096] Example 3: Validation of the indirect ELISA detection method

[0097] 1. Specificity test

[0098] Positive sera for PDCoV, TGEV, CSFV, PRV, PEDV, TGEV, PRRSV, PCV2, and PSV (preserved by the Animal Molecular Pathogen Team of Henan Agricultural University, all with titers of 1 / 400) were detected using the method established in Example 2. Standard positive control groups and standard negative control groups were also established, and the specificity of the method was determined by measuring the absorbance values.

[0099] The results are as follows Figure 9 As shown, this detection method can specifically detect PEDV-RBD antibodies but cannot recognize other antibodies. Therefore, the detection method established in this invention has good specificity.

[0100] 2. Sensitivity Test

[0101] The PEDV standard positive sample (original titer 1 / 400) was serially diluted and tested. Serial dilutions were performed at ratios of 1:2, 1:4, 1:8, ..., until a negative reaction was obtained. Each dilution was repeated in triplicate. Results are as follows: Figure 10 As shown, when PEDV positive samples were diluted to 1:1024 (V / V), all results were positive, while when the positive samples were diluted to 1:2048 (V / V), the detection result was negative. Therefore, the detection method established in this invention has high sensitivity.

[0102] 3. Intra-batch and inter-batch repeatability tests:

[0103] Under the same reaction conditions as described above, 10 samples (5 positive and 5 negative) were randomly selected. The same batch of purified recombinant protein was used to coat the ELISA plate, and the 10 serum samples were tested using the established indirect ELISA method for 3 consecutive days for intra-batch repeatability testing. Three batches of purified recombinant protein were used to coat the ELISA plate, and the same batch of purified recombinant protein was used to coat the 10 serum samples for 3 consecutive days for inter-batch repeatability testing, with each serum sample tested in triplicate. OD was calculated. 450nm The mean, standard deviation, and coefficient of variation were used to verify the repeatability of the method.

[0104] The results are shown in Table 3. The coefficients of variation for intra-batch and inter-batch repeated tests were 1.895%-7.637% and 3.233%-9.6367%, respectively, both less than 10%. The detection method of the present invention has good repeatability and stability.

[0105] Table 3 Detection Results

[0106]

[0107]

[0108] 4. Clinical Application

[0109] (1) 92 clinically randomly collected pig serum samples were compared and identified using commercially available kits, and 43 of them were positive and 49 were negative.

[0110] The identified serum samples were tested using the detection method of this invention. The results are shown in Table 4. The results showed that 56 serum samples were positive and 36 were negative. The detection results of this invention showed a concordance rate of 85.9% with the commercially available kit (IDEXXPEDVIgA antibody detection kit).

[0111] Table 4 Test Results

[0112]

[0113] (2) 92 samples of pig milk collected randomly in clinical settings were compared and identified using commercially available kits. 91 samples were positive and 1 sample was negative.

[0114] The identified breast milk samples were tested using the detection method of this invention. The results are shown in Table 5. The results showed that 79 samples were positive and 13 samples were negative. The detection results of this invention showed a concordance rate of 86.9% with the commercially available kit (IDEXXPEDVIgA antibody detection kit).

[0115] Table 5 Test Results

[0116]

[0117] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Use of a recombinant PEDV-RBD protein in the preparation of a reagent or kit for detecting porcine epidemic diarrhea virus IgA antibody. The amino acid sequence of the recombinant PEDV-RBD protein is shown as SEQ ID NO. 3; the nucleotide sequence of the encoding gene of the recombinant PEDV-RBD protein is shown as SEQ ID NO.

4.

2. Use according to claim 1, characterized in that, The reagent or kit is used for detecting porcine epidemic diarrhea virus IgA antibody in breast milk or serum.

3. An indirect ELISA kit for detecting IgA antibody to porcine epidemic diarrhea virus, characterized by, The kit contains an enzyme-labeled plate coated with the recombinant PEDV-RBD protein as claimed in claim 1.

4. The kit of claim 3, wherein The coating concentration of the recombinant PEDV-RBD protein is 0.5-2 μg / mL.

5. The kit of claim 3, wherein The blocking solution when coating the recombinant PEDV-RBD protein is 2.5% BSA.

6. The kit of claim 3, wherein The kit further comprises a HRP-labeled goat anti-pig IgA solution, a diluent, a washing solution, a TMB substrate solution and a termination solution.

7. The kit of claim 6, wherein The concentration of the HRP-labeled goat anti-pig IgA solution is 12.5-50 ng / mL.

8. The kit of claim 6, wherein When the kit is used, after adding the TMB substrate solution, the reaction is carried out for 15-30 min, and the termination solution is added to terminate the reaction.

Images