Porcine epidemic diarrhea virus IgG antibody detection kit and preparation method and application thereof

Abstract

The invention provides a porcine epidemic diarrhea virus IgG antibody detection kit and a preparation method and application thereof, the porcine epidemic diarrhea virus IgG antibody detection kit comprises an antigen-coated plate, a sample diluent, a concentrated washing solution, an enzyme-labeled reagent, a developing solution, a stop solution, a positive control and a negative control, the antigen-coated plate is an enzyme-labeled plate coated with PEDV-S protein; the sample diluent is a 1 * PBST solution containing 1% of BSA and 10% of FBS; the concentrated washing liquid is a 20 * PBST solution; the enzyme-labeled reagent is a rabbit anti-pig IgG enzyme-labeled antibody; the color developing solution is a TMB single-component solution; the stop solution is a sulfuric acid solution. The kit provided by the invention has the advantages of high sensitivity, strong specificity, simple process and low manufacturing cost, and can be used for detecting the porcine epidemic diarrhea virus IgG antibody.

Description

Technical Field

[0001] The present invention relates to the fields of virus disease diagnosis technology and animal quarantine technology, and in particular to a detection kit for porcine epidemic diarrhea virus IgG antibody and a preparation method and application thereof. Background Art

[0002] Porcine epidemic diarrhea virus (PEDV) is a highly contagious disease with a high mortality rate, causing huge economic losses to pig farming. Neutralizing antibodies play a huge role in immune protection. Studies have shown that IgG antibodies against PDEV S protein show a high correlation with neutralizing antibodies. It is urgent to develop a detection kit for detecting IgG antibodies to evaluate the neutralizing antibody titer after vaccine immunization, so as to provide a scientific reference for monitoring the immune status of pig herds. Summary of the invention

[0003] The purpose of the present invention is to provide a detection kit for porcine epidemic diarrhea virus IgG antibody and its preparation method and application, which has the advantages of high sensitivity and strong specificity, simple process and low manufacturing cost.

[0004] In a first aspect, the present invention provides a detection kit for porcine epidemic diarrhea virus IgG antibody, the kit comprising an antigen-coated plate, a sample diluent, a concentrated washing solution, an enzyme-labeled reagent, a color developing solution, a stop solution, a positive control and a negative control, wherein:

[0005] The antigen-coated plate is an ELISA plate coated with PEDV-S protein;

[0006] The sample diluent is a 1×PBST (phosphate-buffered saline-Tween buffer) solution containing 1% BSA (bovine serum albumin) and 10% FBS (fetal bovine serum);

[0007] The concentrated washing solution is 20×PBST solution;

[0008] The enzyme-labeled reagent is rabbit anti-pig IgG enzyme-labeled antibody;

[0009] The color developing solution is a TMB single-component solution;

[0010] The stop solution is a sulfuric acid solution.

[0011] Further, the concentration of the PEDV-S protein coated on the antigen-coated plate is 0.2 μg / mL to 1.0 μg / mL;

[0012] The PEDV-S protein was independently expressed in the laboratory. The PEDV S protein sequence was obtained by amplifying the TPY7 strain provided by Zhaoqing Dahuanong Biopharmaceutical Co., Ltd. The amino acid sequence of the PEDV-S protein is shown in SEQ ID NO.1.

[0013] Furthermore, if the OD450nm reading of each negative control well is less than 0.3, the test is established and the result is valid.

[0014] Furthermore, the concentration of the sulfuric acid solution is 2M.

[0015] In a second aspect, the present invention further provides a method for preparing a detection kit for porcine epidemic diarrhea virus IgG antibody, characterized in that the preparation method comprises:

[0016] Prepare antigen-coated plates;

[0017] Prepare sample diluent, concentrated washing solution, enzyme-labeled antibody, color development solution and stop solution respectively;

[0018] Prepare positive and negative controls;

[0019] The prepared and configured antigen-coated plate, sample diluent, concentrated washing solution, enzyme-labeled reagent, color developing solution, stop solution, positive control and negative control are loaded into the detection kit.

[0020] In a third aspect, the present invention also proposes the use of a detection kit for porcine epidemic diarrhea virus IgG antibodies in the detection of porcine epidemic diarrhea virus IgG antibodies.

[0021] Furthermore, the application includes:

[0022] S1. Diluting the serum sample to be tested with a sample diluent;

[0023] S2. Add the diluted serum sample to be tested at 100 μL / well, and set up 2 positive and negative control wells at the same time;

[0024] S3, incubate at 25°C in dark for 30 min;

[0025] S4, dilute the concentrated washing solution, then add 300 μL / well of the diluted washing solution and wash the plate 3 times;

[0026] S5, add 100 μL of enzyme labeling reagent to each well;

[0027] S6. Incubate at 25°C in dark for 55 to 65 minutes;

[0028] S7, dilute the concentrated washing solution, then add 300 μL / well of the diluted washing solution and wash the plate 4 times;

[0029] S8. Add 100 μL / well of color development solution to each well and develop color at 25°C in the dark for 15±1 minutes.

[0030] S9, add 100 μL stop solution to each well and oscillate to mix;

[0031] S10, single wavelength determination, set the wavelength of the microplate reader to 450nm.

[0032] Furthermore, if the OD450nm reading of each negative control well is less than 0.3, the test is established and the result is valid.

[0033] Further, S / P value = (sample OD450nm value - negative control OD450nm average value) / (positive control OD450nm average value - negative control OD450nm average value);

[0034] Positive judgment: Sample S / P value>0.8 is considered positive;

[0035] Negative determination: Sample S / P value ≤ 0.8 is considered negative.

[0036] Furthermore, the serum sample to be tested is diluted 1:300 times with the diluent.

[0037] In summary, according to the above-mentioned porcine epidemic diarrhea virus IgG antibody detection kit, it includes an antigen-coated plate, a sample diluent, a concentrated washing solution, an enzyme-labeled reagent, a color developing solution, a stop solution, a positive control and a negative control. Among them, the antigen-coated plate is an enzyme-labeled plate coated with PEDV-S protein, and the high-purity S full-length protein expressed based on CHO is the coating source, which can effectively reduce non-specific binding, and the coating concentration is low. The enzyme-labeled antibody is a rabbit anti-swine IgG enzyme-labeled antibody, which adopts the indirect enzyme-linked immunosorbent assay principle, and the rabbit anti-swine IgG-HRP has cascade efficiency and high sensitivity. The sample diluent is prepared using a specific formula, which can effectively reduce non-specific binding, and the background value of negative samples is low. In addition, the kit provided by the present invention has high sensitivity, strong specificity, and high compliance rate with the neutralizing antibody titer. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the accompanying drawings:

[0039] Figure 1 (a) is a schematic diagram of the test results on the sample dilution ratio in one embodiment of the present invention;

[0040] Figure 1 (b) is a schematic diagram of the test results on the amount of enzyme conjugate added in one embodiment of the present invention;

[0041] Figure 1 (c) is a schematic diagram of the test results of the amount of TMB color developing solution added in one embodiment of the present invention;

[0042] Figure 1 (d) is a schematic diagram of the test results on the amount of stop solution added in one embodiment of the present invention;

[0043] Figure 1 (e) is a schematic diagram of the experimental results on reaction temperature in one embodiment of the present invention;

[0044] Figure 1 (f) is a schematic diagram of the test results of sample reaction time in one embodiment of the present invention;

[0045] Figure 1 (g) is a schematic diagram of the test results regarding the reaction time of the enzyme conjugate in one embodiment of the present invention;

[0046] Figure 1 (h) is a schematic diagram of the test results of the reaction time of TMB color developing solution in one embodiment of the present invention;

[0047] Figure 1 (i) is a schematic diagram of the test results of the post-termination placement time in one embodiment of the present invention;

[0048] Figure 2 This is a schematic diagram of the specific test results of the kit in one embodiment of the present invention;

[0049] Figure 3 Schematic diagram of comparison between a test kit and a S trimer coated plate in one embodiment of the present invention;

[0050] Figure 4 Schematic diagram of the consistency between the reagent kit and the S trimer coated plate in one embodiment of the present invention. DETAILED DESCRIPTION

[0051] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments. It should be noted that the embodiments and features in the embodiments of the present invention can be combined with each other without conflict.

[0052] The following detailed description is an exemplary description, which is intended to provide further detailed description of the present invention. Unless otherwise specified, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art to which the present invention belongs. The terms used in the present invention are only for describing specific embodiments, and are not intended to limit the exemplary embodiments according to the present invention.

[0053] Example 1

[0054] Preparation of PEDV positive and negative serum

[0055] 1.1) Preparation of negative control

[0056] Animals for preparation: 3 piglets at 3 days old. Blood was collected from the corresponding sows 7 days before delivery for PEDV, CSFV, PDCoV, PoRV, PCV2, PRRSV, and TGEV antibody screening, and healthy piglets born from all negative sows were selected.

[0057] Serum preparation and packaging: Pigs were killed by bleeding from the carotid artery, and the blood was placed in a sterile, clean conical flask. The precipitated serum was transferred to a centrifuge bottle and centrifuged at 3000r / min for 5 minutes. The supernatant was taken and mixed, and then sterilized by filtering with a 0.22μm filter membrane, and quantitatively packaged, 1mL / tube.

[0058] Preparation of negative control: dilute the negative serum 40 times with sample diluent, mix well, filter and sterilize with a 0.22 μm filter membrane, and quantitatively divide into 1 mL / tube or 3 mL / tube.

[0059] 1.2) Preparation of positive control

[0060] Animals for preparation: 3 piglets at 3 days old, blood samples were collected from sows 7 days before farrowing for PEDV, CSFV, PDCoV, PoRV, PCV2, PRRSV, and TGEV antibody screening, and healthy piglets born from all negative sows were selected;

[0061] Take commercial PEDV vaccine and dilute it according to the instructions;

[0062] Three pigs were injected with 1 mL of the recombinant vaccine at multiple points in the neck muscle. A second vaccination was performed 14 days later using the same method and dosage as the first vaccination. Blood was collected every 7 days after the second vaccination, and the serum collected was tested for PEDV antibodies using the neutralization method.

[0063] The serum titer was determined by neutralization method, and pigs with PEDV neutralizing antibodies not less than 1:128 were selected for positive serum preparation;

[0064] Carotid artery bleeding was performed on experimental pigs that met the conditions. The blood of each pig was stored in a sterilized conical flask. The precipitated serum was transferred to a centrifuge bottle and centrifuged at 3000r / min for 5 minutes. The supernatant was taken and mixed, then sterilized by filtration with a 0.22μm filter membrane, and quantitatively divided into 1mL / tube;

[0065] Use sample diluent to dilute the qualified positive serum into multiple ratios of 1:400, 1:800, 1:1600, 1:3200, 1:6400, and 1:12800. Use the manufactured kit for detection. Select the highest dilution multiple with OD450nm value ≥1.0, dilute the positive serum with sample diluent according to the dilution multiple, mix well, filter and sterilize with 0.22μm filter membrane, and quantitatively package in 1mL / tube or 3mL / tube.

[0066] Example 2

[0067] Preparation of porcine epidemic diarrhea virus IgG antibody detection kit (taking the coating concentration of 0.6 μg / mL as an example)

[0068] 2.1) Preparation of antigen-coated plates

[0069] The source and standard of the ELISA plate were purchased from Shenzhen Jincanhua ELISA plate, with a specification of 96 wells. Coating: Dilute the PEDV-S protein to 0.6 μg / mL with coating buffer, add it to the ELISA plate, 100 μL / well, and place it at 4°C for 12-16 hours. Take it out, discard the liquid in the well, wash it 3 times with washing solution, 300 μL / well, and pat it dry.

[0070] The sequence of the PEDV-S protein is as follows:

[0071] MKSLTYFWLFLPVLSTLSLPQDVTRCSANTNFRRFFSKFNVQAPAVVVLG

[0072] GYLPIGENQGVNSTWYCAGQHPTASGVHGIFLSHIRGGHGFEIGISQEPFDPSG

[0073] YQLYLHKATNGNTNATARLRICQFPSIKTLGPTADNDVTTGRNCLFNKAIPAH

[0074] MSEHSVVGITWDNDRVTVFSDKIYHFYFKNDWSRVATKCYNSGGCAMQYV

[0075] YEPTYYMLNVTSAGEDGISYQLCTANCIGYAANVFATEPNGHIPEGFSFNNWF

[0076] ILSNDSTLVHGKVVSNQPLLVNCLLAMPKIYGLGQFFSFNQTIDGVCNGAAVQ

[0077] RAPEALRFNINDTSVILAEGSIVLHTALGTNLSFVCSNSSDPHLATFTIPLGATQ

[0078] VPYYCFLKVDTYNSTVYKFLAVLPPTVREIVITKYGDVYVNGFGYLHLGLLD

[0079] AVTINFTGHGTDDDVSGFWTIASTNFVDALIEVQGTAIQRILYCDDPVSQLKCS

[0080] QVAFDLDDGFYPISSRNLLSHEQPISFVTLPSFNDHSFVNITVSASFGGHSGANL

[0081] IASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLS

[0082] FSKFCVSTNLLASACTIDLFGYPEFGSGVKLTSLYFQFTKGELITGTPKPLEGVT

[0083] DVSFMTLDVCTKYTIYGFKGEGIITLTNSSFLAGVYYTSDSGQLLAFKNVTSG

[0084] AVYSVTPCSFSEQAAYVDDDIVGVISSLSSSTFNSTRELPGFFYHSNDGSNCTEP

[0085] VLVYSNIGVCKSGSIGYVPSQSGQVKIAPTVTGNISIPTNFSMSIRTEYLQLYNT

[0086] PVSVDCATYVCNGNSRCKQLLTQYTAACKTIESALQLSARLESVEVNSMLTIS

[0087] EEALQLATISSFNGDGYNFTNVLGASVYDPASGRVVQKRSFIEDLLFNKVVTN

[0088] GLGTVDEDYKRCSNGRSVADLVCACQYYSGVMVLPGVVDAEKLHMYSASLIG

[0089] GMVLGGFTSAAALPFSYAVQARLNYLALQTDVLQRNQQMLAESFNSAIGNIT

[0090] SAFESVKGAISQTSKGLNTVAHALTKVQEVVNSQGAALTQLTVQLQHNFQAI

[0091] SSSIDDIYSRLDILSADVQIDRLITGRLSALNAFVAQTLTKYTEVQASRKLAQQ

[0092] KVNECVKSQSQRYGFCGGDGEHIFSLVQAAPQGLLFLHTVLVPGDFVDVIAIA

[0093] GLCVNDEIALTLREPGLVLFTHELQNHTATEYFVSSRRMFEPRKPTVSDFVQIE

[0094] SCVVTYVNLLTRDQLPEVIPDYIDVNKTLDEILASLPNRTGPSLSLDVFNATYLN

[0095] LTGEIADLEQRSESLRNTTEELQSLIYNINNTLVDLEWLNRVETYIKWPWWVW

[0096] LIIFIVLIFVVSLLVFCCISTGCCGCCGCCGACFSGCCRGPRLQPYEAFEKVHVQ.

[0097] Blocking: Add blocking solution (weigh 0.27g potassium dihydrogen phosphate, 0.2g potassium chloride, 3.58g sodium dihydrogen phosphate dodecahydrate, 8.0g sodium chloride, dissolve in 800mL double distilled water, add 0.5mL Tween-20, ProClin300 with a final concentration of 0.01% (m / V) and BSA with a final concentration of 2% (m / V), adjust the pH to 7.2 after dissolution, then add double distilled water to 1L, mix well, filter with 0.22μm filter membrane for sterilization, and quantitatively divide into packages) 200μL / well to the ELISA plate, and incubate in a 37℃ wet box for 4 hours. Remove and discard the blocking solution, wash 3 times with washing solution, 300μL / well. Dry the ELISA plate and rack it, and dry it at 37℃ for 30 minutes. Bag the ELISA plate and desiccant into an aluminum foil bag and seal it after vacuum.

[0098] 2.2) Prepare sample diluent, concentrated washing solution, enzyme-labeled antibody, colorimetric solution and stop solution

[0099] 2.2.1) Prepare sample diluent as 1×PBST solution containing 1% BSA and 10% FBS: weigh 0.27 g potassium dihydrogen phosphate, 0.2 g potassium chloride, 3.58 g sodium hydrogen phosphate dodecahydrate, and 8.0 g sodium chloride, dissolve in 800 mL double distilled water, add 0.05% Tween-20 (V / V), a final concentration of 0.1% preservative ProClin300sigma (V / V), and a final concentration of 1% BSA (m / V) and 10% FBS (m / V), add double distilled water to 1 L, mix well, filter through a 0.22 μm filter membrane for sterilization, and quantitatively distribute.

[0100] 2.2.2) Prepare concentrated washing solution as 20×PBST solution: weigh 6.8 g potassium dihydrogen phosphate, 5 g potassium chloride, 89.5 g sodium hydrogen phosphate dodecahydrate, and 198.7 g sodium chloride, dissolve in 800 mL double distilled water, add 1.25% Tween-20 (V / V) and a final concentration of 0.1% preservative ProClin300 (V / V), add double distilled water to 1 L, mix well, filter through a 0.22 μm filter membrane for sterilization, and quantitatively distribute.

[0101] 2.2.3) Prepare the enzyme-labeled reagent as rabbit anti-swine IgG enzyme-labeled antibody: The antibody was purchased from Thermo Fisher Scientific (China) Co., Ltd. The horseradish peroxidase-labeled rabbit anti-swine IgG was diluted 1:30,000 (V / V) with sample diluent, sterilized by filtration with a 0.22 μm filter membrane, and quantitatively packaged.

[0102] 2.2.4) The color developing solution was TMB single-component solution: purchased from Guangzhou Weining Biotechnology Co., Ltd.

[0103] 2.2.5) Stop solution: Slowly add 55 mL of concentrated sulfuric acid into a beaker containing 445 mL of double distilled water, stir to mix, and dispense quantitatively.

[0104] 2.3) Determination of reagent dosage

[0105] 2.3.1) Determination of sample dilution ratio

[0106] Use sample diluent to dilute 5 in-house quality control products (QP1#, QP2#, QP3#, QN1, QN2) from the original multiple. By analyzing the OD value of the in-house quality control products, select the best sample diluent and sample loading amount. Figure 1 (a) It can be seen that when the sample dilution ratio is >1:300, the sensitivity is high but the specificity is poor; when the sample dilution ratio is <1:300, the specificity is good but the sensitivity is low. In summary, in order to ensure the sensitivity and specificity of the kit, the sample dilution ratio is set to 1:300, that is, 299μl of sample diluent is added, and then 1μl of sample is added.

[0107] 2.3.2) Determination of the amount of enzyme conjugate added

[0108] HRP-labeled PEDV antigen was made into enzyme conjugate at a determined concentration, and five internal quality control products, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. When adding enzyme conjugate, 40μl / well, 60μl / well, 80μl / well, 100μl / well, 120μl / well, 140μl / well, 160μl / well, and 180μl / well were added to the enzyme conjugate, respectively. The optimal amount of enzyme conjugate added was determined by the OD value of the internal quality control product. Figure 1 (b) It can be seen that when the amount of enzyme conjugate added is >100μl, the sensitivity is high, but the specificity is poor; when the amount of enzyme conjugate added is <100μl, the sensitivity is low. Based on the above analysis, and in order to ensure product quality, the amount of enzyme conjugate added is set to 100μl.

[0109] 2.3.3) Determination of the amount of TMB colorimetric solution added

[0110] Five internal quality control products, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. When adding TMB colorimetric solution, 50μl, 100μl, 150μl, and 200μl were added to each well, respectively. The optimal amount of TMB colorimetric solution was determined based on the OD value of the internal quality control products. Figure 1 (c) It can be seen that when the amount of TMB colorimetric solution added is >≥100 μl, the OD value tends to be stable, and the amount of TMB colorimetric solution added is set to 100 μl.

[0111] 2.3.4) Determination of the amount of stop solution added

[0112] Five internal quality control products, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. When adding the stop solution, 50μl, 100μl, 150μl, and 200μl were added to each well, respectively. The optimal amount of stop solution was determined based on the OD value of the internal quality control product. Figure 1 (d) It can be seen that when the amount of stop solution added is ≥90μl, the OD values ​​of the five quality control samples have basically no change, indicating that the reaction can be terminated by adding 90μl of stop solution. In order to ensure product quality and take into account the convenience of operation, the amount of stop solution added is set to 100μl.

[0113] 3) Determination of reaction conditions

[0114] 3.1) Determination of the reaction temperature of the coated plate after sample addition

[0115] Five internal quality control products, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. The reactions were performed at 4°C, 16°C, 25°C, and 37°C, respectively. The optimal reaction temperature was determined based on the OD values ​​of the internal quality control products. Figure 1 (e) It can be seen that when the reaction temperature is 16°C and 25°C, the specificity is good but the sensitivity is low; at 37°C, the sensitivity is high but the specificity is poor; at 25°C, the sensitivity is high and the specificity is good. In summary, the reaction temperature is set to 25°C.

[0116] 3.2) Determination of sample reaction time

[0117] Five internal quality control samples, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. After adding samples, they were incubated at 25°C for 15, 20, 25, 30, 45, and 60 minutes, respectively. The optimal sample reaction time was determined based on the OD value of the internal quality control samples. Figure 1 (f) It can be seen that when the sample reaction time is 30 minutes, the sensitivity is high and the specificity is good. Based on the above analysis, the sample reaction time is set to 30 minutes.

[0118] 3.3) Determination of enzyme conjugate reaction time

[0119] Five internal quality control samples, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. After adding the enzyme conjugate, they were incubated at 25°C for 30, 40, 50, 60, 70, and 80 minutes, respectively. The optimal enzyme conjugate reaction time was determined based on the OD value of the internal quality control sample. Figure 1 (g) It can be seen that when the enzyme conjugate reaction time is <60 minutes, the specificity is good but the sensitivity is low; when the reaction time is >60 minutes, the sensitivity is high but the specificity is poor. Based on the above analysis and in order to ensure product quality, the enzyme conjugate reaction time is set to 60 minutes.

[0120] 3.4) Determination of TMB colorimetric solution reaction time

[0121] Five internal quality control samples, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. After adding substrate A solution and substrate B solution, they were incubated at 25°C for 5, 10, 15, 20, 25, and 30 minutes, respectively. The optimal TMB colorimetric solution reaction time was determined based on the OD value of the internal quality control samples. Figure 1 (h) It can be seen that when the reaction time of TMB colorimetric solution is ≥10 minutes, the OD values ​​of the three positive quality control products tend to be flat, and the OD values ​​of the two negative quality control products are less than 0.1, indicating good specificity. Based on the above analysis, and in order to ensure product quality, the reaction time of TMB colorimetric solution is set to 15 minutes.

[0122] 3.5) Stability after reaction termination

[0123] Five internal quality control products, QP1#, QP2#, QP3#, QN1, and QN2, were used as research materials. The OD values ​​were read at 0, 5, 10, 15, 20, and 25 minutes after the addition of the stop solution. The OD values ​​of the internal quality control products were used to determine the effective time for reading the value after the reaction was terminated. Figure 1 (i) It can be seen that when the standing time after termination is ≥15 minutes, the OD values ​​of the three positive quality control products begin to decrease significantly. Therefore, in order to ensure the accuracy of the experimental results, the values ​​should be read within 15 minutes after termination.

[0124] 4) Preparation of positive and negative controls

[0125] 5) The prepared and configured antigen-coated plate, sample diluent, concentrated washing solution, enzyme-labeled reagent, colorimetric solution, stop solution, positive control and negative control are loaded into the detection kit. The main components of the detection kit are shown in Table 1.

[0126] Table 1 Main components of the test kit

[0127] Main ingredients Specification quantity Main ingredients Specification quantity Sealed coated plate 96-well 5 pieces Concentrated detergent 200mL 1 bottle Positive Control 5mL 1 bottle TMB colorimetric solution 60mL 1 bottle Negative control 5mL 1 bottle Stop Solution 50mL 1 bottle Sample diluent 150mL 1 bottle sealing film open 5 Enzyme Labeling Reagents 50mL 1 bottle manual share 1

[0128] The above disclosure is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

[0129] Example 3

[0130] Specificity test

[0131] The established ELISA method was used to detect five known PEDV, PRV, CSFV, PPV, PRRSV and PDCoV positive sera respectively under the optimal conditions, and the IDEXX PEDV kit was used as a control to analyze the specificity of the kit. Figure 2 The results showed that except for the positive PEDV serum test, the positive serum tests of other pathogens were all negative, indicating that the kit had good specificity and no cross-reaction with other positive sera.

[0132] Example 4

[0133] Repeatability test

[0134] Three ELISA plates coated at the same time were used for intra-batch repeat tests, and seven different samples were tested under the same conditions and the test results were statistically analyzed. The test results showed that the CV value of the intra-batch repeat test was less than 5%, proving that the method has a certain degree of repeatability within the batch.

[0135] Table 2 Intra-batch coefficient of variation

[0136]

[0137] Three ELISA plates coated at different times were used for batch repetition test, and seven different samples were tested under the same conditions and the test results were statistically analyzed. The test results showed that the CV value of the batch repetition test was less than 5%, proving that the method has certain reproducibility between batches.

[0138] Table 3 Inter-batch coefficient of variation

[0139]

[0140] Example 5 Comparison with S trimer protein coated plate

[0141] The plate coated with S trimer protein as antigen was compared with the self-developed kit under the same parameters as the self-developed kit. 88 serum samples with different antibody titers were tested at the same time. The test results are as follows Figure 3 and Figure 4 As shown, the self-developed kit has good consistency with the S trimer protein coated plate, and the consistency coefficient (R 2 ) is 0.9644, which indicates that the full-length S coating source has the same activity as the S trimer and can be used as a good coating source for IgG antibody determination.

[0142] Example 6 Comparison with commercial kits

[0143] According to clinical experience, when the neutralizing antibody titer is greater than 1:128, the corresponding S / P value is greater than 2.0 (standard 1). 88 serum samples with different antibody titers were tested simultaneously using a self-developed kit and a commercial kit. The test results in Figure 4 show that the self-developed kit and the commercial kit have a similar compliance rate with the neutralizing antibody titer standard 1, which is about 81%. However, the S / P of serum samples with neutralizing antibody titers greater than 1:128 corresponding to the commercial kit are concentrated between 2.4 and 2.8, with no significant difference, and cannot well reflect the true relationship between neutralizing antibody titer and S / P. The test results of the self-developed kit show that the S / P values ​​of samples greater than 1:128 are between 2.1 and 3.9, and the S / P value differences of samples with different neutralizing antibody titers are more significant than those of the commercial kit, which better reflects the relationship between neutralizing antibody titer and S / P value.

[0144] Table 4 Comparison of self-developed kits and commercial kits

[0145]

[0146]

[0147] Example 7 Comparison with neutralizing antibody titer

[0148] The established ELISA kit was used to test 88 serum samples with different antibody titers, and the results were compared with those of the neutralizing antibody titers. The test results in Table 5 show that the self-developed kit has good consistency with the neutralizing antibody titers, with a consistency coefficient (R 2 ) is 0.6917.

[0149] Table 5 Comparison with neutralizing antibody titer

[0150]

[0151]

[0152] According to clinical experience, the neutralizing antibody titer is greater than 1:128, and the corresponding S / P value is greater than 2.0 (standard 1). An ELISA kit was established to detect 88 serum samples with different antibody titers, and compared with the test results of the neutralizing antibody titer. The test results in Table 4 show that the self-developed kit has a compliance rate of 81.8% with the neutralizing antibody titer (neutralizing antibody titer is greater than 1:128, and the corresponding S / P value is greater than 2.0). The S / P value has a good correlation coefficient with the neutralizing antibody titer, which is suitable for the determination of serum IgG antibody levels.

[0153] Based on the linear relationship between the S / P value and the neutralizing antibody titer and the compliance rate (neutralizing antibody titer greater than 1:128, corresponding to an S / P value greater than 2.0), this kit can be used for the evaluation of PEDV serum neutralizing antibodies, and the test result S / P value has a good guiding significance for clinical production.

[0154] The porcine epidemic diarrhea virus IgG antibody detection kit constructed by the present invention is used to detect porcine epidemic diarrhea virus IgG antibodies induced after pigs are immunized with porcine epidemic diarrhea vaccine or pigs are infected with porcine epidemic diarrhea. At the same time, the titer of serum neutralizing antibodies can be evaluated, which has a good guiding significance for clinical production.

[0155] In addition, the beneficial effects obtained by the present invention also include:

[0156] 1. The present invention uses a recombinant porcine epidemic diarrhea virus protein (S protein) that can be stably expressed as a coating antigen to establish a simple and specific indirect ELISA method.

[0157] 2. The present invention also provides a specific, sensitive, rapid and easy-to-operate kit for detecting porcine epidemic diarrhea virus IgG antibodies.

[0158] It is known from common technical knowledge that the present invention can be implemented by other embodiments that do not deviate from its spirit or essential features. Therefore, the above disclosed embodiments are only illustrative in all respects and are not exclusive. All changes within the scope of the present invention or within the scope equivalent to the present invention are included in the present invention.

[0159] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, ordinary technicians in the relevant field should understand that the specific implementation methods of the present invention can still be modified or replaced by equivalents. Any modification or equivalent replacement that does not depart from the spirit and scope of the present invention should be covered within the scope of protection of the claims of the present invention.

Claims

1. A detection kit for porcine epidemic diarrhea virus IgG antibody, characterized in that: The kit includes an antigen-coated plate, a sample diluent, a concentrated washing solution, an enzyme-labeled reagent, a color developing solution, a stop solution, a positive control, and a negative control, wherein: The antigen-coated plate is an ELISA plate coated with PEDV-S protein; The sample diluent is a 1×PBST solution containing 1% BSA and 10% FBS; The concentrated washing solution is 20×PBST solution; The enzyme-labeled reagent is rabbit anti-pig IgG enzyme-labeled antibody; The color developing solution is a TMB single-component solution; The stop solution is a sulfuric acid solution.

2. The detection kit for porcine epidemic diarrhea virus IgG antibody according to claim 1, characterized in that: The concentration of the PEDV-S protein coated on the antigen-coated plate is 0.2 μg / mL to 1.0 μg / mL; The amino acid sequence of the PEDV-S protein is shown in SEQ ID NO.

1.

3. The detection kit for porcine epidemic diarrhea virus IgG antibody according to claim 1, characterized in that: If the OD450nm reading of each negative control well is less than 0.3, the test is established and the result is valid.

4. The detection kit for porcine epidemic diarrhea virus IgG antibody according to claim 1, characterized in that: The concentration of the sulfuric acid solution was 2M.

5. A method for preparing a detection kit for porcine epidemic diarrhea virus IgG antibody according to any one of claims 1 to 4, characterized in that: The preparation method comprises: Prepare antigen-coated plates; Prepare sample diluent, concentrated washing solution, enzyme-labeled antibody, color development solution and stop solution respectively; Prepare positive and negative controls; The prepared and configured antigen-coated plate, sample diluent, concentrated washing solution, enzyme-labeled reagent, color developing solution, stop solution, positive control and negative control are loaded into the detection kit.

6. Use of the detection kit for porcine epidemic diarrhea virus IgG antibody according to any one of claims 1 to 4 in the detection of porcine epidemic diarrhea virus IgG antibody.

7. The use according to claim 6, characterized in that: The applications include: S1. Diluting the serum sample to be tested with a sample diluent; S2. Add the diluted serum sample to be tested at 100 μL / well, and set up 2 positive and negative control wells at the same time; S3, incubate at 25°C in dark for 30 min; S4, dilute the concentrated washing solution, then add 300 μL / well of the diluted washing solution and wash the plate 3 times; S5, add 100 μL of enzyme labeling reagent to each well; S6. Incubate at 25°C in dark for 55 to 65 minutes; S7, dilute the concentrated washing solution, then add 300 μL / well of the diluted washing solution and wash the plate 4 times; S8. Add 100 μL / well of color development solution to each well and develop color at 25°C in the dark for 15±1 minutes. S9, add 100 μL stop solution to each well and oscillate to mix; S10, single wavelength determination, set the wavelength of the microplate reader to 450nm.

8. The use according to claim 7, characterized in that: If the OD450nm reading of each negative control well is less than 0.3, the test is established and the result is valid.

9. The use according to claim 7, characterized in that: S / P value = (sample OD450nm value - negative control OD450nm average value) / (positive control OD450nm average value - negative control OD450nm average value); Positive judgment: Sample S / P value>0.8 is considered positive; Negative determination: Sample S / P value ≤ 0.8 is considered negative.

10. The use according to claim 6, characterized in that: The serum sample to be tested was diluted 1:300 with diluent.

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