Preparation and use of sandwich ELISA antibody detection kit for porcine epidemic diarrhea virus

Abstract

The present invention discloses a method for using a sandwich ELISA antibody detection kit for porcine epidemic diarrhea virus, comprising the following steps: (1) coating purified porcine epidemic diarrhea virus recombinant S1 protein to obtain a diluted antigen solution, and adding the solution to an ELISA plate for coating overnight; (2) adding a blocking solution to block the ELISA plate; (3) adding a sample to be tested, a positive control sample, and a negative control sample to the antigen-coated ELISA plate, respectively, and then adding an enzyme-labeled antigen for co-incubation, wherein the enzyme-labeled antigen is a porcine epidemic diarrhea virus S1 protein labeled with horseradish peroxidase; (4) adding a substrate color development solution after the co-incubation is completed, terminating the reaction after the color development reaction is completed, and using an ELISA instrument to determine the result. The present invention provides a method for using a specific, sensitive, rapid, and easy-to-operate kit for detecting porcine epidemic diarrhea virus antibodies.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a preparation and use method of a porcine epidemic diarrhea virus sandwich ELISA antibody detection kit. Background Art

[0002] Porcine Epidemic Diarrhea Virus (PEDV) can cause porcine epidemic diarrhea, which is manifested by diarrhea, vomiting and dehydration. It is one of the important pathogens that cause diarrhea and death in piglets. Pigs in other stages can continue to carry the virus after infection, causing serious economic losses to the pig industry around the world. At present, the main prevention and control measures are to immunize sows with inactivated vaccines and attenuated vaccines, so that sows can produce maternal antibodies to protect piglets. Therefore, detecting anti-PEDV antibodies in sow milk, blood and piglet blood is an effective means to diagnose infection or evaluate the immunogenicity of vaccines. At present, the main serological diagnostic methods for PEDV are virus neutralization test, indirect immunofluorescence (IFA), fluorescent microsphere immunoassay (FMIA) and enzyme-linked immunosorbent assay (ELISA). Among them, ELISA is widely used in the detection of human and animal diseases because of its advantages of simple operation, strong specificity and high sensitivity.

[0003] PEDV belongs to the genus Alphacoronavirus of the Coronaviridae family. The S protein is a type I membrane glycoprotein located on the viral envelope. It is the largest structural protein of PEDV and plays a variety of biological functions. According to the different functions played by its structure, it is divided into two domains, S1 and S2. The neutralizing epitope region is mainly the S1 subunit. PEDV binds to specific receptors on the surface of intestinal villus epithelial cells through the S1 protein to achieve the invasion of the virus into host cells; the S1 protein is the main antigenic protein of PEDV, which can induce the body to produce specific neutralizing antibodies, and the S1 protein has multiple linear epitopes and B cell epitopes. These characteristics make the S1 protein a dominant protein in the diagnosis of PEDV. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for using a sandwich ELISA antibody detection kit for porcine epidemic diarrhea virus, which is simple, rapid, highly sensitive and highly specific.

[0005] In order to achieve the above technical effects, the present invention provides a method for using a porcine epidemic diarrhea virus sandwich ELISA antibody detection kit, comprising the following steps:

[0006] (1) coating the purified porcine epidemic diarrhea virus recombinant S1 protein to obtain a diluted antigen solution, adding the solution to an ELISA plate for coating overnight;

[0007] (2) Add blocking solution to block the ELISA plate;

[0008] (3) adding the sample to be tested, the positive control sample and the negative control sample to the antigen-coated ELISA plate respectively, and then adding the enzyme-labeled antigen for co-incubation, wherein the enzyme-labeled antigen is the porcine epidemic diarrhea virus S1 protein labeled with horseradish peroxidase;

[0009] (4) After the co-incubation is completed, the substrate color development solution is added, and after the color development reaction is completed, the reaction is terminated and the result is measured using an enzyme reader.

[0010] As an improvement of the above technical solution, the temperature of the co-incubation in step (3) is 35°C to 39°C, and the time is 30min to 60min.

[0011] As an improvement of the above technical solution, in step (1), the concentration of the antigen solution coated on the ELISA plate is 0.1 μg / mL to 1.0 μg / mL; 80 μL to 120 μL of the antigen solution is added to each well of the ELISA plate.

[0012] As an improvement of the above technical solution, the temperature of the closed ELISA plate in step (2) is 35° C. to 39° C., and the time is 1 h to 9 h.

[0013] As an improvement of the above technical solution, the sample to be tested in step (3) is diluted serum or milk to be tested; the dilution of the serum to be tested is 1:(20-160); the dilution of the milk to be tested is 1:(150-1200).

[0014] As an improvement of the above technical solution, the enzyme-labeled antigen in step (3) is diluted before being added, and the dilution ratio of the enzyme-labeled antigen is 1:(5000-50000).

[0015] As an improvement of the above technical solution, the time of the color development reaction in step (4) is 5 minutes to 20 minutes.

[0016] As an improvement of the above technical solution, in step (3), the positive control sample is anti-porcine epidemic diarrhea virus pig serum; and the negative control sample is negative specific pathogen-free pig serum.

[0017] Implementing the embodiments of the present invention has the following beneficial effects:

[0018] The present invention adopts porcine epidemic diarrhea virus S1 protein as a capture antigen, horseradish peroxidase-labeled porcine epidemic diarrhea virus S1 protein as an enzyme-labeled antigen, and provides a method for using a porcine epidemic diarrhea virus sandwich ELISA antibody detection kit through coordinated optimization of reaction steps, reaction conditions and reagents.

[0019] The relative sensitivity, relative specificity and total compliance rate of the ELISA antibody detection kit established by the present invention can all reach more than 95%, and the kit has strong specificity and does not react with PRV, CSFV, PPV and PRRSV positive serum, and the intra-batch and inter-batch repeat coefficients of variation are both less than 8%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effect of the antigen coating concentration and serum dilution on the P / N value in Example 1 of the present invention;

[0021] Figure 2 The effect of the antigen coating concentration and the emulsion dilution on the P / N value in Example 1 of the present invention;

[0022] Figure 3 The effect of the blocking solution and the first incubation time on the P / N value in Example 1 of the present invention;

[0023] Figure 4 is the effect of the sample diluent of Example 1 of the present invention on the P / N value;

[0024] Figure 5 The effect of the dilution of the enzyme-labeled antigen on the P / N value in Example 1 of the present invention;

[0025] Figure 6 The effect of the second incubation time on the P / N value in Example 1 of the present invention;

[0026] Figure 7 It is a graph of specificity test results of Example 4 of the present invention. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with specific embodiments.

[0028] The embodiment of the present invention provides a method for using a porcine epidemic diarrhea virus sandwich ELISA antibody detection kit, comprising the following steps:

[0029] (1) The purified porcine epidemic diarrhea virus recombinant S1 protein was coated with a coating solution to obtain a diluted antigen solution, which was added to an ELISA plate for coating overnight.

[0030] The porcine epidemic diarrhea virus S1 protein was developed and provided by Guangzhou Hanteng Biotechnology Co., Ltd.

[0031] In one embodiment, the concentration of the antigen solution coated on the ELISA plate is 0.1μg / mL to 1.0μg / mL, and preferably, the concentration of the antigen solution coated on the ELISA plate is 0.3μg / mL to 0.6μg / mL. Exemplarily, the concentration of the antigen solution coated on the ELISA plate is 0.3μg / mL, 0.4μg / mL, 0.5μg / mL or 0.6μg / mL, but is not limited thereto. Optimally, according to the P / N value, 0.5μg / mL is selected as the final coating concentration of the kit. 80μL to 120μL of antigen solution is added to each well of the ELISA plate, and optimally, 100μL of antigen solution is added to each well of the ELISA plate. The porcine epidemic diarrhea virus S1 protein used in the present invention has high purity and strong affinity, so the antigen coating concentration can be reduced, thereby reducing the amount of antigen used, reducing costs while not affecting the detection effect.

[0032] The coating solution can be a 0.05M carbonate buffer solution with a pH of 9.6.

[0033] (2) After discarding the coating solution, add blocking solution to block the ELISA plate. After completion, discard the blocking solution to obtain an antigen-coated ELISA plate.

[0034] In one embodiment, washing is performed after discarding the coating solution and the blocking solution, and the washing solution is a PBS solution containing 0.01% to 0.1% Tween-20 (ie, PBST solution).

[0035] In one embodiment, the blocking solution is a PBST solution containing 1% to 5% BSA or 1% to 5% skim milk powder. Exemplarily, the blocking solution is a PBST solution containing 1% BSA, a PBST solution containing 3% BSA, a PBST solution containing 5% BSA, a PBST solution containing 1% skim milk powder, or a PBST solution containing 5% skim milk powder, but is not limited thereto.

[0036] The temperature of the blocked ELISA plate is 35°C to 39°C, and the time is 1h to 9h. The optimal temperature of the blocked ELISA plate is 37°C, and the time is 9h. By optimizing the temperature and time of the blocked ELISA plate, the sensitivity, specificity and stability of the kit can be further improved.

[0037] (3) adding the sample to be tested, the positive control sample and the negative control sample to the antigen-coated ELISA plate respectively, and then adding the enzyme-labeled antigen for co-incubation, wherein the enzyme-labeled antigen is the porcine epidemic diarrhea virus S1 protein labeled with horseradish peroxidase.

[0038] Specifically, the sample to be tested is a diluted serum to be tested or milk to be tested, the positive control sample is anti-PEDV pig serum; the negative control sample is negative SPF (Specific Pathogen Free) pig serum. The dilution of the serum to be tested is 1: (20-160), preferably, the dilution of the serum to be tested is 1: (30-80). Exemplarily, the dilution of the serum to be tested is 1: 30, 1: 40, 1: 50, 1: 60, 1: 70 or 1: 80, but not limited thereto. The dilution of the milk to be tested is 1: (150-1200), preferably, the dilution of the milk to be tested is 1: (300-600). Exemplarily, the dilution of the milk to be tested is 1: 300, 1: 400, 1: 450, 1: 500, 1: 550 or 1: 600, but not limited thereto. The sample to be tested is diluted with a sample diluent, wherein the diluent is a PBST solution containing 0.5% to 1.5% BSA and 5% to 15% PBS. Exemplarily, the diluent is a PBST solution containing 1% BSA and 5% PBS, a PBST solution containing 1% BSA and 10% PBS, or a PBST solution containing 1% BSA and 15% PBS, but is not limited thereto.

[0039] In one embodiment, the enzyme-labeled antigen is diluted before being added, and the dilution of the enzyme-labeled antigen is 1:(5000-50000), preferably, the dilution of the enzyme-labeled antigen is 1:(10000-30000). Exemplarily, the dilution of the enzyme-labeled antigen is 1:10000, 1:15000, 1:20000, 1:25000 or 1:30000, but not limited thereto.

[0040] In one embodiment, the temperature of the co-incubation is 35°C to 39°C, and the time is 30min to 60min. Exemplarily, the time of the co-incubation is 30min, 40min, 45min, 50min or 60min, but not limited thereto. Optimally, the temperature of the co-incubation is 37°C and the time is 45min. Compared with adding the sample to be tested and then adding the enzyme-labeled antigen for incubation, the co-incubation of the sample to be tested and the enzyme-labeled antigen simplifies the operation steps, and the detection is simpler and time-saving. Moreover, since the porcine epidemic diarrhea virus S1 protein used in the present invention has high purity and strong affinity, the co-incubation of the sample to be tested and the enzyme-labeled antigen can not only significantly shorten the detection time, but also will not produce the situation that the sample to be tested competes with the capture antigen and the enzyme-labeled antigen due to the high concentration of the sample to be tested, so that it is difficult to effectively form a sandwich conjugate, and the proportion of weak positive or false negative under the co-incubation method is reduced, and the detection effect is good.

[0041] The ELISA antibody detection kit established by the present invention has a small amount of antigen coating during use, which reduces costs. In the detection process, the sample to be detected and the enzyme-labeled antigen are added and incubated together, which simplifies the operation steps and makes the detection more simple, time-saving and stable.

[0042] (4) After the co-incubation is completed, the substrate color development solution is added, and after the color development reaction is completed, the reaction is terminated and the result is measured using an enzyme reader.

[0043] In one embodiment, 80 μL to 120 μL of substrate colorimetric solution is added to each well, and the colorimetric reaction is carried out at 35°C to 39°C for 5 min to 15 min. The optimal temperature for the colorimetric reaction is 37°C for 15 min. The substrate colorimetric solution is a TMB single-component substrate colorimetric solution. The stop solution is a 1M to 2M H2SO4 aqueous solution; optimally, the stop solution is a 2M H2SO4 aqueous solution.

[0044] After the color development reaction is completed, the absorbance of the sample to be tested, the positive control sample and the negative control sample at a single wavelength OD450mm is measured by an enzyme marker, the S / P value is calculated by the following formula, and the infection status of the sample is determined based on the S / P value.

[0045] S / P value = (Sample-NC average) / (PC average-NC average)

[0046] In the formula, PC is the average OD450 value of the positive control samples; NC is the average OD450 value of the negative control samples; Sample is the average OD450 value of the samples to be tested.

[0047] The present invention is further described below with reference to specific embodiments.

[0048] Example 1

[0049] Selection of the best reaction conditions for the kit:

[0050] 1) Determination of the optimal antigen coating concentration and serum and milk dilution:

[0051] The optimal antigen coating concentration was determined by the square array titration method. Figure 1 and Figure 2 As shown, when the antigen coating concentration is 0.5 μg / mL, the P / N value is the largest, indicating that this concentration is the optimal antigen coating concentration; the serum and milk dilutions are determined according to the P / N values, and the preferred serum dilution is 1:40 and the milk dilution is 1:300.

[0052] 2) Determination of the optimal blocking solution and blocking time:

[0053] PBST containing 1% BSA, 2% BSA, 5% BSA and 5% skim milk powder was placed in a constant temperature incubator at 37°C for 1h, 3h, 6h and 9h respectively. The best blocking solution and blocking time were determined according to the P / N value. The combination with the largest P / N was selected to determine the best blocking solution and blocking time. The results are shown in Figure 3 As shown, when the PBST solution containing 5% skimmed milk powder was used for blocking for 9 h, the P / N value was the largest.

[0054] 3) Determination of the optimal sample dilution:

[0055] The sample diluents were respectively used as PBST solution containing 1% BSA and 5% FBS, PBST solution containing 1% BSA and 10% FBS, and PBST solution containing 1% BSA and 15% FBS, and the reaction was carried out at 37°C. The sample diluent corresponding to the maximum P / N value was selected. Figure 4 As shown, when PBST solution containing 1% BSA and 10% FBS was used as the sample diluent, the P / N value was the largest.

[0056] 4) Determination of the optimal enzyme-labeled antigen dilution:

[0057] Add HRP (horseradish peroxidase) labeled S1 protein and dilute it with PBST to a dilution of 1:5000, 1:10000, 1:20000, 1:40000 and 1:50000, incubate in a 37°C constant temperature incubator, determine the best enzyme-labeled antigen dilution based on the P / N value, and select the combination with the largest P / N value as the best enzyme-labeled antigen dilution. Figure 5 As shown, when the dilution of the enzyme-labeled antigen is 1:20000, the P / N value is the largest.

[0058] 5) Determination of the optimal co-incubation time:

[0059] After adding the sample to be tested and the enzyme-labeled antigen, incubate at 37°C for 30 min, 45 min and 60 min respectively, and select the incubation time with the maximum OD value. Figure 6 As shown in Figures 6a, 6b and 6c, when the co-incubation time is 45 min to 60 min, the OD value is larger. Preferably, the co-incubation time is 45 min.

[0060] 6) Determination of the optimal color development time:

[0061] After adding the substrate color developing solution, the reaction was carried out at 37°C for 5min, 10min, 15min and 20min respectively, and then 100μL / well of 2M H2SO4 was added to terminate the reaction, and the color development time corresponding to the maximum P / N value was selected. The results are shown in Table 1. When the substrate reaction time is 15min to 20min, the P / N value is large and the detection function can be realized. Preferably, the color development reaction time is 15min.

[0062] Table 1 Effect of color development time on P / N value

[0063]

[0064] Therefore, the optimal reaction conditions finally determined were: antigen coating concentration of 0.5 μg / ml, serum dilution of 1:40, milk dilution of 1:300, enzyme-labeled antigen dilution of 1:20000; PBST solution containing 5% skimmed milk powder was used as blocking solution; PBST solution containing 1% BSA and 10% FBS was used as sample diluent; the incubation time of the sample to be tested and the enzyme-labeled antigen was 45 minutes, and the color development time was 15 minutes.

[0065] Example 2

[0066] Preparation of positive control samples and negative control samples:

[0067] Twelve 3-week-old SPF pigs were divided into two groups, infected with PEDV-S1 and a PBS control group. Blood was collected and serum was separated on the 7th, 14th, 21st, 28th and 35th days after immunization.

[0068] Table 2 Preparation of positive control samples and negative control samples

[0069]

[0070] Example 3

[0071] Determination of positive and negative criteria:

[0072] 46 negative sera and 45 positive sera were selected, and the serum antibody OD450 value was detected using the optimized ELISA reaction system. The results were analyzed using GraphPad priam, and it was concluded that when the S / P value ≥ 0.3, it was positive, and when the S / P value < 0.3, it was negative, the sensitivity was 100%, and the specificity was 100%, which was used as the critical standard for negative and positive judgment of the ELISA method.

[0073] Table 3 Determination of positive and negative determination criteria

[0074]

[0075]

[0076] Example 4

[0077] Specificity test:

[0078] The optimal conditions of the established ELISA method were used to test 5 known PEDV, PRV, CSFV, PPV, and PRRSV positive sera, and the IDEXX PEDV kit was used as a control to analyze the specificity of the kit. Figure 7 As shown, except for the positive PEDV serum test, the positive serum tests of other pathogens were all negative, indicating that the kit has good specificity and no cross reaction with other positive sera.

[0079] Example 5

[0080] Repeatability test:

[0081] Three ELISA plates coated at the same time were taken 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 are shown in Table 4, from which it can be seen that the intra-batch repeat test CV value of the kit provided by the present invention is less than 8%, and the coefficient of variation (CV) = (standard deviation / mean value) × 100%, proving that the method has a certain repeatability within the batch.

[0082] Table 4 Intra-batch repeated tests

[0083]

[0084] Three ELISA plates coated at different times were taken for batch repetition test, and six different samples were tested under the same conditions and the test results were statistically analyzed. The test results are shown in Table 5, from which it can be seen that the batch repetition test CV value of the kit provided by the present invention is less than 8%, proving that the method has certain repeatability between batches.

[0085] Table 5 Batch repeat test

[0086]

[0087]

[0088] Example 6

[0089] Compliance test:

[0090] The preferred method of Example 1 was used to detect 55 serum samples with different antibody titers. The results were compared with the detection results of neutralizing antibody titers to analyze the sensitivity, specificity and compliance rate of the method. The results are shown in Table 6.

[0091] Relative sensitivity (%) = {number of positives / (number of positives + number of false negatives)} × 100%

[0092] Relative specificity (%) = {Number of negatives / (Number of negatives + Number of false positives)} × 100%

[0093] Total compliance rate (%) = {(positive number + negative number) / total number of tests} × 100%

[0094] Table 6 Compliance test

[0095]

[0096] The experimental results show that 55 serum samples with different antibody titers were tested using the ELISA plate and detection method coated with PEDV-S1 protein established by the present invention, and the test results were 35 positive and 19 negative, and the virus neutralizing antibody titer test results were 28 positive and 25 negative. According to calculations, the relative sensitivity, relative specificity and total compliance rate of the ELISA method established by the present invention are 97.22%, 95.00% and 98.18%, respectively. The antibody detection kit based on the ELISA plate coated with PEDV-S1 protein involved in the present invention has high sensitivity and specificity, and has a high compliance rate with the virus neutralizing antibody titer results.

[0097] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for using a sandwich ELISA antibody detection kit for porcine epidemic diarrhea virus for non-diagnostic purposes, characterized in that: The following steps are involved: (1) coating the purified porcine epidemic diarrhea virus recombinant S1 protein to obtain a diluted antigen solution, adding it to an ELISA plate for coating overnight; the concentration of the antigen solution coated on the ELISA plate was 0.5 μg / mL; (2) Add blocking solution to block the ELISA plate; (3) adding the sample to be tested, the positive control sample and the negative control sample to the antigen-coated ELISA plate respectively, and then adding the enzyme-labeled antigen for co-incubation, the co-incubation time is 45 minutes, the enzyme-labeled antigen is porcine epidemic diarrhea virus S1 protein labeled with horseradish peroxidase, and the enzyme-labeled antigen is diluted before addition, and the dilution of the enzyme-labeled antigen is 1:20000; (4) After the co-incubation is completed, the substrate color development solution is added, and after the color development reaction is completed, the reaction is terminated and the result is measured using an enzyme reader.

2. The method of use according to claim 1, characterized in that: The temperature of the co-incubation in step (3) is 35°C to 39°C.

3. The method of use according to claim 1, characterized in that: In step (1), add 80 μL to 120 μL of antigen solution to each well of the ELISA plate.

4. The method of use according to claim 1, characterized in that: The temperature of the closed ELISA plate in step (2) is 35° C. to 39° C., and the time is 1 h to 9 h.

5. The method of use according to claim 1, characterized in that: The sample to be tested in step (3) is diluted serum or milk; the dilution of the serum to be tested is 1:(20-160); the dilution of the milk to be tested is 1:(150-1200).

6. The method of use according to claim 1, characterized in that: The time of the color development reaction in step (4) is 5 min to 20 min.

7. The method of use according to claim 1, characterized in that: In step (3), the positive control sample is anti-porcine epidemic diarrhea virus pig serum; the negative control sample is negative specific pathogen-free pig serum.

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