Indirect ELISA (enzyme-linked immuno sorbent assay) detection kit based on Senecavirus type A 3A protein and application of indirect ELISA detection kit

Abstract

The invention belongs to the technical field of biological detection, and particularly discloses an indirect ELISA (enzyme-linked immuno sorbent assay) detection kit based on Senecavirus type A 3A protein and application of the indirect ELISA detection kit. The Seneca virus 3A protein is used as the coating antigen for the first time, the antibody level of Seneca virus in serum can be rapidly detected, and infected animals and immune animals can be accurately distinguished. By detecting the anti-3A antibody of the swinery, the immune condition of the swinery can be more intuitively known, and the method has important significance on diagnosis of Seneca virus diseases and regulation and management of a Seneca virus vaccine immune program of the swinery. The kit provided by the invention can rapidly detect whether a clinical serum sample contains the Senecavirus antibody or not, the final coincidence rate of the kit and a commercial kit and an IFA clinical sample detection method is as high as 100%, the sensitivity and the detection rate are both higher than those of the commercial kit, the kit has good stability at 4 DEG C, and the storage life can at least reach 90 days.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to an indirect ELISA detection kit based on type A Seneca virus 3A protein and an application thereof. Background Art

[0002] Seneca virus (SVA) is an infectious disease mainly caused by pigs characterized by vesicular lesions on the mouth and feet. Its pathogenesis is very similar to the Class I infectious diseases announced by the Ministry of Agriculture and Rural Affairs, such as foot-and-mouth disease, swine vesicular disease, and swine vesicular stomatitis, and it is difficult to distinguish them clinically. The incidence of Seneca virus disease is high, and pigs of all ages can be infected with the disease, especially newborn piglets aged 1-4 days, with a mortality rate of up to 70%-80%. Adult pigs often show subclinical symptoms such as fever, lameness, lethargy, reduced feed intake, and vesicular lesions on the mouth, nose, and hooves, which greatly reduces the overall immune defense of the pig herd and increases the risk of secondary infection in pigs.

[0003] At present, the Seneca virus ELISA antibody detection method mostly uses structural proteins such as VP1 and VP2 as coating antigens. For example, a Seneca virus ELISA antibody detection kit disclosed in Chinese patent application CN108761074A (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences) uses an enzyme label plate pre-coated with Seneca virus VP1 protein. By optimizing the coating amount and detection conditions of VP1 protein, the antibody level of Seneca virus in serum can be quickly detected, and the test results are stable. The method has high sensitivity, good specificity and repeatability, and is suitable for monitoring the antibody level of Seneca virus, which is conducive to mastering the antibody status of Seneca virus disease in pigs. In addition, clinically, small RNA virus family viruses such as foot-and-mouth disease virus subunit vaccines all use structural proteins that can produce neutralizing antibodies as immunogens. Immunized animals only produce antibodies against the structural proteins, and do not produce antibodies against non-structural proteins such as 3A, 3C, and 3D. Animals naturally infected with Seneca virus can not only produce antibodies against structural proteins, but also produce antibodies against non-structural proteins such as 3A and 3C. Therefore, the detection of anti-3A antibodies in animals can distinguish infected animals from immune animals. In addition, 3A protein is expressed in both the early and late stages of viral infection and has good immunogenicity, making it an effective target for the development of Seneca virus detection reagents and Seneca virus disease diagnostic tools.

[0004] At present, there is no report on the indirect ELISA antibody detection kit and detection method for Seneca virus 3A protein. Therefore, the development of antibody detection reagents and methods for Seneca virus 3A protein is of great significance for the prevention and immune evaluation of Seneca virus disease. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an indirect ELISA detection kit based on type A Seneca virus 3A protein, which uses Seneca virus 3A protein as a coating antigen for the first time, and can not only quickly detect the antibody level of Seneca virus in serum, but also accurately distinguish infected animals from immune animals.

[0006] At the same time, the present invention also provides an application of an indirect ELISA detection kit based on type A Seneca virus 3A protein, which improves the sensitivity, specificity and stability of the detection by optimizing the detection conditions.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] An indirect ELISA detection kit based on type A Seneca virus 3A protein, comprising:

[0009] The coating antigen is Seneca virus 3A protein.

[0010] As a preferred embodiment of the present invention, the amino acid sequence of the Seneca virus 3A protein is as follows:

[0011] SPNENDDTPVDEALGRVLTPTAVDEALVDLAPDADPVGRLAILAKLGLALAAVTPGLIILAVGLYKYFSGSDTDQEETETEEPAKAPRSE (SEQ ID NO: 1).

[0012] As a preferred embodiment of the present invention, the coating antigen is pre-coated on an ELISA plate.

[0013] Specifically, the method for preparing the ELISA plate pre-coated with antigen comprises the following steps:

[0014] 1) Add the coated antigen to the ELISA plate and coat at 30-45°C for 1-5 hours;

[0015] 2) Add blocking solution to the ELISA plate and block at 30-45°C for 0.5-2h.

[0016] Specifically, in step 1), the coated antigen is diluted with a coating solution and then added to the ELISA plate. The coating solution is a carbonate buffer, preferably a 0.05 M carbonate buffer (CB, pH 9.6).

[0017] Specifically, in step 1), the coating concentration of the coating antigen is 1-5 μg / mL, preferably 2 μg / mL.

[0018] Specifically, in step 1), coating is carried out at 37° C. for 3 hours.

[0019] Specifically, in step 2), the blocking solution is a BSA solution, preferably a 2% BSA solution.

[0020] Specifically, in step 2), the cells were blocked at 37° C. for 1 h.

[0021] As a preferred embodiment of the present invention, the detection kit further comprises one or more of a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color developing solution, a stop solution, a SVA positive serum control, a SVA negative serum control, and the like.

[0022] Specifically, the sample diluent is PBS buffer.

[0023] Specifically, the washing solution is PBST buffer.

[0024] Specifically, the enzyme-labeled secondary antibody is HRP-goat anti-pig IgG, which is a commercially available product.

[0025] Specifically, the color developing solution is TMB single-component color developing solution, which is a commercially available product.

[0026] Specifically, the stop solution is 2M H2SO4.

[0027] An application of an indirect ELISA detection kit based on type A Seneca virus 3A protein, comprising the following steps:

[0028] (1) Add the sample to be tested to the ELISA plate pre-coated with antigen and incubate at 30-45°C for 0.5-2.5h;

[0029] (2) Add enzyme-labeled secondary antibody and incubate at 30-45°C for 0.5-1.5 h;

[0030] (3) Add color developing solution and develop color at room temperature in the dark;

[0031] (4) Add stop solution to stop color development;

[0032] (5) Detection of OD 450nm value, to determine whether there is Seneca virus antibody in the sample to be tested;

[0033] The judging criteria are as follows:

[0034] OD of sample to be tested 450nm ≥0.48, judged as positive;

[0035] OD of sample to be tested 450nm ≤0.41, judged as negative;

[0036] 0.41<OD of the sample to be tested 450nm <0.48, judged as suspicious.

[0037] As a preferred embodiment of the present invention, in step (1), the sample to be tested is diluted with a sample diluent before being added to the ELISA plate, and the dilution ratio is 1:50-200, preferably 1:100.

[0038] As a preferred embodiment of the present invention, in step (1), the incubation is carried out at 37° C. for 1 h.

[0039] As a preferred embodiment of the present invention, in step (2), the enzyme-labeled secondary antibody is diluted with an enzyme-labeled diluent before being added to the enzyme-labeled plate, and the dilution ratio is 1:4000-16000, preferably 1:8000.

[0040] As a preferred embodiment of the present invention, in step (2), the incubation is carried out at 37° C. for 45 min.

[0041] As a preferred embodiment of the present invention, in step (3), the color development time is 5-20 minutes, preferably 10 minutes.

[0042] As a preferred embodiment of the present invention, in steps (2) and (3), before adding the enzyme-labeled secondary antibody or the color developing solution, the sample is washed with a washing solution at least 3 times, preferably 4 times.

[0043] As a preferred embodiment of the present invention, in step (5), the sample determined to be suspicious is retested, and the retest result OD 450nm ≥0.48 was considered positive, OD 450nm A value of ≤0.41 was considered negative.

[0044] Beneficial effects of the present invention:

[0045] The present invention provides an indirect ELISA detection kit based on type A Seneca virus 3A protein, which uses Seneca virus 3A protein as a coating antigen for the first time, and can not only quickly detect the antibody level of Seneca virus in serum, but also accurately distinguish infected animals from immune animals. By detecting anti-3A antibodies in pig herds, the immune status of the pig herd can be more intuitively understood, which is of great significance for the diagnosis of Seneca virus disease and the regulation and management of the Seneca virus vaccine immunization program of the pig herd. Experimental results show that the kit provided by the present invention can quickly detect whether Seneca virus antibodies are contained in clinical serum samples, and the compliance rate with commercial detection kits and Western blot experiments is as high as 98.37%, and the sensitivity and detection rate are higher than those of commercial detection kits.

[0046] The detection kit provided by the present invention has good stability at 4°C, a shelf life of at least 60 days, strong detection specificity and good repeatability, is particularly suitable for Seneca virus antibody detection in clinical serum samples, and has good clinical application prospects and high economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the SDS-PAGE result of prokaryotic expression of Seneca virus 3A protein in the experimental example.

[0048] Figure 2 Shown are the Western blot results of Seneca virus 3A protein and SVA positive serum in the experimental example.

[0049] Figure 3 This is the experimental result of the best coating solution in the experimental example.

[0050] Figure 4 The experimental results of the best coating conditions in the experimental example.

[0051] Figure 5 This is the experimental result of the best blocking solution in the experimental example.

[0052] Figure 6 This is the experimental result of the optimal blocking concentration in the experimental example.

[0053] Figure 7 This is the experimental result of the best sealing condition in the experimental example.

[0054] Figure 8 This is the experimental result of the best sample dilution in the experimental example.

[0055] Fig. 9 This is the experimental result of the optimal serum reaction time in the experimental example.

[0056] Fig.10 This is the experimental result of the optimal dilution ratio of the enzyme-labeled secondary antibody in the experimental example.

[0057] Fig.11 This is the experimental result of the optimal reaction time of the enzyme-labeled secondary antibody in the experimental example.

[0058] Fig.12 This is the experimental result of the best color development time in the experimental example.

[0059] Fig.13 This is the experimental result of the critical value of the 3A protein indirect ELISA detection method in the experimental example.

[0060] Fig.14 These are the experimental results of the specificity of the indirect ELISA detection method for 3A protein in the experimental example.

[0061] Fig.15 This is the experimental result of the sensitivity of the 3A protein indirect ELISA detection method in the experimental example.

[0062] Fig.16 This is the experimental result of the sensitivity of the commercial ELISA kit in the experimental example.

[0063] Fig.17 These are the experimental results of IFA testing of 18 retested serum samples in the experimental example.

[0064] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the above drawings obtained in the experimental examples are briefly introduced. It should be understood that the above drawings only show some experimental results of the present invention and should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work. DETAILED DESCRIPTION

[0065] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments and experimental examples. It should be understood by those skilled in the art that the embodiments are only used to illustrate the technical solution of the present invention and should not be regarded as limiting the scope of protection of the present invention. Based on the following embodiments, other technical solutions obtained by ordinary technicians in the field without paying creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the scope of protection of the present invention.

[0066] Unless otherwise specified, the experimental methods used in the embodiments and experimental examples are conventional methods in the art.

[0067] Unless otherwise specified, the raw materials, reagents, instruments, etc. used in the embodiments and experimental examples are commonly used in the art and are available to the public (for example, purchased through commercial channels).

[0068] Unless otherwise specified, the terms, abbreviations, etc. involved in the embodiments and experimental examples have the conventional meanings in the art.

[0069] Example 1

[0070] This embodiment provides an indirect ELISA detection kit based on type A Seneca virus 3A protein, including: an ELISA plate pre-coated with an antigen, a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color developing solution, a stop solution, an SVA positive serum control and an SVA negative serum control, and the specific composition is as follows:

[0071] Pre-coated ELISA plate: The coated antigen is Seneca virus 3A protein, and its amino acid sequence is as follows:

[0072] SPNENDDTPVDEALGRVLTPTAVDEALVDLAPDADPVGRLAILAKLGLALAAVTPGLIILAVGLYKYFSGSDTDQEETETEEPAKAPRSE(SEQ ID NO: 1);

[0073] Sample diluent: PBS buffer;

[0074] Washing solution: PBST buffer;

[0075] Enzyme-labeled secondary antibody: HRP-goat anti-swine IgG, commercially available;

[0076] Colorimetric solution: TMB single-component colorimetric solution, commercially available;

[0077] Stop solution: 2M H2SO4;

[0078] SVA positive serum control and SVA negative serum control.

[0079] The preparation method of the ELISA plate pre-coated with antigen in this embodiment is as follows: first dilute the coating antigen with a coating solution to a final concentration of 2 μg / mL, add the diluted ELISA plate to the plate, coat it at 37° C. for 3 h, discard the coating solution, wash it twice with PBS, spin dry it, and pat it dry on dry absorbent paper; after coating, block it by adding 150 mL of 2% BSA blocking solution to each well, and block it at 37° C. for 1 h; discard the blocking solution to obtain the plate.

[0080] Example 2

[0081] This embodiment provides an application of an indirect ELISA detection kit based on type A Seneca virus 3A protein (same as in Example 1), comprising the following steps:

[0082] (1) Serum samples to be tested, SVA-positive serum controls, and SVA-negative serum controls diluted with PBS (1:100) were added to the ELISA plate pre-coated with antigens, respectively, and incubated at 37°C for 1 h. The samples were then discarded, washed 4 times with PBST, and patted dry on dry absorbent paper.

[0083] (2) Use enzyme-labeled diluent to dilute the enzyme-labeled secondary antibody at a ratio of 1:8000, then add the diluted enzyme-labeled secondary antibody to the ELISA plate and incubate at 37°C for 45 min. After the incubation, discard the enzyme-labeled secondary antibody, wash with PBST 4 times, and pat dry on dry absorbent paper;

[0084] (3) Add TMB single-component colorimetric solution to the ELISA plate and color develop for 10 min at room temperature in the dark;

[0085] (4) After color development is completed, add stop solution to the ELISA plate to stop color development;

[0086] (5) Detect OD using an ELISA reader 450nm value, to determine whether there is Seneca virus antibody in the serum sample to be tested;

[0087] The judging criteria are as follows:

[0088] OD of sample to be tested 450nm≥0.48, judged as positive;

[0089] OD of sample to be tested 450nm ≤0.41, judged as negative;

[0090] 0.41<OD of the sample to be tested 450nm <0.48, judged as suspicious.

[0091] The results showed that the OD of the samples to be tested 450nm ≥0.48, it is judged as positive, that is, Seneca virus is present in the serum sample to be tested.

[0092] In other embodiments of the present invention, the composition of the indirect ELISA detection kit and the technical parameters in the detection method can also be arbitrarily adjusted and set within a given range without substantially affecting the final detection performance.

[0093] Experimental example

[0094] 1. Experimental Materials

[0095] 1. 0.2M carbonate buffer solution (CB)

[0096] Weigh 1.59 g of sodium carbonate and 2.93 g of sodium bicarbonate, dissolve in 250 mL of deionized water, and store at 4°C until completely dissolved. Dilute 4 times with deionized water to make 0.05 M carbonate buffer (pH 9.6).

[0097] 2. 0.2M phosphate buffer solution (PB, pH 7.5)

[0098] Weigh 20.43 g of disodium hydrogen phosphate dodecahydrate and 22.27 g of sodium dihydrogen phosphate dihydrate, dissolve them in deionized water, make up to 1 L, and store at 4°C for later use.

[0099] 3. 0.1 M Tirs-HCl buffer (TB, pH 7.5)

[0100] Weigh 12.1 g of Tris and add it to 1 L of deionized water. Add concentrated hydrochloric acid dropwise until the pH value is 7.5.

[0101] 4. 1%-5% BSA

[0102] Weigh 1-5g bovine serum albumin (BSA) and 5g sucrose, dissolve in 80mL 0.01M PBS, add 100μL PC-300, mix until completely dissolved, dilute to 100mL, and store at 4℃ for later use.

[0103] 5. 1%-5% Casein

[0104] Weigh 1-5g casein and 5g sucrose, dissolve in 80mL 0.01M PBS, add 100μL PC-300, mix until completely dissolved, dilute to 100mL, and store at 4℃ for later use.

[0105] 6. 1%-5% skim milk

[0106] Weigh 1-5g skim milk powder and 5g sucrose, dissolve in 80mL 0.01M PBS, add 100μL PC-300, mix until completely dissolved, dilute to 100mL, and store at 4℃ for later use.

[0107] 2. Experimental Methods

[0108] 1. Expression and verification of Seneca virus 3A protein

[0109] The Seneca virus 3A protein was prepared by prokaryotic expression, and the cells were collected after induction of expression, and the samples were boiled and subjected to SDS-PAGE. At the same time, the purified Seneca virus 3A protein was identified by Western blot.

[0110] 2. Optimization of indirect ELISA detection method

[0111] (1) Determination of the dilution ratio of SVA positive and negative serum and the optimal coating concentration of 3A protein

[0112] The chessboard method was used to dilute the purified 3A protein to a final concentration of 4, 2, 1, 0.5, and 0.25 μg / mL. The SVA positive and negative serum were diluted with PBS at a ratio of 1:50, 1:100, 1:200, and 1:400 to react with the coated 3A protein at different final concentrations. The remaining steps were the same as the conventional indirect ELISA method. When the P / N value was the largest, the primary antibody dilution ratio and the optimal coating concentration of the 3A protein were determined.

[0113] (2) Determination of the optimal coating solution

[0114] Subsequent experiments were conducted based on the optimized SVA positive and negative serum dilution ratios and 3A protein coating concentrations. Different antigen coating solutions were used: 0.05M carbonate buffer (pH 9.6), 0.2M phosphate buffer (pH 7.5), and 0.1M Tris-HCl buffer (pH 7.5). The optimal coating solution was determined using the single control variable method. The optimal coating solution was determined based on the P / N value obtained by the final calculation.

[0115] (3) Determination of optimal coating conditions

[0116] According to the optimal conditions determined above, coating conditions at 4°C for 20 h and 37°C for 3 h were set to analyze the thermal stability of the antigen, and the coating condition with the smallest P / N value was selected as the optimal coating condition.

[0117] (4) Determination of the optimal blocking solution

[0118] According to the above optimized experimental conditions, different blocking solutions (1% BSA, 5% BSA, 1% skim milk, 5% skim milk, 1% casein, 5% casein) were used for blocking. The P / N value was compared based on the final test data to determine the best blocking solution. In order to further determine the best concentration of the blocking solution, different concentrations of 1%, 2%, 3%, 4%, and 5% blocking solutions were set for experiments, and the best concentration of the blocking solution was determined by comparing the P / N value.

[0119] (5) Determination of optimal sealing conditions

[0120] According to the above optimized experimental conditions, 5 groups of different blocking conditions were set, namely 4℃20h, 37℃1h, 37℃2h, 37℃3h, 37℃4h. The P / N value was calculated according to the test results to determine the best blocking conditions.

[0121] (6) Determination of the optimal primary antibody dilution

[0122] According to the optimal conditions determined above, PBS and PBST were used to dilute SVA positive and negative sera respectively, and the P / N values ​​were compared according to the test data. The dilution with the largest P / N value was selected as the optimal primary antibody dilution.

[0123] (7) Determination of the optimal primary antibody reaction time

[0124] According to the optimal conditions determined above, 5 groups of different primary antibody reaction times were set, namely 0.5h, 1h, 1.5h, 2h, and 2.5h. The P / N value was calculated based on the test results to determine the optimal primary antibody reaction time.

[0125] (8) Determination of the optimal secondary antibody dilution ratio

[0126] According to the optimal conditions determined above, 5 groups of different secondary antibody dilution ratios were set, and the enzyme-labeled secondary antibody was diluted with enzyme-labeled diluent at a ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000. The P / N value was calculated according to the test results to determine the optimal secondary antibody dilution ratio.

[0127] (9) Determination of optimal secondary antibody reaction conditions

[0128] According to the above optimized experimental conditions, 5 groups of different reaction times were set, and the enzyme-labeled secondary antibody was incubated at 37°C for 0.25h, 0.5h, 0.75h, 1h, and 1.5h, respectively. The P / N value was calculated according to the final test results to determine the optimal secondary antibody reaction time.

[0129] (10) Determination of color development time

[0130] According to the optimized experimental conditions, at room temperature and in the dark, four substrate action times of 5min, 10min, 15min, 20min, and 25min were set respectively. The P / N value was compared based on the test data to determine the optimal color development time.

[0131] (11) Determination of critical value

[0132] According to the indirect ELISA method established above and the optimized reaction conditions, 95 negative sera with known background were selected for detection. 450nm Read the values, perform statistical analysis on the results, and calculate the average value and standard deviation (SD), SD, judged as positive, SD, judged as negative, SD<sample to be tested SD, judged as suspicious.

[0133] 3. Evaluation of indirect ELISA detection method

[0134] (1) Specificity experiment

[0135] The indirect ELISA method established above was used to detect FMDV, PRRSV, CSFV, PRV, AstV, ASFV, PEDV, PDCoV, and blank control. At the same time, SVA positive and negative sera were set up as controls to verify the specificity of the method established above.

[0136] (2) Sensitivity test

[0137] The SVA-positive and -negative sera were diluted at 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, 1:256, 1:512, 1:1024, 1:2048, 1:4096, and 1:8192, respectively, and the indirect ELISA method established above and the commercial ELISA kit were used for detection to evaluate the sensitivity of the method.

[0138] (3) Repeatability experiment

[0139] Using the indirect ELISA detection method established above, three sets of ELISA plates coated in the same batch and three sets of ELISA plates coated in different batches were used to detect 4 SVA-positive pig serum samples and 4 SVA-negative pig serum samples, respectively. A set of duplicate controls was set for each group of samples. The results were analyzed by OD 450nm The intra-batch and inter-batch coefficients of variation (CV) of different samples were counted and analyzed to evaluate the repeatability of the method.

[0140] (4) Shelf life and stability test

[0141] Using the indirect ELISA method established above, the ELISA plates coated with the same batch were stored at 4°C and taken out for testing after 10, 20, 30, 40, 50, 60, 70, 80, and 90 days, and the OD values ​​of SVA positive and negative sera and blank control were read at each test. 450nm value, and analyze the changes in the test results to determine its shelf life and stability.

[0142] (5) Comparison of clinical sample testing

[0143] The indirect ELISA method and commercial ELISA kit were used to simultaneously test 123 clinical pig serum samples. The sera that did not meet the requirements were further verified by Western blot. The final compliance rate was calculated by comparing the test results, and the clinical detection effect of the indirect ELISA method established above was evaluated.

[0144] 3. Experimental Results

[0145] 1. Expression and verification of Seneca virus 3A protein

[0146] The Seneca virus 3A protein was prepared by prokaryotic expression, and the results of SDS-PAGE and Western blot identification were as follows: Figure 1 , Figure 2 As shown, it was determined that the Seneca virus 3A protein was successfully expressed.

[0147] 2. Optimization of indirect ELISA detection method

[0148] (1) Determination of the dilution ratio of SVA positive and negative serum and the optimal coating concentration of 3A protein

[0149] Using the chessboard titration method, when the SVA positive serum OD 450nm Values ​​close to 1, SVA negative serum OD 450nm The optimal coating concentration and primary antibody concentration are determined when the value is less than 0.3 and the P / N value is the largest. As can be seen from Table 1, the optimal coating concentration of 3A is 2 μg / mL, and the optimal primary antibody dilution multiple is 1:100.

[0150] Table 1 Determination of optimal antigen coating concentration and optimal antibody working concentration by chessboard titration method

[0151]

[0152] (2) Determination of the optimal coating solution

[0153] According to the coating concentration and serum dilution ratio determined above, different coating solutions were selected to coat the antigen. The results are as follows: Figure 3 As shown in the figure, when 0.05M CB was used to coat 3A protein, the P / N value was the highest, indicating that 0.05M CB was the best protein coating solution.

[0154] (3) Determination of optimal coating conditions

[0155] The antigen was coated at the optimal coating concentration, and the experiment was completed after coating under different coating conditions. The P / N value was calculated, such as Figure 4 As shown, the P / N value of the ELISA plate coated at 37°C for 3 hours is 13.71, and the P / N value of the ELISA plate coated at 4°C for 20 hours is 9.99, indicating that the antigen has good thermal stability and the coating effect is better at 37°C.

[0156] (4) Determination of the optimal blocking solution

[0157] After the antigen is coated on the ELISA plate, different blocking solutions are used to block the ELISA plate and detect standard SVA positive and negative sera, such as Figure 5 As shown in the figure, the P / N values ​​corresponding to the blocking solutions of 1% BSA and 5% BSA are the largest, indicating that the corresponding detection effects are the best. In order to further determine the optimal concentration of the blocking solution, five groups of BSA blocking solutions with different concentrations of 1%, 2%, 3%, 4%, and 5% were set for experiments. Figure 6 As shown in the figure, by comparing the P / N values, 2% BSA was determined to be the best blocking solution.

[0158] (5) Determination of optimal sealing conditions

[0159] In order to explore the optimal blocking conditions, five different blocking conditions were set at 37°C for 1 h, 37°C for 2 h, 37°C for 3 h, 37°C for 4 h, and 4°C for 20 h. Figure 7 As shown, the P / N value corresponding to 37°C for 1 hour is the largest, and 37°C for 1 hour is determined to be the optimal blocking condition for this detection method.

[0160] (6) Determination of the optimal primary antibody dilution

[0161] According to the optimized conditions, PBS and PBST were used to dilute SVA positive and negative serum for experiments, such as Figure 8As shown in the figure, by comparison, it was found that the P / N value measured by PBS was higher than that of PBST, so PBS was selected as the best primary antibody dilution solution.

[0162] (7) Determination of the optimal primary antibody reaction time

[0163] Set the primary antibody reaction time to 0.5h, 1h, 1.5h, 2h, and 2.5h, and compare the P / N value of each group of experimental results. Fig. 9 As shown, although the P / N of the reaction time of 1h and 1.5h is not much different, in order to shorten the reaction time of the kit, 1h is selected as the optimal reaction time of the serum.

[0164] (8) Determination of the optimal secondary antibody dilution ratio

[0165] According to the optimized conditions above, the enzyme-labeled secondary antibody was diluted at a ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 1:32000, and the P / N value of each group of experimental results was compared. Fig.10 As shown, when the secondary antibody was diluted at a ratio of 1:8000, the P / N value was the largest, so the optimal dilution ratio of the secondary antibody was 1:8000.

[0166] (9) Determination of optimal secondary antibody reaction conditions

[0167] According to the above optimized experimental conditions, the enzyme-labeled secondary antibody was incubated at 37°C for 0.25h, 0.5h, 0.75h, 1h, and 1.5h, respectively. Fig.11 As shown in the figure, when the secondary antibody reaction time is 45 minutes, the P / N value of the experimental results is the largest, so the secondary antibody reaction at 37°C for 45 minutes is the best condition.

[0168] (10) Determination of color development time

[0169] In the indirect ELISA experiment, the above optimized conditions were used, TMB single-component colorimetric solution was added, and the cells were placed at room temperature and protected from light for 5 min, 10 min, and 15 min, respectively. Fig.12 As shown, when the color development time is 10 minutes, the P / N value is the largest, and the optimal action time of the color development solution is determined to be 10 minutes.

[0170] (11) Determination of critical value

[0171] 95 SVA-negative sera were tested by indirect ELISA method and their P / N values ​​were calculated. Fig.13 As shown, the calculated was 0.27, SD was 0.07, and the SD is 0.48, SD is 0.41, so the positive and negative judgment criteria of the indirect ELISA method are determined as follows:

[0172] OD of sample to be tested 450nm ≥0.48, judged as positive;

[0173] OD of sample to be tested 450nm ≤0.41, judged as negative;

[0174] 0.41<OD of the sample to be tested 450nm <0.48, judged as suspicious.

[0175] Suspicious samples were retested, and the retest results were OD 450nm ≥0.48 was considered positive, OD 450nm A value of ≤0.41 was considered negative.

[0176] In summary, the detection conditions of the optimized indirect ELISA method are shown in Table 2.

[0177] Table 2 Optimization conditions of indirect ELISA detection method for Seneca virus 3A protein

[0178]

[0179] 3. Evaluation of indirect ELISA detection method

[0180] (1) Specificity experiment

[0181] The established indirect ELISA method was used to detect FMDV, PRRSV, CSFV, PRV, AstV, ASFV, PEDV, PDCoV, and SVA-positive pig sera, while SVA-positive and -negative sera were used as controls, e.g. Fig.14 As shown, only the SVA positive serum was positive, and the other 8 sera were negative, indicating that the detection method has good specificity.

[0182] (2) Sensitivity test

[0183] SVA positive and negative serum were diluted from 1:2 to 1:8192, and tested simultaneously with the above-established indirect ELISA detection method and commercial ELISA kit, and the corresponding critical value was used as the basis for positive judgment. Fig.15 , Fig.16 As shown, the maximum dilution factor of the antibody in the indirect ELISA detection method established above reaches 1:3072, while the maximum dilution factor of the antibody in the commercial ELISA kit is 1:32. The results show that the sensitivity of the indirect ELISA antibody detection method established above is much higher than that of the commercial kit.

[0184] (3) Repeatability experiment

[0185] As can be seen from Table 3, the coefficients of variation of the eight serum samples in repeated experiments within and between batches were all less than 10%, indicating that the indirect ELISA detection method has a small degree of variation and good intra-batch and inter-batch repeatability.

[0186] Table 3 Repeatability test results of indirect ELISA detection method

[0187]

[0188] (4) Shelf life and stability test

[0189] By calculating the mean value and dispersion coefficient of SVA positive and negative serum and blank control group, the coefficient of variation (CV) of these three groups was 10.69%, 14.4% and 8.14% respectively. As shown in Table 4, the experimental results show that the indirect ELISA plate has good stability at 4°C and can be stored for at least 90 days.

[0190] Table 4 Indirect ELISA test reagent stability test results

[0191]

[0192] (5) Comparison of clinical sample testing

[0193] The above-established indirect ELISA detection method and the commercial kit were used to simultaneously detect 178 clinical pig serum samples. As shown in Tables 5 and 6, the commercial kit detected 18 positive samples and 160 negative samples, and the 3A protein indirect ELISA method detected 34 positive samples and 144 negative samples, with a consistency rate of 88.76% (158 / 178). In order to further determine the consistency of the positive detection rate between the commercial kit and the 3A protein indirect ELISA method, the reaction between the non-compliant serum samples and Seneca virus was verified by IFA. Fig.17 As shown, the IFA results of 18 sera that were negative by the commercial kit and positive by the 3A protein indirect ELISA method were all positive. At the same time, the IFA results of 2 sera that were positive by the 3A protein indirect ELISA method and negative by the commercial kit were all negative. The IFA test results were consistent with the test results of the 3A protein indirect ELISA method established above.

[0194] Table 5 Comparison of clinical sample test results between indirect ELISA method and SVA commercial kit

[0195]

[0196] Table 6 Comparison of the test results of the indirect ELISA method and the IFA clinical sample detection method

[0197]

[0198] In summary, the final compliance rate of the indirect ELISA detection method established by the present invention with the commercial kit and the IFA clinical sample detection method is 100% (178 / 178). This shows that the indirect ELISA detection method established by the present invention can accurately identify Seneca virus antibodies in the sample to be tested, and has a higher detection rate than the commercial ELISA kit, and is suitable for the rapid detection of Seneca virus antibodies in clinical serum samples.

[0199] Based on the above experimental results, the indirect ELISA detection kit and detection method based on type A Seneca virus 3A protein provided by the present invention can effectively detect whether clinical serum samples contain Seneca virus antibodies, and the final compliance rate with the commercial ELISA kit and the IFA clinical sample detection method is 100% (178 / 178), and the sensitivity and detection rate are better than the commercial ELISA kit. In addition, the detection kit provided by the present invention has good stability at 4°C, a shelf life of at least 90 days, strong detection specificity and good repeatability, and can be applied to the detection of Seneca virus antibodies in clinical serum samples, with good clinical application prospects and high economic value.

[0200] Although the technical solution of the present invention has been described in detail above with general descriptions, specific implementation methods and test examples, it should be noted that the embodiments and test examples are only used to illustrate the technical solution and technical effects of the present invention, and should not be regarded as any limitation on the protection scope of the present invention. Simple deformation, modification or improvement based on the technical concept of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. An indirect ELISA detection kit based on type A Seneca virus 3A protein, characterized in that: The detection kit comprises: The coating antigen is Seneca virus 3A protein.

2. The indirect ELISA detection kit according to claim 1, characterized in that: The amino acid sequence of the Seneca virus 3A protein is shown in SEQ ID NO: 1; And / or, the coating antigen is pre-coated on an ELISA plate; The method for preparing the ELISA plate pre-coated with antigen comprises the following steps: 1) Add the coating antigen to the ELISA plate and coat at 30-45°C for 1-5 hours; 2) Add blocking solution to the ELISA plate and block at 30-45°C for 0.5-2h.

3. The indirect ELISA detection kit according to claim 2, characterized in that: In step 1), the coated antigen is diluted with a coating solution and then added to the ELISA plate, wherein the coating solution is a carbonate buffer; And / or, in step 1), the coating concentration of the coating antigen is 1-5 μg / mL; And / or, in step 1), coating is carried out at 37°C for 3 hours.

4. The indirect ELISA detection kit according to claim 2, characterized in that: In step 2), the blocking solution is a BSA solution; And / or, in step 2), blocking is performed at 37°C for 1 h.

5. The indirect ELISA detection kit according to claim 1, characterized in that: The detection kit also includes one or more of a sample diluent, a washing solution, an enzyme-labeled secondary antibody, a color developing solution, a stop solution, an SVA positive serum control, and an SVA negative serum control.

6. The indirect ELISA detection kit according to claim 5, characterized in that: The sample diluent is PBS buffer; And / or, the washing solution is PBST buffer; And / or, the enzyme-labeled secondary antibody is HRP-goat anti-pig IgG; And / or, the color developing solution is a TMB single-component color developing solution; And / or, the stop solution is 2M H2SO4.

7. Use of an indirect ELISA detection kit as described in any one of claims 1 to 6, characterized in that: The following steps are involved: (1) Add the sample to be tested to the ELISA plate pre-coated with antigen and incubate at 30-45°C for 0.5-2.5h; (2) Add enzyme-labeled secondary antibody and incubate at 30-45°C for 0.5-1.5 h; (3) Add color developing solution and develop color at room temperature in the dark; (4) Add stop solution to stop color development; (5) Detection of OD 450nm value, to determine whether there is Seneca virus antibody in the sample to be tested; The judging criteria are as follows: OD of sample to be tested 450nm ≥0.48, judged as positive; OD of sample to be tested 450nm ≤0.41, judged as negative; 0.41<OD of the sample to be tested 450nm <0.48, judged as suspicious.

8. The use according to claim 7, characterized in that: In step (1), the sample to be tested is diluted with a sample diluent before being added to the ELISA plate, and the dilution ratio is 1:50-200; And / or, in step (1), incubate at 37°C for 1 h.

9. The use according to claim 7, characterized in that: In step (2), the enzyme-labeled secondary antibody is diluted with enzyme-labeled diluent before being added to the enzyme-labeled plate, and the dilution ratio is 1:4000-16000; And / or, in step (2), incubate at 37°C for 45 min.

10. The use according to claim 7, characterized in that: In step (3), the color development time is 5-20 minutes.

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