An indirect elisa method for detecting antibodies to foot-and-mouth disease virus sat1

Abstract

The application relates to the technical field of biological detection, and particularly discloses an ELISA kit for detecting foot-and-mouth disease virus SAT1 type antibody. The application provides the ELISA kit for detecting the foot-and-mouth disease virus SAT1 type antibody, which comprises an enzyme-labeled plate coated with foot-and-mouth disease virus SAT1 type P1 protein, enzyme-labeled antibody, positive control serum, negative control serum, sample diluent, washing liquid, substrate developing liquid and termination liquid; the coating concentration of the P1 protein is 0.75-1.5 mu g / mL; the enzyme-labeled antibody is HRP-labeled recombinant fusion golden staphylococcus protein A / streptococcus protein G, and the working concentration of the enzyme-labeled antibody is 25-75 ng / mL. The ELISA kit for detecting the foot-and-mouth disease virus SAT1 type antibody has the advantages of high sensitivity, good accuracy, good repeatability and the like, and has a good use prospect.

Description

Technical Field

[0001] This application relates to the field of biodetection technology, specifically to an indirect ELISA method for detecting SAT1 type antibodies against foot-and-mouth disease virus. Background Technology

[0002] Foot-and-mouth disease (FMD) is an acute, highly contagious infectious disease of cloven-hoofed animals caused by foot-and-mouth disease virus (FMDV). FMDV has seven serotypes: O, A, C, SAT1, SAT2, SAT3, and Asia I. There is no cross-immunity between these serotypes. In recent years, SAT1 serotype outbreaks have occurred in many countries, posing a threat to my country's pig industry and related livestock breeds. Therefore, establishing serological antibody detection methods for SAT1 is of great significance for evaluating immunization efficacy and controlling the epidemic.

[0003] Currently, ELISA is the primary method for FMDV serological detection, with existing methods mostly using VP1 protein as the coating antigen. However, VP1 protein exhibits high variability, with significant sequence differences between different serotypes and strains. Furthermore, when expressed alone, it is prone to problems such as insoluble expression or insufficient retention of conformational epitopes, leading to poor sensitivity, specificity, and stability of detection methods. In addition, existing ELISA products and commercial kits mainly focus on common serotypes such as O and A, with a severe lack of dedicated detection methods for SAT1, making it difficult to meet the urgent need for SAT1 prevention and control.

[0004] Therefore, there is an urgent need to develop a detection kit for SAT1 type foot-and-mouth disease virus antibodies to fill the detection gap for SAT1 type antibodies, while improving the insufficient sensitivity and stability of existing VP1-based ELISA, and providing a reliable technical means for SAT1 type foot-and-mouth disease immune monitoring and epidemic control. Summary of the Invention

[0005] To overcome the current lack of serological detection methods for foot-and-mouth disease virus SAT1 antibodies, insufficient applicability for clinical batch sample testing, and the poor sensitivity, specificity, and stability of existing ELISA methods for VP1 protein, this application provides an indirect ELISA method for detecting foot-and-mouth disease virus SAT1 antibodies.

[0006] In a first aspect, this application provides an ELISA kit for detecting SAT1 type antibodies against foot-and-mouth disease virus, employing the following technical solution: An ELISA kit for detecting SAT1 type antibodies against foot-and-mouth disease virus includes: an enzyme-labeled plate coated with SAT1 type P1 protein of foot-and-mouth disease virus, enzyme-labeled antibody, positive control serum, negative control serum, sample diluent, washing buffer, substrate chromogenic solution and stop solution. The coating concentration of the P1 protein is 0.75-1.5 μg / mL; The enzyme-labeled antibody is an HRP-labeled recombinant fusion of Staphylococcus aureus protein A / Streptococcus protein G, and the working concentration of the enzyme-labeled antibody is 25-75 ng / mL.

[0007] This invention provides an ELISA kit for detecting SAT1 antibodies against foot-and-mouth disease virus. By rationally setting the coating concentration of P1 protein and the working concentration of the enzyme-labeled antibody, this ELISA kit not only achieves highly sensitive detection of SAT1 antibodies against foot-and-mouth disease virus, enabling timely detection of potential infections, but also ensures high specificity, reducing false positive and false negative results. Furthermore, this concentration setting improves the detection stability and repeatability of the kit, ensuring good consistency in results across different batches, thus providing an efficient and accurate solution for the detection of SAT1 antibodies against foot-and-mouth disease virus.

[0008] In this application, limiting the coating concentration of the foot-and-mouth disease virus (FMDV) SAT1 type P1 protein within a specific range ensures an adequate amount of antigen on the ELISA plate, guaranteeing sufficient binding with antibodies in the sample to be tested. If the coating concentration is too low, there will be too few antigen sites available for binding on the ELISA plate, leading to some antibodies failing to bind, resulting in false negatives, reduced sensitivity, and inaccurate detection of FMDV SAT1 type antibodies in low-concentration antibody samples. Conversely, if the coating concentration is too high, excessive antigen may aggregate, affecting the normal binding space and affinity between antigen and antibody, and potentially increasing the probability of non-specific binding, leading to false positives and interfering with the accuracy and reliability of the test. Therefore, this application strictly controls the P1 protein coating concentration within the aforementioned range, finding a balance between sensitivity and specificity, significantly improving the reliability of the test results, which is of great significance for immune assessment.

[0009] This application uses HRP-labeled recombinant fusion Staphylococcus aureus protein A / Streptococcus protein G as the enzyme-labeled antibody. HRP-labeled recombinant fusion Staphylococcus aureus protein A / Streptococcus protein G possesses unique structural and biological characteristics, enabling it to efficiently recognize and bind to the antibody to be detected. This application found that the working concentration of the enzyme-labeled antibody affects the detection performance of the kit. When the working concentration is too low, the amount of enzyme-labeled antibody is relatively small, which may not be sufficient to react with the antibody bound to the ELISA plate, resulting in insufficient signal intensity, inaccurate detection results, and a high risk of missing positive samples. Conversely, when the working concentration is too high, it increases costs, and excessive enzyme-labeled antibody may induce non-specific binding, generating background interference, which also affects the accuracy of the detection results. Therefore, this application controls the working concentration of the enzyme-labeled antibody within the range of 25-75 ng / mL, ensuring sufficient signal intensity for accurate antibody detection while effectively avoiding interference from non-specific binding, resulting in clearer and more accurate detection results.

[0010] Optionally, the coating concentration of P1 protein is 0.75 μg / mL; the working concentration of the enzyme-labeled antibody is 50 ng / mL.

[0011] Optionally, the P1 protein is expressed using a baculovirus system.

[0012] The P1 protein of this application encompasses four capsid proteins: VP4, VP2, VP3, and VP1. Structurally, it is closer to the natural protein, possessing a complete spatial conformation and abundant antigenic epitopes, which is more conducive to reaction with natural foot-and-mouth disease virus positive sera. In addition, the P1 protein is larger than the VP1 protein, more resistant to protease degradation, and has a more stable protein structure, which is beneficial for extending the shelf life of the kit.

[0013] Optionally, the preparation method of the ELISA plate includes the following steps: diluting the P1 protein with carbonate buffer to obtain a suspension, adding the suspension to the ELISA wells, coating at 2-8℃ for 15-20 hours, and then performing blocking and protection treatments in sequence to obtain an ELISA plate coated with foot-and-mouth disease virus SAT1 type P1 protein.

[0014] The method for preparing the ELISA plate provided in this application enables the P1 protein to adhere well to the plate. Subsequent blocking treatment effectively reduces non-specific binding and prevents other substances from binding to non-target areas on the plate, thereby reducing background interference and making the detection results more accurate. The protective treatment maintains the stability and activity of the P1 protein, preventing denaturation or inactivation during storage and use, further enhancing the performance of the ELISA plate, improving the sensitivity and specificity of detection, and ensuring the reliability and repeatability of the detection results.

[0015] In this application, positive and negative control sera are important references in the detection process. Positive control sera contain known SAT1 antibodies against foot-and-mouth disease virus, while negative control sera do not. By comparing the absorbance values ​​of the test sample with those of the positive and negative control sera, the presence of SAT1 antibodies against foot-and-mouth disease virus in the test sample can be accurately determined.

[0016] Optionally, the blocking uses 0.8-1.5% casein, and the blocking conditions are: blocking at 20-30℃ for 0.5-1.5h; the protection uses a protein protection solution, and the protection conditions are: protection at 20-30℃ for 0.5-1.5h.

[0017] Optionally, the sample diluent is 0.3-1% PBSA; the washing solution is PBST washing solution; the substrate chromogenic solution is TMB substrate solution; and the stop solution is 1.5-2.5 mol / L sulfuric acid.

[0018] Secondly, this application provides a method for detecting foot-and-mouth disease virus SAT1 antibodies for non-disease diagnostic purposes, including the step of detecting foot-and-mouth disease virus SAT1 antibodies using an ELISA kit: (1) Sample addition: Dilute the sample to be tested with sample diluent at a volume ratio of 1:(25-200), add it to each enzyme label well, and incubate at 20-30℃ for 25-40 min; (2) Incubation of enzyme-labeled conjugate: Dilute the enzyme-labeled antibody to the working concentration with enzyme-labeled diluent, add it to each enzyme-labeled well, and incubate at 25-37℃ for 15-60 min; (3) Color development: Add substrate color development solution to each enzyme label well and develop color at 25℃ for 15 min; (4) Termination: Add stop solution to each well to terminate the colorimetric reaction; read the absorbance value of each well at 450 nm wavelength using an enzyme reader and judge the result.

[0019] This application provides a method for detecting SAT1 antibodies against foot-and-mouth disease virus (FMDV) for non-disease diagnostic purposes. First, the sample to be tested is diluted with a sample diluent at a specific volume ratio and incubated at a suitable temperature and time. This allows the sample to fully react with the coated antigen, ensuring antibody capture efficiency and improving detection sensitivity. Second, the enzyme-labeled antibody is diluted and incubated at a specific temperature and time, ensuring precise binding to the antibody bound to the antigen, enhancing signal amplification, and making the detection results more accurate. Then, substrate color development and termination clearly present the colorimetric effect, facilitating observation and instrument readings. The detection method provided in this application, by controlling the dilution concentration and incubation conditions of the sample, effectively improves the sensitivity and specificity of the detection, thereby accurately identifying SAT1 antibodies against FMDV in the sample. This provides a reliable solution for antibody detection in non-disease diagnostic scenarios (such as immune assessment) and can be widely applied in scientific research and immune assessment work.

[0020] In this application, the enzyme-labeled diluent contains 0.3-1% PBSA and 0.1-0.3% sucrose.

[0021] Optionally, in the sample addition step, the dilution ratio of the sample to be tested is 1:100; the incubation temperature is 25°C and the time is 30 min.

[0022] In summary, this application has the following beneficial effects: This application uses the SAT1-type P1 protein expressed by a baculovirus system as the coating protein. Its structure is closer to the natural protein, thus possessing complete biological functions and reacting better with natural foot-and-mouth disease virus positive serum, thereby improving the accuracy and reliability of detection. Furthermore, compared to the VP1 protein, the P1 protein has a larger molecular structure, which makes it more resistant to proteases and able to resist protease degradation. Because the P1 protein has a more stable structure, it effectively reduces performance changes caused by protein degradation and other factors during kit storage, thus extending the shelf life of the kit and ensuring its effectiveness within a certain period. Detailed Implementation

[0023] The present application will be further described in detail below with reference to preparation examples, embodiments, and performance testing. The described embodiments are only possible implementations of the present invention, and are not limited thereto. Those skilled in the art can certainly combine the embodiments of the present invention to obtain other embodiments without creative effort, which are also within the protection scope of the present invention.

[0024] In this embodiment, the SAT1 type P1 protein antigen and positive / negative reference sera were prepared and provided by Beijing Jinno Biotech Co., Ltd.; the HRP-labeled recombinant fusion Staphylococcus aureus protein A / Streptococcus protein G was purchased from Thermo Fisher. Other reagents and solvents used in this application are commercially available. Preparation Example 1

[0025] Preparation Example 1 provides an ELISA kit for detecting SAT1 type antibodies against foot-and-mouth disease virus, comprising two enzyme-labeled plates coated with SAT1 type P1 protein of foot-and-mouth disease virus, 30 mL of enzyme-labeled antibody, 3 mL of positive control serum, 3 mL of negative control serum, 60 mL of sample dilution buffer, 125 mL of washing buffer, 30 mL of substrate chromogenic solution and 20 mL of stop solution. Example 1

[0026] Optimization of the optimal coating concentration of P1 protein antigen and the optimal working concentration of enzyme-labeled antibody A method for detecting SAT1 antibodies against foot-and-mouth disease virus for non-disease diagnostic purposes, comprising the following steps: (1) Preparation of ELISA plates: (1-1) P1 protein was diluted with carbonate buffer (0.05 mol / L, pH 9.6) to obtain P1 protein coating concentrations of 0.25 μg / mL, 0.375 μg / mL, 0.5 μg / mL, 0.75 μg / mL, 1.0 μg / mL and 1.5 μg / mL respectively. The suspensions were added to 96-well microplates, 100 μL per well, and coated at 4 °C for 18 h. The buffer was then discarded. (1-2) Add 300 μL of PBST washing buffer to each well and wash 3 times; (1-3) Next, add 1% casein to each well, block at 25°C for 1 hour, discard the blocking solution, and wash again according to step (1-2); (1-4) Then add protein protection solution to each well, incubate at 25°C for 1 hour, discard the protein protection solution, dry, and obtain an ELISA plate coated with foot-and-mouth disease virus SAT1 type P1 protein.

[0027] (2) Sample addition: FMDV-SAT1 positive reference swine serum and FMDV-SAT1 negative reference swine serum were diluted 1:50 with sample dilution buffer (0.5% PBSA), and 100 μL was added to each well. The mixture was incubated at 25°C for 30 min.

[0028] (3) Incubation of enzyme-labeled conjugates: Dilute HRP-labeled recombinant Staphylococcus aureus protein A / Streptococcus protein G to 100 ng / mL, 75 ng / mL, 50 ng / mL, 37.5 ng / mL and 25 ng / mL with enzyme-labeled dilution buffer (0.5% PBSA + 0.2% sucrose). Add 100 μL to each well, incubate at 25°C for 30 min, spin dry, and wash as in (1-2).

[0029] (4) Color development: Add 100 μL of TMB substrate solution to each well and develop color at 25℃ for 15 min.

[0030] (5) Termination: Add 50 μL of stop solution to each well to terminate the colorimetric reaction; read the absorbance (OD) of each well at 450 nm using a microplate reader. 450nm (Value), and judge the result.

[0031] The detection results of FMDV-SAT1 positive reference swine serum were denoted as P, and the detection results of FMDV-SAT1 negative reference swine serum were denoted as N. The P / N value was calculated. The results are shown in Table 1 below. The antigen coating concentration and enzyme-labeled antibody concentration at which the P / N value was maximized were taken as the optimal antigen coating concentration and the optimal working concentration of enzyme-labeled antibody.

[0032] Table 1. Optimization results of optimal antigen coating concentration and optimal working concentration of enzyme-labeled antibody.

[0033] As shown in Table 1, when the P1 protein coating concentration is controlled within the range of 0.75-1.5 μg / mL and the enzyme-labeled antibody working concentration is controlled within the range of 25-75 ng / mL, the P / N ratio can reach above 5.0. Among these, the highest P / N ratio is achieved when the P1 protein coating concentration is 0.75 μg / mL and the enzyme-labeled antibody working concentration is 50 ng / mL. Therefore, the optimal P1 protein coating concentration is determined to be 0.75 μg / mL, and the optimal enzyme-labeled antibody working concentration is determined to be 50 ng / mL. Example 2

[0034] Optimization of the optimal dilution of the test sample The test was conducted according to the detection method of Example 1, with the difference that: (2) in the sample addition step, the dilution ratios of FMDV-SAT1 positive reference swine serum and FMDV-SAT1 negative reference swine serum were 1:25, 1:50, 1:100, and 1:200. The sample dilution with the highest P / N value was taken as the optimal dilution of the sample to be tested. The results are shown in Table 2 below.

[0035] Note: In Example 2, the P1 protein coating concentration was 0.75 μg / mL and the working concentration of the enzyme-labeled antibody was 50 ng / mL.

[0036] Table 2 Optimization results of the optimal dilution of the samples to be tested

[0037] The results in Table 2 show that diluting the samples to be tested at a ratio of 1:(25-200) yields a P / N value of over 5.0. The P / N value is highest when diluted at a ratio of 1:100. Therefore, the optimal dilution of the samples to be tested is determined to be 1:100. Example 3

[0038] Optimization of optimal reaction conditions for enzyme-labeled antibodies The experiment was conducted according to the detection method of Example 1, with the following differences: (2) In the sample addition step, the dilution ratio of FMDV-SAT1 positive reference pig serum and FMDV-SAT1 negative reference pig serum was 1:100; (3) In the enzyme-labeled conjugate incubation step, the incubation conditions were as follows: 25℃ for 15 min, 25℃ for 30 min, 25℃ for 60 min, 37℃ for 15 min, 37℃ for 30 min, and 37℃ for 60 min. The incubation conditions with the highest P / N value were taken as the optimal reaction conditions for the enzyme-labeled antibody. The results are shown in Table 3 below.

[0039] Note: In Example 3, the P1 protein coating concentration was 0.75 μg / mL and the working concentration of the enzyme-labeled antibody was 50 ng / mL.

[0040] Table 3 Optimization results of enzyme-labeled antibody reaction conditions

[0041] Table 3 shows that the P / N value is the highest when the enzyme-labeled antibody is incubated at 25℃ for 15 min. Therefore, the optimal reaction condition for the enzyme-labeled antibody is determined to be incubation at 25℃ for 15 min. Example 4

[0042] Determination of Cut-off Value in Indirect ELISA Detection Methods Forty-five clinically negative swine serum samples, 37 clinically negative bovine serum samples, and 53 clinically negative sheep serum samples were tested. The mean (M) and standard deviation (SD) of the sample OD values ​​were calculated. The calculated value of M + 3SD was used as the cut-off value for positive and negative results, and the positive / negative determination standard for the detection method provided in this application for detecting swine, bovine, and sheep serum samples was determined: the cut-off value was 0.40. Example 5

[0043] Sensitivity study Samples that tested positive for virus neutralization were tested using the detection method determined in Example 4. The P1 protein coating concentration was 0.75 μg / mL; the working concentration of the enzyme-labeled antibody was 50 ng / mL; the sample dilution was 1:100; incubation was performed at 25°C for 30 min; the enzyme-labeled antibody was incubated at 25°C for 15 min; the cut-off value was 0.40; the neutralization test criteria were referenced from the OIE manual (neutralization titer ≥45 is positive; <16 is negative; 16–32 is questionable, retest ≥16 is considered positive).

[0044] Eighty-seven clinically positive immune sera samples that tested positive for SAT1 neutralizing antibodies were analyzed to determine the sensitivity of this method. The results showed that all 87 samples tested positive, with a positive detection rate of 100%, indicating that this method has good sensitivity.

[0045] Serum samples positive for foot-and-mouth disease virus (FMDV) SAT1 neutralizing antibodies were serially diluted twofold and used for sensitivity testing. Results showed that the limit of detection for FMDV SAT1 antibody-positive serum corresponded to a neutralizing antibody titer of 1:16, indicating good sensitivity. Example 6

[0046] Specificity study Samples that tested negative for the virus neutralization test were tested using the detection method determined in Example 4. The P1 protein coating concentration was 0.75 μg / mL; the working concentration of the enzyme-labeled antibody was 50 ng / mL; the sample dilution was 1:100; incubation was performed at 25°C for 30 min; the enzyme-labeled antibody was incubated at 25°C for 15 min; the cut-off value was 0.40; the neutralization test criteria were referenced from the OIE manual (neutralization titer ≥45 is positive; <16 is negative; 16–32 is questionable, and a retest ≥16 is considered positive).

[0047] Sixty-five clinically negative sera (SAT1 neutralizing antibody negative), positive sera for ovine poxvirus, positive sera for Mycoplasma caprineis subsp. caprine pneumonia, positive sera for bovine viral diarrhea / mucosal disease virus, positive sera for bovine infectious rhinotracheitis virus, positive sera for classical swine fever virus, positive sera for porcine pseudorabies virus, positive sera for porcine reproductive and respiratory syndrome virus, positive sera for porcine circovirus type 2, and positive sera for porcine parvovirus were collected and tested to determine the detection specificity of this method. The results showed that the detection method provided in this application was negative for all 65 clinically negative sera and 9 sera with positive antibodies against other pathogens, indicating that the detection method provided in this application has good specificity. Example 7

[0048] Reproducibility studies (1) The detection method determined in Example 4 was used to detect 13 clinically positive immune sera that tested positive by the virus neutralization test. The P1 protein coating concentration was 0.75 μg / mL; the working concentration of the enzyme-labeled antibody was 50 ng / mL; the sample dilution was 1:100, and the samples were incubated at 25°C for 30 min; the enzyme-labeled antibody was incubated at 25°C for 15 min; the cut-off value was 0.40; each sample was tested 5 times using the same batch of coated plates, and the intra-batch coefficient of variation of this method was calculated. (2) The detection method determined in Example 4 was used to detect 13 clinical positive immune sera that were positive by the virus neutralization test. The P1 protein coating concentration was 0.75 μg / mL and the working concentration of the enzyme-labeled antibody was 50 ng / mL. Each sample was tested using 3 different batches of coated plates. The inter-batch coefficient of variation of this method was calculated and the results are shown in Table 4.

[0049] Table 4. Results of repeatability studies using indirect ELISA

[0050] Table 4 shows that the detection method provided in this application has good repeatability.

[0051] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An ELISA kit for detecting SAT1 type antibodies against foot-and-mouth disease virus, characterized in that, include: Enzyme-labeled plate coated with foot-and-mouth disease virus SAT1 type P1 protein, enzyme-labeled antibody, positive control serum, negative control serum, sample diluent, washing buffer, substrate chromogenic solution and stop solution; The coating concentration of the P1 protein is 0.75-1.5 μg / mL; The enzyme-labeled antibody is an HRP-labeled recombinant fusion of Staphylococcus aureus protein A / Streptococcus protein G, and the working concentration of the enzyme-labeled antibody is 25-75 ng / mL.

2. The ELISA kit according to claim 1, characterized in that, The coating concentration of P1 protein was 0.75 μg / mL; the working concentration of the enzyme-labeled antibody was 50 ng / mL.

3. The ELISA kit according to claim 1, characterized in that, The P1 protein was expressed using a baculovirus system.

4. The ELISA kit according to claim 1, characterized in that, The preparation method of the ELISA plate includes the following steps: diluting P1 protein with carbonate buffer to obtain a suspension, adding the suspension to the ELISA wells, coating at 2-8℃ for 15-20h, and then performing blocking and protection treatments in sequence to obtain an ELISA plate coated with foot-and-mouth disease virus SAT1 type P1 protein.

5. The ELISA kit according to claim 4, characterized in that, The blocking uses 0.8-1.5% casein, and the blocking conditions are: 20-30℃ for 0.5-1.5h; the protection uses a protein protection solution, and the protection conditions are: 20-30℃ for 0.5-1.5h.

6. The ELISA kit according to any one of claims 1-5, characterized in that, The sample dilution solution is 0.3-1% PBSA; the washing solution is PBST washing solution; the substrate chromogenic solution is TMB substrate solution; and the stop solution is 1.5-2.5 mol / L sulfuric acid.

7. A method for detecting SAT1 type antibodies against foot-and-mouth disease virus for non-disease diagnostic purposes, characterized in that, Includes the step of detecting SAT1 type antibodies against foot-and-mouth disease virus using the ELISA kit according to any one of claims 1-6: (1) Sample addition: Dilute the sample to be tested with sample diluent at a volume ratio of 1:(25-200), add it to each enzyme label well, and incubate at 20-30℃ for 25-40 min; (2) Incubation of enzyme-labeled conjugate: Dilute the enzyme-labeled antibody to the working concentration with enzyme-labeled diluent, add it to each enzyme-labeled well, and incubate at 25-37℃ for 15-60 min; (3) Color development: Add substrate color development solution to each enzyme label well and develop color at 25℃ for 15 min; (4) Termination: Add stop solution to each well to terminate the colorimetric reaction; read the absorbance value of each well at 450 nm wavelength using an enzyme reader and judge the result.

8. The detection method according to claim 7, characterized in that, In the sample addition step, the dilution ratio of the sample to be tested is 1:100; the incubation temperature is 25℃ and the time is 30min.

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