Competitive ELISA detection method for ASFV detection

Abstract

The invention belongs to the technical field of animal epidemic disease control, and particularly relates to a competitive ELISA detection method for ASFV detection. The method comprises the following steps: sealing a spare plate, adding a sample, detecting, developing, judging and the like. Based on the two prepared novel probes (based on a streptavidin magnetic bead modified African swine fever virus K205R protein probe and a specific monoclonal antibody-HRP fusion probe), a competitive ELISA detection method for detecting ASFV is constructed, and the ASFV can be well detected in combination with optimization of related experimental detection conditions. Preliminary experiment results show that the ELISA detection method provided by the invention has good reproducibility and specificity, does not need to use a commercial secondary antibody and an excessive washing process, can greatly simplify operation steps and detection time, and has good practical value and technical significance for rapid and convenient determination of ASFV and early prevention and control of ASF.

Description

Technical Field

[0001] The present application belongs to the technical field of animal disease prevention and control, and specifically relates to a competitive ELISA detection method for ASFV detection. Background Art

[0002] African swine fever (ASF) is an acute, contagious disease caused by the African swine fever virus (ASFV) that can be transmitted between domestic pigs and wild boars. Once infected, it can cause a fatality rate of up to 100%, making it a serious threat. However, due to the lack of a safe and effective vaccine, accurate laboratory diagnosis is crucial for the prevention and control of ASF.

[0003] Research on ASFV has shown that it is a double-stranded DNA virus with a genome size of approximately 170-194 kb, containing 151-174 open reading frames (ORFs), encoding 150-200 viral proteins. Among these, p12 (O61R), p17 (D117L), p30 (CP204L), p54 (E183L), and p72 (B646L) have been identified as playing key roles in viral replication, immune evasion, and pathogen transmission. Therefore, using these viral proteins as targets, researchers are developing a range of diagnostic viral detection methods and even antiviral agents, with the goal of developing effective ASFV detection and control methods. Summary of the Invention

[0004] The purpose of this application is to provide a competitive ELISA detection method for the detection of African swine fever virus (ASFV), thereby laying a certain technical foundation for the prevention and control of African swine fever (ASF).

[0005] The technical solutions adopted in this application are briefly introduced as follows.

[0006] A competitive ELISA method for ASFV detection, the method specifically comprising the following steps: (1) Blocking: Use a 96-well ELISA plate as the test container. Add 200 μL of 5% BSA solution to each well and incubate at 37°C for 2 h. After blocking, wash three times with PBST and pat dry. Then proceed directly to subsequent testing or store at 4°C for later use. (2) Sample addition and detection: Add 50 μL of serum sample to the ELISA plate blocked in step (1), and then add 50 μL of MBs-SA-K205R probe and 50 μL of mAb-HRP probe to each well; The concentration of the MBs-SA-K205R probe is: 0.5-8 μg / mL (preferably 0.5 μg / mL); The mAb-HRP probe is diluted at a ratio of 1:500 to 8000 (preferably 1:1000) based on the mAb antibody titer; PBS was used as blank control; After adding the sample, shake gently to mix, then incubate at room temperature for 10 to 60 minutes (preferably 30 minutes); (3) Color development and judgment After the reaction, the ELISA plate was magnetically separated for 10 seconds, the supernatant was discarded, and the precipitate was washed four times with PBST buffer to remove unbound components. After washing and patting dry, 100 μL of TMB colorimetric solution was added, and the color reaction was carried out at room temperature in the dark for 10 to 30 minutes (preferably 15 minutes). After the reaction was completed, 50 μL of reaction stop solution (2 M sulfuric acid to stop color development) was added. Finally, the OD value was measured at 450 nm using a microplate reader with PBS buffer as the control, and the inhibition percentage (PI) was calculated as follows: PI = [(1-OD450 nm positive value (P)) / negative value (N)] × 100%.

[0007] When the ASFV detection is applied using the competitive ELISA detection method, the critical value (mean plus three times the standard deviation) (X+3SD) = 32.02%; that is, when the PI value of a sample reaches ≥32.02%, the serum sample is judged to be positive; otherwise, it is considered negative.

[0008] The MBs-SA-K205R probe is prepared by coupling the biotinylated K205R protein with MBs-SA. The specific preparation process is as follows: (1) Biotin-labeled K205R protein First, dissolve 1.0 mg of Sulfo-NHS-LC-Biotin in 180 μL of sterile water to prepare a 10 mM biotin solution. Simultaneously, K205R protein was dissolved in PBS (pH = 7.2) to prepare a 1 mg / mL solution for later use; Subsequently, 13.3 μL of the above biotin solution was added to 1 mL of the K205R protein solution at a 20-fold molar ratio; after mixing evenly, the mixture was reacted at room temperature (about 25°C) in the dark for 1 hour; After the reaction, the cells were dialyzed against PBS overnight to completely remove the unreacted and hydrolyzed biotin reagent; After dialysis, the biotinylated K205R protein solution is obtained, which can be aliquoted and used for subsequent experiments or stored at -80°C for future use; (2) Streptavidin magnetic beads MBs-SA are coupled to the biotinylated K205R protein. First, 1 mL of streptavidin magnetic beads (MBs-SA) solution (10 mg / mL) was taken. After magnetic separation, the beads were washed three times with PBST buffer; and finally, the beads were resuspended in 1 mL of PBST buffer. Subsequently, the magnetic beads were added to 100 μL of biotinylated K205R protein solution (protein concentration was 1 mg / mL) and incubated at room temperature (about 25°C) in the dark for 1.5 h. After incubation, the obtained magnetic beads (i.e., MBs-SA-K205R probe) were washed three times with PBST buffer, and then resuspended in 1 mL of PBST buffer (final concentration was adjusted to 1 mg / mL), and directly used for subsequent applications or stored in the dark at 4°C for future use.

[0009] The mAb-HRP probe is an anti-K205R specific monoclonal antibody mAb-HRP fusion probe, which is prepared by labeling the monoclonal antibody (mAb) with horseradish peroxidase (HRP) using the sodium periodate method. The specific preparation method is as follows: First, dissolve 5 mg of horseradish peroxidase (HRP) in 1 mL of deionized water, add 200 μL of 0.05 M sodium periodate (NaIO4) solution (freshly prepared), and stir slowly for 30 minutes in the dark. After stirring, the solution was transferred to a dialysis bag and dialyzed overnight at 4°C in PBS buffer (pH = 7.4); After dialysis, add 1M NaHCO3 solution and adjust the pH to about 9.5; then immediately add 10 mg of monoclonal antibody (concentration 4 mg / mL) and stir at 4°C in the dark for about 16 hours; After the reaction is completed, add 235 μL of 10 mg / mL sodium borohydride (NaBH4) solution (to be prepared fresh), mix well, and let it stand at 4°C for 2 hours; After standing, add an equal amount of saturated ammonium sulfate solution to the mixture and precipitate at 4°C for 30 min; After precipitation, centrifuge at 12000 r / min for 10 min, discard the supernatant, and resuspend the pellet in PBS and dialyze overnight at 4°C against PBS buffer (pH = 7.4); After dialysis, the dialysate was centrifuged at 1200 r / min for 10 min. The supernatant obtained after centrifugation was the HRP-labeled antibody solution (i.e., mAb-HRP probe solution), which was aliquoted and used for subsequent experiments or stored at -80°C for future use.

[0010] Previous studies have shown that K205R is an early expressed protein during the amplification of ASFV infection. Based on its antigenicity, this protein can be used for early serological diagnosis of ASFV. Based on the usefulness of this protein and the fact that NHS-biotin reacts with amino groups on proteins to form stable amide bonds, the inventors modified it with biotin. Furthermore, due to the high cost-effectiveness and convenience of biomagnetic separation technology, the inventors used streptavidin magnetic beads (MBs-SA) as a carrier. Taking advantage of the extremely high affinity between biotin and streptavidin, they prepared a composite probe, MBs-SA-K205R, which is a biotinylated protein coupled to streptavidin magnetic beads. (The streptavidin magnetic beads (MBs-SA) were coupled to the biotinylated K205R protein primarily to increase its specific surface area and improve its suspension in solution.) Furthermore, a monoclonal antibody (mAb) targeting the K205R protein was labeled with HRP using the sodium periodate method to prepare a mAb-HRP probe.

[0011] In general, this application is based on the two prepared novel probes (based on streptavidin magnetic beads modified African swine fever virus K205R protein probe and specific monoclonal antibody-HRP fusion probe) to construct a competitive ELISA detection method for detecting ASFV, and combined with the optimization of relevant experimental detection conditions, it can achieve better detection of ASFV. Preliminary experimental results show that the ELISA detection method provided by this application has good reproducibility (both intra-batch and inter-batch reproducibility are less than 10%) and specificity, and does not require the use of commercial secondary antibodies and excessive washing processes, which can greatly simplify the operation steps and detection time. It has good practical value and technical significance for the rapid and convenient determination of ASFV and the early prevention and control of ASF. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 These are the relevant test results during the preparation of the MBs-SA-K205R probe; A is the TEM image of streptavidin magnetic beads MBs-SA; B is the TEM image of the prepared MBs-SA-K205R probe; C is the particle size detection result of streptavidin magnetic beads MBs-SA and the prepared MBs-SA-K205R probe; D is the determination results of streptavidin magnetic beads MBs-SA and the prepared MBs-SA-K205R probe using the indirect ELISA method; E is the stability test result of the prepared MBs-SA-K205R probe after being placed for different time periods; Figure 2is the relevant detection result of the prepared mAb-HRP probe; wherein: A is the UV-visible scanning spectrum of mAb-HRP probe; B is the indirect ELISA method to evaluate the binding affinity between mAb-HRP probe and K205R protein; Figure 3 These are the relevant experimental results during the reaction parameter optimization process; among them: The upper left figure shows the test results under different serum dilution conditions; The lower left figure shows the detection results under different incubation reaction times; The lower right figure shows the test results under different color development times; Figure 4 To determine the cutoff value of the established competitive ELISA test method; Figure 5 To evaluate the specificity of the constructed ELISA detection method; A is the test result related to ASFV antibody-negative serum samples; B shows the test results of antisera against ASFV+, ASFV-, PRV+, PRRSV+, PEDV+, and CSFV+. DETAILED DESCRIPTION

[0013] The present application will be further explained below with reference to the following examples. Before introducing the specific examples, a brief description of the experimental background of some of the following examples is given below.

[0014] Biomaterials: ASFV-positive serum, porcine circovirus type 2 (PCV2), classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus (PRV)-positive serum were purchased from the China Veterinary Drug Administration; Because the applicant and the inventor belong to professional scientific teaching and research institutions and professional researchers, the inventor has collected and preserved relevant ASFV-negative serum and serum samples for clinical testing from cooperative units; The K205R protein and the monoclonal antibody targeting ASFV K205R (antibody titer is 1:2048000) are the inventors' previous scientific research results (see the applicant and the inventors' previous relevant public literature reports). Therefore, relevant samples are preserved; Main reagents (test kits) and equipment: Sulfo-NHS-LC-Biotin kit and 96-well ELISA plate were purchased from ThermoFisher Scientific (Waltham, Massachusetts, USA); Horseradish peroxidase (HRP) was purchased from Sigma-Aldrich (St. Louis, MO, USA); Bovine serum albumin (BSA) was purchased from Beijing Solebow Technology Co., Ltd. (Beijing, China); The 96-well magnetic plate and streptavidin magnetic beads (MBs-SA, the product is in solution form) were purchased from Nanjing Donna Biotechnology Co., Ltd. (Nanjing, China).

[0015] Example 1 The main technical approach of this application is to construct two probes: a streptavidin-magnetic bead-modified African swine fever virus K205R protein probe and a specific monoclonal antibody-HRP fusion probe. These probes are then used to establish a competitive ELISA assay. This example first briefly describes the construction of the two probes.

[0016] (1) Streptavidin magnetic beads modified with African swine fever virus K205R protein probe The construction of a streptavidin magnetic bead-modified African swine fever virus K205R protein probe involves two steps: first, labeling the K205R protein with biotin. Then, the streptavidin magnetic beads are coupled to the biotinylated K205R protein to complete the modification. The principle behind this process is that the amino group (-NH2) on the K205R protein reacts with the -NHS (N-hydroxysuccinimide ester) on the biotin to form a stable amide bond, thereby attaching the biotin molecule to the K205R protein. The biotin-streptavidin system is then used to couple the biotinylated K205R protein to MBs-SA to create the MBs-SA-K205R probe. The detailed process is described below.

[0017] (1) Biotin-labeled K205R protein First, dissolve 1.0 mg of Sulfo-NHS-LC-Biotin in 180 μL of sterile water to prepare a 10 mM biotin solution. Simultaneously, K205R protein was dissolved in PBS (pH = 7.2) to prepare a 1 mg / mL solution for later use; Subsequently, 13.3 μL of the above biotin solution was added to 1 mL of 1 mg / mL K205R protein solution at a 20-fold molar ratio; after mixing evenly, the mixture was reacted at room temperature (around 25°C) in the dark for 1 h; After the reaction, the cells were dialyzed against PBS overnight to completely remove the unreacted and hydrolyzed biotin reagent; After dialysis, the biotinylated K205R protein solution is obtained, which can be aliquoted and used for subsequent experiments or stored at -80°C for future use.

[0018] (2) Conjugation of Streptavidin Magnetic Beads MBs-SA with Biotinylated K205R Protein First, 1 mL of Streptavidin Magnetic Beads (MBs-SA) solution (concentration of 10 mg / mL) was taken. After magnetic separation, the magnetic beads were washed three times with PBST buffer and finally resuspended in 1 mL of PBST buffer. Subsequently, the magnetic beads were added to 100 μL of biotinylated K205R protein solution (protein concentration 1 mg / mL) and incubated at room temperature (about 25°C) in the dark for 1.5 h. After incubation, the obtained magnetic beads (i.e., MBs-SA-K205R probe) were washed three times with PBST buffer, resuspended in 1 mL of PBST buffer (and the concentration was adjusted to 1 mg / mL), and directly used for subsequent applications or stored in the dark at 4°C for future use.

[0019] The inventors further characterized the final MBs-SA-K205R probe (i.e., streptavidin magnetic beads modified with African swine fever virus K205R protein probe) using a transmission electron microscope (TEM, JEM-f200, JEOL Ltd, Tokyo, Japan) and a laser particle size analyzer (DLS, ZEN3700, Malvern Panalytical Ltd, Malvern, UK), and preliminarily measured its performance using an indirect ELISA method. The specific results are briefly described below.

[0020] (A) Performance parameter characterization The electron microscopy results of different samples are as follows Figure 1 ( Figure 1 A. Figure 1 Analysis shows that after coupling, there are tiny protrusions on the surface of MBs-SA-K205R, which indicates that the K205R protein is successfully attached to MBs-SA, that is, the preparation of the relevant probe is successful.

[0021] Further particle size test results ( Figure 1 C) shows that the particle size of MBs-SA is about 900 nm, while the constructed MBs-SA-K205R probe is about 1100 nm. The particle size of MBs-SA-K205R is significantly larger than that of MBs-SA. This result also indicates that the K205R protein and MBs-SA are successfully coupled into a whole.

[0022] (B) Indirect ELISA test The detection method is as follows: The prepared MBs-SA-K205R probe sample was diluted to 0.6 μg / mL with PBS, and 100 μL of mAb targeting K205R protein (1:1000) was added. After gently shaking, the sample was incubated at 37°C for 60 min. After the reaction, the wells were magnetically separated and washed three times with PBST buffer. After being patted dry, 100 μL of HRP-conjugated goat anti-mouse IgG secondary antibody (1:5000) was added to each well and reacted at 37°C for 45 min. After the reaction, magnetic separation was performed, the plates were washed four times with PBST buffer, and after being gently patted dry, 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB, Solarbio) color development solution was added to each well and incubated in the dark at room temperature for 15 min; Finally, 50 μL of 2 M sulfuric acid stop solution was added to each well, and the OD450 nm value was read using a multifunctional microplate reader (Spark 10 M, Tecan Trading AG, Mnnedorf, SU) (the significant difference between OD values ​​was determined by t-test).

[0023] Test results such as Figure 1 ( Figure 1 As shown in D), analysis shows that only the MBs-SA-K205R probe reacts with the monoclonal antibody mAb, while the streptavidin magnetic beads MBs-SA alone do not react with the monoclonal antibody mAb. This also confirms that the prepared MBs-SA-K205R probe is as expected.

[0024] Furthermore, the prepared probe was stored at 4°C and samples were taken regularly according to the above method to evaluate the stability of the probe under long-term storage. Figure 1 ( Figure 1 E). It can be seen that the OD value obtained in the indirect ELISA test did not change significantly over 112 days. This result indicates that the prepared MBs-SA-K205R probe can be stored stably for a long time.

[0025] (2) Specific monoclonal antibody-HRP fusion probe (mAb-HRP probe) Monoclonal antibodies (mAbs) were labeled with horseradish peroxidase (HRP) using the sodium periodate method. The specific operation method is as follows.

[0026] First, dissolve 5 mg of horseradish peroxidase (HRP) in 1 mL of deionized water, add 200 μL of 0.05 M sodium periodate (NaIO4) solution (freshly prepared), and stir slowly for 30 minutes in the dark. After stirring, the solution was transferred to a dialysis bag and dialyzed overnight at 4°C in PBS buffer (pH = 7.4); After dialysis, add 1M NaHCO3 solution and adjust the pH to about 9.5; then immediately add 10 mg of monoclonal antibody (concentration 4 mg / mL) and stir at 4°C in the dark for about 16 hours; After the reaction is completed, add 235 μL of 10 mg / mL sodium borohydride (NaBH4) solution (to be prepared fresh), mix well, and let it stand at 4°C for 2 hours; After standing, add an equal amount of saturated ammonium sulfate solution to the mixture and precipitate at 4°C for 30 min; After precipitation, centrifuge at 12000 r / min for 10 min, discard the supernatant, and resuspend the pellet in PBS and dialyze overnight at 4°C against PBS buffer (pH = 7.4); After dialysis, the dialysate was centrifuged at 1200 r / min for 10 min, and the supernatant obtained after centrifugation was the HRP-labeled antibody solution (i.e., mAb-HRP probe solution) (concentration 4 mg / mL; Figure 2 The titer of the labeled antibody in B is 1:7000, and the OD value is still above 1. The optimal dilution concentration after optimization in the subsequent detection method establishment is 1:1000). After packaging, use it for subsequent experiments or store it at -80℃ for future use.

[0027] The inventors used a UV spectrophotometer to detect the absorption peak of the prepared mAb-HRP probe (to assess whether the antibody was successfully labeled with HRP) and also used a direct ELISA method to detect it. The specific results are briefly described below.

[0028] (A) Absorption peak detection The absorption peak curve detection results of the prepared mAb-HRP probe are as follows Figure 2 ( Figure 2 As shown in Figure A, the mAb-HRP probe's absorption peak curve combines the absorption peaks of HRP and mAb, peaking at approximately 275 nm and 400 nm, respectively. This result demonstrates that HRP and mAb are successfully coupled to form a new entity, and the new mAb-HRP probe possesses the original properties of both HRP and mAb.

[0029] (B) Direct ELISA First, the MBs-SA-K205R probe prepared above was diluted to 0.6 μg / mL with PBS, and 100 μL per well was added to the ELISA plate; The prepared mAb-HRP probe was then diluted at a ratio of 1:500, 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, and 1:7000, with 100 μL per well. After gently shaking, the cells were reacted at room temperature (about 25°C) for 45 minutes. After the reaction, the cells were washed four times with PBST and TMB colorimetric solution was directly added to react in the dark for 15 minutes. Finally, 2MH2SO4 stop solution was added and the OD450nm value was read using a microplate reader.

[0030] Test results such as Figure 2 ( Figure 2 B). The analysis shows that the mAb-HRP probe has a high affinity for the K205R protein. This also indicates that the conjugation of HRP to the monoclonal antibody mAb does not affect the affinity between the mAb and the K205R protein. Example 2 Based on the probes constructed in Example 1 (MBs-SA-K205R probe, mAb-HRP probe), the inventors further constructed a competitive ELISA detection method and optimized the relevant reaction parameters, as briefly described below.

[0031] (1) Closed spare plate Use a 96-well ELISA plate as the detection container, add 200 μL of 5% BSA solution to each well, and incubate at 37°C for 2 hours. After incubation, wash three times with PBST and pat dry before proceeding directly to subsequent detection or storing at 4°C for future use.

[0032] (2) Sampling and testing Add 50 μL of serum sample (ASFV-positive serum or test sample) to the ELISA plate blocked in step (1), and then add 50 μL of MBs-SA-K205R probe and 50 μL of mAb-HRP probe to each well; PBS was used as blank control; After adding the sample, shake gently to mix, and then incubate the reaction at room temperature (about 25°C). (3) Color development and judgment After the reaction, the ELISA plate was magnetically separated for 10 seconds, the supernatant was discarded, and the precipitate was washed four times with PBST buffer to remove unbound components. After washing and patting dry, 100 μL of TMB colorimetric solution was added and the color reaction was carried out at room temperature in the dark. After the reaction was completed, 50 μL of reaction stop solution (2 M sulfuric acid was added to stop the color development) was added. Finally, PBS buffer was used as a control, and the OD value was measured at 450 nm using a microplate reader to calculate the inhibition percentage (PI), PI = [(1-OD450 nm positive value (P)) / negative value (N)] × 100%.

[0033] Based on the above reaction process, the inventors have optimized the relevant reaction parameters, and the specific details are briefly described as follows.

[0034] (1) Probe concentration To determine the optimal dilution concentration of the MBs-SA-K205R probe and the optimal dilution of the mAb-HRP probe, the inventors conducted experimental exploration using the checkerboard method, and the specific details are briefly described below.

[0035] Refer to the above operation flow: In step (2), the MBs-SA-K205R probe was diluted to different concentrations of 0.5-8 μg / mL with PBS buffer, and the mAb-HRP probe was diluted at a ratio of 1:500 to 1:8000; In step (2), the ASFV-positive serum was not diluted; the reaction was incubated for 30 minutes; In step (3), the color reaction was carried out for 15 minutes; Finally, the optimal dilution parameters were determined by measuring the OD value and calculating the inhibition rate.

[0036] The inhibition rate determination results under different combinations of MBs-SA-K205R dosage and McAb-HRP dilution are shown in Table 1 below.

[0037] Table 1. Inhibition rates at different MBs-SA-K205R dosages and McAb-HRP dilution combinations The results in the table above show that the PI value is maximized when the MBs-SA-K205R probe dosage is 0.5 μg / mL (i.e., 50 μL, concentration is 1 μg / mL) and the mAb-HRP probe dilution is 1:1000. Therefore, subsequent experiments were conducted using this concentration and dilution ratio.

[0038] (2) Dilution of test sample Refer to the above process: in step (ii), the concentration of MBs-SA-K205R probe is 0.5 μg / mL, and the dilution of mAb-HRP probe is 1:1000; In step (2), the ASFV-positive serum was diluted with PBS buffer at a volume ratio of 1:1, 1:2, 1:5, 1:10, and 1:20, respectively. After determining the above conditions, the serum was incubated at room temperature for 30 minutes. In step (3), the color reaction was carried out for 15 minutes; Finally, the optimal dilution parameters were determined by measuring the OD value and calculating the inhibition rate.

[0039] The results are as follows Figure 3 ( Figure 3 As shown in the upper left figure in the middle, it can be seen that the PI value can reach the maximum when the serum is not diluted.

[0040] (3) Incubation reaction time Refer to the above process: in step (ii), the concentration of MBs-SA-K205R probe is 0.5 μg / mL, and the dilution of mAb-HRP probe is 1:1000; In step (ii), ASFV-positive serum was not diluted, and the incubation reaction was adjusted to 10, 20, 30, 40, 50, and 60 min respectively; In step (3), the color reaction was carried out for 15 minutes; Finally, the optimal dilution parameters were determined by measuring the OD value and calculating the inhibition rate.

[0041] The results are as follows Figure 3 ( Figure 3 As shown in the lower left figure, it can be seen that in the case of undiluted serum, the best effect is achieved when the incubation time is 30 minutes.

[0042] (4) Color development time Refer to the above process: in step (ii), the concentration of MBs-SA-K205R probe is 0.5 μg / mL, and the dilution of mAb-HRP probe is 1:1000; In step (2), ASFV-positive serum was incubated undiluted for 30 min; In step (3), the color development reaction time was adjusted to: 10, 15, 20, 25, and 30 min respectively; Finally, the optimal dilution parameters were determined by measuring the OD value and calculating the inhibition rate.

[0043] The results are as follows Figure 3 ( Figure 3 It can be seen that the best overall effect is achieved when the color reaction lasts for 15 minutes.

[0044] Based on the above-mentioned optimized reaction parameters, the inventors conducted a test and evaluation on the relevant characteristics of the competitive ELISA detection method established in this application, and the specific details are briefly described as follows.

[0045] (A) Detection critical value of the ELISA detection method constructed in this application Referring to the above optimized reaction parameters and reaction conditions, 50 ASFV-negative sera were used as samples for testing, and the critical value was set as the average PI of 50 negative pig sera plus 3 standard deviations (SD); and the judgment criteria were set as: When the sample inhibition rate is ≤X+3SD, it is judged as negative, and ≥X+3SD, it is positive.

[0046] Test results such as Figure 4 As shown in the figure. Analysis of the test data reveals that the average PI value (X) of the 115 serum samples was 12.25%, with a standard deviation (SD) of 6.59%. The critical value (mean plus three times the standard deviation) (X + 3SD) = 32.02%. In other words, a serum sample is considered positive when its PI value reaches ≥32.02%; otherwise, it is considered negative.

[0047] (B) Specificity With reference to the optimized reaction parameters and conditions described above, the distribution of PI values ​​was analyzed using 118 known ASFV-negative sera as samples. In addition, reactions were performed using serum antibodies against common porcine viruses, including ASFV(-), PRRSV(+), PCV(+), CSFV(+), and PRV(+), to evaluate the specificity of the detection method of this application.

[0048] Test results such as Figure 5 As shown. The analysis shows that: for 118 known negative pig serum samples, the results show that all samples are negative. In addition, further analysis results show that ( Figure 5 A), of the 118 samples, 61 samples had a CV% lower than 10%, and 2 samples had a PI value between 30% and 32.02%. This further demonstrates that the detection method constructed in this application and the related index settings have good accuracy. When testing different serum antibody samples ( Figure 5 B) As can be seen, except for ASF-positive sera, the PI values ​​of other pig sera did not exceed 32.02%. This result shows that the set index can accurately distinguish antibodies against ASFV and other types of viruses.

[0049] (C) Repetitive situations The coefficient of variation (CV) was calculated based on 5 positive and 5 negative test results to assess the inter-assay (between plates) and intra-assay (within plates) reproducibility.

[0050] The results of intra- and inter-batch reproducibility tests are shown in Table 2. Analysis shows that the coefficient of variation (i.e., the ratio of standard deviation to mean) for all samples was less than 10%, indicating that the method has good reproducibility.

[0051] Table 2. Intra-assay and inter-assay reproducibility (X: mean of 4 replicates; SD: standard deviation of 4 replicates; CV: coefficient of variation).

[0052] (D) Comparison with commercial kits The existing commercial African swine fever virus blocking ELISA antibody detection kit (a product of Qingdao Lijian Biotechnology Co., Ltd., the technical principle of this product is to use blocking ELISA technology to detect African swine fever virus p30 antibodies in pig serum) was used as a control (refer to its instructions for operation), and 160 randomly obtained clinical pig serum samples were used as test samples, and their detection results were compared with the detection method constructed in this application.

[0053] The comprehensive comparison results are shown in Table 3 below.

[0054] Table 3. Comparison of the detection results of the present detection method and the commercial kit

[0055] As can be seen from the results in the table above, the detection results of the present application's detection method are highly consistent with those of the commercial kit at a rate of 98.1%. This result indicates that the detection method constructed in this application has good prospects for commercial application. More importantly, compared with existing commercial kits, this application does not require the use of secondary antibodies, so not only is the preparation cost lower, but the detection time can be shortened, thus having greater practical value.

Claims

1. A competitive ELISA method for detecting ASFV, characterized in that: The method comprises the following steps: (1) Closed spare plate The ELISA plate was used as the detection container, and each well was sealed with BSA solution before use; (2) Sampling and testing Add 50 μL of the serum sample to be tested to the ELISA plate blocked in step (1), and then add 50 μL of the MBs-SA-K205R probe and 50 μL of the mAb-HRP probe to each well; The concentration of the MBs-SA-K205R probe is: 0.5-8 μg / mL; The mAb-HRP probe is diluted at a ratio of 1:500 to 8000 based on the mAb antibody titer; PBS was used as blank control; After adding the sample, shake gently to mix, then incubate the reaction for 10 to 60 minutes; (3) Color development and judgment After the reaction is completed, the ELISA plate is magnetically separated, the supernatant is discarded, the precipitate is washed and patted dry, and TMB color development solution is added. The color reaction is carried out in the dark for 10 to 30 minutes. After the reaction is completed, the stop solution is added. Finally, PBS buffer was used as a control, and the OD value was measured at 450 nm using a microplate reader to calculate the inhibition percentage PI, PI = [(1-OD450 nm positive value (P)) / negative value (N)] × 100%.

2. The competitive ELISA method for ASFV detection according to claim 1, wherein In step (2), the concentration of the MBs-SA-K205R probe was 0.5 μg / mL; the mAb-HRP probe was diluted at a ratio of 1:1000.

3. The competitive ELISA detection method for ASFV detection according to claim 1, characterized in that In step (2), the reaction was incubated for 30 min.

4. The competitive ELISA detection method for ASFV detection according to claim 1, characterized in that In step (3), the color reaction was carried out in the dark for 15 minutes.

5. The competitive ELISA detection method for ASFV detection according to claim 1, characterized in that In step (3), when the PI value of a sample is ≥32.02%, the serum sample is judged as positive; otherwise, it is judged as negative.

6. The competitive ELISA detection method for ASFV detection according to claim 1, characterized in that In step (ii), the MBs-SA-K205R probe is prepared by coupling the biotinylated K205R protein with MBs-SA; the specific preparation process is as follows: (1) Biotin-labeled K205R protein First, dissolve Sulfo-NHS-LC-Biotin in sterile water to prepare a 10 mM biotin solution for later use. Simultaneously, dissolve K205R protein in PBS to prepare a 1 mg / mL solution for later use. Subsequently, the biotin solution was added to the K205R protein solution at a 20-fold molar ratio; after mixing evenly, the reaction was carried out in the dark; After the reaction is completed, dialysis treatment is performed; After dialysis is completed, the result is a biotinylated K205R protein solution; (2) Streptavidin magnetic beads MBs-SA are coupled with biotinylated K205R protein First, the streptavidin magnetic beads MBs-SA were cleaned and added to the biotinylated K205R protein solution prepared in step (1) and incubated for 1 to 2 hours; After the incubation is completed, the obtained magnetic beads are washed to obtain the MBs-SA-K205R probe.

7. The competitive ELISA detection method for ASFV detection according to claim 1, characterized in that In step (ii), the mAb-HRP probe is an anti-K205R specific monoclonal antibody mAb-HRP fusion probe, and the monoclonal antibody mAb is labeled with horseradish peroxidase HRP using the sodium periodate method. The specific preparation method is: First, horseradish peroxidase HRP was dissolved in deionized water, and sodium periodate NaIO4 solution was added and treated in the dark for no less than 30 min. After the light-proof treatment, the solution was transferred to a dialysis bag and dialyzed overnight; After dialysis, add NaHCO3 solution and adjust the pH to 9.5; then immediately add monoclonal antibody and stir at 4°C in the dark for 12 to 24 hours; After the reaction is completed, sodium borohydride (NaBH4) solution is added, mixed evenly, and allowed to stand at 4°C for 1 to 3 hours; After standing, add an equal amount of saturated ammonium sulfate solution to the mixture and precipitate at 4°C for 20-40 minutes; After precipitation, centrifuge, discard the supernatant, resuspend the pellet and dialyze overnight; After dialysis, the dialysate was centrifuged, and the resulting supernatant was the mAb-HRP probe solution.

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