An indirect elisa kit for detecting antibodies against african swine fever virus p22

Abstract

The present application relates to the technical field of virus detection, and more particularly to an indirect ELISA detection kit for African swine fever virus P22 antibody, which comprises an enzyme-labeled plate coated with TF-P22 protein, a sample diluent, a goat anti-pig enzyme-labeled secondary antibody, a color developing solution and a termination solution. P22 is a high-efficiency transcription and expression structural protein in the early stage of ASFV infection, which can activate the host humoral immune response in the early stage of viral infection. Experimental verification shows that P22 specific serum antibody can be detected at 8-9 days after challenge, which is 2-3 days earlier than the antibody turning positive time of traditional p72 antigen target. This early serological conversion feature provides reliable technical support for realizing "early detection and early disposal" of ASF infection, significantly prolongs the golden window period of epidemic intervention, and has key significance for blocking the hidden transmission of the virus in pig populations.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and more specifically, to an indirect ELISA detection kit for African swine fever virus P22 antibody. Background Technology

[0002] African swine fever (ASF) is a highly contagious and deadly infectious disease of pigs caused by the African swine fever virus (ASFV). Both domestic and wild pigs are susceptible. Clinically, it is characterized by high fever, respiratory distress, systemic hemorrhage, and significant splenomegaly. The mortality rate of acute infection is as high as 100%. Since its introduction from Africa in 1957, the disease has gradually spread to Europe and Eurasia. It was first introduced into my country in 2018 and has rapidly spread to most provinces across the country, causing devastating damage to the pig farming industry and seriously threatening the security of the global food supply chain and the stability of the biosecurity system.

[0003] African swine fever virus (ASFV), the sole member of the African swine fever virus family, is a large double-stranded DNA enveloped virus with a genome size of approximately 180–190 kb. It encodes over 150 viral proteins, exhibiting a complex genetic structure, diverse genotypes, and rich immune evasion mechanisms. Currently, there is no commercially available, safe, and effective ASF vaccine globally, and disease control heavily relies on accurate early diagnosis, strict biosafety management, and timely culling of diseased and dead pigs. Therefore, in-depth analysis of the structure and function of key ASFV proteins has significant theoretical and applied value for developing novel diagnostic markers and vaccine candidate antigens.

[0004] Viral structural proteins are core components mediating viral assembly, host interaction, and inducing the body's immune response, and are also key targets for diagnostic reagents and subunit vaccine design. Among them, the P22 protein encoded by the KP177R gene, as an important structural protein expressed early in ASFV, possesses outstanding characteristics such as high conservation, strong immunodominance, and early expression, playing a crucial role in viral invasion of host cells and immune activation. In recent years, the P22 protein has become a hot target in the fields of basic research and application translation of ASFV. Developing gene engineering expression, antigen epitope screening, and detection methods around it is of great practical significance for overcoming current bottlenecks in ASF prevention and control technology. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, one aspect of the present invention is to provide an indirect ELISA detection kit for African swine fever virus P22 antibody, the detection kit comprising an enzyme-labeled plate coated with TF-P22 protein, sample diluent, goat anti-swine enzyme-labeled secondary antibody, chromogenic solution and stop solution.

[0006] Preferably, the ELISA plate coated with TF-P22 protein is specifically made with purified and desalted TF-P22 protein.

[0007] Preferably, the TF-P22 protein is a recombinant protein.

[0008] Preferably, the TF-P22 protein is the ASFV-TF-P22 gene sequence obtained by inserting a TF fusion tag into an ASFV epidemic strain. The target gene for expressing the protein is inserted into the pET-28a vector to synthesize a recombinant plasmid. The synthesized recombinant plasmid is transformed into the BL21(DE3) expression strain. After the bacterial culture and induction expression are completed, the Escherichia coli cells are collected, and the soluble expressed protein is directly purified after the supernatant is collected by lysing.

[0009] Preferably, the amino acid sequence of the ASFV-TF-P22 gene sequence is SEQ ID NO.1.

[0010] Preferably, the sample diluent is 1×PBS.

[0011] Preferably, the sample diluent is African swine fever virus serum.

[0012] Preferably, the goat anti-pig enzyme-labeled secondary antibody is an HRP-labeled goat anti-pig antibody (IgG).

[0013] Preferably, the test kit further includes a buffer solution containing 0.05% Tween-20 phosphate buffer.

[0014] Preferably, the test kit further includes a blocking solution, wherein the blocking solution is 1% chicken oocyte albumin.

[0015] The beneficial effects of this invention are as follows: With broad-spectrum applicability, it breaks through the limitations of traditional target strain differences: The P22 protein is encoded by the KP177R gene. Its core amino acid sequence and three major functional B-cell epitopes are highly conserved in the major prevalent type I and II strains worldwide, with 100% homology. Even in ancient African genotype strains, there are only a few substitutions at non-critical sites, and the overall antigenicity remains stable. Compared with traditional antigens such as p72 and p54, which are easily affected by strain variations, the P22 protein has outstanding cross-strain broad-spectrum recognition ability, which can effectively adapt to the detection and immunization needs of the mainstream epidemic areas around the world and the current prevalent strains in my country, and significantly reduce the risk of missed detection due to target mutations.

[0016] Early diagnosis offers significant advantages and effectively extends the window period for epidemic prevention and control: P22, as a structural protein that is efficiently transcribed and expressed in the early stages of ASFV infection, can activate the host's humoral immune response in the early stages of viral infection. Experimental verification shows that P22-specific serum antibodies can be detected 8-9 days after challenge, which is 2-3 days earlier than the antibody seroconversion time of traditional antigen targets such as p72. This early serological conversion characteristic provides reliable technical support for achieving "early detection and early treatment" of ASF infection, significantly extends the golden window period for epidemic intervention, and is of key significance for blocking the covert transmission of the virus in pig herds.

[0017] With excellent detection specificity and effective avoidance of cross-reaction interference: Immunological validation shows that the P22 protein has extremely strong immunospecificity and exhibits no cross-immune reaction with common swine pathogens such as classical swine fever virus, porcine reproductive and respiratory syndrome virus (PRRSV), and pseudorabies virus. The serological detection method based on P22 can effectively eliminate non-specific interference from antibodies against other common diseases in farms, significantly reduce the false positive rate, and significantly improve the accuracy and reliability of detection results, providing a more precise technical tool for disease eradication and biosecurity tiered management in large-scale pig farms.

[0018] Additional aspects and advantages of the invention will become apparent from the description which follows, or may be learned by practice of the invention. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is an SDS-PAGE electrophoresis pattern of TF-P22 recombinant protein expression in an embodiment of the present invention; Figure 2 This is an SDS-PAGE electrophoresis pattern of the purified TF-P22 recombinant protein from an embodiment of the present invention. Figure 3 This is an SDS-PAGE electrophoresis pattern of recombinant MBP-P22 protein expression in an embodiment of the present invention; Figure 4 This is an SDS-PAGE electrophoresis pattern of the purified MBP-P22 recombinant protein from an embodiment of the present invention. Figure 5 This is an SDS-PAGE electrophoresis pattern of the DsbA-P22 recombinant protein expression in an embodiment of the present invention. Figure 6 This is an SDS-PAGE electrophoresis pattern of the purified DsbA-P22 recombinant protein from an embodiment of the present invention. Figure 7This is an SDS-PAGE electrophoresis pattern of three recombinant proteins, TF-P22, MBP-P22, and DsbA-P22, after purification and desalting according to embodiments of the present invention. Figure 8 The optimal cut-off value is determined by ROC curve analysis in this embodiment of the invention. Figure 9 This is a specific detection and analysis method in the ELISA method of this invention. Detailed Implementation

[0020] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0021] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be implemented in other ways different from those described herein. Therefore, the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0022] 1.1 Expression and purification of ASFV-TF-P22 protein The ASFV epidemic strain (MK333180) was analyzed using the Editseq program in DNAStar software. The ASFV-TF-P22 gene sequence obtained by inserting the TF fusion tag was obtained. The amino acid sequence is SEQ ID NO.1. Finally, the target gene of the expressed protein was inserted into the pET-28a vector to synthesize a recombinant plasmid. The synthesized recombinant plasmid was transformed into the BL21(DE3) expression strain.

[0023] Transformation process: 1 μL of recombinant plasmid was added to competent bacteria, placed on ice for 10 min, subjected to heat shock at 42℃ for 90 s (without shaking), and then placed on ice for 10 min. 900 μL of antibiotic-free LB liquid medium was added, and the culture was incubated at 37℃ for 220 rpm / min for approximately 1-2 h. The culture was then centrifuged at 3000 rpm / min for 4 min, and 800 μL of supernatant was gently discarded (the amount of supernatant retained depends on the degree of plate dryness). The competent bacteria were spread onto LB solid medium containing antibiotics and incubated overnight at 37℃. The next day, single colonies were picked and inoculated into liquid LB medium at a 1:100 ratio, and cultured at 37℃ for 220 rpm / min until the bacterial concentration reached approximately 0.6-0.8 at OD600 nm (approximately 2 h). IPTG was added to a final concentration of 1 mM / L, and the culture was induced at 37℃ for 220 rpm for 12 h in a constant temperature shaker. Protein expression levels were verified by SDS-PAGE. Figure 1 (SDS-PAGE verification of TF-P22 protein expression shown) The TF-P22 recombinant expression strain was cultured in 2L2YT+G medium at 37℃ and 220 rpm. When the OD600nm value reached 0.6~0.8, IPTG at a final concentration of 1mM / mL was added to induce recombinant protein expression. The induction conditions were 20℃ for 16 hours. After cell culture and induction expression, E. coli cells were collected, and the soluble expressed protein was directly purified after lysing and collecting the supernatant. The collected supernatant solution was purified by affinity chromatography (e.g., using a GEHisTrapFF column) and desalting enzyme digestion (e.g., using a Sephadex G-25 Fine column).

[0024] Purification process: In affinity chromatography purification, the collected supernatant sample solution is diluted, filtered, and loaded onto the substrate. It is first washed with equilibration buffer, then eluted with elution buffer. The elution peak is collected to obtain the purified affinity-tagged recombinant protein; (e.g.) Figure 2 (SDS-PAGE verification of TF-P22 protein purification shown) The affinity-purified sample was desalted, and the sample concentration was detected by a UV spectrophotometer at A280nm to obtain stable high-purity protein.

[0025] 1.2 Expression and purification of ASFV-MBP-P22 protein The ASFV epidemic strain (MK333180) was analyzed using the Editseq program in DNAStar software. The ASFV-MBP-P22 gene sequence obtained by inserting the MBP fusion tag was obtained. The amino acid sequence is SEQ ID NO.2. Finally, the target gene of the expressed protein was inserted into the pET-28a vector to synthesize a recombinant plasmid. The synthesized recombinant plasmid was transformed into the BL21(DE3) expression strain.

[0026] Transformation process: 1 μL of recombinant plasmid was added to competent bacteria, placed on ice for 10 min, and subjected to heat shock at 42°C for 90 s (without shaking), followed by an ice bath for 10 min. 900 μL of antibiotic-free LB liquid medium was added, and the culture was incubated at 37°C for 220 rpm / min for approximately 1-2 h. The culture was then centrifuged at 3000 rpm / min for 4 min, and 800 μL of supernatant was gently discarded (the amount of supernatant retained depends on the degree of plate dryness). The competent bacteria were spread onto LB solid medium containing antibiotics and incubated overnight at 37°C. The next day, single colonies were picked and inoculated into liquid LB medium at a 1:100 ratio, and cultured at 37°C for 220 rpm / min until the bacterial concentration reached approximately 0.6-0.8 at OD600 nm (approximately 2 h). IPTG was added to a final concentration of 1 mM / L, and the culture was induced at 37°C for 220 rpm in a shaker for 12 h. Protein expression and SDS-PAGE verification were then performed (e.g., ...). Figure 3 (SDS-PAGE verification of MBP-P22 protein expression shown).

[0027] The MBP-P22 recombinant expression strain was cultured in 2L of 2YT+G medium at 37℃ and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG was added at a final concentration of 1mM / mL to induce recombinant protein expression. The induction conditions were 20℃ for 16 hours. After cell culture and induction, E. coli cells were collected, and the soluble expressed protein was lysed, and the supernatant was directly purified. The collected supernatant was purified by affinity chromatography (e.g., using a GE HisTrap FF column) and desalting enzyme digestion (e.g., using a Sephadex G-25 Fine column).

[0028] Purification process: In affinity chromatography purification, the collected supernatant sample solution is diluted, filtered, and loaded onto the substrate. It is first washed with equilibration buffer, then eluted with elution buffer. The elution peak is collected to obtain the purified affinity-tagged recombinant protein; (e.g.) Figure 4 (As shown in the SDS-PAGE verification of MBP-P22 protein purification) The affinity-purified sample was desalted, and the sample concentration was detected by a UV spectrophotometer at A280nm to obtain stable high-purity protein.

[0029] 1.3 Expression and purification of ASFV-DsbA-P22 protein The ASFV epidemic strain (MK333180) was analyzed using the Editseq program in DNAStar software. The ASFV-DsbA-P22 gene sequence obtained by inserting a TF fusion tag was obtained. The amino acid sequence is SEQ ID NO.3. Finally, the target gene of the expressed protein was inserted into the pET-28a vector to synthesize a recombinant plasmid. The synthesized recombinant plasmid was transformed into the BL21(DE3) expression strain.

[0030] Transformation process: 1 μL of recombinant plasmid was added to competent bacteria, placed on ice for 10 min, subjected to heat shock at 42℃ for 90 s (without shaking), and then placed on ice for 10 min. 900 μL of antibiotic-free LB liquid medium was added, and the culture was incubated at 37℃ for 220 rpm / min for approximately 1-2 h. The culture was then centrifuged at 3000 rpm / min for 4 min, and 800 μL of supernatant was gently discarded (the amount of supernatant retained depends on the degree of plate dryness). The competent bacteria were spread onto LB solid medium containing antibiotics and incubated overnight at 37℃. The next day, single colonies were picked and inoculated into liquid LB medium at a 1:100 ratio, and cultured at 37℃ for 220 rpm / min until the bacterial concentration reached approximately 0.6-0.8 at OD600nm (approximately 2 h). IPTG was added to a final concentration of 1 mM / L, and the culture was induced at 37℃ for 220 rpm in a shaker for 12 h. Protein expression and SDS-PAGE verification were then performed. (e.g.) Figure 5(SDS-PAGE verification of DsbA-P22 protein expression shown) The DsbA-P22 recombinant expression strain was cultured in 2L of 2YT+G medium at 37℃ and 220 rpm. When the OD600nm value reached 0.6-0.8, IPTG at a final concentration of 1mM / mL was added to induce recombinant protein expression. The induction conditions were 20℃ for 16 hours. After cell culture and induction, E. coli cells were collected, and the soluble expressed protein was lysed, and the supernatant was directly purified. The collected supernatant solution was purified by affinity chromatography (e.g., using a GE HisTrap FF column) and desalting enzyme digestion (e.g., using a Sephadex G-25 Fine column).

[0031] Purification process: In affinity chromatography purification, the collected supernatant sample solution is diluted, filtered, and loaded onto the substrate. It is first washed with equilibration buffer, then eluted with elution buffer. The elution peak is collected to obtain the purified affinity-tagged recombinant protein; (e.g.) Figure 6 (SDS-PAGE verification of DsbA-P22 protein purification shown) The affinity-purified sample was desalted, and the sample concentration was detected by UV spectrophotometer A280nm to obtain stable high-purity protein.

[0032] Compared with the recombinant protein linked with the TF tag, the recombinant protein linked with the MBP tag and the DsbA tag produced a significantly higher expression level of the target protein TF tag recombinant protein than the MBP and DsbA fusion expression mode, as shown in Table 1 below.

[0033] Table 1. Summary and comparison of P22 protein expression levels

[0034] Recombinant proteins constructed using three fusion tags—TF-P22, MBP-P22, and DsbA-P22—were purified, desalted, and then used for evaluation in the detection of African swine fever virus. (e.g., Figure 7 (SDS-PAGE verification of the three recombinant proteins shown) The purified and desalted recombinant proteins were used as coating antigens to prepare ELISA plates to detect the level of African swine fever virus antibodies in porcine serum, and various conditions affecting the experiment were optimized.

[0035] 2.1 Antigen Coating Concentration and Optimal Serum Dilution Matrix titration was used to dilute the purified recombinant proteins TF-P22, MBP-P22, and DsbA-P22 to concentrations of 10 μg / mL, 8 μg / mL, 6 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, and 0.25 μg / mL, respectively, with 100 μL added to each well of a 96-well ELISA plate and incubated overnight at 4°C. African swine fever virus positive and negative sera were diluted to 1:12.5, 1:25, 1:50, 1:100, 1:200, and 1:400, respectively, with 100 μL added to each well and incubated at 37°C for 45 minutes. Goat anti-pig IgG-HRP secondary antibody was diluted to 1:100,000 (100 μL / well) and added to each well, and incubated at 37°C for 45 minutes. Finally, chromogenic buffer (100 μL / well) was added. After developing the enzyme at 37℃ in the dark for 15 min, add stop solution (50 μL / well) to terminate the reaction. Immediately read the value at OD450nm using a microplate reader. Optimal antigen coating concentration and serum dilution factor were determined based on the P / N ratio. The results showed that for TF-P22 antigen coating concentration of 6 μg / mL and serum dilution of 1:100, the maximum P / N ratio was 14.449; for MBP-P22 antigen coating concentration of 6 μg / mL and serum dilution of 1:100, the maximum P / N ratio was 8.487; and for DsbA-P22 antigen coating concentration of 8 μg / mL and serum dilution of 1:100, the maximum P / N ratio was 8.455. These results are shown in Table 2-4 below.

[0036] Table 2. Detection results of optimal antigen dilution and optimal sample dilution for TF-P22 recombinant protein.

[0037] Table 3. Detection results of optimal antigen dilution and optimal sample dilution for MBP-P22 recombinant protein.

[0038] Table 4. Detection results of optimal antigen dilution and optimal sample dilution for DsbA-P22 recombinant protein.

[0039] By recombining P22 with three fusion tags, expressing, purifying, and analyzing the antigen reactivity of TF-P22, the expression level of TF-P22 was the highest, with 6.58 mg of purified protein obtained per gram of bacterial cells. After desalting, the purification level of TF-P22 protein was high, and the P / N value of the reaction with African swine fever virus negative and positive sera was the highest at 14.449. Therefore, we will apply for the development and research of ELISA methods using TF-P22 recombinant protein as the coating antigen.

[0040] 2.2 Determination of the original packaging time The enzyme-labeled plates were coated with the determined optimal antigen concentration under the following conditions: 4℃ for 8 hours, 4℃ for 16 hours, and 4℃ for 24 hours. Then, ELISA was performed. The coating method with the highest P / N ratio was determined to be the optimal coating method. The results showed that the coating effect was the best at 4℃ for 16 hours. The results are shown in Table 5.

[0041] Table 5. Results of antigen coating time detection

[0042] 2.3 The accuracy of the sealing fluid ELISA assays were performed using 1% chicken oocyte albumin, 1% BSA, and 5% skim milk powder as blocking solutions under optimized experimental conditions. The solution with the highest P / N ratio was determined to be the optimal blocking solution. The results showed that 1% chicken oocyte albumin had the best blocking effect, as shown in Table 6.

[0043] Table 6. Detection results of the optimal sealing fluid

[0044] 2.4 Determination of the optimal reaction time for the sample African swine fever virus negative and positive sera were subjected to ELISA at 37°C for 30 min, 45 min, and 60 min, respectively. The optimal reaction time for the primary antibody was determined by the highest P / N ratio. In this invention, the optimal serum reaction time was 30 min, and the recommended reaction time for subsequent samples was 30 min. The results are shown in Table 7.

[0045] Table 7. Detection results of optimal reaction time for samples

[0046] 2.5 Determination of the optimal dilution factor for enzyme-labeled antibodies HRP-labeled goat anti-swine IgG was diluted 10,000, 20,000, 40,000, 80,000, 160,000, and 320,000 times, respectively, and ELISA was performed using African swine fever virus positive and negative sera. In this invention, the P / N value reached its maximum when the enzyme-labeled antibody was diluted 80,000 times, with a value of 19.514. The results are shown in Table 8.

[0047] Table 8. Detection results of the optimal working concentration of enzyme-labeled antibody

[0048] 2.6 Determination of optimal reaction conditions for enzyme-labeled antibody solution African swine fever virus negative and positive sera were incubated at 37°C for 30 min, and then the enzyme-labeled antibody solution was subjected to ELISA at 37°C for 30 min, 45 min, and 60 min, respectively. The results showed that the P / N value reached its maximum of 17.532 when the enzyme-labeled antibody solution was incubated for 30 min. The results are shown in Table 9.

[0049] Table 9. Detection results of optimal reaction time for enzyme-labeled antibodies

[0050] In summary, through the above optimized screening process, the optimal steps for ELISA detection using the recombinant protein described in this application are finally determined as follows: Coating: The purified recombinant protein TF-P22 was diluted with coating buffer to a final concentration of 6 μg / mL. 100 μL was added to each well and coated at 4°C for 16 hours. The mixture was then washed three times with PBST buffer (0.05% Tween-20 phosphate buffer) and patted dry. Blocking: Add 200 μL of PBST blocking buffer containing 1% chicken ovalbumin to each well, incubate at 37°C for 1 h, wash 3 times with PBST buffer, and pat dry; Add the serum to be tested: Add 100 μL to each well, dilute the porcine serum 1:100 with PBS, incubate at 37°C for 30 minutes, wash 3 times with PBST buffer, and pat dry; Add enzyme-labeled secondary antibody: Add 100 μL of goat anti-pig IgG-HRP enzyme diluted 1:80000 with PBS to each well, incubate at 37°C for 30 minutes, wash 3 times with PBST buffer, and pat dry; Add colorimetric reagent: Add 100 μL of colorimetric reagent to each well and incubate at 37°C for 15 minutes; Add stop solution: Add 50 μL of stop solution to each well and measure OD450 nm using an ELISA reader.

[0051] 2.7 Determination of the optimal ELISA cutoff value Under optimal working concentration conditions, 136 clinical serum samples of African swine fever virus were detected by indirect ELISA, including 80 negative sera, 24 positive sera, and 32 weakly positive sera. The S / P values ​​of the 136 samples were entered into MedCalc software, ROC curve analysis was selected, and the ROC curves were output. The cut-off value was selected when the software's sensitivity and specificity reached their maximum. The results are shown in Table 10.

[0052] Table 10. Results of 136 clinical serum tests

[0053]

[0054] Based on the S / P values ​​of 136 serum samples, the data were input into MedCalc software. ROC curve analysis was performed, and the ROC curves were output. The cutoff value was set at 0.265, where both sensitivity and specificity reached a maximum of 98.5%. The cutoff value results are shown in the figure below. Considering the ease of clinical data analysis, the final cutoff value was determined to be 0.3. (See details...) Figure 8 .

[0055] 3. Methodological Validation of Indirect ELISA 3.1 Sensitivity Analysis Twenty sensitivity control sera were diluted at 1:100, 1:1000, and 1:4000 to serve as test samples. Three batches of laboratory products were used for testing to determine the testing standards for different sensitivity dilutions. This kit showed a positive result at a 1000-fold dilution, but all results were negative at 2000-fold and 4000-fold dilutions. Therefore, the limit of detection for this kit is 1:1000. The results are shown in Table 11.

[0056] Table 11. Sensitivity control serum dilution factors

[0057] 3.2 Detection of serum in early infection The purified TF-P22 protein was used to coat an ELISA plate, and porcine serum was detected on days 1, 3, 5, 7, 9, 12, and 14 after African swine fever virus challenge. The commercially available ID.Vet African swine fever virus antibody ELISA kit was used for simultaneous detection. The results showed that the kit of this invention could detect antibody seroconversion on day 9 after challenge, and the ID.Vet kit detected antibody seroconversion on day 14 after challenge. The results are shown in Table 12, indicating that the kit of this application has a significant advantage in early warning of African swine fever virus infection.

[0058] Table 12. Serum test results after African swine fever challenge

[0059] 3.3 Specificity test The purified TF-P22 protein was used as the diagnostic antigen to coat ELISA samples for detection of swine fever virus-positive sera, porcine reproductive and respiratory syndrome virus-positive sera, porcine pseudorabies-positive sera, porcine circovirus type 2-positive sera, and Escherichia coli BL21(DE3-positive sera. The detection OD450nm was used. The results showed that none of the above five specific positive sera reacted with the TF-P22 antigen, indicating that the kit proposed in this application has good specificity. Figure 9 As shown.

[0060] 3.4 Repeatability Test This study selected 5 known positive, 5 weakly positive, and 5 negative clinical samples as sample trays for the reproducibility study. From three batches of laboratory products SY01, SY02, and SY03, 5 kits were randomly selected from each batch, numbered SY01-1, SY01-2, SY01-3, SY01-4, SY01-5; SY02-1, SY02-2, SY02-3, SY02-4, SY02-5; and SY03-1, SY03-2, SY03-3, SY03-4, SY03-5. Fifteen samples from each batch were tested, with each sample tested five times. The results showed that the intra-batch coefficient of variation ranged from 0.63% to 7.44%, and the inter-batch coefficient of variation ranged from 0.26% to 8.33%. Both intra-batch and inter-batch coefficients of variation were less than 10.00%, indicating good reproducibility of the kits. The results are shown in Tables 13-14.

[0061] Table 13. Results of Intra-Batch Repeatability Testing

[0062] Table 14. Results of inter-batch repeatability testing

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the invention by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the invention should be included within the scope of protection of the invention.

Claims

1. An indirect ELISA detection kit for African swine fever virus P22 antibody, characterized in that: The detection kit includes an ELISA plate coated with TF-P22 protein, sample dilution buffer, goat anti-pig ELISA-labeled secondary antibody, chromogenic solution, and stop solution.

2. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The enzyme-labeled plate coated with TF-P22 protein specifically uses purified and desalted TF-P22 protein.

3. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The TF-P22 protein is a recombinant protein.

4. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 3, characterized in that: The TF-P22 protein is the ASFV-TF-P22 gene sequence obtained by inserting a TF fusion tag into an ASFV epidemic strain. The target gene for expressing the protein is inserted into the pET-28a vector to synthesize a recombinant plasmid. The synthesized recombinant plasmid is transformed into the BL21 expression strain. After the bacterial culture and induction expression are completed, the E. coli cells are collected, and the soluble expressed protein is directly purified after the supernatant is collected by lysing.

5. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 4, characterized in that: The amino acid sequence of the ASFV-TF-P22 gene is SEQ ID NO.

1.

6. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The sample diluent was 1×PBS.

7. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The sample diluent is African swine fever virus serum.

8. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The goat anti-pig enzyme-labeled secondary antibody is an HRP-labeled goat anti-pig antibody.

9. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The test kit also includes a buffer solution containing 0.05% Tween-20 phosphate buffer.

10. The African swine fever virus P22 antibody indirect ELISA detection kit according to claim 1, characterized in that: The test kit also includes a blocking solution, which is 1% chicken oocyte albumin.

Images