An indirect elisa antibody detection kit for bovine nodular skin disease and a preparation method thereof

Abstract

This invention discloses an indirect ELISA antibody detection kit for bovine nodular dermatitis and its preparation method, belonging to the field of animal infectious disease detection technology. The indirect ELISA antibody detection kit for bovine nodular dermatitis provided by this invention is the first to use LSDV-ORF122 protein as the coating antigen to coat the ELISA plate, enabling high accuracy and high specificity in detecting antibodies produced after immunization with LSDV vaccine or infection with bovine nodular dermatitis virus.

Description

Technical Field

[0001] This invention belongs to the field of animal infectious disease detection technology, specifically relating to an indirect ELISA antibody detection kit for bovine nodular skin disease and its preparation method. Background Technology

[0002] Lumpy skin disease (LSD) in cattle is a viral infectious disease caused by the bovine nodular skin disease virus (LSDV), also known as bovine bumpy skin disease, bovine nodular dermatitis, and bovine nodular rash. LSD is an acute or subacute contagious disease in cattle, with a morbidity rate between 5% and 45% and a mortality rate as high as 10%. Clinically, the main symptoms include fever, emaciation, swollen lymph nodes, widespread nodules on the skin (mucous membranes, organs), and skin edema. In addition, LSDV can cause primary and secondary pneumonia, abortion in cows, and temporary or permanent infertility in bulls, posing a significant threat to the cattle industry and is listed as a notifiable animal disease by the World Organisation for Animal Health (OIE).

[0003] Currently, there are many methods for detecting the pathogen of this disease, including virus isolation and identification, PCR molecular detection, virus neutralization assay, immunofluorescence, and electron microscopy. However, these methods have drawbacks such as complex operation, high cost, the need for biosafety laboratories, and limited applicability at the grassroots level, making it difficult to conduct epidemiological surveys and monitoring of the disease in the field. Serological antibody detection methods, due to their simplicity and speed, have been widely used at the grassroots level for disease screening. Currently, the main strategy for controlling bovine nodular dermatitis is a combination of vaccination and culling. Therefore, serological antibody detection has become a key technology for monitoring natural infection and evaluating the effectiveness of vaccination. However, at present, there is no serological antibody detection kit specifically for bovine nodular dermatitis. Therefore, developing a simple, rapid, widely applicable serological method that can detect all infected cattle and immunized cattle is particularly important. Summary of the Invention

[0004] In view of one or more problems existing in the prior art, one aspect of the present invention provides an indirect ELISA antibody detection kit for bovine nodular dermatitis, comprising: an enzyme-labeled plate coated with LSDV-ORF122 protein.

[0005] In some embodiments, the amino acid sequence of the LSDV-ORF122 protein is shown in SEQ ID NO:2 of the sequence listing.

[0006] In some embodiments, the nucleotide sequence of the gene encoding the LSDV-ORF122 protein is shown in SEQ ID NO:1 in the sequence listing.

[0007] In some embodiments, the LSDV-ORF122 protein is obtained by: introducing the encoding gene of the LSDV-ORF122 protein shown in SEQ ID NO:1 into recipient E. coli cells to obtain recombinant E. coli cells; inducing expression in the recombinant E. coli cells and collecting the induced expression recombinant E. coli cells; lysing the induced expression recombinant E. coli cells to obtain inclusion bodies; denaturing the inclusion bodies to obtain a denatured sample; refolding the denatured sample to obtain a refolded sample; and purifying the refolded sample to obtain purified LSDV-ORF122 protein.

[0008] In some embodiments, the denaturation is performed in a denaturation buffer consisting of 20 mM Tris, 6 M Gua-HCl, 20 mM DTT, and pH 8.5. The renaturation is performed in a renaturation buffer consisting of 50 mM Tris, 1 M Gua-HCl, 0.75 mM Arg, 3 mM GSH, 1 mM GSSG, and pH 8.5. The purification involves performing Ni strain affinity chromatography on the renatured protein and collecting the elution peak. The chromatographic medium used for the affinity chromatography is GE Ni strain, and the elution buffer is 20 mM Tris, 0.5 M NaCl, 0.5 M imidazole, and pH 8.0.

[0009] In some embodiments, the indirect ELISA antibody detection kit further includes: sample diluent, enzyme-labeled secondary antibody, and chromogenic substrate.

[0010] In some embodiments, the sample diluent is 1×PBS, and the enzyme-labeled secondary antibody is an HRP-labeled goat anti-bovine antibody.

[0011] In some embodiments, the indirect ELISA antibody detection kit further includes an enzyme-labeled antibody diluent, a phosphate buffer containing Tween, a TMB chromogenic solution, and a stop solution (2% sulfuric acid solution).

[0012] Another aspect of the present invention provides a method for preparing an indirect ELISA antibody detection kit for bovine nodular dermatitis, comprising coating an enzyme-labeled plate with a coating solution containing LSDV-ORF122 protein and blocking it to obtain an enzyme-labeled plate coated with LSDV-ORF122 protein, thereby preparing the indirect ELISA antibody detection kit.

[0013] In some embodiments, the concentration of the coating solution containing LSDV-ORF122 protein is 2-10 μg / mL, optionally 4-6 μg / mL, and further optionally 6 μg / mL.

[0014] In some embodiments, the coating conditions are: overnight coating at 4±0.5°C.

[0015] In some embodiments, the blocking operation is as follows: add PBST blocking solution containing 0.8-1.2% BSA to the wells of the ELISA plate coated with LSDV-ORF122 protein, and incubate at 37±0.5℃ for 110-130 min.

[0016] In another aspect, the present invention provides an indirect ELISA antibody detection method for bovine nodular dermatitis, comprising the following steps:

[0017] 1) Add the sample to be tested to the wells of the ELISA plate in the above indirect ELISA antibody detection kit, and wash it after a period of time.

[0018] 2) Add enzyme-labeled secondary antibody to the wells from step 1), allow it to react for a period of time, and then wash.

[0019] 3) Add TMB developer to the wells from step 2) and allow it to react for a period of time; and

[0020] 4) Add the stop solution to the wells from step 3) and measure the OD450 value using an ELISA reader.

[0021] In some embodiments, the sample to be tested in step 1) is bovine serum diluted 1:200-1:400 with PBS, or optionally 1:200-1:300, and the incubation conditions are 37°C for 60-90 min, or optionally 60-70 min.

[0022] In some embodiments, the enzyme-labeled secondary antibody in step 2) is goat anti-bovine IgG / horseradish peroxidase diluted 1:60000-1:80000 times with PBS, and the reaction conditions are 37°C for 60-90 min, or 60-70 min.

[0023] In some implementations, the conditions for the action described in step 3) are: 37°C for 15 minutes.

[0024] In some embodiments, the terminating solution in step 4) is a 2% sulfuric acid solution.

[0025] In another aspect, the present invention provides an enzyme-labeled plate for indirect ELISA antibody detection of bovine nodular dermatitis, which is coated with LSDV-ORF122 protein.

[0026] The indirect ELISA antibody detection kit for bovine nodular dermatitis based on the above technical solution is the first to use LSDV-ORF122 protein as the coating antigen to coat the ELISA plate. It can detect antibodies produced after immunization with LSDV vaccine or infection with bovine nodular dermatitis virus with high accuracy and high specificity. It can meet the detection needs of LSDV infection, outbreak and prevention and control in my country at this stage, and can also be used to evaluate the immunization effect of LSDV vaccine. Attached Figure Description

[0027] Figure 1 Electrophoresis diagram for identifying protein expression in recombinant Escherichia coli pET32a-LSDV122.

[0028] Figure 2 The purification curve and identification electrophoresis diagram of recombinant Escherichia coli pET32a-LSDV122 protein.

[0029] Figure 3 Electrophoresis diagram for identifying protein expression in recombinant Escherichia coli pET32a-GTPV117.

[0030] Figure 4 The purification curve and identification electrophoresis diagram of recombinant Escherichia coli pET32a-GTPV117 protein.

[0031] Figure 5 Electrophoresis diagram for identifying protein expression in recombinant Escherichia coli Rosetta-p32.

[0032] Figure 6 Electrophoresis image for purification and identification of recombinant Escherichia coli Rosetta-p32 protein.

[0033] Figure 7 The reaction of LSDV122 protein with three positive sera: LSDV, BVDV, and IBRV. Detailed Implementation

[0034] The present invention aims to provide a simple, rapid, widely applicable, and serological antibody detection method for bovine nodular skin disease, and its preparation method.

[0035] The present invention will be described in detail through the following specific embodiments.

[0036] Unless otherwise specified, the methods used in the following embodiments are conventional methods. The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials for this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0037] The embodiments are implemented based on the technical solution of the present invention, and provide detailed implementation methods and specific operation processes. The embodiments will help to understand the present invention, but should not be regarded as limiting the content of the present invention.

[0038] Example 1: Expression and purification of LSDV-ORF122 protein (named LSDV122)

[0039] Referring to the LSDV gene coding sequence (NC_003027.1) in GenBank, the LSDV-ORF122 gene sequence (SEQ ID NO:1 in the sequence listing) was obtained by analyzing and sequencing using the Editseq program in DNAStar software. The amino acid sequence it encodes was then deduced (SEQ ID NO:2 in the sequence listing). Finally, the target gene expressing the protein was inserted into the pET-32a vector and sent to the company to synthesize the recombinant plasmid. The synthesized recombinant plasmid was transformed into the BL21(DE3) expression strain to obtain recombinant Escherichia coli pET32a-LSDV122. The transformation process was as follows: 1 μL of recombinant plasmid was added to competent bacteria, placed on ice for 10 min, subjected to heat shock at 42°C 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°C and 220 rpm / min for about 1-2 h; 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 dryness of the plate); the competent bacteria were spread on LB solid medium containing antibiotics and incubated overnight at 37°C. The following day, single colonies were picked and inoculated into liquid LB medium at a 1:100 ratio. The culture was carried out at 37°C and 220 rpm / min until the bacterial concentration reached approximately 0.4-0.6 (about 2 hours). IPTG was then added to a final concentration of 1 mM / L, and the mixture was induced in a shaker at 37°C and 220 rpm for 12 hours. Protein expression and SDS-PAGE verification were performed, and the results are as follows: Figure 1As shown, lane BI contains whole-cell protein of recombinant Escherichia coli pET32a-LSDV122 before induction, lane TCL contains whole-cell protein of recombinant Escherichia coli pET32a-LSDV122 after induction, lane SUP contains the lysed supernatant of recombinant Escherichia coli pET32a-LSDV122 after induction, lane IBS contains the lysed precipitate of recombinant Escherichia coli pET32a-LSDV122 after induction, and lane M contains protein molecular weight standards with a molecular weight of 10-180 kDa.

[0040] The expressed protein was collected by centrifugation, and the cells were washed twice with sterile PBS and lysed in an ice-water mixing bath. After centrifugation, 5 g of inclusion bodies were collected and added to 50 mL of denaturation buffer (20 mM Tris, 6 M Gua-HCl, 20 mM DTT, pH 8.5) at a ratio of 1 g:10 mL (w / v). The protein was allowed to dissolve at room temperature for 3 h. The supernatant was collected by centrifugation at 12000 rpm for 15 min to obtain the denatured sample. 50 mL of the denatured sample was added to 500 mL of renaturation buffer (50 mM Tris, 1 M Gua-HCl, 0.75 mM Arg, 3 mM GSH, 1 mM GSSG, pH 8.5) and renatured overnight at 4 °C. The renatured sample was then desalted using a G25 desalting column (desalting buffer: 20 mM Tris + 0.3 M NaCl, pH 8.0). The protein sample desalted from G25 was subjected to affinity chromatography on an AKTA Purifier (GE 5mL pre-packed column). The column was pre-equilibrated with buffer A (20mM Tris + 0.5M NaCl, pH 8.0) before loading. After loading, equilibration was performed with 5 column volumes of buffer A, followed by linear gradient elution with buffer B (20mM Tris + 0.5M NaCl + 0.5M imidazole, pH 8.0). The elution peak of the renatured protein was collected. The purified protein was validated by SDS-PAGE, and the results are shown below. Figure 2 As shown, panel A represents the purification curve of pET32a-LSDV122 protein, panel B represents the SDS-PAGE validation results of the purified pET32a-LSDV122 protein, and the lanes are: stock solution (pET32a-LSDV122 precipitate), lane FT (affinity chromatography flow-through sample), lanes EL1, EL2, and EL3 (affinity elution samples), and lane M (protein molecular weight standard with a molecular weight of 10-180 kDa).

[0041] Example 2: Expression and purification of GTPV-ORF117 protein (named GTPV117)

[0042] Referring to the GTPV gene coding sequence (NC_004003.1) in GenBank, the GTPV-ORF117 gene sequence (SEQ ID NO:3 in the sequence listing) was obtained by using the Editseq program in DNAStar software. The amino acid sequence it encodes was deduced (SEQ ID NO:4 in the sequence listing). Finally, the target gene expressing the protein was inserted into the pET-32a vector and sent to the company to synthesize the recombinant plasmid. The synthesized recombinant plasmid was transformed into the BL21(DE3) expression strain to obtain recombinant Escherichia coli pET32a-GTPV117. The transformation process was as follows: 1 μL of recombinant plasmid was added to competent bacteria, placed on ice for 10 min, subjected to heat shock at 42°C 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°C and 220 rpm / min for about 1-2 h; 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 dryness of the plate); the competent bacteria were spread on LB solid medium containing antibiotics and incubated overnight at 37°C. The following day, single colonies were picked and inoculated into liquid LB medium at a 1:100 ratio. The culture was carried out at 37°C and 220 rpm / min until the bacterial concentration reached approximately 0.4-0.6 (about 2 hours). IPTG was then added to a final concentration of 1 mM / L, and the mixture was induced in a shaker at 37°C and 220 rpm for 12 hours. Protein expression and SDS-PAGE verification were performed, and the results are as follows: Figure 3 As shown, lane BI represents the whole-cell protein of recombinant E. coli pET32a-GTPV117 before induction, lane TCL represents the whole-cell protein of recombinant E. coli pET32a-GTPV117 after induction, lane SUP represents the lysed supernatant of recombinant E. coli pET32a-GTPV117 after induction, lane IBS represents the lysed precipitate of recombinant E. coli pET32a-GTPV117 after induction, and lane M represents the molecular weight standard of proteins with a molecular weight of 10-180 kD.

[0043] The expressed protein was centrifuged to collect bacterial cells, washed twice with sterile PBS, and lysed in an ice-water mixing bath. After centrifugation, 5g of inclusion bodies were taken out and added to 50mL of denaturing buffer (with the same composition as in Example 1) at a ratio of 1g:10mL (w / v), and the protein was allowed to dissolve at room temperature for 3h. The supernatant was collected by centrifugation at 12000rpm for 15min to obtain the denatured sample. 50mL of the denatured sample was added to 500mL of refolding buffer (with the same composition as in Example 1), and refolded overnight at 4℃. The refolded sample was then desalted using a G25 desalting column. The G25 desalted protein sample was subjected to affinity chromatography on an AKTA Purifier (GE 5mL pre-packed column). The column was pre-equilibrated with buffer A (with the same composition as in Example 1) before loading the sample. After loading, the column was equilibrated with 5 column volumes of buffer A, and then eluted with a linear gradient of buffer B (with the same composition as in Example 1). The elution peak of the refolded protein was collected. The purified protein was validated by SDS-PAGE, and the results are as follows: Figure 4 As shown, panel A represents the purification curve of pET32a-GTPV117 protein, panel B represents the SDS-PAGE validation results of the purified pET32a-GTPV117 protein, and the lanes show the precipitate of expressed pET32a-GTPV117, lane FT is the flow-through sample of affinity chromatography, lanes EL1 and EL2 are the affinity elution samples, and lane M is the molecular weight standard of protein with a molecular weight of 10-180 kDa.

[0044] Example 3: P32 protein expression and verification

[0045] Referring to the P32 gene coding sequence (JN596275.1) in GenBank, the P32 gene sequence obtained by sequencing was analyzed using the Editseq program in DNAStar software (shown as SEQ ID NO:5 in the sequence listing), and its encoded amino acid sequence was deduced (shown as SEQ ID NO:6 in the sequence listing). Finally, the target gene expressing the protein was inserted into the pET-Sumo vector and sent to Beijing Zhongmei Taihe Company to synthesize the recombinant plasmid. The synthesized recombinant plasmid was transformed into the Rosetta expression strain to obtain recombinant Escherichia coli Rosetta-p32. The transformation process was as follows: 1 μL of recombinant plasmid was added to competent bacteria, placed on ice for 10 min, subjected to heat shock at 42°C 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°C and 220 rpm / min for about 1-2 h; 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 dryness of the plate); the competent bacteria were spread on LB solid medium containing antibiotics and incubated overnight at 37°C. The following day, single colonies were picked and inoculated into liquid LB medium at a 1:100 ratio. The culture was carried out at 37°C and 220 rpm / min until the bacterial concentration reached approximately 0.4-0.6 (about 2 hours). IPTG was then added to a final concentration of 1 mM / L, and the mixture was induced in a shaker at 37°C and 220 rpm for 12 hours. Protein expression and SDS-PAGE verification were performed, and the results are as follows: Figure 5 As shown, lane P32 BI contains whole-cell protein of recombinant E. coli Rosetta-p32 before induction, lane P32 TCL contains whole-cell protein of recombinant E. coli Rosetta-p32 after induction, lane P32 SUP contains the lysed supernatant of recombinant E. coli Rosetta-p32 after induction, lane P32 IBS contains the lysed precipitate of recombinant E. coli Rosetta-p32 after induction, and lane M contains protein molecular weight standards with a molecular weight of 10-180 kD.

[0046] The expressed protein was collected by centrifugation, and the cells were washed twice with sterile PBS and lysed in an ice-water mixing bath. After centrifugation, 5 g of inclusion bodies were collected and added to 50 mL of denaturing buffer (20 mM Tris, 8 M Urea, 20 mM DTT, pH 8.5) at a ratio of 1 g:10 mL (w / v). The protein was allowed to dissolve at room temperature for 5 h. The supernatant was collected by centrifugation at 12000 rpm for 15 min to obtain the denatured sample. 50 mL of the denatured sample was added to 500 mL of renaturation buffer (50 mM Tris, 2 M Urea, 10% sucrose, 1 mM EDTA, 3 mM GSH, 1 mM GSSG, 0.4 M NaCl, pH 8.5) and renatured overnight at 4 °C. The renatured sample was then desalted using a G25 desalting column (desalting buffer: 20 mM Tris + 0.3 M NaCl + 2 M Urea, pH 8.0). The protein sample desalted from G25 was subjected to affinity chromatography on an AKTA Purifier (GE 5mL pre-packed column). The column was pre-equilibrated with buffer A (20mM Tris + 0.15M NaCl, pH 8.0) before loading. After loading, equilibration was performed with 5 column volumes of buffer A, followed by linear gradient elution with buffer B (20mM Tris + 0.15M NaCl + 0.5M imidazole, pH 8.0). The elution peak of the renatured protein was collected. The purified protein was validated by SDS-PAGE, and the results are shown below. Figure 6 As shown, lane P32 stock solution is the precipitate of expressed Rosetta-p32, lane P32 FT is the flow-through sample for affinity chromatography, lane P32 purified to affinity elution sample, and lane M is the protein molecular weight standard with a molecular weight of 10-180kD.

[0047] Example 4: Evaluation of the use of three antigens, P32, LSDV122, and GTPV117 (GTPV sheep pox virus), for bovine sarcoidosis antibody detection.

[0048] ELISA plates were prepared using the three recombinant proteins (P32, LSDV122, and GTPV117) purified in Examples 1-3 above as coating antigens. The levels of LSDV antibodies in bovine serum were detected, and various conditions affecting the experiment were optimized to select the best coating antigen. Specifically:

[0049] 4.1 Antigen Coating Concentration and Optimal Serum Dilution

[0050] Matrix titration was used to dilute the purified P32, LSDV122, and GTPV117 antigens 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. 100 μL of each antigen was added to a 96-well ELISA plate and incubated overnight at 4°C. LSDV positive and negative sera were diluted to concentrations of 1:50, 1:100, 1:200, and 1:400, respectively. After release, 100 μL of the antibody was added to each well and incubated at 37°C for 1 h. Goat anti-bovine IgG-HRP secondary antibody was diluted to 1:50000 (100 μL / well) and added to the wells, then incubated at 37°C for 1 h. TMB (100 μL / well) was added and the mixture was incubated at room temperature in the dark for 15 min. Then, stop solution (50 μL / well) was added to terminate the reaction. Immediately afterward, the OD450 reading was measured using a microplate reader. The optimal antigen coating concentration and serum dilution factor were determined based on the P / N ratio and the OD450 reading of positive samples (ideally close to 1). The results are shown in Table 1 below.

[0051] According to the results shown in Table 1, the maximum P / N value of LSDV122 antigen coating concentration of 6 μg / mL and serum dilution of 1:200 is 2.538; the maximum P / N value of GTPV117 antigen coating concentration of 2 μg / mL and serum dilution of 1:50 is 1.948; and the maximum P / N value of P32 antigen coating concentration of 0.5 μg / mL and serum dilution of 1:50 is 1.585. Therefore, LSDV122 was selected as the optimal coating antigen. As shown in Table 1, when LSDV122 antigen is selected as the coating antigen, the P / N value at a serum dilution of 1:200 is relatively large at any LSDV122 antigen coating concentration, followed by the P / N value at a serum dilution of 1:400. Therefore, the serum dilution is determined to be 1:200-1:400, preferably 1:200-1:300. Furthermore, when the serum dilution is 1:200 and the LSDV122 antigen coating concentration is 2-10 μg / mL, the P / N value is not less than [value missing]. 2.191. In particular, when the LSDV122 antigen coating concentration is 4-6 μg / mL, the P / N value is not less than 2.360, and the OD450 value of the positive sample is close to 1. When the LSDV122 antigen coating concentration is 6 μg / mL, the P / N value is relatively large, at 2.538, and the OD450 value of the positive sample is 1.198, which is close to 1. Therefore, the LSDV122 antigen coating concentration is determined to be 2-10 μg / mL, preferably 4-6 μg / mL, and more preferably 6 μg / mL.

[0052] Table 1: P / N ratios of P32, LSDV122, and GTPV117 as coating antigens at different coating concentrations and serum dilutions.

[0053]

[0054]

[0055] 4.2 Determination of LSDV122 Packaging Method

[0056] Using the optimal coating antigen LSDV122 determined in step 4.1 above, the ELISA plate was coated at an antigen concentration of 6 μg / mL. The coating conditions were: overnight at 4°C, 2 h at room temperature followed by overnight at 4°C, 2 h at 37°C followed by overnight at 4°C, and 2 h at 37°C. Then, ELISA was performed (goat anti-bovine IgG-HRP secondary antibody was diluted to 1:70000 (100 μL / well) and added to the wells). The coating method with the highest P / N ratio was determined as the optimal coating method. The results are shown in Table 2 below.

[0057] As shown in Table 2, overnight coating at 4℃ yields the best coating effect. Therefore, the following coating method can be used when coating LSDV122 antigen: overnight coating at 4±0.5℃ to prepare ELISA plates coated with LSDV122 antigen.

[0058] Table 2: Determination of LSDV122 Packaging Method

[0059]

[0060] 4.3 Determination of the sealing fluid

[0061] BSA and skim milk powder were used as blocking solutions at concentrations of 1%, 2%, and 5% (in PBST), respectively. ELISA plates coated with LSDV122 antigen were prepared according to the optimized experimental conditions described in steps 4.1 (the coating antigen was determined to be LSDV122 antigen, the concentration of the coating antigen was 6 μg / mL, and the serum dilution was 1:200) and 4.2 (coating overnight at 4°C). The blocking solution was incubated at 37°C for 2 hours. ELISA assays were then performed (goat anti-bovine IgG-HRP secondary antibody was diluted to 1:70000 (100 μL / well) and added to the wells). The blocking solution with the highest P / N ratio was determined as the optimal blocking solution. The results are shown in Table 3 below.

[0062] As shown in Table 3 below, the blocking effect of PBST containing 1% BSA is the best. Therefore, PBST containing about 1% BSA is selected as the blocking solution. For example, PBST blocking solution containing 0.8-1.2% BSA can be used at 37±0.5℃ for 110-130 min.

[0063] Table 3: Determination of Sealing Fluid

[0064]

[0065] 4.4 Determination of the optimal interaction time between antigen and antibody

[0066] Following the optimized conditions outlined in steps 4.1 (the coating antigen was determined to be LSDV122 antigen, the concentration of the coating antigen was 6 μg / mL, and the serum dilution was 1:200), 4.2 (coating overnight at 4℃), and 4.3 (incubating at 37℃ for 2 h with PBST blocking buffer containing 1% BSA), ELISA plates coated with LSDV122 antigen were prepared. ELISA assays were then performed (goat anti-bovine IgG-HRP secondary antibody was diluted to 1:70000 (100 μL / well) and added to the wells). Positive and negative sera were incubated at 37℃ for 30 min, 45 min, 60 min, and 90 min, respectively. The optimal antigen-antibody reaction time was determined based on the highest P / N ratio. The results are shown in Table 4 below.

[0067] As shown in Table 4 below, the optimal antigen-antibody reaction time is 60 min, followed by 90 min. Therefore, the recommended optimal antigen-antibody reaction time is 60-90 min, preferably 60-70 min, and the reaction temperature is 37±0.5℃.

[0068] Table 4: Determination of the optimal interaction time between antigen and antibody

[0069]

[0070]

[0071] In summary, through the above optimized steps, the specific steps for ELISA detection using the fusion antigen (LSDV122) described in this invention can be determined as follows:

[0072] (1) Coating: The purified recombinant protein LSDV122 was diluted with coating buffer to 2-10 μg / mL, or 4-6 μg / mL, or even 6 μg / mL. 100 μL was added to each ELISA well and coated overnight at 4±0.5℃. The mixture was washed with PBST buffer for 5 min and repeated three times.

[0073] (2) Blocking: Add 100 μL of PBST blocking buffer containing 0.8-1.2% BSA to each ELISA well, incubate at 37±0.5℃ for 110-130 min, wash with PBST buffer for 5 min, and repeat three times;

[0074] (3) Add the test sample: Add 100 μL of the test sample (e.g., bovine serum) diluted 1:200-1:400 with PBS to each ELISA well, or 1:200-1:300. Incubate at 37±0.5℃ for 60-90 min, or 60-70 min. Wash with PBST buffer for 5 min. Repeat three times.

[0075] (4) Add enzyme-labeled secondary antibody: Add 100 μL of goat anti-bovine IgG / horseradish peroxidase diluted 1:60000-1:80000 times with PBS to each ELISA well, or 1:70000 times diluted. Incubate at 37±0.5℃ for 60-90 min, or 60-70 min. Wash with PBST buffer for 5 min. Repeat three times.

[0076] (5) Add colorimetric solution: Add 100 μL TMB colorimetric solution to each ELISA well and incubate at 37°C for 15 min;

[0077] (6) Add stop solution: Add 50 μL of stop solution to each ELISA well and measure the OD450 value using a microplate reader. The stop solution is a 2% sulfuric acid solution.

[0078] 4.5 Determination of the optimal ELISA cutoff value

[0079] Under optimal working concentration conditions, ELISA plates prepared using three recombinant proteins (P32, LSDV122, and GTPV117) as coating antigens were used to detect 30 LSDV antibody-negative bovine serum samples using indirect ELISA according to the detection method determined in step 4.4 above, in order to determine the optimal ELISA cutoff value. The results are shown in Table 5 below.

[0080] As shown in Table 5, the positive cutoff value (X+3SD) for the LSDV122 coated plate was 0.725, while that for the GTPV117 coated plate was 1.16, and for the P32 coated plate was 1.05. This indicates that the GTPV117 and P32 coated plates showed higher detection values ​​for negative samples in this experiment, while the LSDV122 coated plate showed lower values ​​for the same samples, further demonstrating that LSDV122 is the better choice as the coating antigen.

[0081] Table 5: P32, LSDV122, and GTPV117 are the OD values ​​of LSDV antibody-negative bovine serum under the coated antigen.

[0082]

[0083]

[0084] Example 5: Methodological validation of indirect ELISA using LSDV122 as the coating antigen

[0085] 5.1 Specificity test

[0086] An ELISA kit prepared using LSDV122 antigen as the coating antigen was used to detect bovine BVDV positive serum, bovine IBRV positive serum, and bovine LSDV positive serum, respectively. The specific detection method is as described in step 4.4 of Example 4. The detection results are expressed as OD450 values, and the results are as follows: Figure 7 As shown.

[0087] Depend on Figure 7 The results show that the LSDV122-coated antigen reacts significantly with bovine LSDV-positive serum, but does not react with bovine BVDV-positive serum or bovine IBRV-positive serum. This indicates that the ELISA kit prepared using LSDV122 antigen as the coating antigen provided by this invention can detect LSDV antibodies in bovine LSDV-positive serum with high specificity, without reacting with other virus-positive serum.

[0088] 5.2 Repeatability Test

[0089] An ELISA kit prepared using LSDV122 antigen as the coating antigen was used to test the same serum sample on days 1, 3, and 7. A total of 10 serum samples (including bovine LSDV positive and bovine LSDV negative serum) were selected for the experiment. The specific detection method is described in step 4.4 of Example 4. The results are shown in Table 6 below. As can be seen from the results in Table 6, the coefficient of variation of the detection results for each serum sample was between 0.8% and 7.1%, all less than 10%, indicating that the established ELISA detection method has good intra-batch stability.

[0090] Ten serum samples were tested using ELISA plates coated with LSDV122 protein from three different batches. The specific detection method is described in step 4.4 of Example 4. The results are shown in Table 7. Table 7 shows that the coefficients of variation ranged from 1.9% to 9.5%, all less than 10%, indicating good batch-to-batch stability of the established ELISA detection method.

[0091] Table 6: Results of Intra-Batch Repeatability Tests

[0092]

[0093]

[0094] Table 7: Results of inter-batch repeatability tests

[0095]

[0096] 5.3 Accuracy (comparison with other kits)

[0097] Using the indirect ELISA method established in this experiment (see step 4.4 in Example 4 for details) and the BOSTONE bovine sarcoidosis antibody detection kit, 100 serum samples with known backgrounds (including 25 naturally / laboratory-infected bovine LSDV positive sera, 25 bovine LSDV positive sera immunized with LSDV vaccine, and 50 bovine LSDV negative sera) were simultaneously tested. The results are shown in Table 8 below.

[0098] As shown in Table 8, the indirect ELISA detection method established in this invention detected 51 positive serum samples and 49 negative serum samples. The BOSTONE bovine sarcoidosis antibody detection kit detected 50 positive serum samples and 50 negative serum samples. Among them, 47 serum samples were positive for both positive and negative serum samples, and 46 serum samples were negative for both positive and negative serum samples. Therefore, the concordance rate between the two methods is 93%, indicating that the ELISA detection method established in this invention has high accuracy.

[0099] Table 8: Accuracy Analysis Results

[0100]

[0101] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. An indirect ELISA antibody detection kit for bovine nodular dermatitis, comprising: The enzyme-labeled plate coated with LSDV-ORF122 protein, the enzyme-labeled secondary antibody, and the chromogenic substrate.

2. The indirect ELISA antibody detection kit according to claim 1, wherein the amino acid sequence of the LSDV-ORF122 protein is shown in SEQ ID NO:2 in the sequence listing.

3. The indirect ELISA antibody detection kit according to claim 1, wherein the nucleotide sequence of the gene encoding the LSDV-ORF122 protein is shown in SEQ ID NO:1 in the sequence listing.

4. The indirect ELISA antibody detection kit according to claim 1, further comprising a sample diluent.

5. The indirect ELISA antibody detection kit according to claim 4, wherein the sample diluent is 1×PBS.

6. The indirect ELISA antibody detection kit according to claim 1, wherein the enzyme-labeled secondary antibody is an HRP-labeled goat anti-bovine antibody.

7. The indirect ELISA antibody detection kit according to any one of claims 1-6, further comprising an enzyme-labeled antibody diluent, a phosphate buffer containing Tween, a TMB chromogenic solution, and a stop solution.

8. The indirect ELISA antibody detection kit according to claim 7, wherein the stop solution is a 2% sulfuric acid solution.

9. A method for preparing the indirect ELISA antibody detection kit according to any one of claims 1-8, comprising: The coating solution containing LSDV-ORF122 protein was coated onto an ELISA plate and then blocked to obtain an ELISA plate coated with LSDV-ORF122 protein. The indirect ELISA antibody detection kit was prepared from the plate, along with an enzyme-labeled secondary antibody and a chromogenic substrate.

10. The preparation method according to claim 9, wherein the concentration of the coating solution containing LSDV-ORF122 protein is 2-10 μg / mL; and / or The coating conditions are: overnight coating at 4±0.5℃; and / or The blocking procedure is as follows: add PBST blocking solution containing 0.8-1.2% BSA to the wells of the ELISA plate coated with LSDV-ORF122 protein, and incubate at 37±0.5℃ for 110-130 min.

11. The preparation method according to claim 9, wherein the concentration of the coating solution containing LSDV-ORF122 protein is 4-6 μg / mL.

12. The preparation method according to claim 9, wherein the concentration of the coating solution containing LSDV-ORF122 protein is 6 μg / mL.

13. An indirect ELISA antibody detection method for bovine nodular dermatitis, comprising the following steps: 1) Add the sample to be tested into the well of the ELISA antibody detection kit according to any one of claims 1-8, and wash it after reacting for a period of time; 2) Add enzyme-labeled secondary antibody to the wells from step 1), allow it to react for a period of time, and then wash. 3) Add TMB developer to the wells from step 2) and allow it to react for a period of time; and 4) Add the stop solution to the wells from step 3) and measure the OD450 value using an ELISA reader.

14. The indirect ELISA antibody detection method for bovine nodular dermatitis according to claim 13, The sample to be tested in step 1) is bovine serum diluted 1:200-1:400 with PBS, and the incubation conditions are 37℃ for 60-90 min; and / or The enzyme-labeled secondary antibody mentioned in step 2) is goat anti-bovine IgG / horseradish peroxidase diluted 1:60,000-1:80,000 with PBS, and the incubation conditions are 37℃ for 60-90 min; and / or The conditions for the action described in step 3) are: 37°C for 15 min; and / or The terminating solution mentioned in step 4) is a 2% sulfuric acid solution.

15. The indirect ELISA antibody detection method for bovine nodular dermatitis according to claim 13, wherein the sample to be tested in step 1) is bovine serum diluted 1:200-1:300 with PBS.

16. The indirect ELISA antibody detection method for bovine nodular dermatitis according to claim 13, wherein the conditions for action in step 1) are 37°C for 60-70 min.

17. The indirect ELISA antibody detection method for bovine nodular dermatitis according to claim 13, wherein the conditions for action in step 2) are 37°C for 60-70 min.

18. An enzyme-labeled plate for indirect ELISA antibody detection of bovine nodular dermatitis, coated with LSDV-ORF122 protein.

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