A double antibody sandwich ELISA kit for detecting LSDV and a preparation method thereof

Abstract

The application provides a double antibody sandwich ELISA detection kit for lumpy skin disease virus (LSDV), and belongs to the technical field of detection. The double antibody sandwich ELISA kit for detecting LSDV is established based on two rabbit-derived monoclonal antibodies (B5-27 and B5-67) against the B5 protein of LSDV. Experiments show that the kit has the advantages of good specificity, high sensitivity, good repeatability and batch detection, and can provide a tool for preventing and controlling lumpy skin disease.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a double-antibody sandwich ELISA kit for detecting LSDV and its preparation method. Background Technology

[0002] Bovine lumpy skin disease (LSD) is an infectious disease of cattle caused by bovine lumpyskin disease virus (LSDV). The initial symptoms of LSDV infection in cattle are mainly the appearance of multiple nodules on the skin, similar to the symptoms of bovine pseudonodular dermatitis, bovine herpetic papillitis, and pseudopox. Therefore, clinical observation alone is insufficient to diagnose the disease; laboratory diagnostic methods are necessary for confirmation.

[0003] Currently, laboratory diagnostic methods primarily rely on the detection of nucleic acids, antibodies, and antigens to confirm LSDV. Enzyme-linked immunosorbent assay (ELISA) is characterized by high specificity, high sensitivity, and ease of operation, and is suitable for large-scale detection. Compared to indirect ELISA, sandwich ELISA specifically recognizes antigens using two different antibodies, thus offering higher specificity and eliminating concerns about biosafety. However, the current lack of highly specific, high-affinity monoclonal antibodies significantly limits the application of double-antibody sandwich ELISA kits in the diagnosis of bovine nodular dermatitis. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a monoclonal antibody B5-27 against LSDV B5 protein, which has strong detection specificity and sensitivity, providing a basis for the diagnosis of bovine nodular dermatitis.

[0005] This invention provides a monoclonal antibody B5-27 against bovine nodular dermatosis virus B5 protein, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO:1; The amino acid sequence of the light chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO:2.

[0006] The present invention provides a monoclonal antibody composition against bovine nodular dermatosis virus B5 protein, comprising the monoclonal antibody B5-27 and monoclonal antibody B5-67; The amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO:3; The amino acid sequence of the light chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO:4.

[0007] This invention provides the use of the monoclonal antibody B5-27 or the monoclonal antibody composition in the preparation of a kit for diagnosing bovine nodular dermatosis virus infection.

[0008] Preferably, the kit is prepared using at least one of the following immunoassay techniques: enzyme-linked immunosorbent assay (ELISA), colloidal gold immunoassay, immunofluorescence assay, chemiluminescence immunoassay, and immunoblotting.

[0009] This invention provides a kit for diagnosing bovine nodular dermatitis virus infection, comprising the monoclonal antibody B5-27 and any one of the following components: monoclonal antibody B5-67 and bovine nodular dermatitis virus B5 protein in the monoclonal antibody composition.

[0010] Preferably, the kit is a sandwich ELISA kit; The sandwich ELISA kit includes an ELISA plate coated with the monoclonal antibody B5-27 and monoclonal antibody B5-67 as the detection antibody.

[0011] Preferably, the coating concentration of the monoclonal antibody B5-27 is 8~11 μg / mL.

[0012] Preferably, the kit further includes at least one of the following: washing solution, horseradish peroxidase, colorimetric solution, and stop solution.

[0013] Preferably, the monoclonal antibody B5-67 further includes a biotin label; the horseradish peroxidase is labeled with streptavidin.

[0014] The present invention provides a method for preparing the kit, wherein the working solution of monoclonal antibody B5-27 is added to each detection well of the ELISA plate and coated at 4-8°C for 10-14 hours to obtain the coated ELISA plate; The coated ELISA plate was washed to remove moisture and then sealed to obtain an ELISA plate coated with the monoclonal antibody B5-27. The ELISA plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 are packaged into a kit. Preferably, the sealing solution is a BSA solution with a mass percentage of 1.8% to 2.2%; The sealing temperature is 36~38℃; the sealing time is 80~100min.

[0015] This invention provides a monoclonal antibody B5-27 against bovine nodular dermatophyte virus (B5) protein. The amino acid sequence of the heavy chain variable region of monoclonal antibody B5-27 is shown in SEQ ID NO:1; the amino acid sequence of the light chain variable region of monoclonal antibody B5-27 is shown in SEQ ID NO:2. Monoclonal antibody B5-27 of this invention is obtained by immunizing New Zealand rabbits with recombinantly expressed LSDVB5 protein as an immunogen, and then screening the obtained monoclonal B cells through several ELISA tests. Compared with other monoclonal antibodies screened in the same batch, the monoclonal antibody B5-27 exhibits good binding affinity to inactivated virus and MBP-B5 protein, and is positive only for bovine nodular dermatitis virus (below 0.169), while being negative for other common bovine pathogenic viruses (Bovine viral diarrhea virus (BVDV), Bovine enteroviruses (BEV), Infectious bovine rhinotracheitis (IBRV), and Bovine parainfluenza virus (BPIV)). Therefore, the monoclonal antibody B5-27 possesses high detection specificity and strong affinity, and can be used as a coating antibody for the clinical detection of bovine nodular dermatitis.

[0016] This invention provides a monoclonal antibody composition against bovine nodular dermatitis virus (B5) protein, comprising monoclonal antibody B5-27 and monoclonal antibody B5-67; ​​the amino acid sequence of the heavy chain variable region of monoclonal antibody B5-67 is shown in SEQ ID NO:3; the amino acid sequence of the light chain variable region of monoclonal antibody B5-67 is shown in SEQ ID NO:4. Monoclonal antibody B5-67 was obtained by immunizing New Zealand rabbits with recombinantly expressed LSDV B5 protein as an immunogen, and the resulting single B cells were screened through several ELISA tests. Compared with other monoclonal antibodies screened in the same batch, monoclonal antibody B5-67 exhibits a strong affinity for the B5 protein. Furthermore, pairwise combination detection of the screened candidate monoclonal antibodies showed that, compared with other combinations, the present invention, using monoclonal antibodies B5-27 and B5-67, exhibits the best detection effect against inactivated bovine nodular dermatitis virus and against MBP-B5 protein. Therefore, the combination of two monoclonal antibodies enables the sandwich ELISA method to achieve high detection sensitivity for bovine nodular dermatitis virus infection. Experiments show that the limit of detection for the double-antibody sandwich ELISA prepared based on the aforementioned monoclonal antibody composition is 65.625 PFU / mL. Attached Figure Description

[0017] Figure 1This is a graph showing the SDS-PAGE electrophoresis results of purified MBP-B5 protein, monoclonal antibodies B5-27 and B5-67; Figure 2 This is a graph showing the results of Western blot analysis of B5-27 and B5-67 antibodies; Figure 3 This is the standard curve for detecting B5 protein using a double-antibody sandwich ELISA. Figure 4 This is the standard curve for detecting inactivated LSDV viral particles using a double-antibody sandwich ELISA. Figure 5 This is a graph showing the results of specificity analysis of a double-antibody sandwich ELISA. Figure 6 This is a graph showing the results of sensitivity analysis of a double-antibody sandwich ELISA. Detailed Implementation

[0018] This invention provides a monoclonal antibody B5-27 against LSDV B5 protein. The amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO:1 (CQSLEESGGRLVTPGTPLTLTCTVSGIDLSTATMGWVRQAPGKGLEWIGIINRVGSTYYAHWAKGRFTISKASSTTVDLKIASPTTEDTATYFCTRGWPMFGIWGPGTLVTVSS); the amino acid sequence of the light chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO:2 (DGDMTQTPASVSAAVGGTVTINCQSSQSVYDNNWLAWYQQKPGQPPKLLIGYTSTLASGVPSRFSGSGAGTQFTLTISGVQCDDAATYYCQGGYSDNIIPFGGGTEVVVKGDP).

[0019] In this invention, the monoclonal antibody B5-27 is obtained by using bovine nodular dermatitis virus (LSDV) B5 protein as an immunogen, through single B cells isolated from immunized animals, followed by ELISA screening and single-cell sequencing. The B5 protein is a conserved membrane protein of LSDV, related to viral virulence, and plays a crucial role in viral envelope formation. The B5 protein is also a major target of anti-LSDV neutralizing antibodies. The B5 protein is highly immunogenic and can elicit a strong immune response. This invention does not impose any particular limitations on the methods for isolating single B cells and screening via ELISA; any method well-known in the art for single B cell preparation and ELISA detection (using both B5 protein and MBP-B5 protein as detection targets) can be used. After two rounds of ELISA screening, a total of eight monoclonal antibodies were obtained. The eight monoclonal antibodies were combined in pairs, and the optimal combination was found to be B5-27 as the coating antigen and B5-67 as the detection antigen. However, the detection results of B5-67 as the coating antigen and B5-27 as the detection antigen were not as good as the above combination, and the detection results of other monoclonal antibody combinations were not ideal.

[0020] In this invention, the amino acid sequence of the heavy chain of the monoclonal antibody B5-27 is as follows: (QCQSLEESGGRLVTPGTPLTLTCTVSGIDLSTATMGWVRQAPGKGLEWIGIINRVGSTYYAHWAKGRFTISKASSTTVDLKIASPTTEDTATYFCTRGWPMFGIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSDTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSV) The amino acid sequence of the light chain of the monoclonal antibody B5-27 is shown in SEQ ID NO(FIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK). ID NO:6 (QSSQSVYDNNWLAWYQQKPGQPPKLLIGYTSTLASGVPSRFSGSGAGTQFTLTISGVQCDDAATYYCQGGYSDNIIPFGGGTEVVVKGDPVAPTV LIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC).

[0021] In this invention, the preparation method of the monoclonal antibody B5-27 is preferably carried out by in vitro recombinant expression. In this embodiment, the recombinant expression method is preferably carried out using a eukaryotic expression system. Specifically, recombinant plasmids containing the heavy chain coding gene and recombinant plasmids containing the light chain coding gene are transfected into eukaryotic cells in suspension culture under the action of a transfection reagent, cultured further, and the cell supernatant is collected, purified, and the monoclonal antibody is obtained. The nucleotide sequence of the heavy chain coding gene is shown in SEQ ID NO:11. The nucleotide sequence of the light chain coding gene is shown in SEQ ID NO:12. The backbone vector of the recombinant plasmid is preferably a pTT5 vector. The recombinant plasmid is preferably obtained by whole-gene synthesis.

[0022] This invention provides a monoclonal antibody composition against bovine nodular dermatosis virus B5 protein, comprising monoclonal antibody B5-27 and monoclonal antibody B5-67; ​​the amino acid sequence of the heavy chain variable region of monoclonal antibody B5-67 is shown in SEQ ID NO:3 (CQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIGRSGNTWYASWVKGRFTIFKTSTTVDLKITSPTTEDTATYFCARASGSTYYTEDYYFNIWGPGTLVTVSS); the amino acid sequence of the light chain variable region of monoclonal antibody B5-67 is shown in SEQ ID NO:4 (DGDMTQTPASVSAAVGGTVAINCQSSQSVYNNNLLSWYQQMPGQPPKLLIYDASSLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADFWAFGGGTEVVVK).

[0023] In this invention, the amino acid sequence of the heavy chain of the monoclonal antibody B5-67 is as shown in SEQ ID NO:7 (QCQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIGRSGNTWYASWVKGRFTIFKTSTTVDLKITSPTTEDTATYFCARASGSTYYTEDYYFNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSDTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLG GPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK) is shown; the amino acid sequence of the light chain of the monoclonal antibody B5-67 is shown in SEQ. As shown in ID NO:8 (DGDMTQTPASVSAAVGGTVAINCQSSQSVYNNNLLSWYQQMPGQPPKLLIYDASSLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADFWAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC), the monoclonal antibody B5-67 exhibits stronger binding affinity to the B5 protein compared to other monoclonal antibodies screened in the same batch.

[0024] In this invention, the preparation method of the monoclonal antibody B5-67 is the same as that of the monoclonal antibody B5-27. The nucleotide sequence of the coding gene for the heavy chain of the monoclonal antibody B5-67 is shown in SEQ ID NO:13. The nucleotide sequence of the coding gene for the light chain of the monoclonal antibody B5-67 is shown in SEQ ID NO:14.

[0025] This invention provides the use of the monoclonal antibody B5-27 or the monoclonal antibody composition in the preparation of a kit for diagnosing bovine nodular dermatosis virus infection.

[0026] In this invention, the kit is preferably prepared using at least one of the following immunoassay techniques: enzyme-linked immunosorbent assay (ELISA), colloidal gold immunoassay, immunofluorescence assay, chemiluminescence immunoassay, and immunoblotting assay.

[0027] This invention provides a kit for diagnosing bovine nodular dermatitis virus infection, comprising the monoclonal antibody B5-27 and any one of the following components: monoclonal antibody B5-67 and bovine nodular dermatitis virus B5 protein in the monoclonal antibody composition.

[0028] In this invention, when the monoclonal antibody B5-27 is used in the preparation of a kit for diagnosing bovine nodular dermatitis virus infection, it can be combined with protein B to prepare a bovine nodular dermatitis virus infection detection kit based on a competitive assay. When the monoclonal antibody composition is used in the preparation of a kit for diagnosing bovine nodular dermatitis virus infection, the bovine nodular dermatitis virus infection detection kit can be prepared based on a double-antibody sandwich assay.

[0029] In this invention, the kit is preferably a sandwich ELISA kit. The sandwich ELISA kit preferably comprises an ELISA plate coated with the monoclonal antibody B5-27 and monoclonal antibody B5-67 as the detection antibody.

[0030] In this invention, the preferred coating concentration of monoclonal antibody B5-27 is 8-11 μg / mL, and can be 10 μg / mL. The preferred solvent for monoclonal antibody B5-27 is ELISA coating buffer (C1050, Solarbio). In an embodiment of this invention, the coating concentration of monoclonal antibody B5-27 was optimized. Monoclonal antibody B5-27 at concentrations of 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, and 10 μg / mL was used as the coating antibody to coat an ELISA plate. Then, monoclonal antibody B5-67 at concentrations of 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, and 0.8 μg / mL were added for ELISA detection. The highest P / N value was used as the screening criterion. The results showed that the optimal concentration of the coating antibody was 8-10 μg / mL, and the optimal concentration of the detection antibody was 0.1 μg / mL.

[0031] In this invention, the kit preferably further includes at least one of the following: a washing solution, horseradish peroxidase, a chromogenic solution, and a stop solution. The monoclonal antibody B5-67 preferably also includes a biotin label. The washing solution is preferably PBST. The washing solution is used to remove unreacted components during the detection process.

[0032] In this invention, the horseradish peroxidase is labeled with streptavidin. The preferred dilution of the streptavidin-labeled horseradish peroxidase is 1:500, and the optimal incubation time is 15 min. This invention optimized the horseradish peroxidase-labeled streptavidin dilutions of 1:500, 1:1000, 1:2500 / 1:5000, and simultaneously set the incubation times to 15 min, 30 min, 45 min, and 60 min, respectively. The results showed that at a dilution of 1:500 and an incubation time of 15 min, the P / N value was 17.467, which was better than other combinations.

[0033] In this invention, the colorimetric solution is preferably determined according to the type of enzyme. When the enzyme is horseradish peroxidase, the colorimetric solution is TMB colorimetric solution. The stop solution is a strong acid or strong base solution, such as a 2M sulfuric acid aqueous solution.

[0034] The present invention provides a method for preparing the kit, wherein the working solution of monoclonal antibody B5-27 is added to each detection well of the ELISA plate and coated at 4-8°C for 10-14 hours to obtain the coated ELISA plate; The coated ELISA plate was washed to remove moisture and then sealed to obtain an ELISA plate coated with the monoclonal antibody B5-27. The ELISA plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 are packaged into a kit.

[0035] In this invention, the working solution of monoclonal antibody B5-27 is added to each detection well of an ELISA plate, and the plate is coated at 4-8°C for 10-14 hours to obtain the coated ELISA plate.

[0036] In this invention, the ELISA plate is preferably a transparent 96-well ELISA plate. The concentration of the working solution of the monoclonal antibody B5-27 is 8-11 μg / mL, and can be 10 μg / mL. The amount of the working solution of the monoclonal antibody B5-27 added is preferably 100 μL / well. The coating temperature is preferably 4-6℃, and can be 4℃. The coating time is preferably 11-13 h, and can be 12 h.

[0037] After obtaining the coated ELISA plate, the present invention washes the coated ELISA plate to remove moisture, and then blocks it to obtain an ELISA plate coated with the monoclonal antibody B5-27.

[0038] In this invention, the washing solution is a washing liquid. The washing is preferably performed 2-4 times, but can be performed 3 times. The purpose of washing is to remove monoclonal antibody B5-27 that is not coated on the ELISA plate. The preferred method for removing moisture is to pat the ELISA plate dry and then allow it to air dry naturally. The blocking solution is preferably a BSA solution with a mass percentage of 1.8%-2.2%, but can be a 2.0% BSA solution.

[0039] The sealing temperature is preferably 36-38℃, and can be 37℃. The sealing time is preferably 80-100 min, and can be 85-95 min, or 90 min. In this embodiment of the invention, the sealing solvent and time are optimized to further improve the detection sensitivity. 5% skim milk powder, 2% BSA, 2.5% gelatin, and 2% mannose are used as sealing solutions, with sealing times of 30 min, 60 min, 90 min, and 120 min, respectively. To optimize the closure time, the maximum P / N value was used as the screening criterion. The results showed that the P / N value of the 2% BSA scheme was 14.812, while the P / N value of the scheme with a closure time of 90 min was 14.0407, which was better than other experimental groups.

[0040] In this invention, the detection method of the reagent kit preferably includes the following steps: (1) Dilute the positive sample to 1×10 4 PFU / mL was added to the microplate at 100 μL / well as a positive control, and PBST was used as a negative control. At the same time, the test samples were taken and tested, with 100 μL / well added to the microplate and incubated at 37 ℃ for 2 h. (2) Dilute the biotin-labeled B5-67 antibody to 0.1 μg / mL with PBST, add it to the enzyme-labeled plate obtained in step (1) at a rate of 100 μL / well, and incubate at 37℃ for 0.5 h. (3) HRP-labeled streptavidin was diluted 1:500 with PBST and added to the microplate in step (2) at a rate of 100 μL / well, and incubated at 37°C for 0.5 h. (4) Take equal amounts of solution A and solution B of the two-component TMB colorimetric solution, mix them well, and add them to the enzyme-labeled plate obtained in step (3) at an addition rate of 100 μL / well. Incubate at room temperature for 10 min. (5) Add 50 μL of 2 M sulfuric acid aqueous solution to the microplate obtained in step (4) to stop the reaction, and measure the OD of each well using a microplate reader. 450 nm ; (6) Result judgment criteria: When the positive control OD 450 nm >0.169, and the OD of the negative control. 450 nmWhen <0.169, if the OD of the sample is detected... 450 nm If the value is ≥ 0.169, it is considered positive; otherwise, it is considered negative.

[0041] In this invention, the incubation time in step (1) is preferably 2 hours. In this embodiment of the invention, in order to further improve the detection sensitivity and accuracy, the incubation time of the sample was optimized. The results showed that when incubated at 37 °C for 30 min, 60 min, 90 min and 120 min, the P / N ratio gradually increased with the extension of the incubation time, and the incubation time of 120 min was the optimal time.

[0042] In this invention, the incubation time of the biotin-labeled B5-67 antibody in step (2) is preferably 20-40 min, or 30 min. In this embodiment of the invention, in order to further improve the detection sensitivity and accuracy, the incubation time of the biotin-labeled B5-67 antibody was optimized. The results showed that the optimal incubation time for the detection antibody was 30 min.

[0043] In this invention, the incubation time of HRP-labeled streptavidin in step (3) is preferably 13-17 min, and can be 15 min. In this embodiment of the invention, in order to further improve the detection sensitivity and accuracy, the incubation time of HRP-labeled streptavidin was optimized. The results showed that as the incubation time increased, the P / N value gradually decreased, and the optimal incubation time of HRP-labeled streptavidin was 15 min.

[0044] In this invention, the preferred development time of the TMB colorimetric solution is 10 min or 20 min. In this embodiment, the P / N value is highest when the development time is 20 min. However, the P / N value is also relatively high when the development time is 10 min, and the OD of the negative control is also low. 450nm The value is less than 0.8.

[0045] In this invention, the limit of detection (LOD) of the double-antibody sandwich ELISA kit is 65.625 PFU / mL. The double-antibody sandwich ELISA kit exhibits good repeatability, with intra-assay repeatability coefficients of variation ranging from 0.93% to 5.34%, and inter-assay repeatability coefficients of variation ranging from 3.46% to 10.13%, as shown in Table 16. When clinical samples were tested using the double-antibody sandwich ELISA kit, the concordance rate with that of fluorescence PCR was 95.08%.

[0046] The following detailed description, in conjunction with embodiments, of a double-antibody sandwich ELISA kit for detecting LSDV and its preparation method provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0047] Example 1 Screening of anti-LSDV monoclonal antibodies and optimal antibody pairing The purified B5 protein (SEQ ID NO:9: LCDLNKCCYPPSIKNGYIYNEKTEYNIGSNVTFFCGNNTRGVSYTLVGEKNIICEKDGKWNKEFPVCKIIRCRFPALQNGFVNGIPDSRKFYYESEVSFSCKPGFVLIGTKYSVCGINSSWIPKVPICSRDNITYNKIYINKVNIDDNFFNQINNSNTYYFDKILQINNVNR) was expressed in eukaryotes. The sequence of the encoding gene is CTGTGCGACCTGAACAAGTGCTGTTACCCCCCTAGCATCAAGAACGGCTACATCTACAATGAGAAGACCGAGTACAATATCGGCAGCAACGTGACCTTTTTCTGCGGCAATAACACAAGGGGCGTGAGCTACACACTGGTGGGCGAGAAGAATATCATCTGCGAGAA GGACGGCAAGTGGAACAAGGAGTTCCCCGTGTGCAAGATCATCAGATGCAGATTTCCCGCCCTGCAGAATGGCTTTGTGAATGGCATCCCCGATAGCAGAAAGTTTTACTACGAGTCCGAGGTGTCCTTTTCCTGTAAGCCCGGCTTCGTGCTGATCGGCACCAAGTACAGCGTGTGTGGCATCAATTCCTCCTGGATTCCTAAGGTGCCTATCTGTTCCAGGGACAATATCACCTACAATAAGATCTACATCAACAAGGTGAACATCGATGACAACTTCTTCAACCAGATCAACAACAGCAACACCTACTACTTTGACAAGATCCTGCAGATCAATAATGTGAACAGA (SEQ ID NO:10) was used as an immunogen and completely mixed with Freund's adjuvant at a volume ratio of 1:1. The mixture was then injected at multiple sites to immunize New Zealand rabbits. After four immunizations, spleen cells were collected from the spleen and prepared into a single-cell suspension. Subsequently, flow cytometry sorting was used to screen a total of 96 B cell clones. The supernatant from these 96 single B cell clones was used for the first ELISA screening, detecting B5 protein and MBP-B5 protein (B5 protein with an added MBP tag). The specific steps are as follows: 1. Coat a plate with ELISA coating buffer at 4°C overnight with a concentration of 1 μg / mL of B5 protein or MBP-B5 protein.

[0048] 2. Discard the liquid in the wells, wash three times with PBST for 3 minutes each time, and pat dry the liquid in the wells after the last wash.

[0049] 3. Add 2% BSA solution to the well and seal at 37°C for 1 hour.

[0050] 4. Discard the liquid in the hole and repeat step 2.

[0051] 5. Add the supernatant of the single B cell clone to the well and incubate at 37°C for 2 hours.

[0052] 6. Discard the liquid in the hole and repeat step 2.

[0053] 7. Add HRP-labeled rabbit secondary antibody to the wells and incubate at 37°C for 1 hour.

[0054] 8. Discard the liquid in the hole and repeat step 2.

[0055] 9. Add TMB colorimetric solution to the wells and let it stand at room temperature in the dark for 10 minutes.

[0056] 10. Add stop solution to the well and measure OD. 450 value.

[0057] The results are shown in Table 1.

[0058] Table 1 Results of the first screening

[0059] Select plates 27 (coordinates 2 horizontally, 7 vertically), 28 (coordinates 2 horizontally, 8 vertically), 31 (coordinates 3 horizontally, 1 vertically), 57 (coordinates 5 horizontally, 7 vertically), and 67 (coordinates 6 horizontally, 7 vertically) from plate 1, and plates 17 (coordinates 1 horizontally, 7 vertically) and 18 (coordinates 1 horizontally, 8 vertically) from plate 2. Cells 24 (coordinates 2 horizontally, 4 vertically), 26 (coordinates 2 horizontally, 6 vertically), 37 (coordinates 3 horizontally, 7 vertically), 42 (coordinates 4 horizontally, 2 vertically), 44 (coordinates 4 horizontally, 4 vertically), 47 (coordinates 4 horizontally, 7 vertically), and 57 (coordinates 5 horizontally, 7 vertically) underwent a second ELISA screening. The cell detection results in plate 1 are shown in Table 2, and the cell detection results in plate 2 are shown in Table 3.

[0060] Table 2 Results of the second sorting (plate 1)

[0061] The results in Table 2 show that 67-4, 28-7, and 27-3 have better effects.

[0062] Table 3 Results of the second sorting (plate 2)

[0063] Table 4 Results of the second sorting (plate 2)

[0064] The results in Tables 3 and 4 show that antibodies 57-5, 24-2, 26-7, 42-1, and 18-9 performed well. Based on the combined results from both plates, eight antibodies were ultimately selected: 67, 27, 57, 24, 26, 18, 42, and 28.

[0065] These eight antibodies were paired up, and LSDV inactivated virus, cell lysate, and MBP-B5 protein were detected separately. The results are shown in Table 5.

[0066] Table 5. Results of Antibody Pairing Detection

[0067]

[0068] Example 2 Preparation methods of coated antibody B5-27 and biotin-labeled detection antibody B5-67 1. Antibody expression through suspension culture Add 20 mL of suspension cell culture medium to a 125 mL Erlenmeyer flask to revive HEK293F cells (gifted by Yantai Teco Biotechnology Co., Ltd.), ensuring an initial cell density of 0.4 × 10⁻⁶ cells. 6 –0.6×10 6 The cells were then incubated at 37 °C, 5% CO2, and 110 rpm in a constant temperature shaker. When the cell density exceeded 2 × 10⁻⁶ cells / mL... 6 Passage should be performed when the cell density reaches 2 × 10⁶ cells / mL. After two passages, prepare for transfection. On the day of transfection, the cell density in the shake flask should reach 2 × 10⁶ cells / mL. 6 Cells / mL. Take two 1.5 mL centrifuge tubes, A and B. Add 250 μL of sterile PBS, 10 μg of heavy chain expression plasmid, and 10 μg of light chain expression plasmid to tube A to prepare plasmid dilution. Add 250 μL of sterile PBS and 60 μL of polyethyleneimine (PEI, 1 mg / mL) to tube B to prepare PEI dilution. Then add the PEI dilution to the plasmid dilution, mix well, and let stand at room temperature for 20 min. Then add the plasmid / PEI mixture to the suspended cells and continue culturing for 144 h, adding cell culture medium every two days. After culturing, centrifuge to collect the cell supernatant, aliquot, and store at -20 ℃.

[0069] 2. Affinity chromatography for antibody purification The protein concentration in the cell supernatant was measured using the BCA kit. The required volume of chromatography media was calculated based on the loading capacity of Protein A+G chromatography media, and the column was packed. The pre-packed column was washed and equilibrated with 20 column volumes of equilibration wash buffer. An appropriate amount of supernatant was filtered through a 0.22 μm filter and loaded onto the column. The pre-packed column was washed with 40 column volumes of equilibration wash buffer to remove non-specifically adsorbed proteins. When collecting eluted antibodies, 1.2 mL of neutralization buffer was added to the collection tube beforehand, and then 12 mL of elution buffer was used to elute the bound antibodies. After collecting all the eluent, it was concentrated and desalted using an ultrafiltration tube. The chromatography column was then washed and reequilibrated.

[0070] 3. Biotinylation of antibodies The antibody to be biotinylated was diluted to 1 mg / mL with 1×PBS buffer, and 1 mg of biotinylate succinimide was dissolved in 1 mL of DMSO. 1 mL of the antibody solution was taken, and 120 µL of biotinylate succinimide solution was added. The mixture was then placed on a shaker and incubated for 2 h. After the reaction was complete, 9.6 µL of 1 mol / L NH4Cl was added to terminate the reaction. Finally, free biotin was removed using an ultrafiltration tube, and the resulting biotin-labeled antibody was stored at -20 °C.

[0071] Example 3 Establishment and optimization of the sandwich ELISA method 1. Basic steps of the sandwich ELISA method (1) Coating: Dilute B5-27 to 10 μg / mL with coating buffer, add 100 μL / well to the microplate, and coat overnight at 4 ℃.

[0072] (2) Washing: Discard the coating solution in the plate, add 200 μL of PBST to the plate and wash on a shaker for 5 min. Repeat three times.

[0073] (3) Sealing: Add 5% skim milk at 100 μL / well to the plate and let it stand in a 37 ℃ incubator for 2 h.

[0074] (4) Washing: Same as step (2).

[0075] (5) Sample addition: Add 100 μL of sample to the plate at a rate of 100 μL / well, and then incubate at 37 °C for 1 h.

[0076] (6) Washing: Same as step (2).

[0077] (7) Add detection antibody: Dilute biotin-labeled detection antibody B5-67 to 1 μg / mL with PBST, add 100 μL / well to the plate, and incubate at 37 °C for 1 h.

[0078] (8) Washing: Same as step (2).

[0079] (9) Add HRP-labeled streptavidin: Dilute HRP-labeled streptavidin 1:5000 with PBST and add 100 μL / well to the plate. Incubate at 37 °C for 30 min.

[0080] (10) Washing: Same as step (2).

[0081] (11) Color development: Take equal amounts of solution A and solution B of the two-component TMB color development solution, mix them well, and add 100 μL / well to the plate. React at room temperature in the dark for 10 min.

[0082] (12) Termination: Add 2 M H2SO4, 50 μL / well to terminate the reaction.

[0083] (13) Reading: On the microplate reader, read the OD value. 450nm The value of .

[0084] 2. Optimization of the sandwich ELISA method 2.1 Determination of the optimal concentrations of coating and detection antibodies Using the checkerboard method, the coating antibody was diluted to 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL, and 10 μg / mL, and 100 μL / well was coated onto an ELISA plate. The plate was incubated overnight at 4 °C. After adding the sample, the plate was incubated at 37 °C for 60 min. Then, 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL, and 0.8 μg / mL of detection antibody were added, respectively, and ELISA was performed under the previous conditions, with the highest P / N value being considered the optimal condition.

[0085] The experimental results are shown in Table 6. The optimal concentration of the coating antibody was determined to be 10 μg / mL, and the optimal concentration of the detection antibody was determined to be 0.1 μg / mL.

[0086] Table 6. Determination of the optimal concentrations of coating and detection antibodies.

[0087] 2.2 Determination of the optimal sealing solution Blocking was performed using 5% skim milk, 2% BSA, 2% trehalose, and 2.5% gelatin, respectively, under the optimal conditions determined in 2.1, and incubated at 37 °C for 1 h. ELISA was then performed under the previously determined conditions, with the highest P / N value considered the optimal condition.

[0088] As shown in Table 7, the optimal sealing solution is 2% BSA.

[0089] Table 7 Determination of the Optimal Sealing Fluid

[0090] 2.3 Determination of the optimal closure time Using the optimal conditions determined in 2.1-2.2, block at 37 ℃ for 30 min, 60 min, 90 min, and 120 min. Perform ELISA under the previously determined conditions, using the highest P / N value as the optimal condition.

[0091] As shown in Table 8, the optimal closure time is 90 min.

[0092] Table 8 Determination of Optimal Closure Time

[0093] 2.4 Determination of the optimal incubation time for samples Under the optimal conditions determined in 2.1-2.3, after adding the sample, incubate at 37 °C for 30 min, 60 min, 90 min and 120 min respectively, and perform ELISA according to the above steps, with the largest P / N value as the optimal condition.

[0094] As shown in Table 9, the optimal incubation time for the samples is 120 min.

[0095] Table 9 Determination of Optimal Incubation Time for Samples

[0096] 2.5 Determination of the optimal incubation time for antibody detection Under the optimal conditions determined in 2.1-2.4, after adding the detection antibody, incubate at 37 °C for 30 min, 60 min, 90 min and 120 min respectively, and perform ELISA according to the above steps, with the largest P / N value as the optimal condition.

[0097] As shown in Table 10, the optimal incubation time for the detection antibody is 30 min.

[0098] Table 10 Determination of the optimal incubation time for antibody detection

[0099] 2.6 Determination of the optimal dilution and optimal incubation time for HRP-labeled streptavidin: Under the optimal conditions determined in 2.1-2.5, HRP-labeled streptavidin was added at different dilutions and incubated at 37 °C for 15 min, 30 min, 45 min and 60 min, respectively. ELISA was performed according to the aforementioned steps, with the largest P / N value being taken as the optimal condition.

[0100] As shown in Table 11, the optimal dilution of HRP-labeled streptavidin is 1:500, and the optimal incubation time is 15 min.

[0101] Table 11 Determination of the optimal dilution and optimal incubation time for HRP-labeled streptavidin

[0102] 2.7 Determination of Optimal Color Development Time Under the optimal conditions determined in 2.1-2.6, after adding the chromogenic solution, perform ELISA for 5 min, 10 min, 15 min and 20 min respectively, following the steps described above, and use the largest P / N value as the optimal condition.

[0103] Table 12 shows that the P / N value was highest when the color development time was 20 min. However, the P / N value was also high when the color development time was 10 min, and the OD of the negative control was also high. 450nm The value is less than 0.8. Therefore, we chose 10 minutes as the optimal color development time.

[0104] Table 12 Determination of Optimal Color Development Time

[0105] 2.8 Determining the Cut-off Value Using the optimal conditions determined in 2.1-2.7, 32 negative clinical samples were tested using ELISA following the aforementioned steps. The results are shown in Table 13. According to the formula, the cut-off value = 2.1 × mean value = 0.169.

[0106] Table 13 Determination of Cut-off Value

[0107] 3. Evaluation of the sandwich ELISA method 3.1 Establishment of the standard curve Purified MBP-B5 protein was serially diluted from 1000 ng / mL to 0.488 ng / mL. Inactivated LSDV virus was serially diluted eight times, and then detected using an optimized double-antibody sandwich ELISA. The results are shown in Tables 14 and 15. OD is plotted as protein concentration and LSDV dilution on the x-axis. 450nm The values ​​are used as the ordinate to establish a four-parameter fitting equation and determine the linear range. Finally, the logarithm of protein concentration and the base-2 logarithm of LSDV dilution are used as the abscissa, and OD is calculated. 450nm The value is the ordinate, and a linear equation is established. The result is as follows: Figure 3 and Figure 4 As shown, the ELISA exhibits a good linear relationship when detecting the protein and LSDV inactivated virus.

[0108] Table 14 MBP-B5 protein detection results

[0109] Table 15 LSDV Detection Results

[0110] 3.1 Specificity evaluation LSDV and other bovine viruses stored in our laboratory were detected using an optimized ELISA, such as bovine viral diarrhea virus (BVDV), bovine enteroviruses (BEV), infectious bovine rhinotracheitis virus (IBRV), and bovine parainfluenza virus (BPIV). The results are as follows: Figure 5 As shown in the figure. The results showed that only LSDV tested positive, while the OD values ​​of the other viruses were all below 0.169, and were therefore judged as negative. This indicates that the double-antibody sandwich ELISA method established in this invention has good specificity.

[0111] 3.2 Sensitivity Evaluation The inactivated LSDV sample was serially diluted 2-fold (2 4 -2 11 ), and were detected separately using the optimized ELISA, and the results are as follows: Figure 6 As shown, the highest dilution at which a positive result was determined was 1:256. The concentration of the stock virus solution was 1.68 × 10⁻⁶. 4 If the viral concentration is PFU / mL, then the lowest detectable viral concentration for this double-antibody sandwich ELISA is 65.625 PFU / mL.

[0112] 3.3 Repeatability Evaluation Seven clinical samples were tested using ELISA plates coated in the same batch and different batches, respectively, for intra-batch and inter-batch repeatability tests. The results are shown in Tables 16 and 17. Table 16 shows that the coefficient of variation for intra-batch repeatability was 0.93%–5.34%, and Table 17 shows that the coefficient of variation for inter-batch repeatability was 3.46%–10.13%.

[0113] Table 16 Intra-batch repeatability tests

[0114] Table 17 Inter-batch repeatability experiments

[0115] 3.4 Clinical Sample Testing Table 18 Clinical Sample Detection

[0116] Twenty-six blood samples and 35 tissue samples were tested using real-time fluorescence PCR and the double-antibody sandwich ELISA method established in this invention, respectively. The real-time fluorescence PCR detection method is as follows: After nucleic acid extraction from the samples, real-time fluorescence PCR was performed. The reaction system was as follows: 4 μL sample, 0.4 μL upstream primer, 0.4 μL downstream primer, 10 μL dye-based quantitative PCR premix, and 5.2 μL water. The upstream primer was TGGGAAAAGGTAGAAAAATCAGGAGG (SEQ ID NO:15), and the downstream primer was ATCCGCATCGGCATACGATT (SEQ ID NO:16). The reaction conditions were: 94 ℃ for 30 s, 94 ℃ for 5 s, and 60 ℃ for 30 s, with 45 cycles at the last two temperatures. The positive criterion was: a Cq value < 37 was considered positive, and a Cq value ≥ 37 was considered negative.

[0117] The test results are shown in Table 18. As can be seen from Table 18, the double-antibody sandwich ELISA method established in this invention has a high concordance rate with real-time fluorescent PCR, indicating good detection accuracy.

[0118] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A monoclonal antibody B5-27 against the B5 protein of the bovine dermatophilus virus, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO:1; The amino acid sequence of the light chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO:

2.

2. A monoclonal antibody composition against B5 protein of bovine dermatophilus, characterized in that, Includes monoclonal antibody B5-27 and monoclonal antibody B5-67 as described in claim 1; The amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO:3; The amino acid sequence of the light chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO:

4.

3. The use of the monoclonal antibody B5-27 of claim 1 or the monoclonal antibody composition of claim 2 in the preparation of a kit for diagnosing bovine nodular dermatosis virus infection.

4. Use according to claim 3, characterized in that, The kit is prepared using at least one of the following immunoassay techniques: enzyme-linked immunosorbent assay (ELISA), colloidal gold immunoassay, immunofluorescence assay, chemiluminescence immunoassay, and immunoblotting.

5. A reagent kit for diagnosing bovine nodular dermatitis virus infection, characterized in that, It includes the monoclonal antibody B5-27 of claim 1 and any one of the following components: monoclonal antibody B5-67 and bovine nodular dermatosis virus B5 protein in the monoclonal antibody composition of claim 2.

6. The kit of claim 5, wherein The kit is a sandwich ELISA kit; The sandwich ELISA kit includes an ELISA plate coated with the monoclonal antibody B5-27 and monoclonal antibody B5-67 as the detection antibody.

7. The kit of claim 6, wherein The coating concentration of the monoclonal antibody B5-27 is 8~11 μg / mL.

8. The kit of claim 6, wherein The kit also includes at least one of the following: washing solution, horseradish peroxidase, colorimetric solution, and stop solution.

9. The kit of claim 8, wherein The monoclonal antibody B5-67 also includes a biotin label; the horseradish peroxidase is labeled with streptavidin.

10. Process for the preparation of a kit according to any one of claims 5 to 9, characterized in that, The working solution of monoclonal antibody B5-27 was added to each well of the ELISA plate and coated at 4-8℃ for 10-14 hours to obtain the coated ELISA plate. The coated ELISA plate was washed to remove moisture and then sealed to obtain an ELISA plate coated with the monoclonal antibody B5-27. The ELISA plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 are packaged into a kit. The sealing solution is a BSA solution with a mass percentage of 1.8% to 2.2%; The sealing temperature is 36~38℃; the sealing time is 80~100min.

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