Double-antibody sandwich ELISA kit for detecting LSDV and preparation method thereof

Abstract

The invention provides a double antibody sandwich ELISA (Enzyme-Linked Immunosorbent Assay) detection kit for a bovine nodular skin disease virus (LSDV), and belongs to the technical field of detection. According to the invention, on the basis of two rabbit monoclonal antibodies (B5-27 and B5-67) aiming at the LSDV B5 protein, the double-antibody sandwich ELISA kit for detecting the LSDV is established. 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 bovine nodular dermatosis.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a double-antibody sandwich ELISA kit for detecting LSDV and a preparation method thereof. Background Art

[0002] Lumpy skin disease (LSD) is an infectious disease of cattle caused by the lumpyskin disease virus (LSDV). The initial symptoms of LSDV infection in cattle are the appearance of multiple skin nodules, similar to those seen in other diseases such as pseudonodular dermatosis, herpetic mastitis, and pseudocowpox. Therefore, clinical observation alone cannot confirm the underlying disease; laboratory diagnosis is essential for definitive diagnosis.

[0003] Currently, laboratory diagnostic methods primarily rely on the detection of nucleic acids, antibodies, and antigens to confirm the diagnosis of LSDV. The enzyme-linked immunosorbent assay (ELISA) is characterized by high specificity, strong sensitivity, ease of operation, and suitability for large-scale testing. Compared to indirect ELISA, sandwich ELISA uses two different antibodies to specifically recognize antigens, resulting in higher specificity and no need to consider biosafety issues. However, the current lack of highly specific and high-affinity monoclonal antibodies has significantly limited the use of double-antibody sandwich ELISA kits in the diagnosis of bovine nodular dermatitis. Summary of the Invention

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

[0005] The present invention provides a monoclonal antibody B5-27 against the B5 protein of bovine lumpy skin disease virus, wherein the amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO: 1;

[0006] The amino acid sequence of the light chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO: 2.

[0007] The present invention provides a monoclonal antibody composition against bovine lumpy skin disease virus B5 protein, comprising the monoclonal antibody B5-27 and the monoclonal antibody B5-67;

[0008] The amino acid sequence of the heavy chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO: 3;

[0009] The amino acid sequence of the light chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO: 4.

[0010] The present invention provides use of the monoclonal antibody B5-27 or the monoclonal antibody composition in preparing a kit for diagnosing bovine lumpy skin disease virus infection.

[0011] Preferably, the kit is prepared by at least one of the following immunoassay techniques: enzyme-linked immunosorbent assay, colloidal gold immunoassay, immunofluorescence assay, chemiimmunoluminescence assay and immunoblotting assay.

[0012] The present invention provides a kit for diagnosing bovine lumpy skin disease virus infection, comprising the monoclonal antibody B5-27 and any one of the following components: the monoclonal antibody B5-67 in the monoclonal antibody composition and the bovine lumpy skin disease virus B5 protein.

[0013] Preferably, the kit is a sandwich ELISA kit;

[0014] The sandwich ELISA kit comprises an enzyme labeling plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 as a detection antibody.

[0015] Preferably, the coating concentration of the monoclonal antibody B5-27 is 8-11 μg / mL.

[0016] Preferably, the kit further comprises at least one of the following: a washing solution, horseradish peroxidase, a color developing solution, and a stop solution.

[0017] Preferably, the monoclonal antibody B5-67 further comprises a biotin label; and the horseradish peroxidase is labeled with streptavidin.

[0018] The present invention provides a method for preparing the kit, comprising adding a working solution of the monoclonal antibody B5-27 to each detection well of an ELISA plate, coating the plate at 4 to 8° C. for 10 to 14 hours, and obtaining a coated ELISA plate.

[0019] Washing the coated ELISA plate to remove moisture, and blocking the plate to obtain an ELISA plate coated with the monoclonal antibody B5-27;

[0020] packaging the ELISA plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 into a kit;

[0021] Preferably, the blocking solution is a BSA solution with a mass percentage of 1.8% to 2.2%;

[0022] The sealing temperature is 36-38° C.; the sealing time is 80-100 minutes.

[0023] The present invention provides a monoclonal antibody B5-27 against the B5 protein of bovine lumpy skin disease virus. 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. The monoclonal antibody B5-27 of the present invention was obtained by immunizing New Zealand rabbits with recombinantly expressed LSDVB5 protein as an immunogen, and then screening the obtained single B cells through multiple ELISA tests. Compared with other monoclonal antibodies screened in the same batch, the monoclonal antibody B5-27 has good binding affinity for inactivated viruses and MBP-B5 protein, and is only positive for bovine lumpy skin disease virus (below 0.169), while testing negative for other common bovine pathogenic viruses (bovine viral diarrhea virus (BVDV), bovine enteroviruses (BEV), infectious bovine rhinotracheitis virus (IBRV), and bovine parainfluenza virus (BPIV)). This shows that the monoclonal antibody B5-27 has the characteristics of high detection specificity and strong affinity, and can be used as a coating antibody for the clinical detection of bovine lumpy skin disease.

[0024] The present invention provides a monoclonal antibody composition against bovine lumpy skin disease 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. The monoclonal antibody B5-67 is obtained by immunizing New Zealand rabbits with recombinantly expressed LSDV B5 protein as an immunogen, and the obtained single B cells are screened several times by ELISA. Compared with other monoclonal antibodies screened in the same batch, the monoclonal antibody B5-67 has the characteristic of strong binding affinity to the B5 protein, and the screened candidate monoclonal antibodies are tested in pairs. The results show that compared with other combinations, the monoclonal antibody B5-27 and the monoclonal antibody B5-67 of the present invention have the best detection effect on inactivated bovine lumpy skin disease virus and MBP-B5 protein. Therefore, the combination of the two monoclonal antibodies enables the sandwich method to achieve a high sensitivity for detecting bovine lumpy dermatitis virus infection. Experiments have shown that the minimum detection limit of the double antibody sandwich ELISA prepared based on the monoclonal antibody composition is 65.625 PFU / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the SDS-PAGE electrophoresis test result of the purified MBP-B5 protein and the monoclonal antibodies B5-27 and B5-67;

[0026] Figure 2 This is the Western Blot test result of B5-27 and B5-67 antibodies;

[0027] Figure 3 This is the standard curve for the double-antibody sandwich ELISA to detect B5 protein;

[0028] Figure 4 This is the standard curve for detecting inactivated LSDV virions using double-antibody sandwich ELISA;

[0029] Figure 5 This is a graph showing the specificity analysis results of a double-antibody sandwich ELISA;

[0030] Figure 6 This is a graph showing the sensitivity analysis results of the double-antibody sandwich ELISA. DETAILED DESCRIPTION

[0031] The present invention provides a monoclonal antibody B5-27 against LSDV 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 (CQSLEESGGRLVTPGTPLTLTCTVSGIDLSTATMGWVRQAPGKGLEWIGIIN RVGSTYYAHWAKGRFTISKASSTTVDLKIASPTTEDTATYFCTRGWPMFGIWG PGTLVTVSS); the amino acid sequence of the light chain variable region of the monoclonal antibody B5-27 is shown in SEQ ID NO: 2 (DGDMTQTPASVSAAVGGTVTINCQSSQSVYDNNWLAWYQQKPGQPPKLLI GYTSTLASGVPSRFSGSGAGTQFTLTISGVQCDDAATYYCQGGYSDNIIPFGG GTEVVVKGDP).

[0032] In the present invention, the monoclonal antibody B5-27 is obtained by using the B5 protein of bovine nodular dermatitis virus as an immunogen, and is obtained by single-cell sequencing after ELISA screening of single B cells isolated from immunized animals. The B5 protein is a conserved membrane protein of LSDV, which is related to the virulence of the virus and plays a vital role in the formation of the viral envelope. The B5 protein is also the main target of anti-LSDV neutralizing antibodies. The immunogenicity of the B5 protein is very strong and can cause a strong immune response. The present invention has no special restrictions on the method of isolating single B cells and ELISA screening, and can adopt the single B cell preparation and ELISA detection methods well known in the art (using B5 protein and MBP-B5 protein as detection objects, respectively). After two screenings, the ELISA screening obtained a total of 8 monoclonal antibodies. The eight monoclonal antibodies were combined in pairs, and the best combination was obtained, which was B5-27 as the coating antigen and B5-67 as the detection antigen. 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 scheme, and the detection results of other monoclonal antibody combinations were not ideal.

[0033] In the present invention, the amino acid sequence of the heavy chain of the monoclonal antibody B5-27 is as shown in SEQ ID NO: 5 (QCQSLEESGGRLVTPGTPLTLTCTVSGIDLSTATMGWVRQAPGKGLEWIGII NRVGSTYYAHWAKGRFTISKASSTTVDLKIASPTTEDTATYFCTRGWPMFGIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVT WNSDTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQ DDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGF YPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK); the amino acid sequence of the light chain of the monoclonal antibody B5-27 is as SEQ ID NO:6(QSSQSVYDNNWLAWYQQKPGQPPKLLIGYTSTLASGVPSRFSGSGAGTQF TLTISGVQCDDAATYYCQGGYSDNIIPFGGGTEVVVKGDPVAPTVLIFPPAAD QVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLS STLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC).

[0034] In the present invention, the preparation method of the monoclonal antibody B5-27 is preferably prepared by an in vitro recombinant expression method. In this embodiment, the recombinant expression method is preferably carried out using a eukaryotic expression system, specifically, a recombinant plasmid containing the heavy chain encoding gene and a recombinant plasmid containing the light chain encoding gene, respectively, are transferred to eukaryotic cells in suspension culture under the action of a transfection reagent, the culture is continued, and the cell supernatant is collected and purified to obtain the monoclonal antibody. The nucleotide sequence of the heavy chain encoding gene is shown in SEQ ID NO:7. The nucleotide sequence of the light chain encoding gene is shown in SEQ ID NO:8. The backbone vector of the recombinant plasmid is preferably a pTT5 vector. The recombinant plasmid is preferably obtained by whole gene synthesis.

[0035] The present invention provides a monoclonal antibody composition against bovine lumpy skin disease virus B5 protein, comprising the monoclonal antibody B5-27 and the 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 (CQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIIG RSGNTWYASWVKGRFTIFKTSTTVDLKITSPTTEDTATYFCARASGSTYYTED YYFNIWGPGTLVTVSS); the amino acid sequence of the light chain variable region of the monoclonal antibody B5-67 is shown in SEQ ID NO: 4 (DGDMTQTPASVSAAVGGTVAINCQSSQSVYNNNLLSWYQQMPGQPPKLLI YDASSLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADFWAFGGGTEVVVK).

[0036] In the present invention, the amino acid sequence of the heavy chain of the monoclonal antibody B5-67 is as shown in SEQ ID NO: 7 (QCQSLEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGII GRSGNTWYASWVKGRFTIFKTSTTVDLKITSPTTEDTATYFCARASGSTYYTEDYYFNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSDTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVD VSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK); the amino acid sequence of the light chain of the monoclonal antibody B5-67 is shown in SEQ ID NO: 8 (DGDMTQTPASVSAAVGGTVAINCQSSQSVYNNNLLSWYQQMPGQPPKLLI YDASSLASGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCLGGYDDDADFWAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC). Compared with other monoclonal antibodies screened in the same batch, the monoclonal antibody B5-67 has stronger binding ability to B5 protein.

[0037] In the present invention, the preparation method of the monoclonal antibody B5-67 is the same as the preparation method of the monoclonal antibody B5-27. The nucleotide sequence of the gene encoding the heavy chain of the monoclonal antibody B5-67 is shown in SEQ ID NO: 11. The nucleotide sequence of the gene encoding the light chain of the monoclonal antibody B5-67 is shown in SEQ ID NO: 12.

[0038] The present invention provides use of the monoclonal antibody B5-27 or the monoclonal antibody composition in preparing a kit for diagnosing bovine lumpy skin disease virus infection.

[0039] In the present invention, the kit is preferably prepared by at least one of the following immunoassay techniques: enzyme-linked immunosorbent assay, colloidal gold immunoassay, immunofluorescence assay, chemiimmunoluminescence assay and immunoblotting assay.

[0040] The present invention provides a kit for diagnosing bovine lumpy skin disease virus infection, comprising the monoclonal antibody B5-27 and any one of the following components: the monoclonal antibody B5-67 in the monoclonal antibody composition and the bovine lumpy skin disease virus B5 protein.

[0041] In the present invention, when the monoclonal antibody B5-27 is used in the preparation of a kit for diagnosing bovine lumpy skin disease virus infection, it can be combined with protein B to prepare a bovine lumpy skin disease virus infection detection kit based on a competitive method. When the monoclonal antibody composition is used in the preparation of a kit for diagnosing bovine lumpy skin disease virus infection, a bovine lumpy skin disease virus infection detection kit can be prepared based on a double antibody sandwich method.

[0042] In the present invention, the kit is preferably a sandwich ELISA kit, which preferably comprises an enzyme-labeled plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 as a detection antibody.

[0043] In the present invention, the coating concentration of the monoclonal antibody B5-27 is preferably 8 to 11 μg / mL, and can be 10 μg / mL. The solvent of the monoclonal antibody B5-27 is preferably an ELISA coating solution (C1050, Solebol). In an embodiment of the present invention, the coating concentration of the monoclonal antibody B5-27 is optimized, and the monoclonal antibody B5-27 with a concentration of 1 μg / mL, 2 μg / mL, 5 μg / mL, 8 μg / mL and 10 μg / mL is coated on an enzyme-labeled plate as a coating antibody, and then the monoclonal antibody B5-67 with a concentration of 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL and 0.8 μg / mL is added for ELISA detection, with the maximum P / N value as the screening criterion. The results show that the optimal concentration of the coating antibody is 8 to 10 μg / mL, and the optimal concentration of the detection antibody is 0.1 μg / mL.

[0044] In the present invention, the kit preferably further comprises at least one of the following: a washing solution, horseradish peroxidase, a color developing solution, and a stop solution. The monoclonal antibody B5-67 preferably also comprises a biotin label. The washing solution is preferably PBST. The washing solution is used to remove unreacted components during the detection process.

[0045] In the present invention, the horseradish peroxidase is labeled with streptavidin. The dilution of the streptavidin-labeled horseradish peroxidase is preferably 1:500, and the optimal incubation time is 15 minutes. The present invention optimizes the dilution of the horseradish peroxidase-labeled streptavidin to 1:500, 1:1000, 1:2500 / 1:5000, and sets the incubation time to 15 minutes, 30 minutes, 45 minutes, and 60 minutes, respectively. The results show that when the dilution is 1:500 and the incubation time is 15 minutes, the P / N value is 17.467, which is better than other combination detection effects.

[0046] In the present invention, the color developing solution is preferably determined according to the type of enzyme. When the enzyme is horseradish peroxidase, the color developing solution is TMB color developing solution. The stop solution is a strong acid or strong base solution, such as a 2M sulfuric acid aqueous solution.

[0047] The present invention provides a method for preparing the kit, comprising adding a working solution of the monoclonal antibody B5-27 to each detection well of an ELISA plate, coating the plate at 4 to 8° C. for 10 to 14 hours, and obtaining a coated ELISA plate.

[0048] Washing the coated ELISA plate to remove moisture, and blocking the plate to obtain an ELISA plate coated with the monoclonal antibody B5-27;

[0049] The ELISA plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 are packaged into a kit.

[0050] The invention adds the working solution of the monoclonal antibody B5-27 into each detection well of the enzyme labeling plate, and coats the plate at 4-8° C. for 10-14 hours to obtain the coated enzyme labeling plate.

[0051] In the present 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 to 11 μg / mL, and may 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 to 6°C, and may be 4°C. The coating time is preferably 11 to 13 hours, and may be 12 hours.

[0052] After obtaining the coated ELISA plate, the present invention washes the coated ELISA plate, removes moisture, and seals the plate to obtain an ELISA plate coated with the monoclonal antibody B5-27.

[0053] In the present invention, the washing solution is a washing solution. The number of washes is preferably 2 to 4, and can be 3. The purpose of the wash is to remove monoclonal antibody B5-27 that is not coated on the ELISA plate. The method for removing moisture is preferably to pat the ELISA plate dry and then air dry it. The blocking solution is preferably a BSA solution with a mass percentage of 1.8% to 2.2%. It can be a 2.0% BSA solution.

[0054] The blocking temperature is preferably 36-38°C, and can be 37°C. The blocking time is preferably 80-100 min, and can be 85-95 min, or 90 min. In an embodiment of the present invention, in order to further improve the detection sensitivity, the blocking solvent and time are optimized. 5% skim milk powder, 2% BSA, 2.5% gelatin and 2% mannose were used as blocking solutions, and 30 min, 60 min, 90 min and 120 min were used as blocking times for optimization. The maximum P / N value was used as the screening index. The results showed that the P / N value in the 2% BSA scheme was 14.812, while the P / N value in the scheme with a blocking time of 90 min was 14.0407, which was better than other experimental groups.

[0055] In the present invention, the detection method of the kit preferably comprises the following steps:

[0056] (1) Dilute the positive sample to 1×10 4 PFU / mL, add 100 μL / well to the ELISA plate as a positive control, use PBST as a negative control, and take the sample to be tested at the same time, add 100 μL / well to the ELISA plate and incubate at 37°C for 2 hours;

[0057] (2) Take the biotinylated B5-67 antibody and dilute it to 0.1 μg / mL with PBST. Add it to the ELISA plate obtained in step (1) at a volume of 100 μL / well and incubate at 37°C for 0.5 h.

[0058] (3) HRP-labeled streptavidin was diluted 1:500 with PBST and added to the ELISA plate in step (2) at a volume of 100 μL / well, and incubated at 37°C for 0.5 h;

[0059] (4) Take equal amounts of solution A and solution B of the two-component TMB colorimetric solution, mix them evenly, add them to the ELISA plate obtained in step (3) at a rate of 100 μL / well, and incubate at room temperature for 10 min;

[0060] (5) Add 50 μL / well of 2M sulfuric acid aqueous solution to the ELISA plate obtained in step (4) to terminate the reaction, and measure the OD value of each well using an ELISA reader. 450nm ;

[0061] (6) Result judgment standard: When the positive control OD 450nm >0.169, and the OD of the negative control 450nm <0.169, if the OD of the sample is detected 450nm If the value is ≥0.169, it is judged as positive, otherwise it is judged as negative.

[0062] In the present invention, the incubation time in step (1) is preferably 2 hours. In the present embodiment, 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 minutes, 60 minutes, 90 minutes and 120 minutes, the P / N ratio gradually increased with the extension of the incubation time, and the incubation time of 120 minutes was the best.

[0063] In the present invention, the incubation time of the biotin-labeled B5-67 antibody in step (2) is preferably 20 to 40 minutes, and can be 30 minutes. In an embodiment of the present invention, in order to further improve the detection sensitivity and accuracy, the incubation time of the biotin-labeled B5-67 antibody was optimized, and the results showed that the optimal incubation time for the detection antibody was 30 minutes.

[0064] In the present invention, the incubation time of HRP-labeled streptavidin in step (3) is preferably 13 to 17 minutes, and can be 15 minutes. In the embodiment of the present invention, in order to further improve the detection sensitivity and accuracy, the incubation time of HRP-labeled streptavidin was optimized. The results showed that with the extension of the incubation time, the P / N value gradually decreased, and the optimal incubation time of HRP-labeled streptavidin was 15 minutes.

[0065] In the present invention, the color development time of the TMB color developing solution is preferably 10 min or 20 min. In the embodiment of the present invention, when the color development time is 20 min, the P / N value is the largest. However, when the color development time is 10 min, the P / N value is also high, and the OD value of the negative control is 450nm The value is less than 0.8.

[0066] In the present invention, the double-antibody sandwich ELISA test kit has a minimum detection limit of 65.625 PFU / mL. The double-antibody sandwich ELISA test kit has good detection repeatability. Results show that the intra-assay coefficient of variation is 0.93% to 5.34%, and as shown in Table 16, the inter-assay coefficient of variation is 3.46% to 10.13%. When clinical samples were tested using the double-antibody sandwich ELISA test kit, the results were compared with those of fluorescence PCR, achieving a concordance rate of 95.08%.

[0067] The double antibody sandwich ELISA kit for detecting LSDV and its preparation method provided by the present invention are described in detail below with reference to the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] Screening of monoclonal antibodies against LSDV and optimal antibody pairing

[0070] The B5 protein purified by eukaryotic expression (SEQ ID NO: 9: LCDLNKCCYPPSIKNGYIYNEKTEYNIGSNVTFFCGNNTRGVSYTLVGEKNIICEKDGKWNKEFPVCKIIRCRFPALQNGFVNGIPDSRKFYYESEVSFSCKPGFVLIGTKYSVCGINSSWIPKVPICSRDNITYNKIYINKVNIDDNFFNQINNSNTYYFDKILQINNVNR, 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, and then immunized New Zealand rabbits with multiple injections. After immunization four times, spleen cells were collected and prepared into a single cell suspension. A total of 96 B cells were subsequently screened by flow cytometry. The supernatants of these 96 single B cell clones were used for the first ELISA screening, detecting B5 protein and MBP-B5 protein (B5 protein with an MBP tag added). The specific steps are as follows:

[0071] 1. Coat the B5 protein or MBP-B5 protein at a concentration of 1 μg / mL in an ELISA plate using ELISA coating solution overnight at 4°C.

[0072] 2. Discard the liquid in the wells and wash with PBST three times, 3 minutes each time. After the last wash, pat dry the liquid in the wells.

[0073] 3. Add 2% BSA solution to the wells and block at 37°C for 1 hour.

[0074] 4. Discard the liquid in the wells and repeat step 2.

[0075] 5. Add the supernatant of a single B cell clone to the well and incubate at 37°C for 2 hours.

[0076] 6. Discard the liquid in the wells and repeat step 2.

[0077] 7. Add HRP-labeled rabbit secondary antibody to the wells and incubate at 37°C for 1 hour.

[0078] 8. Discard the liquid in the wells and repeat step 2.

[0079] 9. Add TMB colorimetric solution to the wells and let it react for 10 minutes at room temperature in the dark.

[0080] 10. Add stop solution to the wells and measure OD 450 value.

[0081] The results are shown in Table 1.

[0082] Table 1 Results of the first screening

[0083]

[0084] Select 27 (the coordinates in the enzyme plate are horizontally numbered 2 and vertically numbered 7), 28 (the coordinates in the enzyme plate are horizontally numbered 2 and vertically numbered 8), 31 (the coordinates in the enzyme plate are horizontally numbered 3 and vertically numbered 1), 57 (the coordinates in the enzyme plate are horizontally numbered 5 and vertically numbered 7), 67 (the coordinates in the enzyme plate are horizontally numbered 6 and vertically numbered 7) in plate 1 and 17 (the coordinates in the enzyme plate are horizontally numbered 1 and vertically numbered 7), 18 (the coordinates in the enzyme plate are horizontally numbered 1 and vertically numbered 8) in plate 2. ELISA screening was performed for the second time using the following methods: 24 (the coordinate in the ELISA plate is numbered 2 horizontally and 4 vertically), 26 (the coordinate in the ELISA plate is numbered 2 horizontally and 6 vertically), 37 (the coordinate in the ELISA plate is numbered 3 horizontally and 7 vertically), 42 (the coordinate in the ELISA plate is numbered 4 horizontally and 2 vertically), 44 (the coordinate in the ELISA plate is numbered 4 horizontally and 4 vertically), 47 (the coordinate in the ELISA plate is numbered 4 horizontally and 7 vertically), and 57 (the coordinate in the ELISA plate is numbered 5 horizontally and 7 vertically) were screened. The test results of cells in plate 1 are shown in Table 2, and the test results of cells in plate 2 are shown in Table 3.

[0085] Table 2 Second sorting results (plate 1)

[0086]

[0087] From the results in Table 2, it can be seen that 67-4, 28-7 and 27-3 have better effects.

[0088] Table 3 Second sorting results (plate 2)

[0089]

[0090] Table 4 Second sorting results (plate 2)

[0091]

[0092]

[0093] From the results in Tables 3 and 4, we can see that 57-5, 24-2, 26-7, 42-1, and 18-9 have the best results. Combining the test results of the two panels, we finally screened out 8 antibodies: 67, 27, 57, 24, 26, 18, 42, and 28.

[0094] These 8 antibodies were paired in pairs and used to detect LSDV inactivated virus, cell lysate and MBP-B5 protein, respectively. The detection results are shown in Table 5.

[0095] Table 5 Antibody pairing test results

[0096]

[0097]

[0098]

[0099] Example 2

[0100] Preparation method of coated antibody B5-27 and biotin-labeled detection antibody B5-67

[0101] 1. Suspension Culture to Express Antibodies

[0102] 20 mL of suspension cell culture medium was added to a 125 mL triangular shake flask to revive HEK293F cells (donated by Yantai Teke Biotechnology Co., Ltd.) and the initial cell density was maintained at 0.4 × 10 6 -0.6×10 6 cells / mL. The resuspended cells were then cultured in a constant temperature shaker at 37°C, 5% CO2, and 110 rpm. When the cell density was greater than 2×10 6 cells / mL. After 2 passages, prepare for transfection. On the day of transfection, the cell density in the shake flask should reach 2×10 6 cells / mL. Take two 1.5mL centrifuge tubes A and B, add 250μL sterile PBS, 10μg of plasmid expressing heavy chain and 10μg of plasmid expressing light chain to tube A to make plasmid dilution solution, and add 250μL sterile PBS and 60μL polyethyleneimine (Polyethylenimine, PEI, 1mg / mL) to tube B to make PEI dilution solution. Then add the PEI dilution solution to the plasmid dilution solution, mix well and let it stand at room temperature for 20 minutes. Then add the plasmid / PEI mixture to the suspended cultured cells and continue to culture for 144 hours. During this period, add cell culture medium feed solution every two days. After the culture is completed, centrifuge to collect the cell supernatant, package it, and store it at -20℃.

[0103] 2. Antibody purification by affinity chromatography

[0104] Use the BCA kit to measure the protein concentration in the cell supernatant, calculate the required volume of chromatography medium according to the loading capacity of the ProteinA+G chromatography medium, and load the column; use 20 column volumes of equilibrium wash buffer to wash the equilibrated pre-loaded column; take an appropriate amount of supernatant, filter it with a 0.22μm filter membrane, and then load the sample; use 40 column volumes of equilibrium wash buffer to wash the pre-loaded column to remove non-specifically adsorbed impurities; when collecting the eluted antibody, add 1.2mL of neutralization buffer to the collection tube in advance, and then elute the bound antibody with 12mL of elution buffer. After collecting all the eluate, concentrate and desalt it with an ultrafiltration tube; then wash and re-equilibrate the chromatography column.

[0105] 3. Biotin Labeling of Antibodies

[0106] Dilute the antibody to be biotinylated to 1 mg / mL in 1× PBS buffer. Dissolve 1 mg of biotin succinimidyl ester in 1 mL of DMSO. Add 120 μL of biotin succinimidyl ester solution to 1 mL of the antibody solution. Place the tube on a shaker and shake for 2 hours. After the reaction is complete, add 9.6 μL of 1 mol / L NH4Cl to terminate the reaction. Finally, remove free biotin using an ultrafiltration tube and store the resulting biotin-labeled antibody at -20°C.

[0107] Example 3

[0108] Establishment and optimization of sandwich ELISA method

[0109] 1. Basic steps of sandwich ELISA method

[0110] (1) Coating: Dilute B5-27 to 10 μg / mL with coating solution, add 100 μL / well to the ELISA plate, and coat overnight at 4°C.

[0111] (2) Washing: Discard the coating solution in the plate, add PBST to the plate at 200 μL / well, shake on a shaker for 5 minutes, and repeat three times.

[0112] (3) Blocking: 5% skim milk was added to the plate at 100 μL / well and incubated at 37°C for 2 h.

[0113] (4) Washing: Same as step (2).

[0114] (5) Sample addition: 100 μL / well of the sample was added to the plate, followed by incubation at 37°C for 1 h.

[0115] (6) Washing: Same as step (2).

[0116] (7) Add detection antibody: Dilute the biotinylated 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.

[0117] (8) Washing: Same as step (2).

[0118] (9) Add HRP-labeled streptavidin: Dilute HRP-labeled streptavidin 1:5000 with PBST, add 100 μL / well to the plate, and incubate at 37°C for 30 min.

[0119] (10) Washing: Same as step (2).

[0120] (11) Color development: Take equal amounts of solution A and solution B of the two-component TMB color development solution, mix them evenly, add 100 μL / well to the plate, and react at room temperature in the dark for 10 min.

[0121] (12) Termination: Add 2M H2SO4, 50 μL / well, to terminate the reaction.

[0122] (13) Reading: Read the OD on the microplate reader. 450nm value.

[0123] 2. Optimization of Sandwich ELISA Method

[0124] 2.1 Determination of the optimal concentrations of coating and detection antibodies

[0125] 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 on the ELISA plate and incubated at 4°C overnight. After adding the sample, it was incubated at 37°C for 60 minutes, and then 0.1 μg / mL, 0.2 μg / mL, 0.4 μg / mL and 0.8 μg / mL of detection antibody were added respectively. ELISA was performed according to the previous conditions, and the maximum P / N value was taken as the optimal condition.

[0126] The experimental results are shown in Table 6. It was finally determined that the optimal concentration of the coating antibody was 10 μg / mL and the optimal concentration of the detection antibody was 0.1 μg / mL.

[0127] Table 6 Determination of the optimal concentrations of coating antibodies and detection antibodies

[0128]

[0129]

[0130] 2.2 Determination of the optimal blocking solution

[0131] Using the optimal conditions determined in 2.1, block with 5% skim milk, 2% BSA, 2% trehalose, and 2.5% gelatin and incubate at 37°C for 1 hour. Perform ELISA according to the previous conditions, and use the highest P / N value as the optimal condition.

[0132] From the results in Table 7, it can be seen that the best blocking solution is 2% BSA.

[0133] Table 7 Determination of the optimal blocking solution

[0134] Blocking buffer type 5% skim milk powder 2% BSA 2.5% gelatin 2% mannose P 1.449 1.459 1.459 1.3805 N 0.1085 0.0985 0.0985 0.0995 P / N 13.35484 14.81218 13.7619 13.87437

[0135] 2.3 Determination of the optimal closure time

[0136] Block at 37°C for 30, 60, 90, and 120 minutes using the optimal conditions determined in 2.1-2.2. Perform ELISA under the same conditions as above, and use the highest P / N value as the optimal condition.

[0137] From the results in Table 8, it can be seen that the optimal sealing time is 90 min.

[0138] Table 8 Determination of optimal sealing time

[0139] Closure time 0.5h 1h 1.5h 2h P 1.1945 1.086 1.2075 1.023 N 0.087 0.0885 0.086 0.083 P / N 13.72989 12.27119 14.0407 12.3253

[0140] 2.4 Determination of the optimal incubation time for samples

[0141] Using the optimal conditions determined in 2.1-2.3, add the sample and incubate at 37°C for 30 min, 60 min, 90 min, and 120 min, respectively. Perform ELISA according to the above steps, and take the maximum P / N value as the optimal condition.

[0142] From the results in Table 9, it can be seen that the optimal incubation time for the sample is 120 min.

[0143] Table 9 Determination of the optimal incubation time of samples

[0144] Sample incubation time 0.5h 1h 1.5h 2h P 0.843 0.908 0.9405 1.0435 N 0.096 0.0965 0.0935 0.0945 P / N 8.78125 9.409326 10.05882 11.04233

[0145] 2.5 Determination of the optimal incubation time for detection antibodies

[0146] Using the optimal conditions determined in 2.1-2.4, add the detection antibody and incubate at 37°C for 30 min, 60 min, 90 min, and 120 min, respectively. Perform ELISA according to the above steps, and take the maximum P / N value as the optimal condition.

[0147] From the results in Table 10, it can be seen that the optimal incubation time for detection antibodies is 30 min.

[0148] Table 10 Determination of the optimal incubation time for detection antibodies

[0149] Detection antibody binding time 0.5h 1h 1.5h 2h P 1.5945 1.51 1.598 1.554 N 0.099 0.0975 0.113 0.124 P / N 16.10606 15.48718 14.14159 12.53226

[0150] 2.6 Determination of the optimal dilution and incubation time of HRP-labeled streptavidin:

[0151] Using the optimal conditions determined in 2.1-2.5, add HRP-labeled streptavidin at different dilutions and incubate at 37°C for 15 min, 30 min, 45 min, and 60 min, respectively. Perform ELISA according to the above steps, and take the maximum P / N value as the optimal condition.

[0152] From the results in Table 11, it can be seen that the optimal dilution of HRP-labeled streptavidin is 1:500, and the optimal incubation time is 15 min.

[0153] Table 11 Determination of the optimal dilution and optimal incubation time of HRP-labeled streptavidin

[0154]

[0155] 2.7 Determination of the optimal color development time

[0156] Using the optimal conditions determined in 2.1-2.6, add the color developing solution and color for 5 min, 10 min, 15 min, and 20 min, respectively. Perform ELISA according to the above steps, and take the maximum P / N value as the optimal condition.

[0157] The results in Table 12 show that the P / N value is the largest when the color development time is 20 minutes. However, when the color development time is 10 minutes, the P / N value is also high, and the OD value of the negative control is 450nm The value is less than 0.8. Therefore, we choose 10 min as the optimal color development time.

[0158] Table 12 Determination of optimal color development time

[0159]

[0160]

[0161] 2.8 Determination of Cut-off Value

[0162] Using the optimal conditions determined in 2.1-2.7, 32 negative clinical samples were tested by ELISA according to the aforementioned steps. The test results are shown in Table 13. According to the formula, cut-off value = 2.1 × average value = 0.169.

[0163] Table 13 Determination of Cut-off value

[0164]

[0165] 3. Evaluation of Sandwich ELISA Method

[0166] 3.1 Establishment of standard curve

[0167] The purified MBP-B5 protein was diluted two-fold from 1000 ng / mL to 0.488 ng / mL, and the inactivated LSDV virus was diluted eight times, and then tested using the optimized double antibody sandwich ELISA. The test results are shown in Tables 14 and 15. The protein concentration and LSDV dilution were used as the horizontal axis, and the OD 450nm The value is the vertical axis, and a four-parameter fitting equation is established to determine the linear range. Finally, the logarithm of protein concentration and LSDV dilution with base 2 is used as the horizontal axis, OD 450nm The value is the vertical coordinate, and a linear equation is established. The result is as follows Figure 3 and Figure 4 As shown, it can be seen that the ELISA has a good linear relationship when detecting protein and LSDV inactivated virus.

[0168] Table 14 MBP-B5 protein detection results

[0169] Protein concentration 1000 500 250 125 62.5 31.25 15.625 7.813 lg (protein concentration) 3.000 2.699 2.398 2.097 1.796 1.495 1.194 0.893 <![CDATA[OD 450nm ]]> 2.213 2.112 2.086 1.983 1.818 1.599 1.406 1.096

[0170] Table 15 LSDV test results

[0171]

[0172]

[0173] 3.1 Specificity evaluation

[0174] The optimized ELISA was used to detect LSDV and other cattle disease viruses stored in our laboratory, such as bovine viral diarrhea virus (BVDV), bovine enteroviruses (BEV), infectious bovine rhinotracheitis virus (IBRV), and bovine parainfluenza virus (BPIV). The test results were as follows: Figure 5 The results showed that only LSDV was positive, while the OD values ​​of the other viruses were all below 0.169, which was judged to be negative. This shows that the double antibody sandwich ELISA method established by the present invention has good specificity.

[0175] 3.2 Sensitivity evaluation

[0176] The inactivated LSDV sample was diluted 2-fold in series (2 4 -2 11 ), and were tested with the optimized ELISA, and the test results were as follows Figure 6 As shown, the maximum dilution for positive result is 1:256. The concentration of virus stock solution is 1.68×10 4 PFU / mL, the minimum detectable virus concentration of the double-antibody sandwich ELISA is 65.625 PFU / mL.

[0177] 3.3 Repeatability evaluation

[0178] Intra-assay and inter-assay reproducibility tests were performed on seven clinical samples using ELISA plates coated from the same and different batches. The results are shown in Tables 16 and 17. Table 16 shows that the intra-assay coefficient of variation (CV) ranged from 0.93% to 5.34%, while Table 17 shows that the inter-assay coefficient of variation (CV) ranged from 3.46% to 10.13%.

[0179] Table 16 Intra-batch repeatability experiment

[0180]

[0181] Table 17 Inter-batch repeatability experiment

[0182]

[0183] 3.4 Clinical sample testing

[0184] Table 18 Clinical sample testing

[0185]

[0186] 26 blood samples and 35 tissue samples were tested using real-time fluorescence PCR and the double antibody sandwich ELISA method established in the present invention. The real-time fluorescence PCR detection method is as follows:

[0187] After nucleic acid extraction, real-time fluorescence PCR was performed using the following reaction system: 4 μL sample, 0.4 μL upstream primer, 0.4 μL downstream primer, 10 μL dye-based fluorescence 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°C for 30 seconds, 94°C for 5 seconds, and 60°C for 30 seconds, with 45 cycles of the latter two temperatures. A positive result was determined when the Cq value was <37, and a negative result was determined when the Cq value was ≥37.

[0188] The test results are shown in Table 18. As shown in Table 18, the double antibody sandwich ELISA method established in the present invention has a high consistency rate with real-time fluorescence PCR, indicating that the detection accuracy is good.

[0189] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A monoclonal antibody B5-27 against the B5 protein of bovine lumpy skin disease 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 bovine lumpy skin disease virus B5 protein, characterized in that: Comprising the monoclonal antibody B5-27 and the monoclonal antibody B5-67 according to 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. Use of the monoclonal antibody B5-27 according to claim 1 or the monoclonal antibody composition according to claim 2 in the preparation of a kit for diagnosing bovine lumpy skin disease virus infection.

4. The application according to claim 3, characterized in that The kit is prepared by at least one of the following immunoassay technologies: enzyme-linked immunosorbent assay technology, colloidal gold immunoassay technology, immunofluorescence assay technology, chemiimmunoluminescence assay technology and immunoblotting assay technology.

5. A kit for diagnosing bovine lumpy skin disease virus infection, characterized in that: The invention comprises the monoclonal antibody B5-27 according to claim 1 and any one of the following components: the monoclonal antibody B5-67 and the bovine lumpy skin disease virus B5 protein in the monoclonal antibody composition according to claim 2.

6. The kit according to claim 5, characterized in that The kit is a sandwich ELISA kit; The sandwich ELISA kit comprises an enzyme labeling plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 as a detection antibody.

7. The kit according to claim 6, characterized in that The coating concentration of the monoclonal antibody B5-27 is 8-11 μg / mL.

8. The kit according to claim 6, characterized in that The kit further comprises at least one of the following: a washing solution, horseradish peroxidase, a color developing solution, and a stop solution.

9. The kit according to claim 6, characterized in that The monoclonal antibody B5-67 further includes a biotin label; and the horseradish peroxidase is labeled with streptavidin.

10. The method for preparing the kit according to any one of claims 5 to 9, characterized in that: Add the working solution of monoclonal antibody B5-27 to each detection well of the ELISA plate, and coat at 4-8°C for 10-14 hours to obtain a coated ELISA plate; Washing the coated ELISA plate to remove moisture, and blocking the plate to obtain an ELISA plate coated with the monoclonal antibody B5-27; packaging the ELISA plate coated with the monoclonal antibody B5-27 and the monoclonal antibody B5-67 into a kit; Preferably, the blocking solution is a BSA solution with a mass percentage of 1.8% to 2.2%; The sealing temperature is 36-38° C.; the sealing time is 80-100 minutes.

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