CN116836270B - Monoclonal antibody of anti-bluetongue virus VP7 protein, preparation method and application - Google Patents
Monoclonal antibody of anti-bluetongue virus VP7 protein, preparation method and application Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54306—Solid-phase reaction mechanisms
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
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Abstract
The invention relates to the technical field of biology, in particular to a monoclonal antibody of anti-bluetongue virus VP7 protein, a preparation method and application. The invention screens and obtains a positive hybridoma cell line which efficiently secretes anti-bluetongue virus VP7 protein monoclonal antibody, and successfully obtains the heavy chain and light chain variable region nucleotide sequences of the monoclonal antibody through RT-PCR technology. The monoclonal antibody not only has good affinity reaction capability with the VP7 protein expressed by recombination, but also can perform specific reaction with the natural VP7 protein in bluetongue virus infected cells; the monoclonal antibody is used as a competitive antibody to establish a bluetongue virus competitive ELISA antibody detection method, and provides tools for detection and diagnosis of bluetongue and epidemiological investigation.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a monoclonal antibody of anti-bluetongue virus VP7 protein, a preparation method and application thereof.
Background
Bluetongue is a disease caused by Bluetongue virus (Bluetongue virus) and is transmitted mainly by blood sucking biting midges of the genus culicoides, in ruminants such as sheep, cattle, deer, etc. Sheep are most susceptible to the disease, and clinical symptoms of sheep are mainly marked by depression of spirit, rise of body temperature, congestion of facial edema, ulcer of oral mucosa and nasal mucosa or congestion. World animal health organization (WOAH) lists it as a disease that must be reported. The bluetongue virus is a double stranded RNA virus belonging to the genus circovirus of the reoviridae family. The genome consists of ten RNA fragments with different sizes, and totally encodes 7 structural proteins and 4 non-structural proteins. The serotypes of BTV are numerous, and although there is a cross-reaction between different serotypes, no vaccine is available to treat the disease, so early diagnosis and prevention is an important means of preventing the disease from becoming prevalent. Structural protein VP7 is highly conserved among different serotypes, and amino acid homology is over 94%, and is an antigen for establishing a serogroup specific diagnostic method. The monoclonal antibody has high specificity aiming at specific single epitope, and has the characteristics of high purity and high sensitivity, and can be used for establishing a serological diagnosis method of pathogen.
With the ongoing variation of bluetongue viruses, at least 29 serotypes have now been found, with no or poor cross protection between the different serotypes. There are several methods for detecting bluetongue, but each method has its advantages and disadvantages. The invention provides a positive hybridoma cell line capable of efficiently secreting monoclonal antibodies, and a monoclonal antibody of anti-bluetongue virus VP7 protein is obtained. The monoclonal antibody has good affinity with VP7 protein, can react specifically with the bluetongue virus VP7 protein, and provides an important basis for identification and diagnosis methods of bluetongue virus.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a monoclonal antibody of anti-bluetongue virus VP7 protein, a preparation method and application, and specifically comprises the following contents:
in a first aspect, the invention provides a monoclonal antibody against the bluetongue virus VP7 protein, the monoclonal antibody comprising an antibody heavy chain and an antibody light chain;
the variable region CDR of the heavy chain of the antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.5, a CDR2 with an amino acid sequence shown as SEQ ID NO.6 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 7;
the variable region CDR of the antibody light chain comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 10.
Preferably, the amino acid sequence of the variable region of the heavy chain is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the antibody light chain is shown as SEQ ID NO. 3.
In a second aspect, the invention provides a nucleic acid encoding an antibody heavy chain and an antibody light chain of the monoclonal antibody of the first aspect above.
Preferably, the variable region of the heavy chain of the antibody has a gene sequence shown in SEQ ID NO.2 and the variable region of the light chain of the antibody has a gene sequence shown in SEQ ID NO. 4.
In a third aspect, the present invention provides the use of a monoclonal antibody as described in the first aspect in the preparation of a reagent, or a test strip, or a kit for detecting bluetongue virus.
In a fourth aspect, the present invention provides an immunoconjugate comprising:
(i) The monoclonal antibody of the first aspect;
(ii) And a coupling moiety selected from the group consisting of: a detectable label, a drug, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.
In a fifth aspect, the present invention provides a bluetongue virus ELISA detection kit comprising a monoclonal antibody according to the first aspect.
Preferably, the kit comprises an ELISA plate, a bluetongue virus antigen, a blocking solution, a diluent, the monoclonal antibody, an ELISA secondary antibody, a washing solution, a color reagent and a stop solution.
Preferably, the bluetongue virus antigen is selected from the group consisting of bluetongue virus VP7 recombinant proteins;
preferably, the blocking solution is a PBST buffer containing 5% nonfat dry milk;
preferably, the diluent is 0.01M PBS pH7.2;
preferably, the wash solution is a PBST buffer.
Preferably, the enzyme-labeled secondary antibody is an HRP-labeled goat anti-mouse secondary antibody.
In a sixth aspect, the present invention provides the use of a monoclonal antibody according to the first aspect above for in vitro detection of bluetongue virus for non-disease diagnosis purposes.
In a seventh aspect, the present invention provides a blue tongue virus ELISA detection method for the purpose of non-disease diagnosis, said method comprising the steps of:
(1) Coating: diluting the expressed BTV VP7 recombinant protein to 0.5 mug/mL with CBS buffer, coating 100 mug/hole into an ELISA plate, and coating at 4 ℃ overnight; washing the plate 5 times with PBST buffer; the CBS buffer was 0.05M carbonate-bicarbonate buffer ph9.6;
(2) Closing: blocking the ELISA plate with PBST buffer containing 5% skimmed milk powder, 200 μl/well, and incubating at 37deg.C for 1 hr; washing the plate 5 times with PBST buffer;
(3) And (3) detection: the dilutions were added to the elisa plate, 50 μl/well, and the serum sample to be tested 1:4, adding the diluted solution into an ELISA plate A hole, 50 mu L/hole, and performing double dilution (A-H; 1:8-1:1024); incubation is carried out for 30min at 37 ℃, and the plate is washed 5 times by PBST buffer solution; the dilution was 0.01M PBS pH7.2;
(4) Adding a competing antibody: diluting the monoclonal antibody of the first aspect by 0.5 mu g/mL, adding the diluted monoclonal antibody into an ELISA plate, incubating the ELISA plate at 100 mu L/well for 30min at 37 ℃, and washing the plate with PBST buffer solution for 5 times; the dilution was 0.01M PBS pH7.2;
(4) Adding enzyme-labeled secondary antibodies: 1, 1:40000 diluted HRP-labeled goat anti-mouse secondary antibody was added to the ELISA plate, 100. Mu.L/well, and incubated at 37℃for 30min; washing the plate 5 times with PBST buffer; the dilution was 0.01M PBS pH7.2;
(5) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; 100. Mu.L/well of stop solution was added and OD was read 450 Is a value of (2);
the judging method comprises the following steps: PI% = (1-sample OD/negative OD) ×100%, positive when PI is greater than or equal to 60%, negative when PI is less than 60%, negative OD is greater than 0.3, and less than 2.0.
The beneficial effects of the invention are as follows: (1) the invention provides a monoclonal antibody of a bluetongue virus structural protein VP 7; (2) immunoblotting experiments show that the polypeptide reacts with structural protein VP7 specifically; (3) the monoclonal antibody can be specifically combined with the bluetongue virus in the infected cells, and can be used for detecting and diagnosing the bluetongue virus; (4) compared with the existing antibody detection method, the method has better sensitivity and specificity, and is suitable for detecting and diagnosing the bluetongue virus.
Drawings
SDS-PAGE identification of the purified monoclonal antibody 2D7 of FIG. 1;
FIG. 2 identification of monoclonal antibody 2D7 by cellular immunofluorescence;
FIG. 3 Western Blot of monoclonal antibody 2D 7.
Detailed Description
The following detailed description of embodiments of the invention is provided for the purpose of illustration only and is not to be construed as limiting the invention. In addition, all reagents employed in the examples below are commercially available or may be synthesized in accordance with text or known methods, and are readily available to those skilled in the art for reaction conditions not listed, if not explicitly stated.
EXAMPLE 1 preparation of monoclonal antibodies
1. Preparation of hybridoma cells
1.1 immunization of mice
6 female BALB/c mice with the age of 6-8 weeks are taken; injecting the expressed BTV VP7 recombinant protein into the immune; after the VP7 protein and equivalent Freund's complete adjuvant are fully emulsified for the first immunization, carrying out subcutaneous immune injection on the mice, wherein the injection is 50 mug/mouse; after 14d, performing a second immunization injection, and after full emulsification of Freund's incomplete adjuvant and VP7 protein, subcutaneously immunizing and injecting the mice with 50 mug/mouse; after 14d, performing a third immunization injection, fully emulsifying Freund's incomplete adjuvant and VP7 protein, and subcutaneously immunizing a mouse with 50 mug/mouse; after 10d, the tail of the mice is cut off to collect blood, and the serum antibody titer is detected by an indirect ELISA method. When the antibody level reaches the requirement, at the 14d after the third immunization, the immunized mice with the highest titer are selected for boosting immunization, and the dosage is 50 mug/mouse without adjuvant by intraperitoneal injection; after 3d, mouse spleen cells were aseptically harvested and then fused with SP2/0 cells.
1.2 cell fusion
Cell fusion was performed in a sterile environment, and prepared SP2/0 cells and mouse spleen cells were prepared according to 1:5, adding 20mL of incomplete culture medium, centrifuging at 1000rpm for 10min, and discarding the supernatant. Lightly beating the bottom of the centrifuge tube with fingers to disperse the precipitated cells, fusing the cells in a water bath at 37deg.C with 1mL of 50% PEG preheated at 37deg.C while gently stirring, standing for 1min to allow the PEG to fully exert its effect, terminating the fusion with 20mL of DMEM medium, centrifuging at 1000rpm for 5min, re-suspending the fused cells with HAT medium, inoculating into 96-well cell culture plates, and placing 96-well plates in 5% CO at 37deg.C 2 Culturing in an incubator.
1.3 selection of hybridoma cells
HAT medium was used 7 days after fusion, HT medium was used 7-14 days later, and ordinary complete medium was used 14 days later when the area of the clones reached 1/3-1/2 of the area of the culture wells, at which time the medium for all the cloned growth wells was examined. And (5) timely cloning the cells with the detected specific antibody positive holes. Subcloning adopts a double-ratio dilution method, after subcloning for 7 days, detecting culture supernatant by an indirect ELISA method, selecting a single positive hole of a cell mass, continuing subcloning, repeating for 3 times, and screening out a stable hybridoma cell strain.
The specific detection method comprises the following steps:
(1) Coating: the expressed BTV VP7 recombinant protein was diluted to 0.5. Mu.g/mL with CBS buffer (0.05M carbonate-bicarbonate buffer pH 9.6), 100. Mu.L/well was added to the ELISA plate, and coated overnight at 4 ℃; plates were washed 5 times with PBST buffer.
(2) Closing: blocking the ELISA plate with PBST buffer containing 5% skimmed milk powder, 200 μl/well, and incubating at 37deg.C for 1 hr; plates were washed 5 times with PBST buffer.
(3) And (3) detection: cell culture supernatants of the above hybridoma cells were added to an ELISA plate at 100. Mu.L/well, and incubated with non-immunized mouse serum as a negative control at 37℃for 1 hour, and the plate was washed 5 times with PBST buffer.
(4) Adding enzyme-labeled secondary antibodies: 1, 1: an HRP-labeled goat anti-mouse secondary antibody diluted with 40000 (0.01M PBS pH7.2 as a diluent) was added to the ELISA plate, 100. Mu.L/well, and incubated at 37℃for 1h; plates were washed 5 times with PBST buffer.
(5) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; 100. Mu.L/well of stop solution was added and OD was read 450 Wherein 2D7 is a monoclonal antibody secreted by the hybridoma cell line described above, as shown in table 1 below.
Table 1 OD 450 Values of (2)
| 2D7 | Negative control |
| 3.6555 | 0.0952 |
| 2.9486 | 0.0660 |
2. Preparation of monoclonal antibodies
2.1 preparation of monoclonal antibody ascites
2 BALB/c mice were prepared, and paraffin oil was injected intraperitoneally into the mice in advance, 0.5 mL/mouse, before the hybridoma was inoculated. After 7 days, the prepared hybridoma cells were inoculated with the number of cells being 10 6 On the left and right sides, on the seventh day, the abdomen of the mice is observed to be obviously enlarged, and when the mice are touched by hands, the skin has tension, so that ascites can be extracted.
2.2 purification of monoclonal antibodies
Centrifuging the collected ascites, removing sediment, and collecting supernatant. Antibodies were then purified using Protein G columns.
The specific purification steps are as follows:
(1) Pretreatment of a chromatographic column: the equilibrium chromatography column was washed with 10 column volumes of PBS.
(2) Sample loading: the collected ascites was diluted with 2 volumes of PBS, pH was adjusted, and then slowly added to the column.
(3) Washing: washed with 10 column volumes of PBS until no protein was detected in the effluent.
(4) Antibody elution: antibody elution was performed with 0.1M glycine and the eluate was collected until no protein was detected.
(5) pH adjustment: the eluted product was pH-adjusted to neutral with saturated sodium carbonate.
(6) Sample concentration: concentrating by ultrafiltration with 10kDa ultrafiltration tube to about 1-5 mL.
The purified monoclonal antibody was subjected to SDS-PAGE.
As shown in FIG. 1, the purity of the purified monoclonal antibody 2D7 is more than 95%, so that the requirements of clinical application can be met.
3. Identification of monoclonal antibodies
Immunofluorescence detection of anti-BTV VP7 monoclonal antibody 2D 7: immunofluorescence analysis was performed on BTV-1-infected BSR cells using the prepared anti-BTV VP7 monoclonal antibody 2D 7. Spreading BSR cells on a slide in advance one day, infecting the cells with BTV-1, culturing at 37deg.C for 24 hr, discarding cell culture supernatant, fixing with 4% paraformaldehyde for 10min, washing with PBS for 3 times, allowing 0.5% Triton X-100 to act for 10min, washing with PBS for 3 times, blocking with 3% BSA for 1 hr, absorbing old solution, adding monoclonal antibody 2D7,4 deg.C overnight for incubation, then transfecting nuclei with Hoechst 33342 for 10min, washing with PBS for 3 times, adding goat anti-mouse IgG (Alexa488 incubation for 1h at room temperature in the dark was performed, and images were observed and collected after sealing.
As shown in FIG. 2, the BSR cells infected with BTV-1 were fluorescent, while the control was not. The results show that: the anti-BTV-1 VP7 monoclonal antibody disclosed by the invention can be combined with cells infected with BTV-1, but not combined with uninfected cells.
Immunoblot detection of anti-BTV-1 VP7 monoclonal antibody 2D 7: the purified BTV VP7 protein is prepared into Western Blot, the primary antibody is incubated with the prepared anti-BTV VP7 monoclonal antibody 2D7,4 ℃ overnight, PBST is washed 3 times for 10 minutes each time, the secondary antibody is incubated with an HRP-labeled goat anti-mouse secondary antibody (1:10000) for 1h at room temperature, PBST is washed 3 times, and finally, color development and observation are carried out.
As shown in FIG. 3, the monoclonal antibody 2D7 prepared by the invention can specifically react with the recombinant VP7 protein.
4. Cloning and analysis of monoclonal antibody heavy and light chain variable region genes
4.1 amplification of monoclonal antibody heavy and light chain variable regions
The total RNA of the hybridoma cells secreting monoclonal antibody 2D7 is extracted by a TRIzol cleavage method, and then reverse transcription is carried out by using a reverse transcription kit to synthesize cDNA. And (3) carrying out PCR amplification by taking the obtained cDNA as a template, and connecting an amplification product to a carrier for sequencing to obtain the heavy chain and light chain variable region nucleotide sequences of the antibody.
The coding DNA sequence of the heavy chain variable region of the monoclonal antibody is as follows: GCGGTGCAGCTTCAGGA GTCAGGACCTAGCCTCGTGAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCGACTCCATCACCAGTGGTTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAGTACATGGGGTACGTAAGCTACAGTGGTAGCACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATCACTCGAGACACATCCAAGAACCGGTACTACCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAAATGGGAGGATTACGACCCCTTTGGGTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA (SEQ ID NO. 2);
the coding DNA sequence of the light chain variable region of the monoclonal antibody is as follows: GACATCCAGATGACACAGTCT CCATCCTCACTGTCTGCATCTCTGGGAGGCAAAGTCACCATCACTTGCAAGGCAAGCCAAGACATTAACAAGTATATAGCTTGGTACCAACACAAGCCTGGAAAAGGTCCTAGGCTACTCATACATTACACATCTACATTACAGCCAGGCATCCCATCAAGGTTCAGTGGAAGTGGGTCTGGGAGAGATTATTCCTTCAGCATCAGCAACCTGGAGCCTGAAGATATTGCAACTTATTATTGTCTACAGTATGATAATCTGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO. 4);
from the codon encoding rules, it can be seen that: the amino acid sequence of the heavy chain variable region of the monoclonal antibody is as follows: AV QLQESGPSLVKPSQTLSLTCSVTGDSITSGYWNWIRKFPGNKLEYMGYVSYSGSTYYNPSL KSRISITRDTSKNRYYLQLNSVTTEDTATYYCAKWEDYDPFGYWGQGTLVTVSA (SEQ ID NO. 1);
the amino acid sequence of the light chain variable region of the monoclonal antibody is as follows: DIQMTQSPSSLSASLGGKVTITC KASQDINKYIAWYQHKPGKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYY CLQYDNLWTFGGGTKLEIK (SEQ ID NO. 3).
4.2 determination of CDR regions
The sequences of the light chain and heavy chain variable regions of the monoclonal antibody 2D7 obtained by sequencing are analyzed on an abysis website to obtain CDR regions.
The 3 Complementarity Determining Region (CDR) sequences of the heavy chain variable region are shown below:
CDR1: SGYWN (shown in SEQ ID NO. 5);
CDR2: YVSYSGSTYYNPSLKS (SEQ ID NO. 6);
CDR3: WEDYDPFGY (SEQ ID NO. 7);
the 3 Complementarity Determining Region (CDR) sequences of the light chain variable region are shown below:
CDR1: KASQDINKYIA (SEQ ID NO. 8);
CDR2: YTS QP (SEQ ID NO. 9);
CDR3: LQYDLWT (shown in SEQ ID NO. 10).
Example 2 method for establishing competitive ELISA detection Using monoclonal antibodies to the VP7 protein of bluetongue Virus
1. Establishment of detection method
(1) Coating: the expressed BTV VP7 recombinant protein was diluted to 0.5. Mu.g/mL with CBS buffer (0.05M carbonate-bicarbonate buffer pH 9.6), 100. Mu.L/well coated into an ELISA plate, coated overnight at 4 ℃; plates were washed 5 times with PBST buffer.
(2) Closing: blocking the ELISA plate with PBST buffer containing 5% skimmed milk powder, 200 μl/well, and incubating at 37deg.C for 1 hr; plates were washed 5 times with PBST buffer.
(3) And (3) detection: the dilution (0.01M PBS pH 7.2) was added to the ELISA plate at 50. Mu.L/well, and the serum sample 1 to be tested was: 4, adding the diluted solution into an ELISA plate A hole, 50 mu L/hole, and performing double dilution (A-H; 1:8-1:1024); incubate at 37℃for 30min and wash the plates 5 times with PBST buffer.
(4) Adding a competing antibody: after dilution of 0.5. Mu.g/mL of the monoclonal antibody 2D7 prepared in the examples of the present application (dilution of 0.01M PBS pH 7.2), the solution was added to the ELISA plate, incubated at 37℃for 30min and the plate was washed 5 times with PBST buffer.
(4) Adding enzyme-labeled secondary antibodies: 1, 1: a40000 diluted (0.01M PBS pH 7.2) HRP-labeled goat anti-mouse secondary antibody was added to the ELISA plate, incubated at 37℃for 30min at 100. Mu.L/well, and the plate was washed 5 times with PBST buffer.
(5) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; 100. Mu.L/well of stop solution was added and OD was read 450 Is a value of (2).
The judging method comprises the following steps: PI% = (1-sample OD/negative OD) ×100%, positive when PI is no less than 60%, negative OD is greater than 0.3, and less than 2.0.
2. Competition ELISA specificity experiments
The ELISA experimental method established by the embodiment is used for detecting positive serum of the mouth sore, the sheep pox, the sheep peste des petits ruminants and the like, and evaluating the specificity of the blue tongue virus competition ELISA detection method.
The results are shown in Table 2, only the bluetongue serum test results were positive, while the other pathogenic positive serum test results were negative. The method for detecting the bluetongue virus competition ELISA established by utilizing the monoclonal antibody 2D7 has good specificity.
TABLE 2 specificity results of blue tongue Virus Competition ELISA detection methods
| OD 450 Value of | PI％ | |
| Sore mouth | 1.7468 | 13.5％ |
| Sheep pox disease | 1.9678 | 2.5％ |
| Peste des petits ruminants | 1.7556 | 13％ |
| Bluetongue disease | 0.5775 | 66.8％ |
3. Sensitivity test
Bluish tongue virus positive serum was prepared according to 1: 8. 1: 16. 1: 32. 1: 64. serial dilutions were performed to assess the sensitivity of the bluetongue virus competition ELISA detection method.
The results are shown in Table 3, when serum samples were run 1: the detection result is positive when 64 is diluted, which indicates that the blue tongue virus competition ELISA detection method established by the monoclonal antibody 2D7 has good sensibility.
TABLE 3 sensitivity results of blue tongue Virus Competition ELISA detection methods
| OD 450 Value of | PI％ | |
| 1：8 | 0.3642 | 81.9％ |
| 1：16 | 0.4697 | 76.7％ |
| 1：32 | 0.6035 | 70.1％ |
| 1：64 | 0.7749 | 61.6％ |
4. Compliance rate experiment
11 parts of known trace neutralization test BTV positive serum (1-11) and 11 parts of BTV negative serum (12-22) were tested by the competition ELISA test method established in the example.
The results are shown in table 4, and indicate that the blue tongue virus competition ELISA detection method established by using the monoclonal antibody 2D7 has good accuracy, and the coincidence rate of the detection result reaches 100%.
TABLE 4 compliance rate results of blue tongue Virus Competition ELISA detection methods
| Numbering device | OD 450 | PI％ | Numbering device | OD 450 | PI％ |
| 1 | 0.3999 | 78.7％ | 12 | 1.8778 | -7.8％ |
| 2 | 0.5236 | 72.1％ | 13 | 1.8669 | -7.1％ |
| 3 | 0.489 | 74％ | 14 | 1.8796 | 1.5％ |
| 4 | 0.6523 | 65.3％ | 15 | 1.8077 | -3.7％ |
| 5 | 0.7425 | 74.2％ | 16 | 1.8495 | -6.1％ |
| 6 | 0.7253 | 72.5％ | 17 | 1.8901 | 0.9％ |
| 7 | 0.6821 | 63％ | 18 | 1.8961 | 0.6％ |
| 8 | 0.6831 | 66.1％ | 19 | 1.6868 | 8.5％ |
| 9 | 0.3642 | 81.9％ | 20 | 1.9004 | 0.4％ |
| 10 | 0.5989 | 70.3％ | 21 | 1.9087 | 0 |
| 11 | 0.6413 | 63.1％ | 22 | 1.9331 | -1.2％ |
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A monoclonal antibody against the bluetongue virus VP7 protein, wherein the monoclonal antibody comprises an antibody heavy chain and an antibody light chain;
the variable region CDR of the heavy chain of the antibody comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.5, a CDR2 with an amino acid sequence shown as SEQ ID NO.6 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 7;
the variable region CDR of the antibody light chain comprises a CDR1 with an amino acid sequence shown as SEQ ID NO.8, a CDR2 with an amino acid sequence shown as SEQ ID NO.9 and a CDR3 with an amino acid sequence shown as SEQ ID NO. 10.
2. The monoclonal antibody of claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID No.1 and the amino acid sequence of the variable region of the light chain of the antibody is shown in SEQ ID No. 3.
3. A nucleic acid encoding the antibody heavy and light chains of the monoclonal antibody of claim 1 or 2.
4. The nucleic acid of claim 3, wherein the sequence of the gene encoding the variable region of the heavy chain of said antibody is shown in SEQ ID No.2 and the sequence of the gene encoding the variable region of the light chain of said antibody is shown in SEQ ID No. 4.
5. The use of the monoclonal antibody according to claim 1 or 2 for the preparation of a reagent, or a test strip, or a kit for the detection of bluetongue virus.
6. An immunoconjugate, the immunoconjugate comprising:
(i) The monoclonal antibody of claim 1 or 2;
(ii) And a coupling moiety selected from the group consisting of: a detectable label, a drug, a gold nanoparticle/nanorod, a nanomagnetic particle, a viral coat protein or VLP, or a combination thereof.
7. A bluetongue virus ELISA detection kit, comprising the monoclonal antibody of claim 1 or 2.
8. The ELISA detection kit as claimed in claim 7, wherein the kit comprises an ELISA plate, a bluetongue virus antigen, a blocking solution, a diluent, the monoclonal antibody as claimed in claim 1 or 2, an ELISA secondary antibody, a washing solution, a color reagent and a stop solution.
9. The ELISA test kit of claim 7, wherein the bluetongue virus antigen is selected from the group consisting of bluetongue virus VP7 recombinant proteins.
10. Use of a monoclonal antibody according to claim 1 or 2 in an in vitro assay of bluetongue virus for the purpose of non-disease diagnosis.
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