CN117700537A - Monoclonal antibody with good affinity reaction capacity for VP7 protein of African horse sickness virus and application - Google Patents
Monoclonal antibody with good affinity reaction capacity for VP7 protein of African horse sickness virus and application Download PDFInfo
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
The invention belongs to the technical field of immunology and in-vitro diagnosis, and particularly relates to a monoclonal antibody with good affinity reaction capability of a Vp7 protein of an African horse sickness virus and application thereof. According to the invention, after a recombinant expressed African horse sickness virus VP7 protein is immunized for 3 times on a mouse, spleen and myeloma cell SP2/0 of the mouse are taken for fusion, and hybridoma cell strain 5D3 is finally screened out, so that a monoclonal antibody with good reactivity is obtained. The nucleotide sequence of the heavy chain and light chain variable region of the monoclonal antibody is successfully obtained by reverse transcription PCR technology. The monoclonal antibody and the recombinant expressed African horse sickness virus VP7 protein have good affinity reaction capacity, and a competition ELISA detection method for the African horse sickness virus VP7 protein is established as a competition antibody, so that technical support is provided for effectively preventing and controlling the African horse sickness.
Description
Technical Field
The invention belongs to the technical field of immunology and in-vitro diagnosis, and particularly relates to a monoclonal antibody with good affinity reaction capability of a Vp7 protein of an African horse sickness virus and application thereof.
Background
African horse sickness is a non-contact acute or subacute infectious disease of equine animals caused by African horse sickness virus (African horse sickness virus, AHSV). The disease is transmitted mainly by biting midges. Horses are most susceptible in equine animals, of which young horses are most susceptible, and donkey and mule are second most susceptible, and are generally asymptomatic after zebra infection, and may be their storage hosts, playing an important role in the transmission of the disease. Clinical symptoms of african horse sickness can be classified into 4 types, lung type, heart type, mixed type and fever type. All of the initial symptoms manifest as fever, and the lungs are usually choked to death by respiratory tract serous fluid a few hours after the onset of clinical symptoms. Heart type is mainly manifested as edema of cheek, orbit, neck and chest, late conjunctival and lingual bleeding points, and finally heart failure. The clinical symptoms of both lung and heart types are mixed and are often found in post-mortem dissections. The fever type clinical symptoms are not obvious, and symptoms such as slight anorexia, mental depression and the like can appear. World animal health organization (WOAH) lists it as a disease that must be reported, and China lists it as a type of animal disease.
The African horse sickness virus belongs to the genus Cyclovirus of the reoviridae family, the genome of which comprises 10 double stranded RNA fragments of different sizes, encoding 7 structural proteins (VP 1-VP 7) and 4 non-structural proteins (NS 1-NS 4), the outer capsid consisting of structural proteins VP2 and VP5, wherein the VP2 protein is a serotype specific antigen, and the inner capsid consisting of structural proteins VP3 and VP7, wherein the VP7 protein is a serogroup specific antigen. The current research finds that the African horse sickness virus has 9 serotypes, and the ELISA method based on VP7 protein can be used for detecting the specific antibody of the African horse sickness virus group, and is suitable for detection of large-scale samples and epidemiological investigation.
The current method for detecting African horse sickness antibodies mainly comprises competition ELISA, virus neutralization test, agar gel immune diffusion reaction and immunofluorescence test. The present invention screens and obtains a monoclonal antibody aiming at AHSV VP7 protein and variable region sequences of heavy chain and light chain thereof, and establishes an AHSV competitive ELISA antibody detection method by using the antibody as a competitive antibody.
Disclosure of Invention
Aiming at the technical problems, the invention provides a monoclonal antibody with good affinity reaction capacity for the VP7 protein of the African horse sickness virus and application, and the monoclonal antibody has the characteristics of high titer, strong affinity and good specificity, and can specifically bind with the VP7 protein of the African horse sickness virus.
The method specifically comprises the following steps:
in a first aspect, the present invention provides a monoclonal antibody to VP7 protein of the african horse sickness virus, the monoclonal antibody comprising an antibody heavy chain and an antibody light chain;
the variable region CDR of the heavy chain of the monoclonal 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 monoclonal 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 of the antibody is shown as SEQ ID NO.1, and the amino acid sequence of the variable region of the light chain of the antibody 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 invention provides an application of the monoclonal antibody in the first aspect in preparing a reagent, a test strip or a kit for detecting african horse sickness virus.
In a fourth aspect, the invention provides an ELISA kit for African horse sickness virus, which is characterized in that the kit comprises the monoclonal antibody in the first aspect.
Preferably, the kit further comprises an ELISA plate, a sealing solution, a diluent, an ELISA secondary antibody, a washing solution, a color reagent and a stop solution.
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 HRP-labeled goat anti-mouse IgG.
In a fifth aspect, the present invention provides the use of a monoclonal antibody according to the first aspect above for in vitro detection of african horse sickness virus for the purpose of non-disease diagnosis.
In a sixth aspect, the present invention provides a method for ELISA detection of African horse sickness virus for the purpose of diagnosis of a non-disease, said method comprising the steps of:
(1) Coating: diluting the expressed AHSV VP7 recombinant protein to 0.5 mug/mL by using a CBS buffer solution, coating 100 mug/hole into an ELISA plate, and coating at 4 ℃ overnight; washing the plate 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; washing the plate 5 times with PBST buffer;
(3) And (3) detection: adding a horse serum sample to be detected, standard positive serum and standard negative serum into an ELISA plate, incubating for 1h at 37 ℃ with 100 μl/hole, and washing the plate with PBST buffer solution for 5 times;
(4) Then adding 1:1000 dilution of the monoclonal antibody of the first aspect, incubating for 1h at 37 ℃, and washing the plate with PBST buffer for 5 times;
(5) Adding enzyme-labeled secondary antibodies: 1, 1:40000 diluted HRP-labeled goat anti-mouse IgG was added to the ELISA plate and incubated at 37℃for 1h at 100. Mu.L/well; washing the plate 5 times with PBST buffer;
(6) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; add 100. Mu.l/well stop solution and read OD 450 Is a value of (2);
(7) Determination result: blocking rate (BP) = (negative control OD) 450 Sample OD 450 ) /(negative control OD) 450 Positive control OD 450 ) X 100%; the positive control is less than 0.2, and the negative control is more than 2.0; when BP is less than or equal to 45%, the result is negative; when BP is more than or equal to 50%, the result is positive; when 45%<BP<At 50%, the results were suspicious.
Preferably, the CBS buffer is 0.05M carbonate-bicarbonate buffer, ph9.6.
Preferably, the diluent is 0.01M PBS pH7.2.
The beneficial effects of the invention are as follows: (1) the invention provides a monoclonal antibody for resisting VP7 protein of African horse sickness virus; (2) immunoblotting experiments show that the recombinant VP7 protein reacts with specificity; (3) the monoclonal antibody can specifically react with recombinant VP7 protein expressed in eukaryotic cells; (4) compared with the existing antibody detection method, the method has better sensitivity and specificity, and is suitable for detecting and diagnosing the African horse sickness virus.
Drawings
FIG. 1 identification of molecular weight and purity of AHSV VP 7-resistant monoclonal antibodies;
FIG. 2 immunofluorescence detection of AHSV VP 7-resistant monoclonal antibodies;
FIG. 3 immunoblot detection of anti-AHSV VP7 monoclonal antibodies.
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, as would be readily available to one skilled in the art for reaction conditions not listed, if not explicitly stated.
EXAMPLE 1 preparation of monoclonal antibodies to the VP7 protein of African horse sickness Virus
1. Expression and purification of African horse sickness virus VP7 recombinant protein
The S7 gene sequence for expressing the VP7 protein of the African horse sickness virus is constructed on a pET-28a vector, then the gene sequence is transformed into BL21 (DE 3) competent cells, the cell is cultured at 37 ℃ and 220rpm for about 3 hours to reach an OD value of 0.6-0.8, and then the cell is induced by adding IPTG with a final concentration of 0.5 mu M at 16 ℃ for 16 hours. And collecting thalli, performing ultrasonic crushing and cracking, and purifying the supernatant after ultrasonic cracking by using a nickel column.
2. Immunization of mice
6 female BALB/c mice, 6-8 weeks old, were immunized with purified AHSV VP7 recombinant protein. Primary immunization VP7 recombinant protein was emulsified with an equal amount of freund's complete adjuvant, injected subcutaneously on the back, 50 μg/dose. The first immunization was followed by full emulsification with the VP7 recombinant protein, subcutaneous injection, 50. Mu.g/dose, with equal amount of Freund's incomplete adjuvant at day 14 and day 28. The serum antibody titer of the mice is detected by indirect ELISA, when the antibody level reaches the requirement, the mice are injected into the abdominal cavity for enhancing immunity, and after 3d of immunization, spleen cells of the mice are aseptically collected and fused with SP2/0 cells.
3. Cell fusion
Prepared SP2/0 cells and mouse spleen cells were prepared according to 1: ratio of 5Example mixing, adding 20mL of incomplete medium, centrifuging at 1000rpm/min for 10min, and discarding the supernatant. Lightly beating the bottom of the centrifuge tube with finger to disperse the precipitated cells, then fusing the cells with 1mL of 50% PEG preheated at 37deg.C while gently stirring, standing for 1min, stopping fusing with 20mL of DMEM medium, centrifuging at 1000rpm for 5min, re-suspending the fused cells with HAT medium, inoculating into 96-well cell culture plate, placing 96-well plate at 37deg.C with 5% CO 2 Culturing in an incubator.
4. Screening and subcloning 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, and when the area of the clone reached 1/3-1/2 of the area of the culture well, the medium of all clone 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 the process for 3 times, and screening out a stable hybridoma cell strain 5D3.
The specific method for indirect ELISA detection comprises the following steps:
(1) Coating: AHSV VP7 recombinant protein was diluted to 0.5. Mu.g/mL, 100. Mu.L/well in CBS buffer (0.05M carbonate-bicarbonate buffer, pH 9.6) and coated onto ELISA plates overnight at 4 ℃. Plates were washed 5 times with PBST buffer.
(2) Closing: the ELISA plates were blocked with 5% nonfat milk in PBST buffer, 200. Mu.L/well, and incubated at 37℃for 1h. Plates were washed 5 times with PBST buffer.
(3) And (3) detection: the hybridoma cell culture supernatant to be detected was added to the ELISA plate at 100. Mu.L/well, while the cell culture supernatant of normal SP2/0 cells was used as a negative control, incubated at 37℃for 1h, and the plate was washed 5 times with PBST buffer.
(4) Adding enzyme-labeled secondary antibodies: 1, 1: a40000 dilution (dilution of 0.01M PBS pH 7.2) of HRP-labeled goat anti-mouse IgG 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: avoidance with TMB color developing solutionLight color development, 100. Mu.L/well, incubation at 37℃for 10min; 100. Mu.L/well of stop solution was added and OD was read 450 Is a value of (2).
OD 450 The results of the values of (a) are shown in Table 1, and the results show that the hybridoma cells (designated as 5D3 cell line) have good reactivity with VP7 recombinant protein.
TABLE 1OD 450 Detection result
| Group of | OD 450 |
| 5D3 cell lines | 3.658 |
| Negative control | 0.092 |
5. Preparation of mouse ascites antibody
2 female BALB/c mice of 6-8 weeks old were prepared and liquid paraffin was injected intraperitoneally, 0.5 mL/mouse. After 7 days, inoculate 10 6 The hybridoma cells obtained in the step 4 are observed, the abdomen of the mouse is observed to be obviously enlarged about 7-10 days, and when the hybridoma cells are touched by hands, the skin has tension, so that ascites can be extracted. The collected ascites was centrifuged to remove the precipitate and the supernatant was collected. And then purifying the ascites by using a Protein G column to obtain the high-titer and high-purity 5D3 monoclonal antibody. The concentration of the purified monoclonal antibody was 1.14mg/mL.
The specific purification steps are as follows:
(1) Pretreatment of a chromatographic column: the column volume of 10 times deionized water is washed 3-5 times, and then the column volume of 10 times PBS is washed 3-5 times.
(2) Loading: the ascites was diluted with PBS and filtered through a 022. Mu.L filter.
(3) Washing: PBS was washed until no protein flow was detected (G250 did not change blue).
(4) Antibody elution: elution with 0.1m ph3.0 glycine, the eluate was collected and the eluted product was checked with G250 until it did not turn blue.
(5) pH value adjustment: saturated sodium carbonate adjusts the pH of the eluted product to neutral.
(6) Sample concentration: and (3) a 10kDa ultrafiltration tube, and concentrating the solution to about 1 to 5 mL.
Example 2 identification of monoclonal antibodies against the VP7 protein of African horse sickness Virus
1. Identification of molecular weight and purity of AHSV VP 7-resistant monoclonal antibody
Performing molecular weight and purity identification of the antibody by adopting an SDS-PAGE method; the monoclonal antibody purified in example 1 was prepared, then subjected to SDS-PAGE, loaded with 10. Mu.L per well, stained with Coomassie blue solution for 1h after the electrophoresis was completed, and then decolorized. The results are shown in FIG. 1. The result shows that the monoclonal antibody is successfully purified, the purity is more than 95%, and the application requirement can be met.
2. Determination of the titers of AHSV VP 7-resistant monoclonal antibodies
Determining the titer of the monoclonal antibody by using indirect ELISA, taking AHSV VP7 recombinant protein as a coating antigen, coating at a concentration of 0.5 mug/mL, 100 mug/hole and 4 ℃ overnight; PBST washes the plate 3 times; adding 5% skimmed milk powder for sealing, 200 μl/well, incubating at 37deg.C for 2h, and washing the PBST plate 3 times; respectively adding 100 mu L of a sample to be tested diluted by a ratio (the dilution is 0.01M PBS pH 7.2), incubating for 1h at 37 ℃, and washing the PBST plate for 3 times; adding 1: HRP-labeled goat anti-mouse IgG diluted 10000 (0.01 m PBS ph7.2 as diluent), 100 μl/well, incubated 40min at 37 ℃, pbst washed 5 times; 100. Mu.L/well of substrate solution was added, developed in the dark at 37℃for 10-15min, and 100. Mu.L/well of stop solution was added. Reading OD of each well 450 The value is judged by the ratio (P/N) of positive antibody to negative antibody being greater than 2.1.
The results are shown in Table 2, and the titer of the monoclonal antibody can reach 1:1638400.
TABLE 2 OD of determination of AHSV VP 7-resistant monoclonal antibody titers 450 Value of
| Concentration of primary antibody | OD 450 Value of |
| 1：1600 | 2.0397 |
| 1：3200 | 1.9733 |
| 1：6400 | 1.6844 |
| 1：12800 | 1.4823 |
| 1：25600 | 1.1258 |
| 1：51200 | 0.9729 |
| 1：102400 | 0.8237 |
| 1：204800 | 0.7568 |
| 1：409600 | 0.6072 |
| 1：819200 | 0.5169 |
| 1：1638400 | 0.4479 |
| Negative control | 0.0655 |
3. Immunofluorescence detection of AHSV VP 7-resistant monoclonal antibodies
Immunofluorescence analysis was performed on BSR cells transfected with recombinant plasmids expressing AHSV VP7 (pcdna3.1-AHSV VP7, prepared by conventional means) using the prepared anti-AHSV VP7 monoclonal antibodies. The BSR cells were climbing-up, recombinant plasmid pcDNA3.1-AHSV VP7 was transfected into the BSR cells by liposome 3000, incubated at 37℃for 24 hours, cell culture supernatant was discarded, fixed with 4% paraformaldehyde for 10min, washed 3 times with PBS, then subjected to the action of 0.5% Triton X-100 for 10min, washed 3 times with PBS, blocked with 3% BSA for 1 hour, after the old solution was removed by suction, anti-AHSV VP7 monoclonal antibody (1:1000) prepared in example 1 was added, incubated overnight at 4℃and then nuclei were stained with Hoechst 33342 for 10min, washed with PBS for 3 times, goat anti-mouse IgG (Alexa was added488 Incubation for 1h at room temperature in the dark, and after sealing, observation and image collection.
As shown in FIG. 2, the BSR cells transfected with the recombinant plasmid pcDNA3.1-AHSV VP7 showed green fluorescence, while the cells transfected with the empty vector pcDNA3.1 did not fluoresce, which indicated that the AHSV VP 7-resistant monoclonal antibodies were able to specifically bind to VP7 protein expressed by eukaryotic cells.
4. Immunoblot detection of AHSV VP 7-resistant monoclonal antibodies
Immunoblotting was performed with a preparation of expressed AHSV VP7 recombinant protein, the primary antibody was incubated with the AHSV VP 7-resistant monoclonal antibody prepared in example 1 (1:1000), washed with PBST 3 times at room temperature for 10 minutes each time, with HRP-labeled goat anti-mouse IgG (1:10000), incubated at room temperature for 1h, washed with PBST 3 times, and finally visualized.
As shown in FIG. 3, the AHSV VP 7-resistant monoclonal antibodies prepared in the present application are capable of specifically reacting with VP7 recombinant proteins in immunoblotting experiments.
5. Cloning and analysis of monoclonal antibody heavy and light chain variable region genes
(1) Amplification of monoclonal antibody heavy and light chain variable regions
The total RNA of the AHSV VP7 monoclonal antibody-secreting 5D3 hybridoma cells prepared in example 1 was extracted by TRIzol cleavage method, then cDNA was synthesized by reverse transcription, and then PCR amplification was performed using the cDNA as a template to obtain the heavy and light chain variable region nucleotide sequences of the antibody.
Sequencing results show that the nucleotide sequence of the heavy chain variable region of the AHSV VP 7-resistant monoclonal antibody is as follows: GAGGTCCA ACTGCAACAGTCTGGCCCTGAGCTGGTGAAGCCTGGAGCTTCACTGAAGATATCCTGCAAGGCTTCTGGTTACTCATTCTCTGGCTACACCGTTAACTGGGTGAAGCAGAGCCACGGAAAGACCCTTGAGTGGATTGGACTTATTCTTCCTTACAATGGTGGTACTACCTACAACCAGAAGTTCACGGGCAAGGCCACTTTAACTGTCGACAAGTCATCCAGCACAGCCTACATGGAACTCCTCAGTCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGGGAGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA (SEQ ID NO. 2);
the amino acid sequence of the heavy chain variable region of the AHSV VP 7-resistant monoclonal antibody is as follows: EVQLQQSGPELVKPGASLKIS CKASGYSFSGYTVNWVKQSHGKTLEWIGLILPYNGGTTYNQKFTGKATLTVDKSSSTAYM ELLSLTSEDSAVYYCARGGDYWGQGTTLTVSS (SEQ ID NO. 1);
the nucleotide sequence of the light chain variable region of the AHSV VP 7-resistant monoclonal antibody is as follows: GATGTTGTGGTGACCCAGAC TCCACTCACTTTGTCGGTTACCTTTGGACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAAATAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTCAACTGGACTCTGGAGTCCCTGACAGGTTCACTGGCACTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA (SEQ ID NO. 4);
the amino acid sequence of the light chain variable region of the AHSV VP 7-resistant monoclonal antibody is as follows: DVVVTQTPLTLSVTFGQPASISC KSSQSLLNSDGKTYLNWLLQRPGQSPKRLIYLVSQLDSGVPDRFTGTGSGTDFTLKISRVEA EDLGVYYCWQGTHFPWTFGGGTKLEIK (SEQ ID NO. 3).
(2) Determination of CDR regions
Analyzing the sequences of heavy chain and light chain variable regions of the AHSV VP7 resistant monoclonal antibody obtained by sequencing, wherein the amino acid sequences of 3 CDR regions of the heavy chain variable region are respectively as follows:
CDR1: GYTVN (shown in SEQ ID NO. 5);
CDR2: LILPYNGGTTYNQKFTG (SEQ ID NO. 6);
CDR3: GGDY (shown in SEQ ID NO. 7);
the amino acid sequences of the 3 CDR regions of the light chain variable region are respectively:
CDR1: KSSQSLLNSDGKTYLN (SEQ ID NO. 8);
CDR2: LVSQLDS (shown in SEQ ID NO. 9);
CDR3: WQGTHFPWT (SEQ ID NO. 10).
Example 3 establishment of African horse sickness competition ELISA diagnosis method based on AHSV VP 7-resistant monoclonal antibody 1. Establishment of ELISA diagnosis method
The prepared african horse sickness VP7 recombinant protein was coated overnight at 4℃at a concentration of 0.5. Mu.g/mL and 100. Mu.l/well. African horse sickness standard positive serum and standard negative serum are stored in the laboratory, and the prepared AHSV VP 7-resistant monoclonal antibody is used as a competitive antibody to establish a diagnosis method.
The specific detection method comprises the following steps:
(1) Coating: the expressed AHSV 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 ℃; washing the plate 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; washing the plate 5 times with PBST buffer;
(3) And (3) detection: adding a horse serum sample to be detected, standard positive serum and standard negative serum into an ELISA plate, incubating for 1h at 37 ℃ with 100 μl/hole, and washing the plate with PBST buffer solution for 5 times;
(4) Then, the AHSV VP 7-resistant monoclonal antibody prepared in example 1 was added at a dilution of 1:1000 (0.01M PBS, pH 7.2), incubated at 37℃for 1h, and the plate was washed 5 times with PBST buffer;
(5) Adding enzyme-labeled secondary antibodies: 1, 1: HRP-labeled goat anti-mouse IgG diluted with 40000 (0.01M PBS, pH 7.2) was added to the ELISA plate, 100. Mu.L/well, and incubated at 37℃for 1h; washing the plate 5 times with PBST buffer;
(6) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; add 100. Mu.l/well stop solution and read OD 450 Is a value of (2).
(7) Determination result: blocking rate (BP) = (negative control OD) 450 Sample OD 450 ) /(negative control OD) 450 Positive control OD 450 ) X 100%; the positive control is less than 0.2, and the negative control is more than 2.0; when BP is less than or equal to 45%, the result is negative; when BP is more than or equal to 50%, the result is positive; when 45%<BP<At 50%, the results were suspicious.
2. Sensitivity test
African horse sickness positive serum was serially diluted 1:2, 1:4, 1:8, 1:16 to assess the sensitivity of African horse sickness competition ELISA detection methods. The results are shown in Table 3, and the detection results are positive when the serum sample is subjected to 1:16 dilution, which indicates that the African horse sickness competition ELISA detection method established by using the monoclonal antibody 5D3 has good sensitivity.
TABLE 3 sensitivity results of African horse sickness competition ELISA detection method
| Dilution factor | OD 450 | BP |
| 1:2 | 0.328 | 94.3％ |
| 1:4 | 0.497 | 87％ |
| 1:8 | 0.864 | 71.5％ |
| 1:16 | 1.15 | 59.4％ |
3. Compliance rate experiment
The competition ELISA assay established in this example was used to detect 5 parts (1-5) of AHSV positive serum and 5 parts (6-10) of AHSV negative serum stored in the laboratory. The results are shown in Table 4, and indicate that the coincidence rate of the detection results of the African horse sickness competition ELISA method established by using the monoclonal antibody 5D3 disclosed in the application reaches 100%.
TABLE 4 coincidence rate results of African horse sickness competition ELISA detection method
| Sample of | OD 450 | BP |
| 1 | 0.2627 | 96.9％ |
| 2 | 0.3399 | 93.7％ |
| 3 | 0.8508 | 72.1％ |
| 4 | 0.5145 | 86.3％ |
| 5 | 1.2142 | 56.8％ |
| 6 | 1.5377 | 43.1％ |
| 7 | 1.7933 | 32.4％ |
| 8 | 2.494 | 2.85％ |
| 9 | 2.5274 | 1.4％ |
| 10 | 2.4048 | 6.6％ |
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 to VP7 protein of an african horse sickness virus, characterized in that 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 in the preparation of a reagent, or a test strip, or a kit for detecting african horse sickness virus.
6. An african horse sickness virus ELISA detection kit comprising the monoclonal antibody of claim 1 or 2.
7. The ELISA detection kit of claim 6, wherein the kit further comprises an ELISA plate, a blocking solution, a diluent, an ELISA secondary antibody, a washing solution, a color-developing agent and a stop solution.
8. Use of a monoclonal antibody according to claim 1 or 2 for in vitro detection of african horse sickness virus for the purpose of diagnosis of non-disease.
9. A method of ELISA detection of african horse sickness virus for the purpose of non-disease diagnosis, the method comprising: the African horse sickness virus VP7 recombinant protein coats an ELISA plate; closing; adding a horse serum sample to be detected, standard positive serum and standard negative serum into an ELISA plate, and adding the monoclonal antibody of claim 1 or 2 for incubation; adding enzyme-labeled secondary antibodies for incubation; color development, reading OD 450 Is a value of (2); and judging the detection result.
10. The method of detection according to claim 9, wherein the method comprises the steps of:
(1) Coating: diluting the recombinant protein of the African horse sickness virus VP7 into 0.5 mug/mL with CBS buffer solution, coating 100 mug/hole into an ELISA plate, and coating at 4 ℃ overnight; washing the plate 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; washing the plate 5 times with PBST buffer;
(3) And (3) detection: adding a horse serum sample to be detected, standard positive serum and standard negative serum into an ELISA plate, incubating for 1h at 37 ℃ with 100 μl/hole, and washing the plate with PBST buffer solution for 5 times;
(4) Then adding 1:1000 dilution of the monoclonal antibody of claim 1 or 2, incubating for 1h at 37 ℃, and washing the plate 5 times with PBST buffer;
(5) Adding enzyme-labeled secondary antibodies: 1, 1:40000 diluted HRP-labeled goat anti-mouse IgG was added to the ELISA plate and incubated at 37℃for 1h at 100. Mu.L/well; washing the plate 5 times with PBST buffer;
(6) Color development: developing color with TMB color development liquid in dark place, 100 μl/hole, and incubating at 37deg.C for 10min; add 100. Mu.l/well stop solution and read OD 450 Is a value of (2);
(7) Determination result: blocking rate (BP) = (negative control OD) 450 Sample OD 450 ) /(negative control OD) 450 Positive control OD 450 ) X 100%; the positive control is less than 0.2, and the negative control is more than 2.0; when BP is less than or equal to 45%, the result is negative; when BP is more than or equal to 50%, the result is positive; when 45%<BP<At 50%, the results were suspicious.
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