CN110927390A - ELISA method and kit for detecting African swine fever CD2v protein antibody and application - Google Patents
ELISA method and kit for detecting African swine fever CD2v protein antibody and application Download PDFInfo
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Abstract
The invention discloses an ELISA method for detecting an African swine fever CD2v protein antibody, a kit and application. The ELISA method is a double-antigen sandwich method. By the method, an ELISA kit for detecting the African swine fever CD2v protein antibody is obtained, and the kit comprises a horseradish peroxidase-labeled CD2v antigen and a CD2v protein-coated ELISA plate. The kit designed by the invention can be used for screening a large amount of African swine fever virus antibodies, can also be used for identifying African swine fever vaccine strains and wild strains in immune swinery with CD2v deletion strains, has quick and sensitive detection and good specificity, is very suitable for large-area quick detection and general investigation of African swine fever epidemic situation and purification and eradication of African swine fever in a pig farm, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of animal medical animal epidemic disease molecular biological diagnosis, and particularly relates to an ELISA method for detecting an African swine fever CD2v protein antibody, a kit and application.
Background
African Swine Fever (ASF) is a highly contagious disease with high lethality caused by African Swine Fever Virus (ASFV) infecting domestic and wild pigs. Clinically, it is characterized by high fever, extensive bleeding of internal organs, splenomegaly and black color due to bleeding. There are many African swine fever virus genotypes, and 24 genotypes are currently known. So far, no commercial vaccine exists for the disease, and epidemic prevention and control mainly depends on enhancing the biosafety management of a feeding farm, limiting the transregional flow of live pigs and pig products, adopting treatment modes such as herding and killing of diseased animals and the like, and often causing huge economic loss. The world animal health organization lists the African swine fever as an animal epidemic disease which is reported by the legal method, and China lists the African swine fever as a type of animal epidemic disease.
In 1921, Montgomery first reported the development of African swine fever in Kenya. Since 2007, African swine fever has spread and prevailed in several countries in the middle east Europe, particularly Russia and its surrounding areas. In the 3 th year 2017, the African swine fever epidemic situation occurs in the far east region of Russia in Ieculzke, in the 8 th year 2018, the African swine fever epidemic situation is found for the first time in Shenbei region in Shenyang city of Liaoning province of China, and through nucleic acid detection and sequence comparison analysis, the strain and the popular Grougia strain (Georgia 2007) in Russia and China and eastern Europe countries belong to the same clade and belong to a gene II type strain. Subsequently, the disease shows diffusion and popularity in multiple provinces (autonomous regions and direct prefectures) of Henan, Jiangsu, inner Mongolia, Heilongjiang, Zhejiang, Anhui and the like in China, billions of yuan of economic loss is caused to the pig industry in China, and the disease still spreads at present.
Since ASF can cause devastating hits in the swine industry, studies on ASF vaccines have never been interrupted. So far, a plurality of ASFV gene deletion vaccine candidate strains are obtained internationally by adopting a genetic engineering means, and the attenuated vaccine candidate strains are obtained by a method of weakening cells by passage and separating attenuated strains from the nature, and MGF and CD2v gene deletion strains are mostly studied in gene deletion vaccine. In 2017 abroad, the BA71 strain (genotype I) with CD2v deletion is constructed to be sufficiently weakened, and the survival rate of immunized pigs is all 100% (Monteagoudo, P.L, et al BA71DeltaCD2: a New Recombinant Live infected after infection of Swine with Cross-Protective Capacities. J Virol,2017.91 (21)). The CD2v and MGF double-gene-deleted pig is constructed in 2019, so that the immune pig can be protected by 100% (Zhanyan et al, construction of African swine fever virus gene-deleted vaccine strain and immune protection characteristics, Chinese veterinary science report, 2019.39(8), 1421-.
The patent CN102236017A discloses an indirect ELISA kit for detecting an African swine fever virus antibody and application thereof, and the indirect ELISA kit for detecting the African swine fever virus antibody is developed by an enzyme-linked immunosorbent assay technology on the basis of a prokaryotic expression recombinant protein. However, the kit can be only used for screening a large amount of African swine fever virus antibodies, and cannot identify vaccine strains and wild strains. Therefore, in the future, it is urgent to identify vaccine strains and wild strains with the use of large quantities of vaccines.
Disclosure of Invention
The primary object of the application is to provide an ELISA method for detecting an African swine fever CD2v protein antibody.
The invention also aims to provide a double-antigen sandwich ELISA kit for detecting the African swine fever virus CD2v antibody. The kit can be used for large-scale screening of African swine fever virus antibodies and for identifying African swine fever vaccine strains and wild strains in immune swinery with CD2v deletion strains.
The invention further aims to provide application of the double-antigen sandwich ELISA kit in detection of the African swine fever virus CD2v antibody.
The above object of the present invention is achieved by the following technical solutions:
an ELISA method for detecting an African swine fever CD2v protein antibody comprises the following steps:
(1) coating the recombinant African swine fever virus CD2v protein in a microporous plate, then sealing, adding a sample to be tested, a standard positive control and a standard negative control into different micropores for reaction, and washing the plate;
(2) adding HRP marked CD2v antigen for reaction, and washing the plate; adding a color developing solution for color developing reaction, and adding a stopping solution to stop the reaction;
(3) measuring the absorbance value of each hole at the wavelength of 450 nm; and according to the measurement result, carrying out qualitative or quantitative judgment on the sample to be measured.
The coating amount of the African swine fever virus CD2v protein in the step (1) is preferably 0.06 mu g/hole.
The coating conditions in step (1) are preferably 4 ℃ overnight.
The blocking in the step (1) is preferably performed by using 1-5% (w/v) bovine serum albumin solution, 200 mu L/hole and standing for 12 hours at 4 ℃.
The reaction in step (1) is preferably incubated at 37 ℃ for 1 hour.
The number of times of plate washing in the step (1) is preferably 3-5.
The amount of HRP-labeled CD2v antigen added in step (2) is preferably 0.02. mu.g/well.
The reaction conditions for the reaction of adding the HRP-labeled CD2v antigen in step (2) are preferably incubation at 37 ℃ for 30 min.
The number of times of plate washing in the step (2) is preferably 3-5.
The condition of the color reaction in the step (2) is preferably room temperature and light-shielding reaction for 10-15 minutes.
The qualitative judgment is to judge whether the sample to be detected is infected by the African swine fever virus, and the method specifically comprises the following steps: and determining the serum sample to be detected to be positive if the OD450 is more than 0.4, and determining the serum sample to be detected to be negative if the OD450 is not more than 0.4.
A double-antigen sandwich ELISA kit for detecting an African swine fever virus CD2v antibody comprises an ELISA plate coated by a CD2v protein and a CD2v antigen marked by horseradish peroxidase (HRP). By means of the enzyme-labeled antibody, a double-antigen sandwich ELISA method is established for detecting the African swine fever virus CD2v antibody.
The horseradish peroxidase (HRP) labeled CD2v antigen is preferably prepared by a sodium periodate method; more preferably by HRP labeling kit.
The indirect ELISA kit for detecting the African swine fever virus antibody further comprises at least one of substrate developing solution, stop solution, positive control, negative control, serum diluent and 10-fold concentrated washing solution.
The ELISA plate is preferably a detachable ELISA plate.
The number of the enzyme label plates is preferably 2.
The standard of the ELISA plate is preferably 8 holes multiplied by 12 strips.
The CD2v protein-coated ELISA plate is preferably prepared by the following steps: adding 0.6. mu.g/mL CD2v protein solution into blank enzyme label plate at 100. mu.L/well by using 0.05M carbonate buffer solution with pH9.6 as coating buffer solution, and coating overnight at 4 ℃; then, the coating solution was discarded, and 5% (w/v) BSA solution was added at 200. mu.L/well, allowed to stand at 4 ℃ for 12 hours, washed, dried at room temperature, packed in a bag, added with a desiccant, and stored under vacuum.
The composition of the substrate color developing solution is preferably as follows: 0.5mL of tetramethylbiphenyl ethanol solution with the concentration of 2mg/mL, 10mL of substrate buffer solution and 0.75% H2O2，32μL；
The composition of the substrate buffer was as follows: na in a concentration of 0.2mol/L2HPO425.7mL of the solution, 24.3mL of 0.1M citric acid, and 50mL of distilled water.
The stop solution comprises the following components: 178.3mL of distilled water and 21.7mL of 98% concentrated sulfuric acid.
The 10-fold concentrated washing solution is PBS (pH7.4, 1.5M).
The serum diluent consists of the following components: bovine serum albumin (1 g) was added to a volume of 100mL with 0.15M PBS (pH7.4).
The positive control is at least one of a CD2v antibody and a sample containing a CD2v antibody.
The negative control was a sample without CD2v antibody.
The double-antigen sandwich ELISA kit is applied to detection of an African swine fever virus CD2v protein antibody, and is mainly used for identifying whether the African swine fever virus infection exists in pigs in a CD2v deletion strain immune swinery or for antibody detection of the African swine fever virus of the non-immune swinery.
Compared with the prior art, the invention has the following beneficial effects and advantages:
the kit provided by the invention adopts recombinant CD2v protein expressed by CHO-K1 cells as a coating antigen, and detects the antibody of the African swine fever CD2v protein in pig serum according to the double-antigen sandwich ELISA principle. The kit designed by the invention can be used for screening a large amount of African swine fever virus antibodies, can also be used for identifying African swine fever vaccine strains and wild strains in immune swinery with CD2v deletion strains, has quick and sensitive detection and good specificity, is very suitable for large-area quick detection and general investigation of African swine fever epidemic situation and purification and eradication of African swine fever in a pig farm, and has wide application prospect.
Drawings
FIG. 1 is a graph showing the results of indirect Immunofluorescence (IFA) assay of CHO-CD2v cell line stably expressing CD2v gene.
FIG. 2 is a titer chart of a CD2v protein immunized New Zealand white rabbit antibody detected by an established double antigen sandwich ELISA kit; wherein, R1-R5 respectively represent 5 immunized New Zealand white rabbits, and R6-R8 represent 3 unimmunized New Zealand white rabbits.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
Example 1: preparation of CD2v antigen
1. Expression of African swine fever CD2v protein
According to a CD2v gene sequence of an African Swine Fever (ASFV) Chinese epidemic strain Pig/HLJ/2018(GenBank: MK333180.1) in GenBank, a CD2v whole gene is artificially synthesized to be used as a template, a pair of primers CD2v-F/CD2v-R are designed to be amplified by PCR and cloned to a eukaryotic expression vector pIRESpuro2 (purchased from Clontech in the United states), a recombinant plasmid pIRES-CD2v is constructed, CHO-K1 cells (purchased from the cell resource center of Shanghai Life sciences research institute of China academy of sciences) are transfected after sequencing verification, puromycin screening and 2-3 rounds of single cell cloning are carried out, and the identification is carried out by indirect Immunofluorescence (IFA). The specific identification method comprises the following steps: the cells of the single cell clone are transferred to a 12-hole cell culture plate, cultured into a compact monolayer and fixed by 4% paraformaldehyde, because the tail end of the expressed protein is provided with 6 histidine tags, the cells can react with histidine tag antibodies (purchased from Thermo company in the United states), after washing for 3 times, FITC-labeled goat anti-mouse IgG is added for reaction, and then the result is observed by a fluorescence microscope (shown in figure 1), the result shows that green fluorescence exists in the cells, and the obtained cell strain is a cell strain stably expressing a CD2v gene and is named as a CHO-CD2v cell strain.
Wherein, the primers used are:
CD2v-F:5'-GACGCTAGCATGATATACTTATTTTTTTAATA-3'；
CD2v-R:5'-GTCGCGGCCGCGTGGTGGTGGTGGTGGTGAATAATTCTATCTACGTGAATAAGC-3'；
the underlined part is an introduced restriction enzyme site, CD2v-F is an ASFV CD2v gene upstream amplification primer, and the introduced restriction enzyme site is Nhe I; CD2v-R is ASFV CD2v gene downstream amplification primer, the introduced restriction enzyme cutting site is Not I, simultaneously, 6 histidine (6 XHis) coding bases are introduced into the downstream primer for the purification of CD2v expression protein, and the primer is synthesized by the company Limited in the biological engineering (Shanghai).
2. Purification of African swine fever CD2v protein
The CHO-CD2v cell line prepared as described above was cultured in a culture solution F12 containing 10% (v/v) fetal bovine serum (both fetal bovine serum and F12 are purchased from Gibico, USA) at 37 ℃ under 5% CO2Performing large-scale suspension culture in an incubator for 3d, centrifuging to collect cells, discarding supernatant, washing the resuspended cells with PBS, performing ultrasonic lysis (ultrasonic 1s, interval 1s, total 10min), centrifuging at 12000rpm, collecting supernatant, and performing SDS-PAGE analysis. When the protein is purified, a nickel ion affinity chromatographic column (purchased from Thermo company in America) is used for purification, the operation is carried out according to the specification of the nickel ion affinity chromatographic column, SDS-PAGE and western blotting are used for identification, a primary antibody used in western blotting experiments is African swine fever positive serum (which is detected as positive by an African swine fever antibody detection kit produced by Ingenasa company in Guangdong province), a secondary antibody is rabbit anti-pig IgG marked by HRP, and the result shows that the protein is a single strip and is consistent with the theoretical predicted value in size and has better antigenicity. By adopting a commercialized kit PierceTMThe BCA Protein Assay Kit (purchased from Thermo company, USA) measures the content of purified Protein, and the concentration of a standard substance (provided by the Kit) and the corresponding OD value are shown in Table 1, wherein A-H are standard substances with different dilution times, and the concentration unit of the standard substance is as follows: μ g/mL. The OD value of the purified CD2v protein was 1.917, and its concentration was 1.5mg/mL compared with the standard.
Table 1: purified protein content, standard substance concentration and corresponding OD value
HRP labeling of CD2v protein antigen
The HRP labeling of the CD2v protein antigen is carried out by adopting a commercial HRP labeling kit (purchased from Beijing Boolong immune technology Co., Ltd.), the CD2v protein is fully dialyzed by PBS (0.01M, pH7.4) before labeling to remove imidazole, Tris and other substances in an elution buffer solution during protein purification, then the concentration is adjusted to 2mg/ml, then the labeling is carried out according to the kit instruction, an equal volume of label preserving fluid (provided by the kit) is added after the labeling is finished, the labeling is fully and uniformly mixed, and the mixture is stored at the temperature of minus 20 ℃.
4. Preparation of standard ASFV positive serum and standard ASFV negative serum
In the ELISA detection process, in order to establish validity verification and negative-positive judgment standards of the kit and avoid artificial operation errors, the invention develops standard ASFV antibody positive control serum and standard ASFV antibody negative control serum for optimization of detection conditions and judgment of detection results.
4.1 preparation of Standard Positive serum
Taking 5 New Zealand white rabbits (purchased from the center of laboratory animals in Guangdong province) and weighing 2-3 kg. For the first immunization, 1mL of an emulsion of Freund's Complete Adjuvant (FCA) (purchased from Sigma, USA) mixed with recombinant CD2v protein in equal volume ratio was drawn up by syringe and 0.5mL was injected into the medial thigh muscle. After 2 weeks, carrying out second immunization, injecting 1mL of emulsion mixed by Freund incomplete adjuvant and recombinant CD2v protein in equal volume ratio at the same dose and part, respectively collecting 0.5-1.0 mL of blood from ear vein at 0d, 7d, 14d, 21d and 28d, separating serum, and detecting serum titer by ELISA method, as shown in figure 2; as can be seen from the results of fig. 2, antibodies were detected in new zealand white rabbits one week after immunization, and were significantly elevated after the second immunization, while non-immunized new zealand white rabbits remained negative all the time.
Collecting blood by heart blood collection method at 35d, separating after separating out serum, centrifuging at 3000r/min for 15min, collecting supernatant, adding thimerosal with final concentration of 0.01% for antisepsis, packaging, and storing at-20 deg.C.
4.2 preparation of Standard negative serum
A clean-grade experimental pig (purchased from southern medical university laboratory animal center) is taken as a blood-taking pig, the blood-taking precursor condition is good, the body temperature, the appetite and the stool and urine are normal, and any vaccine is not immunized and no other diseases exist. After disinfection, the anterior vena cava is used for blood collection, serum is separated, and one ten thousandth of thimerosal is added for preservation. Packaging into sterile tubes at a volume of 0.5ML per tube, and storing at-20 deg.C.
Example 2 establishment of double antigen sandwich ELISA kit for detecting African Swine fever Virus CD2v antibody
1. Detection principle of the kit of the invention
The invention adopts a double-antigen sandwich method, coats recombinant African swine fever virus CD2v protein in a micropore plate, then seals the enzyme label plate by 1% (w/v) BSA solution, and adds a sample to be detected and a standard positive control and a standard negative control. The African swine fever virus CD2v antibody in the sample or the standard can react with CD2v antigen coated in an enzyme label plate, after HRP-labeled CD2v antigen is added, the enzyme-labeled antigen is combined with the CD2v antigen-antibody complex after the previous reaction, horseradish peroxidase (HRP) substrate TMB is added for color development, stop solution is added for stopping the reaction, the absorbance value of each hole is measured by an enzyme label instrument under the wavelength of 450nm, and the value of 0D is in direct proportion to the content of the African swine fever virus CD2v antibody in the sample to be measured.
2. Compositions of the kits of the invention
2.1 optimal preparation method of enzyme-labeled plate
The purified recombinant CD2v protein prepared in example 1 was diluted with 0.05M carbonate buffer (pH 9.6) as a coating buffer and added to a microplate at 100. mu.L/well, and the content of CD2v protein in each well was 0.06. mu.g. Coating overnight at 4 deg.C, the next day, discarding the coating solution, adding blocking solution of 1% (w/v) BSA at 200. mu.L/well, standing at 4 deg.C for 12 hr, washing and spin-drying. Drying at room temperature, packaging, adding desiccant, and vacuum preserving.
2.2 working reagent preparation
Washing solution (pH7.4, 1.5M PBS): KH (Perkin Elmer)2PO40.2g，Na2HPO4-12H2O2.9 g, NaCl 8.0g, KC10.2g and Tween-20 (0.05%) 0.5mL, adding distilled water to constant volume of 100mL to obtain 10 times of washing solution as storage solution, and diluting by 10 times when in use.
Serum diluent: 1g of bovine serum albumin, and adding a washing buffer solution to 100 mL;
substrate buffer (ph5.0 citric acid phosphate): 0.2M Na2HPO425.7mL, 24.3mL of 0.1M citric acid, and 50mL of distilled water;
TMB (tetramethylbiphenyl) use solution: TMB (10mg/5mL absolute ethanol) 0.5mL, substrate buffer 10mL, 0.75% H2O，32μL；
Stop solution (2M H)2SO4): 178.3mL of distilled water, 21.7mL of concentrated sulfuric acid (98%) was added dropwise.
2.3 construction of double-antigen sandwich ELISA kit for detecting African swine fever
An enzyme linked immunosorbent assay kit for detecting the African swine fever CD2v antibody is constructed, and comprises the following components:
the kit comprises a 96-well enzyme label plate coated by CD2v recombinant protein, a CD2v antigen protein marked by horseradish peroxidase (HRP), a standard positive control, a standard negative control, a concentrated washing solution, a serum diluent, a TMB substrate, a stop solution and a product specification.
EXAMPLE 3 detection of African swine fever Virus CD2v antibody in samples
1. Detection with the kit prepared above
1) Diluting the serum to be detected, the positive control and the negative control by serum diluent according to a proportion, adding 100 mu L of the ELISA plate in the embodiment 2 into each hole, incubating for 1 hour at 37 ℃, discarding the solution, and washing for 3-5 times by using a washing solution.
2) 100 mu L (0.2 mu g/ml) of HRP-labeled CD2v antigen protein is added into each hole, incubation is carried out for 30min at 37 ℃, and the solution is discarded and washed 3-5 times by washing liquid.
3) Adding 100 mu L of TMB substrate solution into each hole, and reacting for 10-15 minutes at room temperature in a dark place.
4) Add 100. mu.L of 2M H per well2SO4And stopping the reaction, and detecting the absorbance value of 450nm by using an enzyme-labeling instrument.
2. Analysis of detection results
1) Judgment standard of negative and positive samples
Detecting 100 parts of negative serum by using the established double-antigen sandwich ELISA method, repeating 2 holes for each part of serum, performing ELISA determination according to the established ELISA program, reading an OD450 value, and calculating an OD450 average value and a standard deviation of 100 parts of serum;
according to the statistical principle, when the mean of the OD450 values is +3 × the standard deviation, the serum sample can be judged to be positive at the level of 99.9%, therefore, the negative-positive critical value is set to be equal to the mean of the OD450 values of the negative serum +3 × the standard deviation, the negative-positive critical value is 0.4, under the specified experimental conditions, the OD450 value is greater than the critical value, namely, the serum sample to be detected has OD450>0.4, and the serum sample is judged to be positive, otherwise, the serum sample is negative.
2) Specificity test
Taking positive serum of porcine reproductive and respiratory syndrome virus antibody (PRRS-Ab), positive serum of porcine circovirus type 2 antibody (PCV2-Ab), positive serum of porcine foot and mouth disease antibody (FMDV-Ab) and positive serum of porcine pseudorabies virus antibody (PRV-Ab) (detected as positive by a related antibody kit produced by American IDEXX company), detecting by using the established double-antigen sandwich ELISA kit, and displaying the results of OD (OD) of the samples of the positive serum of porcine reproductive and respiratory syndrome virus antibody (PRRS-Ab), the positive serum of porcine circovirus type 2 antibody (PCV2-Ab), the positive serum of porcine foot and mouth disease antibody (FMDV-Ab) and the positive serum of porcine pseudorabies virus antibody (PRV-Ab)450The values are all less than 0.2 (see table 2), and the results show that the double-antigen sandwich ELISA kit established by the invention has good specificity.
Table 2: OD value corresponding to 100-fold dilution of serum samples infected with different viruses
3) Results of sample testing
The CD2v protein immune New Zealand white rabbits collect blood and separate serum, the established double-antigen sandwich ELISA kit is used for detection, and the result shows that 7d antibody of 5 new Zealand white rabbits after immune changes positive (OD)450>0.4), the 21d antibody after the second immunization is obviously raised, and the highest titer is reached when the blood collection is died. The CD2v serum from immunized New Zealand white rabbits had a high OD value of above 0.4, while the OD value of the uninmmunized 3 New Zealand white rabbits was below 0.2 (see FIG. 2 for the results).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> institute of animal health of academy of agricultural sciences of Guangdong province
<120> ELISA method, kit and application for detecting African swine fever CD2v protein antibody
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<170>SIPOSequenceListing 1.0
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<213> Artificial Sequence (Artificial Sequence)
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gacgctagca tgatatactt atttttttaa ta 32
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<213> Artificial Sequence (Artificial Sequence)
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gtcgcggccg cgtggtggtg gtggtggtga ataattctat ctacgtgaat aagc 54
Claims (10)
1. An ELISA method for detecting an African swine fever CD2v protein antibody is characterized by comprising the following steps:
(1) coating the recombinant African swine fever virus CD2v protein in a microporous plate, then sealing, adding a sample to be tested, a standard positive control and a standard negative control into different micropores for reaction, and washing the plate;
(2) adding HRP marked CD2v antigen for reaction, and washing the plate; adding a color developing solution for color developing reaction, and adding a stopping solution to stop the reaction;
(3) measuring the absorbance value of each hole at the wavelength of 450 nm; and according to the measurement result, carrying out qualitative or quantitative judgment on the sample to be measured.
2. The ELISA method for detecting antibody against African swine fever CD2v protein according to claim 1, wherein the ELISA method comprises the following steps:
the coating amount of the African swine fever virus CD2v protein in the step (1) is 0.06 mu g/hole;
the amount of HRP-labeled CD2v antigen added in step (2) was 0.02. mu.g/well.
3. The ELISA method for detecting antibody against African swine fever CD2v protein according to claim 1, wherein the ELISA method comprises the following steps: the qualitative judgment criteria are as follows: and determining the serum sample to be detected to be positive if the OD450 is more than 0.4, and determining the serum sample to be detected to be negative if the OD450 is not more than 0.4.
4. A double-antigen sandwich ELISA kit for detecting an African swine fever virus CD2v antibody is characterized in that: comprises an ELISA plate coated by CD2v protein and a CD2v antigen marked by horseradish peroxidase.
5. The dual antigen sandwich ELISA kit of claim 4, wherein: also comprises at least one of substrate color development liquid, stop solution, positive control, negative control, serum diluent and 10-fold concentrated washing liquid.
6. The dual antigen sandwich ELISA kit according to claim 4 or 5, characterized in that: the CD2v protein-coated ELISA plate is prepared by the following steps: adding 0.6. mu.g/L CD2v protein solution into blank enzyme label plate at 100. mu.L/well by using 0.05M carbonate buffer solution with pH9.6 as coating buffer solution, and coating overnight at 4 ℃; then, the coating solution was discarded, and 5% (w/v) BSA solution was added at 200. mu.L/well, allowed to stand at 4 ℃ for 12 hours, washed, dried at room temperature, packed in a bag, added with a desiccant, and stored under vacuum.
7. The double-antigen sandwich ELISA kit according to claim 5, wherein the substrate color-developing solution comprises: 0.5mL of tetramethylbiphenyl ethanol solution with the concentration of 2mg/mL, 10mL of substrate buffer solution and 0.75% H2O2，32μL；
Wherein the substrate is bufferedThe composition of the liquid was as follows: na in a concentration of 0.2mol/L2HPO425.7mL of the solution, 24.3mL of 0.1M citric acid, and 50mL of distilled water.
8. The dual antigen sandwich ELISA kit of claim 5, wherein:
the positive control is at least one of a CD2v antibody and a sample containing a CD2v antibody;
the negative control was a sample without CD2v antibody.
9. The use of the double-antigen sandwich ELISA kit of any one of claims 1-8 for detecting African swine fever virus CD2v protein antibody.
10. Use according to claim 9, characterized in that: the double-antigen sandwich ELISA kit is used for identifying whether the pigs in a CD2v deletion strain immune swinery have African swine fever virus infection or for detecting the antibody of the African swine fever virus of the non-immune swinery.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518174A (en) * | 2020-05-12 | 2020-08-11 | 浙江鼎持生物制品有限公司 | Optimized African swine fever CD2v protein and high-efficiency expression method and application thereof |
| CN111537741A (en) * | 2020-05-24 | 2020-08-14 | 广州敏捷生物技术有限公司 | Double-antigen sandwich immunofluorescence chromatography kit for detecting African swine fever virus CD2v protein antibody |
| CN111793128A (en) * | 2020-07-30 | 2020-10-20 | 北京世纪元亨动物防疫技术有限公司 | Hybridoma cell line and monoclonal antibody for resisting African swine fever virus CD2v protein |
| CN111961120A (en) * | 2020-09-01 | 2020-11-20 | 中牧实业股份有限公司 | African swine fever virus MGFs and CD2v ELISA antibody detection kit |
| CN112877348A (en) * | 2021-01-08 | 2021-06-01 | 中国农业大学 | African swine fever virus CD2v extracellular domain recombinant protein and application thereof |
| CN113563461A (en) * | 2021-06-29 | 2021-10-29 | 中国检验检疫科学研究院 | Competitive monoclonal antibody based on African swine fever virus CD2v protein, kit and application thereof |
| CN114152748A (en) * | 2021-10-29 | 2022-03-08 | 浙江理工大学绍兴生物医药研究院有限公司 | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof |
| CN114773483A (en) * | 2022-01-19 | 2022-07-22 | 湖南农业大学 | African swine fever virus antigen recombinant tandem epitope protein, construction method and application thereof, and antibody ELISA kit |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104360062A (en) * | 2014-11-28 | 2015-02-18 | 山东创新药物研发有限公司 | Application of peptidylarginine deiminase 1 (PAD1) to preparation of reagent for clinical diagnosis of tumors |
| CN106896224A (en) * | 2017-03-15 | 2017-06-27 | 广州万联生物科技有限公司 | A kind of swine fever novel antibodies ELISA detection kit |
| CN110078801A (en) * | 2019-05-22 | 2019-08-02 | 青岛易邦生物工程有限公司 | A kind of Chinese hamster ovary celI strain of high efficient expression African swine fever CD2V albumen |
| CN110551853A (en) * | 2019-09-19 | 2019-12-10 | 华南农业大学 | Triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strain and gene deletion strain |
-
2019
- 2019-12-16 CN CN201911293735.7A patent/CN110927390A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104360062A (en) * | 2014-11-28 | 2015-02-18 | 山东创新药物研发有限公司 | Application of peptidylarginine deiminase 1 (PAD1) to preparation of reagent for clinical diagnosis of tumors |
| CN106896224A (en) * | 2017-03-15 | 2017-06-27 | 广州万联生物科技有限公司 | A kind of swine fever novel antibodies ELISA detection kit |
| CN110078801A (en) * | 2019-05-22 | 2019-08-02 | 青岛易邦生物工程有限公司 | A kind of Chinese hamster ovary celI strain of high efficient expression African swine fever CD2V albumen |
| CN110551853A (en) * | 2019-09-19 | 2019-12-10 | 华南农业大学 | Triple PCR detection primer and kit for rapidly distinguishing African swine fever virus wild strain and gene deletion strain |
Non-Patent Citations (2)
| Title |
|---|
| 张艳艳 等: "非洲猪瘟病毒基因缺失疫苗株的构建和免疫保护特性", 《中国兽医学报》 * |
| 郑辉 等: "PRV SD株gE基因主要抗原区域原核表达及间接ELISA方法的建立", 《中国动物检疫》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518174A (en) * | 2020-05-12 | 2020-08-11 | 浙江鼎持生物制品有限公司 | Optimized African swine fever CD2v protein and high-efficiency expression method and application thereof |
| CN111518174B (en) * | 2020-05-12 | 2022-08-26 | 浙江鼎持生物制品有限公司 | Optimized African swine fever CD2v protein and high-efficiency expression method and application thereof |
| CN111537741A (en) * | 2020-05-24 | 2020-08-14 | 广州敏捷生物技术有限公司 | Double-antigen sandwich immunofluorescence chromatography kit for detecting African swine fever virus CD2v protein antibody |
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| CN111793128B (en) * | 2020-07-30 | 2022-04-08 | 北京世纪元亨动物防疫技术有限公司 | Monoclonal antibody of anti-African swine fever virus CD2v protein and application thereof |
| CN111961120A (en) * | 2020-09-01 | 2020-11-20 | 中牧实业股份有限公司 | African swine fever virus MGFs and CD2v ELISA antibody detection kit |
| CN112877348A (en) * | 2021-01-08 | 2021-06-01 | 中国农业大学 | African swine fever virus CD2v extracellular domain recombinant protein and application thereof |
| CN113563461A (en) * | 2021-06-29 | 2021-10-29 | 中国检验检疫科学研究院 | Competitive monoclonal antibody based on African swine fever virus CD2v protein, kit and application thereof |
| CN114152748A (en) * | 2021-10-29 | 2022-03-08 | 浙江理工大学绍兴生物医药研究院有限公司 | Double-antibody sandwich ELISA diagnostic kit for detecting African swine fever virus and method thereof |
| CN114773483A (en) * | 2022-01-19 | 2022-07-22 | 湖南农业大学 | African swine fever virus antigen recombinant tandem epitope protein, construction method and application thereof, and antibody ELISA kit |
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