CN116068192A - ELISA detection kit for detecting African swine fever virus antibody and application of protein L - Google Patents
ELISA detection kit for detecting African swine fever virus antibody and application of protein L Download PDFInfo
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Abstract
The invention relates to the technical field of immunodetection, and discloses an ELISA detection kit for detecting African swine fever virus antibodies and application of proteol. The ELISA detection kit for detecting the African swine fever virus antibody is prepared by combining the protein L and the African swine fever virus capsid protein P72 trimer, can rapidly and specifically detect the African swine fever virus antibody in serum, has extremely high reaction sensitivity, is simple and convenient to operate, has low cost and stable reaction result, and is suitable for monitoring and checking African swine fever of a pig farm in large-scale cultivation, and the kit is easy to popularize and apply in a large scale.
Description
The application is a divisional application of an ELISA detection kit for detecting African swine fever virus antibody, wherein the application is 24 days of 2022 and 01, the application number is 202210078482.7, and the application of protein L.
Technical Field
The application relates to the technical field of immunodetection, in particular to an ELISA detection kit for detecting African swine fever virus antibodies and application of proteol.
Background
African swine fever (African swine fever, ASF) is an acute and virulent infectious disease of pigs caused by infection with African swine fever virus (African swine fevervirus, ASFV). The disease can cause symptoms of high fever, reticuloendothelial system hemorrhage, visible mucous membrane redness and the like of pigs clinically, and the disease course of the virulent strain infected pigs is short, and the death rate is up to 100%.
African swine fever virus is a double-stranded DNA virus with a complex multilayer structure. The 5-layer structure of the membrane comprises a similar core, a core shell, an inner membrane, a capsid and an outer capsule membrane from inside to outside. African swine fever virus may encode more than 200 proteins, wherein the major component P72 protein of the capsid is about 33% of the total mass of African swine fever virus. Previously, indirect ELISA antibody detection kits developed based on P30 and P54 proteins have been reported. Among them, because of the difficulty in preparing the full-length P72 trimeric protein, the indirect ELISA kit based on the P72 protein on the market uses the P72 truncate, and no report of preparing the detection kit by using the full-length P72 trimeric protein is available. Previously, we have successfully expressed P72 trimeric proteins in large quantities in the Saccharomyces cerevisiae expression system and in exactly the same structure as the native structure (patent number: 202110522777.4).
OIE recommends diagnostic techniques for African swine fever virus including etiology detection and serology testing. Among them, ELISA is an international trade test method prescribed by OIE. The indirect ELISA method based on the P72 full-length trimeric protein has the advantages of high specificity and strong sensitivity, and simultaneously, the characteristics of ELISA multi-sample and high-throughput detection are also suitable for being implemented in a large-scale farm.
Disclosure of Invention
Therefore, the invention aims to provide an ELISA kit for detecting African swine fever virus antibodies, which has higher specificity, sensitivity and accuracy when used for detecting the African swine fever virus antibodies;
it is another object of the present invention to provide the use of protein L in the preparation of ELISA kits for detecting African swine fever virus antibodies, such as for preparing enzyme conjugates.
In order to solve the technical problems or at least partially solve the technical problems/achieve the purposes, the invention provides an ELISA detection kit for detecting African swine fever virus antibodies, which comprises an ELISA plate coated with African swine fever virus capsid protein P72 trimer and a protein L enzyme conjugate.
According to the invention, on the basis of the existing achievement of the early-stage P72 full-length trimer protein, the development of an African swine fever virus antibody ELISA detection product is carried out, and the research of the invention discovers that the P72 trimer of the African swine fever virus capsid protein is taken as a coating antigen and combined with a protein L enzyme conjugate, positive serum can be detected in 64000 times by indirect ELISA detection, and positive serum can be detected only in 16000 times, 8000 times and 8000 times by conventional sensitivity taking P72 unordered folding monomer protein, P54 protein and P30 protein as the coating antigen;
furthermore, detection using the protein L/A/G enzyme conjugate and the goat anti-porcine IgG enzyme conjugate provided that the same P72 trimer coated antigen was used, and the results showed that 64000 fold diluted positive serum could be detected using the protein L enzyme conjugate, and that only 32000 fold diluted positive serum could be detected using the other three enzyme conjugates. In view of the above excellent technical effects, the invention provides application of protein L in preparing an ELISA detection kit for detecting African swine fever virus antibodies, preferably application of combination of protein L and African swine fever virus capsid protein P72 trimer in preparing an ELISA detection kit for detecting African swine fever virus antibodies. In a specific embodiment of the invention, the protein L is used to prepare an enzyme conjugate in a kit.
The African swine fever virus capsid protein P72 trimer in the kit of the invention realizes the high copy expression of the P72 trimer protein BY inserting an African swine fever virus specific P72 protein gene into a saccharomyces cerevisiae BY4743 genome BY utilizing a CRISPR-Cas9 technology, and the relevant contents are all described in CN202110522777.4, and the contents can be also described as the invention. The recombinant P72 protein which can be assembled correctly to form a trimer configuration without relying on auxiliary protein B602L is prepared according to the preparation method of CN 202110522777.4. The sequence of the African swine fever virus capsid protein P72 is shown as SEQ ID NO. 1 or a protein tag sequence is added on the protein sequence shown as SEQ ID NO. 1. In the specific embodiment of the invention, the protein tag is Twin-Strep-tag, and is fused at the N end or the C end of the P72 protein.
The protein L enzyme conjugate in the kit is enzyme-labeled protein L; preferably, the enzyme is horseradish peroxidase, and the working concentration of the protein L enzyme conjugate is 1:5000-10000, and in a specific embodiment of the invention, the working concentration is 1:8000.
Meanwhile, the kit also comprises any one or more than two of the following components:
enzyme conjugate dilutions, washes, substrates, sample dilutions, stop solution, negative and positive controls. Wherein the substrate is selected according to the enzyme selected, and in the specific embodiment of the invention, the enzyme is horseradish peroxidase, and the substrate is selected as substrate A solution 3,3', 5' -tetramethyl benzidine solution and substrate B solution peroxide solution.
Preferably, the enzyme conjugate diluent is PBST containing BSA; in a specific embodiment of the invention, the enzyme conjugate diluent is PBST containing 1% bsa.
Preferably, the sample diluent is PBST containing Fetal Bovine Serum (FBS); in a specific embodiment of the invention, the sample diluent is PBST containing 1% fetal bovine serum.
Preferably, the positive control is normal pig serum immunized with the African swine fever virus capsid protein P72 trimer, and the negative control is normal pig serum without the African swine fever virus capsid protein P72 trimer.
The kit is adopted to detect the verified African swine fever virus antibody positive serum, porcine circovirus antibody positive serum, porcine blue ear virus antibody positive serum, porcine pseudorabies virus antibody positive serum, swine fever virus antibody positive serum and African swine fever virus antibody negative serum respectively, and the results show that the antigen has good specificity when being used for detecting the African swine fever virus antibody.
In addition, 92 clinical serum samples to be tested are respectively detected by taking the same batch of indirect ELISA kit, and the detection results are observed and compared with the detection data of the international universal kit ID VET indirect ELISA kit. The detection data of the ID VET indirect ELISA kit show that: of 92 serum samples, 57 were positive and 35 were negative; the invention has the advantages of positive coincidence of 60 parts, negative coincidence of 32 parts, positive coincidence rate of 100 percent (57/57), negative coincidence rate of 91.4 percent (32/35) and total coincidence rate of 96.7 percent (89/92). The antigen is proved to have good accuracy when being used for detecting the African swine fever virus antibody.
According to the technical scheme, the protein L enzyme conjugate and the African swine fever virus capsid protein P72 trimer coating antigen are used for preparing the ELISA detection kit for detecting the African swine fever virus antibody in a combined way, the kit can rapidly and specifically detect the African swine fever virus antibody in serum, has extremely high reaction sensitivity, is simple and convenient to operate, has low cost and stable reaction result, and is suitable for monitoring and checking African swine fever of a pig farm in large-scale cultivation, and is easy to popularize and apply in a large scale.
Detailed Description
The invention discloses an ELISA detection kit for detecting African swine fever virus antibodies and application of protein L, and a person skilled in the art can refer to the content of the ELISA detection kit and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the products, processes and applications of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the products, processes and applications described herein without departing from the spirit and scope of the invention. It will be apparent that the described embodiments are some, but not all, of the embodiments of the present application. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present application based on the embodiments herein.
It should be noted that, in this document, relational terms such as "first" and "second," "step 1" and "step 2," "S1" and "S2," and "(1)" and "(2)" and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a process, method, article, or apparatus that comprises the element.
The invention specifically provides an indirect ELISA kit for detecting African swine fever virus antibodies, which comprises the following components: coating plate, enzyme conjugate diluent, 25x concentrated wash, substrate a, substrate B, sample diluent, stop solution, negative control, positive control, serum dilution plate, cover plate membrane, and instructions;
wherein, the coating plate is coated with African swine fever virus capsid protein P72 trimer with a sequence shown in SEQ ID NO. 1, and is used for determining the specific binding of the recombinant protein with African swine fever virus antibody positive serum;
the enzyme conjugate is protein L marked by horseradish peroxidase, and the concentration is 1: 5000-10000;
the 25X concentrated wash solution was 25X PBST, and the enzyme conjugate diluent was PBST containing 1% bsa; the sample diluent is PBST containing 1% of fetal bovine serum, and the stop solution is 2M H 2 SO 4 The substrate A solution is 3,3', 5' -tetramethyl benzidine solution, and the substrate B solution is peroxide solution.
Meanwhile, the invention also provides a using method of the kit:
(1) Diluting serum to be detected by using a sample diluent, adding the diluted serum into a coating plate, and adding 100 mu L of the diluted serum into each hole; meanwhile, 100 mu L of each of the negative control and the positive control is added, the coating plate is sealed by a cover plate film, and the coating plate is incubated for 30min at 37 ℃.
(2) The 25 Xconcentrated wash was diluted to 1 Xwash and the coated plate was washed 3 times with shaking, each beat dry.
(3) The enzyme conjugate was diluted 1:100 with enzyme conjugate dilution, added to the coated plate at 100. Mu.L/well, the coated plate was sealed with cover plate membrane and incubated at 37℃for 30min.
(4) Oscillating and washing the coating plate 3 times by using 1X washing liquid, and beating to dry each time;
(5) 1:1 mixing substrate A solution and substrate B solution, adding into a coated plate at a rate of 100 mu L/hole, sealing the coated plate with a cover plate film, and incubating at 37 ℃ for 10min. Stop solution was added to the coating plate at 50. Mu.L/well and OD was read on an ELISA reader 450nm Is a light absorption value of (2);
(6) And (3) result judgment: when positive control OD 450nm Not less than 1.0, and negative control OD 450nm Results were valid < 0.2; (sample OD) 450nm Value-negative control OD 450nm Value)/(positive control OD 450nm Value-negative control OD 450nm The value) is more than or equal to 0.2, and the result is judged to be positive; (sample OD) 450nm Value-negative control OD 450nm Value)/(positive control OD 450nm Value-negative control OD 450nm When the value is less than 0.1, the sample is negative, and the OD is less than or equal to 0.1 (sample OD) 450nm Value-negative control OD 450nm Value)/(positive control OD 450nm Value-negative control OD 450nm And if the value) is less than 0.2, judging as suspected.
In each of the comparative experiments provided by the present invention, unless otherwise specified, other experimental conditions, materials, etc. were kept consistent to allow for comparability, except for the differences noted in each group.
The ELISA detection kit for detecting African swine fever virus antibodies provided by the invention is further described below.
Example 1: preparation of African swine fever virus P72 trimer protein
1. Preparation of recombinant expression strain of African swine fever virus P72 trimer
The invention designs a P72 protein sequence based on a P72 protein three-dimensional conformation by bioinformatics and structural biology methods, and synthesizes a P72 (strep-tag) gene sequence of African swine fever virus by using a chemical synthesis method. The P72 gene was constructed with the yeast GAL1 promoter and ADH1 terminator on a plasmid to form a P72 protein gene expression cassette. The P72 protein gene expression cassette with the homologous recombination arm is amplified by PCR as a repair template. The CRISPR-Cas9 technology is utilized to coexpress the gRNA of recognition GGATTTAGGAATCCATAAAA (SEQ ID NO: 2), and a P72 protein gene expression cassette is inserted into a Saccharomyces cerevisiae multicopy Ty2 retrotransposon in a homologous recombination mode to realize gene multicopy expression. Cloning and screening the repair template and pCas-ty2 plasmid co-transformed Saccharomyces cerevisiae BY4743 BY using a plate lacking URA. Screening out monoclonal, identifying copy number by qPCR method, and selecting high copy strain for expression detection.
2. Expression of recombinant African swine fever virus P72 trimer protein
High-copy positive monoclonal saccharomyces cerevisiae colonies were selected and inoculated into YPD liquid medium and cultured overnight at 30 ℃. And (3) taking the bacterial liquid cultured overnight, and performing 100-time expansion culture on the bacterial liquid in YPD+2% glucose liquid culture medium, and culturing at 30 ℃ for 60 hours until the OD600 reaches 3.0, thus obtaining the required protein.
3. Purification of recombinant proteins
1L of the Saccharomyces cerevisiae culture was centrifuged at 6000rpm for 10min, and the precipitate was collected. The pellet was resuspended in 50ml buffer W (IBA), and then the yeast cells were disrupted by high pressure homogenizer, centrifuged at 17000rpm for 60 minutes at 4℃and the supernatant was filtered through a 0.45 μm filter and purified. Fusion protein affinity purification is carried out by using Strep-Tactin XT gravity-flow column (IBA), and the main operation steps are as follows:
a. the column was equilibrated with 10mL bufferW (IBA), and the supernatant after filtration was applied to the column. Then slowly flows out by gravity flow, and the operation is repeated twice.
b. The protein was washed with 20mL bufferW (IBA) to remove the contaminating proteins.
c. The desired target protein was eluted by elution bufferBXT (IBA), and the eluate was collected at 1 mL/tube.
d. The protein of interest was collected and concentrated using a Merck-Millipore 30KD concentration tube.
e. Molecular sieves (Superdex S-200, GE) were equilibrated with FPLC at 4℃at a flow rate of 1ml/min. The proteins were batch loaded onto the loading loop and the P72 protein was further purified using molecular sieves.
f. The collected samples were analyzed by SDS-PAGE, and a single band was present in the purified SDS-PAGE result, and the size was about 73kD. The protein of interest was concentrated using a Merck-Millipore 30KD concentration tube and then assayed for protein concentration by the BCA method.
Example 2: establishment of the indirect ELISA kit for detecting African swine fever virus antibody
1. Determination of optimal coating concentration
Coating the purified recombinant P72 trimer protein with 1 mug/mL, 2.5 mug/mL, 5 mug/mL and 10 mug/mL respectively, coating a 96-well ELISA plate at the temperature of 100 mug/well and 4 ℃ overnight, washing 3 times by PBST the next day, then using 1% BSA as a sealing liquid, sealing for 2 hours at the temperature of 200 mug/well and 37 ℃, using an ASFV antibody positive serum sample as a primary antibody, using an ASFV antibody negative serum sample as a negative control, incubating for 30 minutes at the temperature of 37 ℃, washing the plate by PBST for 3 times and beating to dry; adding enzyme conjugate diluted 1:8000, incubating at 37 ℃ for 30min, washing the plate 3 times by PBST, and beating to dryness; 100 mu L of TMB substrate solution is added into each hole, the color development is carried out for 10min at 37 ℃, 50 mu L of stop solution is used for stopping the reaction, and an enzyme-labeling instrument is used for reading OD of each hole 450nm The P/N value is calculated and the optimal coating concentration is determined.
2. Determination of optimal sealing liquid
Purified recombinant P72 trimeric protein was added at 5. Mu.g/mL, 100. Mu.LThe 96-well ELISA plate is coated overnight at 4 ℃ in the well, PBST is used for washing 3 times in the next day, then 1% BSA, 1% OVA, 1% gelatin and 5% skimmed milk powder are respectively used as blocking liquid, 200 mu L/well is blocked for 2 hours at 37 ℃, ASFV antibody positive serum sample is used as primary antibody, ASFV antibody negative serum sample is used as negative control, incubation is carried out at 37 ℃ for 30min, and PBST is used for washing 3 times and drying is carried out; adding enzyme conjugate diluted 1:8000, incubating at 37 ℃ for 30min, washing the plate 3 times by PBST, and beating to dryness; 100 mu L of TMB substrate solution is added into each hole, the color development is carried out for 10min at 37 ℃, 50 mu L of stop solution is used for stopping the reaction, and an enzyme-labeling instrument is used for reading OD of each hole 450nm And calculating the P/N value, and determining the optimal sealing liquid.
3. Determination of optimal serum dilutions
The test is carried out according to the determined optimal indirect ELISA parameters, the sample serum is diluted by 1:100 respectively by PBST containing 1% BSA, PBST containing 1% fetal bovine serum, PBST and PBST containing 1% rabbit serum, 100 mu L/well is added into a 96-well ELISA plate for reaction, other reference 1 indirect ELISA steps are carried out, and the ELISA instrument reads the OD of each well after color development 450nm The P/N value was calculated and the optimal serum dilution was determined.
4. Determination of optimal enzyme conjugate dilutions
The enzyme conjugate was diluted to a titer of 1:8000 with 1% BSA in PBST, 1% fetal bovine serum in PBST, and 1% rabbit serum in PBST, and the wells OD was read by a microplate reader after development according to the optimal indirect ELISA parameters determined above, other reference 1 indirect ELISA procedure 450nm The P/N value was calculated and the optimal enzyme conjugate dilution was determined.
5. Determination of optimal enzyme conjugate working titers
Experiments were performed according to the optimal indirect ELISA parameters determined above, diluting the enzyme conjugate to titers of 1:8000, 1:10000 and 1:12000, setting 2 replicates, other reference 1 indirect ELISA steps, and reading the OD of each well with an ELISA reader after color development 450nm P/N values were calculated to determine the optimal enzyme conjugate working titers.
6. Determination of the critical value
5 parts of serum which is detected as being negative to ASFV by the commercial ELISA kit are tested according to an indirect ELISA procedure under the optimal reaction conditionTesting, calculating sample OD 450nm Average value of (2)
And Standard Deviation (SD).
The best parameters for the indirect ELISA reaction are summarized in Table 1.
TABLE 1 parameters related to indirect ELISA reactions
Example 3: indirect ELISA kit for detecting African swine fever virus antibody based on African swine fever virus P72 trimer
1. Determination of preparation process of indirect ELISA detection kit
An indirect ELISA kit for detecting African swine fever virus antibodies was prepared by the following steps:
(1) Coating: the ELISA plate is coated with recombinant African swine fever virus P72 trimer protein at a concentration of 5ug/ml, 100 uL/well, coated overnight at 4deg.C, washed 3 times with PBST the next day, and patted dry.
(2) Closing: blocking with 1% BSA, 200. Mu.L/well, incubation for 2h at 37℃and washing 3 times with PBST followed by spin-drying. Drying at 37 ℃ for 2 hours, and then vacuum sealing for standby.
2. Determination of the operating procedure of an indirect ELISA detection method
(1) Diluting serum to be detected by using serum diluent, adding the diluted serum into a coating plate, and adding 100 mu L of the diluted serum into each hole; meanwhile, 100 mu L of each of the negative control and the positive control is added, the coating plate is sealed by a cover plate film, and the coating plate is incubated for 30min at 37 ℃.
(2) The 25 Xconcentrated wash was diluted to 1 Xwash and the coated plate was washed 3 times with shaking, each beat dry.
(3) Protein L enzyme conjugate was diluted 1:100 with enzyme conjugate dilution, added to the coated plate at 100. Mu.L/well, the coated plate was sealed with cover plate membrane and incubated at 37℃for 30min.
(4) Oscillating and washing the coating plate 3 times by using 1X washing liquid, and beating to dry each time;
(5)1：1 mixing substrate A solution and substrate B solution, adding into a coated plate at a rate of 100 mu L/hole, sealing the coated plate with a cover plate film, and incubating at 37 ℃ for 10min. Stop solution was added to the coating plate at 50. Mu.L/well and OD was read on an ELISA reader 450nm Is a light absorption value of (2);
and (3) result judgment: when positive control OD 450nm Not less than 1.0, and negative control OD 450nm Results were valid < 0.2; s/p= (sample OD 450nm Value-negative control OD 450nm Value)/(positive control OD 450nm Value-negative control OD 450nm Value), when S/P is more than or equal to 0.2, judging positive; S/P is less than 0.1, and is judged as negative, and S/P is less than or equal to 0.1 and less than or equal to 0.2, and is judged as suspected.
Example 4: sensitivity detection
1. Contrast of different coating antigens
Known african swine fever virus antibody positive sera were made 1:100, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000 and 1:128000 are diluted, and the indirect ELISA kit based on the P72 trimeric protein and the indirect ELISA kit based on the P30, P54 and P72 unordered folding monomeric proteins prepared by the invention are respectively used for detection, and the results show that the sensitivity of the indirect ELISA kit based on the African swine fever virus antibody prepared by the P72 trimeric protein is highest and can reach 1:64000 times, and the results are shown in Table 2.
TABLE 2 sensitivity of different kits to African swine fever virus antibody positive serum at different dilution factors
| P72 trimer (S/P) | P72 unordered folding monomer protein (S/P) | P54(S/P) | P30(S/P) | |
| 1:100 | 1.33 | 0.932 | 0.905 | 0.939 |
| 1:1000 | 0.973 | 0.71 | 0.676 | 0.704 |
| 1:2000 | 0.922 | 0.642 | 0.598 | 0.622 |
| 1:4000 | 0.826 | 0.428 | 0.380 | 0.404 |
| 1:8000 | 0.744 | 0.302 | 0.223 | 0.257 |
| 1:16000 | 0.605 | 0.21 | 0.107 | 0.145 |
| 1:32000 | 0.403 | 0.083 | 0.036 | 0.073 |
| 1:64000 | 0.2 | 0.043 | 0.010 | 0.016 |
| 1:12800 | 0.131 | 0.007 | 0.003 | 0.004 |
| NegativeSerum | 0.005 | 0.002 | 0.000 | 0.002 |
2. Comparison of different enzyme conjugates
Staphylococcus aureus protein a (Staphylococcalprotein A, SPA), streptococcal protein G (Streptococcal protein G, SPG) and streptococcus pyogenes protein L (Peptostreptococcus proteinL, PPL) are typical immunoglobulin binding proteins that bind immunoglobulins in most mammals, but in different ways and binding profiles. The indirect ELISA kit based on the P72 trimer Protein prepared by the invention is compared with different enzyme-labeled secondary antibodies (goat anti-pig IgG-HRP, protein A-HRP and Protein G-HRP), and the result shows that the sensitivity based on the Protein L-HRP is highest and can reach 1:64000 times, and the result is shown in Table 3.
TABLE 3 sensitivity of kit containing different second enzyme-labeled antibodies to African swine fever virus antibody positive serum at different dilution factors
3. Post-challenge detection
Seven week old SPF long white pigs were offered by the Harbin veterinary institute of agricultural sciences, china, laboratory animal center, and were confirmed to be free of PCV, CSFV, PRRSV and PrV infection. Intramuscular inoculation of experimental pigs with low dose African swine fever virus HLJ/HRB1/20 strain with an inoculation dose of 10 3 TCID 50 Head. Blood is collected through the anterior vena cava on the 0 th day, the 3 rd day, the 5 th day, the 7 th day, the 9 th day, the 11 th day and the 13 th day after immunization, serum is separated, the indirect ELISA kit based on the P72 trimer protein prepared by the invention is used for detecting antibodies, the positive antibodies can be detected on the 9 th day, and the time for detecting the antibodies by the other different coating antigens and enzyme conjugate controls is longer than 9 days.
Example 5: specificity test
And detecting the verified African swine fever virus antibody positive serum, porcine circovirus antibody positive serum, porcine blue ear virus antibody positive serum, porcine pseudorabies virus antibody positive serum, swine fever virus antibody positive serum and African swine fever virus antibody negative serum by using an established indirect ELISA detection method, and carrying out statistical analysis on the obtained test result data. Unless the antibody positive serum of the African swine fever virus is negative, the result of the test is negative, which indicates that the ASFV indirect ELISA method using the recombinant P72 trimer protein as the diagnosis antigen has better specificity, and the result is shown in Table 4.
TABLE 4 specificity test results
Example 6: repeatability test
Taking ELISA detection plates coated with recombinant antigens in different batches, respectively detecting 5 ASFV positive serum and 5 ASFV negative serum, and carrying out statistical analysis on the results by making 5 repeated holes in parallel on each serum sample, wherein the results show that the detection results of the in-batch repeatability test and the in-batch repeatability test are consistent with the virus neutralization test results, and the indirect ELISA detection method has good repeatability and accuracy.
Example 7: compliance test with commercial ELISA kit
92 clinical serum samples to be tested are respectively detected by taking the same batch of indirect ELISA kit, and the result is observed and compared with the detection data of the international universal kit ID VET indirect ELISA kit. The detection data of the ID VET indirect ELISA kit show that 92 serum samples are 57 positive and 35 negative; the African swine fever virus antibody physical examination test paper card prepared by the invention has positive coincidence of 60 parts, negative coincidence of 32 parts, positive coincidence rate of 100 percent (57/57), negative coincidence rate of 91.4 percent (32/35) and total coincidence rate of 96.7 percent (89/92). The antigen is proved to have good accuracy when being used for detecting the African swine fever virus antibody.
The foregoing is only a specific embodiment of the invention to enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
- The application of protein L and African swine fever virus capsid protein P72 trimer in preparing a kit for detecting African swine fever virus antibodies is characterized in that the sequence of the African swine fever virus capsid protein P72 trimer is shown as SEQ ID NO. 1 or a tag sequence is added on the basis of the sequence.
- 2. The use according to claim 1, wherein the protein L is used for preparing an enzyme conjugate in a kit.
- 3. The use according to claim 2, wherein the protein L enzyme conjugate is a protein L horseradish peroxidase conjugate.
- 4. The use according to any one of claims 1 to 3, wherein the kit further comprises any one or more of an enzyme conjugate diluent, a wash solution, a substrate, a sample diluent, a stop solution, a negative control and a positive control.
- 5. The use according to claim 4, wherein the enzyme conjugate diluent is PBST containing BSA.
- 6. The use according to claim 4, wherein the sample diluent is PBST containing fetal bovine serum.
- 7. The use according to claim 4, wherein the positive control is normal porcine serum immunized with the african swine fever virus capsid protein P72 trimer.
- 8. The use according to claim 4, wherein the negative control is normal porcine serum of the unimmunized african swine fever virus capsid protein P72 trimer.
- 9. An ELISA detection kit for detecting African swine fever virus antibodies is characterized by comprising an ELISA plate coated with African swine fever virus capsid protein P72 trimer and a protein L enzyme conjugate; the sequence of the African swine fever virus capsid protein P72 trimer is shown in SEQ ID NO. 1 or a tag sequence is added on the basis of the sequence.
- 10. The kit of claim 9, wherein the protein L enzyme conjugate is a protein L horseradish peroxidase conjugate.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6162903A (en) * | 1992-05-07 | 2000-12-19 | Actinova Limited | Immunoglobulin binding proteins derived from L protein and their uses |
| WO2007007792A1 (en) * | 2005-07-12 | 2007-01-18 | Eisai R & D Management Co., Ltd. | Detection method and detection kit for antibody |
| CN104870015A (en) * | 2012-11-05 | 2015-08-26 | 德勒尼克斯治疗股份公司 | Binding members to IL-1 beta |
| CN105242043A (en) * | 2015-10-19 | 2016-01-13 | 东北农业大学 | Multi-species universal ELISA kit for differential diagnosis of foot and mouth disease virus infection |
| CN112979765A (en) * | 2021-05-13 | 2021-06-18 | 中国农业大学 | African swine fever virus capsid protein P72, and preparation method and application thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2019524639A (en) * | 2016-05-04 | 2019-09-05 | ザ チルドレンズ メディカル センター コーポレーション | Methods for purifying avidin-like proteins and their fusion proteins |
| CN111315407B (en) * | 2018-09-11 | 2023-05-02 | 上海市公共卫生临床中心 | Broad-spectrum anti-influenza vaccine immunogen and application thereof |
| CN113721035B (en) * | 2021-11-03 | 2022-02-11 | 中国农业大学 | Colloidal gold immunochromatographic test paper card for detecting African swine fever virus antibody |
-
2022
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6162903A (en) * | 1992-05-07 | 2000-12-19 | Actinova Limited | Immunoglobulin binding proteins derived from L protein and their uses |
| WO2007007792A1 (en) * | 2005-07-12 | 2007-01-18 | Eisai R & D Management Co., Ltd. | Detection method and detection kit for antibody |
| CN104870015A (en) * | 2012-11-05 | 2015-08-26 | 德勒尼克斯治疗股份公司 | Binding members to IL-1 beta |
| CN105242043A (en) * | 2015-10-19 | 2016-01-13 | 东北农业大学 | Multi-species universal ELISA kit for differential diagnosis of foot and mouth disease virus infection |
| CN112979765A (en) * | 2021-05-13 | 2021-06-18 | 中国农业大学 | African swine fever virus capsid protein P72, and preparation method and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| ANGEL ALBERTO JUSTIZ 等: "Bacterial proteins and their proposed interactions with Fc OR Fab fragments of immunoglobulins", BIOCHEM PHYSIOL, vol. 3, no. 4, pages 2 * |
| ANGEL JUSTIZ-VAILLANT 等: "Use of immunoglobulin-binding bacterial proteins in immunodetection", BIORXIV, pages 8 - 9 * |
| M. DE CHATEAU等: "On the interaction between protein L and immunoglobulins of various mammalian species", SCAND J IMMUNOL, vol. 37, pages 5 * |
| SIGMA-ALDRICH: "protein L-peroxidase from peptostreptococcus magnus, recombinant expressed in Escherichia coli", Retrieved from the Internet <URL:https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/346/472/p3226dat.pdf> * |
| 李民友 等: "应用蛋白质L检测和纯化培养上清液中的单链抗体", 免疫学杂志, vol. 20, no. 3, pages 231 - 233 * |
| 王兴龙 等: "基于表面电荷对抗体分子状态调控的研究", 高校化学工程学报, vol. 35, no. 5, pages 851 - 852 * |
| 霍方珍: "抗盐酸克伦特罗单链抗体的高效表达及免疫学活性研究", 中国优秀硕士学位论文全文库, no. 2007, pages 68 * |
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