CN114113634A - 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 an African swine fever virus antibody. The kit can quickly 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, is suitable for monitoring and troubleshooting work of African swine fever in large-scale breeding, and is easy to popularize and apply in a large scale.
Description
Technical Field
The application relates to the technical field of immunodetection, in particular to an ELISA detection kit for detecting an African swine fever virus antibody and application of protein L.
Background
African Swine Fever (ASF) is an acute, virulent infectious disease of swine caused by African Swine Fever Virus (ASFV) infection. The disease can cause symptoms of hyperpyrexia, reticuloendothelial system hemorrhage, visible mucous membrane redness and the like of pigs clinically, the disease course of virulent strain infected pigs is short, and the death rate is up to 100%.
The African swine fever virus is a double-stranded DNA virus with an envelope and has a complex multi-layer structure. The 5-layer structure of the artificial marble comprises a similar core, a nuclear shell, an inner membrane, a capsid and an outer cyst membrane from inside to outside in sequence. The African swine fever virus can code more than 200 proteins, wherein the main component P72 protein of the capsid accounts for about 33% of the total mass of the African swine fever virus. Heretofore, indirect ELISA antibody detection kits developed based on P30 and P54 proteins have been reported. Because the P72 full-length trimer protein is difficult to prepare, the commercial indirect ELISA kits based on the P72 protein all use P72 truncated bodies, and no report exists on the preparation and detection kit of the P72 full-length trimer protein. Previously, we have succeeded in expressing P72 trimer protein in large quantities in the Saccharomyces cerevisiae expression system and the structure is identical to the natural structure (patent No.: 202110522777.4).
OIE recommends diagnostic techniques for African swine fever virus including pathogenic testing and serological testing. Among them, ELISA is an international trade test method specified by OIE. The indirect ELISA method based on the P72 full-length trimer protein has the advantages of high specificity and strong sensitivity, and meanwhile, the characteristics of ELISA multi-sample and high-throughput detection are also suitable for being implemented in large-scale farms.
Disclosure of Invention
In view of the above, the present invention provides an ELISA kit for detecting african swine fever virus antibody, which has high specificity, sensitivity and accuracy when used for detecting the african swine fever virus antibody;
another object of the present invention is to provide the use of protein L in the preparation of an ELISA kit for detecting antibodies to African swine fever virus, such as the preparation of enzyme conjugates.
To solve the above technical problem/achieve the above object or at least partially solve the above technical problem/achieve the above object, the present invention provides an ELISA detection kit for detecting african swine fever virus antibodies, comprising an ELISA plate coated with african swine fever virus capsid protein P72 trimer and a protein L enzyme conjugate.
According to the invention, the research of the African swine fever virus antibody ELISA detection product is carried out on the basis of the prior P72 full-length trimer protein, and the research discovers that 64000 times of diluted positive serum can be detected by using the African swine fever virus capsid protein P72 trimer as a coating antigen and combining with a protein L enzyme conjugate according to indirect ELISA detection, and only 16000 times, 8000 times and 8000 times of diluted positive serum can be detected by using the conventional sensitivity which uses the P72 disordered folding monomer protein, the P54 protein and the P30 protein as the coating antigens;
moreover, on the premise that the same P72 trimer is coated with antigen, the detection is carried out by using a protein L/A/G enzyme conjugate and a goat anti-pig IgG enzyme conjugate, and the result shows that 64000 times of diluted positive serum can be detected by using the protein L enzyme conjugate, and only 32000 times of diluted positive serum can be detected by using the other three enzyme conjugates. In view of the above-mentioned excellent technical effects, the present invention provides the use of protein L in the preparation of ELISA detection kit for detecting African swine fever virus antibody, preferably the use of protein L and African swine fever virus capsid protein P72 trimer in combination in the preparation of ELISA detection kit for detecting African swine fever virus antibody.
The African swine fever virus capsid protein P72 trimer in the kit realizes the high copy expression of the protein of the P72 trimer BY inserting the African swine fever virus specific P72 protein gene into the genome of Saccharomyces cerevisiae BY4743 BY using the CRISPR-Cas9 technology, and related contents are all recorded in CN202110522777.4, and all the records can also be used as the contents recorded in the invention. The recombinant P72 protein which can be correctly assembled to form a trimer configuration without depending on the auxiliary protein B602L is prepared and obtained 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 a specific embodiment of the invention, the protein tag is Twin-Strep-tag fused at the N-terminal or C-terminal 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 protein L enzyme conjugate has a working concentration of 1:5000-10000, and in the 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 diluent, wash, substrate, sample diluent, stop buffer, negative control and positive control. In the specific embodiment of the invention, the enzyme is selected to be horseradish peroxidase, and then the substrates are selected to be substrate A liquid 3,3 ', 5, 5' -tetramethylbenzidine solution and substrate B liquid peroxide solution.
Preferably, the enzyme conjugate diluent is PBST containing BSA; in a particular embodiment of the invention the enzyme conjugate diluent is PBST with 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 swine serum immunized with the African swine fever virus capsid protein P72 trimer, and the negative control is normal swine serum immunized with the African swine fever virus capsid protein P72 trimer.
The kit is adopted to detect verified African swine fever virus antibody positive serum, porcine circovirus antibody positive serum, porcine reproductive and respiratory syndrome 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 result shows 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 detected are respectively detected by taking the same batch number 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 parts positive and 35 parts negative; the number of positive matches of the invention is 60, the number of negative matches is 32, the positive match rate is 100% (57/57), the negative match rate is 91.4% (32/35), and the total match rate is 96.7% (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 envelope antigen are combined to prepare the ELISA detection kit for detecting the African swine fever virus antibody, the kit can quickly and specifically detect the African swine fever virus antibody in serum, is extremely high in reaction sensitivity, simple and convenient to operate, low in cost and stable in reaction result, is suitable for monitoring and troubleshooting work of African swine fever in large-scale breeding, 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. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the products, processes and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations and modifications, or appropriate alterations and combinations, of the products, processes and applications described herein may be made and used without departing from the spirit, scope and ambit of the invention. It should be apparent that the described embodiments are only some of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
It is noted that, in this document, relational terms such as "first" and "second", "step 1" and "step 2", "S1" and "S2" and "(1)" and "(2)" etc. 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. Also, 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 an … …" does not exclude the presence of other identical 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: coating plate, enzyme conjugate diluent, 25 Xconcentrated washing solution, substrate A solution, substrate B solution, sample diluent, stop solution, negative control, positive control, serum dilution plate, cover plate membrane and instructions;
wherein, the coating plate is coated with an African swine fever virus capsid protein P72 trimer with a sequence shown in SEQ ID NO. 1 and is used for determining the specific combination of the recombinant protein and the African swine fever virus antibody positive serum;
the enzyme conjugate is horse radish peroxidase-labeled protein L, and the concentration of the enzyme conjugate is 1: 5000-;
the 25 × concentrated wash was 25 × PBST, and the enzyme conjugate diluent was PBST with 1% BSA; the sample diluent is PBST containing 1% fetal calf serum, and the stop solution is 2M H2SO4The substrate A solution is a 3,3 ', 5, 5' -tetramethylbenzidine solution, and the substrate B solution is a peroxide solution.
Meanwhile, the invention also provides a using method of the kit, which comprises the following steps:
(1) diluting the serum to be detected by using a sample diluent, adding the diluted serum into a coated plate, and adding 100uL of the diluted serum into each hole; meanwhile, 100uL of each of the negative and positive controls was added, the coated plate was sealed with a cover plate, and incubated at 37 ℃ for 30 min.
(2) The 25 Xconcentrated wash was diluted to 1 Xwash and the coated plate was washed 3 times with shaking, each time patting dry.
(3) Diluting the enzyme conjugate with enzyme conjugate diluent at a ratio of 1:100, adding to the coated plate at a concentration of 100 uL/well, sealing the coated plate with a cover plate, and incubating at 37 deg.C for 30 min.
(4) Shaking and washing the coated plate for 3 times by using 1 × washing solution, and drying each time;
(5)1: 1 mixing the substrate A solution and the substrate B solution, adding the mixture into a coated plate at a rate of 100 uL/hole, sealing the coated plate with a cover plate film, and incubating at 37 ℃ for 10 min. Adding stop solution into the coated plate at 50 uL/well, and reading OD on a microplate reader450nmThe light absorption value of (a);
(6) and (4) judging a result: when positive control OD450nmNot less than 1.0 and negative control OD450nmThe results are valid when the value is less than 0.2; (sample OD)450nmValue-negative control OD450nmvalue)/(Positive control OD450nmValue-negative control OD450nmValue) is more than or equal to 0.2, the result is judged to be positive; (sample OD)450nmValue-negative control OD450nmvalue)/(Positive control OD450nmValue-negative control OD450nmWhen the value is less than 0.1, the sample is judged to be negative, and the sample OD is less than or equal to 0.1450nmValue-negative control OD450nmvalue)/(Positive control OD450nmValue-negative control OD450nmValue) < 0.2, the sample is judged to be suspected.
In each group comparison experiment provided by the invention, unless particularly stated, other experiment conditions, materials and the like are consistent except for the differences indicated by each group so as to have comparability.
The ELISA detection kit for detecting African swine fever virus antibody provided by the invention is further explained 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 the three-dimensional conformation of the P72 protein through 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, yeast GAL1 promoter and ADH1 terminator are constructed on a plasmid to form a P72 protein gene expression cassette. The P72 protein gene expression cassette with homologous recombination arms is amplified by PCR and used as a repair template. The method is characterized in that a CRISPR-Cas9 technology is utilized to co-express a gRNA for recognizing GGATTTAGGAATCCATAAAA, and a P72 protein gene expression cassette is inserted into a multiple-copy Ty2 retrotransposon of saccharomyces cerevisiae in a homologous recombination mode to realize gene multiple-copy expression. The repair template and the pCas-ty2 plasmid were co-transformed into Saccharomyces cerevisiae BY4743, and the URA-deficient plates were used for cloning screening. Screening out monoclonals, identifying copy number by a qPCR method, and selecting high-copy strains 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 overnight cultured bacterial liquid to perform 100-fold amplification culture in YPD +2% glucose liquid culture medium, and culturing at 30 ℃ for 60 hours until the OD600 reaches 3.0 to obtain the required protein.
3. Purification of recombinant proteins
Taking 1L of Saccharomyces cerevisiae culture, 6000 rpm, centrifuging for 10min, and collecting precipitate. The precipitate was resuspended in 50ml buffer W (IBA), then the yeast cells were disrupted by a high-pressure homogenizer, centrifuged at 17000rpm at 4 ℃ for 60 minutes, and the supernatant was filtered through a 0.45 μm filter and purified. The fusion protein affinity purification is carried out by using a Strep-Tactin XT gradient-flow column (IBA), and the main operation steps are as follows:
a. the column was equilibrated with 10ml of buffer W (IBA), and the filtered supernatant was applied to the column. Then slowly flowed out by gravity flow, and the operation was repeated twice.
b. The resulting mixture was washed with 20 mL buffer W (IBA) to remove the contaminating proteins.
c. The desired target protein was eluted with elution buffer BXT (IBA), and the eluate was collected in 1 ml/tube.
d. The protein of interest was collected and concentrated using a Merck-Millipore 30KD concentration tube.
e. Molecular sieves were equilibrated with FPLC (Superdex S-200, GE) at 4 ℃ and a flow rate of 1 ml/min. The protein was loaded in batches 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 with a size of about 73kD appeared on the purified SDS-PAGE. The protein of interest was concentrated using a Merck-Millipore 30KD concentration tube, and then the protein concentration was measured by the BCA method.
Example 2: establishment of indirect ELISA kit for detecting African swine fever virus antibody
1. Determination of optimal coating concentration
Respectively coating a 96-well enzyme label plate with the purified recombinant P72 trimer protein by 1 [ mu ] g/mL, 2.5 [ mu ] g/mL, 5 [ mu ] g/mL and 10 [ mu ] g/mL overnight at 4 ℃ at 100 [ mu ] L/well, washing the plate with PBST for 3 times the next day, then using 1% BSA as a sealing solution, 200 [ mu ] L/well, sealing the plate for 2h at 37 ℃, using an ASFV antibody positive serum sample as a primary antibody, using an ASFV antibody negative serum sample as a negative control, incubating the plate for 30min at 37 ℃, and washing the plate with PBST for 3 times and patting the plate dry; adding enzyme conjugate diluted at 1:8000, incubating at 37 deg.C for 30min, washing plate with PBST for 3 times, and drying; adding 100 muL TMB substrate solution into each well, developing for 10min at 37 ℃, stopping the reaction by using 50 muL stop solution, and reading OD of each well by using an enzyme-labeling instrument450nmAnd calculating the P/N value and determining the optimal coating concentration.
2. Determination of optimal confining liquid
Coating a 96-well enzyme label plate with purified recombinant P72 trimer protein 5 mug/mL overnight at 4 ℃ according to 100 mug L/well, washing the plate with PBST for 3 times the next day, then respectively using 1% BSA, 1% OVA, 1% gelatin and 5% skimmed milk powder as sealing liquid, sealing the plate with PBST for 2h at 37 ℃, using an ASFV antibody positive serum sample as a primary antibody and using an ASFV antibody negative serum sample as a negative control, incubating the plate for 30min at 37 ℃, and washing the plate with PBST for 3 times and patting the plate dry; adding enzyme conjugate diluted at 1:8000, incubating at 37 deg.C for 30min, washing plate with PBST for 3 times, and drying; adding 100 muL TMB substrate solution into each well, developing for 10min at 37 ℃, stopping the reaction by using 50 muL stop solution, and reading OD of each well by using an enzyme-labeling instrument450nmAnd calculating the P/N value to determine the optimal confining liquid.
3. Determination of optimal serum dilutions
Performing test according to the determined optimal indirect ELISA parameters, diluting sample serum by PBST containing 1% BSA, PBST containing 1% fetal calf serum, PBST and PBST containing 1% rabbit serum according to a ratio of 1:100, adding 100 muL/well into 96-well ELISA plate for reaction, performing indirect ELISA step according to other reference 1, and reading OD (optical density) of each well by an ELISA reader after color development450nmAnd calculating the P/N value and determining the optimal serum dilution.
4. Determination of optimal enzyme conjugate dilutions
The assay was performed according to the above-identified optimal indirect ELISA parameters by diluting the enzyme conjugate with 1% BSA in PBST, 1% fetal bovine serum in PBST, PBST and 1% rabbit serum in PBST to a titer of 1:8000, and reading the OD of each well with a developed microplate reader after the other reference 1 indirect ELISA steps450nmAnd calculating the P/N value to determine the optimal enzyme conjugate dilution.
5. Determination of the working Titers of the optimal enzyme conjugates
Performing test according to the determined optimal indirect ELISA parameters, diluting enzyme conjugate to titer of 1:8000, 1:10000 and 1:12000, repeating 2 times, performing indirect ELISA steps with reference to 1, and reading OD of each well with enzyme-labeling instrument after development450nmAnd calculating the P/N value to determine the optimal working titer of the enzyme conjugate.
6. Determination of a threshold value
5 parts of serum which is detected as ASFV negative by a commercial ELISA kit are tested according to an indirect ELISA program under the optimal reaction condition, and the OD of a sample is calculated450nmAverage value of (a) <
) And Standard Deviation (SD).
The optimal parameters of the indirect ELISA reaction are shown in Table 1.
TABLE 1
| Optimum coating concentration | Sealing liquid | Serum diluent | Enzyme conjugate dilution | Enzyme conjugate working titer |
| 5µg/mL | 1 % BSA | 1% fetal bovine serum | 1% BSA | 1:8000 |
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 is prepared by the following steps:
(1) coating: the recombinant African swine fever virus P72 tripolymer protein is used for coating an enzyme label plate, the coating concentration is 5ug/ml, the dosage is 100 uL/hole, the enzyme label plate is coated at 4 ℃ overnight, and the enzyme label plate is washed 3 times by PBST the next day and patted dry.
(2) And (3) sealing: blocking with 1% BSA, 200 uL/well, incubation at 37 ℃ for 2h, washing 3 times with PBST, and spin-drying. Dried at 37 ℃ for 2h and then blocked in vacuo for further use.
2. Determination of the procedure of the Indirect ELISA detection method
(1) Diluting the serum to be detected by using a serum diluent, adding the diluted serum into a coated plate, and adding 100uL of the diluted serum into each hole; meanwhile, 100uL of each of the negative and positive controls was added, the coated plate was sealed with a cover plate, and incubated at 37 ℃ for 30 min.
(2) The 25 Xconcentrated wash was diluted to 1 Xwash and the coated plate was washed 3 times with shaking, each time patting dry.
(3) The protein L enzyme conjugate was diluted 1:100 with the enzyme conjugate diluent, added to the coated plate at 100 uL/well, the coated plate was sealed with a cover plate film, and incubated at 37 ℃ for 30 min.
(4) Shaking and washing the coated plate for 3 times by using 1 × washing solution, and drying each time;
(5)1: 1 mixing the substrate A solution and the substrate B solution, adding the mixture into a coated plate at a rate of 100 uL/hole, sealing the coated plate with a cover plate film, and incubating at 37 ℃ for 10 min. Adding stop solution into the coated plate at 50 uL/well, and reading OD on a microplate reader450nmThe light absorption value of (a);
and (4) judging a result: when positive control OD450nmNot less than 1.0 and negative control OD450nmThe results are valid when the value is less than 0.2; S/P = (sample OD)450nmValue-negative control OD450nmvalue)/(Positive control OD450nmValue-negative control OD450nmValue), when the S/P is more than or equal to 0.2, judging the test result to be positive; if S/P is less than 0.1, the sample is judged to be negative, and if S/P is more than or equal to 0.1 and less than 0.2, the sample is judged to be suspected.
Example 4: sensitivity detection
1. Comparison of different envelope antigens
The known African swine fever virus antibody positive serum is subjected to 1:100, 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000 and 1:64000 and 1:128000, and the indirect ELISA kit based on the P72 tripolymer protein and the indirect ELISA kit based on the P30, P54 and P72 disordered folding monomer protein, which are prepared by the invention, are respectively used for detection, and the result shows that the African swine fever virus antibody indirect ELISA kit prepared based on the P72 tripolymer protein has the highest sensitivity which can reach 1:64000 times, and the result is shown in Table 2.
TABLE 2
| P72 trimer (S/P) | P72 disordered fold 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 |
| 1:128000 | 0.131 | 0.007 | 0.003 | 0.004 |
| Negative Serum | 0.005 | 0.002 | 0.000 | 0.002 |
2. Comparison of different enzyme conjugates
Staphylococcus aureus protein A (SPA), Streptococcal Protein G (SPG) and Peptostreptococcus magnus protein L (PPL) are typical immunoglobulin-binding proteins that bind most mammalian immunoglobulins, but in different ways and 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
| Protein L-HRP（S/P） | Goat anti-pig IgG-HRP (S/P) | Protein A-HRP（S/P） | Protein G-HRP（S/P） | |
| 1:100 | 1.33 | 1.003 | 1.041 | 1.051 |
| 1:1000 | 0.973 | 0.833 | 0.908 | 0.898 |
| 1:2000 | 0.922 | 0.751 | 0.802 | 0.768 |
| 1:4000 | 0.826 | 0.601 | 0.663 | 0.622 |
| 1:8000 | 0.744 | 0.376 | 0.496 | 0.465 |
| 1:16000 | 0.605 | 0.305 | 0.329 | 0.315 |
| 1:32000 | 0.403 | 0.213 | 0.244 | 0.233 |
| 1:64000 | 0.2 | 0.121 | 0.155 | 0.150 |
| 1:128000 | 0.131 | 0.040 | 0.075 | 0.064 |
| Negative Serum | 0.005 | 0.002 | 0.010 | 0.009 |
3. Post-challenge detection
Seven week old SPF long white pigs were provided by the Experimental animal center of Harbin veterinary institute, national academy of agricultural sciences, and were verified to be free of PCV, CSFV, PRRSV and PrV infections. The experimental pig is inoculated with the African swine fever virus HLJ/HRB1/20 strain with low dose in the muscle, and the inoculation dose is 103 TCID50Head of the device. 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, blood is collected through an anterior vena cava, serum is separated, the indirect ELISA kit based on the P72 trimer protein is used for antibody detection, antibody positivity can be detected on the 9 th day, and the time for detecting the antibody by other different envelope 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 reproductive and respiratory syndrome 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 the established indirect ELISA detection method, and performing statistical analysis on the obtained test result data. Except for the positive serum of the African swine fever virus antibody, the test results are negative, which shows that the ASFV indirect ELISA method which takes the recombinant P72 tripolymer protein as the diagnostic antigen has better specificity, and the results are shown in Table 4.
TABLE 4 results of specificity test
| Serum sample | P72 trimer (S/P) |
| African swine fever antibody positive serum | 1.330 |
| Porcine circovirus antibody positive serum | 0.006 |
| Porcine reproductive and respiratory syndrome virus antibody positive serum | 0.003 |
| Porcine pseudorabies virus antibody positive serum | 0.002 |
| Hog cholera virus antibody positive serum | 0.009 |
| African swine fever antibody negative serum | 0.005 |
Example 6: repeatability test
And (3) taking ELISA detection plates coated with recombinant antigens in different batches, respectively detecting 5 ASFV positive serums and 5 ASFV negative serums, parallelly making 5 repeated holes on each serum sample, and carrying out statistical analysis on the result, wherein the result shows that the detection results of the in-batch repeatability test and the in-batch repeatability test are consistent and are consistent with the virus neutralization test result, and the indirect ELISA detection method has good repeatability and accuracy.
Example 7: compliance test with commercial ELISA kit
And (3) respectively detecting 92 clinical to-be-detected serum samples by taking the same batch number indirect ELISA kit, and comparing the observed results with 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 parts positive and 35 parts negative; the African swine fever virus antibody detection test paper card prepared by the invention has 60 positive coincidence numbers, 32 negative coincidence numbers, 100 percent of positive coincidence rate (57/57), 91.4 percent of negative coincidence rate (32/35) and 96.7 percent of total coincidence rate (89/92). The antigen is proved to have good accuracy when being used for detecting the African swine fever virus antibody.
The foregoing are merely exemplary embodiments of the present invention, which enable those skilled in the art to understand or practice the present 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.
Sequence listing
<110> university of agriculture in China
Harbin Institute of veterinary medicine, Chinese Academy of Agricultural Sciences
<120> ELISA detection kit for detecting African swine fever virus antibody and application of protein L
<130> HT217540
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 646
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Met Ala Ser Gly Gly Ala Phe Cys Leu Ile Ala Asn Asp Gly Lys Ala
1 5 10 15
Asp Lys Ile Ile Leu Ala Gln Asp Leu Leu Asn Ser Arg Ile Ser Asn
20 25 30
Ile Lys Asn Val Asn Lys Ser Tyr Gly Lys Pro Asp Pro Glu Pro Thr
35 40 45
Leu Ser Gln Ile Glu Glu Thr His Leu Val His Phe Asn Ala His Phe
50 55 60
Lys Pro Tyr Val Pro Val Gly Phe Glu Tyr Asn Lys Val Arg Pro His
65 70 75 80
Thr Gly Thr Pro Thr Pro Gly Asn Lys Leu Thr Phe Gly Ile Pro Gln
85 90 95
Tyr Gly Asp Phe Phe His Asp Met Val Gly His His Ile Leu Gly Ala
100 105 110
Cys His Ser Ser Trp Gln Asp Ala Pro Ile Gln Gly Thr Ser Gln Met
115 120 125
Gly Ala His Gly Gln Leu Gln Thr Phe Pro Arg Asn Gly Tyr Asp Trp
130 135 140
Asp Asn Gln Thr Pro Leu Glu Gly Ala Val Tyr Thr Leu Val Asp Pro
145 150 155 160
Phe Gly Arg Pro Ile Val Pro Gly Thr Lys Asn Ala Tyr Arg Asn Leu
165 170 175
Val Tyr Tyr Cys Glu Tyr Pro Gly Glu Arg Leu Tyr Glu Asn Val Arg
180 185 190
Phe Asp Val Pro Gly Asn Ser Leu Asp Glu Tyr Ser Ser Asp Val Thr
195 200 205
Thr Leu Val Arg Lys Phe Cys Pro Pro Gly Asp Pro Met Thr Gly Tyr
210 215 220
Lys His Leu Val Gly Gln Glu Val Ser Val Glu Gly Thr Ser Gly Pro
225 230 235 240
Leu Leu Cys Asn Ile His Asp Leu His Lys Pro His Gln Ser Lys Pro
245 250 255
Ile Leu Thr Asp Glu Asn Asp Thr Gln Arg Thr Cys Ser His Thr Asn
260 265 270
Pro Lys Phe Leu Ser Gln His Phe Pro Glu Asn Ser His Asn Ile Gln
275 280 285
Thr Ala Gly Lys Gln Asp Ile Thr Pro Ile Thr Asp Ala Thr Tyr Leu
290 295 300
Asp Ile Arg Arg Asn Val His Tyr Ser Cys Asn Gly Pro Gln Thr Pro
305 310 315 320
Lys Tyr Tyr Gln Pro Pro Leu Ala Leu Trp Ile Lys Leu Arg Phe Trp
325 330 335
Pro Asn Glu Asn Val Asn Pro Ala Ile Pro Ser Val Ser Ile Pro Phe
340 345 350
Gly Glu Arg Phe Ile Thr Ile Lys Leu Ala Ser Gln Lys Asp Leu Val
355 360 365
Asn Glu Phe Pro Gly Leu Phe Val Arg Gln Ser Arg Phe Ile Ala Gly
370 375 380
Arg Pro Ser Arg Arg Asn Ile Arg Phe Lys Pro Trp Phe Ile Pro Gly
385 390 395 400
Val Ile Asn Glu Ile Ser Leu Thr Asn Asn Glu Leu Tyr Ile Asn Asn
405 410 415
Leu Phe Val Thr Pro Glu Ile His Asn Leu Phe Val Lys Arg Val Arg
420 425 430
Phe Ser Leu Ile Arg Val His Lys Thr Gln Val Thr His Thr Asn Asn
435 440 445
Pro His His Asp Glu Lys Leu Met Ser Ala Leu Lys Trp Pro Ile Glu
450 455 460
Tyr Met Phe Ile Gly Leu Lys Pro Thr Trp Asn Ile Pro Asp Gln Asn
465 470 475 480
Pro Pro Gln His Arg Asp Trp His Lys Phe Gly His Val Val Asn Ala
485 490 495
Ile Met Gln Pro Thr His His Ala Glu Ile Ser Phe Pro Asp Arg Asp
500 505 510
Thr Ala Leu Pro Asp Ala Cys Ser Ser Ile Ser Asp Ile Ser Pro Val
515 520 525
Thr Tyr Pro Ile Thr Leu Pro Ile Ile Lys Asn Ile Ser Val Thr Ala
530 535 540
Pro Gly Ile Asn Leu Ile Asp Lys Phe Pro Ser Lys Phe Cys Ser Ser
545 550 555 560
Tyr Ile Pro Phe Pro Pro Gly Gly Asn Ala Ile Lys Thr Pro Asp Asp
565 570 575
Pro Gly Ala Met Met Ile Thr Phe Ala Leu Lys Pro Arg Glu Glu Tyr
580 585 590
Gln Pro Ser Gly His Ile Asn Val Ser Arg Ala Arg Glu Phe Tyr Ile
595 600 605
Ser Trp Asp Thr Asp Tyr Val Gly Ser Pro Thr Thr Ala Asp Leu Val
610 615 620
Val Ser Ala Ser Ala Ile Asn Phe Leu Leu Leu Gln Asn Gly Ser Ala
625 630 635 640
Val Leu Arg Tyr Ser Thr
645
Claims (10)
1. An ELISA detection kit for detecting an African swine fever virus antibody is characterized by comprising an ELISA plate coated with an African swine fever virus capsid protein P72 tripolymer and a protein L enzyme conjugate.
2. The kit according to claim 1, wherein the African swine fever virus capsid protein P72 trimer has the sequence shown in SEQ ID NO 1 or is added with a tag sequence on the basis thereof.
3. The kit of claim 1, wherein said protein L enzyme conjugate is a protein L horseradish peroxidase conjugate.
4. The kit according to any one of claims 1 to 3, further comprising any one or more than two of the following components:
enzyme conjugate diluent, wash, substrate, sample diluent, stop buffer, negative control and positive control.
5. The kit of claim 4, wherein the enzyme conjugate diluent is PBST containing BSA.
6. The kit of claim 4, wherein the sample diluent is PBST containing fetal bovine serum.
7. The kit of claim 4, wherein the positive control is normal swine serum immunized with the capsid protein P72 trimer of African swine fever virus.
8. The kit of claim 4, wherein the negative control is normal swine serum of capsid protein P72 trimer of non-immune African swine fever virus.
Application of protein L in preparation of ELISA detection kit for detecting African swine fever virus antibody.
10. The use according to claim 9, wherein the protein L is used for the preparation of an enzyme conjugate in a kit.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210078482.7A CN114113634A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
| CN202210926456.5A CN116068192A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210078482.7A CN114113634A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210926456.5A Division CN116068192A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114113634A true CN114113634A (en) | 2022-03-01 |
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| CN202210926456.5A Pending CN116068192A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
| CN202210078482.7A Pending CN114113634A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
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| CN202210926456.5A Pending CN116068192A (en) | 2022-01-24 | 2022-01-24 | ELISA detection kit for detecting African swine fever virus antibody and application of protein L |
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| CN (2) | CN116068192A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115032398A (en) * | 2022-06-23 | 2022-09-09 | 北京阳光德美医药科技有限公司 | Method for quantitatively detecting content of antibody in biological sample |
Citations (5)
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|---|---|---|---|---|
| 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 |
| US20190119332A1 (en) * | 2016-05-04 | 2019-04-25 | The Children's Medical Center Corporation | Methods to purify avidin-like proteins and fusion proteins thereof |
| US20210100892A1 (en) * | 2018-09-11 | 2021-04-08 | Jian Qing XU | Universal vaccines against immunogens of pathogenic organisms that provide organism-specific and cross-group protection |
| 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 |
| CN113721035A (en) * | 2021-11-03 | 2021-11-30 | 中国农业大学 | Colloidal gold immunochromatographic test paper card for detecting African swine fever virus antibody |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2135208C (en) * | 1992-05-07 | 2009-06-30 | Angus Robert Trowern | 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 |
| MX2015005384A (en) * | 2012-11-05 | 2015-10-09 | Delenex Therapeutics Ag | Binding members to il-1 beta. |
-
2022
- 2022-01-24 CN CN202210926456.5A patent/CN116068192A/en active Pending
- 2022-01-24 CN CN202210078482.7A patent/CN114113634A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| US20190119332A1 (en) * | 2016-05-04 | 2019-04-25 | The Children's Medical Center Corporation | Methods to purify avidin-like proteins and fusion proteins thereof |
| US20210100892A1 (en) * | 2018-09-11 | 2021-04-08 | Jian Qing XU | Universal vaccines against immunogens of pathogenic organisms that provide organism-specific and cross-group protection |
| 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 |
| CN113721035A (en) * | 2021-11-03 | 2021-11-30 | 中国农业大学 | Colloidal gold immunochromatographic test paper card for detecting African swine fever virus antibody |
Non-Patent Citations (4)
| Title |
|---|
| FORTEBIO: "Dip and Read™ Protein L biosensors", 《WWW.FORTEBIO.COM》 * |
| GUSTAV RODRIGO等: "Antibody Fragments and Their Purification by Protein L Affinity Chromatography", 《ANTIBODIES》 * |
| M. DE CHATEAU等: "On the Interaction between Protein L and Immunoglobulins of Various Mammalian Species", 《SCAND. J. IMMUNOL.》 * |
| 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》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115032398A (en) * | 2022-06-23 | 2022-09-09 | 北京阳光德美医药科技有限公司 | Method for quantitatively detecting content of antibody in biological sample |
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| CN116068192A (en) | 2023-05-05 |
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Application publication date: 20220301 |