CN117209569A - African swine fever virus p72 protein ELISA antibody detection kit - Google Patents
African swine fever virus p72 protein ELISA antibody detection kit Download PDFInfo
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- Peptides Or Proteins (AREA)
Abstract
The invention discloses an African swine fever virus p72 protein ELISA antibody detection kit. The kit comprises an ELISA plate, positive control serum, negative control serum, an ELISA secondary antibody, a sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the ELISA plate is coated with an African swine fever virus P72 epitope polypeptide composition. The antigen epitope polypeptide composition is one or more than two of polypeptide shown in a sequence 1 in a sequence table, polypeptide shown in a sequence 2 in the sequence table and polypeptide shown in a sequence 3 in the sequence table. The kit uses the chemically synthesized antigen peptide coated ELISA plate, has less antigen consumption and high sensitivity and specificity, and can efficiently detect whether the African swine fever virus antibody is infected. The kit disclosed by the invention has the advantages of high sensitivity, good specificity, convenience in operation and good market prospect.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to an African swine fever virus p72 protein ELISA antibody detection kit.
Background
African swine fever (African swine fever, ASF) is an animal epidemic that must be reported by the world animal health organization (World Organisation for Animal Health, WOAH), an acute, febrile, highly contagious infectious disease of pigs caused by African swine fever virus (African swine fever virus, ASFV) infection, characterized clinically by hyperthermia, reticuloendothelial system hemorrhage and high mortality. Since the first report of African swine fever in 2018, the high lethality and high infectivity of African swine fever cause great loss to pig industry in China, and greatly influence pig supply in China. At present, no vaccine is available for ASF, the elimination of the disease can only be realized through strict detection and killing, the serum antibody is monitored at high flux and low cost, and the method is an important link for controlling epidemic spreading and tracking epidemic sources, so that first-line epidemic prevention personnel can make correct diagnosis on site when epidemic outbreaks occur, and effective precautions are taken. The ELISA (enzyme-linked immunosorbent assay) has the characteristics of high sensitivity, good specificity, simple and convenient operation, capability of rapidly detecting a large number of samples with high flux, and the like, is suitable for detecting epidemic diseases, and is particularly important for developing an ELISA detection kit for African swine fever antibodies for diagnosis and prevention of ASF.
ASFV is a double-stranded DNA virus with a capsule membrane, the average particle diameter is about 200nm, the size of the virus genome is 170-190 kb, the virus has more than 150 main open reading frames, 150-200 proteins can be encoded, a virus core of 80nm comprises the virus genome and nucleoprotein p10 and pA104R, and the virus core is wrapped by a virus capsid composed of p35, p15, p150, p37, p34 and p14 proteins; surrounding the viral capsid are two layers of lipid molecules, the inner envelope consisting of p54, p17, pE248R and p12, the capsid structure containing p72, pE120R and pB438L; the virus budds through the plasma membrane and is released from the host cell, and the outer envelope obtained in this process contains the proteins p24, CD2v, p30 and p12. The p72 protein is one of the main structural proteins of the virus, has stable antigenicity, can be used for serodiagnosis of ASF, can detect antibodies generated by ASFV, and provides a detection method for prevention and control of ASF.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting an African swine fever virus p72 antibody, which utilizes an African swine fever virus p72 epitope polypeptide as a coating antigen, and establishes an indirect ELISA method with good specificity, sensitivity and repeatability for detecting whether an African swine fever virus antibody is contained in pig serum.
In order to achieve the above purpose, the African swine fever virus p72 epitope polypeptide composition with better reactivity is firstly screened and obtained, and the African swine fever virus p72 epitope polypeptide composition provided by the invention is any combination of two or more than two of the polypeptides shown in the sequence 1 in the sequence table, the sequence 2 in the sequence table and the sequence 3 in the sequence table. When the polypeptide composition is two of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3, the mass ratio of the two polypeptides is (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1, a step of; when the polypeptide composition is three of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3, the mass ratio of any three polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1.
the invention also claims an African swine fever virus p72 epitope polypeptide which is a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table or a polypeptide shown in a sequence 3 in the sequence table.
The invention relates to an African swine fever virus p72 protein ELISA antibody detection kit, which comprises an enzyme-linked reaction plate, positive control serum, negative control serum, an enzyme-labeled secondary antibody, a sample diluent, 20-time concentrated washing liquid, substrate liquid A, substrate liquid B and stop solution, wherein the enzyme-linked reaction plate is coated with an African swine fever virus p72 epitope polypeptide composition.
The ELISA plate is a detachable 96-hole ELISA plate; the polypeptides in the African swine fever virus p72 epitope polypeptide composition are obtained through chemical and artificial synthesis.
The optimal preparation method and the condition of the ELISA plate are that the African swine fever virus p72 epitope polypeptide composition is dissolved in a carbonate solution with the pH of 9.6, then the solution is added into a 96-hole polystyrene ELISA plate, 150ng of polypeptide or the polypeptide composition is placed at the temperature of 2-8 ℃ for 8-12 hours, the polypeptide antigen and the ELISA plate are fully combined, then PBS buffer solution containing 10mg/ml Bovine Serum Albumin (BSA) with the pH of 7.4 is added according to 300 mu l/hole, the sealing treatment is carried out at the temperature of 37 ℃ for 2-3 hours, and after the drying, the ELISA plate is dried and then sealed and stored at the temperature of 4 ℃.
The positive control serum is African swine fever virus antibody positive serum, and can be specifically African swine fever positive serum (CVCC number is Z286) purchased from Chinese veterinary medicine monitoring institute and used after being properly diluted by a sample diluent.
The negative control serum was pig serum free of Specific Pathogen (SPF).
The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-pig IgG antibody.
The substrate solution A is a citric acid phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide, the substrate solution B is a tetramethyl benzidine solution with the concentration of 0.2mg/ml, and the substrate solution A and the substrate solution B are mixed in a ratio of 1:1 when the substrate solution A is used. The stop solution is 2mol/L sulfuric acid solution.
The kit also comprises a sample diluent and a 20-time concentrated washing liquid; the sample diluent is phosphate buffer solution with the value of 0.01M, pH and 7.4 and containing 5mg/ml casein; the concentrated washing solution is phosphate buffer solution with pH value of 7.4 and 0.01M containing Tween-20 with concentration of 0.8% -1.2% (ml/ml).
The detection program of the kit provided by the invention is as follows:
1. balance: taking the kit out of the refrigeration environment, and standing at room temperature for 30min for standby; the liquid reagent is mixed evenly before use.
2. Preparing liquid: diluting the concentrated washing solution with distilled water or deionized water for 20 times to obtain a washing buffer solution;
3. setting: 2 negative control wells and 2 positive control wells, the remainder being wells for samples to be tested.
4. Pre-diluting a sample to be tested: sample serum to be tested was prepared using sample dilutions according to 1: 20. The negative control serum and the positive control serum were used as they are.
5. Sample adding: each well was pre-set with 100. Mu.l of diluted test sample. The time span of the sample adding process should be as short as possible.
6. Incubation: shaking and mixing uniformly, and placing in a 37 ℃ incubator for reaction for 30min.
7. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, the washing solution was discarded, and the plate was washed continuously 4 times and then dried by pipetting.
8. Adding enzyme: mu.l horseradish peroxidase was added to each well to label the rabbit anti-pig IgG antibody.
9. Incubation: placing in a 37 ℃ incubator for reaction for 30min.
10. Washing the plate: the reaction solution was discarded, 300. Mu.l of diluted washing buffer was added to each well, the washing solution was discarded, and the plate was washed continuously 4 times and then dried by pipetting.
11. Adding 100 μl of substrate working solution (substrate working solution A and substrate working solution B are mixed in equal amounts to obtain substrate working solution, and mixing immediately before use), shaking, mixing, placing in a 37 deg.C incubator, and reacting for 15min in dark place.
12. 50 μl of the color development stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well 450nm Value (the reaction plate with stop solution should read OD within 15 min) 450nm Values).
And (3) judging a detection result:
1. negative control OD 450nm The average value should be less than or equal to 0.15, otherwise, the method is ineffective.
2. The positive control should have a value between 1.0 and 2.5 per test, otherwise it is ineffective.
3. Calculating a critical value: critical value = 0.17 x positive control OD 450nm Average value of values.
Determination of OD by serum to be examined 450nm The value greater than or equal to the critical value is determined asPositive; determination of OD by serum to be examined 450nm Value of<The threshold value is judged as negative.
The kit provided by the invention can be used for detecting the African swine fever virus antibody so as to judge whether the African swine fever virus antibody generated after infection exists in the detected animal.
The application of the kit in preparing the kit for detecting whether the African swine fever virus is infected or not also belongs to the protection scope of the invention;
the invention has the positive effects that: the invention adopts bioinformatics method to accurately analyze the antigen epitope of African swine fever virus, and screens out peptide segments suitable for ELISA detection from main antigen epitope on p72 protein. The peptide segment integrates antigen epitope and has the advantages of high sensitivity and strong specificity.
Meanwhile, an advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, as the coating antigen used in the kit is a chemically synthesized polypeptide, the kit does not contain mixed protein, has high purity, and further improves the efficiency of detecting the African swine fever virus antibody so as to judge whether the detected animal is infected with the African swine fever virus.
In a word, the kit adopts an antigen peptide coating enzyme-linked reaction plate of a main antigen site of a chemically synthesized structural protein p72, has less antigen consumption, high sensitivity and strong specificity, and can effectively detect antibodies generated after African swine fever virus infection so as to judge whether an animal to be detected is infected with African swine fever virus. Experimental results show that the kit provided by the invention has the advantages of good repeatability, strong specificity and high sensitivity. Can meet the needs of people of different levels, and has wide market prospect and good economic and social benefits.
The diagnosis kit for the African swine fever virus ELISA test is used for detecting whether animals are infected with African swine fever virus, and is beneficial to the establishment of an African swine fever virus prevention and control system in China.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of African swine fever virus p72 protein ELISA antibody detection kit coated antigen the present test uses bioinformatics method to accurately analyze the main epitope of p72 protein of Pic/HLJ/2018 strain sequence (MK 333180.1) published by GenBank, screens out proper peptide segments, synthesizes three peptides with full-automatic polypeptide synthesizer, the sequences are shown as sequence 1, sequence 2 and sequence 3 in sequence table respectively, and prepares coated antigen with purity of about 80%, which can cover the main neutralization epitope of African swine fever virus, and improve the detection rate of antibody positive. The method for synthesizing the polypeptides can be a conventional method, and the three polypeptides of the invention are synthesized by the following method and serve as coating precursors of the kit.
The coated antigen of the present invention may be prepared using a Applied Biosystem full-automatic polypeptide synthesizer (model 433A). Fmoc (9-fluoroethylene carbonyl, 9-fluorenylmethoxycarbonyl) modified amino acid was used by Merrifield solid phase synthesis, using Rink Amide MBHA resin as solid phase carrier. The production process includes the steps of polypeptide antigen solid phase synthesis, polypeptide cleavage and identification, antigen purification, freeze drying and preservation. The following description will be given respectively:
1. solid phase synthesis of coated antigen
1. Preparation of synthetic reagents
The amino acid sequences of the synthesized coating antigen are shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3.
SEQ ID NO:1：NIRFKPWFIPGVINEISLTNNELYI
SEQ ID NO:2：FQDRDTALPDACSSISDISPVTYPIT
SEQ ID NO:3：TFALKPREEYQPSGHINVSR
Appropriate Fmoc modified amino acids (available from NOVA) were prepared according to the coating antigen sequence and the scale of synthesis and added to the corresponding Carridge. The resin 5g was weighed according to the required synthesis scale, placed in a reaction chamber, and the upper and lower lids were screwed down and labeled, and the name, lot number, TARE of the reaction chamber and the weight of the resin were recorded. The reaction chamber was loaded into the synthesizer. The appropriate amounts of synthetic reagents were formulated and placed into corresponding reagent bottles, including 100% NMP, 3% AIM (caproyl imidazole), 35% PIP (piperidine), 100% MeOH (methanol), etc.
2. Detection of synthesizer state
Checking 433A whether the polypeptide synthesis instrument runs normally, and running a Run Self Test program after starting up, wherein the instrument automatically checks whether various indexes are normal. Additionally, it was checked whether nitrogen was sufficient and the system gauge pressure was normal (433A normal gauge pressure 10.2 psi). The performance of the instrument should be known prior to synthesis, so the flow rate of each synthetic reagent is measured. 433A synthesizer: sending Flow Rate1-18 to synthesizer, selecting Main Menu-Module Test-find Module A, moduleD, moduleI, moduleI, module A according to Prer or next) -measure or observe according to Start-according to more, if the Flow is not proper, regulating the lower valve pressure until the requirement is met (specific detection requirement see Table 1 below).
TABLE 1 flow rate detection Standard table for polypeptide synthesizer
| Reagent(s) | Bottle number | Module | Standard range |
| 35％Piperidine | 1 | A | 1.0～1.2ml |
| 3％AIM | 4 | D | 1.0～1.2ml |
| 100％MeOH | 9 | I | 3.5～4.0ml |
| DIC | 8 | I | 0.45～0.55g |
| 100％NMP | 10 | A | 2.6～2.8ml |
3. Coated antigen synthesis begins
The amino acid sequence to be synthesized in the procedure of 433A synthesizer was sent Std Fmoc 1.0Sol DIC90 to the synthesizer. File-New-Sequence-edit Sequence of synthetic peptide, save. File-New-Run, check Chemistry for Std Fmoc 1.0Sol DIC 90; whether Sequence is a stored name; setting the Cycles; and (5) preserving. And finally, sending the obtained product to a synthesizer.
Main Menu-Cycle Monitor-begin, start running.
4. Coated antigen synthesis
The Fmoc group is removed, the electron-withdrawing effect of the fluorene ring system of the Fmoc group enables the 9-H to be acidic and easy to be removed by weak base, piperidine (PIP) is used for attacking the 9-H during reaction, beta is eliminated to form dibenzofluorenene, and the dibenzofluorenene is easy to be attacked by secondary cyclic amine to form a stable addition product. After removal of the Fnov group, the "-NH2" group is exposed for the synthesis reaction. The activated next Fmoc group protected amino acid and 1-Hydroxybenzotriazole (HOBT) were then added to the reactor.
The above polypeptide sequence is synthesized by sequentially repeating the synthesis steps from the C terminal to the N terminal according to a specific sequence (the synthesizer automatically completes the synthesis according to the program, and the specific circulation steps are shown in the following table 2). And (5) observing and recording the dosage and the running condition of the reagent during the process.
TABLE 2 coating antigen Synthesis cycle procedure
5. End of coating antigen synthesis
The synthesizer will automatically stop after the coated antigen synthesis is completed and the peptide resin (peptide now also attached to the resin) is essentially washed clean. Then the reactor is taken down from the polypeptide synthesizer, the peptide resin is washed 3 times by using 100 percent methanol, and then the peptide resin is dried in a fume hood, and then the peptide resin is completely transferred into a brown polyethylene bottle and is put into a refrigerator with the temperature of minus 20 ℃ and sealed by a sealing film for standby.
2. Cleavage and identification of coating antigen
1. Cleavage of polypeptide antigen
The polypeptide obtained by the above reaction is bound to the solid phase carrier by chemical bond, and the polypeptide must be separated from the solid phase carrier by acidolysis with a specific organic strong acid. The acid hydrolysis also removes the protecting groups on the functional groups of each amino acid. The method comprises the following steps:
the synthesized polypeptide resin (referring to the peptide also attached to the resin) was removed from the freezer and placed in a 2L round bottom flask, 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), 10ml Tripropylsilane (TIS) and magnetic stirrer were added to the flask in a fume hood, and the flask was then stably placed on a magnetic stirrer and stirred at room temperature for 1h until the reaction was complete. After the reaction is finished, the TFA in the crude product is removed by continuous evaporation for 30-120 min by using a rotary evaporator with a cold trap. And then, repeatedly cleaning the crude product of the polypeptide antigen by using Dimethylformamide (DMF), and finally, filtering the mixed resin by using a sand core funnel to obtain the coating antigen.
2. Identification of coating antigen
After the synthesis of the polypeptide antigen, qualitative and quantitative analysis is carried out by using matrix-assisted laser desorption/flight time mass spectrometry (MODAL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC), and the synthesized peptide is identified by using common amino acid analysis.
3. Coated antigen purification
The cyclized polypeptide antigen is ultrafiltered by using a circulating tangential filter membrane package (Tangential Flow Device circulating tangential filter membrane package produced by PALL company and a peristaltic pump matched with the circulating tangential filter membrane package), the polypeptide antigen can not pass through a filter membrane with a certain aperture as a macromolecule, and small molecular impurities formed or introduced in the early synthesis process and the later cyclization reaction can pass through the filter membrane. Then sterilizing by a filter with the aperture of 0.2 mu m, subpackaging the obtained solution into sterile plastic bottles, and labeling. The label is marked with the name, number, production lot number, concentration, production date, shelf life and preservation condition of the polypeptide, and the polypeptide is packaged and stored at-20 ℃ or-40 ℃ for standby.
4. Freeze drying of coated antigen
In order to facilitate long-term storage and transport, the coated antigen needs to be freeze-dried to obtain the polypeptide in a solid state. And (3) placing the pre-frozen coated antigen on a Labconco freeze dryer for drying to obtain the coated antigen in a solid state. After packaging, a label is attached, and the name, the number, the production lot number, the concentration, the production date, the storage life and the storage condition of the polypeptide are marked on the label.
Example 2 preparation of African swine fever Virus p72 protein ELISA antibody detection kit
The African swine fever virus p72 protein ELISA antibody detection kit comprises:
(1) A 96-hole removable polystyrene enzyme-linked reaction plate coated with African swine fever virus p72 protein antigen; 2X 96 wells.
(2) Positive control serum: african swine fever positive serum (CVCC No. Z286) purchased from China veterinary drug monitoring institute was properly diluted with sample dilution to serve as positive control serum for the kit (1 tube, 1.5 ml/tube).
(3) Negative control serum: pig serum without Specific Pathogen (SPF) was used as negative control serum for the kit (1 tube, 1.5 ml/tube).
(4) Enzyme-labeled secondary antibody: is prepared by diluting horseradish peroxidase-labeled rabbit anti-pig IgG (available from sigma company under the product number A5670) as a stock solution at a ratio of 1:30000, and 2 bottles (12 ml/bottle).
(5) Sample dilution: 1 bottle (24 ml/bottle) of phosphate buffer with a value of 7.4 of 0.01M, pH containing 5mg/ml casein.
(6) Substrate solution A: phosphate buffer (1 bottle, 12 ml/bottle) of citric acid containing 0.6mg/ml urea hydrogen peroxide
(7) Substrate solution B: a solution of 0.2mg/ml Tetramethylbenzidine (TMB) (1 bottle, 12 ml/bottle).
(8) Stop solution: 2mol/L sulfuric acid solution (1 bottle, 12 ml/bottle).
(9) 20-fold concentrated washing solution: phosphate buffer (50 ml/bottle, 2 bottles) containing Tween-20 at a concentration of 0.8% -1.2% (ml/ml) and having a pH of 7.4.
Serum dilution plates (2, 96 wells/block) can also be included in the kit as needed for dilution of serum samples.
The preparation method of the 96-hole removable polystyrene enzyme-linked reaction plate coated with the African swine fever virus p72 protein antigen comprises the following steps: 1. dissolving the polypeptide antigen prepared in the embodiment 1 in a carbonate solution with the pH value of 9.6, adding the solution to a 96-well polystyrene enzyme-linked reaction plate, adding 150ng of the polypeptide (seven groups of treatment are arranged, 150ng of the polypeptide shown in the sequence 1 in each well of ZM202301, 150ng of the polypeptide shown in the sequence 2 in each well of ZM202302, 150ng of the polypeptide shown in the sequence 3 in each well of ZM202303, 75ng of the polypeptide shown in the sequence 1 in each well of ZM202304 and 75ng of the polypeptide shown in the sequence 2 in each well of ZM202305, 75ng of the polypeptide shown in the sequence 2 in each well of 75ng of the polypeptide shown in the sequence 3 in each well of ZM202306, 50ng of the polypeptide shown in the sequence 2 in each well of the sequence 3 in each well of ZM 202307), placing the solution at the temperature of 2-8 ℃ for full binding the polypeptide antigen and the enzyme-linked reaction plate, adding the polypeptide containing 10mg/ml of bovine serum albumin (BSA at the temperature of 4 ℃ in each well of 300 mu l/well, sealing the solution at the temperature of 7.37 ℃ for sealing, sealing the solution, and preserving after the enzyme-linked reaction plate is dried.
Example 3 sensitivity test
1. Using method of African swine fever virus p72 protein ELISA antibody detection kit
1. Balance: taking the kit out of the refrigeration environment, and standing at room temperature for 30min for standby; the liquid reagent is mixed evenly before use.
2. Preparing liquid: diluting the concentrated washing solution with distilled water or deionized water for 20 times to obtain a washing buffer solution;
3. setting: 2 negative control wells and 2 positive control wells, the remainder being wells for samples to be tested.
4. Pre-diluting a sample to be tested: sample serum to be tested was prepared using sample dilutions according to 1: 20.
5. Sample adding: each well was pre-set with 100. Mu.l of diluted test sample. The time span of the sample adding process should be as short as possible.
6. Incubation: shaking and mixing uniformly, and placing in a 37 ℃ incubator for reaction for 30min.
7. Washing the plate: the reaction solution was discarded, 300. Mu.l of the diluted washing buffer was added to each well, the washing solution was discarded, and the plate was washed continuously 4 times and then dried by pipetting.
8. Adding enzyme: mu.l horseradish peroxidase was added to each well to label the rabbit anti-pig IgG antibody.
9. Incubation: placing in a 37 ℃ incubator for reaction for 30min.
10. Washing the plate: the reaction solution was discarded, 300. Mu.l of diluted washing buffer was added to each well, the washing solution was discarded, and the plate was washed continuously 4 times and then dried by pipetting.
11. Adding 100 μl of substrate working solution (substrate working solution A and substrate working solution B are mixed in equal amounts to obtain substrate working solution, and mixing immediately before use), shaking, mixing, placing in a 37 deg.C incubator, and reacting for 15min in dark place.
12. 50 μl of the color development stop solution was added to each well, and the reaction was stopped by shaking and mixing.
13. Determination of OD per well 450nm Value (the reaction plate with stop solution should read OD within 15 min) 450nm Values).
And (3) judging a detection result:
1. negative control OD 450nm The average value should be less than or equal to 0.15, otherwiseAnd (3) invalidating.
2. The positive control should have a value between 1.0 and 2.5 per test, otherwise it is ineffective.
3. Calculating a critical value: critical value = 0.17 x positive control OD 450nm Average value of values.
Determination of OD by serum to be examined 450nm If the value is more than or equal to the critical value, judging the value as positive; determination of OD by serum to be examined 450nm Value of<The threshold value is judged as negative.
2. Sensitivity test
Seven batches of African swine fever virus p72 protein ELISA antibody detection kits (batches ZM 202301-ZM 202307) prepared according to the method of the embodiment 2 are used for detecting 24 parts of pig serum to be detected, which is collected in a pig farm, respectively according to the method of using the African swine fever virus p72 protein ELISA antibody detection kits, the experimental results are shown in Table 3, 12 parts of African swine fever virus p72 protein ELISA antibody detection kits of ZM202301 batch numbers of the invention are totally detected, and the sensitivity of the kit to 24 parts of serum to be detected is 50%; 14 parts of African swine fever virus p72 protein ELISA antibody detection kit of ZM202302 batch number is detected, and the sensitivity of the kit to 24 parts of serum to be detected is 58.3%; 16 parts of African swine fever virus p72 protein ELISA antibody detection kit of ZM202303 batch number is detected, and the sensitivity of the kit to 24 parts of serum to be detected is 66.6%; the African swine fever virus p72 protein ELISA antibody detection kit of ZM202304 batch number detects 17 parts in total, and the sensitivity of the kit to 24 parts of serum to be detected is 70.8%; the African swine fever virus p72 protein ELISA antibody detection kit of ZM202305 batch number detects 20 parts in total, and the sensitivity of the kit to 24 parts of serum to be detected is 83.3%; the African swine fever virus p72 protein ELISA antibody detection kit of ZM202306 batch number detects 19 parts in total, and the sensitivity of the kit to 24 parts of serum to be detected is 79.2%; 22 parts of African swine fever virus p72 protein ELISA antibody detection kit of ZM202307 lot number disclosed by the invention is detected, and the sensitivity of the kit to 24 parts of serum to be detected is 95.8%.
TABLE 3 sensitivity test results
| Kit lot number | Detection rate of | Sensitivity to |
| ZM202301 (sequence 1) | 12/24 | 50％ |
| ZM202302 (sequence 2) | 14/24 | 58.3％ |
| ZM202303 (sequence 3) | 16/24 | 66.6％ |
| ZM202304 (SEQ ID NO: 1+SEQ ID NO: 2) | 17/24 | 70.8％ |
| ZM202305 (SEQ ID NO: 2+SEQ ID NO: 3) | 20/24 | 83.3％ |
| ZM202306 (SEQ ID NO: 1+SEQ ID NO: 3) | 19/24 | 79.2％ |
| ZM202307 (SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3) | 23/24 | 95.8％ |
Example 4 specificity test
20 parts of healthy pig serum, 2 parts of swine fever positive serum (CSF), 2 parts of pig foot-and-mouth disease virus type O (FMD-O) positive serum, and 2 parts of pig foot-and-mouth disease virus type A (FMD-A) positive serum were each tested according to the method of using the African swine fever virus p72 protein ELISA antibody test kit described in example 3 using the seven kits of example 2.
The specific detection results of the kit are shown in the following table (table 4), and the detection results of 20 healthy pig serum show that the specificity of all batches of the kit is 100.0%. The detection results of 2 swine fever positive serum CSF), 2 swine foot-and-mouth disease virus type O (FMD-O) positive serum and 2 swine foot-and-mouth disease virus type A (FMD-A) positive serum are all negative, so that the specificity of seven batches of kit for detecting 6 relevant pathogenic positive serum is 100%.
TABLE 4 specific detection results of African swine fever virus p72 protein ELISA antibody detection kit
Example 5 compliance test with import kit
40 parts of pig serum to be detected are detected by using the African swine fever virus p72 protein ELISA antibody detection kit prepared in the embodiment 3 and the African swine fever antibody detection kit of certain company in Spain.
The coincidence rate test results show (table 5), the sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202301) to 40 parts of pig serum to be detected is 40%, the sensitivity of the import kit is 72.5%, and the detection results of the two methods are 23 parts. Therefore, the compliance of the kit of the invention with the inlet kit was 57.5%.
The sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202302) to 40 parts of pig serum to be detected is 42.5%, the sensitivity of the imported kit is 72.5%, and the detection results of the two methods are 24 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 60%.
The sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202303) to 40 parts of pig serum to be detected is 45%, the sensitivity of the imported kit is 72.5%, and the detection results of the two methods are consistent and 25 parts. Therefore, the compliance of the kit of the invention with the inlet kit was 62.5%.
The sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202304) to 40 parts of pig serum to be detected is 52.5%, the sensitivity of the imported kit is 72.5%, and the detection results of the two methods are 26 parts in accordance. Therefore, the compliance of the kit of the invention with the inlet kit is 65%.
The sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202305) to 40 parts of pig serum to be detected is 62.5%, the sensitivity of the imported kit is 72.5%, and the detection result of the two methods is 31 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 80%.
The sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202306) to 40 parts of pig serum to be detected is 60%, the sensitivity of the imported kit is 72.5%, and the detection results of the two methods are consistent and are 29 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 77.5%.
The sensitivity of the African swine fever virus p72 protein ELISA antibody detection kit (batch number is ZM 202307) to 40 parts of pig serum to be detected is 75%, the sensitivity of the imported kit is 72.5%, and the detection results of the two methods are consistent and are 30 parts. Therefore, the coincidence rate of the kit and the imported kit is 90%, the accuracy of the detection result is high, and the kit can be used for detecting African swine fever antibodies.
Table 5 results of compliance test
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Claims (10)
1. The African swine fever virus P72 epitope polypeptide composition is any combination of two or more of a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table and a polypeptide shown in a sequence 3 in the sequence table.
2. The african swine fever virus P72 epitope polypeptide composition of claim 1, wherein: when the polypeptide composition is two of the polypeptide shown in the sequence 1 and the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3, the mass ratio of the two polypeptides is (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1, a step of; when the polypeptide composition is three of the polypeptide shown in the sequence 1, the polypeptide shown in the sequence 2 and the polypeptide shown in the sequence 3, the mass ratio of the three polypeptides is (0.5-1.5): (0.5-1.5): (0.5-1.5); preferably, they have a mass ratio of 1:1:1.
3. the African swine fever virus P72 epitope polypeptide is a polypeptide shown in a sequence 1 in a sequence table, a polypeptide shown in a sequence 2 in the sequence table or a polypeptide shown in a sequence 3 in the sequence table.
4. An African swine fever virus P72 protein ELISA antibody detection kit comprises an enzyme-linked reaction plate, positive control serum, negative control serum and an enzyme-labeled secondary antibody, wherein the enzyme-linked reaction plate is coated with the African swine fever virus P72 epitope polypeptide composition of claim 1 or 2 or the African swine fever virus P72 epitope polypeptide shown in claim 3.
5. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the ELISA plate is a detachable 96-hole ELISA plate; the African swine fever virus P72 epitope polypeptide is obtained through chemical and artificial synthesis.
6. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the method for obtaining the ELISA plate comprises the steps of dissolving the African swine fever virus P72 epitope polypeptide composition of claim 1 or 2 or the African swine fever virus P72 epitope polypeptide of claim 3 in 100 μl of a carbonate solution with the pH of 9.6, adding the solution into a 96-well polystyrene ELISA plate, placing 150ng of the African swine fever virus P72 epitope polypeptide composition of claim 1 or 2 or the African swine fever virus P72 epitope polypeptide of claim 3 in each well at the temperature of 2-8 ℃ for 8-12 hours, fully combining the African swine fever virus P72 epitope polypeptide composition with the ELISA plate, adding PBS buffer with the bovine serum albumin of 0.01g/ml at the pH of 7.4 according to 300 μl/well, sealing at the temperature of 37 ℃ for 2-3 hours, drying the ELISA plate, and hermetically preserving at the temperature of 4 ℃.
7. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the positive control serum is African swine fever virus antibody positive serum.
8. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the negative control serum is pig serum without specific pathogen; the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-pig IgG antibody.
9. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the kit also comprises a substrate solution A, a substrate solution B and a stop solution; the substrate solution A is a citric acid phosphate buffer solution containing 0.6mg/ml of urea hydrogen peroxide, the substrate solution B is a tetramethyl benzidine solution of 0.2mg/ml, and the two solutions are mixed in a ratio of 1:1 when in use; the stop solution is 2mol/L sulfuric acid solution.
10. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the kit also comprises a sample diluent and a 20-time concentrated washing liquid; the sample diluent is phosphate buffer solution with 0.01mol/L, pH value of 7.4 and containing 5mg/ml casein; the concentrated washing liquid is phosphate buffer solution with the pH value of 7.4 and 0.01mol/L of Tween-20 with the volume percentage concentration of 0.8-1.2%.
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