Porcine Reproductive and Respiratory Syndrome ELISA Antibody Detection Kit

Abstract

The present invention discloses a porcine reproductive and respiratory syndrome ELISA antibody detection kit and its application. The kit includes an enzyme-labeled plate, positive control serum, negative control serum, enzyme-labeled secondary antibody, sample diluent, 20-fold concentrated washing solution, substrate solution A, substrate solution B, and termination solution. Among them, the enzyme-labeled plate is coated with a porcine reproductive and respiratory syndrome antigen epitope polypeptide composition. The antigen epitope polypeptide composition is one or any combination of two or more of the polypeptides shown in Sequence 1 in the sequence listing, Sequence 2 in the sequence listing, and the polypeptide shown in Sequence 3 in the sequence listing. This kit uses chemically synthesized antigen peptides to coat the enzyme-labeled plate, with less antigen consumption, high sensitivity and specificity, and can efficiently detect whether there are porcine reproductive and respiratory syndrome antibodies. The kit of the present invention has high sensitivity, good specificity, and convenient operation, and has a good market prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and more specifically, the present invention relates to a kit for detecting Porcine reproductive and respiratory syndrome ELISA antibodies. Background Art

[0002] The pathogen of Porcine reproductive and respiratory syndrome (PRRS) is Porcine reproductive and respiratory syndrome virus (PRRSV). This disease mainly causes reproductive disorders in breeding pigs and severe respiratory diseases in piglets. It is a highly contagious viral infectious disease, commonly known as "Porcine blue ear disease" in China. After virus infection, breeding sows mainly show reproductive disorders such as anorexia, fever, abortion, weak piglets, and mummified fetuses. Infected piglets mainly show elevated body temperature, respiratory system diseases, high morbidity, and high mortality. After boars are infected, it can lead to decreased appetite, listlessness, and decreased semen quality. After fattening pigs are infected, most of the diseases are mild.

[0003] Porcine reproductive and respiratory syndrome seriously affects the global pig industry. In 1996, China first reported and isolated Porcine reproductive and respiratory syndrome virus. In order to better prevent and control the Porcine reproductive and respiratory syndrome epidemic in China, it is very important to conduct research on relevant diagnostic techniques.

[0004] Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to the genus Arterivirus of the family Arteriviridae and is an enveloped single-stranded positive-sense RNA virus. The genotypes of PRRSV vary widely and are currently mainly divided into two genotypes: PRRSV-1 (European genotype) and PRRSV-2 (North American genotype). The full-length genome of PRRSV is approximately 15 kb, with a cap structure at the 5' end and a polyA tail at the 3' end. Excluding the untranslated regions (UTR), it contains at least 10 open reading frames (ORFs) encoding 16 non-structural proteins and 7 structural proteins, which express nucleocapsid protein (N), membrane matrix protein (M), envelope glycoproteins (GP2 - GP5), and non-glycosylated envelope protein E. Research shows that N protein and GP5 protein are the main structural proteins. In particular, the N protein is the most abundant protein in PRRSV, containing 4 - 5 important antigenic domains, rich in epitopes that can be recognized by the immune system, and is often used as a target for the detection of virus-specific antibodies and disease diagnosis. GP5 is a glycosylated envelope protein and is the main part of the virus envelope. The GP5 protein contains multiple antigenic epitopes, including both linear epitopes and conformational epitopes, and also has neutralizing epitopes in the extracellular region. It is also involved in the adsorption and invasion of the virus into host cells. As a main structural protein of PRRSV, the GP5 protein plays an important role in the pathogenic and immune processes.

[0005] Currently, the serological detection techniques for PRRSV mainly include virus neutralization test (VNT), immunoperoxidase monolayer assay (IPMA), indirect immunofluorescent antibody test (IFA), and enzyme-linked immunosorbent assay (ELISA), etc. Among them, ELISA is widely used for the detection of PRRSV antibodies. Summary of the Invention

[0006] The purpose of the present invention is to provide an indirect ELISA detection kit for detecting porcine reproductive and respiratory syndrome antibodies. The kit uses antigenic epitope polypeptides of porcine reproductive and respiratory syndrome N protein and GP5 protein as the coating antigen, and establishes an indirect ELISA method with good specificity, sensitivity, and repeatability for detecting whether there are porcine reproductive and respiratory syndrome-specific antibodies in porcine serum.

[0007] To achieve the above object, the present invention first screened and obtained a porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition with excellent performance. The porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition provided by the present invention is one or any combination of two or more of the polypeptides shown in Sequence 1 in the sequence listing, the polypeptides shown in Sequence 2 in the sequence listing, and the polypeptides shown in Sequence 3 in the sequence listing. When the polypeptide composition is two of the polypeptides shown in Sequence 1, Sequence 2, and Sequence 3, the mass ratio of the two polypeptides is (0.5 - 1.5):(0.5 - 1.5); preferably, their mass ratio is 1:1; when the polypeptide composition is three of the polypeptides shown in Sequence 1, Sequence 2, and Sequence 3, the mass ratio of any three polypeptides is (0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5); preferably, their mass ratio is 1:1:1;

[0008] When the polypeptide composition consists of the polypeptides shown in Sequence 1, Sequence 2, and Sequence 3, their mass ratio is (0.5 - 1.5):(0.5 - 1.5):(0.5 - 1.5); preferably, their mass ratio is 1:1:1.

[0009] The present invention also claims protection for a porcine reproductive and respiratory syndrome antigenic epitope polypeptide, which is the polypeptide shown in Sequence 1 in the sequence listing, the polypeptide shown in Sequence 2 in the sequence listing, or the polypeptide shown in Sequence 3 in the sequence listing.

[0010] The porcine reproductive and respiratory syndrome ELISA antibody detection kit of the present invention includes an enzyme-linked reaction plate, positive control serum, negative control serum, enzyme-labeled secondary antibody, sample diluent, 20-fold concentrated washing solution, substrate solution A, substrate solution B, and termination solution, wherein the enzyme-linked reaction plate is coated with a porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition.

[0011] The enzyme-linked reaction plate is a detachable 96-well microtiter plate; the polypeptides in the porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition are all chemically artificially synthesized.

[0012] The best preparation method and conditions of the enzyme-linked reaction plate are to dissolve the porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition in 100 ul of carbonate solution with a pH of 9.6, and then add it to a 96-well polystyrene enzyme-linked reaction plate, 150 ng of the above polypeptide composition or the above polypeptide per well, place it at 2 - 8 °C for 8 - 12 hours to allow the polypeptide antigen to fully bind to the enzyme-linked reaction plate, and then add 300 μl / well of PBS buffer solution containing 10 mg / ml bovine serum albumin (BSA) with a pH of 7.4, and perform a blocking treatment at 37 °C for 2 - 3 hours. After centrifuging to dryness, store it sealed at 4 °C after the enzyme-linked reaction plate is dried.

[0013] The positive control serum is the highly immune serum prepared after multiple immunizations with the live porcine reproductive and respiratory syndrome vaccine, and is used as the positive control serum of the kit after being appropriately diluted with the sample diluent.

[0014] The negative control serum is the serum of healthy pigs (serum without porcine reproductive and respiratory syndrome antibodies).

[0015] The enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-pig IgG antibody.

[0016] The substrate solution A is a citrate phosphate buffer containing 0.6 mg / ml of urea hydrogen peroxide, and the substrate solution B is a 0.2 mg / ml solution of tetramethylbenzidine. When in use, the two are mixed in a ratio of 1:1. The stop solution is a 2 mol / L sulfuric acid solution.

[0017] The kit also includes a sample diluent and a 20-fold concentrated washing solution; the sample diluent is a 0.01 M phosphate buffer with a pH value of 7.4 containing 5 mg / ml of casein; the concentrated washing solution is a 0.01 M phosphate buffer with a pH value of 7.4 containing 0.8% - 1.2% (ml / ml) of Tween-20.

[0018] The detection procedure of the kit of the present invention is as follows:

[0019] 1. Equilibration: Take out the kit from the refrigerated environment, place it at room temperature for 30 min for standby; mix the liquid reagents before use.

[0020] 2. Preparation of solution: Dilute the concentrated washing solution 20-fold with distilled water or deionized water to obtain the washing buffer solution;

[0021] 3. Setting: Set up 2 negative control wells and 2 positive control wells, and the rest are wells for test samples.

[0022] 4. Pre-dilution of test specimens: Dilute the test sample serum, negative control serum, and positive control serum to be tested with the sample diluent in a ratio of 1:20.

[0023] 5. Sample addition: Add 100 μl of the diluted test sample to each well according to the pre-setting. The time span during the sample addition process should be as short as possible.

[0024] 6. Incubation: Mix well by shaking, place it in an incubator at 37 °C, and react for 30 min.

[0025] 7. Plate washing: Discard the reaction solution, add 300 μl of the diluted washing buffer solution to each well, soak for 15 s, discard the washing solution, and wash the plate continuously 4 times and then pat dry.

[0026] 8. Enzyme addition: Add 100 μl of horseradish peroxidase-labeled rabbit anti-pig IgG antibody to each well.

[0027] 9. Incubation: Place in an incubator at 37°C and react for 30 min.

[0028] 10. Plate washing: Discard the reaction solution, add 300 μl of diluted washing buffer to each well, soak for 15 s, discard the washing solution by shaking off, and pat dry after washing the plate 4 times continuously.

[0029] 11. Color development: Add 100 μl of substrate working solution to each well (mix equal amounts of substrate solution A and substrate solution B to obtain the substrate working solution, prepare it freshly before use), mix well by shaking, place in an incubator at 37°C, and react for 15 min in the dark.

[0030] 12. Add 50 μl of color development stop solution to each well and mix well by shaking to terminate the reaction.

[0031] 13. Measure the OD of each well 450nm value (the reaction plate with the stop solution added should be read for the OD 450nm value within 15 min).

[0032] Judgment of test results:

[0033] 1. The average OD of the negative control 450nm should be ≤ 0.15, otherwise it is invalid.

[0034] 2. Each test value of the positive control should be between 1.0 and 2.5, otherwise it is invalid.

[0035] 3. Calculation of the critical value: Critical value = 0.17 × average value of the OD of the positive control 450nm value.

[0036] The OD of the test serum to be measured 450nm value ≥ critical value is judged as positive; the OD of the test serum to be measured 450nm value < critical value is judged as negative.

[0037] The above-mentioned kit of the present invention can be used to detect antibodies against porcine reproductive and respiratory syndrome to determine whether the animals to be tested have antibodies against porcine reproductive and respiratory syndrome produced after wild virus infection or vaccine immunization.

[0038] The positive effects of the present invention are as follows: The present invention uses bioinformatics methods to accurately analyze the antigenic epitopes of porcine reproductive and respiratory syndrome, and screens out peptide segments suitable for ELISA detection from the main antigenic epitopes on the N protein and GP5 protein. This peptide segment concentrates the antigenic epitopes and has the advantages of high sensitivity and strong specificity.

[0039] At the same time, advanced solid-phase synthetic peptide technology is used to synthesize polypeptide antigens for the preparation of coated enzyme-linked reaction plates.

[0040] In addition, since the coated antigen used in the kit is a chemically synthesized polypeptide, free of heteroproteins and with high purity, it further improves the efficiency of detecting antibodies against porcine reproductive and respiratory syndrome, so as to determine whether the tested animal has antibodies against porcine reproductive and respiratory syndrome produced after wild virus infection or vaccine immunization.

[0041] In summary, this kit uses antigen peptides that chemically synthesize the main antigenic sites of N protein and GP5 protein to coat the enzyme-linked reaction plate. With less antigen consumption, high sensitivity, and strong specificity, it can effectively detect specific antibodies against porcine reproductive and respiratory syndrome to determine whether the tested animal has antibodies against porcine reproductive and respiratory syndrome produced after wild virus infection or vaccine immunization. The experimental results show that the kit of the present invention has good repeatability, strong specificity, and high sensitivity. It can meet the needs of personnel at different levels and has broad market prospects and good economic and social benefits.

[0042] The enzyme-linked immunosorbent assay diagnostic kit for porcine reproductive and respiratory syndrome involved in the present invention is used to detect whether the animal has antibodies against porcine reproductive and respiratory syndrome produced after wild virus infection or vaccine immunization, which is beneficial to the establishment of the prevention and control system for porcine reproductive and respiratory syndrome in China. Specific Embodiments

[0043] The methods in the following examples are all conventional methods unless otherwise specified.

[0044] Example 1. Preparation of the Coated Antigen of the ELISA Antibody Detection Kit for Porcine Reproductive and Respiratory Syndrome

[0045] In this experiment, bioinformatics methods were used to accurately analyze the main antigenic epitopes of the N protein and GP5 protein of Porcine reproductive and respiratory syndrome virus strain CH-1a (AY032626.1) published in GenBank, and appropriate peptide segments were screened out. Three peptides were synthesized respectively using an automatic polypeptide synthesizer, and the sequences are shown as Sequence 1, Sequence 2, and Sequence 3 in the sequence listing, to prepare a coated antigen with a purity of about 80%, which can cover the main neutralizing antigenic epitopes of porcine reproductive and respiratory syndrome and improve the detection rate of positive antibodies. The polypeptide synthesis method can be a conventional method. The following method is used in the present invention to synthesize the three polypeptides of the present invention as the coated antigen of the kit of the present invention.

[0046] The coated antigen of the present invention can be prepared using an Applied Biosystem fully automatic peptide synthesizer (model 433A). Using the Merrifield solid-phase synthesis method, Fmoc (9-fluorenylmethyloxycarbonyl) modified amino acids are used, and Rink Amide MBHA resin is used as the solid-phase carrier. The production process includes five parts: solid-phase synthesis of the peptide antigen, cleavage and identification of the peptide, purification of the antigen, freeze-drying, and preservation. The following is a description of each part:

[0047] I. Solid-phase synthesis of the coated antigen

[0048] 1. Preparation of synthesis reagents

[0049] The amino acid sequences of the coated antigens to be synthesized are shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.

[0050] SEQ ID NO:1: MSWRYSCTRYTNFLLDTKGRLYRWRSPVIVEKGGKVEVEGHLIDL;

[0051] SEQ ID NO:2: SRGKGPGKKNKKKSPEKPHFPLATEDDVRHHFTPSER;

[0052] SEQ ID NO:3: NGKQRKKKKGNGQPVN.

[0053] Prepare appropriate Fmoc-modified amino acids (purchased from NOVA Company) according to the coated antigen sequence and the synthesis scale, and add them to the corresponding Cartridge. Also, weigh 5 g of resin according to the required synthesis scale, put it into the reaction chamber, tighten the upper and lower lids, attach a label, and record the name, batch number of the synthesized peptide, the TARE of the reaction chamber, and the weight of the weighed resin. Install the reaction chamber into the synthesizer. Prepare an appropriate amount of synthesis reagents, including 100% NMP, 3% AIM (acetylimidazole), 35% PIP (piperidine), 100% MeOH (methanol), etc., and place them in the corresponding reagent bottles.

[0054] 2. Detection of the synthesizer status

[0055] Check whether the 433A polypeptide synthesizer is running properly. After powering on, run the Run Self Test program to check whether all indicators of the instrument self-test are normal. In addition, check whether the nitrogen is sufficient and whether the system gauge pressure is normal (the normal gauge pressure of 433A is 10.2 psi). Before synthesis, the performance of the instrument should be understood, so the flow rate of each synthesis reagent should be measured. 433A synthesizer: Send Flow Rate1-18 to the synthesizer, select Main Menu—Module Test—press Prer or next to find Module A, ModuleD, ModuleI, ModuleI, Module A)—press Start—press more to measure or observe. If the flow rate is inappropriate, adjust the valve pressure until the requirement is met (see Table 1 below for specific detection requirements).

[0056] Table 1 Flow Rate Detection Standard Table of Polypeptide Synthesizer

[0057] Reagent 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

[0058] 3. Coating antigen synthesis begins

[0059] In the program of the 433A synthesizer, send the amino acid sequence to be synthesized, Std Fmoc 1.0Sol DIC90, to the synthesizer. File - New - Sequence - edit the sequence of the synthetic peptide and save. File - New - Run, check whether Chemistry is Std Fmoc 1.0SolDIC 90; whether Sequence is the saved name; set Cycles; save. Finally, send it to the synthesizer.

[0060] Main Menu—Cycle Monitor—begin, start running.

[0061] 4. Coating antigen synthesis is in progress

[0062] Removal of the Fmoc group. The electron-withdrawing effect of the fluorenyl ring system of the Fmoc group makes 9-H acidic and easily removed by a weaker base. During the reaction, piperidine (PIP) attacks 9-H, and β-elimination forms diphenylfluorenylene, which is easily attacked by a secondary cyclic amine to form a stable adduct. After the removal of the Fmov group, the "-NH2" group is exposed for the synthesis reaction. Then add the next activated Fmoc group-protected amino acid and 1-hydroxybenzotriazole (HOBT) to the reactor.

[0063] For the polypeptide sequence as described above, during synthesis, it starts from the C-terminus to the N-terminus, and the synthesis steps are continuously repeated in a specific order (the synthesizer automatically completes according to the program, and the specific cycle steps are shown in Table 2 below). During this period, record the reagent usage and operation status.

[0064] Table 2 Coated Antigen Synthesis Cycle Steps

[0065]

[0066] 5. Completion of Coated Antigen Synthesis

[0067] After the synthesis of the coated antigen is completed, the synthesizer will automatically stop, and the peptide resin (the peptide is still attached to the resin) is basically washed clean. Then remove the reactor from the polypeptide synthesizer, wash the peptide resin 3 times with 100% methanol, dry it in the fume hood, then transfer all the polypeptide resin to a brown polyethylene bottle, put it in a -20°C refrigerator, and seal it with a sealing film for standby.

[0068] II. Cleavage and Identification of Coated Antigen

[0069] 1. Cleavage of Polypeptide Antigen

[0070] The polypeptide obtained through the above reaction is bound to the solid phase carrier through a chemical bond, and the polypeptide must be separated from the solid phase carrier by acidolysis with a specific strong organic acid. At the same time, the protecting groups on each amino acid functional group are removed. The steps are as follows:

[0071] Take out the synthesized polypeptide resin (referring to the peptide still attached to the resin) from the refrigerator, put it into a 2L round-bottom flask, add 90 ml of trifluoroacetic acid (TFA), 10 ml of tripropylsilane (TIS), and a magnetic stir bar into the flask in the fume hood, then steadily place the flask on a magnetic stirrer and continuously stir at room temperature for 1 h until the reaction is complete. After the reaction is over, use a rotary evaporator with a cold trap to continuously evaporate for 30 - 120 min to remove TFA from the crude product. Then wash the crude polypeptide antigen with dimethylformamide (DMF) multiple times, and finally filter out the mixed resin with a sintered glass funnel to obtain the coated antigen.

[0072] 2. Identification of Coated Antigen

[0073] After the synthesis of the polypeptide antigen is completed, matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) and reverse-phase high-performance liquid chromatography (RP-HPLC) are used for qualitative and quantitative analysis, and common amino acid analysis is used to identify the synthesized peptide.

[0074] 3. Purification of Coated Antigen

[0075] Ultrafiltrate the cyclized polypeptide antigen using a tangential flow filtration membrane cassette (Tangential Flow Device tangential flow filtration membrane cassette produced by PALL Corporation and the peristaltic pump supporting it). As a macromolecule, the polypeptide antigen cannot pass through a filter membrane with a certain pore size, while the small molecule impurities formed or introduced during the previous synthesis process and the subsequent cyclization reaction can pass through the filter membrane. Then, sterilize it through a 0.2 μm pore size filter, and dispense the finally obtained solution into sterile plastic bottles and label them. The label shall indicate the name, number, production batch number, concentration, production date, storage period, and storage conditions of the polypeptide. After dispensing, store it at -20 °C or -40 °C for standby.

[0076] 4. Freeze-dry the coated antigen

[0077] For long-term storage and transportation, it is necessary to freeze-dry the coated antigen to obtain a solid-state polypeptide. Place the pre-frozen coated antigen on a freeze dryer of Labconco for drying to obtain a solid-state coated antigen. After packaging, label it. The label shall indicate the name, number, production batch number, concentration, production date, storage period, and storage conditions of the polypeptide.

[0078] Example 2. Preparation of the positive control serum for the porcine reproductive and respiratory syndrome ELISA antibody detection kit

[0079] 1. The commercialized vaccine, live porcine reproductive and respiratory syndrome vaccine (R98 strain), is a product of China Animal Husbandry Industry Co., Ltd.

[0080] 2. Animal immunization The immunized animals are pigs, and they are immunized by intramuscular injection behind the ear root. Immunize them with the above-mentioned commercialized live porcine reproductive and respiratory syndrome vaccine. Immunize in 3 times, at 0, 3, and 6 weeks respectively. The immunization doses are 1 dose, 2 doses, and 4 doses respectively.

[0081] 3. Serum preparation Collect a small amount of blood, and measure the antibody titer using the IDEXX porcine reproductive and respiratory syndrome virus antibody detection ELISA kit. When the detected antibody titer of the porcine serum reaches a strong positive as detected by the IDEXX porcine reproductive and respiratory syndrome virus antibody detection ELISA kit, collect blood from the anterior vena cava of the pig, centrifuge and separate the serum (store it at -80 °C for standby). Further measure the titer by indirect ELISA, and dilute it appropriately with the sample diluent to make the OD value between 1.0 and 2.5, which is the positive control serum of the kit.

[0082] Example 3. Preparation of the porcine reproductive and respiratory syndrome ELISA antibody detection kit

[0083] The porcine reproductive and respiratory syndrome ELISA antibody detection kit includes:

[0084] (1) 96-well detachable polystyrene ELISA plates coated with synthetic peptide antigen of porcine reproductive and respiratory syndrome; 2 × 96 wells.

[0085] (2) Positive control serum: Prepared according to the method of Example 2, the hyperimmune serum prepared by immunizing with live porcine reproductive and respiratory syndrome vaccine multiple times is diluted with sample diluent and used as the positive control serum of the kit (1 tube, 1.5 ml / tube).

[0086] (3) Negative control serum: Healthy pig serum (serum without porcine reproductive and respiratory syndrome antibody) is used as the negative control serum of the kit (1 tube, 1.5 ml / tube).

[0087] (4) Enzyme-labeled secondary antibody: Prepared by diluting rabbit anti-pig IgG labeled with horseradish peroxidase (purchased from sigma company, product number A5670) as the stock solution at 1:50000, 2 bottles (12 ml / bottle).

[0088] (5) Sample diluent: 0.01M phosphate buffer solution with a pH value of 7.4 containing 5 mg / ml casein, 1 bottle (24 ml / bottle).

[0089] (6) Substrate solution A: Citrate phosphate buffer solution containing 0.6 mg / ml urea hydrogen peroxide (1 bottle, 12 ml / bottle)

[0090] (7) Substrate solution B: 0.2 mg / ml tetramethylbenzidine (TMB) solution (1 bottle, 12 ml / bottle).

[0091] (8) Stop solution: 2 mol / L sulfuric acid solution (1 bottle, 12 ml / bottle).

[0092] (9) 20-fold concentrated washing solution: 0.01M phosphate buffer solution with a pH value of 7.4 containing 0.8% - 1.2% (ml / ml) Tween-20 (50 ml / bottle, 2 bottles).

[0093] According to needs, the kit may also include serum dilution plates (2 pieces, 96 wells / piece) for diluting serum samples.

[0094] According to needs, the kit may also include a sample addition groove (1 piece) for holding various liquid reagents.

[0095] Among them, the preparation method of a 96-well detachable polystyrene ELISA plate coated with the synthetic peptide antigen of porcine reproductive and respiratory syndrome is as follows: 1. Dissolve the polypeptide antigen prepared in Example 1 in 100 μl of carbonate solution with a pH of 9.6, and then add it to the 96-well polystyrene ELISA plate, with 150 ng of polypeptide in each well (seven batches are set respectively. Among them, Kit ZM202301 adds 150 ng of the polypeptide shown in Sequence 1 in the sequence listing, Kit ZM202302 adds 150 ng of the polypeptide shown in Sequence 2 in the sequence listing, Kit ZM202303 adds 150 ng of the polypeptide shown in Sequence 3 in the sequence listing, 50 ng, Kit ZM202304 adds 75 ng of the polypeptide shown in Sequence 1 in the sequence listing and 75 ng of the polypeptide shown in Sequence 2 in the sequence listing, Kit ZM202305 adds 75 ng of the polypeptide shown in Sequence 2 in the sequence listing and 75 ng of the polypeptide shown in Sequence 3 in the sequence listing, Kit ZM202306 adds 75 ng of the polypeptide shown in Sequence 1 in the sequence listing and 75 ng of the polypeptide shown in Sequence 3 in the sequence listing, Kit ZM202307 adds 50 ng of the polypeptide shown in Sequence 1 in the sequence listing, 50 ng of the polypeptide shown in Sequence 2 in the sequence listing and 50 ng of the polypeptide shown in Sequence 3 in the sequence listing), place it at 2-8°C for 8-12 hours to allow the polypeptide antigen to fully bind to the ELISA plate, then add 300 μl / well of PBS buffer containing 10 mg / ml bovine serum albumin (BSA) with a pH of 7.4, perform a blocking treatment at 37°C for 2-3 hours, after draining, seal and store at 4°C after the ELISA plate is dried.

[0096] Example 4. Sensitivity Test

[0097] I. Usage Method of Porcine Reproductive and Respiratory Syndrome ELISA Antibody Detection Kit

[0098] 1. Equilibration: Take out the kit from the refrigerated environment, place it at room temperature for 30 min for standby; mix the liquid reagents well before use.

[0099] 2. Solution Preparation: Dilute the concentrated washing solution 20-fold with distilled water or deionized water to obtain the washing buffer; 3. Setting: Set 2 negative control wells and 2 positive control wells, and the rest are wells for test samples.

[0100] 4. Pre-dilution of Test Specimens: Dilute the test sample serum, negative control serum, and positive control serum to be tested with the sample diluent at a ratio of 1:20.

[0101] 5. Sample Addition: Add 100 μl of the diluted test sample to each well according to the pre-setting. The time span during the sample addition process should be as short as possible.

[0102] 6. Incubation: Vortex and mix well, place it in an incubator at 37°C, and react for 30 min.

[0103] 7. Plate washing: Discard the reaction solution. Add 300 μl of diluted washing buffer to each well, soak for 15 s, discard the washing solution, wash the plate 4 times continuously and then pat dry.

[0104] 8. Enzyme addition: Add 100 μl of horseradish peroxidase-labeled rabbit anti-pig IgG antibody to each well.

[0105] 9. Incubation: Place in an incubator at 37 °C and react for 30 min.

[0106] 10. Plate washing: Discard the reaction solution. Add 300 μl of diluted washing buffer to each well, soak for 15 s, discard the washing solution, wash the plate 4 times continuously and then pat dry.

[0107] 11. Color development: Add 100 μl of substrate working solution to each well (mix equal amounts of substrate solution A and substrate solution B to obtain the substrate working solution, prepare it freshly before use), shake well, place in an incubator at 37 °C, and react in the dark for 15 min.

[0108] 12. Add 50 μl of color development termination solution to each well, shake well to terminate the reaction.

[0109] 13. Measure the OD 450nm value of each well (the reaction plate with the termination solution added should be read within 15 min for the OD 450nm value).

[0110] Judgment of test results:

[0111] 1. The average OD 450nm value of the negative control should be ≤ 0.15, otherwise it is invalid.

[0112] 2. Each test value of the positive control should be between 1.0 and 2.5, otherwise it is invalid.

[0113] 3. Calculation of the critical value: Critical value = 0.17 × average OD 450nm value of the positive control.

[0114] The OD 450nm value of the serum to be tested is ≥ the critical value, which is judged as positive; the OD 450nm value of the serum to be tested < the critical value, which is judged as negative.

[0115] II. Sensitivity test

[0116] Using seven batches of porcine reproductive and respiratory syndrome ELISA antibody detection kits (batch numbers ZM202301 to ZM202307) prepared by the method of Example 3, 21 samples of porcine serum to be tested collected from pig farms were respectively detected according to the above-mentioned method for using the porcine reproductive and respiratory syndrome ELISA antibody detection kit. The experimental results are shown in Table 3. The porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202301 of the present invention detected a total of 11 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 52.4%; the porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202302 of the present invention detected a total of 13 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 61.9%; the porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202303 of the present invention detected a total of 9 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 42.9%; the porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202304 of the present invention detected a total of 18 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 85.7%; the porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202305 of the present invention detected a total of 16 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 76.2%; the porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202306 of the present invention detected a total of 17 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 81.0%; the porcine reproductive and respiratory syndrome ELISA antibody detection kit with batch number ZM202307 of the present invention detected a total of 20 samples, and the sensitivity of this kit to 21 samples of serum to be tested was 95.2%.

[0117] Table 3 Sensitivity test results

[0118] Kit Lot Number Detection Rate Sensitivity ZM202301 (Sequence 1) 11 / 21 52.4％ ZM202302 (Sequence 2) 13 / 21 61.9％ ZM202303 (Sequence 3) 9 / 21 42.9％ ZM202304 (Sequence 1 + Sequence 2) 18 / 21 85.7％ ZM202305 (Sequence 2 + Sequence 3) 16 / 21 76.2％ ZM202306 (Sequence 1 + Sequence 3) 17 / 21 81.0％ ZM202307 (Sequence 1 + Sequence 2 + Sequence 3) 20 / 21 95.2％

[0119] Example 5, Specificity test

[0120] Using the seven batches of kits in Example 2, 20 samples of healthy porcine serum, 2 samples of porcine classical swine fever positive serum (CSF), 2 samples of porcine circovirus type 2 (PCV2) positive serum, and 2 samples of foot-and-mouth disease virus (FMD) positive serum were respectively detected according to the method for using the porcine reproductive and respiratory syndrome ELISA antibody detection kit described in Example 3.

[0121] The specific detection results of the kit are shown in the following table (Table 4). The detection results of 20 healthy pig sera show that the specificity of all batches of the kit is 100.0%. The detection results of 2 classical swine fever positive sera (CSF), 2 porcine circovirus type 2 (PCV2) positive sera, and 2 foot-and-mouth disease virus (FMD) positive sera are all negative. Therefore, the specificity of the seven batches of the kit for the detection of these 6 relevant pathogen positive sera is 100%.

[0122] Table 4 Specific detection results of porcine reproductive and respiratory syndrome ELISA antibody detection kit

[0123]

[0124]

[0125] Example 6. Concordance test with imported kits

[0126] The porcine reproductive and respiratory syndrome ELISA antibody detection kit prepared in Example 3 and a porcine reproductive and respiratory syndrome antibody detection kit of a certain company in the United States were used to detect 45 pig sera to be tested respectively.

[0127] The results of the concordance test show (Table 5) that the sensitivity of the porcine reproductive and respiratory syndrome ELISA antibody detection kit (batch number ZM202301) for 45 pig sera to be tested is 42.2%, and the sensitivity of the imported kit is 84.4%. The results of the two methods are consistent for 24 samples. Therefore, the concordance rate of the kit of the present invention with the imported kit is 53.3%.

[0128] The sensitivity of the porcine reproductive and respiratory syndrome ELISA antibody detection kit (batch number ZM202302) for 45 pig sera to be tested is 48.9%, and the sensitivity of the imported kit is 84.4%. The results of the two methods are consistent for 25 samples. Therefore, the concordance rate of the kit of the present invention with the imported kit is 55.6%.

[0129] The sensitivity of the porcine reproductive and respiratory syndrome ELISA antibody detection kit (batch number ZM202303) for 45 pig sera to be tested is 42.2%, and the sensitivity of the imported kit is 84.4%. The results of the two methods are consistent for 24 samples. Therefore, the concordance rate of the kit of the present invention with the imported kit is 53.3%.

[0130] The sensitivity of the porcine reproductive and respiratory syndrome ELISA antibody detection kit (batch number ZM202304) for 45 pig sera to be tested is 68.9%, and the sensitivity of the imported kit is 84.4%. The results of the two methods are consistent for 34 samples. Therefore, the concordance rate of the kit of the present invention with the imported kit is 75.6%.

[0131] The sensitivity of the Porcine Reproductive and Respiratory Syndrome ELISA antibody detection kit (batch number ZM202305) to 45 porcine sera to be detected was 64.4%, and the sensitivity of the imported kit was 84.4%. The results of the two methods were consistent for 34 samples. Therefore, the coincidence rate of the kit of the present invention and the imported kit was 75.6%.

[0132] The sensitivity of the Porcine Reproductive and Respiratory Syndrome ELISA antibody detection kit (batch number ZM202306) to 45 porcine sera to be detected was 64.4%, and the sensitivity of the imported kit was 84.4%. The results of the two methods were consistent for 32 samples. Therefore, the coincidence rate of the kit of the present invention and the imported kit was 71.1%.

[0133] The sensitivity of the Porcine Reproductive and Respiratory Syndrome ELISA antibody detection kit (batch number ZM202307) to 45 porcine sera to be detected was 86.7%, and the sensitivity of the imported kit was 84.4%. The results of the two methods were consistent for 42 samples. Therefore, the coincidence rate of the kit of the present invention and the imported kit was 93.3%, and the accuracy of the detection results was relatively high, which could be used for the detection of antibodies against Porcine Reproductive and Respiratory Syndrome.

[0134] Table 5 Results of the coincidence rate test

[0135]

[0136]

Claims

1. A porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition, which is composed of the polypeptide shown in Sequence 1 in the sequence listing, the polypeptide shown in Sequence 2 in the sequence listing, and the polypeptide shown in Sequence 3 in the sequence listing.

2. The porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition according to claim 1, wherein: The mass ratio of the polypeptide shown in Sequence 1, the polypeptide shown in Sequence 2, and the polypeptide shown in Sequence 3 is (0.5~1.5):(0.5~1.5):(0.5~1.5).

3. The porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition according to claim 2, wherein: The mass ratio of the polypeptide shown in Sequence 1, the polypeptide shown in Sequence 2, and the polypeptide shown in Sequence 3 is 1:1:

1.

4. A porcine reproductive and respiratory syndrome ELISA antibody detection kit, which includes an enzyme-linked reaction plate, positive control serum, negative control serum, and enzyme-labeled secondary antibody, wherein the enzyme-linked reaction plate is coated with the porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition according to any one of claims 1-3.

5. The porcine reproductive and respiratory syndrome ELISA antibody detection kit according to claim 4, wherein: The enzyme-linked reaction plate is a detachable 96-well enzyme-labeled plate; the porcine reproductive and respiratory syndrome antigenic epitope polypeptide is obtained by chemical artificial synthesis.

6. The porcine reproductive and respiratory syndrome ELISA antibody detection kit according to claim 4, wherein: The method for obtaining the enzyme-linked reaction plate is to dissolve the porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition according to any one of claims 1-3 in a carbonate solution with a pH of 9.6, and then add it to a 96-well polystyrene enzyme-linked reaction plate, 150 ng of the porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition per well, and place it at 2-8 °C for 8-12 hours to allow the porcine reproductive and respiratory syndrome antigenic epitope polypeptide composition to fully bind to the enzyme-linked reaction plate. Then, add a PBS buffer solution containing 10 mg / ml bovine serum albumin with a pH of 7.4 at 300 μl / well, and perform a blocking treatment at 37 °C for 2-3 hours. After centrifuging to dryness, store it sealed at 4 °C after the enzyme-linked reaction plate is dried.

7. The porcine reproductive and respiratory syndrome ELISA antibody detection kit according to claim 4, wherein: The positive control serum is a hyperimmune serum prepared after multiple immunizations with the porcine reproductive and respiratory syndrome live vaccine.

8. The porcine reproductive and respiratory syndrome ELISA antibody detection kit according to claim 4, characterized in that: The negative control serum is healthy pig serum; the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-pig IgG antibody.

9. The porcine reproductive and respiratory syndrome ELISA antibody detection kit according to claim 4, characterized in that: The kit also includes substrate solution A, substrate solution B, and termination solution; substrate solution A is a citrate phosphate buffer solution containing 0.6 mg / ml hydrogen peroxide urea, substrate solution B is a 0.2 mg / ml tetramethylbenzidine solution, and when used, the two are mixed in a ratio of 1:1; the termination solution is a 2 mol / L sulfuric acid solution.

10. The porcine reproductive and respiratory syndrome ELISA antibody detection kit according to claim 4, wherein: The kit also includes a sample diluent and a 20-fold concentrated washing solution; the sample diluent is a phosphate buffer solution containing 5 mg / ml casein with a concentration of 0.01 mol / L and a pH value of 7.4; the concentrated washing solution is a phosphate buffer solution containing 0.8% - 1.2% (v / v) Tween-20 with a concentration of 0.01 mol / L and a pH value of 7.4.

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