CN116482356A - ELISA antibody detection kit for porcine reproductive and respiratory syndrome - Google Patents
ELISA antibody detection kit for porcine reproductive and respiratory syndrome Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses an ELISA antibody detection kit for porcine reproductive and respiratory syndrome and application thereof. 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 antigen epitope polypeptide composition of porcine reproductive and respiratory syndrome. The antigen epitope polypeptide composition is one or any combination of 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 antibodies of the porcine reproductive and respiratory syndrome exist. 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 ELISA antibody detection kit for porcine reproductive and respiratory syndrome.
Background
The pathogen of the porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS) is porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), which mainly causes the reproductive disorder of breeding pigs and the serious respiratory disease of piglets, is a highly infectious viral infectious disease, and is commonly called as 'porcine reproductive and respiratory syndrome' in China. After virus infection, breeding sows mainly show dysplasia such as anorexia, fever, abortion, weaning, mummy embryo and the like, and infected piglets mainly show body temperature rise, respiratory diseases, high morbidity and high mortality. The boar can cause appetite reduction, mental depression and semen quality reduction after infection, and most of fattening pigs have mild disease occurrence after infection.
Pig breeding and respiratory syndrome seriously affects the global pig industry, and in 1996, pig breeding and respiratory syndrome viruses are reported and separated for the first time in China, and in summer 2006, epidemic caused by highly pathogenic pig breeding and respiratory syndrome viruses (HP-PRRSV) is outbreaked, so that huge losses are brought to cultivation in China. At present, the epidemic situation of the pig breeding and respiratory syndrome in China is very complex, and the important immunosuppressive diseases seriously endangering the breeding industry in China are still very important for better preventing and controlling the epidemic situation of the pig breeding and respiratory syndrome in China and researching related diagnosis technologies.
PRRSV belongs to the genus Arterivirus (Arterivirus) of the arterividae (Arteriviridae), and is a enveloped single-stranded positive-strand RNA virus. There is a wide variation in PRRSV genotypes, which are currently largely divided into two genotypes: PRRSV-1 (european gene) and PRRSV-2 (north american genotype). PRRSV genome is about 15kb in full length, has a cap structure at the 5 'end and a polyA tail at the 3' end, removes the non-coding region (UTR), contains at least 10 ORFs, encodes 16 non-structural proteins and 7 structural proteins, and expresses nucleoprotein (N), membrane matrix protein (Membrane, M), envelope protein (GP 2-GP 5) and non-glycosylated envelope E protein (envelope protein, E), respectively. The research shows that the N protein and the GP5 protein are main structural proteins, especially the N protein is the protein with highest abundance in PRRSV, contains 4-5 important antigen structural domains, is rich in epitopes capable of being recognized by an immune system, and is often used as a target for virus specific antibody detection and disease diagnosis. GP5 is glycosylated envelope protein, is the main part of the envelope of virus, and GP5 protein contains a plurality of antigen epitopes, wherein the antigen epitopes have linear epitopes and conformational epitopes, and the antigen epitopes have neutralizing epitopes in extracellular regions, and are involved in the adsorption and invasion processes of the virus to host cells. The GP5 protein serves as the major structural protein of PRRSV and plays an important role in both pathogenic and immune processes.
Currently, serological detection techniques for PRRSV mainly include virus neutralization assay (Virus neutralization test, VNT), immunoperoxidase monolayer assay (immunoperoxidase monolayer assay, IPMA), indirect immunofluorescent antibody assay (indirect immunofluorescent antibody test, IFA)), and enzyme-linked immunosorbent assay (enzymelinked immunosorbent assay, ELISA), among others, wherein ELISA is widely used for detecting PRRSV antibodies.
Disclosure of Invention
The invention aims to provide an indirect ELISA detection kit for detecting antibodies of porcine reproductive and respiratory syndrome, which utilizes antigen epitope polypeptides of N protein and GP5 protein of porcine reproductive and respiratory syndrome as coating antigens, establishes an indirect ELISA method with good specificity, sensibility and repeatability, and is used for detecting whether the porcine reproductive and respiratory syndrome specific antibodies are contained in porcine serum.
In order to achieve the above purpose, the invention firstly screens out the epitope polypeptide composition of the porcine reproductive and respiratory syndrome, which has excellent performance, and the epitope polypeptide composition of the porcine reproductive and respiratory syndrome provided by the invention is any combination of one or more than two of the polypeptides shown in the sequence 1 in the sequence table, the polypeptides shown in the sequence 2 in the sequence table and the polypeptides shown in 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, a step of;
when the polypeptide composition consists 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 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 the epitope polypeptide of the porcine reproductive and respiratory syndrome, 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 ELISA antibody detection kit for the porcine reproductive and respiratory syndrome comprises an enzyme-linked reaction plate, positive control serum, negative control serum, enzyme-labeled secondary antibodies, 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 epitope polypeptide composition of the porcine reproductive and respiratory syndrome antigen.
The ELISA plate is a detachable 96-hole ELISA plate; the polypeptides in the antigen epitope polypeptide composition of the porcine reproductive and respiratory syndrome are obtained by chemical artificial synthesis.
The optimal preparation method and conditions of the ELISA plate are that the pig breeding and respiratory syndrome antigen epitope polypeptide composition is dissolved in 100ul of carbonate solution with pH of 9.6, then added into a 96-well polystyrene ELISA plate, 150ng of the polypeptide composition or the polypeptide is placed at 2-8 ℃ for 8-12 hours per well, polypeptide antigen and the ELISA plate are fully combined, then 300 ul/well of PBS buffer solution containing 10mg/ml Bovine Serum Albumin (BSA) pH7.4 is added, sealing treatment is carried out at 37 ℃ for 2-3 hours, and after spin-drying, the ELISA plate is dried and sealed and stored at 4 ℃.
The positive control serum is hyperimmune serum prepared after the porcine reproductive and respiratory syndrome live vaccine is immunized for a plurality of times, and is used as positive control serum of the kit after being properly diluted by a sample diluent.
The negative control serum was healthy pig serum (serum free of antibodies against porcine reproductive and respiratory syndrome).
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 detected, negative control serum and positive control serum are subjected to a sample dilution according to a ratio of 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 mixture was immersed for 15 seconds, the washing solution was discarded, and the plate was washed continuously for 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 the diluted washing buffer was added to each well, immersed for 15 seconds, the washing solution was thrown away, and the plate was washed continuously for 4 times and then dried by shaking.
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 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.
The kit can be used for detecting the porcine reproductive and respiratory syndrome antibody so as to judge whether the detected animal has the porcine reproductive and respiratory syndrome antibody generated after wild virus infection or vaccine immunization.
The invention has the positive effects that: the invention adopts bioinformatics method to accurately analyze the epitope of the porcine reproductive and respiratory syndrome, and screens out peptide segments suitable for ELISA detection from main epitope on N protein and GP5 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, the coating antigen used in the kit is a chemically synthesized polypeptide, does not contain mixed protein, has high purity, and further improves the efficiency of detecting the porcine reproductive and respiratory syndrome antibody so as to judge whether the detected animal has the porcine reproductive and respiratory syndrome antibody generated after wild virus infection or vaccine immunization.
In a word, the kit adopts an enzyme-linked reaction plate coated with antigen peptide of main antigen sites of chemically synthesized N protein and GP5 protein, has less antigen consumption, high sensitivity and strong specificity, and can effectively detect the specific antibody of the porcine reproductive and respiratory syndrome so as to judge whether the detected animal has the antibody of the porcine reproductive and respiratory syndrome generated after wild virus infection or vaccine immunization. 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 ELISA of the pig breeding and respiratory syndrome is used for detecting whether an animal has wild virus infection or pig breeding and respiratory syndrome antibodies generated after vaccine immunization, and is beneficial to the establishment of a pig breeding and respiratory syndrome prevention and control system in China.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of coated antigen for ELISA antibody detection kit for porcine reproductive and respiratory syndrome
The experiment adopts a bioinformatics method to accurately analyze main epitopes of N protein and GP5 protein of Porcine reproductive and respiratory syndrome virus strain CH-1a (AY 032626.1) published by GenBank, screens out proper peptide segments, synthesizes three peptides respectively by a full-automatic polypeptide synthesizer, and prepares coating antigens with the purity of about 80% respectively as shown by sequence 1, sequence 2 and sequence 3 in a sequence table, can cover main neutralizing epitopes of porcine reproductive and respiratory syndrome, and improves the detection rate of positive antibodies. 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：MSWRYSCTRYTNFLLDTKGRLYRWRSPVIVEKGGKVEVEGHLIDL；
SEQ ID NO:2：SRGKGPGKKNKKKSPEKPHFPLATEDDVRHHFTPSER；
SEQ ID NO:3：NGKQRKKKKGNGQPVN。
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
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 positive control serum for ELISA antibody detection kit for porcine reproductive and respiratory syndrome
1. Commercial vaccine live vaccine for porcine reproductive and respiratory syndrome (R98 strain), product of Zhongmu practice Co., ltd.
2. The animal immune animal is pig, the pig is immunized by intramuscular injection after auricular root, the commercial pig breeding and respiratory syndrome live vaccine is adopted for immunization, 3 times of immunization are respectively carried out at 0, 3 and 6 weeks, and the immunization doses are 1 part, 2 parts and 4 parts respectively.
3. The serum is prepared into a small amount for blood collection, an antibody titer is measured by using an ELISA kit for detecting the antibody of the American Edison porcine reproductive and respiratory syndrome virus, when the detected antibody of the serum of the pig reaches the strong positive value detected by the ELISA kit for detecting the antibody of the American Edison porcine reproductive and respiratory syndrome virus, the blood collection of the anterior vena cava is carried out on the pig, and the serum is centrifuged and separated for standby (-80 ℃). Further indirectly ELISA to measure titer, and properly diluting with sample diluent to make OD value between 1.0-2.5, namely positive control serum of the kit.
Example 3 preparation of ELISA antibody detection kit for porcine reproductive and respiratory syndrome
ELISA antibody detection kit for porcine reproductive and respiratory syndrome comprises:
(1) 96-hole removable polystyrene enzyme-linked reaction plate coated with synthetic peptide antigen of porcine reproductive and respiratory syndrome; 2X 96 wells.
(2) Positive control serum: the hyperimmune serum prepared by immunizing a live vaccine against porcine reproductive and respiratory syndrome for a plurality of times was diluted with a sample diluent as a positive control serum (1 tube, 1.5 ml/tube) of the kit according to the method of example 2.
(3) Negative control serum: is healthy pig serum (without pig breeding and respiratory syndrome antibody serum) and is used as negative control serum (1 tube, 1.5 ml/tube) of the kit.
(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 by 1:50000, 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.
According to the requirement, the kit can also be provided with 1 sample adding groove for containing each liquid reagent.
The preparation method of the 96-hole removable polystyrene enzyme-linked reaction plate coated with the synthetic peptide antigen of the porcine reproductive and respiratory syndrome comprises the following steps: 1. the polypeptide antigen prepared in example 1 was dissolved in 100ul of a carbonate solution having a pH of 9.6, and then added to a 96-well polystyrene enzyme-linked reaction plate, 150ng of polypeptide per well (seven batches were set up, respectively, wherein, kit ZM202301 was added with the polypeptide shown in sequence 1 in the 150ng sequence table, kit ZM202302 was added with the polypeptide shown in sequence 2 in the 150ng sequence table, kit ZM202303 was added with the polypeptide shown in sequence 3 in the 150ng sequence table, 50ng, kit ZM202304 was added with the polypeptide shown in sequence 1 in the 75ng sequence table and the polypeptide shown in sequence 2 in the 75ng sequence table, kit ZM202305 was added with the polypeptide shown in sequence 2 in the 75ng sequence table and the polypeptide shown in sequence 3 in the 75ng sequence table, adding 75ng of the polypeptide shown in the sequence 1 in the sequence table and 75ng of the polypeptide shown in the sequence 3 in the sequence table into the kit ZM202306, adding 50ng of the polypeptide shown in the sequence 1 in the sequence table, 50ng of the polypeptide shown in the sequence 2 in the sequence table and 50ng of the polypeptide shown in the sequence 3 in the sequence table into the kit ZM202307, placing the kit at 2-8 ℃ for 8-12 hours to fully combine the polypeptide antigen with an enzyme-linked reaction plate, adding a PBS buffer solution containing 10mg/ml Bovine Serum Albumin (BSA) pH7.4 according to 300 mu l/hole, sealing the kit at 37 ℃ for 2-3 hours, spin-drying the kit, and sealing the kit at 4 ℃ after the enzyme-linked reaction plate is dried.
Example 4 sensitivity test
1. Application method of ELISA antibody detection kit for porcine reproductive and respiratory syndrome
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 detected, negative control serum and positive control serum are subjected to a sample dilution according to a ratio of 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 mixture was immersed for 15 seconds, the washing solution was discarded, and the plate was washed continuously for 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 the diluted washing buffer was added to each well, immersed for 15 seconds, the washing solution was thrown away, and the plate was washed continuously for 4 times and then dried by shaking.
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 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 pig breeding and respiratory syndrome ELISA antibody detection kits (batches ZM 202301-ZM 202307) prepared according to the method of example 3 are used for detecting 21 parts of pig serum to be detected, which is collected in a pig farm, according to the method of using the pig breeding and respiratory syndrome ELISA antibody detection kit, the experimental results are shown in Table 3, 11 parts of pig breeding and respiratory syndrome ELISA antibody detection kits of ZM202301 batch number of the invention are detected in total, and the sensitivity of the kit to 21 parts of serum to be detected is 52.4%; 13 parts of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome, which is used for detecting the ZM202302 lot number, are detected, and the sensitivity of the kit to 21 parts of serum to be detected is 61.9%; 9 parts of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome, which is the ZM202303 lot number, are detected totally, and the sensitivity of the kit to 21 parts of serum to be detected is 42.9%; 18 parts of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome, which is used for detecting the ZM202304 lot number, are detected, and the sensitivity of the kit to 21 parts of serum to be detected is 85.7%; 16 parts of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome, which is the ZM202305 lot number, are detected totally, and the sensitivity of the kit to 21 parts of serum to be detected is 76.2%; the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome, ZM202306 lot number, disclosed by the invention, is used for detecting 17 parts in total, and the sensitivity of the kit to 21 parts of serum to be detected is 81.0%; the ELISA antibody detection kit for the ZM202307 batch number pig breeding and respiratory syndrome detects 20 parts, and the sensitivity of the kit to 21 parts of serum to be detected is 95.2%.
TABLE 3 sensitivity test results
| Kit lot number | Detection rate of | Sensitivity to |
| ZM202301 (sequence 1) | 11/21 | 52.4％ |
| ZM202302 (sequence 2) | 13/21 | 61.9％ |
| ZM202303 (sequence 3) | 9/21 | 42.9％ |
| ZM202304 (SEQ ID NO: 1+SEQ ID NO: 2) | 18/21 | 85.7％ |
| ZM202305 (SEQ ID NO: 2+SEQ ID NO: 3) | 16/21 | 76.2％ |
| ZM202306 (SEQ ID NO: 1+SEQ ID NO: 3) | 17/21 | 81.0％ |
| ZM202307 (SEQ ID NO: 1+SEQ ID NO: 2+SEQ ID NO: 3) | 20/21 | 95.2％ |
Example 5 specificity test
20 parts of healthy pig serum, 2 parts of swine fever positive serum (CSF), 2 parts of porcine circovirus type 2 (PCV 2) positive serum, 2 parts of porcine foot-and-mouth disease virus (FMD) positive serum were each tested according to the method of use of the ELISA antibody detection kit for porcine reproductive and respiratory syndrome described in example 3 using the seven-lot kit 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 parts of swine fever positive serum (CSF), 2 parts of porcine circovirus type 2 (PCV 2) positive serum and 2 parts of porcine foot-and-mouth disease virus (FMD) positive serum are all negative, so that the specificity of the seven batches of kit for detecting the 6 relevant pathogenic positive serum is 100%.
TABLE 4 ELISA antibody detection kit specific detection results for porcine reproductive and respiratory syndrome
Example 6 compliance test with import kit
45 parts of pig serum to be detected are detected by using the ELISA antibody detection kit for pig reproduction and respiratory syndrome, which is prepared in example 3, and the ELISA antibody detection kit for pig reproduction and respiratory syndrome of certain company in the United states.
The coincidence rate test results show (table 5), the sensitivity of the ELISA antibody detection kit for porcine reproductive and respiratory syndrome (batch number is ZM 202301) to 45 parts of pig serum to be detected is 42.2%, the sensitivity of the imported kit is 84.4%, 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 53.3%.
The sensitivity of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome (batch number is ZM 202302) to 45 parts of the porcine serum to be detected is 48.9%, the sensitivity of the imported kit is 84.4%, and the detection results of the two methods are consistent with each other by 25 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 55.6%.
The sensitivity of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome (batch number is ZM 202303) to 45 parts of pig serum to be detected is 42.2%, the sensitivity of the imported kit is 84.4%, and the detection results of the two methods are consistent and are 24 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 53.3%.
The sensitivity of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome (batch number is ZM 202304) to 45 parts of pig serum to be detected is 68.9%, the sensitivity of the imported kit is 84.4%, and the detection results of the two methods are 34 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 75.6%.
The sensitivity of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome (batch number is ZM 202305) to 45 parts of pig serum to be detected is 64.4%, the sensitivity of the imported kit is 84.4%, and the detection results of the two methods are 34 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 75.6%.
The sensitivity of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome (batch number is ZM 202306) to 45 parts of pig serum to be detected is 64.4%, the sensitivity of the imported kit is 84.4%, and the detection results of the two methods are consistent and are 32 parts. Therefore, the coincidence rate of the kit of the invention and the imported kit is 71.1%.
The sensitivity of the ELISA antibody detection kit for the porcine reproductive and respiratory syndrome (batch number ZM 202307) to 45 parts of the porcine serum to be detected is 86.7%, the sensitivity of the imported kit is 84.4%, and the detection results of the two methods are consistent and are 42 parts. Therefore, the coincidence rate of the kit and the imported kit is 93.3%, the accuracy of the detection result is high, and the kit can be used for detecting antibodies of porcine reproductive and respiratory syndrome.
Table 5 results of compliance test
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Claims (10)
1. The antigen epitope polypeptide composition of the porcine reproductive and respiratory syndrome is any combination of 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.
2. The porcine reproductive and respiratory syndrome epitope polypeptide composition of claim 1, wherein: when the polypeptide composition is one 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. an ELISA antibody detection kit for porcine reproductive and respiratory syndrome, which comprises an ELISA reaction plate, positive control serum, negative control serum and an ELISA secondary antibody, wherein the ELISA reaction plate is coated with the antigen epitope polypeptide composition for porcine reproductive and respiratory syndrome of claim 1 or 2 or the antigen epitope polypeptide for porcine reproductive and respiratory syndrome of claim 10.
4. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the ELISA plate is a detachable 96-hole ELISA plate; the antigen epitope polypeptide of the porcine reproductive and respiratory syndrome is obtained by chemical artificial synthesis.
5. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the method for obtaining the ELISA plate comprises the steps of dissolving the pig breeding and respiration syndrome antigen epitope polypeptide composition as claimed in claim 1 or 2 or the pig breeding and respiration syndrome antigen epitope polypeptide as claimed in claim 3 in a carbonate solution with the pH of 9.6, adding the solution into a 96-hole polystyrene ELISA plate, placing 150ng of the pig breeding and respiration syndrome antigen epitope polypeptide composition as claimed in claim 1 or 2 or the pig breeding and respiration syndrome antigen epitope polypeptide as claimed in claim 10 in each hole at 2-8 ℃ for 8-12 hours, fully combining the pig breeding and respiration syndrome antigen epitope polypeptide composition with the ELISA plate, adding a PBS buffer solution containing 10mg/ml bovine serum albumin with the pH of 7.4 according to 300 mu l/hole, sealing at 37 ℃ for 2-3 hours, drying the ELISA plate, and sealing and preserving at 4 ℃.
6. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the positive control serum is the hyperimmune serum prepared after the porcine reproductive and respiratory syndrome live vaccine is immunized for a plurality of times.
7. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: the negative control serum is healthy pig serum; the enzyme-labeled secondary antibody is a horseradish peroxidase-labeled rabbit anti-pig IgG antibody.
8. The enzyme-linked immunosorbent assay kit according to claim 3, wherein: 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.
9. 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%.
10. The antigen epitope polypeptide of the porcine reproductive and respiratory syndrome 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.
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