CN110669112A - Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus - Google Patents
Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus Download PDFInfo
- Publication number
- CN110669112A CN110669112A CN201911085713.1A CN201911085713A CN110669112A CN 110669112 A CN110669112 A CN 110669112A CN 201911085713 A CN201911085713 A CN 201911085713A CN 110669112 A CN110669112 A CN 110669112A
- Authority
- CN
- China
- Prior art keywords
- mouth disease
- foot
- enzyme
- disease virus
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- 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
- G01N33/54306—Solid-phase reaction mechanisms
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/32011—Picornaviridae
- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
- C12N2770/32122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Physics & Mathematics (AREA)
- Pathology (AREA)
- Organic Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses an enzyme-linked immunoassay kit for a swine foot-and-mouth disease virus A-type antibody. The kit comprises an enzyme-linked reaction plate coated by foot-and-mouth disease virus A-type antigen epitope polypeptide and an enzyme-labeled anti-antibody; the foot-and-mouth disease virus A-type epitope polypeptide is a polypeptide shown as a sequence 1 in a sequence table or a polypeptide shown as a sequence 2. The kit uses a chemically synthesized antigen peptide to coat the reaction plate, has small antigen dosage and high sensitivity and specificity, and can efficiently detect whether the foot-and-mouth disease virus infection exists. The kit has the advantages of good specificity, sensitivity, high efficiency and good market prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an enzyme-linked immunoassay kit for a foot-and-mouth disease virus A-type antibody, in particular to an enzyme-linked immunoassay kit for a swine foot-and-mouth disease virus A-type antibody.
Background
Foot-and-mouth disease (FMD) is a virulent infectious disease of artiodactyl animals caused by FMDV (FMDV) infection. Foot and mouth disease has a low mortality rate, but the infection rate is high. The main symptoms of the onset are: blisters occur on the oral mucosa, tongue, lip, nasoscope, hoof fork, nipple, etc., which are broken to form spots, and the edge of the hoof shell of the sick livestock is cracked or peeled off in the lying place and in the severe place. Different animals have slightly different symptoms, and pregnant cows can be aborted and then have reduced fecundity; the pig has broken hoof as the main symptom; goat and sheep are generally milder symptomatic than cattle. Foot and mouth disease infectivity is high, spread rapidly, infect pig, ox, sheep etc. livestock often lead to the death of young animal, adult animal's productivity drops sharply, seriously jeopardize the development of animal husbandry and the production supply of animal products. The animal disease is widely distributed in the world, can infect more than 70 kinds of domestic and wild animals, has extremely high incidence (about 100 percent), seriously affects the animal husbandry production and International trade, is highly valued by various countries, and is listed as a legal report animal disease by the world animal health Organization (OIE).
Foot-and-mouth disease virus (FMDV) is a member of the picornaviridae (picornavi-ridae), foot-and-mouth disease virus (aphthvirus) family, and consists of a single-stranded, plus-stranded RNA of about 8500 nucleotides in length and a capsid protein. The viral capsid is composed of 60 copies each of 4 structural proteins, VP1, VP2, VP3 and VP4, of which VP1 and VP3 are the major immunizing antigens. Foot and Mouth Disease Virus (FMDV) has the characteristics of polymorphism, changeability and the like, has 7 serotypes, namely A, O, C, SATI, SATII, SATIII and AsiaI types, each type can be divided into a plurality of subtypes, and more than 70 subtypes are discovered at present. Since antigenic drift occurs continuously, subtypes cannot be strictly distinguished. Almost no cross immune reaction exists among serotypes, and the antigenicity of different strains in the same type also changes to a certain extent, which brings great difficulty to the diagnosis and prevention of foot-and-mouth disease.
The prevention and control of foot-and-mouth disease is mainly vaccine immunization, slaughter infection and suspicious herd. The forced immunization is carried out on the foot-and-mouth disease in China, the centralized immunization is carried out once in spring and autumn every year, and the livestock in the newly-supplemented fence is timely supplemented and immunized between two times. In the actual production, how to identify and diagnose whether susceptible animals are subjected to vaccine immunization or infected with foot-and-mouth disease virus is very important for the establishment, inspection and quarantine of a foot-and-mouth disease epidemic prevention system. Serological techniques for the diagnosis of foot-and-mouth disease are Complement Fixation Test (CFT), enzyme linked immunosorbent assay (ELISA), Virus Neutralization Test (VNT), indirect hemagglutination test (IHA), etc., of which ELISA is the most commonly used method. The basic principle of ELISA is that an antigen or an antibody is adsorbed to a solid phase carrier, an immunoenzymatic reaction is performed on the carrier, and the result is determined by naked eyes or a spectrophotometer after the color development of a substrate. The ELISA method has the characteristics of specificity, sensitivity, rapidness, simplicity, good reliability and the like, can be automatically operated, can quickly detect a large number of samples, is increasingly emphasized by people in FMD diagnosis, and is one of the conventional methods for internationally detecting whether susceptible animals such as pigs and cows are immunized by vaccines or infected by viruses.
The indirect ELISA method is simple in process, easy to operate and high in sensitivity. De et al, Italy 1997, established an indirect capture ELISA based on FMDV, which uses monoclonal antibodies to capture FMDV expressed in E.coli, and used to detect a large number of vaccinated and infected animals, all challenged animals had OD values in ELISA greater than 0.2, and over 99% of vaccinated animals had negative ELISA (i.e., less than 0.2). The above data show that the method is highly sensitive. Specificity tests prove that the specificity of the method is over 99.5%. The method can detect the antibody 8 days after immunization, and can still detect the antibody after 1 year. An indirect ELISA for distinguishing FMDV infected animals from vaccine-immunized animals was established by Callicarpa nudiflora, etc. in 2003, and the ELISA uses FMDV3ABC expressed by Escherichia coli as an antigen. The test result proves that the ELISA method has higher accuracy than the virus infection-associated antigen agarose gel immunodiffusion test (VIAAAGID).
Foot-and-mouth disease is a major animal epidemic disease related to national economic safety, and related key technologies should be firmly mastered in hands of people. Once we have the independent intellectual property right, the economic safety of China can be guaranteed.
Disclosure of Invention
The invention aims to provide a novel enzyme-linked immunoassay kit for differential diagnosis of foot-and-mouth disease A-type antibodies.
Based on the above, the invention provides the A-type epitope polypeptide of the swine foot-and-mouth disease virus, which is a polypeptide shown as a sequence 1 or a polypeptide shown as a sequence 2 in a sequence table.
Further, the invention also provides a swine foot-and-mouth disease virus A-type antigen epitope polypeptide composition which is a polypeptide shown as a sequence 1 and a polypeptide shown as a sequence 2 in a sequence table.
The mass ratio of the polypeptide shown in the sequence 1 to the polypeptide shown in the sequence 2 is (0.5-1.5): (0.5 to 1.5); preferably, the mass ratio of the components is 1: 1.
furthermore, the enzyme-linked immunosorbent assay kit for the swine foot-and-mouth disease virus A-type antibody provided by the invention comprises an enzyme-linked immunosorbent assay plate coated by the swine foot-and-mouth disease virus A-type epitope polypeptide or the swine foot-and-mouth disease virus epitope polypeptide composition and an enzyme-labeled anti-antibody.
The related coating antigen is produced by adopting a solid-phase chemical synthesis method, contains main antigen site polypeptide of the porcine foot-and-mouth disease virus, and can be specifically combined with an antibody generated after the infection of the foot-and-mouth disease virus.
The labeled enzyme of the enzyme-labeled anti-antibody is horseradish peroxidase; the enzyme-labeled anti-antibody is an enzyme-labeled rabbit anti-pig IgG polyclonal antibody, and is prepared by diluting a commercial enzyme-labeled rabbit anti-pig IgG polyclonal antibody (purchased from sigma company, product number A5670) serving as a stock solution at a ratio of 1: 30000.
The enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the pig foot-and-mouth disease virus antigen epitope polypeptide is obtained by chemical artificial synthesis.
The invention is realized by the following modes: the detachable 96-hole polystyrene enzyme-linked reaction plate is coated by the swine foot-and-mouth disease virus A-type antigen epitope polypeptide or the swine foot-and-mouth disease virus A-type antigen epitope polypeptide composition. Meanwhile, the kit contains rabbit anti-pig IgG polyclonal antibody enzyme conjugate labeled by horseradish peroxidase, negative control serum, positive control serum, sample diluent, TMB substrate solution, 20-time concentrated washing solution, stop solution and other components. Detecting the antibody in the sample by an indirect enzyme-linked immunosorbent assay (ELISA) method.
The optimal preparation conditions of the involved enzyme-linked reaction plate are as follows: when coating, dissolving the polypeptide antigen in glycine-hydrochloric acid buffer solution with pH2.2, adding the solution into a 96-hole polystyrene enzyme-linked reaction plate, wherein 200ng of antigen is placed in each hole overnight (8-12 h) at 2-8 ℃ so that the polypeptide antigen is fully combined with the enzyme-linked reaction plate. Then adding a blocking solution (PBS buffer solution containing 1% (g/ml) of Bovine Serum Albumin (BSA) and 0.5% of casein (g/ml) and having pH of 7.4) into the mixture according to 300 mu l/hole, blocking the mixture at 37 ℃ for 2-3 h, washing the mixture, then drying the mixture, and sealing the mixture for storage at 2-8 ℃ after the enzyme-linked reaction plate is dried.
The kit contains a color development liquid and a stop solution; the color development solution consists of a substrate solution A and a substrate solution B, wherein the substrate solution A is a citrate phosphate buffer solution containing 0.06% (g/ml) of urea hydrogen peroxide, and the substrate solution B is a tetramethylbenzidine solution of 0.2 mg/ml; when in use, the ratio of the two is 1:1, and mixing. The stop solution is a 2mol/L sulfuric acid solution.
The kit also comprises negative control serum and positive control serum; the negative control serum is prepared by diluting healthy pig serum by 1:20 times with a sample diluent; the positive control serum is prepared by using a type A (AF/72 strain) for foot-and-mouth disease to challenge animals;
the related negative control serum is specifically obtained by isolating and observing healthy pigs for 7 days, aseptically collecting blood from the anterior vena cava, centrifuging and separating serum, inactivating at 56 ℃ for 30 minutes, diluting with a sample diluent at a ratio of 1:20 times after the serum is qualified, and aseptically and quantitatively subpackaging (1.5 ml/tube) to serve as the negative control serum of the kit.
The positive control serum is prepared by appropriately diluting a sample diluent according to the titer of positive stock serum. The positive stock serum is obtained by using afd/72 strain to challenge animal, monitoring serum antibody level 35 days after challenge, and determining whether the serum antibody level meets OD450nmWhen the value is more than or equal to 2.0, the anterior vena cava aseptically collects blood, centrifuges and separates serum, inactivates for 30 minutes at 56 ℃, and is used as positive stock serum after passing the inspection. The positive stock serum was diluted appropriately with a sample diluent (to make it satisfactory for OD detection by ELISA method)450nmThe value is between 0.9 and 2.0) and is used as the positive control serum of the kit.
The kit also comprises a sample diluent and a 20-time concentrated washing solution; the sample diluent was PBS buffer (filter diameter 0.2 μm filter) of pH 7.4 containing 0.5% (g/ml) casein; the 20-fold concentrated wash was PBS buffer pH 7.4 (sterilized through a 0.2 μm filter) containing 0.5% (ml/ml) Tween-20.
The detection program of the kit of the invention is as follows:
1. balancing: the kit is taken out from the refrigeration environment, is used after being balanced for 30 minutes at room temperature, and is uniformly mixed before the liquid reagent is used.
2. Before the washing solution is prepared and used, 20 times of the concentrated washing solution is recovered to room temperature, if precipitates exist, the concentrated washing solution is dissolved in water bath at 37 ℃, and then the concentrated washing solution is diluted by 20 times of deionized water for standby.
3. The sample is diluted in a serum dilution plate according to the proportion of 1:20 to be detected, and the negative and positive control serum is diluted and can be directly used.
4. Sample adding: and (3) taking an antigen coated plate, adding the diluted serum to be detected, a negative control and a positive control into the antigen coated plate, and keeping the concentration at 100 mu l/hole. Each serum to be detected is provided with 1 hole, the negative control and the positive control are respectively provided with 2 holes, and the time span of the sample adding process is required to be as short as possible.
5. And (3) incubation: shaking and mixing evenly, and incubating for 30 minutes in an incubator at 37 ℃.
6. Washing the plate: the reaction solution was discarded, 300. mu.l of diluted washing solution was added to each well, soaked for 15 seconds, the washing solution was discarded, the plate was washed continuously for 5 times, and then patted dry.
7. Adding an enzyme: mu.l of horseradish peroxidase labeled rabbit anti-pig IgG antibody was added to each well.
8. And (3) incubation: incubate at 37 ℃ for 30 minutes.
9. Washing the plate: discarding the reaction solution, adding 300 μ l of diluted washing solution into each hole, soaking for 15 seconds, throwing away the washing solution, continuously washing the plate for 5 times, and then drying by beating.
10. Color development: and adding 100 mul of substrate working solution (substrate working solution is prepared by mixing substrate solution A and substrate solution B in equal amount) into each hole, shaking and mixing uniformly, and placing in an incubator at 37 ℃ for reacting for 15 minutes in a dark place.
11. And (4) terminating: 50. mu.l of each chromogenic stop solution was added to each well, and the reaction was stopped by shaking and mixing.
12. And (3) determination: OD 450nm was measured in each well after zeroing with a blank control well.
Determination of the detection result of the kit of the present invention:
1. the negative control OD mean should be less than or equal to 0.15, otherwise it is not effective.
2. The positive controls should each be between 0.9 and 2.0, otherwise they are invalid.
3. Calculation of the critical value: the cut-off value was 0.17 × positive control mean OD value.
4. And (4) judging a result: the sample is judged to be positive if the OD value is larger than or equal to the critical value; determination of OD value of sample
If < critical value, the result is negative.
The kit can be used for detecting the foot-and-mouth disease virus A, and particularly can be used for detecting the pig foot-and-mouth disease virus caused by the pig foot-and-mouth disease virus A.
The invention has the positive effects that: the invention adopts a bioinformatics method to accurately analyze the epitope, screens out the peptide segment suitable for ELISA detection, and has the advantages of high sensitivity and strong specificity.
Meanwhile, the advanced solid-phase peptide synthesis technology is adopted to synthesize the polypeptide antigen for preparing the coated enzyme-labeled reaction plate.
In addition, because the coating antigen used in the kit is a chemically synthesized polypeptide, the kit does not contain hybrid protein, has high purity, and further improves the efficiency of detecting the foot-and-mouth disease antibody so as to judge whether the detected animal is infected with the foot-and-mouth disease virus.
In a word, the kit adopts a chemically synthesized antigen peptide to coat the reaction plate, has small antigen dosage and high sensitivity and specificity, and can efficiently identify whether the animals susceptible to the foot-and-mouth disease are infected with the virus or are immunized by the vaccine. The kit has the advantages of good specificity, sensitivity, high efficiency and good market prospect. The experimental result shows that the kit has good repeatability, strong specificity and high sensitivity. The method can effectively detect the antibodies generated after the foot-and-mouth disease virus infection so as to judge whether the detected animals are infected with the foot-and-mouth disease virus. Can meet the requirements of personnel at different levels, and has wide market prospect and good economic and social benefits.
The swine foot-and-mouth disease virus antibody enzyme-linked immunoassay kit is used for differential diagnosis of swine vaccine immunity and virus infection of animals susceptible to foot-and-mouth disease, is favorable for reducing the loss caused by foot-and-mouth disease outbreak in China, and is also favorable for establishing a foot-and-mouth disease prevention and control system in China.
Detailed Description
The methods in the following examples are conventional methods unless otherwise specified.
Example 1 preparation of envelope antigen of swine foot-and-mouth disease Virus type A antibody enzyme-linked immunoassay kit
The invention analyzes the sequence of the foot-and-mouth disease A type strain which is popular in recent years, precisely analyzes the epitope by adopting a bioinformatics method, and screens out the peptide segment which is suitable for ELISA detection, namely the foot-and-mouth disease virus A type epitope polypeptide from the main epitope, wherein the sequence of the peptide segment is shown as sequence 1 and sequence 2 in a sequence table. The polypeptide composition is used as a coating antigen of the kit to prepare the swine foot-and-mouth disease virus A-type antibody enzyme-linked immunoassay kit.
The coated antigen of the present invention can be prepared using an Applied Biosystem full-automatic polypeptide synthesizer (model 433A). The method applies a Merrifield solid phase synthesis method, adopts Fmoc (9-fluoromethylenecarbonyl, 9-fluorenylmethyloxycarbonyl) modified amino acid, and uses Rink Amide MBHA resin as a solid phase carrier. The production process comprises five parts of polypeptide antigen solid phase synthesis, polypeptide cracking and identification, antigen purification, freeze drying and storage. The following are described separately:
first, solid phase synthesis of envelope antigen
1. Preparation of synthetic reagents
The synthetic envelope antigen amino acid sequence is as follows:
sequence 1: SADPVTTTVATGESADPVRGDSGPLAA
Sequence 2: ATEIRELLVTVYSGTSKYAQLPASFNF
Appropriate Fmoc-modified amino acids (purchased from NOVA) were prepared according to the coating antigen sequence and synthesis scale and added to the corresponding amino acid vials. Similarly, 5g of the resin was weighed on the desired synthesis scale, placed in the reaction chamber, the upper and lower caps were tightened, labeled, and the name and batch number of the synthesized peptide, TARE in the reaction chamber, and the weight of the resin were recorded. The reaction chamber was loaded into the synthesizer. Preparing appropriate amount of synthetic reagent including N-methylpyrrolidone (NMP), hexanoyl imidazole (AIM), piperidine (PIP), methanol, etc. into corresponding reagent bottles.
2. Detection of synthesizer state
Checking whether the polypeptide synthesis instrument is normally operated. After the computer is started, a Run Self Test program is operated, and the instrument Self-checks whether each index is normal. In addition, whether the nitrogen is sufficient or not and whether the gauge pressure of the system is normal or not are checked. The flow rate of each synthesis reagent is measured because the performance of the instrument is known prior to synthesis. Sending Flow Rate1-18 to synthesizer, selecting MainMenu-Module Test-finding Module A, Module D, Module I, Module A) according to Prer or next-measuring or observing according to Start-more, if the Flow is not proper, adjusting the lower valve pressure until the requirement is reached.
3. Initiation of coating antigen Synthesis
The amino acid sequence to be synthesized was sent to the Std Fmoc 1.0Sol DIC90 on the synthesizer in the 433A synthesizer program. File-New-Sequence-editing the Sequence of the synthetic peptide, and storing. File-New-Run, check Chemistry; whether Sequence is the stored name; setting Cycles; and (5) storing. And finally sending the data to a synthesizer.
Main Menu-Cycle Monitor-begin, run.
4. Coating antigen synthesis
The polypeptide sequence is synthesized by starting from the C-terminal to the N-terminal, and repeating the following steps in sequence according to a given sequence:
(1) deprotection reaction: placing the amino resin in NMP solution of piperidine with the volume percentage of 15-30%, and reacting at 20-28 ℃ for 25-40 minutes to remove the Fmoc protective group on the amino resin;
(2) washing: drying with nitrogen, and washing amino resin with NMP;
(3) condensation reaction: adding HOBT, DIC and Fmoc protected amino acid, and reacting at 20-28 deg.C for 0.5-2.5 hr;
(4) washing: drying with nitrogen, and washing amino resin with NMP;
(5) and (3) blocking reaction: adding 1.5-4% by weight of an NMP solution of acetyl imidazole according to volume percentage, and reacting for 20-40 minutes at 20-28 ℃.
5. End of coating antigen Synthesis
The synthesizer will automatically stop after the synthesis of the coated antigen is complete and the peptide resin (peptide now also attached to the resin) is essentially washed clean. Then taking off the reactor from the polypeptide synthesizer, washing the peptide resin with 100% methanol for 3 times, drying in a fume hood, transferring all the polypeptide resin into a brown polyethylene bottle, placing into a refrigerator at-20 deg.C, and sealing with a sealing film for use.
Secondly, the cleavage and identification of the envelope antigen
1. Cleavage of polypeptide antigens
Preparing a lysate according to a volume ratio (trifluoroacetic acid (TFA)/Triisopropylsilane (TIS)/phenol/water (85/8/6/1)), taking out the synthesized polypeptide resin from a refrigerator, placing the polypeptide resin into a round-bottom flask, adding the prepared lysate and a magnetic stirrer into the flask in a fume hood, then stably placing the flask on a magnetic stirrer, and continuously stirring for 1 hour at room temperature until the reaction is completed. After the reaction is finished, the TFA in the crude product is removed by continuous evaporation for 30-120 minutes by using a rotary evaporator with a cold trap. And collecting and precipitating the polypeptide by using ether, washing a crude product of the polypeptide antigen for multiple times by using Dimethylformamide (DMF), and finally filtering the mixed resin by using a sand core funnel to obtain the polypeptide antigen.
2. Identification of coating antigens
After the polypeptide antigen is synthesized, qualitative and quantitative analysis is carried out by matrix-assisted laser desorption time of flight mass spectrometry (MODAL-TOF) and reversed-phase high-pressure liquid chromatography (RP-HPLC), and the synthesized peptide is identified by common amino acid analysis.
3. Purification of coating antigen
The cyclized polypeptide antigen is ultrafiltered by using a circulating Tangential filtration membrane (a Tangential Flow Device circulating Tangential filtration membrane manufactured by PALL company and a peristaltic pump matched with the circulating Tangential filtration membrane), the polypeptide antigen as a macromolecule can not pass through a filtration membrane with a certain pore diameter, and small molecular impurities formed or introduced in the early synthesis process and the later cyclization reaction can pass through the filtration membrane. Then sterilizing with 0.2 μm filter membrane, packaging the obtained solution into sterile plastic bottles, and labeling. The name, serial number, production lot number, concentration, production date, storage life and storage condition of the polypeptide are marked on the label, and the label is subpackaged and stored at-20 ℃ or-40 ℃ for later use.
4. Freeze drying of coated antigens
For long-term storage and transportation, 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 freeze dryer of Labconco for drying to obtain the coated antigen in a solid state. And (5) labeling after packaging. The name, number, production lot number, concentration, production date, shelf life and storage conditions of the polypeptide are indicated on the label.
Example 2 composition of enzyme-linked immunoassay kit for swine foot-and-mouth disease virus antibody
Evaluation of polypeptide epitope
In order to improve the positive detection rate of the detection method, 27 parts of swine foot-and-mouth disease type A virus infected serum are respectively detected by a conventional indirect ELISA method by respectively combining the screened epitope polypeptide sequence 1(Seq1), the sequence 2(Seq2) or the mixed peptide (MIX) of 2 mixed peptides as coating antigens (1.0 mu g/ml/peptide). The result shows that the positive detection rates of the sequence 1, the sequence 2 and the mixed peptide on 35 infected serums are 81.5%, 92.3% and 100%, respectively, and the result shows that the mixed peptide can completely detect the infected serums, and the positive detection rate is obviously higher than that of a single peptide, so that the mixed peptide mixed with 2 peptides is used as the coating antigen of the swine foot-and-mouth disease virus ELISA antibody detection kit.
Preparation of enzyme-linked immunoassay kit for swine foot-and-mouth disease virus antibody
The pig foot-and-mouth disease virus antibody enzyme-linked immunoassay kit comprises:
(1) the 96-hole detachable polystyrene enzyme linked reaction plate coated with the pig foot-and-mouth disease virus epitope chemosynthetic polypeptide, 96 holes/block, 2 blocks/box.
(2) Positive control serum: is prepared by preparing serum after animal is attacked by afd/72 strain, and diluting with sample diluent (to make it meet the requirement of detecting OD by ELISA method)450nmValue between 0.9 and 2.0) as positive control serum for the kit, 1 tube (1.5 ml).
(3) Negative control serum: is prepared by diluting healthy pig serum with sample diluent at a ratio of 1:20 times, and is used as negative control serum of the kit, and the volume of the kit is 1 tube (1.5 ml).
(4) Horseradish peroxidase-labeled rabbit anti-porcine IgG polyclonal antibody, 2 flasks (12ml each).
(5) Sample diluent: PBS buffer pH 7.4 containing 0.5% (g/ml) casein, filtered through 0.2 μm filter, 1 vial (24 ml).
(6) Substrate solution A: is a citrate phosphate buffer containing 0.06% (g/ml) of urea hydrogen peroxide, 1 bottle (12 ml).
(7) Substrate solution B: is a 0.2mg/ml Tetramethylbenzidine (TMB) solution in 1 vial (12 ml).
(8)20 times of concentrated washing solution: PBS buffer containing 0.5% (ml/ml) Tween-20pH 7.4, sterilized through 0.2 μm filter, 2 vials (50 ml each).
(9) Color development termination liquid: 2mol/L sulfuric acid solution, 1 bottle (12 ml).
(10) Serum dilution plate: the reaction plate is a detachable polystyrene enzyme-linked reaction plate, 96 holes/block and 2 blocks/box.
The preparation method of the 96-hole detachable polystyrene enzyme-linked reaction plate coated with the synthetic peptide coating antigen comprises the following steps: the polypeptide antigen prepared in example 1 is dissolved in glycine-hydrochloric acid buffer solution with pH2.2, and then added to a 96-well polystyrene enzyme-linked reaction plate, wherein 200ng of antigen (100 ng of polypeptide shown in sequence 1 in a sequence table and 100ng of polypeptide shown in sequence 2 in the sequence table) is added to each well, and the solution is placed at 2-8 ℃ overnight to allow the polypeptide antigen to be fully combined with the enzyme-linked reaction plate. Then adding a sealing solution (PBS buffer solution containing 1% (g/ml) of Bovine Serum Albumin (BSA) and 0.5% of casein (g/ml) and having pH of 7.4) into each hole by 300 mu l/hole, sealing for 2-3 h at 37 ℃, washing with a washing buffer solution (20 times of concentrated washing solution is diluted by distilled water or deionized water by 20 times to obtain the washing buffer solution), drying, and sealing and storing at 2-8 ℃ after the enzyme-linked reaction plate is dried.
Example 3 detection method of pig foot-and-mouth disease virus antibody ELISA kit
1.1 sample preparation animal whole blood is taken, after blood coagulation, centrifugation is carried out for 10 minutes at 3500r/min, supernatant is collected, blood can also be collected, after coagulation, serum is naturally separated out, and the serum is required to be clear without hemolysis.
1.2 before the washing solution is prepared, the 20 times of concentrated washing solution is returned to the room temperature, if the precipitate exists, the concentrated washing solution is dissolved in water bath at 37 ℃, and then the concentrated washing solution is diluted by 20 times of deionized water for standby.
1.3 dilution of the sample into a serum dilution plate, the serum to be detected is diluted according to the proportion of 1:20, and the negative and positive control serum is diluted and can be directly used.
1.4 operating procedure
1.4.1 adding sample and taking the antigen coated plate, adding the diluted serum to be detected, the negative control and the positive control into the antigen coated plate, and 100 mu l/hole. Each serum to be detected is provided with 1 hole, the negative control and the positive control are respectively provided with 2 holes, and the time span of the sample adding process is required to be as short as possible.
1.4.2 incubation, shaking and mixing (without overflow), and incubating in a 37 ℃ incubator for 30 minutes.
1.4.3 washing the plate, discarding the reaction solution, adding 300 μ l of washing solution into each hole, soaking for 15 seconds, throwing away the washing solution, continuously washing the plate for 5 times, and then patting dry.
1.4.4 enzyme addition 100. mu.l rabbit anti-porcine enzyme-labeled secondary antibody was added to each well.
1.4.5 incubation and 37 ℃ incubator incubation for 30 minutes.
1.4.6 washing the plate, discarding the reaction solution, adding 300 mu l of washing solution into each hole, soaking for 15 seconds, throwing away the washing solution, continuously washing the plate for 5 times, and then patting dry.
1.4.7 mu.l of substrate working solution (substrate working solution is obtained by mixing substrate solution A and substrate solution B in equal amount, the substrate working solution is prepared at present), shaking and mixing uniformly, and placing in a 37 ℃ incubator to react for 15 minutes in a dark place.
1.4.8 the reaction was stopped by adding 50. mu.l of a color developing stopping solution to each well, shaking and mixing, and the results were measured within 15 minutes.
2 determining the OD of each well on the microplate reader450nmThe value is obtained.
2.1 the conditions under which the test is true are: positive control well OD450nmThe value should be between 0.9 and 2.0, and the negative control well OD450nmThe values are all less than or equal to 0.15;
2.2 Cut-off value (Cut-off value) OD of Positive control well450nmMean × 0.17;
2.3 determination of OD in the serum to be examined450nmIf the value is larger than or equal to the critical value, judging the value as positive; determination of OD in serum to be examined450nmValue of<The critical value is judged to be negative.
2.4 when the kit is matched with an ELISA antibody detection kit for the structural protein corresponding to the foot-and-mouth disease of pigs, the final judgment is carried out according to the following standards.
Example 4 sensitivity test of enzyme-linked immunoassay kit for swine foot-and-mouth disease virus antibody
1. Sensitivity test
Using the three batches of swine foot-and-mouth disease virus antibody enzyme-linked immunoassay kit (batches ZmNS701, ZmNS702, and ZmNS703, antigens used) prepared in example 2, 200 swine serum infected with swine foot-and-mouth disease virus A was detected and the sensitivity of the kit was evaluated in accordance with the method of using the swine foot-and-mouth disease virus antibody enzyme-linked immunoassay kit in example 3.
The detection result of 200 parts of swine foot-and-mouth disease virus A-type infected serum shows that the positive detection rate of the kit is 99.5%.
2. Minimum detection limit test
Randomly drawing 10 parts of serum (including 4 parts of strong positive serum, 4 parts of weak positive serum and 2 parts of negative serum) and 1 part of sensitivity quality control serum for dilution in a multiple ratio, simultaneously detecting by using 3 batches of kits trial-produced in a laboratory and a foot-and-mouth disease virus antibody detection kit of Prionics corporation of Switzerland, detecting and comparing the minimum detection limit of the two, and displaying that the kit can detect the positive serum diluted by 1:320 times and the kit of Prionics corporation can detect the positive serum diluted by 1:160 times, so the kit has higher sensitivity.
Example 5 specificity test of ELISA kit for swine foot-and-mouth disease Virus antibody
The three kits in the embodiment 4 are used for detecting the positive sera of relevant pathogens such as swine fever, porcine circovirus, porcine reproductive and respiratory syndrome and the like according to the using method of the swine foot-and-mouth disease virus antibody enzyme-linked immunoassay kit in the embodiment 3, the results are all negative, and the results show that the sera of pigs infected with the diseases do not have cross reaction on the kit. The detection result of 300 healthy pig sera shows that the specificity of 3 kits is 99.0%. The detection result of 90 parts of swine aftosa A vaccine immune serum shows that the specificity of 3 kits is 97.8%. The above results prove that the kit has high specificity.
Example 6 shelf life test of enzyme-linked immunoassay kit for swine foot-and-mouth disease virus antibody
The actual storage life test operation of the pig foot-and-mouth disease virus antibody enzyme-linked immunoassay kit is as follows:
in 3 batches of swine foot-and-mouth disease virus antibody enzyme-linked immunoassay kits trial-produced in a laboratory, 12 kits are randomly extracted from each batch, stored at 2-8 ℃, and taken out for detection in 0, 3, 6, 9, 12 and 15 months respectively. The results show that: after the kit is stored at 2-8 ℃ for 0, 3, 6, 9, 12 and 15 months, the property and sterility test of the kit meet the test standard of the kit; the sensitivity test of the kit meets the standard; the specificity detection 18 specific quality control serums are all negative. In consideration of uncertain factors in practical application, in order to ensure that the kit can achieve the optimal use effect, the storage period of the kit is temporarily set to be 2-8 ℃ for 12 months.
Sequence listing
<110> Zhongmu industries GmbH
Enzyme-linked immunoassay kit for <120> swine foot-and-mouth disease virus A-type antibody
<130>WHOI190095
<170>Patent-In 3.5
<160>2
<210>1
<211>27
<212>PRT
<213> Artificial sequence (Artificial sequence)
<400>1
Ser Ala Asp Pro Val Thr Thr Thr Val Ala Thr Gly Glu Ser Ala Asp
1 5 10 15
Pro Val Arg Gly Asp Ser Gly Pro Leu Ala Ala
20 25
<210>2
<211>27
<212>PRT
<213> Artificial sequence (Artificial sequence)
<400>2
Ala Thr Glu Ile Arg Glu Leu Leu Val Thr Val Tyr Ser Gly Thr Ser
1 5 10 15
Lys Tyr Ala Gln Leu Pro Ala Ser Phe Asn Phe
20 25
Claims (10)
1. The A-type epitope polypeptide of the swine foot-and-mouth disease virus is a polypeptide shown as a sequence 1 or a polypeptide shown as a sequence 2 in a sequence table.
2. A pig foot-and-mouth disease virus A-type antigen epitope polypeptide composition is a polypeptide shown as a sequence 1 and a polypeptide shown as a sequence 2 in a sequence table.
3. The pig foot-and-mouth disease virus type A epitope polypeptide composition according to claim 2, wherein the mass ratio of the polypeptide shown in sequence 1 to the polypeptide shown in sequence 2 is (0.5-1.5): (0.5 to 1.5).
4. An enzyme-linked immunoassay kit for a swine foot-and-mouth disease virus A-type antibody comprises an enzyme-linked reaction plate coated by swine foot-and-mouth disease virus antigen epitope polypeptide and an enzyme-labeled anti-antibody; the pig foot-and-mouth disease virus epitope polypeptide is the pig foot-and-mouth disease virus epitope polypeptide composition of claim 1.
5. The kit of claim 4, wherein: the labeled enzyme of the enzyme-labeled anti-antibody is horseradish peroxidase; the enzyme-labeled anti-antibody is an enzyme-labeled rabbit anti-porcine IgG polyclonal antibody.
6. The kit according to claim 2 or 3, characterized in that: the enzyme-linked reaction plate is a detachable 96-hole enzyme label plate; the foot-and-mouth disease virus antigen epitope polypeptide is obtained by chemical artificial synthesis.
7. The kit according to claim 3 or 4, characterized in that: the method for obtaining the enzyme-linked reaction plate coated by the swine foot-and-mouth disease virus A-type epitope polypeptide composition comprises the steps of dissolving the swine foot-and-mouth disease virus A-type epitope polypeptide in 100 mu l of glycine-hydrochloric acid buffer solution with the pH value of 2.2, adding the solution into a 96-hole polystyrene enzyme-linked reaction plate, placing 200ng of the swine foot-and-mouth disease virus A-type epitope polypeptide composition in each hole at the temperature of 2-8 ℃ for 8-12 hours to enable the swine foot-and-mouth disease virus epitope polypeptide composition to be fully combined with the enzyme-linked reaction plate, then adding PBS buffer solution containing 1% (g/ml) of bovine serum albumin with the pH value of 7.4 into each hole according to 300 mu l, sealing and processing at the temperature of 37 ℃ for 2-3 hours, washing, drying, and sealing and storing at the temperature of 2-8 ℃ after the enzyme-.
8. The kit according to any one of claims 1 to 5, characterized in that: the kit also contains a developing solution and a stop solution; the color development solution consists of a substrate solution A and a substrate solution B, wherein the substrate solution A is a citrate phosphate buffer solution containing 0.06% (g/ml) of urea hydrogen peroxide, and the substrate solution B is a tetramethylbenzidine solution of 0.2 mg/ml; the stop solution is a 2mol/L sulfuric acid solution.
9. The kit of claim 6, wherein: the kit also comprises negative control serum and positive control serum; the negative control serum is prepared by diluting healthy pig serum by 1:20 times with a sample diluent; the positive control serum is prepared by properly diluting serum prepared by attacking animals with foot-and-mouth disease A type suckling mouse toxin with sample diluent; and/or, the kit further comprises a sample diluent and a 20-fold concentrated washing solution; the sample diluent was PBS buffer pH 7.4 containing 0.5% (g/ml) casein; the 20-fold concentrated washing solution was 0.01mol/L, pH 7.4, and contained 0.5% (ml/ml) of Tween-20 in phosphate buffer.
10. The use of the polypeptide of claim 1 in the preparation of a kit for detecting infection with animal type a foot and mouth disease virus; wherein the animal A-type foot-and-mouth disease virus disease is a pig A-type foot-and-mouth disease virus disease.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911085713.1A CN110669112B (en) | 2019-11-08 | 2019-11-08 | Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911085713.1A CN110669112B (en) | 2019-11-08 | 2019-11-08 | Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110669112A true CN110669112A (en) | 2020-01-10 |
| CN110669112B CN110669112B (en) | 2020-12-01 |
Family
ID=69086549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911085713.1A Active CN110669112B (en) | 2019-11-08 | 2019-11-08 | Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110669112B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103193869A (en) * | 2013-03-29 | 2013-07-10 | 中国农业科学院兰州兽医研究所 | Cattle food-and-mouth disease virus A type synthetic peptide and preparation and application thereof |
| CN106432434A (en) * | 2016-08-29 | 2017-02-22 | 中牧实业股份有限公司 | Enzyme-linked immunosorbent assay kit for detecting foot and mouth disease A-type structural protein VP1 antibody |
| CN107513101A (en) * | 2017-09-18 | 2017-12-26 | 中牧实业股份有限公司 | Swine foot-and-mouth disease virus non-structural protein antibody ELISA immunity detection reagent |
-
2019
- 2019-11-08 CN CN201911085713.1A patent/CN110669112B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103193869A (en) * | 2013-03-29 | 2013-07-10 | 中国农业科学院兰州兽医研究所 | Cattle food-and-mouth disease virus A type synthetic peptide and preparation and application thereof |
| CN106432434A (en) * | 2016-08-29 | 2017-02-22 | 中牧实业股份有限公司 | Enzyme-linked immunosorbent assay kit for detecting foot and mouth disease A-type structural protein VP1 antibody |
| CN107513101A (en) * | 2017-09-18 | 2017-12-26 | 中牧实业股份有限公司 | Swine foot-and-mouth disease virus non-structural protein antibody ELISA immunity detection reagent |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110669112B (en) | 2020-12-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110618279B (en) | African swine fever virus epitope antigen polypeptide and application thereof | |
| CN109627293B (en) | Seikaga valley virus structural protein epitope polypeptide and application thereof | |
| CN110642925B (en) | African swine fever virus synthetic peptide ELISA antibody detection kit | |
| CN109485703B (en) | Foot-and-mouth disease A-type structural protein VP1 antigen epitope polypeptide and application thereof | |
| CN103864906B (en) | Foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent | |
| CN111961120B (en) | African swine fever virus MGFs and CD2v ELISA antibody detection kit | |
| CN107513101B (en) | Pig foot-and-mouth disease virus non-structural protein antibody enzyme-linked immunoassay kit | |
| CN103408641B (en) | Foot and mouth disease virus structural protein antibody enzyme-linked immunosorbent assay kit | |
| CN104478998B (en) | Swine foot-and-mouth disease virus nonstructural protein 3A BC antibody ELISA immunity detection reagents | |
| CN109627292B (en) | Porcine circovirus type 3 epitope polypeptide and application thereof | |
| CN109239355B (en) | Enzyme-linked immunoassay kit for avian influenza virus H9 subtype antibody | |
| Yoon et al. | Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses | |
| CN104961809B (en) | The O-shaped Structural protein VP1 antibody ELISA immunity detection reagent of ox aftosa | |
| CN108802384B (en) | ELISA antibody detection kit for porcine circovirus type 2 synthetic peptide | |
| CN110669112B (en) | Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus | |
| CN107991481A (en) | It is a kind of to detect porcine pseudorabies virus and the bigeminy blocking ELISA antibody assay kits of foot and mouth disease virus and its application | |
| CN107991499B (en) | Enzyme-linked immunoassay kit for porcine parvovirus structural protein antibody | |
| CN112213493A (en) | Peste des petits ruminants detection kit capable of distinguishing vaccine immunity from natural infection | |
| CN117143205A (en) | ELISA antibody detection kit and application thereof | |
| CN115993449A (en) | Niu Jiejie skin disease virus ELISA antibody detection kit | |
| CN113999293B (en) | Antibody specifically binding with novel coronavirus S protein and application thereof | |
| SE470074B (en) | Method for diagnosis of picorene and / or flavivirus infection, peptides, diagnostic antigens and vaccine composition with these peptides | |
| CN116165379B (en) | Swine fever virus identification and detection kit and preparation method and application thereof | |
| CN117209569A (en) | African swine fever virus p72 protein ELISA antibody detection kit | |
| CN117783523A (en) | Rabbit hemorrhagic disease virus 2 type structural protein VP60 antibody enzyme-linked immunosorbent assay kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |