CN109485703A - A kind of foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide and its application - Google Patents
A kind of foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide and its application Download PDFInfo
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Abstract
The invention discloses a kind of foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide and its applications.A kind of foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide is polypeptide shown in sequence 3 in polypeptide shown in sequence 2 or sequence table in sequence table.The present invention is coated with reaction plate using the above-mentioned VP1 Antigenic Peptide of chemical synthesis, and antigen dosage is few, sensitivity and specificity are high, can efficiently detect whether to exist infection or it is immune after the foot and mouth disease A-type virus Structural protein VP1 antibody that generates.Kit specificity of the present invention is good, sensitive, efficient, has good market prospects.
Description
The application is that application No. is " 201610751029.2 ", a kind of entitled " foot-and-mouth disease a type Structural protein VP1s
The divisional application of the patent application of antibody ELISA immunity detection reagent "
Technical field
The invention belongs to field of biotechnology, more particularly it relates to a kind of Foot-and-mouth disease antibody
Enzyme-linked immunologic detecting kit, especially foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent.
Background technique
Aftosa (Foot and Mouth Disease, FMD) is by foot and mouth disease virus (Foot-and-mouth
Disease virus, FMDV) caused by infect acute, hot, high degree in contact infectiousness based on artiodactyl and quickly remote
The animal epidemic of propagation, spread speed strong with infectiousness is fast, endangers serious and famous (Xie Qingge, 2004).World animal
Health organization (OIE) is classified as many animals that must be notified to and suffers from one of infectious disease altogether, and China is classified as a kind of animal and passed
It catches an illness.
Aftosa infectiousness is high, propagates rapidly, and the livestocks such as infection pig, ox, sheep often result in cub death, adults production
Ability sharply declines.The disease is propagated rapidly, can cause it is global be very popular, huge economic damage not only is caused to animal husbandry
It loses, and seriously affects the international trade and reputation of animals and animal product.Due to the significant damage of FMD, U.S.'s authority
" livestock contagious disease " is early in just statement in version in 1981: FMD is not only economy disease, while being also political epidemic disease.
Foot and mouth disease virus belongs to Picornaviridae, Hostis.By the 20 symmetrical capsid of face body of "false" and
Viral nucleic acid is constituted, and it is not stringent positive 20 face body that the entire shape of the virion, which is spherical,.Completely virion includes
Capsid and RNA two parts, capsid is by each 60 molecular compositions of 4 kinds of structural proteins such as VP1, VP2, VP3 and VP4.Structural protein VP1
It is largely exposed to virion surface, is to determine the main component of virus antigenicity, and stimulation body generates neutralizing antibody
Major protein.VP1 variability is maximum in 4 kinds of structural proteins, and VP2 is more conservative, and VP4 is most conservative.The function of viral capsid proteins
Can have: protection RNA degrades from nuclease；Identify particular cellular receptors, it is resolved that host range and tissue tropism；Determine disease
The antigenicity of poison；Release and transmitting viral RNA are entered in permissive cell by cell membrane；Guidance selection and packaging virus RNA.
The aftosa serum detection method that OIE in 2004 is announced has 3 kinds: virus neutralization tests (VNT), liquid phase block
ELISA (LPB-ELISA), solid phase competitive ELISA (SPC-ELISA), wherein virus neutralization tests is the most classical in 3 kinds of methods
Method, be evaluate other methods gold standard.1996, aftosa world reference laboratory established liquid phase blocking
ELISA method, by constantly improve and perfecting, it has also become the method for the extensive Serologic detection in various countries is exempted from for detecting animal
Epidemic disease antibody, assessment animal immune situation, progress serosurvey etc., the sensibility, specificity and stability of this method exist always
It gains public acceptance in the world, is to use most wide aftosa serum detection method at present.2001, aftosa world reference experiment
Room establishes solid phase competitive ELISA method, and this method is special other than the traditional advantage with Liquid-phase blocking ELISA method
Property it is more preferable, operate more simple (its operating time is only 3.5h), 2004 by OIE be asserted aftosa serum investigate most
New recommended method.
Aftosa is the great animal epidemic for being related to economic security of the country, currently, most developing countries and area
The prevalence of aftosa is all controlled by the method for vaccine inoculation.China prevention and control FMD mainly takes vaccine immunity and antibody detection
Based on Synthetical prevention policy, drove entirety antibody level is improved by vaccine inoculation to prevent the infection and propagation of aftosa.
Therefore quick diagnosis, pig and the ox Efficacy evaluation of infection of foot-and-mouth disease, natural infection and the antidiastole of vaccine immunity become
The Some Key Technologies of prevention and control aftosa.
Summary of the invention
The object of the present invention is to provide a kind of new based on enzyme-linked immunosorbent assay, for detecting pig, being in cow's serum
The no kit containing foot and mouth disease A-type virus structural proteins antibody.The present invention utilize VP1 structural proteins, establish a species specificity,
Sensibility and reproducible indirect ELISA method mention for pig, the detection of ox aftosa immune antiboidy and the diagnosis of infection antibody
For a kind of simple and effective new method, technical support is provided for aftosa Synthetical prevention.
In order to achieve the above object, the present invention screens the antigen epitope polypeptide being had excellent performance first, and the present invention mentions
The foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide of confession is polypeptide shown in sequence 1 in sequence table, sequence in sequence table
Polypeptide shown in sequence 3 in column 2 or sequence table.
Further, the present invention provides a kind of foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide composition,
Its effective component is shown in sequence 3 in the polypeptide of sequence 2 in polypeptide shown in sequence 1 in sequence table, sequence table and sequence table
One or more of polypeptide any combination.
Foot-and-mouth disease antibody ELISA immunity detection reagent of the invention, including foot and mouth disease A-type virus knot
The coated enzyme-linked reaction plate of structure albumen VP1 antigen epitope polypeptide and ELIAS secondary antibody；The foot and mouth disease A-type virus Structural protein VP1
Antigen epitope polypeptide is sequence 3 in polypeptide shown in sequence 2 in polypeptide shown in sequence 1 in sequence table, sequence table and sequence table
Shown in polypeptide.
In Foot-and-mouth disease antibody ELISA immunity detection reagent of the invention, sequence 1 in the sequence table
Shown in polypeptide, in sequence table in polypeptide shown in sequence 2 and sequence table the mass ratio of polypeptide shown in sequence 3 be 0.5~
2.0:0.5~2.0:1, preferably 1:1:1.Related envelope antigen is produced using the method for solid-state chemical reaction method, and coating is anti-
Major antigenic sites of the original containing Foot-and-mouth disease VP1, can with generate after mouth disease virus infection or after immune
Structural protein VP1 antibody specific bond.The marker enzyme of the ELIAS secondary antibody is horseradish peroxidase or alkaline phosphatase；It is described
ELIAS secondary antibody is that enzyme marks staphylococcal protein A；The ELIAS secondary antibody is preferably the staphylococcus A of horseradish peroxidase-labeled
Albumen.
The enzyme-linked reaction plate is detachable 96 hole elisa Plates；The foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide
It is obtained for chemistry is artificial synthesized.
The plate of the coated enzyme-linked reaction plate of foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide most preferably prepares item
Part is: the foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide being dissolved in the carbonate solution of the pH9.6 of 100 μ l, then
96 hole polystyrene enzyme-linked reaction plates are added to, every every peptide 50ng antigen in hole was placed at 37 DEG C of placements 2~4h, then 4~8 DEG C
Night combines polypeptide antigen sufficiently with enzyme-linked reaction plate, is then added according to 300 holes μ l/ and contains 1% (g/ml) bovine serum albumin
The PBS buffer solution (pH=7.4) of white (BSA), 37 DEG C of 2~3h of Seal treatment, with washing buffer (concentrated cleaning solution distilled water
Or 20 times of dilutions of deionized water obtain washing buffer) it dries after washing, it is sealed for 4 DEG C after enzyme-linked reaction plate is dry.
Contain developing solution and terminate liquid in the kit；When marker enzyme is horseradish peroxidase, developing solution is by developing the color
Liquid A liquid and developing solution B liquid composition, the developing solution A liquid are the citrate-phosphate buffer of the hydrogen peroxide urea containing 0.6mg/ml
Liquid；The developing solution B liquid is tetramethyl benzidine (TMB) solution of 0.2mg/ml, is mixed both when use with the ratio of 1:1；
When marker enzyme is alkaline phosphatase, developing solution is 4- nitrophenols phosphate buffer；The terminate liquid is the sulfuric acid of 2mol/L
Solution.
The kit further includes negative control sera, positive control serum；The negative control sera is with no mouth hoof
The normal swine serum of epidemic disease antibody；The positive control serum is more with the foot and mouth disease A-type virus Structural protein VP1 epitope
Peptide is the serum that immunogen immune pig obtains；
Related positive control serum is specifically with the envelope antigen of above-mentioned artificial chemistry synthesis (foot-and-mouth disease a type disease
Malicious Structural protein VP1 antigen epitope polypeptide) height is prepared as immunogene (be immunized 25~35 ages in days without specific pathogeny body pig) exempts from blood
Clearly, 0.2 μm of filter membrane degerming, the positive control serum as kit are crossed.
Related negative control sera is with the normal swine serum of no foot-and-mouth disease antibody, excessively 0.22 μm of filter membrane degerming, work
For kit negative control sera.
The kit further includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid is to contain 0.5% (g/ml) junket
The PBS buffer solution (crossing 0.22 μm of filter membrane) that 0.01M, pH of albumen are 7.4；Concentrated cleaning solution: 0.01M, pH 7.4 contains
The phosphate of 0.8%~1.2% (ml/ml, preferably 0.5%ml/ml) Tween-20 and 0.05% (g/ml) Sodium azide preservative
Buffer (passes through 0.22 μm of filter membrane degerming).
The detection program of kit of the present invention are as follows:
1, it balances: kit being taken out from cold storage environment, it is spare to set equilibrium at room temperature 30min；The preceding mixing of liquid reagent.
2, match liquid: the 20 times of dilutions of concentrated cleaning solution distilled water or deionized water are obtained into washing buffer；
3, set: 2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4, sample pre-dilution to be measured: use sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to the dilution proportion of 1:20.
5, be loaded: 2 negative control holes respectively add 100 μ l negative controls, and 2 Positive control wells respectively add 100 μ l positive controls；
Remaining hole is respectively by presetting plus the 100 diluted samples to be tested of μ l.Being loaded process time span should be short as far as possible.
6, incubate: concussion mixes, and sets in 37 DEG C of incubators or water-bath, reacts 30min.
7, board-washing: discarding reaction solution, and every hole adds the washing buffer after 300 μ l dilution, impregnates 15s, get rid of abandoning washing lotion.Continuously
It is patted dry after board-washing 4 times.
8, enzyme: each hole adds the staphylococcal protein A of 100 μ l horseradish peroxidase-labeleds.
9, it incubates: setting in 37 DEG C of incubators or water-bath, react 30min.
10, board-washing: discarding reaction solution, and the 300 μ l of washing buffer after dilution is added in every hole, impregnates 15s, gets rid of abandoning washing
Liquid.It is patted dry after continuous board-washing 4 times.
11, develop the color every hole is separately added into the substrate developing solution of 100 μ l (wherein solution A and solution B is with volume ratio for 1:1's
Ratio mixing).It covers, reacts 15min in 37 DEG C of insulating boxs；
12, every hole is separately added into 50 μ l terminate liquids and terminates reaction, mixes；
13, the OD in every hole is measured450(OD should be read to value in 15min by adding the reaction plate of terminate liquid450Value).
The judgement of testing result:
1, negative control OD450Average value should≤0.15, otherwise in vain.
2, each detected value of positive control should be between 0.9-2.3, otherwise in vain.
3, the calculating of critical value: cow's serum critical value=0.15 × positive control OD450It is worth average value
Swine serum critical value=0.18 × positive control OD450It is worth average value
Serum to be checked measures OD450Value >=critical value person is judged to the positive；Serum to be checked measures OD450Value < critical value person is judged to
It is negative.
Mentioned reagent box of the invention can be used for detecting foot-and-mouth disease a type virus structural protein antibody, to judge tested animal
The foot and mouth disease A-type virus Structural protein VP1 antibody generated with the presence or absence of infection or after being immunized, wherein animal aftosa viral disease
For pig, hostis pecoris disease, it is specifically as follows hoof-and-mouth disease viral disease caused by pig, ox foot and mouth disease A-type virus.
Application in the kit that preparation detects whether infection animal hoof-and-mouth disease viral disease also belongs to protection of the invention
Range；
The positive effect of the present invention is: the present invention is carried out using epitope of the bioinformatics method to structural proteins
Accurate Analysis filters out the peptide fragment of suitable ELISA detection from the Main Antigenic on VP1 albumen.The peptide fragment is concentrated
Epitope has the advantages that sensitivity height, high specificity.
Meanwhile it being tried using advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen for being coated with enzyme reaction plate and preparation
Positive control serum in agent box.
In addition, the envelope antigen as used in kit is chemically synthesized polypeptide, foreign protein, purity is high, into one are free of
Step improves the efficiency of detection aftosa structural proteins antibody, to judge whether tested animal infects foot and mouth disease virus or inoculation epidemic disease
Seedling.
In short, this kit is coated with integrated enzyme reaction using the Antigenic Peptide of chemical synthesis Structural protein VP1 major antigenic sites
Plate, antigen dosage is few, high sensitivity, high specificity, after mouth disease virus infection or inoculation inactivated vaccine can be effectively detected
The Structural protein VP1 antibody of generation, to judge whether tested animal infects foot and mouth disease virus or vaccine inoculation, with foot and mouth disease virus
The combination of non-structural protein antibody assay kit, can distinguish mouth disease virus infection animal and vaccine immunity animal.Experimental result
Show that kit of the invention is reproducible, high specificity, high sensitivity.The needs of different levels personnel are able to satisfy, are had wide
Wealthy market prospects and good economical, societal benefits.
Foot-and-mouth disease ELISA diagnostic kit according to the present invention is dynamic for detecting
Whether object infects foot and mouth disease virus or inoculation aftosa vaccine, advantageously reduces loss brought by the outburst of China's aftosa,
Be conducive to the foundation of China's aftosa prevention and control system.
Specific embodiment
Method in following embodiments is unless otherwise instructed conventional method.
The preparation of embodiment 1, foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent envelope antigen
This test is directed to the difference of domestic aftosa prevalence strain, using bioinformatics method to foot and mouth disease virus A type
The multiple hypotypes of VP1 structural proteins carry out Accurate Analysis, filter out suitable peptide fragment, are respectively synthesized out with full-automatic polypeptide synthetic instrument
According to 3 peptides of popular strain sequence design, sequence is respectively that purity is made shown in sequence 1 in sequence table, sequence 2 and sequence 3
The more full envelope antigen of about 90% update, can adapt to the characteristic that foot and mouth disease virus is constantly mutated, improves the inspection of antibody positive
Extracting rate.Polypeptide synthesis method can be conventional method, and the present invention synthesizes three polypeptides of the invention with the following method, as this hair
The coating antigen of bright kit.
Applied Biosystem full-automatic polypeptide synthetic instrument (model 433A) system can be used in envelope antigen of the invention
It is standby.With Merrifield solid-phase synthesis, using Fmoc (9-fluorenylmethyloxycarbonyl, 9- fluorenes first
Oxygen carbonyl) modification amino acid, use Rink Amide MBHA resin as solid phase carrier.Production process includes polypeptide antigen
Synthesis in solid state, polypeptide cleavage and identification, five parts of antigen purification, freeze-drying and preservation.It is illustrated individually below:
One, envelope antigen synthesis in solid state
1, the preparation of synthetic agent
Envelope antigen amino acid sequence is synthesized as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.
Prepare the amino acid (purchased from NOVA company) of suitable Fmoc modification according to envelope antigen sequence and synthesis scale,
It is added into corresponding Cartridge.Equally synthesis scale claims resin 5g as required, is put into reaction chamber, upper and lower lid is tightened,
Labelling records the title of synthesized peptide, lot number, the weight of the TARE of reaction chamber and alleged resin.Reaction chamber is packed into and is synthesized
Instrument.Prepare suitable synthetic agent include 100% NMP, 3% AIM (acyl imidazoles), 35% PIP (piperidines), 100%
MeOH (methanol) etc. be placed into corresponding reagent bottle.
2, the detection of synthesizer state
It checks 433A Peptide systhesis instrument whether normal operation, after booting, runs Run Self Test program, instrument is certainly
Whether normal examine indices.In addition check whether nitrogen is sufficient, whether normal (the normal gauge pressure of 433A of system gauge pressure
10.2psi).The performance of reply instrument is had gained some understanding before synthesis, so to be measured to the flow velocity of every kind of synthetic agent.433A
Synthesizer: Flow Rate1-18 is sent to synthesizer, Main Menu-Module Test-is selected to look for by Prer or next
Module A, ModuleD, ModuleI, ModuleI, Module A)-measured or observed by more by Start-, if
Flow is improper, then adjusts lower valve pressure, until reaching requirement (specific testing requirements see the table below 1).
1. Peptide synthesizer flow rate detection standard scale of table
| Reagent | Bottle number | Module | Critical field |
| 35%Piperidine | 1 | A | 1.0~1.2ml |
| 3%AIM | 4 | D | 1.0~1.2ml |
| 100%MeOH | 9 | I | 3.5~4.0ml |
| DIC | 8 | I | 0.45~0.55g |
| 100%NMP | 10 | A | 2.6~2.8ml |
3, envelope antigen synthesis starts
The amino acid sequence that will be synthesized in the program of 433A synthesizer send 1.0 Sol DIC90 of Std Fmoc to
On synthesizer.The sequence of File-New-Sequence- Edit and Compose peptide saves.File-New-Run checks that Chemistry is
No is 1.0 Sol DIC 90 of Std Fmoc；Whether Sequence is to be deposited name；Set Cycles；It saves.It is finally sent to
On synthesizer.
Main Menu-Cycle Monitor-begin, brings into operation.
4, envelope antigen synthesis carries out
The removing of Fmoc group, the electron attraction of the fluorenes ring system of Fmoc group make 9-H have acidity, are easily removed compared with weak base
It goes, when reaction is eliminated to form hexichol fluorenes alkene with piperidines (PIP) attack 9-H, β, it is easy to be formed by second level cyclammonium attack stable
Addition product."-NH is exposed after the removing of Fmov group2" group to be to carry out synthetic reaction.Then the next of activation is added
In the amino acid and I-hydroxybenzotriazole (1-hydroxybenzotriazole, HOBT) to reactor of Fmoc radical protection.
Such as above-mentioned polypeptide sequence, synthesis when is since C-terminal to N-terminal, according to specific sequence, successively constantly
Repeat synthesis step (synthesis is automatically performed by program, specific circulation step such as the following table 2).Period observes and records reagent dosage and fortune
Market condition.
2. envelope antigen of table synthesizes circulation step
5, envelope antigen synthesis terminates
Synthesizer will be automatically stopped after envelope antigen synthesizes, and peptide resin (peptide is connected on resin) washs substantially
Completely.Then reactor is removed from Peptide synthesizer, then after washing peptide resin 3 times with 100% methanol, is blown in draught cupboard
It is dry, then polypeptide resin is fully transferred in the polyethylene bottle of brown, is put into -20 DEG C of refrigerators, sealed membrane sealing is spare.
Two, the cracking and identification of envelope antigen
1, the cracking of polypeptide antigen
It through the obtained polypeptide of above-mentioned reaction is chemically bound together with solid phase carrier, it is necessary to by specific
The acidolysis of organic acid polypeptide is separated with solid phase carrier.Also the guarantor on each amino acid functional group is eliminated while acidolysis
Protect base.Steps are as follows:
The peptide resin (polypeptide is connected on resin) that synthesis is taken out out of refrigerator, is put into the round-bottomed flask of a 2L,
The tripropyl silane of 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), 10ml is added in draught cupboard into flask
(TIS) and magnetic stir bar, then steadily flask is placed on magnetic stirring apparatus, at room temperature persistently stirring 1h to having reacted
Entirely.After reaction, the TFA in 30~120min removing crude product is persistently evaporated using the Rotary Evaporators with cold-trap.Then
The crude product of polypeptide antigen is cleaned multiple times with dimethylformamide (DMF), finally by the resin mixed sand core funnel mistake
It filters out and, both obtain envelope antigen.
2, the identification of envelope antigen
Polypeptide antigen is high with substance assistant laser desorpted winged examination time mass spectrum method (MODAL-TOF) and reverse phase after synthesizing
Pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis, and synthesized peptide is identified with common amino acid analysis.
3, envelope antigen purifies
Polypeptide antigen after cyclisation is carried out ultrafiltration using circulating tangential flow filtration film packet (to be produced with PALL company
The circulating tangential flow filtration film packet of Tangential Flow Device and peristaltic pump matched with its), polypeptide antigen is as big
Molecule cannot be by the filter membrane of certain pore size, and the small molecule that synthesis process early period and later period cyclization are formed or introduced is miscellaneous
Matter can then pass through filter membrane.Then passing through aperture again is 0.2 μm of filter degerming, and last acquired solution is dispensed into aseptic plastic
It is labelled in bottle.Title, number, product batch number, concentration, date of manufacture, pot-life and the preservation of polypeptide are indicated on label
Condition, after packing, be stored in -20 DEG C or -40 DEG C it is spare.
4, envelope antigen is freeze-dried
For the ease of long-term preservation and transport, need for envelope antigen to be freeze-dried to obtain the more of solid state
Peptide.The envelope antigen freezed in advance is placed on the freeze drier of Labconco and is dried, solid state is obtained
Envelope antigen.It is labelled after packaging.Title, number, product batch number, the concentration, date of manufacture, preservation of polypeptide are indicated on label
Time limit and preservation condition.
The preparation of embodiment 2, foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent
Foot-and-mouth disease antibody ELISA immunity detection reagent includes:
(1) it is coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of foot-and-mouth disease virus antigen；2 × 96 holes.
(2) staphylococcal protein A (being purchased from abcam company, article No. ab7456) of horseradish peroxidase-labeled, 2 bottles (each
12ml)。
(3) positive control serum: it is the envelope antigen using artificial chemistry synthesis in embodiment 1 as immunogene, exempts from respectively
The hyper-immune serum that 25~35 age in days of epidemic disease is prepared without specific pathogeny body pig crosses 0.22 μm of filter membrane degerming, and the positive as kit is right
According to serum (1 pipe, 1.5ml/ pipe).
(4) negative control sera: being with the normal swine serum of no foot-and-mouth disease antibody, 0.22 μm of filter membrane degerming excessively, as examination
Agent box negative control sera (1 pipe, 1.5ml/ pipe).
(5) it substrate developing solution: is made of two kinds of solution mixing, solution A is the lemon of the hydrogen peroxide urea containing 0.6mg/ml
Acid phosphoric acid salt buffer (1 bottle, 12ml/ bottles)；Solution B be 0.2mg/ml tetramethyl benzidine (TMB) solution (1 bottle, 12ml/
Bottle)；It is mixed both when use with volume ratio for the ratio of 1:1.
(6) concentrated cleaning solution (20 ×): contain 0.5% (ml/ml) Tween-20 and 0.05% (g/ml) Sodium azide anti-corrosion
The 0.01M phosphate buffer of agent, pH 7.4, after by 0.22 μm of filter membrane degerming (2 bottles, 50ml/ bottles).
(7) terminate liquid: the sulfuric acid solution (1 bottle, 12ml/ bottles) of 2mol/L.
(8) sample diluting liquid: the PBS buffer solution that 0.01M, pH containing 0.5% (g/ml) casein are 7.4 crosses 0.22 μ
M filter membrane (2 bottles, 12ml/ bottles).
As needed, serum can also dilutes plate (2 pieces, 96 holes/block) in kit, the dilution for blood serum sample.
Wherein, be coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of foot-and-mouth disease virus antigen the preparation method comprises the following steps: will
Polypeptide antigen prepared by embodiment 1 is dissolved in the carbonate solution of pH 9.6, is then added to 96 hole polystyrene enzyme-linked reaction plates, often
The every peptide 50ng antigen in hole is (wherein in sequence table in polypeptide shown in sequence 1 and sequence table in polypeptide shown in sequence 2 and sequence table
Polypeptide shown in sequence 3, the mass ratio of three kinds of polypeptides are 1:1:1), 8h is placed at 37 DEG C of placements 2~4h, then 4~8 DEG C, is made
Polypeptide antigen is sufficiently combined with enzyme-linked reaction plate.Then it is added according to 300 μ l/ hole concentration and contains 1% (g/ml) bovine serum albumin(BSA)
(BSA) PBS buffer solution (pH=7.4), 37 DEG C of 2~3h of closing, with washing buffer (concentrated cleaning solution distilled water or go from
The 20 times of dilutions of sub- water obtain washing buffer) it dries after washing, it is sealed for 4 DEG C after reaction plate is dry.
The coincidence rate test of embodiment 3, foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent
One, the application method of foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent
1, it balances: the kit for being stored in 4 DEG C being taken out, is balanced spare to room temperature；The preceding mixing of liquid reagent.
2, match liquid: the 20 times of dilutions of concentrated cleaning solution distilled water or deionized water are obtained into washing buffer；
3, set: 2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4, sample pre-dilution to be measured: use sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to the dilution proportion of 1:20.
5, be loaded: 2 negative control holes respectively add 100 μ l negative controls, and 2 Positive control wells respectively add 100 μ l positive controls；
Remaining hole is respectively by presetting plus the 100 diluted samples to be tested of μ l.Loading time span should be short as far as possible.
6, incubate: concussion mixes, and sets in 37 DEG C of incubators or water-bath, reacts 30min.
7, board-washing: discarding reaction solution, and every hole adds washing buffer obtained in 300 μ l steps 2, impregnates 15s, gets rid of abandoning washing
Liquid.It is patted dry after continuous board-washing 4 times.
8, enzyme: each hole adds the staphylococcal protein A of 100 μ l horseradish peroxidase-labeleds.
9, it incubates: setting in 37 DEG C of incubators or water-bath, react 30min.
10, board-washing: discarding reaction solution, and the 300 μ l of washing buffer after dilution is added in every hole, impregnates 15s, gets rid of abandoning washing
Liquid.It is patted dry after continuous board-washing 4 times.
11, develop the color: every hole is separately added into the substrate developing solution of 100 μ l, and (wherein solution A and solution B are with volume ratio for 1:1's
Ratio mixing).It covers, reacts 15min in 37 DEG C of insulating boxs；
12, every hole is separately added into 100 μ l terminate liquids and terminates reaction, mixes；
13, the OD in every hole is measured450(OD should be read to value in 15min by adding the reaction plate of terminate liquid450Value).
The judgement of testing result:
1, negative control OD450Average value should≤0.15, otherwise in vain.
2, each detected value of positive control should be between 0.9~2.3, otherwise in vain.
3, the calculating of critical value: cow's serum critical value=0.15 × positive control OD450It is worth average value
Swine serum critical value=0.18 × positive control OD450It is worth average value
Serum to be checked measures OD450Value >=critical value person is judged to the positive；Serum to be checked measures OD450Value < face value person is judged to yin
Property.
Two, suckling mouse serum neutralization test (MSN) method
At present domestic common aftosa serum detection method first is that suckling mouse neutralization test (MSN), therefore tried with this
Agent box carries out coincidence rate test with suckling mouse neutralization test.
Material
1. immune serum, Swine serum is immunized in 50 parts, and ((aftosa is O-shaped, A type, Asia I type three for Schweineseuche inactivated vaccine
Bivalent inactivated vaccine, article No. ABB10601730250) immune 28 days latter)；Cow's serum (ox inactivated foot-and-mouth disease vaccine is immunized in 50 parts
21 days after (aftosa is O-shaped, A type, Asia I type tervalence inactivated vaccine, article No. ABB10601730250) is immune), You Zhongmu industry
Limited liability company, biology pharmaceutical factory, Lanzhou provides.
2. healthy 50 parts of Swine serum, healthy 50 parts of cow's serum, are mentioned by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.
For.
3. test is aftosa AF/72 virus with virus, seed culture of viruses is provided by Lanzhou veterinary institute, You Zhongmu industry share
Co., Ltd, biology pharmaceutical factory, Lanzhou saves and uses, and by virus after 6~8 age in days suckling mouses passed for 2 generations, collects virus.With 3~4 days
Age suckling mouse measures and adjusts its malicious valence to 1000LD50/ 0.2ml is placed in -20 DEG C and saves for use.
Suckling mouse serum neutralization test method is as follows: serum PBS being made 2 times of dilutions, takes 1ml and equivalent 100LD50/ ml's
The mixing of AF/72 system virus liquid, is incubated for 1 hour in 37 DEG C, is then inoculated with 3 age in days suckling mouses (0.2ml is subcutaneously injected in neck), every group
(that is, each serum to be checked) 4, while virus control, normal healthy controls and standard positive serum control group are set up, every group 3,
7d is observed, suckling mouse fatality ratio is recorded and is determined as a result, living if suckling mouse 4/4 or 3/4 is strong, serum to be checked is positive (R), if newborn
Mouse 4/4 or 3/4 death, then serum to be checked is negative (-)；If suckling mouse 2/4 is strong living or 2/4 is dead, determine that result is suspicious (±),
Replication need to be doubled, repeats to have 1 group to be positive in test, be then determined as the positive.
Sensibility=judgement positive serum sample number/test serum sample number
Three, coincidence rate test result:
The foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent prepared using embodiment 2 and suckling mouse
Neutralization test (MSN) detects 100 parts of Swine serums (50 parts of immune serum, 50 parts of healthy serum) respectively, and 100 parts of cow's serums are (immune
50 parts of serum, 50 parts of healthy serum).
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent is shown in Table the testing result of Swine serum
3.The sensibility of kit is 98%, and the sensibility of suckling mouse serum neutralization test is 92%, in 50 parts of immune Swine serums, two
Consistent kind method testing result is 47 parts.Therefore, this kit and the coincidence rate of suckling mouse serum neutralization test are 94%, therefore
This kit sensibility with higher.The coincidence rate of both tests to healthy Swine serum is 100%, and testing result is yin
Property (being shown in Table 3).
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent and suckling mouse neutralization test detect altogether
100 parts of Swine serums, wherein 97 parts of Swine serum two methods testing results are consistent, 3 parts of Swine serum testing results are variant, coincidence rate
It is 97%.
3. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent of table and suckling mouse serum neutralization test
Testing result of the method to immune Swine serum and healthy Swine serum
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent is shown in Table the testing result of cow's serum
4.The result shows that the sensibility of kit is 98%, and the sensibility of suckling mouse serum neutralization test is 94%, in 50 parts of immune cattles
In serum, consistent two methods testing result is 48 parts, and therefore, the coincidence rate of this kit and suckling mouse serum neutralization test is
96%, therefore this kit sensibility with higher.The coincidence rate of both tests to healthy cow's serum is 100%, detection
Result is negative (being shown in Table 4).
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent and suckling mouse neutralization test detect altogether
100 parts of cow's serums, wherein 98 parts of cow's serum two methods testing results are consistent, 2 parts of ox serum detection results are variant, coincidence rate
It is 98%.
4. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent of table and suckling mouse serum neutralization test
Testing result of the method to immune cow's serum and healthy cow's serum
The sensitivity tests of embodiment 4, foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent
Sensitivity tests is tried using the foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immune detection in embodiment 3
The detection of agent box detects the experimental result in pig source and ox source serum respectively.Pig infects serum by Zhongmu Industry Co., Ltd Lanzhou
Biological pharmaceutical factory provides, and is Schweineseuche A type virus infection serum.Pig source immune serum is Schweineseuche A type virus synthetic peptide epidemic disease
Seedling (polypeptide sequence of synthetic peptide vaccine is sequence 4 in sequence table) Post-immunisation serum, by Zhongmu Industry Co., Ltd Lanzhou
Biological pharmaceutical factory provides.Cattle infected serum is provided by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd., is ox foot-and-mouth disease a type disease
Poison infection serum.Ox source vaccine immune sera is that (polypeptide sequence of synthetic peptide vaccine is ox foot and mouth disease A-type virus synthetic peptide vaccine
Sequence 5 in sequence table) Post-immunisation serum, it is provided by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd..
The three batches of foot and mouth disease A-type virus structural proteins antibody ELISA immunosorbent adsorption tests diagnosis examination prepared using embodiment 2
Agent box (batch ZMAVP001, ZMAVP002, ZMAVP003), it is anti-according to foot and mouth disease A-type virus Structural protein VP1 in embodiment 3
Body enzyme-linked immunologic detecting kit application method infects serum each 200 parts and pig, Niu Kou to pig, ox foot and mouth disease A-type virus respectively
Each 200 parts of progress sensitivity tests of fever aphthous A type virus synthetic peptide vaccine immune serum, experimental result are shown in Table 5.
Using three polypeptides shown in method as described in example 2 preparation sequence 1, sequence 2 and sequence 3, individually wrap
A type virus structural protein antibody ELISA immunity detection reagent (ZMAVP1-1, ZMAVP1-2, the ZMAVP1- assembled by ELISA Plate
3), right respectively according to foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent application method in embodiment 3
Pig, ox foot and mouth disease A-type virus infect serum each 200 parts and pig, ox foot and mouth disease A-type virus synthetic peptide vaccine immune serum each 200
Part carries out sensitivity tests, and experimental result is shown in Table 5.
The sensibility that ZMAVP001 kit infects Swine serum detection to 200 parts is 196/200 × 100%=98%；It is right
The sensibility of 200 parts of immune Swine serum detections is 198/200 × 100%=99%.ZMAVP002 kit is to 200 parts of infection pigs
The sensibility of serum detection is 197/200 × 100%=98.5%；Sensibility to 200 parts of immune Swine serum detections is 198/
200 × 100%=99%.The sensibility that ZMAVP003 kit infects Swine serum detection to 200 parts is 197/200 × 100%
=98.5%；Sensibility to 200 parts of immune Swine serum detections is 197/200 × 100%=98.5%.
The sensibility that ZMAVP1-1 kit infects Swine serum detection to 200 parts is 193/200 × 100%=96.5%；
Sensibility to 200 parts of immune Swine serum detections is 195/200 × 100%=97.5%.ZMAVP1-2 kit feels 200 parts
The sensibility for contaminating Swine serum detection is 194/200 × 100%=97%；Sensibility to 200 parts of immune Swine serums detections is
194/200 × 100%=97%.ZMAVP1-3 kit to 200 parts infect Swine serums detection sensibility be 194/200 ×
100%=97%；Sensibility to 200 parts of immune Swine serum detections is 193/200 × 100%=96.5%.
The sensibility that ZMAVP001 kit infects ox serum detection to 200 parts is 197/200 × 100%=98.5%；
Sensibility to 200 parts of immune ox serum detections is 196/200 × 100%=98%.ZMAVP002 kit infects 200 parts
The sensibility of ox serum detection is 197/200 × 100%=98.5%；Sensibility to 200 parts of immune ox serum detections is
197/200 × 100%=98.5%.ZMAVP003 kit to 200 parts infect ox serum detections sensibility be 197/200 ×
100%=98.5%；Sensibility to 200 parts of immune ox serum detections is 198/200 × 100%=99%.
The sensibility that ZMAVP1-1 kit infects ox serum detection to 200 parts is 195/200 × 100%=97.5%；
Sensibility to 200 parts of immune ox serum detections is 194/200 × 100%=97%.ZMAVP1-2 kit infects 200 parts
The sensibility of ox serum detection is 194/200 × 100%=97%；Sensibility to 200 parts of immune ox serum detections is 193/
200 × 100%=96.5%.ZMAVP1-3 kit to 200 parts infect ox serum detections sensibility be 193/200 ×
100%=96.5%；Sensibility to 200 parts of immune ox serum detections is 195/200 × 100%=97.5%.
Sensibility of the 5. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent of table to pig, cow's serum
Testing result
The specific test of embodiment 5, foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent
Using three batches of kits in embodiment 4 according to foot and mouth disease A-type virus Structural protein VP1 described in embodiment 3
The application method of antibody ELISA immunity detection reagent is to 200 parts of healthy Swine serum (Zhongmu Industry Co., Ltd Bao Shanchang
There is provided with Lanzhou biology pharmaceutical factory), (Zhongmu Industry Co., Ltd is blue for 2 parts of Schweineseuche Asia I type (Asia1) positive serums
State biology pharmaceutical factory provide), (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. mentions 2 parts of Schweineseuche O-shaped (O) positive serums
For), 2 parts of swine fever positive serums (China Veterinery Drug Inspection Office), 2 parts of swine pox positive serums (in herd industry share limited
Company provides), 2 parts of pig annulus positive serums (Zhongmu Industry Co., Ltd's offer) and 2 parts of pig blue-ear disease positive serums (in
Industry Co., Ltd's offer is provided) it is detected respectively.
The specific detection result such as following table (table 6) of kit is shown.To the testing result table of 200 parts of healthy Swine serums
Bright, the specificity of ZMAVP001 kit is that the specificity of 99.0%, ZMAVP002 kit is 99.5%, ZMAVP003 reagent
The specificity of box is 99.0%.To 2 parts of Schweineseuche Asia I type (Asia I) positive serums, 2 parts Schweineseuche O-shaped (O) positives
Serum, 2 parts of swine fever positive serums, 2 parts of swine pox positive serums, 2 parts of pig annulus positive serums and 2 parts of pig blue-ear disease positive bloods
Clear testing result is illustrated as feminine gender, therefore the specificity that three kits detect this 12 parts of pig positive serums is
100%.
The specific detection result such as following table (table 6) of kit is shown.It is aobvious to the testing result of 200 parts of healthy cow's serums
Show, the specificity of ZMAVP001 kit is that the specificity of 99.0%, ZMAVP002 kit is 99.5%, ZMAVP003 reagent
The specificity of box is 99.5%.It is positive to 2 parts of ox aftosa Asia I type (Asia I) positive serums, 2 parts of ox aftosas O-shaped (O)
Serum, 2 parts of bovine viral diarrhoea positive serums testing result be illustrated as feminine gender, therefore three kits are to this 6 parts of ox sun
Property serum detection specificity be 100%.
6. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent specific detection result of table
Using three polypeptides shown in method as described in example 2 preparation sequence 1, sequence 2 and sequence 3, individually wrap
A type virus structural protein antibody ELISA immunity detection reagent (ZMAVP1-1, ZMAVP1-2, the ZMAVP1- assembled by ELISA Plate
3), right respectively according to foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent application method in embodiment 3
200 parts of healthy Swine serums (Baoshan factory, Zhongmu Industry Co., Ltd and Lanzhou biology pharmaceutical factory provide), 2 parts of Schweineseuche Asia
I type (Asia1) positive serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer), 2 parts Schweineseuche O-shaped (O) sun
Property serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer), (the Chinese veterinary medicament supervision of 2 parts of swine fever positive serums
Institute), 2 parts of swine pox positive serums (Zhongmu Industry Co., Ltd's offer), 2 parts of pig annulus positive serums (in herd industry
Limited liability company provide) and 2 parts of pig blue-ear disease positive serums (Zhongmu Industry Co., Ltd's offer) detected respectively.
The specific detection result such as following table (table 7) of kit is shown.To the testing result table of 200 parts of healthy Swine serums
Bright, the specificity of ZMAVP1-1 kit is that the specificity of 97.0%, ZMAVP1-2 kit is 98.5%, ZMAVP1-3 reagent
The specificity of box is 98.0%.To 2 parts of Schweineseuche Asia I type (Asia I) positive serums, 2 parts Schweineseuche O-shaped (O) positives
Serum, 2 parts of swine fever positive serums, 2 parts of swine pox positive serums, 2 parts of pig annulus positive serums and 2 parts of pig blue-ear disease positive bloods
Clear testing result is illustrated as feminine gender, therefore the specificity that three kits detect this 12 parts of pig positive serums is
100%.
The specific detection result such as following table (table 7) of kit is shown.It is aobvious to the testing result of 200 parts of healthy cow's serums
Show, the specificity of ZMAVP1-1 kit is that the specificity of 98.0%, ZMAVP1-2 kit is 97.5%, ZMAVP1-3 reagent
The specificity of box is 97.5%.It is positive to 2 parts of ox aftosa Asia I type (Asia I) positive serums, 2 parts of ox aftosas O-shaped (O)
Serum, 2 parts of bovine viral diarrhoea positive serums testing result be illustrated as feminine gender, therefore three kits are to this 6 parts of ox sun
Property serum detection specificity be 100%.
The independent peptide specific testing result of table 7.
Sequence table
<110>Zhongmu Industry Co., Ltd
<120>a kind of foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide and its application
<130> WHOI180089
<160> 5
<170> SIPOSequenceListing 1.0
<210> 1
<211> 40
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 1
Tyr Asn Gly Thr Thr Lys Tyr Ser Thr Gly Asn Ala Gly Arg Arg Gly
1 5 10 15
Asp Leu Gly Ser Leu Ala Ala Arg Val Ala Ala Gln Leu Pro Ala Ser
20 25 30
Phe Asn Phe Gly Ala Ile Arg Ala
35 40
<210> 2
<211> 40
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 2
Val Tyr Asn Gly Thr Ser Lys Tyr Ser Ala Pro Ala Thr Arg Arg Gly
1 5 10 15
Asp Leu Gly Ser Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser
20 25 30
Phe Asn Tyr Gly Ala Ile Arg Ala
35 40
<210> 3
<211> 40
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Val Tyr Ser Gly Thr Ser Lys Tyr Ser Ala Ser Gln Asn Arg Arg Gly
1 5 10 15
Asp Leu Gly Pro Leu Ala Ala Arg Leu Ala Ala Gln Leu Pro Ala Ser
20 25 30
Phe Asn Phe Gly Ala Ile Arg Ala
35 40
<210> 4
<211> 60
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Arg Met Glu Ala Ala Lys Thr Gly Ile Glu Ile Ala Tyr Asp Glu Arg
1 5 10 15
Arg Gly Leu Lys Val Tyr Asn Gly Thr Thr Lys Tyr Ser Thr Gly Asn
20 25 30
Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val Ala Ala
35 40 45
Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg
50 55 60
<210> 5
<211> 60
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
Arg Met Glu Ala Lys Lys Thr Gly Ile Glu Ile Ala Gly Asp Glu Arg
1 5 10 15
Arg Gly Leu Lys Val Tyr Asn Gly Thr Thr Lys Tyr Ser Thr Gly Asn
20 25 30
Ala Gly Arg Arg Gly Asp Leu Gly Ser Leu Ala Ala Arg Val Ala Ala
35 40 45
Gln Leu Pro Ala Ser Phe Asn Phe Gly Ala Ile Arg
50 55 60
Claims (10)
1. foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide, for shown in sequence 3 in sequence 2 in sequence table or sequence table
Polypeptide.
2. foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide composition, effective component is sequence 1 in sequence table
Shown in polypeptide, one or more of polypeptide shown in sequence 3 any combination in sequence 2 and sequence table in sequence table.
3. a kind of foot and mouth disease A-type virus structural proteins antibody ELISA immunity detection reagent, including foot and mouth disease A-type virus structure egg
The coated enzyme-linked reaction plate of white VP1 antigen epitope polypeptide and ELIAS secondary antibody；The foot and mouth disease A-type virus Structural protein VP1 antigen
Epitope polypeptide is one or more of polypeptide shown in sequence 3 in polypeptide shown in sequence 2 in sequence table and sequence table composition
Mixed polypeptide.
4. kit according to claim 1, it is characterised in that: polypeptide shown in sequence 2 and sequence in the sequence table
The mass ratio of polypeptide shown in sequence 3 in table are as follows: 0.5~2.0:1；Polypeptide shown in sequence 2 and sequence table in the sequence table
The mass ratio of polypeptide shown in middle sequence 3 is preferably 1:1.
5. according to any one of claim 2~4, it is characterised in that: the marker enzyme of the ELIAS secondary antibody is horseradish peroxidating
Object enzyme or alkaline phosphatase；The ELIAS secondary antibody is that enzyme marks staphylococcal protein A；The ELIAS secondary antibody is preferably horseradish peroxide
The staphylococcal protein A of compound enzyme label.
6. according to the kit of any one of claim 2~4, it is characterised in that: the enzyme-linked reaction plate is detachable 96
Hole elisa Plates；The foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide, which is that chemistry is artificial synthesized, to be obtained.
7. kit according to claim 6, it is characterised in that: the foot and mouth disease A-type virus Structural protein VP1 antigen table
The preparation method of the position coated enzyme-linked reaction plate of polypeptide is by the foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide
It is dissolved in the carbonate solution of the pH 9.6 of 100 μ l, is then added to 96 hole polystyrene enzyme-linked reaction plates, every every peptide of hole 50ng,
Placing 8~12 hours at 37 DEG C of placements 2~4 hours, then 4-8 DEG C combines polypeptide antigen sufficiently with enzyme-linked reaction plate, then presses
According to 300 holes μ l/ be added the PBS buffer solution containing 0.01g/ml bovine serum albumin(BSA) pH7.4,37 DEG C Seal treatment 2~3 hours,
It dries after washing, is sealed for 4 DEG C after enzyme-linked reaction plate is dry.
8. kit according to claim 7, it is characterised in that: also contain developing solution and terminate liquid in the kit；
When marker enzyme is horseradish peroxidase, developing solution is made of developing solution A liquid and developing solution B liquid, the developing solution A liquid be containing
The citrate phosphate buffer of 0.6mg/ml hydrogen peroxide urea, the developing solution B liquid are the tetramethyl biphenyl of 0.2mg/ml
Amine aqueous solution；When marker enzyme is alkaline phosphatase, developing solution is 4- nitrophenols phosphate buffer；The terminate liquid is 2mol/L
Sulfuric acid solution.
9. kit according to claim 8, it is characterised in that: the kit further includes negative control sera, the positive
Control serum；The negative control sera is the normal pig or cow's serum with no foot-and-mouth disease antibody；The positive control serum is
The serum obtained using the A type Foot-and-mouth disease VP1 antigen epitope polypeptide as immunogen immune pig, ox；And/or institute
Stating kit further includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid be the 0.01M containing 0.005g/ml casein,
The PBS buffer solution that pH is 7.4；Concentrated cleaning solution: 0.01M, pH 7.4 is 0.8%~1.2% containing concentration expressed in percentage by volume
The phosphate buffer of Tween-20 and 0.0005g/ml Sodium azide preservative.
10. foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide described in claim 1 is in preparation detection infection or exempts from
Application in the kit of the foot and mouth disease A-type virus Structural protein VP1 antibody generated after epidemic disease；Wherein, animal aftosa viral disease
For pig or ox foot and mouth disease A-type virus disease；Preferably, the animal aftosa poison is mouth caused by pig, ox foot and mouth disease A-type virus
Aphtovirus disease.
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| CN109627293B (en) * | 2017-08-13 | 2021-02-09 | 中牧实业股份有限公司 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
| CN107589269A (en) * | 2017-11-01 | 2018-01-16 | 中国农业科学院兰州兽医研究所 | Detection card for A type antibodies against foot-and-mouth disease virus in Quantitative detection serum |
| CN108931644B (en) * | 2018-07-19 | 2021-09-10 | 河南省农业科学院 | Foot-and-mouth disease virus immune antibody evaluation and infection and immune differential diagnosis dual test strip |
| CN109799343A (en) * | 2018-12-07 | 2019-05-24 | 中国农业科学院兰州兽医研究所 | A type antibodies against foot-and-mouth disease virus detection kit based on virus-like particle |
| CN109856396B (en) * | 2018-12-24 | 2022-02-08 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Enzyme linked immunosorbent assay kit for detecting foot and mouth disease virus infection antibody and application thereof |
| CN109655623A (en) * | 2019-01-31 | 2019-04-19 | 中国农业科学院兰州兽医研究所 | A kind of the visualization quick detection kit and its application of A type antibodies against foot-and-mouth disease virus |
| CN110734478B (en) * | 2019-11-08 | 2020-12-01 | 中牧实业股份有限公司 | Foot-and-mouth disease A type synthetic peptide vaccine and preparation method and application thereof |
| CN110669112B (en) * | 2019-11-08 | 2020-12-01 | 中牧实业股份有限公司 | Enzyme-linked immunoassay kit for A-type antibody of swine foot-and-mouth disease virus |
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| CN104961809A (en) * | 2015-07-10 | 2015-10-07 | 中牧实业股份有限公司 | Foot and mouth disease O-type structure protein VP1 antibody enzyme-linked immune detection reagent kit |
| CN105418738A (en) * | 2015-07-03 | 2016-03-23 | 申联生物医药（上海）有限公司 | A-type antigen polypeptide, fusion antigen polypeptide and vaccine of foot and mouth disease virus |
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| CN105418738A (en) * | 2015-07-03 | 2016-03-23 | 申联生物医药（上海）有限公司 | A-type antigen polypeptide, fusion antigen polypeptide and vaccine of foot and mouth disease virus |
| CN104961809A (en) * | 2015-07-10 | 2015-10-07 | 中牧实业股份有限公司 | Foot and mouth disease O-type structure protein VP1 antibody enzyme-linked immune detection reagent kit |
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| CN106432434B (en) | 2019-11-12 |
| CN109485703B (en) | 2021-07-06 |
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