CN106432434A - Enzyme-linked immunosorbent assay kit for detecting foot and mouth disease A-type structural protein VP1 antibody - Google Patents
Enzyme-linked immunosorbent assay kit for detecting foot and mouth disease A-type structural protein VP1 antibody Download PDFInfo
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Abstract
The invention discloses an enzyme-linked immunosorbent assay kit for detecting a foot and mouth disease A-type structural protein VP1 antibody. The enzyme-linked immunosorbent assay kit comprises an enzyme reaction plate coated with a foot and mouth disease A-type structural protein VP1 epitope peptide and an HRP-conjugated antibody; and the foot and mouth disease A-type viral structural protein VP1 epitope peptide is one or a combination of two or more of a polypeptide shown in a sequence 1, a polypeptide shown in a sequence 2 and a polypeptide shown in a sequence 3 in a sequence table. The reaction plate is coated with the chemically synthetized VP1 antigen peptide, the consumption of antigens is low, the sensitivity and the specificity are high, and the condition whether the foot and mouth disease A-type viral structural protein VP1 antibody exists after infection or immunization is efficiently detected. The kit is high in specificity, is sensitive and efficient, and has an excellent market prospect.
Description
Technical field
A kind of the invention belongs to biological technical field, more particularly it relates to Foot-and-mouth disease antibody
Enzyme-linked immunologic detecting kit, particularly foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent.
Background technology
Foot and mouth disease (Foot and Mouth Disease, FMD) be by foot and mouth disease viruses (Foot-and-mouth
Disease virus, FMDV) cause to infect acute, hot, high degree in contact infectiousness based on artiodactyl and quick remote
The animal epidemic of propagation, so that infectiousness is strong, spread speed fast, endanger serious and famous (Xie Qingge, 2004).World animal
Health organization (OIE) is classified as many animals that must circulate a notice of and is suffered from one of infectious disease altogether, and China is classified as class animal biography
Catch an illness.
Foot and mouth disease infectiousness height, propagates rapidly, and the domestic animal such as infected pigs, cattle, sheep often results in young stock death, and adults are produced
Ability drastically declines.The disease propagate rapid, can cause global be very popular, not only cause huge economic damage to animal husbandry
Lose, and have a strong impact on international trade and the reputation of animals and animal product.Due to the significant damage of FMD, U.S.'s authority
《Livestock contagious disease》Just state in version in 1981:FMD is not only economy disease, while being also political epidemic disease.
Foot and mouth disease viruses belong to Picornaviridae, Hostises.By the symmetrical capsid of 20 face body of "false" and
Viral nucleic acid is constituted, and the whole profile of the virion is spherical, is not strict positive 20 face body.Complete virion includes
Capsid and RNA two parts, capsid is by each 60 molecular compositions of 4 kinds of structural protein such as VP1, VP2, VP3 and VP4.Structural protein VP1
Virion surface is largely exposed to, is the main component for determining virus antigenicity, and stimulate body to produce neutralizing antibody
Major protein.VP1 variability maximum in 4 kinds of structural protein, VP2 is more guarded, and VP4 is most conservative.The work(of viral capsid proteins
Can have:Protection RNA exempts from nuclease degradation；Identification specificity cell receptor, it is resolved that host range and tissue tropism；Determine virus
Antigenicity；Release and transmission viral RNA Z are entered in permissive cell by cell membrane；Instruct and select and packaging virus RNA.
The aftosa serum detection method of OIE announcement in 2004 has 3 kinds：Virus neutralization tests (VNT), liquid phase blocking
ELISA (LPB-ELISA), solid phase competitive ELISA (SPC-ELISA), wherein virus neutralization tests are the most classical in 3 kinds of methods
Method, be evaluate additive method golden standard.1996, foot and mouth disease world reference laboratory established liquid phase blocking
ELISA method, through constantly improve and sound, it has also become various countries' SD method on a large scale, for detecting that animal exempts from
Epidemic disease antibody, assessment animal immune situation, carry out serosurvey etc., the sensitivity of the method, specificity and stability exist always
Gain public acceptance in the world, be to use most wide aftosa serum detection method at present.Calendar year 2001, foot and mouth disease world reference experiment
Room establishes solid phase competitive ELISA method, and the method is in addition to the traditional advantage with LPB-ELISA method, and which is special
Property more preferably, operate more simple (its operating time is only 3.5h), aftosa serum investigation be asserted most by OIE in 2004
New recommendation method.
Foot and mouth disease is the great animal epidemic for being related to economic security of the country, at present, most developing countries and area
All control the popular of foot and mouth disease by vaccinated method.China prevention and control FMD mainly takes vaccine immunity and antibody detection
Based on Synthetical prevention policy, drove entirety antibody horizontal is improved preventing infection and the propagation of foot and mouth disease by vaccination.
Therefore the quick diagnosis of infection of foot-and-mouth disease, pig and cattle Efficacy evaluation, natural infection and the Differential Diagnosiss of vaccine immunity become
The Some Key Technologies of prevention and control foot and mouth disease.
Content of the invention
It is an object of the invention to provide a kind of new based on elisa, for detecting in pig, Ox blood serum be
The no test kit containing foot and mouth disease A-type virus structural protein antibody.The present invention utilize VP1 structural protein, set up a species specificity,
Sensitivity and reproducible indirect ELISA method, are that pig, the detection of cattle foot and mouth disease immune antibody and the diagnosis of infection antibody are carried
For a kind of simple and effective new method, it is that foot and mouth disease Synthetical prevention provides technical support.
In order to achieve the above object, the present invention screens the antigen epitope polypeptide for having obtained excellent performance first, and the present invention is carried
For foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide, be polypeptide in sequence table shown in sequence 1, sequence in sequence table
Polypeptide in row 2 or sequence table shown in sequence 3.
Further, the present invention provides a kind of foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide compositionss,
Its effective ingredient is polypeptide shown in sequence 1 in sequence table, in sequence table in the polypeptide of sequence 2 and sequence table shown in sequence 3
One or more combination in any in polypeptide.
The Foot-and-mouth disease antibody ELISA immunity detection reagent of the present invention, ties including foot and mouth disease A-type virus
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 the polypeptide in the polypeptide in sequence table shown in sequence 1, sequence table shown in sequence 2 and sequence table
Shown polypeptide.
In the Foot-and-mouth disease antibody ELISA immunity detection reagent of the present invention, sequence 1 in the sequence table
The mass ratio of the polypeptide in the polypeptide and sequence table in shown polypeptide, sequence table shown in sequence 2 shown in sequence 3 is 0.5～
2.0:0.5～2.0:1, preferably 1:1:1.Involved envelope antigen is produced using the method for solid-state chemical reaction method, is coated anti-
The former major antigenic sites containing Foot-and-mouth disease VP1, can with mouth disease virus infection after or immunity after produce
Structural protein VP1 antibody specific bond.The marker enzyme of the ELIAS secondary antibody is horseradish peroxidase or alkali phosphatase；Described
ELIAS secondary antibody is enzyme labelling 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
Obtain for chemical synthetic.
The plate of the coated enzyme-linked reaction plate of the foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide most preferably prepare bar
Part is：The foot-and-mouth disease a type Structural protein VP1 antigen epitope polypeptide is dissolved in the carbonate solution of the pH9.6 of 100 μ l, then
96 hole polystyrene enzyme-linked reaction plates are added to, per hole per peptide 50ng antigen, are placed at 37 DEG C and placed at 2～4h, then 4～8 DEG C
At night, polypeptide antigen is made fully to be combined with enzyme-linked reaction plate, then according to 300 μ l/ holes add contains 1% (g/ml) bovine serum albumin
The PBS (pH=7.4) of (BSA) in vain, 37 DEG C of 2～3h of sealing treatment, with lavation buffer solution (concentrated cleaning solution distilled water
Or 20 times of dilutions of deionized water obtain lavation buffer solution) washing after dry, after enzyme-linked reaction plate dry after 4 DEG C of sealing preserves.
Containing nitrite ion and terminate liquid in the test kit；When marker enzyme is for horseradish peroxidase, nitrite ion is by developing the color
Liquid A liquid and nitrite ion B liquid composition, the nitrite ion A liquid is the citrate-phosphate buffer containing 0.6mg/ml hydrogen peroxide urea
Liquid；The nitrite ion B liquid is tetramethyl benzidine (TMB) solution of 0.2mg/ml, with 1 both during use:1 ratio mixing；
When marker enzyme is for alkali phosphatase, nitrite ion is 4- nitrophenols phosphate buffer；The terminate liquid is the sulphuric acid of 2mol/L
Solution.
The test kit also includes negative control sera, positive control serum；The negative control sera is to use no mouth hoof
The normal swine serum of epidemic disease antibody；The positive control serum is many with the foot and mouth disease A-type virus Structural protein VP1 epitope
The serum that peptide is obtained for immunogen immune pig；
Involved positive control serum is envelope antigen (the foot-and-mouth disease a type disease specifically with the synthesis of above-mentioned artificial chemistry
Malicious Structural protein VP1 antigen epitope polypeptide) height to be prepared as immunogen (immune 25～35 ages in days no specific source of disease body pig) exempt from blood
Clearly, 0.2 μm of filter membrane is crossed degerming, as the positive control serum of test kit.
Involved negative control sera is the normal swine serum of use no foot-and-mouth disease antibody, and 0.22 μm of filter membrane excessively is degerming, makees
For test kit negative control sera.
The test kit also includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid be containing 0.5% (g/ml) cheese
0.01M, pH of albumen is 7.4 PBS (crossing 0.22 μm of filter membrane)；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 (degerming through 0.22 μm of filter membrane).
The detection program of test kit of the present invention is：
1st, balance：Test kit is taken out from cold storage environment, puts equilibrium at room temperature 30min standby；The front mixing of liquid reagent.
2nd, join liquid：Concentrated cleaning solution distilled water or 20 times of dilutions of deionized water are obtained lavation buffer solution；
3rd, set：2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4th, specimen pre-dilution to be measured：Using sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to 1：20 dilution proportion.
5th, it is 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 the sample to be tested for presetting plus 100 μ l dilute.Sample-adding process time span should be as far as possible short.
6th, incubate：Concussion is mixed, and is put in 37 DEG C of incubators or water-bath, reacts 30min.
7th, plate is washed：Reactant liquor is discarded, is added the lavation buffer solution after 300 μ l dilution per hole, 15s is soaked, is got rid of and abandon washing liquid.Continuously
Pat dry after washing plate 4 times.
8th, enzyme-added：Each hole adds the staphylococcal protein A of 100 μ l horseradish peroxidase-labeled.
9th, incubate：Put in 37 DEG C of incubators or water-bath, react 30min.
10th, plate is washed：Reactant liquor is discarded, and the 300 μ l of lavation buffer solution after dilution is added per hole, is soaked 15s, get rid of and abandon washing
Liquid.Pat dry after continuously washing plate 4 times.
11st, developing the color the substrate nitrite ion of 100 μ l is separately added into per hole (wherein solution A and solution B are with volume ratio as 1：1
Ratio mixes).Add a cover, in 37 DEG C of calorstats, react 15min；
12nd, 50 μ l terminate liquid terminating reactions are separately added into per hole, mix；
13rd, OD per hole is determined450Value (plus the Sptting plate of terminate liquid should read OD in 15min450Value).
The judgement of testing result：
1st, negative control OD450Meansigma methodss should≤0.15, otherwise invalid.
2nd, each detected value of positive control should be otherwise invalid between 0.9-2.3.
3rd, the calculating of marginal value：Ox blood serum marginal value=0.15 × positive control OD450Value meansigma methodss
Porcine blood serum marginal value=0.18 × positive control OD450Value meansigma methodss
Determination of serum OD to be checked450Value >=marginal value person is judged to the positive；Determination of serum OD to be checked450Value<Marginal value person is judged to
Negative.
The mentioned reagent box of the present invention, can be used to detect foot-and-mouth disease a type virus structural protein antibody, to judge tested animal
With the presence or absence of the foot and mouth disease A-type virus Structural protein VP1 antibody for producing after infection or immunity, wherein, animal aftosa viral disease
For pig, hostis pecoris disease, pig is specifically as follows, hoof-and-mouth disease viral disease that cattle foot and mouth disease A-type virus cause.
Application in the test kit for detecting whether infection animal hoof-and-mouth disease viral disease is prepared falls within the protection of the present invention
Scope；
The positive effect of the present invention is：The present invention is carried out to the epitope of structural protein using bioinformatics method
Accurate Analysis, filter out the peptide fragment of suitable ELISA detection on the Main Antigenic from VP1 albumen.The peptide fragment is concentrated
Epitope, have the advantages that susceptiveness height, high specificity.
Meanwhile, it is used for being coated enzyme reaction plate using advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen and prepares examination
Positive control serum in agent box.
Further, since the envelope antigen used in test kit is chemically synthesized polypeptide, without foreign protein, purity height, enter one
Step improves the efficiency of detection foot and mouth disease structural protein antibody, to judge whether tested animal infects foot and mouth disease viruses or inoculation epidemic disease
Seedling.
In a word, this test kit is coated integrated enzyme reaction using the antigenic peptides of chemosynthesis Structural protein VP1 major antigenic sites
Plate, antigen consumption is few, sensitivity is high, high specificity, after can effectively detecting mouth disease virus infection or inoculation inactivated vaccine
The Structural protein VP1 antibody of generation, to judge whether tested animal infects foot and mouth disease viruses or vaccination, with foot and mouth disease viruses
Non-structural protein antibody assay kit is combined, and can distinguish mouth disease virus infection animal and vaccine immunity animal.Experimental result
Show, the test kit of the present invention is reproducible, high specificity, sensitivity height.The needs of different levels personnel can be met, with wide
Wealthy market prospect and good economical, societal benefits.
Foot-and-mouth disease ELISA diagnostic kit involved in the present invention is dynamic for detecting
Whether thing infects foot and mouth disease viruses or inoculation foot-and-mouth disease vaccine, advantageously reduces the brought loss of China's foot and mouth disease outburst,
Be conducive to the foundation of China's foot and mouth disease prevention and control system.
Specific embodiment
Method in following embodiments, if no special instructions, is conventional method.
Embodiment 1, the preparation of foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent envelope antigen
This test for the difference of domestic foot and mouth disease epidemic isolates, using bioinformatics method to foot and mouth disease viruses A type
The multiple hypotypes of VP1 structural protein carry out Accurate Analysis, filter out suitable peptide fragment, are respectively synthesized out with full-automatic polypeptide synthetic instrument
According to 3 peptides of epidemic isolates sequential design, sequence is respectively shown in sequence 1 in sequence table, sequence 2 and sequence 3, makes purity
About 90% characteristic for updating more full envelope antigen, can adapt to that foot and mouth disease viruses are constantly mutated, improves the inspection of antibody positive
Go out rate.Polypeptide synthesis method can be conventional method, and the present invention makes to synthesize three polypeptides of the present invention with the following method, used as this
The coating antigen of bright test kit.
The envelope antigen of the present invention can be made using Applied Biosystem full-automatic polypeptide synthetic instrument (model 433A)
Standby.With Merrifield solid-phase synthesis, Fmoc (9-fluorenylmethyloxycarbonyl, 9- fluorenes first is used
Oxygen carbonyl) aminoacid modified, Rink Amide mbha resin is used as solid phase carrier.Production process includes polypeptide antigen
Solid phase synthesis, polypeptide cleavage and identification, antigen purification, lyophilization and five parts of preservation.Illustrate individually below：
First, envelope antigen solid phase synthesis
1st, the preparation of synthetic agent
Synthesis envelope antigen aminoacid sequence such as SEQ ID NO:1、SEQ ID NO:2 and SEQ ID NO:Shown in 3.
Prepare the aminoacid (purchased from NOVA company) of suitable Fmoc modification according to envelope antigen sequence and synthesis scale,
In adding to corresponding Cartridge.Equally synthesis scale claims resin 5g on request, is put into reaction chamber, upper and lower lid is tightened,
Label, the title of the synthesized peptide of record, lot number, the weight of the TARE of reaction chamber and alleged resin.Reaction chamber is loaded synthesis
Instrument.Prepare appropriate synthetic agent include 100% NMP, 3% AIM (acyl imidazoles), 35% PIP (piperidines), 100%
MeOH (methanol) etc. be placed in corresponding reagent bottle.
2nd, the detection of synthesizer state
Check whether 433A Peptide systhesis instrument normally runs, after start, Run Self Test program is run, instrument is certainly
Whether inspection indices are normal.In addition check whether nitrogen is sufficient, whether normal (the normal gauge pressure of 433A of system gauge pressure
10.2psi).Before synthesis, the performance of reply instrument is had gained some understanding, so will be measured to the flow velocity of every kind of synthetic agent.433A
Synthesizer：Flow Rate1-18 is sent to synthesizer, select Main Menu Module Test to look for by Prer or next
Module A, ModuleD, ModuleI, ModuleI, Module A) measure by more by Start or observe, if
Flow is improper, then adjust lower valve pressure, until it reaches require (concrete detection requirement 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 |
3rd, envelope antigen synthesis starts
The aminoacid sequence that will synthesize in the program of 433A synthesizer sends Std Fmoc 1.0Sol DIC90 to conjunction
On Cheng Yi.The sequence of File-New-Sequence- Edit and Compose peptide, preserves.Whether File-New-Run, check Chemistry
For Std Fmoc 1.0Sol DIC 90；Whether Sequence is by depositing name；Set Cycles；Preserve.It is finally sent to close
On Cheng Yi.
Main Menu Cycle Monitor begin, brings into operation.
4th, envelope antigen synthesis is carried out
The removing of Fmoc group, the electron attraction of the fluorenes ring system of Fmoc group makes 9-H have acidity, is easily removed compared with weak base
Go, eliminated to form hexichol fluorenes alkene with piperidines (PIP) attack 9-H, β during reaction, it is easy to formed by two-stage ring amine attack stable
Addition product."-NH is exposed after the removing of Fmov group2" group to be to carry out synthetic reaction.It is subsequently adding the next one of activation
The aminoacid of Fmoc radical protection and I-hydroxybenzotriazole (1-hydroxybenzotriazole, HOBT) are to reactor.
Peptide sequence described above, is started to N-terminal from C-terminal when synthesis, according to specific order, successively constantly
Repeat synthesis step (synthesis follow procedure is automatically performed, concrete circulation step such as table 2 below).Period observed and recorded reagent dosage and fortune
Market condition.
2. envelope antigen synthesis cycle step of table
5th, envelope antigen end of synthesis
After envelope antigen end of synthesis, synthesizer will be automatically stopped, and peptide resin (peptide is connected on resin) is washed substantially
Totally.Then remove reactor from Peptide synthesizer, then washed after peptide resin 3 times with 100% methanol, blow in fume hood
Dry, then polypeptide resin is fully transferred in the polyethylene bottle of brown, is put in -20 DEG C of refrigerators, sealing film phonograph seal is standby.
2nd, the cracking of envelope antigen and identification
1st, the cracking of polypeptide antigen
It is chemically bound together with solid phase carrier through the polypeptide obtained by above-mentioned reaction, it is necessary to by specific
The acidolysis of organic acid polypeptide is separated with solid phase carrier.Also guarantor each amino acid functional group on is eliminated while acidolysis
Shield base.Step is as follows：
The peptide resin (polypeptide is connected to resin) of synthesis is taken out from refrigerator, is put in the round-bottomed flask of a 2L,
90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), the tripropyl silane of 10ml is added in fume hood in flask
(TIS) and magnetic stir bar, then stably flask is placed on magnetic stirring apparatuss, under room temperature, continuously stirred 1h is to having reacted
Entirely.After reaction terminates, 30～120min is persistently evaporated using the Rotary Evaporators with cold-trap and remove the TFA in crude product.Then
The crude product of polypeptide antigen is cleaned multiple times with dimethylformamide (DMF), finally by the resin for mixing sand core funnel mistake
Leach and, both obtained envelope antigen.
2nd, the identification of envelope antigen
Polypeptide antigen synthesis winged tries time mass spectrum method (MODAL-TOF) and anti-phase height with substance assistant laser desorpted after finishing
Pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis, identifies synthesized peptide with common amino acid analysises.
3rd, envelope antigen purification
Polypeptide antigen after cyclisation is carried out ultrafiltration (with the production of PALL company using circulating tangential flow filtration film bag
The circulating tangential flow filtration film bag of Tangential Flow Device and the peristaltic pump supporting with which), polypeptide antigen is used as big
Molecule can not be by the filter membrane of certain pore size, and early stage building-up process and later stage cyclization are formed or the small molecule of introduction is miscellaneous
Matter can then pass through filter membrane.Then degerming for 0.2 μm of filter by aperture again, last resulting solution is dispensed into aseptic plastic
In bottle, labelled.Title, numbering, product batch number, concentration, date of manufacture, pot-life and the preservation of polypeptide is indicated on label
Condition, after subpackage, be stored in -20 DEG C or -40 DEG C standby.
4th, envelope antigen lyophilization
For the ease of long-term storage and transport, need for envelope antigen to carry out lyophilization to obtain many of solid state
Peptide.The envelope antigen for freezing in advance is positioned on the freezer dryer of Labconco and is dried, obtain solid state
Envelope antigen.Labelled after packaging.The title of dated polypeptide, numbering, product batch number, concentration, date of manufacture, preservation on label
Time limit and preservation condition.
Embodiment 2, the preparation of 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) the removable polystyrene enzyme-linked reaction plate in 96 holes of foot-and-mouth disease virus antigen is coated with；2 × 96 holes.
(2) staphylococcal protein A (purchased from abcam company, article No. ab7456) of horseradish peroxidase-labeled, 2 bottles (each
12ml).
(3) positive control serum：Be using embodiment 1 in artificial chemistry synthesis envelope antigen as immunogen, exempt from respectively
25～35 age in days of the epidemic disease hyper-immune serum that no prepared by specific source of disease body pig, 0.22 μm of filter membrane excessively is degerming, and the positive as test kit is right
According to serum (1 pipe, 1.5ml/ is managed).
(4) negative control sera：It is the normal swine serum of use no foot-and-mouth disease antibody, 0.22 μm of filter membrane excessively is degerming, used as examination
Agent box negative control sera (1 pipe, 1.5ml/ is managed).
(5) substrate nitrite ion：It is made up of two kinds of solution mixing, solution A is the Fructus Citri Limoniae containing 0.6mg/ml hydrogen peroxide urea
Acid phosphoric acid salt buffer (1 bottle, 12ml/ bottle)；Solution B for 0.2mg/ml tetramethyl benzidine (TMB) solution (1 bottle, 12ml/
Bottle)；With volume ratio as 1 both during use:1 ratio mixing.
(6) concentrated cleaning solution (20 ×)：Containing 0.5% (ml/ml) Tween-20 and 0.05% (g/ml) sodium azide anti-corrosion
The 0.01M phosphate buffer of agent, pH is degerming (2 bottles, 50ml/ bottle) through 0.22 μm of filter membrane 7.4, after being.
(7) terminate liquid：The sulfuric acid solution (1 bottle, 12ml/ bottle) of 2mol/L.
(8) sample diluting liquid：Containing the PBS that 0.5% (g/ml) caseic 0.01M, pH are 7.4,0.22 μ is crossed
M filter membrane (2 bottles, 12ml/ bottle).
As needed, there can also be serum-dilution plate (2 pieces, 96 holes/block) in test kit, for the dilution of blood serum sample.
Wherein, the preparation method for being coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of foot-and-mouth disease virus antigen is：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, per
Hole is per peptide 50ng antigen (in the polypeptide wherein in sequence table shown in sequence 1 and polypeptide shown in sequence in sequence table 2 and sequence table
Polypeptide shown in sequence 3, the mass ratio of three kinds of polypeptides is 1:1:1), place at 2～4h, then 4～8 DEG C at 37 DEG C and 8h is placed, make
Polypeptide antigen is fully combined with enzyme-linked reaction plate.Then according to 300 μ l/ hole concentration are added and contain 1% (g/ml) bovine serum albumin
(BSA) PBS (pH=7.4), 37 DEG C closing 2～3h, with lavation buffer solution (concentrated cleaning solution distilled water or go from
The 20 times of dilutions of sub- water obtain lavation buffer solution) dry after washing, question response plate dries rear 4 DEG C of sealing preserves.
Embodiment 3, the coincidence rate test of foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent
First, the using method of foot-and-mouth disease a type Structural protein VP1 antibody ELISA immunity detection reagent
1st, balance：The test kit for being stored in 4 DEG C is taken out, is balanced standby to room temperature；The front mixing of liquid reagent.
2nd, join liquid：Concentrated cleaning solution distilled water or 20 times of dilutions of deionized water are obtained lavation buffer solution；
3rd, set：2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4th, specimen pre-dilution to be measured：Using sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to 1：20 dilution proportion.
5th, it is 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 the sample to be tested for presetting plus 100 μ l dilute.Loading time span should be as far as possible short.
6th, incubate：Concussion is mixed, and is put in 37 DEG C of incubators or water-bath, reacts 30min.
7th, plate is washed：Reactant liquor is discarded, adds the lavation buffer solution for obtaining in 300 μ l steps 2 per hole, 15s is soaked, is got rid of and abandon washing
Liquid.Pat dry after continuously washing plate 4 times.
8th, enzyme-added：Each hole adds the staphylococcal protein A of 100 μ l horseradish peroxidase-labeled.
9th, incubate：Put in 37 DEG C of incubators or water-bath, react 30min.
10th, plate is washed：Reactant liquor is discarded, and the 300 μ l of lavation buffer solution after dilution is added per hole, is soaked 15s, get rid of and abandon washing
Liquid.Pat dry after continuously washing plate 4 times.
11st, develop the color：(wherein solution A and solution B are with volume ratio as 1 the substrate nitrite ion of 100 μ l to be separately added into per hole：1
Ratio mixes).Add a cover, in 37 DEG C of calorstats, react 15min；
12nd, 100 μ l terminate liquid terminating reactions are separately added into per hole, mix；
13rd, OD per hole is determined450Value (plus the Sptting plate of terminate liquid should read OD in 15min450Value).
The judgement of testing result：
1st, negative control OD450Meansigma methodss should≤0.15, otherwise invalid.
2nd, each detected value of positive control should be otherwise invalid between 0.9～2.3.
3rd, the calculating of marginal value：Ox blood serum marginal value=0.15 × positive control OD450Value meansigma methodss
Porcine blood serum marginal value=0.18 × positive control OD450Value meansigma methodss
Determination of serum OD to be checked450Value >=marginal value person is judged to the positive；Determination of serum OD to be checked450Value<Face value person and be judged to the moon
Property.
2nd, neonatal rat serum neutralization test (MSN) method
One of domestic conventional aftosa serum detection method is neonatal rat neutralization test (MSN) at present, is therefore tried with this
Agent box carries out coincidence rate test with neonatal rat neutralization test.
Material
1. immune serum, ((foot and mouth disease is O-shaped, A type, Asia I type three for Schweineseuche inactivated vaccine for 50 part immunity porcine blood serums
Bivalent inactivated vaccine, article No. ABB10601730250) immune latter 28 days)；50 part immunity Ox blood serum (cattle inactivated foot-and-mouth disease vaccines
(foot and mouth disease is O-shaped, A type, Asia I type tervalence inactivated vaccine, article No. ABB10601730250) immunity latter 21 days), by herd industry
Limited company Lanzhou biology pharmaceutical factory provides.
2. healthy 50 parts of porcine blood serum, healthy 50 parts of Ox blood serum, are carried by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.
For.
3. test virus is foot and mouth disease AF/72 virus, and seed culture of viruses provided by Lanzhou veterinary institute, by herd industry share
Company limited Lanzhou biology pharmaceutical factory preserves and uses, and virus was passed through 6～8 age in days neonatal rats after 2 generations, collects virus.With 3～4 days
Age neonatal rat determines and adjusts its malicious valency to 1000LD50/ 0.2ml, is placed in -20 DEG C of preservations stand-by.
Neonatal rat serum neutralization test method is as follows：Serum is made 2 times of dilutions with PBS, 1ml is taken with equivalent 100LD50/ ml's
The virus liquid mixing of AF/72 system, is incubated 1 hour in 37 DEG C, is then inoculated with 3 age in days neonatal rats (cervical region subcutaneous injection 0.2ml), per group
(that is, each serum to be checked) 4, while set up virus control, normal healthy controls and standard positive serum matched group, per group 3,
Observation 7d, record neonatal rat fatality ratio result of determination, if neonatal rat 4/4 or 3/4 is strong living, serum to be checked is positive (R), if newborn
Mus 4/4 or 3/4 are dead, then serum to be checked for negative (-)；If neonatal rat 2/4 is strong living or 2/4 dead, result of determination suspicious (±),
Replication need to be doubled, in repeating to test, have 1 group to be positive, be then judged to the positive.
Sensitivity=judgement positive serum sample number/test serum sample number
3rd, coincidence rate result of the test：
The foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent for being prepared using embodiment 2 and neonatal rat
Neutralization test (MSN) detects 100 parts of porcine blood serums (50 parts of immune serum, 50 parts of healthy serum), 100 parts of Ox blood serum (immunity respectively
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 to the testing result of porcine blood serum
3.The sensitivity of test kit is 98%, and the sensitivity of neonatal rat serum neutralization test is in 50 parts of immune porcine blood serums two
Kind of method testing result consistent for 47 parts.Therefore, the coincidence rate of this test kit and neonatal rat serum neutralization test is 94%, therefore
This test kit has higher sensitivity.Both coincidence rates of testing to healthy porcine blood serum are 100%, and testing result is the moon
Property (being shown in Table 3).
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent is had altogether with neonatal rat neutralization test and detects
100 parts of porcine blood serums, two methods of wherein 97 parts porcine blood serum testing results are consistent, and 3 parts of porcine blood serum testing results are variant, coincidence rate
For 97%.
3. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent of table and neonatal rat serum neutralization test
Method is to immune porcine blood serum and the testing result of healthy porcine blood serum
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent is shown in Table to the testing result of Ox blood serum
4.As a result show, the sensitivity of test kit is 98%, and the sensitivity of neonatal rat serum neutralization test is 94%, in 50 parts of immune cattles
In serum, two methods testing result consistent for 48 parts, therefore, the coincidence rate of this test kit and neonatal rat serum neutralization test is
96%, therefore this test kit is with higher sensitivity.Both coincidence rates of testing to healthy Ox blood serum are 100%, detection
As a result negative (being shown in Table 4) is.
Foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent is had altogether with neonatal rat neutralization test and detects
100 parts of Ox blood serums, two methods of wherein 98 parts Ox blood serum testing results are consistent, and 2 parts of ox serum detection results are variant, coincidence rate
For 98%.
4. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent of table and neonatal rat serum neutralization test
Method is to immune Ox blood serum and the testing result of healthy Ox blood serum
Embodiment 4, the sensitivity testss of foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent
Sensitivity testss are 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 of pig source and cattle source serum respectively.Pig infection serum is 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 (peptide 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 cattle foot-and-mouth disease a type disease
Poison infection serum.Cattle source vaccine immune sera is that (peptide sequence of synthetic peptide vaccine is cattle foot and mouth disease A-type virus synthetic peptide vaccine
Sequence 5 in sequence table) Post-immunisation serum, provided by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd..
The three batches of foot and mouth disease A-type virus structural protein antibody ELISA immunosorbent adsorption test diagnosis examinations for being prepared using embodiment 2
Agent box (batch ZMAVP001, ZMAVP002, ZMAVP003), anti-according to foot and mouth disease A-type virus Structural protein VP1 in embodiment 3
Body enzyme-linked immunologic detecting kit using method is respectively to pig, cattle foot and mouth disease A-type virus infection serum each 200 parts and pig, Niu Kou
Fever aphthous A type each 200 parts of synthetic peptide vaccine immune serum of virus carries out sensitivity testss, and experimental result is shown in Table 5.
Sequence 1, sequence 2 and three polypeptides shown in sequence 3 are prepared using the method described in embodiment 2, individually wrap
A type virus structural protein antibody ELISA immunity detection reagent (ZMAVP1-1, ZMAVP1-2, the ZMAVP1- for being assembled by ELISA Plate
3), right respectively according to foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent using method in embodiment 3
Pig, cattle foot and mouth disease A-type virus infection serum each 200 parts and pig, cattle foot and mouth disease A-type virus synthetic peptide vaccine immune serum each 200
Part carries out sensitivity testss, and experimental result is shown in Table 5.
ZMAVP001 test kit is 196/200 × 100%=98% to the sensitivity of 200 parts of infected pigs' Virus monitory；Right
The sensitivity of 200 parts of immune swine Virus monitory is 198/200 × 100%=99%.ZMAVP002 test kit is to 200 parts of infected pigs
The sensitivity of Virus monitory is 197/200 × 100%=98.5%；Sensitivity to 200 parts of immune swine Virus monitory is 198/
200 × 100%=99%.ZMAVP003 test kit is 197/200 × 100% to the sensitivity of 200 parts of infected pigs' Virus monitory
=98.5%；Sensitivity to 200 parts of immune swine Virus monitory is 197/200 × 100%=98.5%.
ZMAVP1-1 test kit is 193/200 × 100%=96.5% to the sensitivity of 200 parts of infected pigs' Virus monitory；
Sensitivity to 200 parts of immune swine Virus monitory is 195/200 × 100%=97.5%.ZMAVP1-2 test kit is felt to 200 parts
The sensitivity of dye porcine blood serum detection is 194/200 × 100%=97%；To the sensitivity of 200 parts of immune swine Virus monitory it is
194/200 × 100%=97%.ZMAVP1-3 test kit is 194/200 to the sensitivity of 200 parts of infected pigs' Virus monitory ×
100%=97%；Sensitivity to 200 parts of immune swine Virus monitory is 193/200 × 100%=96.5%.
ZMAVP001 test kit is 197/200 × 100%=98.5% to the sensitivity of 200 parts of infection ox serum detection；
Sensitivity to 200 parts of immune ox serum detection is 196/200 × 100%=98%.ZMAVP002 test kit is infected to 200 parts
The sensitivity of ox serum detection is 197/200 × 100%=98.5%；To the sensitivity of 200 parts of immune ox serum detection it is
197/200 × 100%=98.5%.ZMAVP003 test kit to 200 parts infection ox serum detection sensitivity be 197/200 ×
100%=98.5%；Sensitivity to 200 parts of immune ox serum detection is 198/200 × 100%=99%.
ZMAVP1-1 test kit is 195/200 × 100%=97.5% to the sensitivity of 200 parts of infection ox serum detection；
Sensitivity to 200 parts of immune ox serum detection is 194/200 × 100%=97%.ZMAVP1-2 test kit is infected to 200 parts
The sensitivity of ox serum detection is 194/200 × 100%=97%；Sensitivity to 200 parts of immune ox serum detection is 193/
200 × 100%=96.5%.ZMAVP1-3 test kit to 200 parts infection ox serum detection sensitivity be 193/200 ×
100%=96.5%；Sensitivity to 200 parts of immune ox serum detection is 195/200 × 100%=97.5%.
5. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent of table is to pig, the sensitivity of Ox blood serum
Testing result
Embodiment 5, the specific test of foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent
Using three batches of test kits in embodiment 4 according to the foot and mouth disease A-type virus Structural protein VP1 described in embodiment 3
The using method of antibody ELISA immunity detection reagent is to 200 parts of health porcine blood serum (Zhongmu Industry Co., Ltd Bao Shanchang
There is provided with Lanzhou biology pharmaceutical factory), 2 parts of Schweineseuche Asia I type (Asia1) positive serums (Zhongmu Industry Co., Ltd orchid
State biology pharmaceutical factory provide), (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. carries 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) detected respectively.
The specific detection result such as following table (table 6) of test kit shows.Testing result table to 200 parts of healthy porcine blood serums
Bright, the specificity of ZMAVP001 test kit is 99.5%, ZMAVP003 reagent for the specificity of 99.0%, ZMAVP002 test kit
The specificity of box is 99.0%.To 2 parts of Schweineseuche Asia I types (Asia I) positive serum, 2 parts Schweineseuche O-shaped (O) positive
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 blood
Clear testing result is illustrated as feminine gender, and therefore three test kits are to the specificity of this 12 parts of pig positive serum detections
100%.
The specific detection result such as following table (table 6) of test kit shows.The testing result of 200 parts of healthy Ox blood serums is shown
Show, the specificity of ZMAVP001 test kit is 99.5%, ZMAVP003 reagent for the specificity of 99.0%, ZMAVP002 test kit
The specificity of box is 99.5%.To 2 parts of cattle foot and mouth disease Asia I type (Asia I) positive serums, 2 parts of cattle foot and mouth disease O-shaped (O) positives
Serum, the testing result of 2 parts of bovine viral diarrhoea positive serums are illustrated as feminine gender, and therefore three test kits are to this 6 parts of cattle sun
The specificity of property Virus monitory is 100%.
6. foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent specific detection result of table
Sequence 1, sequence 2 and three polypeptides shown in sequence 3 are prepared using the method described in embodiment 2, individually wrap
A type virus structural protein antibody ELISA immunity detection reagent (ZMAVP1-1, ZMAVP1-2, the ZMAVP1- for being assembled by ELISA Plate
3), right respectively according to foot and mouth disease A-type virus Structural protein VP1 antibody ELISA immunity detection reagent using method in embodiment 3
200 parts of healthy porcine blood serum (Baoshan factory of 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 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 test kit shows.Testing result table to 200 parts of healthy porcine blood serums
Bright, the specificity of ZMAVP1-1 test kit is 98.5%, ZMAVP1-3 reagent for the specificity of 97.0%, ZMAVP1-2 test kit
The specificity of box is 98.0%.To 2 parts of Schweineseuche Asia I types (Asia I) positive serum, 2 parts Schweineseuche O-shaped (O) positive
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 blood
Clear testing result is illustrated as feminine gender, and therefore three test kits are to the specificity of this 12 parts of pig positive serum detections
100%.
The specific detection result such as following table (table 7) of test kit shows.The testing result of 200 parts of healthy Ox blood serums is shown
Show, the specificity of ZMAVP1-1 test kit is 97.5%, ZMAVP1-3 reagent for the specificity of 98.0%, ZMAVP1-2 test kit
The specificity of box is 97.5%.To 2 parts of cattle foot and mouth disease Asia I type (Asia I) positive serums, 2 parts of cattle foot and mouth disease O-shaped (O) positives
Serum, the testing result of 2 parts of bovine viral diarrhoea positive serums are illustrated as feminine gender, and therefore three test kits are to this 6 parts of cattle sun
The specificity of property Virus monitory is 100%.
The independent peptide specific testing result of table 7.
Claims (10)
1. foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide is polypeptide in sequence table shown in sequence 1, in sequence table
Polypeptide in sequence 2 or sequence table shown in sequence 3.
2. foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide compositionss, its effective ingredient is sequence in sequence table 1
One or more combination in any in polypeptide in shown polypeptide, sequence table in sequence 2 and sequence table shown in sequence 3.
3. a kind of foot and mouth disease A-type virus structural protein antibody ELISA immunity detection reagent, including foot and mouth disease A-type virus structure egg
The white coated enzyme-linked reaction plate of VP1 antigen epitope polypeptide and ELIAS secondary antibody；The foot and mouth disease A-type virus Structural protein VP1 antigen
Epitope polypeptide is shown in sequence 3 in the polypeptide in the polypeptide in sequence table shown in sequence 1, sequence table shown in sequence 2 and sequence table
The mixed polypeptide of one or more compositions in polypeptide.
4. test kit according to claim 1, it is characterised in that：Polypeptide, sequence table in the sequence table shown in sequence 1
The mass ratio of the polypeptide in the polypeptide and sequence table shown in middle sequence 2 shown in sequence 3 is 0.5～2.0:0.5～2.0:1；Described
The quality of polypeptide shown in sequence 3 in polypeptide and sequence table in polypeptide and sequence table in sequence table shown in sequence 1 shown in sequence 2
Than being preferably 1:1:1.
5. according to any one in claim 2～4, it is characterised in that：The marker enzyme of the ELIAS secondary antibody is Radix Cochleariae officinalises peroxidating
Thing enzyme or alkali phosphatase；The ELIAS secondary antibody is enzyme labelling staphylococcal protein A；The ELIAS secondary antibody is preferably Radix Cochleariae officinalises peroxide
The staphylococcal protein A of compound enzyme labelling.
6. according to the test kit of any one in 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 is obtained for chemical synthetic.
7. test 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 be by the foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide
The carbonate solution of the pH 9.6 of 100 μ l is dissolved in, 96 hole polystyrene enzyme-linked reaction plates are then added to, per hole 50ng per peptide,
Placing to place at 2～4 hours, then 4-8 DEG C for 37 DEG C makes polypeptide antigen fully be combined with enzyme-linked reaction plate in 8～12 hours, then presses
The PBS containing 0.01g/ml bovine serum albumin pH7.4 is added according to 300 μ l/ holes, 37 DEG C of sealing treatment 2～3 hours,
Dry after washing, 4 DEG C of sealing preserves after enzyme-linked reaction plate dries.
8. test kit according to claim 7, it is characterised in that：Also containing nitrite ion and terminate liquid in the test kit；
When marker enzyme is for horseradish peroxidase, nitrite ion is made up of nitrite ion A liquid and nitrite ion B liquid, the nitrite ion A liquid be containing
The citrate phosphate buffer of 0.6mg/ml hydrogen peroxide urea, the nitrite ion B liquid is the tetramethyl biphenyl of 0.2mg/ml
Amine aqueous solution；When marker enzyme is for alkali phosphatase, nitrite ion is 4- nitrophenols phosphate buffer；The terminate liquid is 2mol/L
Sulfuric acid solution.
9. test kit according to claim 8, it is characterised in that：The test kit also includes negative control sera, the positive
Control serum；The negative control sera is to use the no normal pig of foot-and-mouth disease antibody or Ox blood serum；The positive control serum is
With the serum that the A type Foot-and-mouth disease VP1 antigen epitope polypeptide is obtained as immunogen immune pig, cattle；And/or institute
Stating test kit also includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid be containing the caseic 0.01M of 0.005g/ml,
PH is 7.4 PBS；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. the foot and mouth disease A-type virus Structural protein VP1 antigen epitope polypeptide described in claim 1 is preparing detection infection or is exempting from
Application in the test kit of the foot and mouth disease A-type virus Structural protein VP1 antibody for producing after epidemic disease；Wherein, animal aftosa viral disease
For pig or cattle foot and mouth disease A-type virus disease；Preferably, the malicious mouth for causing for pig, cattle foot and mouth disease A-type virus of the animal aftosa
Aphtovirus disease.
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| CN108931644A (en) * | 2018-07-19 | 2018-12-04 | 河南省农业科学院 | A kind of evaluation of foot and mouth disease virus immune antiboidy and infection diagnose bigeminy test strips with Immune dctection |
| 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 |
| CN109856396A (en) * | 2018-12-24 | 2019-06-07 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Detect enzyme linked immunological kit and its application of mouth disease virus infection antibody |
| 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 |
| CN110669112A (en) * | 2019-11-08 | 2020-01-10 | 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 |
| CN110734478A (en) * | 2019-11-08 | 2020-01-31 | 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 |
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| CN109485703B (en) | 2021-07-06 |
| CN106432434B (en) | 2019-11-12 |
| CN109485703A (en) | 2019-03-19 |
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