CN103864906B - Foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent - Google Patents
Foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent Download PDFInfo
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- CN103864906B CN103864906B CN201410092728.1A CN201410092728A CN103864906B CN 103864906 B CN103864906 B CN 103864906B CN 201410092728 A CN201410092728 A CN 201410092728A CN 103864906 B CN103864906 B CN 103864906B
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- C12N2770/32111—Aphthovirus, e.g. footandmouth disease virus
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Abstract
The invention discloses a kind of foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent.It includes the foot and mouth disease virus non-structural protein coated enzyme-linked reaction plate of 3B antigen epitope polypeptide and enzyme labelling anti antibody；Described foot and mouth disease virus non-structural protein 3B antigen epitope polypeptide is the polypeptide shown in sequence 1 in sequence table.This test kit uses chemosynthesis non-structural protein 3B antigenic peptides to be coated Sptting plate, and antigen consumption is few, susceptiveness and specificity high, can detect whether efficiently to there is mouth disease virus infection.Test kit specificity of the present invention is good, sensitive, efficient, has good market prospect.
Description
Technical field
The invention belongs to biological technical field, more particularly it relates to an Foot-and-mouth disease antibody
Enzyme-linked immunologic detecting kit, particularly Foot-and-mouth disease VP1 antibody ELISA immunity detection reagent.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease, FMD) is widely distributed in world wide, propagates climing internationally
The global prevalence sexually transmitted disease prolonged.World Organization for Animal Health (OIE) 1984 revision zoonosis classification in by mouth
Fever aphthous is classified as first of A class infectious disease.Foot and mouth disease is the strong biography being caused artiodactylous animals to occur by mouth disease virus infection
Catch an illness.The most popular cows and swinery.The fatality rate of foot and mouth disease is the lowest, but infection rate is the highest.Morbidity
Cardinal symptom is: the positions such as oral mucosa, tongue, lip, asoscope, frog, nipple occur vesicle, and ulceration forms speck,
The sleeping ground of ill domestic animal hoof pain, severe patient coffin edge debacle or shelling.The symptom of different animals is slightly different, and cow in calf can
Can miscarry, then cause reproductive capacity to reduce；Pig is then main symptom with broken hoof；The symptom of goat and sheep generally than
Cattle is gentle.Foot and mouth disease infectiousness is high, propagates rapidly, and it is dead that the domestic animal such as infected pigs, cattle, sheep often results in young stock, grows up
Animal production drastically declines, the development of serious harm animal husbandry and the production and supply of livestock products.Stream can be caused simultaneously
The market circulation of row country animal and animal's products, and make its international trade by and big block and restriction,
The loss of tremendous economic is caused to the Animal husbandry production of popular countries and regions.Therefore foot and mouth disease is always by each international politics
The great attention at mansion.
Foot and mouth disease virus (Foot-and-Mouth Disease Virus, FMDV) belongs to picornavirus, this group
Other members of virus include common cold virus and smallpox virus etc..Foot and mouth disease virus (FMDV) have pleiomorphism,
The features such as changeableness.At present it is known that there are 7 serotypes in the whole world: A, O, C, Sat1 (South Africa I type), Sat2 (south
Non-II type), Sat3 (South Africa type III) and AsiaI (Asia I type).Each type divides again some hypotypes, the Asia having now been found that
Type has more than 70.Owing to constantly there is antigenic drift, therefore can not strictly distinguish hypotype.FMDV is by a core
Acid RNA chain and capsid protein composition, geneome RNA total length about 8.5kb, can be directly as messenger RNA, FMDV
Geneome RNA is made up of 5 ' UTR, 3 ' UTR and OFR.5 ' UTR are about 1300nt, containing VPg, two grades
Structure, P10 gene, Poly (c) section and internal ribosome entry site etc. (IRES).3 ' UTR by Poly (A) tail and
Big 92 nt residue compositions, long 172nt between ORF3 ' end and Poly (A) section 5 ' end.Big ORF is by L
Gene (P20a), P1 structural protein gene, P2 and P3 nonstructural protein gene and start codon and termination codon
Son composition.It is arranged in order with the order of L, P1, P2 and P3, is about 6.5Kb.Wherein the 3D section of genome is
The RNA polymerase of coding FMDV, be indispensable composition in virus replication.P1 structural protein gene encodes
Structural protein VP1 main in 4, VP2, VP3 and VP4, they each 60 coat protein constituting FMD.
The coded non-structural protein produced of foot-and-mouth disease virus gene has L, 2A, 2BC, 3AB, 3ABC, 3D etc.,
Wherein the immunogenicity of L albumen is more weak than other non-structural proteins, and infection animal differs and produces antibody surely, even if producing
Antibody, the time of maintenance is the most comparatively short.Virus-infected animal can produce anti-2B antibody, but this antibody is at ELISA
In reaction, repeatability is bad, it is impossible to as detection antibody.2C antibody is fast due to the ratio 3ABC disappeared, and immune cattle
In can detect that 2C antibody, therefore can not be as Testing index.3D, also known as VIA, does not has type
Specificity, detecting its antibody horizontal is to evaluate whether animal contacted the important indicator of antigen, is also that international trade must be examined
Project.It is now recognized that only detection 3D antibody cannot distinguish between infection animal and immune animal, because often containing in vaccine
The non-structural antigen of trace, after repeatedly immunity, also can detect 3D antibody in immune animal body.The poly-egg of 3ABC
Bai Zhong, 3A immunogenicity is the strongest, 3C less immunogenic.Many data think that 3ABC can as the mark infected
Reliability is bigger.Detection 3ABC antibody is the important indicator making a definite diagnosis infection.
At present, the preventing and treating of foot and mouth disease relies primarily on vaccine immunity.Foot and mouth disease is implemented mandatory immunity, annual spring by China
Autumn once concentrates immunity, between twice, new benefit hurdle domestic animal is carried out benefit in time and exempts from.How actual production differentiates
Diagnosis susceptible animal has been by vaccine immunity and has still infected foot and mouth disease virus to foot and mouth disease the epidemic prevention foundation of system, inspection
It is all highly important for testing quarantine.Countries in the world have also been carried out in this field and have been studied widely.ELISA method has spy
The features such as different, sensitive, quick, easy, good reliability, and can be automated operation, and a large amount of sample can be detected rapidly
Product, are increasingly subject to the attention of people in the diagnosis of FMD, have become and have detected susceptible animal pig, cattle etc. in the world
Whether one of vaccine immunity or the conventional method infecting virus.
The De of Italy etc. (1997) develop the elisa technique that indirectly captures based on 3ABC albumen, and can distinguish
Wild virus infection and vaccine virus infect.The Sorensen of Denmark etc. (1998) develop the antigen institute structure with baculovirus expression
The ELISA method built, can detect nonstructural protein 3A B, 3ABC, 3D, wherein 3AB, 3ABC, 3D respectively
ELISA all can detect 7 kinds of serotype antibody of FMDV.The Bruderer (2004) of Germany is also successfully established
3ABC ELISA method.Xie Qingge etc. (2003) utilize the foot and mouth disease virus non-structural protein that colon bacillus is expressed
White 3ABC polypeptide establishes a kind of FMDV that can distinguish as antigen and infects the indirect enzyme-linked immunosorbent with vaccine immunity
Adsorption test.You Yongjin, Zhu Caizhu etc. (2003) set up ELISA based on nonstructural protein 3A BC with same principle
Detection method.
The Sptting plate of existing enzyme-linked immunosorbent assay method based on non-structural protein use is coated is mostly
3ABC, 3AB, and be mostly the envelope antigen that obtains non-structural protein of the method by prokaryotic expression.This type of protokaryon
Containing a small amount of carrier protein and Host Strains albumen in the protein product expressed, it is highly difficult for removing them completely, these
Foreign protein will produce nonspecific combination with detection sample, thus have impact on the accuracy of testing result.
Foot and mouth disease is to be related to the great animal epidemic of economic security of the country, and related key technical should firmly rest in certainly
In own hands.Once we have independent intellectual property right, will be beneficial to ensure the economic security of China.
Summary of the invention
It is an object of the invention to provide a kind of new mirror infected for the immunity of foot and mouth disease susceptible animal pig vaccine with virus
The enzyme-linked immunologic detecting kit not diagnosed.
The foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent of the present invention, including the non-knot of foot and mouth disease virus
The structure albumen coated enzyme-linked reaction plate of 3B antigen epitope polypeptide and enzyme labelling anti antibody；Described foot and mouth disease virus non-structural
Albumen 3B antigen epitope polypeptide is the polypeptide shown in sequence 1 in sequence table.
Involved envelope antigen uses the method for solid-state chemical reaction method to produce, and envelope antigen contains the non-knot of foot and mouth disease virus
The major antigenic sites polypeptide of structure albumen 3B, can be special with the non-structural protein 3B antibody of generation after mouth disease virus infection
Different combination.
The marker enzyme of described enzyme labelling anti antibody is horseradish peroxidase or alkali phosphatase；Described enzyme labelling anti antibody
For the anti-pig IgG of enzyme labelling rabbit；The anti-pig IgG of rabbit that described enzyme labelling anti antibody is preferably horseradish peroxidase-labeled is many
Clonal antibody.
Described enzyme-linked reaction plate is detachable 96 hole ELISA Plate；Described foot and mouth disease virus non-structural protein 3B epitope
Polypeptide obtains for chemistry synthetic.
The present invention is accomplished by: use the artificial chemistry synthesis polypeptide of foot and mouth disease virus non-structural protein 3B
Coated removable 96 hole polystyrene enzyme-linked reaction plates.Meanwhile, containing with horseradish peroxidase-labeled in test kit
The IgG polyclonal antibody enzyme conjugates of the anti-pig of rabbit, negative control sera, positive control serum, sample diluting liquid,
Tmb substrate solution, the component such as 20 × concentrated cleaning solution and stop buffer.With indirect enzyme-linked immunosorbent assay (ELISA)
3B protein antibodies in method detection sample.
The optimum preparating condition of involved enzyme-linked reaction plate is: polypeptide antigen is dissolved in when being coated the carbon of PH9.4
Acid salt solution, is then added to 96 hole polystyrene enzyme-linked reaction plates, every hole 50ng antigen, places 2-4h at 37 DEG C,
4-8 DEG C of left overnight (8-12 hour) makes polypeptide antigen fully be combined with enzyme-linked reaction plate again.Then according to
300ul/ hole adds containing 1%(g/ml) PBS (PH=7.4) of bovine serum albumin (BSA), 37 DEG C
Sealing treatment 1-2h, dries after washing, treats that enzyme-linked reaction plate seals 4 DEG C of preservations after drying.
Containing nitrite ion and stop buffer in described test kit；When marker enzyme is horseradish peroxidase, nitrite ion is by developing the color
Liquid A liquid and nitrite ion B liquid composition, described nitrite ion A liquid is the citric acid containing 0.2% (g/ml) hydrogen peroxide urea
Phosphate buffer, described nitrite ion B liquid is the tetramethyl biphenyl amine aqueous solution of 0.2mg/ml；With 1 both during use:
The ratio mixing of 1.When marker enzyme is alkali phosphatase, nitrite ion is 4-nitrophenols phosphate buffer.Described end
Only liquid is the sulfuric acid solution of 2mol/L.
Described test kit also includes negative control sera, positive control serum；Described negative control sera is for without mouth
The normal swine serum of fever aphthous antibody；Described positive control serum is with described foot and mouth disease virus non-structural protein 3B antigen
Epitope polypeptide is the serum that immunogen immune pig obtains；
The non-structural protein 3B epitope that involved positive control serum specifically synthesizes with artificial chemistry is many
Peptide without specific source of disease body (SPF) pig, is prepared hyper-immune serum as immunogen, immunity 25-35 age in days, is added
1000U/ml streptomycin and penicillin, filter the filter membrane that footpath is 0.2 μm degerming, as the positive control of test kit
Serum.
Involved negative control sera specifically with the normal swine serum without foot-and-mouth disease antibody, adds 1000U/ml
Streptomycin and 1000U/ml penicillin, filter the filter membrane that footpath is 0.2 μm degerming, as test kit negative serum.
Described test kit also includes sample diluting liquid and concentrated cleaning solution；Sample diluting liquid is containing 3% (g/ml) BSA
The PBS that 0.15M, pH are 7.4 (filter footpath be 0.45 μm filter membrane)；Described concentrated cleaning solution
For 0.15M, pH7.4, the phosphate buffer containing 0.5% (ml/ml) Tween-20 (is 0.2 μm through filtering footpath
Filter membrane degerming).
The detection program of test kit of the present invention is:
1. balance: taken out from cold storage environment by test kit, put equilibrium at room temperature and use after 30 minutes, liquid tries
The front mixing of agent.
2. dosing: by concentrated cleaning solution distilled water or 20 times of diluted for use of deionized water.
3. set: staying 1 blank well, 3 negative control holes, and 2 Positive control wells, remaining is for treating
Survey sample aperture.
Specimen pre-dilution the most to be measured: every hole adds 100 μ l Sample dilution in dilution plate hole, is separately added into 5 μ l
Sample to be tested.
5. sample-adding: blank well is not loaded；3 negative control holes respectively add 100 μ l negative controls, and 2 positives are right
100 μ l positive controls are respectively added according to hole；Remaining hole respectively by preset add 100 μ l dilution to be measured
Sample.Sample-adding process time span should be the shortest.
6. incubation: concussion mixing, puts in 37 DEG C of incubators or water-bath, reacts 30 minutes.
7. washing plate: discard reactant liquor, every hole adds the cleaning mixture 300 μ l after dilution, soaks 15 seconds, gets rid of and abandon
Washing liquid.Pat dry after washing plate 4 times continuously.
The most enzyme-added: blank well is the most enzyme-added；Remaining each hole adds the 100 μ l anti-pig IgGs of horseradish peroxidase-labeled rabbit
Antibody (deep red).
9. incubation: put in 37 DEG C of incubators or water-bath, reacts 30 minutes.
10. washing plate: discard reactant liquor, every hole adds the cleaning mixture 300 μ l after dilution, soaks 15 seconds, gets rid of and abandon
Cleaning mixture.Pat dry after washing plate 5 times continuously.
11. colour developings: every hole (including blank control wells) is sequentially added into assay chromogenic substrate solution A, assay chromogenic substrate solution B
Each 50 μ l, concussion mixing, put in 37 DEG C of incubators or water-bath, develop the color 15 minutes.
12. terminate: every hole (including blank control wells) adds the colour developing each 50 μ l of stop buffer, and vibration mixing terminates
Reaction.
13. measure: measure the OD value of each hole 450nm with blank control wells after returning to zero.Also dual wavelength can be used
450nm/630nm measures each hole OD value.
The judgement of the testing result of test kit of the present invention:
1. negative control OD meansigma methods should≤0.2, the most invalid.
2. each detected value of positive control should be the most invalid between 0.6 to 1.8.
3. the calculating of marginal value: marginal value=0.17 × positive control mean OD value.
4. result judges: sample measures OD value >=marginal value person and is judged to the positive；It is < critical that sample measures OD value
Value person is judged to feminine gender (advise to OD value<marginal value, observation followed the tracks of by the sample of>=0.5 times of marginal value).
The positive effect of the present invention is: the present invention uses the bioinformatics method epitope to non-structural protein
Carry out Accurate Analysis, the Main Antigenic from 3B albumen filters out the peptide fragment of applicable ELISA detection.With
Time, use advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen to be used for being coated enzyme reaction plate and preparing in test kit
Positive control serum.
Envelope antigen owing to using in test kit is chemically synthesized polypeptide, and without foreign protein, purity is high, high specificity,
Highly sensitive, therefore can effectively detect foot and mouth disease non-structural protein antibody, to judge whether tested animal infects mouth
Aphtovirus.Test result indicate that, test kit is reproducible, and high specificity is highly sensitive.Can effectively detect
The non-structural protein antibody produced after mouth disease virus infection, to judge whether tested animal infects foot and mouth disease virus.Energy
Meet the needs of different levels personnel, there are wide market prospect and good economical, societal benefits.
This test kit uses chemosynthesis 3B antigenic peptides to be coated Sptting plate, and antigen consumption is few, susceptiveness and specificity high,
Can differentiate that foot and mouth disease susceptible animal pig has infected virus or received vaccine immunity efficiently.Simultaneously can basis
Result carries out the evaluation of immune effect.Test kit specificity of the present invention is good, sensitive, efficient, before having good market
Scape.
Foot and mouth disease virus non-structural protein ELISA diagnostic kit involved in the present invention is used for mouth hoof
The Differential Diagnosis that the immunity of epidemic disease susceptible animal pig vaccine is infected with virus, advantageously reduces what China's foot and mouth disease outburst was brought
Loss, is also beneficial to the foundation of China's foot and mouth disease prevention and control system.
Detailed description of the invention
Method in following embodiment, if no special instructions, is conventional method.
Prepared by embodiment 1, foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent envelope antigen
The present invention uses bioinformatics method the epitope of non-structural protein to be carried out Accurate Analysis, from 3B albumen
On Main Antigenic on filter out applicable ELISA detection peptide fragment, i.e. foot and mouth disease virus non-structural protein 3B
Antigen epitope polypeptide, its sequence is as shown in sequence 1 in sequence table.Using this polypeptide being coated as test kit of the present invention
Former preparation foot and mouth disease virus non-structural protein enzyme-linked immunologic detecting kit.
The envelope antigen of the present invention can use Applied Biosystem full-automatic polypeptide synthetic instrument (model 433A) to make
Standby.Use Merrifield solid-phase synthesis, use Fmoc (9-fluorenylmethyloxycarbonyl, 9-
Fluorenylmethyloxycarbonyl) aminoacid modified, use Rink Amide mbha resin as solid phase carrier.Production process bag
Include polypeptide antigen solid phase synthesis, polypeptide cleavage and qualification, antigen purification, lyophilization and preserve five parts.Below
Illustrate respectively:
One, envelope antigen solid phase synthesis
1, the preparation of synthetic agent
Synthesis envelope antigen aminoacid sequence is as shown in SEQ ID NO:1.
The aminoacid preparing suitable Fmoc modification according to envelope antigen sequence and synthesis scale is (public purchased from NOVA
Department), add to corresponding Cartridge.Synthesis scale weighs resin 5g the most on request, puts into reaction chamber, will
Up and down lid is tightened, and labels, the title of the synthesized peptide of record, lot number, the TARE of reaction chamber and alleged resin
Weight.Reaction chamber is loaded synthesizer.Prepare appropriate synthetic agent include 100% NMP, 3% AIM(
Acyl imidazoles), the PIP(piperidines of 35%), the MeOH(methanol of 100%) etc. be placed in corresponding reagent bottle.
2, the detection of synthesizer state
Check that 433A Peptide systhesis instrument is the most properly functioning, after start, run Run Self Test program, instrument
Device self-inspection indices is the most normal.Additionally checking that nitrogen is the most sufficient, system gauge pressure is the most normal, and (433A is just
Often gauge pressure 10.2psi).Before synthesis, the performance of reply instrument is had gained some understanding, so will be to the stream of every kind of synthetic agent
Speed is measured.433A synthesizer: transmission Flow Rate1-18, to synthesizer, selects Main Menu Module
Test looks for Module A, ModuleD, ModuleI, ModuleI, Module A by Prer or next)
Measure by more by Start or observe, if flow is improper, then regulation lower valve pressure, until it reaches
Require (concrete testing requirement see table 1).
Table 1. Peptide synthesizer flow rate detection standard scale
| 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 aminoacid sequence that will synthesize in the program of 433A synthesizer sends Std Fmoc1.0Sol DIC90
On synthesizer.The sequence of File-New-Sequence-Edit and Compose peptide, preserves.File-New-Run, checks
Whether Chemistry is Std Fmoc1.0Sol DIC90；Whether Sequence is by being deposited name；Set
Cycles；Preserve.It is finally sent on synthesizer.
Main Menu Cycle Monitor begin, brings into operation.
4, 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, easily by relatively
Weak base removes, and with piperidines (PIP) attack 9-H during reaction, β eliminates and forms hexichol fluorenes alkene, it is easy to by two-stage ring
Amine attack forms stable addition product.After the removing of Fmov group, it is anti-to carry out synthesis that "-NH2 " group is exposed
Should.It is subsequently adding aminoacid and the I-hydroxybenzotriazole of the next Fmoc radical protection of activation
(1-hydroxybenzotriazole, HOBT) is in reactor.
Peptide sequence described above, is to start to N end, according to specific order, successively from C end when of synthesis
It is repeated continuously synthesis step (synthesizer follow procedure is automatically performed, concrete circulation step such as table 2 below).Period
Observed and recorded reagent dosage and ruuning situation.
Table 2. envelope antigen synthesis cycle step
5, envelope antigen end of synthesis
After envelope antigen end of synthesis, synthesizer will be automatically stopped, and peptide resin (peptide is additionally attached on resin now)
Basic washes clean.Then from Peptide synthesizer, take off reactor, then wash peptide resin 3 times with 100% methanol
After, dry up in fume hood, then polypeptide resin is fully transferred in the polyethylene bottle of brown, puts into-20 DEG C
In refrigerator, sealed membrane seals standby.
Two, the cracking of envelope antigen and qualification
1, the cracking of polypeptide antigen
Polypeptide obtained by above-mentioned reaction is chemically bound together with solid phase carrier, it is necessary to pass through
The acidolysis of specific organic acid separates polypeptide with solid phase carrier.Also each aminoacid is eliminated while acidolysis
Protection group in functional group.
Step is as follows:
In refrigerator, take out the polypeptide resin (referring to that peptide is additionally attached to resin) of synthesis, put into the round bottom of a 2L
In flask, addition 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA) in flask in fume hood,
The tripropyl silane (TIS) of 10ml and magnetic stick, be the most stably placed on flask on magnetic stirring apparatus,
Room temperature with constant stirs 1 hour to reaction completely.After reaction terminates, the Rotary Evaporators of band cold-trap is used to continue
Evaporate and within 30 to 120 minutes, remove the TFA in thick product.Then the most clear with dimethylformamide (DMF)
Wash the crude product of polypeptide antigen, finally the resin sand core funnel mixed is filtered out, be both coated
Antigen.
2, the qualification of envelope antigen
Time mass spectrum method (MODAL-TOF) is tried with substance assistant laser desorpted flying after polypeptide antigen synthesis
Carry out qualitative and quantitative analysis with reversed-phase high pressure liquid chromatography (RP-HPLC), come with common amino acid analysis
Peptide synthesized by qualification.
3, envelope antigen purification
Use circulating tangential flow filtration film bag to carry out ultrafiltration the polypeptide antigen after cyclisation (to produce with PALL company
Tangential Flow Device circulating tangential flow filtration film bag and the peristaltic pump supporting with it), polypeptide resist
Original work are that macromole 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 molecular weight impurity introduced then can pass through filter membrane.It is that 0.2 μm filter is degerming by aperture the most again, will
Last gained solution is dispensed in aseptic plastic bottle, labelled.On label indicate the title of polypeptide, numbering,
Product batch number, concentration, date of manufacture, pot-life and preservation condition, after subpackage, be stored in-20 DEG C or-40
DEG C standby.
4, envelope antigen lyophilization
For the ease of long-term storage and transport, need envelope antigen is carried out lyophilization to obtain solid state
Polypeptide.The envelope antigen frozen in advance is positioned on the freezer dryer of Labconco and is dried, consolidate
The envelope antigen of body state.After packaging labelled.On label indicate the title of polypeptide, numbering, product batch number,
Concentration, date of manufacture, pot-life and preservation condition.
Embodiment 2, the composition of foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent
Foot and mouth disease virus non-structural protein enzyme-linked immunologic detecting kit includes:
(1) 96 hole removable polystyrene enzyme-linked reaction plates of foot-and-mouth disease virus antigen it are coated with；2 × 96 holes.
(2) rabbit of horseradish peroxidase-labeled anti-pig IgG polyclonal antibody is (purchased from sigma company, article No.
A5670), 2 bottles (each 12ml).
(3) positive control serum: be in embodiment 1 artificial chemistry synthesis envelope antigen as immunogen,
The hyper-immune serum that immunity 25-35 age in days is prepared without specific source of disease body (SPF) pig, adds 1000U/ml streptomycin
With 1000U/ml penicillin, cross 0.2 μm filter membrane degerming, as the positive control serum of test kit.1 pipe (1ml).
(4) negative control sera: be with the normal swine serum without foot-and-mouth disease antibody, add 1000U/ml strepto-
Element and 1000U/ml penicillin, cross 0.2 μm filter membrane degerming, as test kit negative control sera.1 pipe (1.5ml).
(5) substrate nitrite ion: being made up of two kinds of solution mixing, substrate chromophoric solution A is the tetramethyl of 0.2mg/ml
Base benzidine (TMB) solution；Substrate chromophoric solution B is containing 0.2%(g/ml) Fructus Citri Limoniae of hydrogen peroxide urea
Acid phosphoric acid salt delays liquid；Both during use, the ratio with 1:1 mixes.
(6) concentrated cleaning solution (20 ×): containing 0.5%(ml/ml) Tween-20 0.15M PBS buffering
Liquid, pH is 7.4, after degerming through 0.2 μm filter membrane.
(7) sulfuric acid solution of colour developing stop buffer: 2mol/L.1 bottle (12ml).
(8) sample diluting liquid: containing 3%(g/ml) BSA the PBS that 0.15M, pH are 7.4 buffering
Liquid, crosses 0.45um filter membrane.2 bottles (each 12ml).
As required, test kit can also have serum-dilution plate 1 piece (96 hole), for the ladder of blood serum sample
Degree dilution.
Wherein, the preparation method of the 96 hole removable polystyrene enzyme-linked reaction plates being coated with foot-and-mouth disease virus antigen is:
Polypeptide antigen prepared by embodiment 1 is dissolved in the carbonate solution of pH9.4, is then added to 96 hole polystyrenes
Enzyme-linked reaction plate, every hole 50ng antigen, place 2-4 hour at 37 DEG C, then 4-8 DEG C of left overnight makes polypeptide
Antigen is fully combined with enzyme-linked reaction plate.Then according to 300 μ l/ holes add containing 1%(g/ml) bovine serum albumin
(BSA) PBS (pH=7.4), closes 1-2 hour for 37 DEG C, and with lavation buffer solution, (concentration is washed
Wash liquid distilled water or 20 times of dilutions of deionized water obtain lavation buffer solution) washing after dry, treat enzyme-linked reaction plate do
Dry latter 4 DEG C seal preservation.
Embodiment 3, the detection method of foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent
1, balance: test kit is taken out from cold storage environment, puts equilibrium at room temperature and use after 30 minutes, liquid
The front mixing of reagent.
2, dosing: by concentrated cleaning solution distilled water or 20 times of diluted for use of deionized water.
3, set: staying 1 blank well, 3 negative control holes, and 2 Positive control wells, remaining is for treating
Survey sample aperture.
4, specimen pre-dilution to be measured: every hole adds 100 μ l Sample dilution in dilution plate hole, is separately added into 5 μ l
Sample to be tested.
5, sample-adding: blank well is not loaded；3 negative control holes respectively add 100 μ l negative controls, 2 positives
Control wells respectively adds 100 μ l positive controls；Remaining hole respectively by preset add 100 μ l dilution treat
Test sample is originally.Sample-adding process time span should be the shortest.
6, incubation: concussion mixing, puts in 37 DEG C of incubators or water-bath, reacts 30 minutes.
7, washing plate: discard reactant liquor, every hole adds the cleaning mixture 300 μ l after dilution, soaks 15 seconds, gets rid of
Abandon washing liquid.Pat dry after washing plate 4 times continuously.
8, enzyme-added: blank well is the most enzyme-added；Remaining each hole adds 100 μ l rabbit anti-pig IgG antibody horseradish enzyme conjugates
(deep red).
9, incubation: put in 37 DEG C of incubators or water-bath, reacts 30 minutes.
10, washing plate: discard reactant liquor, every hole adds the cleaning mixture 300 μ l after dilution, soaks 15 seconds, gets rid of
Abandon cleaning mixture.Pat dry after washing plate 5 times continuously.
11, colour developing: every hole (including blank control wells) is sequentially added into assay chromogenic substrate solution A, assay chromogenic substrate solution B
Each 50 μ l, concussion mixing, put in 37 DEG C of incubators or water-bath, develop the color 15 minutes.
12, terminate: every hole (including blank control wells) adds the colour developing each 50 μ l of stop buffer, and vibration mixing is eventually
Only reaction.
13, measure: after returning to zero, measure the OD value of each hole 450nm with blank control wells.Also dual wavelength can be used
450nm/630nm measures each hole OD value.
The judgement of the testing result of test kit of the present invention:
(1) negative control OD meansigma methods should≤0.2, the most invalid.
(2) each detected value of positive control should be the most invalid between 0.6 to 1.8.
(3) calculating of marginal value: marginal value=0.17 × positive control mean OD value.
Result judges: sample measures OD value >=marginal value person and is judged to the positive；Sample measures OD value < marginal value person
It is judged to feminine gender (advise to OD value<marginal value, observation followed the tracks of by the sample of>=0.5 times of marginal value).
Embodiment 4, foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent sensitivity tests
Use three batches of foot and mouth disease virus non-structural protein antibody ELISA immunosorbent adsorption test diagnostic reagents of embodiment 2 preparation
Box (batch number ZM002, ZM003, ZM005), according to foot and mouth disease virus non-structural protein antibody ELISA in embodiment 3
Immunity detection reagent using method, 200 parts of Schweineseuche O-shaped viruses are infected serum (in herd industry share limited
Biology pharmaceutical factory, company Lanzhou provides) and 200 parts of foot and mouth disease O type virus synthetic peptide vaccine Post-immunisation serum (Zhongmu Stocks Trading Co.s
Baoshan, Yunnan factory) detection sensitivity test.
Lot number is that the testing result of ZM002 test kit shows that the sensitivity of 200 parts of infected pigs's serum is by this test kit
197/200×100%=98.5%；Be 198/200 to the sensitivity of 200 parts of foot-and-mouth disease vaccine immune swine serum ×
100%=99%.Lot number is that the testing result of ZM003 test kit shows this test kit sensitivity to 200 parts of infected pigs's serum
Property is 196/200 × 100%=98%；Be 197/200 to the sensitivity of 200 parts of foot-and-mouth disease vaccine immune swine serum ×
100%=98.5%.Lot number is the testing result visualizingre agent box of the ZM005 test kit sensitivity to 200 parts of infected pigs's serum
Property is 197/200 × 100%=98.5%；Be 199/200 to the sensitivity of 200 parts of foot-and-mouth disease vaccine immune swine serum ×
100%=99.5%。
The limitation test of embodiment 5, foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent lowest detection
To foot and mouth disease virus 3 batches of non-structural protein antibody ELISA immunity detection reagent (batch number ZM002,
ZM003, ZM005) carry out the mensuration of lowest detection limitation.The positive reference serum of detection, by herd industry share limited
Biology pharmaceutical factory, company Lanzhou provides.Detection negative reference serum, i.e. health pig serum, Zhongmu Industry Co., Ltd
Baoshan factory provides.
Positive reference serum and negative reference serum are carried out respectively 1:21,1:42,1:84,1:168,1:336,
After 1:672,1:1344 and 1:2688 times dilutes, with foot and mouth disease virus non-structural protein enzyme linked immunosorbent detection reagent
Box detects, and determines the lowest detection limitation of this test kit with this.
The lowest detection limitation result of the test of foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent is as follows
Shown in table (table 3,4 and 5).Result shows, foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent
The maximum dilution multiple that can detect positive reference serum is 672 times.
Table 3, the lowest detection limitation test of ZM002 test kit
The lowest detection limitation test of table 4.ZM003 test kit
The lowest detection limitation test of table 5.ZM005 test kit
Embodiment 5, the specific test of foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent
Use three batches of test kits in embodiment 4 according to the foot and mouth disease virus non-structural protein antibody described in embodiment 3
The using method of enzyme-linked immunologic detecting kit is to 600 parts of health pig serum (Zhongmu Industry Co., Ltd Bao Shanchang
There is provided with biology pharmaceutical factory, Lanzhou), (Zhongmu Industry Co., Ltd Lanzhou is biological for 10 parts of swine fever (HC) positive serums
Pharmaceutical factory provide), (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. carries 10 parts of swine pox (SVD) positive serums
For), 10 parts of pig circular ring virus (PCV) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer),
10 parts of pig PRRS virus-positive serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer), 10 parts of pig mouths
Fever aphthous Asia I type (Asia1) positive serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer) and 10
Part Schweineseuche A type (A) positive serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer) is entered respectively
Row detection.
Specific detection result such as following table (table 6) display of test kit, the testing result to 600 parts of health pig serum
Display, the specificity of ZM002 test kit is 99.5%, and the specificity of ZM003 test kit is 99.7%, ZM005 reagent
The specificity of box is 99.7%.To 10 parts of swine fever (HC) positive serums, 10 parts of swine pox (SVD) positive serums,
10 parts of pig circular ring virus (PCV) positive serums, 10 parts of pig PRRS virus-positive serum, 10 parts of Schweineseuche Asia
I type (Asia I) positive serum and 10 parts of Schweineseuche A type (A) positive serum positive serum testing results are the most aobvious
Being shown as negative, therefore three test kits are 100% to the specificity of these 60 parts of positive serum detections.
Table 6. foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent specific detection result
Embodiment 6, foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent storage life are tested
The actual storage life test operation of foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent is as follows: from
In selected tri-lot numbers of ZM407, ZM503 and ZM606, each lot number takes a test kit at random in 2-8 DEG C of preservation,
Take out, according to the non-knot of the foot and mouth disease virus of embodiment 30,3,6,9,12,15,18 and 24 months respectively
The detection method of structure protein antibodies enzyme-linked immunologic detecting kit, (Zhong Mu Lanzhou factory provides to test known serum sample
2 parts of positive porcine blood serums；The positive porcine blood serum in 1 part of pig farm, Shunyi, 1 part of weak positive porcine blood serum, 2 parts of negative porcine blood serums) come
The stability of product is estimated.With statistics software GraphPad(V.5.03) testing result is carried out statistics
Analyze.If absorption value/marginal value (S/C) ratio of positive serum sample group is more than 1, the suction of negative serum sample group
Receipts value/marginal value (S/C) ratio is less than 1, i.e. the requirement of coincidence detection, then it represents that test kit is under this test condition
It is stable.
Testing result (table 7) display of lot number ZM407 test kit, serum sample group S-1 to S-4(positive serum
Group) present absorption value/marginal value (S/C) ratio more than 1, with intended serum sample group acceptable S/C value
Scope 100% is consistent；Negative reaction serum sample group S-5 and S-6 then present the S/C ratio less than 1, and in advance
The serum sample group acceptable S/C value scope 100% of phase is consistent.Therefore this test kit can the stability of 2-8 DEG C
Maintain more than 24 months.
Table 7, the actual storage life result of the test (S/C value) (2-8 DEG C) of lot number ZM407
Testing result (table 8) display of lot number ZM503 test kit, serum sample group S-1 to S-4(positive serum
Group) present absorption value/marginal value (S/C) ratio more than 1, with intended serum sample group acceptable S/C value
Scope 100% is consistent；Negative reaction serum sample group S-5 and S-6 then present the S/C ratio less than 1, and in advance
The serum sample group acceptable S/C value scope 100% of phase is consistent.Therefore this test kit can the stability of 2-8 DEG C
Maintain more than 24 months.
Table 8, the actual storage life result of the test (S/C value) (2-8 DEG C) of lot number ZM503
Testing result (table 9) display of lot number ZM606 test kit, serum sample group S-1 to S-4(positive serum
Group) present absorption value/marginal value (S/C) ratio more than 1, with intended serum sample group acceptable S/C value
Scope 100% is consistent；Negative reaction serum sample group S-5 and S-6 then present the S/C ratio less than 1, and in advance
The serum sample group acceptable S/C value scope 100% of phase is consistent.Therefore this test kit can the stability of 2-8 DEG C
Maintain more than 24 months.
Table 9, the actual storage life result of the test (S/C value) (2-8 DEG C) of lot number ZM606
Claims (15)
1. a peptide species, shown in sequence in sequence table 1.
2. a foot and mouth disease virus non-structural protein antibody ELISA immunity detection reagent, including foot and mouth disease virus non-structural
The coated enzyme-linked reaction plate of albumen 3B antigen epitope polypeptide and enzyme labelling anti antibody；Described foot and mouth disease virus non-structural protein
White 3B antigen epitope polypeptide is in sequence table shown in sequence 1.
Test kit the most according to claim 2, it is characterised in that: the marker enzyme of described enzyme labelling anti antibody is peppery
Root peroxidase or alkali phosphatase；Described enzyme labelling anti antibody is the anti-pig IgG of enzyme labelling rabbit.
Test kit the most according to claim 3, it is characterised in that: described enzyme labelling anti antibody is Radix Cochleariae officinalis peroxidating
The rabbit anti-pig IgG polyclonal antibody of thing enzyme labelling.
5. according to the test kit described in any one in claim 2-4, it is characterised in that: described enzyme-linked reaction plate is
Detachable 96 hole ELISA Plate；Described foot and mouth disease virus non-structural protein 3B antigen epitope polypeptide obtains for chemistry synthetic
Arrive.
Test kit the most according to claim 5, it is characterised in that: described foot and mouth disease virus non-structural protein 3B
The preparation method of the coated enzyme-linked reaction plate of antigen epitope polypeptide is by described foot and mouth disease virus non-structural protein 3B antigen
Epitope polypeptide is dissolved in the carbonate solution of the pH 9.4 of 100 μ l, is then added to 96 hole polystyrene enzyme-linked reaction plates,
Every hole 50ng antigen, places placement at 2-4 hour, then 4-8 DEG C at 37 DEG C and within 8-12 hour, makes polypeptide antigen anti-with enzyme connection
Plate is answered fully to combine, then according to 300 μ l/ holes add the PBS buffering containing 1g/100ml bovine serum albumin pH7.4
Liquid, 37 DEG C of sealing treatment 1-2 hour, dry after washing, treat that dried 4 DEG C of enzyme-linked reaction plate seals and preserves.
Test kit the most according to claim 6, it is characterised in that: possibly together with nitrite ion and end in described test kit
Only liquid；When marker enzyme is horseradish peroxidase, nitrite ion is made up of nitrite ion A liquid and nitrite ion B liquid, described aobvious
Color liquid A liquid is the citrate phosphate buffer containing 0.2g/100ml hydrogen peroxide urea, and described nitrite ion B liquid is
The tetramethyl biphenyl amine aqueous solution of 0.2mg/ml；When marker enzyme is alkali phosphatase, nitrite ion is 4-nitrophenols phosphate
Buffer；Described stop buffer is the sulfuric acid solution of 2mol/L.
Test kit the most according to claim 7, it is characterised in that: described test kit also include negative control sera,
Positive control serum；Described negative control sera is the normal swine serum without foot-and-mouth disease antibody；Described positive control blood
The clear serum for obtaining with Foot-and-mouth disease VP1 antigen epitope polypeptide for immunogen immune pig.
Test kit the most according to claim 8, it is characterised in that: described test kit also include sample diluting liquid and
Concentrated cleaning solution；Sample diluting liquid be 0.15M, the PH containing 3g/100mlBSA be the PBS of 7.4；
Described concentrated cleaning solution is 0.15M, pH 7.4, containing the phosphate of the Tween-20 that volumn concentration is 0.5%
Buffer.
Test kit the most according to claim 2, it is characterised in that: described foot and mouth disease virus is pig O type mouth hoof
Epidemic disease poison.
11. test kits according to claim 10, it is characterised in that: described foot and mouth disease virus is Schweineseuche
OR/80 virus.
Polypeptide described in 12. claim 1 is in preparation detects whether the test kit of infection animal hoof-and-mouth disease viral disease
Application；Wherein, described animal aftosa viral disease is that swine foot-and-mouth disease virus is sick.
The arbitrary described test kit of 13. claim 2-11 detects whether the examination of infection animal hoof-and-mouth disease viral disease in preparation
Application in agent box；Wherein, described animal aftosa viral disease is that swine foot-and-mouth disease virus is sick.
14. according to the application described in claim 12 or 13, it is characterised in that: described animal aftosa viral disease is
The swine foot-and-mouth disease virus that pig O type foot and mouth disease virus causes is sick.
15. application according to claim 14, it is characterised in that: described animal aftosa viral disease is pig mouth hoof
The swine foot-and-mouth disease virus that epidemic disease OR/80 virus causes is sick.
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| CN105445462B (en) * | 2014-09-25 | 2017-04-12 | 金宇保灵生物药品有限公司 | Enzyme linked immunosorbent assay kit for specific quantitative detection of foot and mouth disease O type Guangxi strain antigen and application thereof |
| CN104478998B (en) * | 2014-12-09 | 2018-09-04 | 中牧实业股份有限公司 | Swine foot-and-mouth disease virus nonstructural protein 3A BC antibody ELISA immunity detection reagents |
| CN106754738A (en) * | 2016-12-22 | 2017-05-31 | 中国农业科学院哈尔滨兽医研究所 | The hybridoma cell line of the shared monoclonal antibody 3D9 of secretion foot and mouth disease virus and its application |
| CN107177558B (en) * | 2017-05-17 | 2019-12-20 | 中国农业科学院哈尔滨兽医研究所 | Hybridoma cell line for secreting foot-and-mouth disease virus shared monoclonal antibody 10B10 and application thereof |
| CN107219365A (en) * | 2017-05-27 | 2017-09-29 | 中国农业科学院兰州兽医研究所 | A kind of chemiluminescence detection kit based on foot and mouth disease virus 3B neoepitope Westerns |
| CN107513101B (en) * | 2017-09-18 | 2021-04-13 | 中牧实业股份有限公司 | Pig foot-and-mouth disease virus non-structural protein antibody enzyme-linked immunoassay kit |
| CN108387741A (en) * | 2018-02-26 | 2018-08-10 | 新疆畜牧科学院兽医研究所 | A kind of ELISA detection method and kit of FMDV 3B antibody |
| CN108956987B (en) * | 2018-06-15 | 2021-08-27 | 新兴县国研科技有限公司 | Application of fiber2 protein and recombinant protein thereof in detecting duck type 2 adenovirus antibody in duck serum |
| CN109293748B (en) * | 2018-09-26 | 2021-04-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Foot-and-mouth disease virus non-structural protein 3A epitope peptide and application thereof |
| CN109293747B (en) * | 2018-09-26 | 2021-04-23 | 中国农业科学院哈尔滨兽医研究所（中国动物卫生与流行病学中心哈尔滨分中心） | Foot-and-mouth disease virus non-structural protein 3B epitope peptide and application thereof |
| CN111896744B (en) * | 2020-07-28 | 2022-02-22 | 重庆澳龙生物制品有限公司 | Goat echinococcus antibody ELISA detection kit and preparation method and application thereof |
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| CN102662062B (en) * | 2012-04-17 | 2015-05-27 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) |
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