CN103864906A - Foot and mouth disease virus non-structural protein antibody enzyme-linked immunodetection kit - Google Patents
Foot and mouth disease virus non-structural protein antibody enzyme-linked immunodetection kit Download PDFInfo
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Abstract
The invention discloses a foot and mouth disease virus non-structural protein antibody enzyme-linked immunodetection kit. The kit comprises a foot and mouth disease virus non-structural protein 3B antigen epitope peptide-coated polymerase chain reaction plate and an enzyme-labeled antibody, wherein the foot and mouth disease virus non-structural protein 3B antigen epitope peptide is a polypeptide shown as a sequence 1 in a sequence table. The kit adopts a chemical synthesis non-structural protein 3B antigen peptide coated reaction plate and is small in antigen amount, high in sensitivity and high in specificity, and whether the foot and mouth disease virus infection exists can be efficiently detected. The kit is high in specificity, sensitive and high-efficiency and has good market prospects.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to a kind of Foot-and-mouth disease antibody ELISA immunity detection reagent, particularly Foot-and-mouth disease VP1 antibody ELISA immunity detection reagent.
Background technology
Foot and mouth disease (Foot-and-Mouth Disease, FMD) is extensively to distribute in world wide, the global epidemic infectious diseases of its spread in china internationally.World Organization for Animal Health (OIE) classifies foot and mouth disease first of category-A transmissible disease as in the zoonosis classification of revision in 1984.Foot and mouth disease is to cause by mouth disease virus infection the deadly infectious disease that artiodactylous animals occurs.Be everlasting cows and swinery is popular on a large scale.The lethality rate of foot and mouth disease is very low, but infection rate is very high.The cardinal symptom of morbidity is: blister occurs at the positions such as oral mucosa, tongue, lip, asoscope, frog, nipple, 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 may be miscarried, and then causes reproductivity to reduce; Pig is to break hoof as main symptom; The symptom of goat and sheep is conventionally than Niu Wenhe.Foot and mouth disease infectivity is high, propagates rapidly, and the livestocks such as infected pigs, ox, sheep often cause cub death, and adult animals throughput sharply declines, the development of serious harm livestock industry and the production and supply of livestock product.Can cause the market circulation of popular country animal and animal's products simultaneously, and its international trade is subject to and large blockade and restriction, produce the loss that has caused tremendous economic to the livestock industry of popular countries and regions.Therefore foot and mouth disease is always subject to the great attention of national governments.
Foot and mouth disease virus (Foot-and-Mouth Disease Virus, FMDV) belongs to picornavirus, and this crowd of other viral members comprise common cold virus and variola virus etc.Foot and mouth disease virus (FMDV) has the feature such as polytypism, volatility.At present, there are 7 serotype: A, O, C, Sat1 (South Africa I type), Sat2 (South Africa II type), Sat3 (South Africa III type) and AsiaI (Asia I type) in the known whole world.Each type divides again some hypotypes, and the hypotype of finding at present has more than 70.Due to antigenic drift constantly occurring, therefore can not strictly distinguish hypotype.FMDV is made up of a nucleic acid RNA chain and capsid protein, and the about 8.5kb of geneome RNA total length can be directly as messenger RNA(mRNA), and FMDV geneome RNA is made up of 5 ' UTR, 3 ' UTR and OFR.5 ' UTR is about 1300nt, contains (IRES) such as VPg, secondary structure, P10 gene, Poly (c) section and internal ribosome entry sites.3 ' UTR is made up of 92 nt residues between Poly (A) tail and large ORF3 ' end and Poly (A) section 5 ' end, long 172nt.Large ORF is made up of L gene (P20a), P1 structural protein gene, P2 and P3 nonstructural protein gene and initiator codon and terminator codon.Order with L, P1, P2 and P3 is arranged in order, and is about 6.5Kb.Wherein genomic 3D section is the RNA polymerase of coding FMDV, is indispensable composition in virus replication.Main structural protein VP1, VP2, VP3 and VP4 in P1 structural protein gene coding 4, their each 60 coat protein that form FMD.
The Nonstructural Protein of the coded generation of foot-and-mouth disease virus gene has L, 2A, 2BC, 3AB, 3ABC, 3D etc., wherein the immunogenicity of L albumen than other Nonstructural Proteins a little less than, infection animal differs and produces surely antibody, even if produce antibody, the time maintaining is also shorter.Virus infection animal can produce anti-2B antibody, but this antibody repeatability in ELISA reaction is bad, can not serve as and detect antibody.2C antibody is because the ratio 3ABC disappearing is fast, and 2C antibody can be detected in immune cattle, therefore can not serve as and detect index.3D claims again VIA, there is no type specificity, detect its antibody horizontal and be and evaluate the whether important indicator of contacted antigen of animal, and be also international trade essential items for inspection.Think that at present only detecting 3D antibody can not distinguish infection animal and immune animal, because often contain the non-structural antigens of trace in vaccine, through after immunity repeatedly, 3D antibody in immune animal body, also can be detected.In 3ABC polyprotein, 3A immunogenicity is the strongest, 3C less immunogenic.Many data think that 3ABC is larger as the mark confidence level infecting.Detecting 3ABC antibody is the important indicator of making a definite diagnosis infection.
At present, the control of foot and mouth disease mainly relies on vaccine immunity.China implements mandatory immunity to foot and mouth disease, and once concentrate immunity annual spring and autumn, between twice, new benefit hurdle domestic animal mended and exempted from time.In actual production how differential diagnosis susceptible animal to have carried out vaccine immunity or infected foot and mouth disease virus be all very important to foundation, the inspection and quarantine of foot and mouth disease epidemic prevention system.Research has widely been carried out in Ye Gai field, countries in the world.The features such as that ELISA method has is special, responsive, quick, easy, good reliability, and can be automated operation, and can detect rapidly a large amount of samples, in the diagnosis of FMD, be day by day subject to people's attention, now become and detected in the world the whether vaccine immunity or infect one of viral ordinary method such as susceptible animal pig, ox.
Gondola De etc. (1997) develop based on 3ABC albumen indirectly catch elisa technique, and can distinguish wild virus infection and vaccine virus infects.The Sorensen of Denmark etc. (1998) develop the ELISA method constructed with the antigen of baculovirus expression, can detect respectively nonstructural protein 3A B, 3ABC, 3D, wherein 3AB, 3ABC, 3DELISA all can detect 7 kinds of serotype antiviral antibodies of FMDV.The Bruderer (2004) of Germany has also successfully set up 3ABC ELISA method.Xie Qingge etc. (2003) utilize the foot and mouth disease virus nonstructural protein 3A BC polypeptide that colon bacillus is expressed to set up a kind of indirect enzyme-linked immunosorbent assay that can distinguish FMDV infection and vaccine immunity as antigen.You Yongjin, Zhu Caizhu etc. (2003) set up the ELISA detection method based on nonstructural protein 3A BC with same principle.
That the Sptting plate that the existing enzyme-linked immunosorbent assay method based on Nonstructural Protein uses is coated with is 3ABC, 3AB mostly, and is mostly the envelope antigen that obtains Nonstructural Protein by the method for prokaryotic expression.In the protein product of this type of prokaryotic expression, contain a small amount of carrier proteins and Host Strains albumen, it is very difficult removing them completely, and these foreign proteins will produce nonspecific combination with detecting sample, thereby have affected the accuracy of detected result.
Foot and mouth disease is the great animal epidemic that is related to economic security of the country, and related key technical should firmly rest in own hand.Once we have independent intellectual property right, the economic security that ensures China will be beneficial to.
Summary of the invention
The object of this invention is to provide a kind of new enzyme-linked immunologic detecting kit with the differential diagnosis of virus infection for the immunity of foot and mouth disease susceptible animal pig vaccine.
Foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent of the present invention, comprises enzyme-linked reaction plate and enzyme labelling anti-antibody that foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is coated; Described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is the polypeptide shown in sequence 1 in sequence table.
Related envelope antigen adopts the synthetic method of solid state chemistry to produce, the major antigen site polypeptide that envelope antigen contains foot and mouth disease virus Nonstructural Protein 3B, can with the Nonstructural Protein 3B antibody specific combination that produces after mouth disease virus infection.
The marker enzyme of described enzyme labelling anti-antibody is horseradish peroxidase or alkaline phosphatase; Described enzyme labelling anti-antibody is the anti-pig IgG of enzyme labelling rabbit; The anti-pig IgG polyclonal antibody of rabbit that described enzyme labelling anti-antibody is preferably horseradish peroxidase-labeled.
Described enzyme-linked reaction plate is detachable 96 hole enzyme plates; Described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is that chemical synthetic obtains.
The present invention realizes in the following manner: the coated removable 96 hole polystyrene enzyme-linked reaction plates of the synthetic polypeptide of artificial chemistry that adopt foot and mouth disease virus Nonstructural Protein 3B.Meanwhile, in test kit, contain the IgG polyclonal antibody enzyme conjugates with the anti-pig of rabbit of horseradish peroxidase-labeled, negative control sera, positive control serum, sample diluting liquid, tmb substrate solution, the components such as 20 × concentrated cleaning solution and stop buffer.Detect the 3B protein antibodies in sample with indirect enzyme-linked immunosorbent assay (ELISA) method.
The optimum preparating condition of related enzyme-linked reaction plate is: the carbonate solution that when coated, polypeptide antigen is dissolved in to PH9.4, then be added to 96 hole polystyrene enzyme-linked reaction plates, every hole 50ng antigen, 37 ℃ place 2-4h, then at 4-8 ℃ place spend the night (8-12 hour) make polypeptide antigen and the abundant combination of enzyme-linked reaction plate.Then add containing 1%(g/ml according to 300ul/ hole) the PBS damping fluid (PH=7.4) of bovine serum albumin (BSA), 37 ℃ of sealing treatment 1-2h, dry after washing, after enzyme-linked reaction plate is dry, seal 4 ℃ of preservations.
In described test kit, contain nitrite ion and stop buffer; In the time that marker enzyme is horseradish peroxidase, nitrite ion is made up of nitrite ion A liquid and nitrite ion B liquid, described nitrite ion A liquid is the citric acid phosphoric acid salt buffer containing 0.2% (g/ml) hydrogen peroxide urea, the tetramethyl biphenyl amine aqueous solution that described nitrite ion B liquid is 0.2mg/ml; When use, both mix with the ratio of 1:1.In the time that marker enzyme is alkaline phosphatase, nitrite ion is 4-nitrophenol phosphate buffered saline buffer.Described stop buffer is the sulphuric acid soln of 2mol/L.
Described test kit also comprises negative control sera, positive control serum; Described negative control sera is the normal pig serum of using without foot-and-mouth disease antibody; Described positive control serum is the serum obtaining as immunogen immune pig take described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide;
Related positive control serum is specifically using the synthetic Nonstructural Protein 3B antigen epitope polypeptide of artificial chemistry as immunogen, immunity 25-35 age in days is without specific pathogeny body (SPF) pig, prepare hyper-immune serum, add 1000U/ml Streptomycin sulphate and penicillin, filtering footpath is the filter membrane degerming of 0.2 μ m, as the positive control serum of test kit.
Related negative control sera is specifically used the normal pig serum without foot-and-mouth disease antibody, adds 1000U/ml Streptomycin sulphate and 1000U/ml penicillin, and filtering footpath is the filter membrane degerming of 0.2 μ m, as test kit negative serum.
Described test kit also comprises sample diluting liquid and concentrated cleaning solution; The PBS damping fluid (filtering footpath is 0.45 μ m filter membrane) that sample diluting liquid is 7.4 for the 0.15M, the pH that contain 3% (g/ml) BSA; Described concentrated cleaning solution is 0.15M, pH7.4, the phosphate buffered saline buffer that contains 0.5% (ml/ml) Tween-20 (footpath is the filter membrane degerming of 0.2 μ m after filtration).
The trace routine of test kit of the present invention is:
1. balance: test kit is taken out from cold storage environment, put equilibrium at room temperature and use after 30 minutes, liquid reagent is with front mixing.
2. dosing: by distilled water or 20 times of diluted for use of deionized water for concentrated cleaning solution.
3. set: stay 1 blank well, 3 negative control holes, and 2 positive control holes, all the other are sample to be tested hole.
4. sample to be measured dilutes in advance: add 100 μ l sample diluents toward the interior every hole of dilution plate hole, add respectively 5 μ l samples to be tested.
5. application of sample: blank well is application of sample not; 3 negative control holes respectively add 100 μ l negative controls, and 2 positive control holes respectively add 100 μ l positive controls; All the other holes are respectively by presetting the sample to be tested that adds 100 μ l dilutions.Application of sample process time span should be as far as possible short.
6. incubation: concussion mixes, and puts in 37 ℃ of incubators or water-bath, reacts 30 minutes.
7. wash plate: discard reaction solution, every hole adds the washings 300 μ l after dilution, soaks 15 seconds, gets rid of and abandons washing lotion.After washing continuously plate 4 times, pat dry.
8. enzyme-added: blank well is not enzyme-added; All the other each holes add the 100 μ l horseradish peroxidase-labeled anti-pig IgG antibody of rabbit (deep red).
9. incubation: put in 37 ℃ of incubators or water-bath, react 30 minutes.
10. wash plate: discard reaction solution, every hole adds the washings 300 μ l after dilution, soaks 15 seconds, gets rid of and abandons washings.After washing continuously plate 5 times, pat dry.
11. colour developings: every hole (comprising blank hole) adds the each 50 μ l of chromogenic substrate liquid A, chromogenic substrate liquid B successively, and concussion mixes, and puts in 37 ℃ of incubators or water-bath, develops the color 15 minutes.
12. stop: every hole (comprising blank hole) adds the each 50 μ l of colour developing stop buffer, and vibration mixes termination reaction.
13. measure: by the OD value of measuring each hole 450nm after the zeroing of blank hole.Also can measure each hole OD value with dual wavelength 450nm/630nm.
The judgement of the detected result of test kit of the present invention:
Negative control OD mean value should≤0.2, otherwise invalid.
2. the each detected value of positive control should be between 0.6 to 1.8, otherwise invalid.
3. the calculating of threshold value: the average OD value of threshold value=0.17 × positive control.
4. result is judged: sample is measured OD value >=threshold value person and is judged to the positive; It is critical that sample is measured OD value <
Value person is judged to feminine gender (suggestion is to OD value < threshold value, the sample tracing observation of >=0.5 times of threshold value).Positively effect of the present invention is: the present invention adopts bioinformatics method to carry out Accurate Analysis to the epitope of Nonstructural Protein, and the Main Antigenic from 3B albumen filters out the peptide section that is applicable to ELISA detection use.Meanwhile, adopt advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen for being coated with enzyme reaction plate and preparing the positive control serum of test kit.
Because the envelope antigen using in test kit is chemically synthesized polypeptide,, containing foreign protein, purity is not high, and high specificity is highly sensitive, therefore can effectively detect foot and mouth disease Nonstructural Protein antibody, to judge whether tested animal infects foot and mouth disease virus.Experimental result shows, test kit is reproducible, and high specificity is highly sensitive.Can effectively detect the Nonstructural Protein antibody producing after mouth disease virus infection, to judge whether tested animal infects foot and mouth disease virus.Can meet different levels personnel's needs, there are wide market outlook and good economical, societal benefits.
This test kit adopts the coated Sptting plate of chemosynthesis 3B antigen peptide, antigen consumption is few, susceptibility and specificity high, can differentiate efficiently that foot and mouth disease susceptible animal pig has infected virus or accepted vaccine immunity.Can carry out according to result the evaluation of immune effect simultaneously.Test kit specificity of the present invention is good, responsive, efficient, has good market outlook.
Foot and mouth disease virus Nonstructural Protein ELISA diagnostic kit involved in the present invention is the differential diagnosis with virus infection for the immunity of foot and mouth disease susceptible animal pig vaccine, be conducive to reduce China's foot and mouth disease and break out the loss bringing, be also conducive to the foundation of China's foot and mouth disease prevention and control system.
Embodiment
Method in following embodiment, if no special instructions, is ordinary method.
Embodiment 1, the preparation of foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent envelope antigen
The present invention adopts bioinformatics method to carry out Accurate Analysis to the epitope of Nonstructural Protein, Main Antigenic from 3B albumen filters out the peptide section that is applicable to ELISA detection use, be foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide, its sequence is as shown in sequence in sequence table 1.Coating antigen using this polypeptide as test kit of the present invention is prepared foot and mouth disease virus Nonstructural Protein enzyme-linked immunologic detecting kit.
Envelope antigen of the present invention can use Applied Biosystem full-automatic polypeptide synthetic instrument (model 433A) preparation.Use Merrifield solid-phase synthesis, employing be the amino acid that Fmoc (9-fluorenylmethyloxycarbonyl, 9-fluorenylmethyloxycarbonyl) modifies, use Rink Amide mbha resin as solid phase carrier.Production process comprises polypeptide antigen solid phase synthesis, polypeptide cracking and evaluation, antigen purification, lyophilize and preserves five parts.Below describe respectively:
One, envelope antigen solid phase synthesis
1, the preparation of synthetic agent
Synthetic envelope antigen aminoacid sequence is as shown in SEQ ID NO:1.
Prepare according to envelope antigen sequence and synthetic scale the amino acid (purchased from NOVA company) that suitable Fmoc modifies, be added in corresponding Cartridge.Synthetic scale takes resin 5g equally on request, puts into reaction chamber, and upper and lower lid is tightened, and labels, and records the title of synthesized peptide, lot number, the TARE of reaction chamber and the weight of alleged resin.Pack reaction chamber into synthesizer.Prepare appropriate synthetic agent and comprise 100% NMP, 3% AIM(acyl imidazoles), 35% PIP(piperidines), 100% MeOH(methyl alcohol) etc. be placed in corresponding reagent bottle.
2, the detection of synthesizer state
Check the whether normally operation of 433A Peptide synthesizer device, after start, operation Run Self Test program, whether instrument self checking indices is normal.Check that in addition whether nitrogen is sufficient, normally whether system gauge pressure (the normal gauge pressure 10.2psi of 433A).Before synthetic, the performance of reply instrument is had gained some understanding, so will measure the flow velocity of every kind of synthetic agent.433A synthesizer: send Flow Rate1-18 to synthesizer, select Main Menu-Module Test-look for Module A, ModuleD, ModuleI, ModuleI, Module A by Prer or next)-by Start-measure or observe by more, if flow is improper, regulate lower valve pressure, until reach requirement (concrete testing requirement sees the following form 1).
Table 1. Peptide synthesizer flow rate detection standard meter
| Reagent | Bottle number | Module | Standard range |
| 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 is synthetic starts
Aminoacid sequence that will be synthetic in the program of 433A synthesizer sends Std Fmoc1.0Sol DIC90 to 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.Finally send on synthesizer.
Main Menu-Cycle Monitor-begin, brings into operation.
4, envelope antigen is synthetic carries out
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, and when reaction, with piperidines (PIP) attack 9-H, β eliminates and forms hexichol fluorenes alkene, is easy to be formed stable affixture by the attack of secondary cyclammonium.After the removing of Fmov group, expose " NH2 " group to carry out building-up reactions.Then add amino acid and the I-hydroxybenzotriazole of the next Fmoc radical protection of activation
(1-hydroxybenzotriazole, HOBT) is to reactor.
Peptide sequence described above, is to start the end to N from C end synthetic time, according to specific order, constantly repeats successively synthesis step (synthesizer follow procedure completes automatically, and concrete circulation step is as following table 2).Observed and recorded reagent dosage and running condition during this time.
Table 2. envelope antigen synthesis cycle step
5, envelope antigen end of synthesis
After envelope antigen end of synthesis, synthesizer will stop automatically, and the basic washes clean of peptide resin (peptide is also connected on resin now).Then take off reactor from Peptide synthesizer, then use after 100% methanol wash peptide resin 3 times, in stink cupboard, dry up, then polypeptide resin is all transferred in brown polyethylene bottle, put into-20 ℃ of refrigerators, sealed membrane sealing is for subsequent use.
Two, the cracking of envelope antigen and evaluation
1, the cracking of polypeptide antigen
The polypeptide obtaining through above-mentioned reaction is combined by chemical bonded refractory with solid phase carrier, must polypeptide be separated with solid phase carrier by the acidolysis of specific organic acid.When acidolysis, also remove the protecting group on each amino acid functional group.
Step is as follows:
In refrigerator, take out synthetic polypeptide resin (referring to that peptide is also connected to resin), put into the round-bottomed flask of a 2L, in stink cupboard, in flask, add 90ml trifluoroacetic acid (Trifluoroacetic acid, TFA), tripropyl silane (TIS) and the magnetic stick of 10ml, then stably flask is placed on magnetic stirring apparatus, under room temperature, continues to stir 1 hour to reacting completely.After reaction finishes, use the lasting evaporation of Rotary Evaporators with cold-trap within 30 to 120 minutes, to remove the TFA in thick product.Then use dimethylformamide (DMF) repeatedly to clean the crude product of polypeptide antigen, finally the resin mixing is filtered out with sand core funnel, both obtained envelope antigen.
2, the evaluation of envelope antigen
After polypeptide antigen is synthetic, fly to try time mass spectrum method (MODAL-TOF) and reversed-phase high pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis with substance assistant laser desorpted, identify the peptide of synthesized with common amino acid analysis.
3, envelope antigen purifying
Use circulating tangential flow filtration film bag to carry out ultrafiltration (Tangential Flow Device circulating tangential flow filtration film bag and the peristaltic pump supporting with it produced with PALL company) polypeptide antigen after cyclisation, polypeptide antigen can not be by the filter membrane of certain pore size as macromole, and the small molecular weight impurity that building-up process and later stage cyclization formed or introduced early stage can pass through filter membrane.And then be 0.2 μ m strainer degerming by aperture, last gained solution is divided and installed in aseptic plastic bottle, labelled.On label, indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide, after packing, be stored in-20 ℃ or-40 ℃ for subsequent use.
4, envelope antigen lyophilize
For the ease of long-term storage and transport, envelope antigen need to be carried out to lyophilize to obtain the polypeptide of solid state.The envelope antigen having frozen is in advance positioned on the freeze drier of Labconco and is dried, obtain the envelope antigen of solid state.Labelled after packing.On label, indicate title, numbering, product batch number, concentration, date manufactured, storage life and the preservation condition of polypeptide.
The composition of embodiment 2, foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent
Foot and mouth disease virus Nonstructural Protein enzyme-linked immunologic detecting kit comprises:
(1) be coated with 96 removable polystyrene enzyme-linked reaction plates in hole of foot-and-mouth disease virus antigen; 2 × 96 holes.
(2) the anti-pig IgG polyclonal antibody of the rabbit of horseradish peroxidase-labeled (purchased from sigma company, article No. A5670), 2 bottles (each 12ml).
(3) positive control serum: be using the synthetic envelope antigen of artificial chemistry in embodiment 1 as immunogen, the hyper-immune serum that immunity 25-35 age in days is prepared without specific pathogeny body (SPF) pig, add 1000U/ml Streptomycin sulphate and 1000U/ml penicillin, cross 0.2 μ m filter membrane degerming, as the positive control serum of test kit.1 pipe (1ml).
(4) negative control sera: be the normal pig serum of using without foot-and-mouth disease antibody, add 1000U/ml Streptomycin sulphate and 1000U/ml penicillin, cross 0.2 μ m filter membrane degerming, as test kit negative control sera.1 pipe (1.5ml).
(5) substrate nitrite ion: mix and form by two kinds of solution, tetramethyl benzidine (TMB) solution that substrate chromophoric solution A is 0.2mg/ml; Substrate chromophoric solution B is for containing 0.2%(g/ml) the slow liquid of citric acid phosphoric acid salt of hydrogen peroxide urea; When use, both mix with the ratio of 1:1.
(6) concentrated cleaning solution (20 ×): contain 0.5%(ml/ml) the PBS damping fluid of 0.15M of Tween-20, pH is 7.4, after through 0.2 μ m filter membrane degerming.
(7) sulphuric acid soln of colour developing stop buffer: 2mol/L.1 bottle (12ml).
(8) sample diluting liquid: contain 3%(g/ml) PBS damping fluid that 0.15M, the pH of BSA is 7.4, cross 0.45um filter membrane.2 bottles (each 12ml).
As required, in test kit, can also there is serum 1 of plate of dilution (96 hole), for the gradient dilution of serum sample.
Wherein, the preparation method who is coated with the 96 removable polystyrene enzyme-linked reaction plates in hole of foot-and-mouth disease virus antigen is: polypeptide antigen prepared by embodiment 1 is dissolved in the carbonate solution of pH9.4, then be added to 96 hole polystyrene enzyme-linked reaction plates, every hole 50ng antigen, place 2-4 hour at 37 ℃, then place and spend the night and make polypeptide antigen and the abundant combination of enzyme-linked reaction plate at 4-8 ℃.Then add containing 1%(g/ml according to 300 μ l/ holes) the PBS damping fluid (pH=7.4) of bovine serum albumin (BSA), 37 ℃ of sealing 1-2 hour, with drying after lavation buffer solution (20 times of dilutions of concentrated cleaning solution distilled water or deionized water obtain lavation buffer solution) washing, after enzyme-linked reaction plate is dry, 4 ℃ of sealings are preserved.
The detection method of embodiment 3, foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent
1, balance: test kit is taken out from cold storage environment, put equilibrium at room temperature and use after 30 minutes, liquid reagent is with front mixing.
2, dosing: by distilled water or 20 times of diluted for use of deionized water for concentrated cleaning solution.
3, set: stay 1 blank well, 3 negative control holes, and 2 positive control holes, all the other are sample to be tested hole.
4, sample to be measured dilutes in advance: add 100 μ l sample diluents toward the interior every hole of dilution plate hole, add respectively 5 μ l samples to be tested.
5, application of sample: blank well is application of sample not; 3 negative control holes respectively add 100 μ l negative controls, and 2 positive control holes respectively add 100 μ l positive controls; All the other holes are respectively by presetting the sample to be tested that adds 100 μ l dilutions.Application of sample process time span should be as far as possible short.
6, incubation: concussion mixes, and puts in 37 ℃ of incubators or water-bath, reacts 30 minutes.
7, wash plate: discard reaction solution, every hole adds the washings 300 μ l after dilution, soaks 15 seconds, gets rid of and abandons washing lotion.After washing continuously plate 4 times, pat dry.
8, enzyme-added: blank well is not enzyme-added; All the other each holes add 100 μ l rabbit anti-pig IgG antibody horseradish enzyme conjugates (deep red).
9, incubation: put in 37 ℃ of incubators or water-bath, react 30 minutes.
10, wash plate: discard reaction solution, every hole adds the washings 300 μ l after dilution, soaks 15 seconds, gets rid of and abandons washings.After washing continuously plate 5 times, pat dry.
11, colour developing: every hole (comprising blank hole) adds the each 50 μ l of chromogenic substrate liquid A, chromogenic substrate liquid B successively, and concussion mixes, and puts in 37 ℃ of incubators or water-bath, develops the color 15 minutes.
12, stop: every hole (comprising blank hole) adds the each 50 μ l of colour developing stop buffer, and vibration mixes termination reaction.
13, measure: by the OD value of measuring each hole 450nm after the zeroing of blank hole.Also can measure each hole OD value with dual wavelength 450nm/630nm.
The judgement of the detected result of test kit of the present invention:
(1) negative control OD mean value should≤0.2, otherwise invalid.
(2) the each detected value of positive control should be between 0.6 to 1.8, otherwise invalid.
(3) calculating of threshold value: the average OD value of threshold value=0.17 × positive control.
Result is judged: sample is measured OD value >=threshold value person and is judged to the positive; Sample is measured OD value < threshold value person and is judged to feminine gender (suggestion is to OD value < threshold value, the sample tracing observation of >=0.5 times of threshold value).
Embodiment 4, the sensitivity test of foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent
The three batches of foot and mouth disease virus Nonstructural Protein antibody ELISA immunosorbent adsorption test diagnostic kits (batch number ZM002, ZM003, ZM005) that use embodiment 2 to prepare, according to foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent using method in embodiment 3, to serum (Zhongmu Stocks Trading Co. Baoshan, Yunnan factory) detection sensitivity test after 200 parts of Schweineseuche O-shaped virus infection serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides) and the immunity of 200 parts of foot and mouth disease O C-type virus C synthetic peptide vaccines.
Lot number is that the detected result of ZM002 test kit shows that this test kit is 197/200 × 100%=98.5% to the susceptibility of 200 parts of infected pigs's serum; Be 198/200 × 100%=99% to the susceptibility of 200 parts of aftosa vaccine immune swine serum.Lot number is that the detected result of ZM003 test kit shows that this test kit is 196/200 × 100%=98% to the susceptibility of 200 parts of infected pigs's serum; Be 197/200 × 100%=98.5% to the susceptibility of 200 parts of aftosa vaccine immune swine serum.Lot number is that the detected result visualizingre agent box of ZM005 test kit is 197/200 × 100%=98.5% to the susceptibility of 200 parts of infected pigs's serum; Be 199/200 × 100%=99.5% to the susceptibility of 200 parts of aftosa vaccine immune swine serum.
Embodiment 5, the test of foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent lowest detection limitation
Foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent 3 batches (batch number ZM002, ZM003, ZM005) is carried out to the mensuration of lowest detection limitation.Detect positive reference serum, provided by Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd..Detect negative reference serum, i.e. health pig serum, the Bao Shanchang of Zhongmu Industry Co.,Ltd provides.
Positive reference serum and negative reference serum are carried out respectively after 1:21,1:42,1:84,1:168,1:336,1:672,1:1344 and 1:2688 doubly dilute, detect with foot and mouth disease virus Nonstructural Protein enzyme-linked immunologic detecting kit, determine the lowest detection limitation of this test kit with this.
The lowest detection limitation test-results of foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent is as shown in following table (table 3,4 and 5).Result demonstration, the maximum dilution multiple that foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent can detect positive reference serum is 672 times.
The lowest detection limitation test of table 3, 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
The specific test of embodiment 5, foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent
Use three batches of test kits in embodiment 4 according to the using method of the foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent described in embodiment 3 to 600 parts of health pig serum (the biological pharmaceutical factory in Baoshan factory of Zhongmu Industry Co.,Ltd and Lanzhou provides), 10 parts of swine fevers (HC) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides), 10 parts of swine poxs (SVD) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides), 10 parts of pig circular ring virus (PCV) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides), 10 parts of pig PRRS virus-positive serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides), 10 parts of Schweineseuche Asia I types (Asia1) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides) and 10 parts of Schweineseuche A types (A) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. provides) detect respectively.
The specific detection result of test kit, as following table (table 6) shows, shows the detected result of 600 parts of health pig serum, and the specificity of ZM002 test kit is that the specificity of 99.5%, ZM003 test kit is that the specificity of 99.7%, ZM005 test kit is 99.7%.10 parts of swine fevers (HC) positive serum, 10 parts of swine poxs (SVD) positive serum, 10 parts of pig circular ring virus (PCV) positive serum, 10 parts of pig PRRS virus-positive serum, 10 parts of Schweineseuche Asia I type (Asia I) positive serums and 10 parts of Schweineseuche A types (A) positive serum positive serum detected result are all shown as to feminine gender, and the specificity that therefore three test kits detect these 60 parts of positive serums is 100%.
Table 6. foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent specific detection result
Embodiment 6, the test of foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent preservation period
The actual preservation period test operation of foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent is as follows: from selected ZM407, ZM503 and tri-lot numbers of ZM606, each lot number is got a test kit at random in 2-8 ℃ of preservation, respectively 0,3,6,9,12,15,18 and 24 months take out, according to the detection method of the foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent of embodiment 3, (Zhong Mu Lan Zhouchang provides 2 parts of positive porcine blood serum to test known serum sample; Positive porcine blood serum, 2 parts of negative porcine blood serum a little less than the positive porcine blood serum in 1 part, pig farm, Shunyi, 1 part) stability of product is assessed.With statistics software GraphPad(V.5.03) detected result is carried out to statistical analysis.If absorption value/threshold value (S/C) ratio of positive serum sample group is greater than 1, absorption value/threshold value (S/C) ratio of negative serum sample group is less than 1, meets the requirement of detection, represents that test kit is stable under this test condition.
Detected result (table 7) demonstration of lot number ZM407 test kit, serum sample group S-1 is to S-4(positive serum group) present absorption value/threshold value (S/C) ratio that is greater than 1, be consistent with the acceptable S/C value of the serum sample group scope 100% of expection; Negative reaction serum sample group S-5 and S-6 present the S/C ratio that is less than 1, are consistent with the acceptable S/C value of the serum sample group scope 100% of expection.Therefore this test kit can maintain more than 24 months the stability of 2-8 ℃.
The actual preservation period test-results (S/C value) (2-8 ℃) of table 7, lot number ZM407
Detected result (table 8) demonstration of lot number ZM503 test kit, serum sample group S-1 is to S-4(positive serum group) present absorption value/threshold value (S/C) ratio that is greater than 1, be consistent with the acceptable S/C value of the serum sample group scope 100% of expection; Negative reaction serum sample group S-5 and S-6 present the S/C ratio that is less than 1, are consistent with the acceptable S/C value of the serum sample group scope 100% of expection.Therefore this test kit can maintain more than 24 months the stability of 2-8 ℃.
The actual preservation period test-results (S/C value) (2-8 ℃) of table 8, lot number ZM503
Detected result (table 9) demonstration of lot number ZM606 test kit, serum sample group S-1 is to S-4(positive serum group) present absorption value/threshold value (S/C) ratio that is greater than 1, be consistent with the acceptable S/C value of the serum sample group scope 100% of expection; Negative reaction serum sample group S-5 and S-6 present the S/C ratio that is less than 1, are consistent with the acceptable S/C value of the serum sample group scope 100% of expection.Therefore this test kit can maintain more than 24 months the stability of 2-8 ℃.
The actual preservation period test-results (S/C value) (2-8 ℃) of table 9, lot number ZM606
Claims (10)
1. a peptide species, shown in sequence in sequence table 1.
2. a foot and mouth disease virus Nonstructural Protein antibody ELISA immunity detection reagent, comprises enzyme-linked reaction plate and enzyme labelling anti-antibody that foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is coated; Described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is in sequence table shown in sequence 1.
3. test kit according to claim 2, is characterized in that: the marker enzyme of described enzyme labelling anti-antibody is horseradish peroxidase or alkaline phosphatase; Described enzyme labelling anti-antibody is the anti-pig IgG of enzyme labelling rabbit, the anti-pig IgG polyclonal antibody of rabbit that described enzyme labelling anti-antibody is preferably horseradish peroxidase-labeled.
4. according to the test kit described in claim 2 or 3, it is characterized in that: described enzyme-linked reaction plate is detachable 96 hole enzyme plates; Described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is that chemical synthetic obtains.
5. according to the test kit described in claim 2 or 3, it is characterized in that: the preparation method of the coated enzyme-linked reaction plate of described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is the carbonate solution that described foot and mouth disease virus Nonstructural Protein 3B antigen epitope polypeptide is dissolved in to the pH9.4 of 100 μ l, then be added to 96 hole polystyrene enzyme-linked reaction plates, every hole 50ng antigen, place 2-4 hour at 37 ℃, place at 4-8 ℃ and within 8-12 hour, make polypeptide antigen and the abundant combination of enzyme-linked reaction plate again, then add according to 300 μ l/ holes the PBS damping fluid that contains 1g/100ml bovine serum albumin pH7.4, 37 ℃ of sealing treatment 1-2 hour, after washing, dry, after enzyme-linked reaction plate is dry, 4 ℃ of sealings are preserved.
6. according to the test kit described in any one in claim 1-4, it is characterized in that: in described test kit, also contain nitrite ion and stop buffer; In the time that marker enzyme is horseradish peroxidase, nitrite ion is made up of nitrite ion A liquid and nitrite ion B liquid, described nitrite ion A liquid is the citric acid phosphoric acid salt buffer containing 0.2g/100ml hydrogen peroxide urea, the tetramethyl biphenyl amine aqueous solution that described nitrite ion B liquid is 0.2mg/ml; In the time that marker enzyme is alkaline phosphatase, nitrite ion is 4-nitrophenol phosphate buffered saline buffer; Described stop buffer is the sulphuric acid soln of 2mol/L.
7. test kit according to claim 6, is characterized in that: described test kit also comprises negative control sera, positive control serum; Described negative control sera is the normal pig serum without foot-and-mouth disease antibody; Described positive control serum is the serum obtaining as immunogen immune pig take described Foot-and-mouth disease VP1 antigen epitope polypeptide.
8. test kit according to claim 7, is characterized in that: described test kit also comprises sample diluting liquid and concentrated cleaning solution; Sample diluting liquid is the PBS damping fluid that the 0.15M, the PH that contain 3g/100mlBSA is 7.4; Described concentrated cleaning solution is 0.15M, and pH7.4 contains volumn concentration and be the phosphate buffered saline buffer of 0.5% Tween-20.
9. according to the test kit described in any one in claim 1-8, it is characterized in that: described foot and mouth disease virus is pig O type foot and mouth disease virus; Specifically can be Schweineseuche O R/80 virus.
10. the test kit of polypeptide claimed in claim 1 or claim 2-9 detects the application in the test kit of infection animal hoof-and-mouth disease viral disease whether in preparation; Wherein, animal hoof-and-mouth disease viral disease is swine foot-and-mouth disease virus disease; Be specially the swine foot-and-mouth disease virus disease that pig O type foot and mouth disease virus causes; The swine foot-and-mouth disease virus disease more specifically causing for Schweineseuche O R/80 virus.
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