CN107253978A - Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents - Google Patents
Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents Download PDFInfo
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- CN107253978A CN107253978A CN201710688944.6A CN201710688944A CN107253978A CN 107253978 A CN107253978 A CN 107253978A CN 201710688944 A CN201710688944 A CN 201710688944A CN 107253978 A CN107253978 A CN 107253978A
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Abstract
The invention discloses a kind of Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent.The kit includes ELISA Plate, positive control serum, negative control sera, ELIAS secondary antibody, sample diluting liquid, 20 times of concentrated cleaning solutions, substrate solution A, substrate solution B, terminate liquids, wherein the ELISA Plate is coated with Sai Nika paddy virus structural protein antigen epitope polypeptide compositions.The antigen epitope polypeptide composition is any combination more than one or both of polypeptide shown in sequence 4 in the polypeptide shown in sequence 3 in sequence 2, sequence table in the polypeptide shown in sequence 1 in sequence table, sequence table or sequence table.This kit uses chemical synthesis Antigenic Peptide coated elisa plate, and antigen consumption is few, sensitivity and specificity are high, can efficiently detect whether there is the structural proteins antibody of infection Sai Nika paddy viruses.Kit sensitiveness of the present invention is high, specific good and simple operation, with good market prospects.
Description
Technical field
The invention belongs to technical field of biological, more particularly it relates to a kind of Sai Nika paddy virus structure egg
White antibody ELISA immunity detection reagent.
Background technology
Sai Nika paddy virus (SenecaValleyVirus, SVV), also known as SenecavirusA (SVA) is that pig is primary
The main pathogen of property blister sore (Swineidiopathicvesiculardisease, SIVD).After SVV infection swinerys, although not
Larger politics, economic loss can be caused, but caused blister venereal disease becomes, with foot and mouth disease virus, swine pox, vesiculovirus mouth
The lesion that inflammation, pig vesicle etc. are caused is similar, and certain difficulty is caused to Clinical differential diagnosis.At present, North America, South American region
And Australia has swinery to occur SIVD report.China in 2015 and Brazil occur in that SVV infects swinery and gone out several times
Now serious clinical and dead symptom, causes serious economic loss, and China was also isolated to SVV first in 2016.
SVV is the unique member of Picornaviridae (Picornaviridae) Sai Nika Tobamovirus (Senecavirus),
SVV is sub-thread, normal chain, the RNA virus of non-segmented negative.Carried out by 12 representative Tobamovirus viruses to Picornaviridae
Whole genome sequence, which is compared, to be found, it is nearest that Sai Nika Tobamovirus belongs to genetic affinity with myocarditis virus.SVV is diameter about 27nm's
RNA virus of the icosahedron without cyst membrane single-stranded positive non-segmented negative.Viral genome contains 7280 nucleotides, and an opening is read
Frame contains 6543 nucleotides, encodes the polyprotein of 2181 amino acid, can be divided into 12 polypeptides, constitutes standard
Picornaviridae L-4-3-4 patterns.
As all Picornaviridae members, P1 polypeptides are cracked into VP0, VP3 and VP1 by HRV 3CP, constitute virus
Nucleocapsid.Ripe VP0 is cracked to form VP2 and VP4.Virus VP 1~VP4 protein subunit structural conservations, may be thin with virus
Born of the same parents' preferendum is relevant.The interaction of VP2 and VP4 some regions participates in packaged nucleic acid.
Because China in 2016 was isolated to SVV first, the method for relevant SVV antibody tests is still in studying rank at present
Section, the related research of SVV antibody detection methods mainly has indirect ELISA and competitive ELISA.
The content of the invention
It is an object of the invention to provide a kind of indirect ELISA inspection for being used to detect Sai Nika paddy virus structural protein antibody
Test agent box, the kit is anti-by the use of Sai Nika paddy virus structural protein VP1, VP2 and VP3 antigen epitope polypeptide as coating
Original, establishes a species specificity, sensitiveness and reproducible indirect ELISA method, for detecting in Swine serum whether contain
Sai Nika paddy virus structural protein antibody.
In order to achieve the above object, the Sai Nika paddy virus antigen epitopes that screening first of the invention has obtained excellent performance are more
Peptide combinations, the Sai Nika paddy virus structural protein antigen epitope polypeptide compositions that the present invention is provided, are the institute of sequence 1 in sequence table
One in polypeptide in polypeptide or sequence table in the polypeptide that shows, sequence table in sequence 2, sequence table shown in sequence 3 shown in sequence 4
Plant or two or more any combination.When the peptide composition is the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, sequence
During two kinds in the polypeptide shown in polypeptide and sequence 4 shown in 3, the mass ratio of two kinds of polypeptides is (0.5~1.5)：(0.5~
1.5)；It is preferred that, their mass ratio is 1：1；When the peptide composition is many shown in the polypeptide shown in sequence 1, sequence 2
During three kinds in the polypeptide shown in polypeptide and sequence 4 shown in peptide, sequence 3, the mass ratioes of any three kinds of polypeptides for (0.5~
1.5)：(0.5~1.5)：(0.5~1.5)；It is preferred that, their mass ratio is 1：1：1；
When the peptide composition is as the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, the polypeptide shown in sequence 3 and sequence
When polypeptide shown in row 4 is constituted, their mass ratio is (0.5~1.5)：(0.5~1.5)：(0.5~1.5)：(0.5~
1.5)；It is preferred that, their mass ratio is 1：1：1：1.
The Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents of the present invention, including enzyme-linked reaction plate, sun
Property control serum, negative control sera and ELIAS secondary antibody；Wherein described enzyme-linked reaction plate is coated with Sai Nika paddy virus structure eggs
White antigen epitope polypeptide composition.
The enzyme-linked reaction plate is detachable 96 hole elisa Plates；The Sai Nika paddy virus structural protein antigen epitope polypeptide
Polypeptide in composition, which is that chemistry is artificial synthesized, to be obtained.
The optimal preparation method and condition of the enzyme-linked reaction plate are by the Sai Nika paddy virus structural protein antigen table
Position peptide composition is dissolved in pH 9.6 carbonate solution, is then added to 96 hole polystyrene enzyme-linked reaction plates, many per hole 200ng
Peptide, places 8~12 hours at 2~8 DEG C, polypeptide antigen is fully combined with enzyme-linked reaction plate, then according to the addition of 300 μ l/ holes
PBS containing 1% (g/ml) bovine serum albumin(BSA) (BSA) pH7.4,37 DEG C of Seal treatments 2~3 hours, after drying, is treated
4 DEG C of sealing preserves after enzyme-linked reaction plate is dried.
The positive control serum is the Swine serum gathered after the viral artificial challenge of the Sai Nika paddy；The negative control
Serum is the Swine serum of no-special pathogen (SPF).
The ELIAS secondary antibody is horseradish peroxidase-labeled rabbit-anti pig IgG antibody.
The kit also includes substrate solution A, substrate solution B and terminate liquid, and the substrate solution A is containing 0.06% (g/ml) mistake
The citrate phosphate buffer of urea hydrogen is aoxidized, the substrate solution B is 0.2mg/ml tetramethyl biphenyl amine aqueous solution, when using
Both are with 1:1 ratio mixing.The terminate liquid is 2mol/L sulfuric acid solution.
The kit also includes sample diluting liquid and 20 times of concentrated cleaning solutions；Sample diluting liquid is to contain 0.5% (g/
100ml) 0.01M of casein, pH value are 7.4 phosphate buffer；Concentrated cleaning solution be containing concentration be 0.8%~
1.2% (ml/ml) Tween-20 0.01M, pH value is 7.4 phosphate buffer.
The detection program of kit of the present invention is：
1st, balance：Kit is taken out from cold storage environment, equilibrium at room temperature 30min is put standby；Liquid reagent is mixed with preceding.
2nd, with liquid：The 20 times of dilutions of concentrated cleaning solution distilled water or deionized water are obtained into lavation buffer solution；
3rd, set：2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4th, sample pre-dilution to be measured：Using sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to 1：20 dilution proportion.
5th, it is loaded：Each hole is respectively by the sample to be tested preset plus 100 μ l dilute.Sample-adding process time span should try one's best
It is short.
6th, incubate：Concussion is mixed, and is put in 37 DEG C of incubators, reacts 30min.
7th, board-washing：Reaction solution is discarded, the lavation buffer solution after 300 μ l dilutions is added per hole, 15s is soaked, gets rid of and abandon washing lotion, continuously
Patted dry after board-washing 4 times.
8th, it is enzyme-added：Each hole adds 100 μ l horseradish peroxidase-labeled rabbit-anti pig IgG antibody.
9th, incubate：37 DEG C of incubators are put, 30min is reacted.
10th, board-washing：Reaction solution is discarded, the μ l of lavation buffer solution 300 added per hole after dilution soak 15s, get rid of and abandon washing
Patted dry after liquid, continuous board-washing 4 times.
Substrate solution A and substrate solution B mixed in equal amounts (are substrate work by the 100 μ l substrates working solutions of addition that the 11, develop the color per hole
Liquid, matching while using), concussion is mixed, and is put in 37 DEG C of incubators, lucifuge reaction 15min.
12nd, the colour developing μ l of terminate liquid 50 are added per hole, vibration mixes terminating reaction.
13rd, the OD per hole is determined450nmValue (plus the reaction plate of terminate liquid should read OD in 15min450nmValue).
The judgement of testing result：
1st, negative control OD450nmAverage value should≤0.15, it is otherwise invalid.
2nd, each detected value of positive control should be otherwise invalid between 1.0~2.5.
3rd, the calculating of critical value：Critical value=0.17 × positive control OD450nmIt is worth average value.
Serum to be checked determines OD450nmValue >=critical value person is judged to the positive；Serum to be checked determines OD450nmValue<Critical value person sentences
For feminine gender.
The mentioned reagent box of the present invention, available for detection Sai Nika paddy virus structural protein antibody, to judge tested animal
With the presence or absence of the Sai Nika paddy virus structural protein antibody produced after infection.
Application in the kit for detecting whether infection Sai Nika paddy virosis is prepared falls within the protection model of the present invention
Enclose；
The positive effect of the present invention is：The present invention is using bioinformatics method to Sai Nika paddy virus structural proteins
Epitope carries out Accurate Analysis, and suitable ELISA detections are filtered out from the Main Antigenic on VP1, VP2, VP3 albumen and are used
Peptide fragment.The peptide fragment has concentrated epitope, has the advantages that sensitivity height, high specificity.
Meanwhile, the preparation for being coated with enzyme reaction plate is used for using advanced technology for solid phase synthesis of peptide synthetic polypeptide antigen.
Further, since the envelope antigen used in kit is chemically synthesized polypeptide, without foreign protein, purity is high, enters one
Step improves the efficiency of detection Sai Nika paddy virus structural protein antibody, to judge whether tested animal infects Sai Nika paddy disease
Poison.
In a word, this kit is coated with using the Antigenic Peptide of chemical synthesis Structural protein VP1, VP2, VP3 major antigenic sites
Enzyme-linked reaction plate, antigen consumption is few, sensitivity is high, high specificity, can effectively detect after Sai Nika paddy viral infections
The structural proteins antibody of generation, to judge whether tested animal infects Sai Nika paddy virus-virus.Test result indicates that, the present invention
Kit it is reproducible, high specificity, sensitivity is high.The need for different levels personnel can be met, before wide market
Scape and good economical, societal benefits.
Sai Nika paddy virus structural proteins enzyme-linked immunologic detecting kit involved in the present invention is used for whether detecting animal
Sai Nika paddy virus is infected, is conducive to the foundation of the viral prevention and control system of China Sai Nika paddy.
Embodiment
Method in following embodiments, is conventional method unless otherwise instructed.
The preparation of embodiment 1, Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent envelope antigens
This experiment is using major antigen table of the bioinformatics method to Sai Nika paddy virus structural proteins VP1, VP2, VP3
Position carries out Accurate Analysis, filters out suitable peptide fragment, four peptides are respectively synthesized out with full-automatic polypeptide synthetic instrument, sequence is respectively
In sequence table shown in sequence 1, sequence 2, sequence 3 and sequence 4, the more full envelope antigen of the renewal of purity about 80% is made, can
The main Neutralization and crystallization of Sai Nika paddy virus-virus is covered, the recall rate of antibody positive is improved.Polypeptide synthesis method can be
Conventional method, the present invention makes four polypeptides for synthesizing the present invention with the following method, is used as the coating antigen of kit of the present invention.
Applied Biosystem full-automatic polypeptide synthetic instruments (model 433A) system can be used in the envelope antigen of the present invention
It is standby.With Merrifield solid-phase synthesis, Fmoc (9-fluorenylmethyloxycarbonyl, 9- fluorenes first is used
Oxygen carbonyl) modification amino acid, using Rink Amide mbha resins as solid phase carrier.Production process includes polypeptide antigen
Synthesis in solid state, polypeptide cleavage and identification, antigen purification, five parts of freeze-drying and preservation.Illustrate individually below：
First, envelope antigen synthesis in solid state
1st, the preparation of synthetic agent
Synthesize envelope antigen amino acid sequence such as SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3 and SEQ ID
NO:Shown in 4.
Prepare the amino acid (being purchased from NOVA companies) of suitable Fmoc modifications according to envelope antigen sequence and synthesis scale,
Add into corresponding Cartridge.Equally synthesis scale claims resin 5g on request, is put into reaction chamber, will tighten lid up and down,
The weight of labelling, the title of the synthesized peptide of record, lot number, the TARE of reaction chamber and alleged resin.Reaction chamber is loaded and synthesized
Instrument.Preparing appropriate synthetic agent includes 100% NMP, 3% AIM (acyl imidazoles), 35% PIP (piperidines), 100%
MeOH (methanol) etc. be placed into corresponding reagent bottle.
2nd, the detection of synthesizer state
Check whether 433A Peptide systhesis instrument normally runs, after start, run Run Self Test programs, instrument is certainly
Whether normal examine indices.Check whether nitrogen is sufficient in addition, whether normal (the normal gauge pressures of 433A of system gauge pressure
10.2psi).The performance of reply instrument is had gained some understanding before synthesis, so to be measured to the flow velocity of every kind of synthetic agent.433A
Synthesizer：Flow Rate1-18 are sent to synthesizer, selection Main Menu-Module Test-are looked for by Prer or next
Module A, ModuleD, ModuleI, ModuleI, Module A) measured or observed by more-by Start-, if
Flow is improper, then adjusts lower valve pressure, until reaching requirement (specific detection requirement see the table below 1).
The Peptide synthesizer flow rate detection standard scale of table 1.
| Reagent | Bottle number | Module | Critical field |
| 35%Piperidine | 1 | A | 1.0~1.2ml |
| 3%AIM | 4 | D | 1.0~1.2ml |
| 100%MeOH | 9 | I | 3.5~4.0ml |
| DIC | 8 | I | 0.45~0.55g |
| 100%NMP | 10 | A | 2.6~2.8ml |
3rd, envelope antigen synthesis starts
The amino acid sequence that will be synthesized in the program of 433A synthesizers sends Std Fmoc 1.0Sol DIC90 to conjunction
On Cheng Yi.The sequence of File-New-Sequence- Edit and Compose peptides, is preserved.Whether File-New-Run, check Chemistry
For Std Fmoc 1.0Sol DIC 90；Whether Sequence is to be deposited name；Set Cycles；Preserve.It is finally sent to close
On Cheng Yi.
Main Menu-Cycle Monitor-begin, bring into operation.
4th, envelope antigen synthesis is carried out
The removing of Fmoc groups, the electron attraction of the fluorenes ring system of Fmoc groups makes 9-H have acidity, is easily removed compared with weak base
Go, eliminate to form hexichol fluorenes alkene with piperidines (PIP) attack 9-H, β during reaction, it is easy to which stabilization is formed by two-stage ring amine attack
Addition product."-NH2 " group is exposed after the removing of Fmov groups to carry out synthetic reaction.Then the next of activation is added
In amino acid and I-hydroxybenzotriazole (1-hydroxybenzotriazole, HOBT) to the reactor of Fmoc radical protections.
Peptide sequence described above, is to N-terminal, according to specific order, successively constantly when synthesis since C-terminal
Repeat synthesis step (synthesizer is automatically performed by program, specific circulation step such as table 2 below).Period observed and recorded reagent dosage and
Running situation.
The envelope antigen of table 2. synthesizes circulation step
5th, envelope antigen end of synthesis
Synthesizer will be automatically stopped after envelope antigen end of synthesis, and peptide resin (peptide is additionally attached on resin now) base
This washes clean.Then remove reactor from Peptide synthesizer, then washed with 100% methanol after peptide resin 3 times, in fume hood
Polypeptide resin, is then fully transferred in the polyethylene bottle of brown by interior drying, is put into -20 DEG C of refrigerators, and sealed membrane sealing is standby
With.
2nd, the cracking and identification of envelope antigen
1st, the cracking of polypeptide antigen
It is chemically bound together through the polypeptide obtained by above-mentioned reaction and solid phase carrier, it is necessary to by specific
The acidolysis of organic acid polypeptide is separated with solid phase carrier.Also the guarantor on each amino acid functional group is eliminated while acidolysis
Protect base.Step is as follows：
The polypeptide resin (referring to peptide to be additionally attached on resin) of synthesis is taken out out of refrigerator, 2L round-bottomed flask is put into
It is interior, 90ml trifluoroacetic acids (Trifluoroacetic acid, TFA), 10ml tripropyl are added into flask in fume hood
Flask, is then stably placed on magnetic stirring apparatus by silane (TIS) and magnetic stick, and persistently stirring 1h extremely reacts at room temperature
Completely.After reaction terminates, the TFA in 30~120min removing crude products is persistently evaporated using the Rotary Evaporators with cold-trap.So
The crude product of polypeptide antigen is cleaned multiple times with dimethylformamide (DMF) afterwards, finally by the resin mixed sand core funnel
Filter out, both obtain envelope antigen.
2nd, the identification of envelope antigen
Polypeptide antigen synthesis winged tries time mass spectrum method (MODAL-TOF) and anti-phase height after finishing with substance assistant laser desorpted
Pressure liquid chromatography (RP-HPLC) carries out qualitative and quantitative analysis, and synthesized peptide is identified with common amino acid analysis.
3rd, envelope antigen is purified
The polypeptide antigen after cyclisation is carried out into ultrafiltration using circulating tangential flow filtration film bag (to be produced with PALL companies
The circulating tangential flow filtration film bags of Tangential Flow Device and the peristaltic pump supporting with it), polypeptide antigen is as big
Molecule can not be by the filter membrane of certain pore size, and early stage building-up process and the formation of later stage cyclization or the small molecule introduced are miscellaneous
Matter can then pass through filter membrane.Then it is degerming for 0.2 μm of filter by aperture again, last resulting solution is dispensed into aseptic plastic
It is labelled in bottle.Title, numbering, product batch number, concentration, date of manufacture, pot-life and the preservation of polypeptide are indicated on label
Condition, after packing, be stored in -20 DEG C or -40 DEG C it is standby.
4th, envelope antigen is freeze-dried
For the ease of long-term storage and transport, it is necessary to which envelope antigen is freeze-dried to obtain many of solid state
Peptide.The envelope antigen freezed in advance is positioned on Labconco freeze drier and is dried, solid state is obtained
Envelope antigen.It is labelled after packaging.The title of dated polypeptide, numbering, product batch number, concentration, date of manufacture, preservation on label
Time limit and preservation condition.
The preparation of embodiment 2, Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents include：
(1) it is coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of Sai Nika paddy viral antigens；2 × 96 holes.
(2) positive control serum：It is to gather Swine serum after the viral artificial challenge of Sai Nika paddy, be used as the positive of kit
Control serum (1 pipe, 1.5ml/ pipes).
(3) negative control sera：It is no-special pathogen (SPF) Swine serum, is used as the negative control sera of kit
(1 pipe, 1.5ml/ pipes).
(4) ELIAS secondary antibody：It is with horseradish peroxidase-labeled rabbit-anti pig IgG (being purchased from sigma companies, article No. A5670)
1 is carried out as stoste:It is made after 30000 dilutions, 2 bottles (12ml/ bottles).
(5) sample diluting liquid：For the 0.01M containing 0.5% (g/100ml) casein, the phosphate-buffered that pH value is 7.4
Liquid, 1 bottle (24ml/ bottles).
(6) substrate solution A：For the citrate phosphate buffer (1 bottle, 12ml/ bottles) of the hydrogen peroxide urea containing 0.6mg/ml
(7) substrate solution B：For 0.2mg/ml tetramethyl benzidine (TMB) solution (1 bottle, 12ml/ bottles).
(8) terminate liquid：2mol/L sulfuric acid solution (1 bottle, 12ml/ bottles).
(9) 20 times of concentrated cleaning solutions：For 0.01M, pH containing the Tween-20 that concentration is 0.8%~1.2% (ml/ml)
It is worth the phosphate buffer (50ml/ bottles, 2 bottles) for 7.4.
As needed, can also there are serum-dilution plate (2 pieces, 96 holes/block), the dilution for blood serum sample in kit.
Wherein, the preparation method for being coated with the removable polystyrene enzyme-linked reaction plate in 96 holes of Sai Nika paddy viral antigens is：
1. polypeptide antigen prepared by embodiment 1 is dissolved in pH 9.6 carbonate solution, 96 hole polystyrene integrated enzyme reactions are then added to
Plate, per hole 200ng polypeptides, (polypeptide wherein in sequence table shown in sequence 1 is 50ng, and the polypeptide in sequence table shown in sequence 2 is
Polypeptide in 50ng, sequence table shown in sequence 3 is 50ng, and the polypeptide in sequence table shown in sequence 4 is 50ng), 8 are placed at 2~8 DEG C
~12 hours, polypeptide antigen is fully combined with enzyme-linked reaction plate, then added according to 300 μ l/ holes and contain 1% (g/ml) ox blood
Pure albumen (BSA) pH7.4 PBS, 37 DEG C of Seal treatments 2~3 hours, after drying, after 4 after enzyme-linked reaction plate drying
DEG C sealing preserve.
The sensitivity tests of embodiment 3, Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
First, the application method of Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent
1st, balance：Kit is taken out from cold storage environment, equilibrium at room temperature 30min is put standby；Liquid reagent is mixed with preceding.
2nd, with liquid：The 20 times of dilutions of 20 times of concentrated cleaning solution distilled water or deionized water are obtained into lavation buffer solution；
3rd, set：2 negative control holes and 2 Positive control wells, remaining is sample to be tested hole.
4th, sample pre-dilution to be measured：Using sample diluting liquid by measuring samples serum, negative control sera, positive control blood
Clearly according to 1：20 dilution proportion.
5th, it is loaded：Each hole is respectively by the sample to be tested preset plus 100 μ l dilute.Sample-adding process time span should try one's best
It is short.
6th, incubate：Concussion is mixed, and is put in 37 DEG C of incubators, reacts 30min.
7th, board-washing：Reaction solution is discarded, adds 300 μ l lavation buffer solutions per hole, 15s is soaked, gets rid of and abandon washing lotion, continuous board-washing 4 times
After pat dry.
8th, it is enzyme-added：Each hole adds 100 μ l horseradish peroxidase-labeled rabbit-anti pig IgG antibody.
9th, incubate：37 DEG C of incubators are put, 30min is reacted.
10th, board-washing：Reaction solution is discarded, the μ l of lavation buffer solution 300 are added per hole, 15s is soaked, gets rid of and abandon cleaning solution buffer solution,
Patted dry after continuous board-washing 4 times.
Substrate solution A and substrate solution B mixed in equal amounts (are substrate work by the 100 μ l substrates working solutions of addition that the 11, develop the color per hole
Liquid, matching while using), concussion is mixed, and is put in 37 DEG C of incubators, lucifuge reaction 15min.
12nd, the colour developing μ l of terminate liquid 50 are added per hole, vibration mixes terminating reaction.
13rd, the OD per hole is determined450nmValue (plus the reaction plate of terminate liquid should read OD in 15min450nmValue).
The judgement of testing result：
1st, negative control OD450nmAverage value should≤0.15, it is otherwise invalid.
2nd, each detected value of positive control should be otherwise invalid between 1.0~2.5.
3rd, the calculating of critical value：Critical value=0.17 × positive control OD450nmIt is worth average value.
Serum to be checked determines OD450nmValue >=critical value person is judged to the positive；Serum to be checked determines OD450nmValue<Critical value person sentences
For feminine gender.
2nd, to the sensitivity tests of known positive serum
The three batches of Sai Nika paddy virus structural protein antibody ELISAs immune detections prepared using the method according to embodiment 2 are tried
Agent box (batch ZM2017001, ZM2017002, ZM2017003), respectively in accordance with above-mentioned Sai Nika paddy virus structural protein antibody
Enzyme-linked immunologic detecting kit application method to 35 parts of Swine serum of Sai Nika paddy virus infection, (face by the virus infection of Sai Nika paddy
The Swine serum that different time is gathered after bed symptom) sensitivity tests is carried out, experimental result is shown in Table 3, Sai Nika paddy disease of the invention
Malicious structural proteins antibody ELISA immunity detection reagent detects 34 parts altogether, has 1 part not detect, as a result shows this kit to 35
The sensitiveness of positive serum known to part is 97.1%.
The sensitivity Detection result of the Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents of table 3.
| Kit lot number | Recall rate | Sensitiveness |
| ZM2017001 | 34/35 | 97.1% |
| ZM2017002 | 34/35 | 97.1% |
| ZM2017003 | 34/35 | 97.1% |
3rd, lowest detection limitation experiment
3 parts of the Swine serum of Sai Nika paddy virus structural protein antibody positives is chosen, doubling dilution 1 is carried out:20、1:40~1:
320, the three batches of Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent (batches prepared using embodiment 2
ZM2017001, ZM2017002, ZM2017003), according to the examination of above-mentioned Sai Nika paddy virus structural protein antibody ELISA immune detection
Agent box application method is detected to doubling dilution Swine serum, is as a result shown, kit of the present invention can detect 1:160 times
The positive serum of dilution, wherein, table 4 is the experimental result of one of batch.In table 4, positive control：It is with Sai Nika paddy disease
Swine serum is gathered after malicious artificial challenge, the positive control serum (1 pipe, 1.5ml/ pipes) of kit is used as.Negative control：It is no spy
Determine pathogen (SPF) Swine serum, be used as the negative control sera (1 pipe, 1.5ml/ pipes) of kit.
Critical value (Cut-off values)=0.17 × positive control OD450nmIt is worth average value.
The lowest detection limitation testing inspection knot of the Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents of table 4
Really
The specific test of embodiment 4, Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
Using three batches of kits in embodiment 2 according to the Sai Nika paddy virus structural protein antibody described in embodiment 3
The application method of enzyme-linked immunologic detecting kit is to 50 parts of healthy Swine serum (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.
There is provided), (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd. carries O-shaped (FMD-O) positive serum of 2 parts of swine foot-and-mouth disease virus
For), 2 parts of swine foot-and-mouth disease virus A types (FMD-A) positive serums (Lanzhou Biopharmaceutical Factory, Zhongmu Industry Co, Ltd.'s offer), 2
Part swine pox positive serum (SVD) (Zhongmu Industry Co., Ltd's offer), 2 parts of pig vesicular stomatitis virus (VSV) sun
Property serum (Zhongmu Industry Co., Ltd's offer), is detected respectively.
The specific detection result such as following table (table 5) of kit shows that the testing result to 50 parts of healthy Swine serums is shown,
The specificity of ZM2017001 kits is that the specificity of 100.0%, ZM2017002 kits is 100.0%, ZM2017003 examinations
The specificity of agent box is 100.0%.To O-shaped (FMD-O) positive serum of 2 parts of swine foot-and-mouth disease virus, 2 parts of swine foot-and-mouth disease virus A types
(FMD-A) inspection of positive serum, 2 parts of swine pox (SVD) positive serums, 2 parts of pig vesicular stomatitis virus (VSV) positive serums
Survey result and be illustrated as feminine gender, therefore three kits are to the specificity of this 8 parts of Antigen positive hybridomas Virus monitories of related diseases
100%.
The Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent specific detection results of table 5
The coincidence rate experiment of embodiment 5, Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
First, virus neutralization tests (VNT) method
The generally acknowledged detection method of current Sai Nika paddy virus is virus neutralization tests (VNT), therefore with this kit with disease
Malicious neutralization test carries out coincidence rate experiment.
1. material
1.1 infection Swine serums, 24 parts (serum numbering I1~I24) is clinical symptoms occur after Sai Nika paddy virus infects
The Swine serum gathered afterwards, inactivates through 56 DEG C of 30min, is provided by Zhongmu Industry Co., Ltd.
1.2 healthy Swine serums, 24 parts (serum numbering N1~N24) is inactivated, industry share is herded in be had through 56 DEG C of 30min
Limit company provides.
1.3 experiments virus (SVV viruses) is the PK15 cell toxicants of Sai Nika paddy virus, adjusts its malicious valency extremely
400TCID50/ 0.1ml, is placed in -20 DEG C of preservations stand-by.
2. method virus neutralization tests method is as follows：After inactivating blood serum, doubling dilution (25 μ l/ are carried out with DMEM culture mediums
Hole), each dilution factor does 3 repetitions；Isometric (25 μ l/ holes) 100TCID is added per hole50SVV viral (except control), put
It is incubated 1 hour in 37 DEG C；Then the 100 μ l PK15 cells (2~3 × 10 diluted with DMEM culture mediums are added per hole4), it is placed in
37 DEG C, 5%CO2Incubator culture, observes cytopathy (CPE) after 72 hours.Last repressed dilution factor (90 of CPE
~100% is suppressed) it is considered as virus neutralization tests (VNT) potency, potency >=1:64 are judged to the positive.
2nd, coincidence rate result of the test：
The Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent prepared using embodiment 2 is neutralized with virus
Test (VNT) and detect the viral Swine serum of 24 parts of Sai Nika paddy and 24 parts of healthy serum respectively.
Testing result of the Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent to 24 parts of infection Swine serums
6 are shown in Table, the sensitiveness of kit of the present invention is 95.8%, and the sensitiveness of virus neutralization tests is 87.5%, in 24 parts of infection
In Swine serum, two methods testing result it is consistent for 22 parts.Therefore, kit of the present invention and the coincidence rate of virus neutralization tests
For 91.7%, kit of the present invention has higher sensitiveness.To the test of healthy Swine serum, both coincidence rates are 100%,
Testing result is negative (being shown in Table 6).
Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent has 48 parts of detection altogether with virus neutralization tests
Swine serum, wherein two methods of 46 parts of Swine serum testing results are consistent, 2 parts of Swine serum testing results are variant, and coincidence rate is
95.8%.
The Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent of table 6 and virus neutralization tests method are to infected pigs
The testing result of serum and healthy Swine serum
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deformed, and without departing from the spirit of the present invention, all should belong to the model of appended claims of the present invention
Enclose.
Sequence table
<110>Zhongmu Industry Co., Ltd
<120>Sai Nika paddy virus structural protein antibody ELISA immunity detection reagents
<130> WHOI170051
<160> 4
<210> 1
<211> 37
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 1
Ser Asp Leu Glu Val Thr Val Val Ser Leu Glu Pro Asp Leu Glu Phe
1 5 10 15
Ala Val Gly Trp Phe Pro Ser Gly Ser Glu Tyr Gln Ala Ser Ser Phe
20 25 30
Val Tyr Asp Gln Leu
35
<210> 2
<211> 36
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 2
Thr Lys Ser Asp Pro Pro Ser Ser Ser Thr Asp Gln Pro Thr Thr Thr
1 5 10 15
Phe Thr Ala Ile Asp Arg Trp Tyr Thr Gly Arg Leu Asn Ser Trp Thr
20 25 30
Lys Ala Val Lys
35
<210> 3
<211> 38
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 3
Pro Tyr Ile Gly Glu Thr Pro Thr Gln Ser Ser Glu Thr Gln Asn Ser
1 5 10 15
Trp Thr Leu Leu Val Met Val Leu Val Pro Leu Asp Tyr Lys Glu Gly
20 25 30
Ala Thr Thr Asp Pro Glu
35
<210> 4
<211> 34
<212> PRT
<213>Artificial sequence
<220>
<223>
<400> 4
Thr Leu Ser Glu Ala Met Gln Cys Thr Tyr Ser Ile Trp Asp Ile Gly
1 5 10 15
Leu Asn Ser Ser Trp Thr Phe Val Val Pro Tyr Ile Ser Pro Ser Asp
20 25 30
Tyr Arg
Claims (10)
1. it is polypeptide in sequence table shown in sequence 1, sequence table Sai Nika paddy virus structural protein antigen epitope polypeptide compositions
More than one or both of polypeptide in the polypeptide or sequence table in middle sequence 2, sequence table shown in sequence 3 shown in sequence 4
Any combination.
2. Sai Nika paddy virus structural protein antigen epitope polypeptide composition according to claim 1, it is characterised in that：Work as institute
It is many shown in polypeptide, the polypeptide shown in sequence 2, the polypeptide shown in sequence 3 and sequence 4 shown in sequence 1 to state peptide composition
During two kinds in peptide, the mass ratio of two kinds of polypeptides is (0.5~1.5)：(0.5~1.5)；It is preferred that, their mass ratio is 1：
1；When the peptide composition be the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, the polypeptide shown in sequence 3 and the institute of sequence 4
During three kinds in the polypeptide shown, the mass ratio of any three kinds of polypeptides is (0.5~1.5)：(0.5~1.5)：(0.5~1.5)；It is excellent
Choosing, their mass ratio is 1：1：1；When the peptide composition is as the polypeptide shown in sequence 1, the polypeptide shown in sequence 2, sequence
When polypeptide shown in polypeptide and sequence 4 shown in row 3 is constituted, their mass ratio is (0.5~1.5)：(0.5~1.5)：(0.5
~1.5)：(0.5~1.5)；It is preferred that, their mass ratio is 1：1：1：1.
3. a kind of Sai Nika paddy virus structural protein antibody ELISA immunity detection reagent, including enzyme-linked reaction plate, positive control
The wherein described enzyme-linked reaction plate of serum, negative control sera and ELIAS secondary antibody is coated with the Sai Nika paddy described in claim 1 or 2
Virus structural protein antigen epitope polypeptide composition.
4. enzyme-linked immunologic detecting kit according to claim 3, it is characterised in that：The enzyme-linked reaction plate is detachable
96 hole elisa Plates；Each polypeptide is artificial synthesized for chemistry in the Sai Nika paddy virus structural protein antigen epitope polypeptide composition
Obtain.
5. enzyme-linked immunologic detecting kit according to claim 3, it is characterised in that：The acquisition side of the enzyme-linked reaction plate
Method is the pH 9.6 that Sai Nika paddy virus structural protein antigen epitope polypeptide composition described in claim 1 or 2 is dissolved in 100 μ l
Carbonate solution, 96 hole polystyrene enzyme-linked reaction plates are then added to, per hole 200ng Sai Nika paddy virus structural protein antigens
Epitope polypeptide composition, places 8~12 hours at 2~8 DEG C, makes Sai Nika paddy virus structural protein antigen epitope polypeptide compositions
Fully combined with enzyme-linked reaction plate, then adding the PBS containing 0.01g/ml bovine serum albumin(BSA)s pH7.4 according to 300 μ l/ holes delays
Fliud flushing, 37 DEG C of Seal treatments 2~3 hours, after drying, after 4 DEG C of sealing preserves after enzyme-linked reaction plate drying.
6. enzyme-linked immunologic detecting kit according to claim 3, it is characterised in that：
The positive control serum is the Swine serum gathered after the viral artificial challenge of the Sai Nika paddy；The negative control sera
For the Swine serum of no-special pathogen.
7. enzyme-linked immunologic detecting kit according to claim 3, it is characterised in that：The ELIAS secondary antibody is horseradish peroxide
Compound enzyme marks rabbit-anti pig IgG antibody.
8. enzyme-linked immunologic detecting kit according to claim 3, it is characterised in that：The kit also includes substrate solution
A, substrate solution B and terminate liquid, the substrate solution A are the citrate phosphate buffer of the hydrogen peroxide urea containing 0.0006g/ml,
The substrate solution B is 0.2mg/ml tetramethyl biphenyl amine aqueous solution, with 1 both when using:1 ratio mixing；The terminate liquid
For 2mol/L sulfuric acid solution.
9. kit according to claim 3, it is characterised in that：The kit also includes sample diluting liquid and 20 times dense
Contracting cleaning solution；The phosphate-buffered that sample diluting liquid is the 0.01mol/L containing 0.00005g/ml caseins, pH value is 7.4
Liquid；The 0.01mol/L for the Tween-20 that it is 0.8%~1.2% containing concentration expressed in percentage by volume that concentrated cleaning solution, which is, pH value is 7.4
Phosphate buffer.
10. it is polypeptide in sequence table shown in sequence 1, sequence in sequence table Sai Nika paddy virus structural protein antigen epitope polypeptides
Polypeptide in polypeptide or sequence table in row 2, sequence table shown in sequence 3 shown in sequence 4.
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| CN201910028355.4A CN109627293B (en) | 2017-08-13 | 2017-08-13 | Seikaga valley virus structural protein epitope polypeptide and application thereof |
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| CN108761074A (en) * | 2018-05-23 | 2018-11-06 | 中国农业科学院兰州兽医研究所 | Senecan virus ELISA antibody assay kit and preparation method, application |
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| CN109627293B (en) | 2021-02-09 |
| CN107253978B (en) | 2020-11-20 |
| CN109627293A (en) | 2019-04-16 |
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