CN110174516A - Goose parvovirus VP3 proteantigen ELISA detection kit and detection method and application - Google Patents
Goose parvovirus VP3 proteantigen ELISA detection kit and detection method and application Download PDFInfo
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- CN110174516A CN110174516A CN201910484180.8A CN201910484180A CN110174516A CN 110174516 A CN110174516 A CN 110174516A CN 201910484180 A CN201910484180 A CN 201910484180A CN 110174516 A CN110174516 A CN 110174516A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
Abstract
The invention discloses a kind of goose parvovirus VP3 proteantigen ELISA detection kit and detection methods.It include: anti-goose parvovirus VP3 monoclonal antibody, cleaning solution, zymolyte solution A, zymolyte B solution, terminate liquid and the antigen standard of the ELISA Plate for being coated with the polyclonal antibody of goose parvovirus VP3 albumen, confining liquid, sample diluting liquid, the goose parvovirus VP3 recombinant protein antigen standard items of purifying, HRP label in the kit.Meanwhile the invention also discloses a kind of methods using the goose parvovirus VP3 proteantigen in the kit accurate quantitative analysis test sample.Kit of the invention is in addition to having the advantages that ELISA kit, the defects of also overcoming sensitivity caused by specificity caused by Antigen conformation problem is low and secondary antibody problem, significantly improves the specificity and sensitivity of detection.
Description
Technical field
The present invention relates to a kind of detection kits, and in particular to a kind of goose parvovirus VP3 proteantigen ELISA detection examination
Agent box and its preparation method and application.
Background technique
Goose parvovirus, i.e. Goose Parvovirus, Goose Parvovirus are that a kind of height as caused by goose parvovirus connects
Touching property, acute, Septic blood infectious disease, it is main to encroach on 4-20 age in days young bird goose and muscovy duckling.Young goose and muscovy duckling are with down in spirits, food
Abolish and severe diarrhea being characterized property clinical symptoms are intended to, feature are become with exudative enteritis, the acute septicaemia of whole body, small intestine is last
It is to analyse symptom that section stenosis enteric cavity, which has embolus shape object blocking enteric cavity,.Currently, this disease have every year it is popular to some extent, still for
One of most important infectious disease in the country of goose industry and intensive culture kind duck is supported, heavy losses are caused to aquatic bird aquaculture.
Although having many detection methods at present, such as agar gel diffusion test and AGP test inhibit test, neutralization test
And animal protection test, direct immunofluorescence diagnosis, immunoenzyme spot method, but while these methods can detecte GPV, but
Inconvenient to use there are cumbersome, time-consuming and is not easy the defects of universal.In the prior art, CN104833801B is disclosed
A kind of double-antibody sandwich elisa kit detecting gosling plague antigen, the kit detect GPV using double monoclonal antibody combination technologies
Antigen, the kit fails the marketization so far, and the kit preparation procedure is cumbersome, is only capable of qualitative detection gosling plague antigen
Presence, be not able to satisfy the detection for antigenic content in vaccine product.It is badly in need of studying a kind of efficient, accurate and quantitative inspection thus
The method for surveying GPV.
Summary of the invention
The present invention is can satisfy again in order to improve the specificity and sensitivity of detection for gosling plague VP3 in sample
The qualitative and quantitative detection of albumen provides a kind of detection goose parvovirus VP3 proteantigen ELISA detection kit, and mentions
Its detection method and application are supplied.
In a first aspect, the purpose of the present invention is to provide a kind of goose parvovirus VP3 proteantigen ELISA test agent boxes.
Wherein, the kit includes: the ELISA Plate of the polyclonal antibody of coating goose parvovirus VP3 albumen, confining liquid, sample dilution
Liquid, the goose parvovirus VP3 recombinant protein antigen standard items of purifying, HRP label anti-goose parvovirus VP3 monoclonal antibody,
Cleaning solution, zymolyte solution A, zymolyte B solution and terminate liquid.Wherein, the antigen standard is that the recombination of purifying is rod-shaped
The goose parvovirus VP3 albumen of the formation virus-like particle (VLPs) of expressing viral, the zymolyte solution A contain hydrogen peroxide
Sodium acetate buffer solution, zymolyte B solution are 3,3 ', 5,5 '-tetramethyl biphenyl amide hydrochlorides.
In preferred embodiment of the invention, the polyclonal antibody of the goose parvovirus VP3 albumen is that ProteinA column is pure
Change the goose parvovirus VP3 albumen of the formation virus-like particle (VLPs) of recombinant baculovirus expression as antigen immunization experiment rabbit
Obtained by the high price serum obtained afterwards, the package amount of the polyclonal antibody of the goose parvovirus VP3 albumen is 0.01 μ g-0.5 μ g/
Hole.It is coated with the carbonate buffer solution that dilution is the 0.05ml/L that pH is 9.6.Group, which becomes in every liter of carbonate buffer solution, to be contained
1.59gNa2CO3With 2.93g NaHCO3。
In preferred embodiment of the invention, the confining liquid is selected from cow's serum that mass concentration is 1-10% from albumen (BSA)
Or mass concentration is any or combinations thereof of the skimmed milk power of 1-10%.
In preferred embodiment of the invention, sample diluting liquid is 0.1-10%BSA by mass concentration and contains 0.01-1%
The phosphate buffer of ProClin 300 forms, wherein the concentration of the phosphate buffer solution is 0.01mol/L, pH7.2-
7.4。
In preferred embodiment of the invention, the preparation of the sample diluting liquid are as follows: now prepare pH7.2-7.4, concentration is
The phosphate buffer of 0.01mol/L is added 0.1-10%BSA after 121 DEG C of 20min high pressure sterilizations are cooling and contains 0.01-1%
ProClin 300 to get.
In preferred embodiment of the invention, zymolyte solution A contains the acetate buffer of 0.016% (mass concentration) hydrogen peroxide
Solution, wherein the concentration of sodium acetate solution is 10g/L, pH 5.3；It weighs sodium acetate 10g and is dissolved in 1L purified water, with acetic acid tune
PH is 5.3, and adding 540 μ L concentration is 30%H2O2。
The zymolyte B solution is that 3,3 ', the 5,5 '-tetramethyl biphenyl amine hydrochlorates (TMBHCl) of 1.7mmol/L are molten
Liquid；It weighs 0.47gTMBHCl to be added in the citrate buffer solution for the 0.01mol/L that 1L pH is 3.0, be completely dissolved, i.e.,
?.
In preferred embodiment of the invention, the terminate liquid is the H of 1mol/L2SO4Solution.
In preferred embodiment of the invention, the preparation purified water of the cleaning solution.
In preferred embodiment of the invention, the developing solution is that isometric zymolyte solution A and zymolyte B solution mix, existing
With now matching.
It include antigen standard and negative controls in the kit, wherein antigen mark in preferred embodiment of the invention
Quasi- product are formulated as diluting the goose of the formation virus-like particle (VLPs) of the recombinant baculovirus expression of purifying with sample diluting liquid
Parvovirus VP3 albumen, until 1 μ g/mL is made；Negative controls are formulated as thin without goose with 100 times of sample diluting liquid dilutions
The goose embryo allantoic liquid of small virus is formulated.
In preferred embodiment of the invention, recombinated described in the preparation method of the recombination GPV VP3 albumen in the kit
GPV VP3 albumen is GPVP3/Bac plants of recombinant baculovirus that Yangzhou You Bang biologics Co., Ltd develops, by cultivating place
Chief cell is inoculated with recombinant baculovirus GPVP3/Bac plants, isolates and purifies recombinant baculovirus GPV after expressing GPV VP3 albumen
VP3 albumen.
In a preferred embodiment of the invention, recombinant baculovirus GPV VP3 albumen inactivation after cell culture, pass through
Centrifugation or hollow fibre filtering remove cell fragment, obtain the cells and supernatant containing GPV VP3 albumen, then by hypervelocity from
The heart and cesium chloride density gradient centrifugation obtain the VP3 albumen of the formation virus-like particle of purifying.
Second aspect, the purpose of the present invention is to provide a kind of detection method using the antigen detection kit, tools
Body implementation steps:
(1) measuring samples sample diluting liquid is pressed into gradient dilution, is added into the anti-goose parvovirus VP3 albumen of coating
In the ELISA Plate of polyclonal antibody, every hole adds 100 μ L, while positive controls, negative control group and blank control group is arranged,
The antigen standard that stoste is added in middle positive controls, 1:2,1:4,1:8,1:16,1:32,1:64,1:128 are serially diluted is each
100 μ L, negative control group be added 100 μ L negative controls, blank control group be added 100 μ L sample dilutions, each sample and
Control is loaded 2-6 hole in parallel.
(2) after the completion of being loaded, after ELISA Plate is set 37 DEG C of incubations 1 hour, cleaning solution board-washing 3-5 times, drying.
(3) the anti-goose parvovirus VP3 monoclonal antibody of 100 μ L HRP label is added in every hole, and 37 DEG C are educated 1 hour, washing
Liquid board-washing 3-5 times, drying.
(4) 100 μ L developing solutions are added in every hole, are protected from light colour developing 5~after ten minutes, every hole adds 50 μ L terminate liquids, wherein
The developing solution is mixed in equal volume by zymolyte B solution and zymolyte solution A and is made, and is paid attention to ready-to-use.
(5) ELISA Plate is placed in microplate reader, measures its absorbance value OD in 450nm450nm。
(6) result calculates and determines.
Wherein, the judgement of qualitative results: Cut off (CO value)=negative control absorbance value OD450nm× 2.1 times, sample
Value=sample absorbance value OD450nm/ CO value, wherein sample value > 1 is the positive；Sample value≤1 is feminine gender；Alternatively, quantitative result
Judgement: using 2 be bottom OD value logarithm as X-axis, using 2 be bottom standard items concentration as Y-axis, utilize " EXCEL " software obtain line
The concentration of linearity curve and equation, reference material and sample OD value brought RelPot 4.0 into and calculate sample.
The third aspect, the present invention also provides a kind of goose parvovirus VP3 protein ELISA antigen detection kits for examining
Survey the application of gaggle Goose Parvovirus or gosling plague bacterin product.
In preferred embodiment of the invention, the sample can come from gaggle Goose Parvovirus or gosling plague bacterin
Cloacal swab, the cell adapted poison, flesh tissue pathological material of disease, goose embryo allantoic liquid, GPV vaccine of product, i.e. Goose Parvovirus infection
Antigen it is any.Cloacal swab or flesh tissue pathological material of disease from gosling plague infection are added in 800 μ L sample dilutions,
Multigelation 2 times, wink from 10s, takes supernatant to use before use；Cell adapted poison, multigelation cell, before use wink from
10s takes supernatant to use；Goose embryo allantoic liquid or GPV subunit vaccine antigenic direct sample detection.
Developing solution zymolyte B solution of the present invention is 3,3 ', the 5,5 '-tetramethyl benzidine hydrochloric acid of 1.7mmol/L
Salting liquid；Developing solution zymolyte solution A contains the sodium acetate buffer solution of 0.016% (mass concentration) hydrogen peroxide, wherein acetic acid
The concentration of sodium solution is 10g/L, pH 5.3.
Terminate liquid of the present invention is the H of 1mol/L2SO4Solution.
The preparation purified water of cleaning solution of the present invention.
Antigen standard of the present invention is formulated as diluting the recombinant baculovirus expression of purifying with sample diluting liquid
The goose parvovirus VP3 albumen for forming virus-like particle (VLPs), until 1 μ g/ml is made.
Negative controls of the present invention are formulated as being free of the goose embryo of goose parvovirus with 100 times of sample diluting liquid dilutions
Allantoic fluid is formulated.
Blank control liquid of the present invention is sample diluting liquid.
Unless otherwise indicated, when percentage between liquid and liquid of the present invention, the percentage is volume/body
Product percentage；When percentage between liquid and solid of the present invention, the percentage is volume/weight percentage；This
When inventing the percentage between the solid and liquid, the percentage is weight/volume percent；Remaining is weight/weight
Measure percentage.
Compared with prior art, the present invention has following technical effect that
1. kit provided by the invention is using the goose parvovirus VP3 of the virus-like particle (VLPs) of baculovirus expression
Anti- goose parvovirus VP3 monoclonal antibody of the polyclonal antibody coated elisa plate of albumen development as capture antibody, HRP label
Colour developing antibody is done, and then is formed with double sandwich method enzyme-linked immunosorbent assay detection goose parvovirus and goose parvovirus VP3
Recombinant protein.Kit of the invention is low to also overcome Antigen conformation problem institute in addition to having the advantages that ELISA kit
The defects of sensitivity caused by the low and secondary antibody problem of specificity of cause, significantly improves the specificity of detection, sensitivity and credible
Degree, the concentration of lowest detection to antigen standard are 4ng/ml.
2. containing antigen standard in kit of the present invention, the exact level of GPV VP3 albumen in sample can detecte,
Particularly suitable for the quantitative detection of GPV VP3 gene subunit vaccine, large-scale production and can apply.
Detailed description of the invention
GPV VP3 albumen (VLPs) electron microscopic picture of expression is purified in Fig. 1 embodiment 2.
The preparation technology flow chart of kit in Fig. 2 embodiment 3.
HRP enzyme marks anti-goose GPV VP3 monoclonal antibody and GPV VP3 recombinant protein obtained in Fig. 3 embodiment 3
Westernblot analysis chart, wherein M: pre- dsred protein Marker；1:HRP marks anti-GPV VP3 monoclonal antibody.
The canonical plotting that 6 Plays product of Fig. 4 embodiment are drawn.
Specific embodiment
The present invention is described in detail with embodiment with reference to the accompanying drawing.
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment is briefly described, it should be apparent that, be described below in embodiment be some embodiments of the present invention, for
For those of ordinary skill in the art, without creative efforts, it can also be obtained according to these embodiments
His embodiment.
The great expression of 1 VP3 albumen of embodiment
1, by GPVP3/Bac plants of inoculation Insect cells Sf9s of recombinant baculovirus, 27 DEG C are cultivated 4 days, collect culture, from
The heart takes supernatant to obtain F2 for recombinant baculovirus.
2, above-mentioned F2 is accessed into Insect cells Sf9,27 DEG C of cultures 4 for recombinant baculovirus with the inoculum concentration of MOI=5~10
It, collects culture, and centrifuging and taking supernatant obtains recombination VP3 albumen.
3, it will identify that correct recombinant virus connects poison amount inoculation Sf9 cell mass propgation with MOI=1~10, centrifugation is received
Collect culture solution supernatant, that is, obtains containing a large amount of recombination VP3 albumen.
The purifying of 2 VP3 albumen of embodiment
Take the recombination GPV VP3 albumen supernatant 12000rpm/min centrifugation 30min not inactivated that above-described embodiment 1 is expressed
Afterwards, cell fragment is removed, takes supernatant to be packed into bag filter (molecular weight 8000-12000), polyethylene glycol is used under the conditions of 4 DEG C
6000 4 times of embedding concentrations, are shifted in 30ml centrifuge tube, 30000rpm/min after concentration.4 DEG C of centrifugation 4h, discard supernatant, with a small amount of
PBS hangs tube bottom precipitating, and after 4 DEG C of dissolutions overnight plus cesium chloride is uniformly dissolved rear 10 DEG C of 40000rpm/min centrifugation for 24 hours.With note
Visible band is sucked out in emitter respectively, determines purpose band through SDS-PAGE electrophoresis and identifies purity, egg is quantitatively determined with BCA
Bai Hanliang, and electron-microscope scanning is analyzed, and sees Fig. 1.A small amount of packing, -70 DEG C of preservations.
The preparation of 3 ELISA kit of embodiment
The preparation flow of ELISA kit of the present invention is shown in Fig. 2, specific preparation method the following steps are included:
A. the preparation method of the polyclonal antibody of the anti-goose parvovirus VP3 albumen, includes the following steps:
(1) by the GPV VP3 albumen conventional immunoassay rabbit of aforementioned purifying, when rabbit anteserum ELISA potency reaches 1:10000
When above, rabbit blood is acquired, centrifuging and taking obtains serum.
(2) the anti-GPV VP3 albumen of purifying is made by Protein A column purification in rabbit anteserum made from step (1)
Polyclonal antibody.
(3) 50% neutral glycerine, measurement are added in the polyclonal antibody of the anti-GPV VP3 albumen made from step (2)
After working concentration, -20 DEG C are saved backup.
B. the step of screening positive hybridoma cell with ELISA method:
(1) by the GPV VP3 protein determination protein concentration of purifying, square matrix experiment is done with mouse GPV Positive Sera, really
Determine the best peridium concentration 0.5ug/ml of GPV VP3 albumen.
(2) according to the best peridium concentration 0.5ug/ml of GPV VP3 albumen, after mixing coating with carbonate coating buffer
Closing is stand-by.
(3) 37 DEG C of incubation 1h of hybridoma supernatant are taken, the not immune GPV virus mice serum of setting dilutes 1000 times of conducts
Negative control.1000 times of immune GPV virus mice serum is used as positive control.It is washed after 1h, is incubated for sheep anti-mouse igg ELIAS secondary antibody
(by specification dilution) washing colour developing afterwards.
(4) yin and yang attribute control is set up, and hybridoma supernatant potency arranges in descending order, the hole number for selecting positive readings high.
(5) the GPV allantoic fluid and negative allantoic fluid obtained simultaneously with ultracentrifugation is coated with plank respectively, with above method its
His step is consistent, and in the case where yin and yang attribute is compareed and set up, record can be reacted with GPV totivirus, not reacted with negative allantoic fluid
Hole number.
(6) it records and is reacted with the GPV VP3 albumen of purifying and totivirus, the hole number that do not react with negative allantoic fluid is
Candidate hole.
C. the step of using westernblot method validation positive hybridoma cell:
(1) the GPV VP3 albumen of purifying is subjected to electrophoresis, is transferred on NC film, is cut into item according to swimming lane and is individually positioned in
In ware, carry out stand-by after closing washing.
(2) it is separately added into candidate hybridoma supernatant, is washed away after incubation, sheep anti-mouse igg ELIAS secondary antibody is added, after washing
It is developed the color with sedimentation type TMB.
(3) it under yin and yang attribute establishment condition, is recorded in and occurs the hole number of band at 60KDa for candidate hole, as a result such as Fig. 3 institute
Show.
D. the preparation method of the monoclonal antibody of the anti-goose parvovirus VP3 albumen of horseradish peroxidase (HRP) label,
Include the following steps:
(1) goose parvovirus goose embryo allantoic liquid ultracentrifugation is taken into precipitating, with 1/100 phosphate buffer of original volume
Dissolution precipitating.Balb/c mouse is immunized with after Freund's adjuvant emulsification, when mice serum ELISA potency reaches 1:10000 or more,
Extracting spleen cell and SP2/0 cell are merged with the ratio of 108:1.6 × 107, screen hybridoma with HAT selective medium,
The screening that positive hybridoma cell is carried out with ELISA method and westernblot, by the positive hybridoma cell screened through three
Secondary limiting dilution obtains the hybridoma cell strain of secrete monoclonal antibody and the monoclonal of the anti-GPV VP3 albumen of secretion is anti-
Body.
(2) ascites is prepared in the Balb/c Mice Body for crossing the monoclonal antibody cell infusion sensitization of step (1), ProteinG column is pure
Change, the monoclonal antibody of the anti-GPV VP3 albumen purified, measures protein concentration.
(3) with the sodium periodate oxidizing process of improvement to the monoclonal of the anti-GPV VP3 albumen of the purifying obtained of step (2)
Antibody carries out HRP label, and the monoclonal antibody of the anti-GPV VP3 albumen of horseradish peroxidase (HRP) label is made.
(4) be made the anti-GPV VP3 albumen of horseradish peroxidase (HRP) label monoclonal antibody and it is isometric in
Property glycerol mix, measure working concentration after, -20 DEG C save backup.
E. ELISA Plate hole is uniformly coated with the polyclonal antibody of anti-GPV VP3 albumen, and package amount is the hole 0.01ug-5ug/, packet
The carbonate buffer solution for being 0.05mol/L by buffer, PH9.6 contain 1.59g that is, in 1L solution.
F. confining liquid be BSA that mass concentration is 1-10% or skimmed milk milk still in or combine.
G. in kit other solution preparation:
1. sample diluting liquid: now preparing pH7.2-7.4, the phosphate buffer that concentration is 0.01mol/L, 121 DEG C of 20min
Be added after high pressure sterilization is cooling 0.1-10%BSA and containing 0.01-1%ProClin 300 to get；
2. cleaning solution: the purified water of configuration；
3. zymolyte solution A contains the sodium acetate buffer solution of 0.016% (mass concentration) hydrogen peroxide, preparation weighs second
Sour sodium 10g is dissolved in 1L purified water, is 5.3 with acetic acid tune pH, and adding 540 μ L concentration is 30%H2O2；
4. zymolyte B solution is 3,3',5,5'-tetramethylbenzidine hydrochloride (TMBHCl) solution of 1.7mmol/L,
Its preparation weighs 0.47gTMBHCl and is added in the citrate buffer solution for the 0.01mol/L that 1L pH is 3.0, is completely dissolved；
5. developing solution is obtained with zymolyte B solution and solution A 1:1 are ready-to-use；
6. terminate liquid: taking 54.3ml concentration is 95% concentrated sulfuric acid, adds distilled water to 1000ml, is the H of 1mol/L2SO4It is molten
Liquid to get；
7. antigen standard: antigen standard of the present invention is formulated as diluting the recombination bar of purifying with sample diluting liquid
The goose parvovirus VP3 albumen of the formation virus-like particle (VLPs) of shape expressing viral, until 1 μ g/ml is made；
8. negative controls: 100 times of sample diluting liquid dilutions are formulated without the goose embryo allantoic liquid of goose parvovirus；
9. blank control liquid of the present invention is sample diluting liquid.
Contained VP3 albumen in 4 ELISA kit quantitative detection vaccine antigen of embodiment
(1) resisted to suitable concentration with the capture antibody rabbit-anti GPV VP3 albumen of coating buffer dilution purifying, every hole more
100 μ l, 4 DEG C overnight, washs 3 dryings.
(2) the PBST confining liquid of 1%BSA, every 200 μ l of hole are added in above-mentioned enzyme mark hole, 4 DEG C of closings overnight are washed 3 times and blown
It is dry, add desiccant sealing to be protected from light and is placed in 2-8 DEG C of preservation.
(3) antigen standard of different dilutions and the measuring samples of different dilutions are added in above-mentioned enzyme mark hole, such as
Sample to be checked sample diluting liquid 1:100 is diluted, every hole adds 100 μ L, while positive controls are arranged, negative control group and sky
White control group, wherein positive controls addition stoste, 1:2,1:4,1:8,1:16,1:32,1:64,1:128 are serially diluted anti-
100 μ L negative controls are added in each 100 μ L of primary standard product, negative control group, and 100 μ L sample dilutions are added in blank control group,
2-6 hole of each sample sample-adding parallel with control.37 DEG C of incubation 1h wash 3 dryings.
(4) monoclonal antibody for the anti-GPV VP3 albumen that every hole addition detection antibody-HRP is marked in above-mentioned enzyme mark hole, 37
DEG C be incubated for 1h, wash 3 times drying.
(5) developing solution substrate A liquid and substrate A liquid mix in equal volume, and 100 hole μ l/ of above-mentioned enzyme mark hole, 37 DEG C of colour developing 5- are added
10min.50 hole μ l/ of terminate liquid is added.
(6) ELISA Plate is placed in microplate reader, measures its absorbance value OD in 450nm450nm。
The judgement of quantitative result: using 2 be bottom OD value logarithm as X-axis, using 2 be bottom standard items concentration as Y-axis, obtain line
Linearity curve and equation log2Y=1.6399log2X-2.0273(R2=0.9882, X:OD450nmAverage value, Y: concentration), it will mark
Quasi- product and sample OD value bring the concentration that RelPot 4.0 calculates sample into.
The antigen standard of 1 various concentration of table uses kit test result
Contained VP3 albumen in 5 ELISA kit quantitative detection difference infection multiplicity vaccine antigen of embodiment
It when preparing vaccine antigen, is inoculated with using 3 kinds of difference MOI (0.01,0.1,1), different time VP3 albumen passes through
ELISA quantitative detection.The recombinant baculovirus that GPV VP3 is expressed using 3 kinds of difference MOI (0.01,0.1,1) inoculations, respectively at
Destination protein content after inoculation in 7-11 days sampling quantitative analysis culture supernatants, the results are shown in Table 2.
The different infection multiplicity inoculating two kinds viral protein expression amounts (μ g/ml) of table 2 are analyzed
GPV VP3 albumen the result shows that, is expressed using bioreactor serum free suspension culture by VP3 protein quantification,
Under most suitable MOI (MOI=0.1), expression quantity is up to 113ug/ml.Reach maximum value within 9 days after being inoculated with recombinant baculovirus, i.e.,
It can harvest.
6 specific detection of embodiment
According to detection method as described in example 4, gosling plague infection is detected respectively using kit made from embodiment 3
Allantoic fluid, the allantoic fluid of ND infection, the allantoic fluid of ALV H9 infection, negative allantoic fluid, baculoviral GPV VP3 protein expression
Other protein expression liquid of liquid, baculoviral
After measuring samples are diluted with dilution 1:100, each dilution is added what coating rabbit-anti GPV VP3 albumen resisted more
In ELISA Plate hole, each 3 hole, every 100 μ l of hole.Positive controls, negative control group and blank control group are set simultaneously.Wherein, positive
Property control group each 100 μ L of antigen standard that stoste, 1:2,1:4,1:8,1:16,1:32,1:64,1:128 are serially diluted is added,
100 μ L negative controls are added in negative control group, and 100 μ L sample dilutions are added in blank control group, and each sample and control are flat
3 holes of row sample-adding, testing result are shown in Table 3.
The specific detection result of the kit of the present invention of table 3
The allantoic fluid of gosling plague infection is the positive seen from table 3, and the GPV VP3 albumen of baculovirus expression is the positive；Its
He is feminine gender, it is seen that this kit can specificity differentiation gosling plague, ND, ALV, H9, FAdV 4c, FAdV 8b.
7 sensitivity technique of embodiment
According to detection method described in embodiment 3, the antigen of different dilutions is detected using kit made from embodiment 2
Standard items.
Antigen standard is diluted to stoste, 1:2,1:4,1:8,1:16,1:32,1:64,1:128,1 with sample diluting liquid:
256,1:512 series is added in the mostly anti-ELISA Plate hole of coating rabbit-anti GPV VP3 albumen, each 3 hole, every 100 μ l of hole.Negative control
100 μ L negative controls are added in group, and 100 μ L sample dilutions, each sample sample-adding 3 parallel with control is added in blank control group
Hole.Testing result is shown in Table 4.
The antigen standard testing result of the different dilutions of table 4
By table 4 as it can be seen that the concentration of the lowest detection of kit of the invention to antigen standard is 0.004ug/ml.
8 ELISA kit of embodiment detects different ELD50The gosling plague positive allantoic fluid of/0.2ml
According to detection method as described in example 4, different ELD are detected using kit made from embodiment 350/ 0.2ml's
Gosling plague positive allantoic fluid.Goose embryo positive allantoic fluid is examined to use 3 kinds of differences 102.0ELD50/0.2ml、104.0ELD50/0.2ml、
106.0ELD50/0.2ml.Goose embryo positive allantoic fluid is diluted to stoste, 1:2,1:4,1:8,1:16,1:32,1 with sample diluting liquid:
64,1:128 series is added in the mostly anti-ELISA Plate hole of coating rabbit-anti GPV VP3 albumen, each 3 hole, every 100 μ l of hole.Negative control
100 μ L negative controls are added in group, and 100 μ L sample dilutions, each sample sample-adding 3 parallel with control is added in blank control group
Hole.Testing result is shown in Table 5.
The kit of the present invention of table 5 detects different ELD50The result of/0.2ml goose embryo allantoic liquid
By table 5 as it can be seen that 102.0ELD50/0.2ml、104.0ELD50/0.2ml、106.0ELD50The goose embryo of tri- gradients of/0.2ml
There are extremely significant positive correlations for data measured through the invention for positive allantoic fluid.ELD50/ 0.2ml is higher, and testing result is higher.No
Same ELD50The result surveyed after the doubling dilution of the goose embryo positive allantoic fluid of/0.2ml is variant.To sum up, malicious valence exists
7812ELD50The GPV antigen of/0.2ml or more can measure the positive by kit of the present invention.
The comparative studies of two kinds of goose parvovirus VP3 protein ELISA detection kits of comparative example
1, goose parvovirus VP3 protein ELISA antigen detects the preparation of the sandwich kit of double monoclonal antibodies:
(1) with the anti-GPV VP3 protein monoclonal antibody of capture antibody of coating buffer dilution purifying (with HRP label
The monoclonal antibody clone difference of GPV VP3 albumen) to 0.01 μ g-0.5 μ g/ml of suitable concentration.
(2) preparation of other reagents is with embodiment 3 and step with embodiment 4
2, the comparative experiments of kit of the present invention and double monoclonal antibody detection kits
After measuring samples are diluted with dilution 1:100, anti-more than addition coating rabbit-anti GPV VP3 albumen (or anti-GPV
VP3 protein monoclonal antibody) ELISA Plate hole in, each 3 hole, every 100 μ l of hole.Positive controls, negative control group are set simultaneously
And blank control group.Wherein, stoste is added in positive controls, 1:2,1:4,1:8,1:16,1:32,1:64,1:128 are serially diluted
Each 100 μ L of antigen standard, negative control group be added 100 μ L negative controls, blank control group be added 100 μ L samples dilution
Liquid, 3 holes of each sample sample-adding parallel with control.
The sample detection OD of the more double monoclonal antibody detection kits of kit of the invention as can be seen from Table 6450nmIt is higher, it can
Reliability and sensitivity are stronger, more conducively detection judgement.
The kit of the present invention of table 6 is compared with double monoclonal antibody kit testing results
Mode the above is only the implementation of the present invention is not intended to limit the scope of the invention, all to utilize this
Equivalent structure or equivalent flow shift made by description of the invention and accompanying drawing content, it is relevant to be applied directly or indirectly in other
Technical field is included within the scope of the present invention.
Claims (8)
1. a kind of goose parvovirus VP3 proteantigen ELISA detection kit, which is characterized in that the kit includes: coating
ELISA Plate, confining liquid, sample diluting liquid, antigen standard, the HRP of the polyclonal antibody of goose parvovirus VP3 albumen are marked
Anti- goose parvovirus VP3 monoclonal antibody, cleaning solution, zymolyte solution A, zymolyte B solution and terminate liquid, wherein described
Antigen standard is the goose parvovirus VP3 albumen of the formation virus-like particle (VLPs) of the recombinant baculovirus expression of purifying.
2. detection kit according to claim 1, which is characterized in that the goose parvovirus VP3 albumen it is polyclonal
The package amount of antibody is 0.01 hole μ g-0.5 μ g/.
3. detection kit according to claim 1, which is characterized in that it is 1-10% that the confining liquid, which is selected from mass concentration,
Bovine serum albumin(BSA) (BSA) or mass concentration be 1-10% skimmed milk power it is any or combinations thereof.
4. detection kit according to claim 1, which is characterized in that sample diluting liquid is 0.1-10% by mass concentration
BSA and phosphate buffer composition containing 0.01-1%ProClin 300, wherein the concentration of the phosphate buffer is
0.01mol/L, pH7.2-7.4.
5. detection kit according to claim 1, which is characterized in that the zymolyte solution A contains mass concentration and is
The sodium acetate buffer solution of 0.016% hydrogen peroxide, wherein the concentration of sodium acetate solution is 10g/L, pH 5.3；The enzyme bottom
Object B solution is 3,3 ', the 5,5 '-tetramethyl biphenyl amide hydrochlorides of 1.7mmol/L；The terminate liquid is the H of 1mol/L2SO4
Solution.
6. a kind of method using any kit detection goose parvovirus VP3 proteantigen of claim 1-5, special
Sign is, comprising the following steps:
(1) measuring samples sample diluting liquid is pressed into gradient dilution, is added into more grams of the anti-goose parvovirus VP3 albumen of coating
In the ELISA Plate of grand antibody, every hole adds 100 μ L, while positive controls, negative control group and blank control group, middle-jiao yang, function of the spleen and stomach is arranged
Property control group each 100 μ L of antigen standard that stoste, 1:2,1:4,1:8,1:16,1:32,1:64,1:128 are serially diluted is added,
100 μ L negative controls are added in negative control group, and 100 μ L sample dilutions are added in blank control group, and each sample and control are flat
2-6 hole of row sample-adding；
(2) after the completion of being loaded, after ELISA Plate is set 37 DEG C of incubations 1 hour, cleaning solution board-washing 3-5 times, drying；
(3) the anti-goose parvovirus VP3 monoclonal antibody of 100 μ L HRP label is added in every hole, and 37 DEG C are educated 1 hour, and cleaning solution is washed
Plate 3-5 times, drying；
(4) 100 μ L developing solutions are added in every hole, and after being protected from light colour developing 5-10 minutes, every hole adds 50 μ L terminate liquids, wherein described
Developing solution mixed and be made in equal volume by zymolyte B solution and zymolyte solution A；
(5) ELISA Plate is placed in microplate reader, measures its absorbance value OD in 450nm450nm；
(6) result calculates and determines.
7. according to the method described in claim 6, it is characterized in that, the step (6) includes qualitative results or quantitative result
Determine, wherein the judgement of the qualitative results: Cut off (CO value)=negative control absorbance value OD450nm× 2.1 times, sample
Value=sample absorbance value OD450nm/ CO value, wherein sample value > 1 is the positive；Sample value≤1 is feminine gender；Alternatively, quantitative result
Judgement: sample concentration is calculated with linearity curve and equation.
8. any kit of claim 1-5 is in the application of detection Goose Parvovirus or gosling plague bacterin product.
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