CN206756849U - The indirect ELISA testing kit of Porcine epidemic diarrhea virus N protein and S protein antibody - Google Patents
The indirect ELISA testing kit of Porcine epidemic diarrhea virus N protein and S protein antibody Download PDFInfo
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- CN206756849U CN206756849U CN201720517045.5U CN201720517045U CN206756849U CN 206756849 U CN206756849 U CN 206756849U CN 201720517045 U CN201720517045 U CN 201720517045U CN 206756849 U CN206756849 U CN 206756849U
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The utility model discloses a kind of Porcine epidemic diarrhea virus N protein and the indirect ELISA testing kit of S protein antibody, the kit has a box body, antigen coat reaction plate storage tank is provided with box body, sample dilutes plate storage tank, epidemic diarrhea virus N protein antibody positive comparison liquid hole slot, Porcine epidemic diarrhea virus S protein positive control solution hole slot, Porcine epidemic diarrhea virus N protein and S protein negative controls hole slot, sample diluting liquid hole slot, enzyme conjugates hole slot, concentrated cleaning solution hole slot, TMB nitrite ions, terminate liquid hole slot and operational manual storage tank；Antigen coat reaction plate is divided into detection zone I and detection zone II, and detection zone I and detection zone II are coated with Porcine epidemic diarrhea virus N protein antigen and Porcine epidemic diarrhea virus S protein antigen respectively.This kit can be used for the antidiastole that PEDV wild virus infections and subunit vaccine are immunized and the immune effect for assessing subunit vaccine, have the advantages that high sensitivity, specific good, quick detection batch samples.
Description
Technical field
The utility model belongs to epidemic disease detection technique field for animals, and in particular to a kind of Porcine epidemic diarrhea virus N protein
With the indirect ELISA testing kit of S protein antibody.
Background technology
Pig epidemic diarrhea (Porcine Epidemic Diarrhea, PED) is by Porcine epidemic diarrhea virus
It is a kind of to vomit, suffer from diarrhoea and be dehydrated as cardinal symptom caused by (Porcine Epidemic Diarrhea Virus, PEDV)
Enteric infectious disease.The disease is susceptible to the pig of various age levels, the nurture piglet within especially 7 ages in days, dead after infection
Rate is up to 50%-90%.In recent years, the disease was in rising trend in the incidence of disease in China, the death rate, was caused to pig industry
Heavy economic losses.
PEDV N proteins are made up of 441 amino acid, and molecular weight is about 56kDa.PEDV N proteins and viral genome
RNA is mutually wound virus nucleocapsid, is played an important role in viral RNA building-up process.In addition, N protein is in PEDV
Structural proteins in shared ratio it is maximum, obtain great expression in the cell of infection.Pig is early stage PEDV is infected, in vivo
The high-level antibody of N protein is directed to regard to that can produce.Finally, N protein is very conservative, so N protein is examined in PEDV molecular biology
There is good application prospect in disconnected technology.
For PEDV S proteins to stretch out the 20nm club shape glycoprotein of virion envelope, molecular weight is about 180-220kDa,
About it is made up of 1383 amino acid.According to PEDV and the conserved sequence of other coronavirus S proteins similitude, by PEDV S eggs
Two domains of S1 (1-789aa) and S2 (790-1383aa) are divided into vain, wherein S1 is located at virus surface, and main function is
Identification receptor is simultaneously combined with host cell receptor, and mediates the generation of neutralizing antibody.S2 is mainly responsible for virus envelope and host
The fusion of cell membrane, and viral RNA is imported in host cell, so as to cause the infection of cell.In addition, PEDV S proteins are also
Induce the immunogen protein of host body fluids immune response.Therefore, PEDV S proteins are not only in PEDV diagnostic technique in molecular biology
In also there is good application prospect, and one of be also important albumen of PEDV subunit vaccines research.
With the progress of science and technology, the research using PEDV S proteins as PEDV novel subunit vaccines is also progressively
Promote, after Swinery immunity PEDV S protein subunit vaccines, antibody and wild virus infection are produced in order to distinguish vaccine immunity
Caused antibody (purpose is the purification for instructing pig farm to PEDV and evaluates the immune effect of vaccine), this 2 kinds using different
The coated kit of albumen all can use arrive.But neither one is gone back above existing market can detect for this 2 eggs simultaneously
White antibody assay kit, and when using different kit separate detections respectively, error is larger (to be separated and operates twice, just
2 errors can be produced, and are detected simultaneously in same kit, the error that separate detection is brought will be reduced).
Utility model content
The purpose of this utility model be to provide for it is a kind of quick, effectively while detect Porcine epidemic diarrhea virus N protein
With the indirect ELISA testing kit of S protein antibody, screened for experimental animal, the evaluation of PEDV subunit vaccine immune effects
And PEDV purifications in pig farm provide the instrument of new quickness and high efficiency；The error brought when further reducing separate detection simultaneously.
The utility model provides the indirect ELISA detection examination of a kind of Porcine epidemic diarrhea virus N protein and S protein antibody
Agent box, the kit have a box body (1), and antigen coat reaction plate storage tank (2), sample dilution plate are provided with box body (1)
Storage tank (3), epidemic diarrhea virus N protein antibody positive comparison liquid hole slot (4), Porcine epidemic diarrhea virus S protein are positive
Comparison liquid hole slot (5), Porcine epidemic diarrhea virus N protein and S protein negative controls hole slot (6), sample diluting liquid hole slot
(7), enzyme conjugates hole slot (8), concentrated cleaning solution hole slot (9), TMB nitrite ions (10), terminate liquid hole slot (11) and operating instruction
Book storage tank (12)；Antigen coat reaction plate, sample dilution plate storage tank (3) are placed in antigen coat reaction plate storage tank (2)
Interior placement sample dilutes plate, and (4-11) places the reagent bottle equipped with corresponding solution, operational manual storage tank (12) in each hole slot
Interior placement operation specification；Described antigen coat reaction plate is divided into detection zone I and detection zone II, detection zone I and detection zone II
Porcine epidemic diarrhea virus N protein antigen and Porcine epidemic diarrhea virus S protein antigen are coated with respectively.
In the technical solution of the utility model, it is preferable that the specification of described antigen coat reaction plate is 96 holes, Mei Geshi
Agent box shares 5 blocks of coating plates, and every block of coating plate is equally divided into detection zone I and detection zone II from left to right.
In the technical solution of the utility model, it is preferable that the specification of described sample dilution plate is 96 holes, each kit
Share 5 blocks of sample dilution plates.
In the technical solution of the utility model, it is preferable that described antigen coat reaction plate and sample dilution plate be all can
With 96 orifice plates of dismounting, every block of plate can be dismantled as 12 rows, often arrange 8 holes.
In the technical solution of the utility model, it is preferable that described Porcine epidemic diarrhea virus N protein antibody positive control
Liquid and Porcine epidemic diarrhea virus S protein antibody positive comparison liquid are respectively popular with the pig of 100 times of dilutions of sample diluting liquid
The pig positive serum of diarrhea virus N protein and S protein antibody, dispensed respectively by 2mL/ bottles.
In the technical solution of the utility model, it is preferable that described Porcine epidemic diarrhea virus N protein and S protein antibody
Negative controls are the pig for not containing Porcine epidemic diarrhea virus N protein and S protein antibody with 100 times of dilutions of sample diluting liquid
Negative serum, dispensed by 4ml/ bottles.
In the technical solution of the utility model, it is preferable that described sample diluting liquid is 1%BSA solution, according to 250ml/
Bottle packing.
In the technical solution of the utility model, it is preferable that described enzyme conjugates is with 5,000 times of dilution of sample diluting liquid
Commercialization goat-anti pig IgG ELIAS secondary antibody, by 60mL/ bottles dispense.
In the technical solution of the utility model, it is preferable that described concentrated cleaning solution is 25 × PBST solution, according to
150ml/ bottles dispense.
In the technical solution of the utility model, it is preferable that described TMB nitrite ions are the display liquid of commercialization, by 60mL/
Bottle packing, described terminate liquid is 2M H2SO4Solution, dispensed by 60ml/ bottles.
The utility model uses enzyme linked immunological indirect method, and Porcine epidemic diarrhea virus N protein and S protein antigen are wrapped respectively
By in two detection zones of antigen coated microplate, it can be reacted respectively with the specific antibody in measuring samples, form antigen-antibody
Compound, adds the enzyme conjugates of goat-anti pig IgG secondary antibody, forms " the anti-enzyme of Ag-Ab-two " compound, finally by adding
Enter TMB and show that liquid is developed the color, and read absorbance value (OD450).
Experiment effectiveness judges：Detection could effectively only when meeting following condition：Positive control OD values-negative control
OD values ＞ 0.5；Negative control OD value ＜ 0.15.
As a result calculate and judge：S/P=(sample OD values-negative control OD value)/(positive control OD values-negative control OD
Value)；S/P value >=0.35, it is determined as PEDV antibody positives；S/P values ＜ 0.35, is determined as PEDV negative antibodies.
Detection kit of the present utility model has the following advantages that：(1) the utility model uses the restructuring egg of gene expression
It is white that security is good, is polluted without foreign protein for detection antigen, and with specificity is good, high sensitivity, can mass detection etc. it is excellent
Point；(2) kit of the present utility model can detect Porcine epidemic diarrhea virus N protein and S eggs simultaneously on same plank
Bai Kangti, can further distinguish whether have wild virus infection simultaneously in the immune caused antibody of detection subunit vaccine, therefore this examination
Agent box can be applied to antidiastole, the immune effect of assessment PEDV subunit vaccines that PEDV wild virus infections and subunit vaccine are immunized
Fruit and the purification for instructing pig farm progress PEDV；(3) antigen coated microplate of this kit is demountable, when sample is less
When, temporarily no coating plate can be pulled down, subsequently to use, can further reduce use cost and facilitate terminal to use
The use at family；(4) this kit provide sample dilution plate, can on 96 orifice plates direct dilute sample, so sample-adding when
Directly the sample diluted can be added in the corresponding hole of ELISA Plate using multichannel pipettor, when can so save sample-adding
Time, the time consistency being incubated when ensureing detection as far as possible, further reduce error caused by being loaded and being incubated.
Brief description of the drawings
Fig. 1 is the cross section sectional view of this kit：Wherein 1 is box body, 2 is antigen coat reaction plate storage tank, 3 is sample
Product dilution plate storage tank, 4 be epidemic diarrhea virus N protein antibody positive comparison liquid hole slot, 5 be Porcine epidemic diarrhea virus S
Protein positive comparison liquid hole slot, 6 be Porcine epidemic diarrhea virus N protein and S protein negative controls hole slot, 7 be sample dilution
Fluid apertures groove, 8 be enzyme conjugates hole slot, 9 be concentrated cleaning solution hole slot, 10 be TMB nitrite ions, 11 be terminate liquid hole slot, 12 be behaviour
Explain book storage tank.
Fig. 2 is the structural representation that this kit antigen is coated with reaction plate；Wherein, I areas coating Porcine epidemic diarrhea virus N
Proteantigen, II areas coating Porcine epidemic diarrhea virus S protein antigen.
Embodiment
The utility model is described further below with reference to drawings and examples, embodiment of the present utility model is only used
In explanation the technical solution of the utility model, and non-limiting the utility model.
The reagent and consumptive material that the utility model uses are commercially available prod, wherein：
ELISA Plate and sample dilution plate are purchased from NUNC companies；
Purchased from sigma companies；
Enzyme conjugates is purchased from Earthox companies of the U.S.；
TMB shows that liquid is purchased from Beijing Suo Laibao Science and Technology Ltd.
Embodiment 1：Kit forms
The indirect ELISA testing kit of a kind of Porcine epidemic diarrhea virus N protein and S protein antibody, its cross section are cutd open
For view as shown in figure 1, the kit has a box body (1), box body (1) is interior to be provided with antigen coat reaction plate storage tank (2), sample
Dilute plate storage tank (3), epidemic diarrhea virus N protein antibody positive comparison liquid hole slot (4), Porcine epidemic diarrhea virus S eggs
White positive control solution hole slot (5), Porcine epidemic diarrhea virus N protein and S protein negative controls hole slot (6), sample diluting liquid
Hole slot (7), enzyme conjugates hole slot (8), concentrated cleaning solution hole slot (9), TMB nitrite ions (10), terminate liquid hole slot (11) and operation
Specification storage tank (12)；Antigen coat reaction plate, sample dilution plate storage tank are placed in antigen coat reaction plate storage tank (2)
(3) sample dilution plate is placed in, (4-11) places the reagent bottle equipped with corresponding solution, operational manual storage tank in each hole slot
(12) interior placement operation specification；Described antigen coat reaction plate is divided into detection zone I and detection zone II, detection zone I and detection
Area II is coated with Porcine epidemic diarrhea virus N protein antigen and Porcine epidemic diarrhea virus S protein antigen respectively.
Wherein, the specification of antigen coat reaction plate is 96 holes, and each kit shares 5 blocks of coating plates, every block of coating plate from
Left-to-right is equally divided into detection zone I and detection zone II, specific as shown in Figure 2.The specification of sample dilution plate is 96 holes, each reagent
Box shares 5 blocks of sample dilution plates.Antigen coat reaction plate and sample dilution plate are all demountable 96 orifice plates, and every block of plate all may be used
To dismantle as 12 rows, 8 holes are often arranged.
Embodiment 2：It is prepared by kit
1. prepared by antigen coat reaction plate：Detection zone I and detection zone II is uniformly coated with pig epidemic in ELISA Plate hole
Diarrhea virus N protein and S protein antigen, package amount are 0.5-2 μ g/ holes, the carbonic acid that coating buffer solution is 0.05mol/L pH 9.6
Salt buffer (i.e. Na containing 1.59g in 1L carbonate buffer solutions2CO3With 2.93g NaHCO3), after 2~8 DEG C are stayed overnight, use PBST
Board-washing 3 times；Confining liquid is added after board-washing, confining liquid is the BSA that mass concentration is 1-5% or any or its group of skim milk
Close, 200 μ L/ holes, 37 DEG C of closing 2h, be sealed after drying.
2. sample diluting liquid：Using 1 × PBS as solvent, 1%BSA solution is prepared, is addedMake
It is 0.1% with final concentration (v: v), is dispensed according to 250ml/ bottles.
3. enzyme conjugates：Commercialization goat-anti pig IgG ELIAS secondary antibody is formed according to 1: 5,000 dilution with sample diluting liquid,
Dispensed by 60mL/ bottles.
4. concentrated cleaning solution：For 25 × PBST solution, wherein Tween-20 concentration is 1.25% (v/v), is addedThe use of final concentration (v: v) is 0.1%, is dispensed according to 150ml/ bottles.
5.TMB nitrite ions：Liquid is shown for the TMB of commercialization, is dispensed by 60mL/ bottles.
6. terminate liquid：For 2M H2SO4Solution, the 21.76ml concentrated sulfuric acid is added during preparation in 178.26ml ultra-pure water,
Mix and produce, dispensed by 60ml/ bottles.
7. positive control solution：Respectively with the Porcine epidemic diarrhea virus N protein and S eggs of 100 times of dilutions of sample diluting liquid
Bai Kangti pig positive serum, dispensed respectively by 2mL/ bottles.
8. negative controls：Porcine epidemic diarrhea virus N protein and S eggs are not contained with 100 times of dilutions of sample diluting liquid
Bai Kangti pig negative serum, dispensed by 4ml/ bottles.
9. assembling：By each kit with 5 pieces of antigen coat reaction plate, 5 pieces of plate of sample dilution, Porcine Epidemic Diarrhea
Malicious 1 bottle of N protein antibody positive comparison liquid, 1 bottle of Porcine epidemic diarrhea virus S protein positive control solution, pig Porcine Epidemic Diarrhea
Malicious 1 bottle of N protein and S protein antibody negative controls liquid, 1 bottle of sample diluting liquid, 1 bottle of enzyme conjugates, 1 bottle of concentrated cleaning solution, TMB show
1 part of 1 bottle of color liquid, 1 bottle of terminate liquid and operational manual progress mounted box.
Embodiment 3：Kit test method
1. with sample diluting liquid, 100 times of the dilution in sample dilutes plate (such as takes 297 μ L samples dilutions again by measuring samples
Add 3 μ L measuring samples), detection zone I and detection zone II in antigen coat reaction plate is separately added into, adds 100 μ L per hole, together
When positive controls, negative control group are set, wherein, positive controls add corresponding 100 μ L positive control solutions, negative control
Group adds 100 μ L negative controls, 2 holes of each sample sample-adding parallel with control；
2. after the completion of sample-adding, after antigen coat reaction plate is put into 37 DEG C of incubation 1h, cleaning solution is (in advance by 25 × thickening and washing
Liquid is diluted to 1 ×, as cleaning solution, take 25 × concentrated cleaning solutions of 10ml to be added in 240ml ultra-pure waters during dilution, mix and produce)
Board-washing 3-5 times, dry；
3. adding 100 μ L enzyme conjugates per hole, 37 DEG C of incubation 0.5h, cleaning solution board-washing 3-5 times, dry；
4. adding 100 μ L TMB nitrite ions per hole, room temperature lucifuge colour developing 10-15min, 100 μ L terminate liquids are added per hole；
5. antigen coat reaction plate is placed in ELIASA, its absorbance value OD450 is determined in 450nm；
6. interpretation of result：
Experiment effectiveness judges：Detection could effectively only when meeting following condition：Positive control OD values-negative control
OD values ＞ 0.5；Negative control OD value ＜ 0.15.
As a result calculate and judge：S/P=(sample OD values-negative control OD value)/(positive control OD values-negative control OD
Value)；S/P value >=0.35, it is determined as PEDV antibody positives；S/P values ＜ 0.35, is determined as PEDV negative antibodies.
The utility model is illustrated by above embodiment, it is understood, however, that the utility model and unlimited
In particular example as described herein and embodiment.Purpose herein comprising these particular examples and embodiment is to help
Those of skill in the art are helped to put into practice the utility model.Any those of skill in the art are easy to do not departing from this practicality
It is further improved in the case of new spirit and scope and perfect, therefore the utility model is only weighed by the utility model
The limitation of content and scope that profit requires, its intention cover all the utility model for being included in and being limited by appendix claim
Alternative and equivalent in spirit and scope.
Claims (10)
- A kind of 1. indirect ELISA testing kit of Porcine epidemic diarrhea virus N protein and S protein antibody, it is characterised in that：Institute Stating kit has a box body (1), and antigen coat reaction plate storage tank (2), sample dilution plate storage tank are provided with box body (1) (3), epidemic diarrhea virus N protein antibody positive comparison liquid hole slot (4), Porcine epidemic diarrhea virus S protein positive control solution Hole slot (5), Porcine epidemic diarrhea virus N protein and S protein negative controls hole slot (6), sample diluting liquid hole slot (7), enzyme knot Compound hole slot (8), concentrated cleaning solution hole slot (9), TMB nitrite ions (10), terminate liquid hole slot (11) and operational manual storage tank (12)；Antigen coat reaction plate is placed in antigen coat reaction plate storage tank (2), sample is placed in sample dilution plate storage tank (3) Product dilute plate, and (4-11) places the reagent bottle equipped with corresponding solution in each hole slot, and behaviour is placed in operational manual storage tank (12) Explain book；Described antigen coat reaction plate is divided into detection zone I and detection zone II, and detection zone I and detection zone II are coated with respectively Porcine epidemic diarrhea virus N protein antigen and Porcine epidemic diarrhea virus S protein antigen.
- 2. kit according to claim 1, it is characterised in that：The specification of described antigen coat reaction plate is 96 holes, Each kit shares 5 blocks of coating plates, and every block of coating plate is equally divided into detection zone I and detection zone II from left to right.
- 3. kit according to claim 1, it is characterised in that：The specification of described sample dilution plate is 96 holes, each Kit shares 5 blocks of sample dilution plates.
- 4. according to the kit described in claim 1-3 any claims, it is characterised in that described antigen coat reaction plate All it is demountable 96 orifice plate with sample dilution plate, every block of plate can be dismantled as 12 rows, often arrange 8 holes.
- 5. kit according to claim 1, it is characterised in that：Described Porcine epidemic diarrhea virus N protein antibody sun Property comparison liquid and Porcine epidemic diarrhea virus S protein antibody positive comparison liquid be respectively pigs with the dilution of 100 times of sample diluting liquid The pig positive serum of epidemic diarrhea virus N protein and S protein antibody, dispensed respectively by 2mL/ bottles.
- 6. kit according to claim 1, it is characterised in that：Described Porcine epidemic diarrhea virus N protein and S protein Antibody negative controls liquid is not contain Porcine epidemic diarrhea virus N protein and S protein antibody with 100 times of dilutions of sample diluting liquid Pig negative serum, by 4ml/ bottles dispense.
- 7. kit according to claim 1, it is characterised in that：Described sample diluting liquid is 1%BSA solution, according to 250ml/ bottles dispense.
- 8. kit according to claim 1, it is characterised in that：Described enzyme conjugates is with sample diluting liquid 5,000 The commercialization goat-anti pig IgG ELIAS secondary antibody of dilution again, dispensed by 60mL/ bottles.
- 9. kit according to claim 1, it is characterised in that：Described concentrated cleaning solution is 25 × PBST solution, is pressed Dispensed according to 150ml/ bottles.
- 10. kit according to claim 1, it is characterised in that：Described TMB nitrite ions are the display liquid of commercialization, Dispensed by 60mL/ bottles, described terminate liquid is 2M H2SO4Solution, dispensed by 60ml/ bottles.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201720517045.5U CN206756849U (en) | 2017-05-05 | 2017-05-05 | The indirect ELISA testing kit of Porcine epidemic diarrhea virus N protein and S protein antibody |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201720517045.5U CN206756849U (en) | 2017-05-05 | 2017-05-05 | The indirect ELISA testing kit of Porcine epidemic diarrhea virus N protein and S protein antibody |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109884314A (en) * | 2019-02-26 | 2019-06-14 | 广西壮族自治区兽医研究所 | Porcine epidemic diarrhea virus S2 albumen indirect ELISA antibody detection method and its kit |
| CN111474346A (en) * | 2020-04-15 | 2020-07-31 | 浙江理工大学绍兴生物医药研究院有限公司 | Pig epidemic diarrhea virus IgA and IgG antibody detection kit and preparation method and application thereof |
| CN111999496A (en) * | 2020-08-20 | 2020-11-27 | 必欧瀚生物技术（合肥）有限公司 | SARS-CoV-2 antigen-antibody combined detection kit and its preparation method |
| CN113238048A (en) * | 2021-05-11 | 2021-08-10 | 上海真测生物科技有限公司 | Diagnostic marker and application thereof in distinguishing new coronavirus infection and new coronavirus inactivated vaccination |
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