CN105675866B - For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit - Google Patents
For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit Download PDFInfo
- Publication number
- CN105675866B CN105675866B CN201610212417.3A CN201610212417A CN105675866B CN 105675866 B CN105675866 B CN 105675866B CN 201610212417 A CN201610212417 A CN 201610212417A CN 105675866 B CN105675866 B CN 105675866B
- Authority
- CN
- China
- Prior art keywords
- kit
- liquid
- serum
- antibody
- shaped
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Virology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Peptides Or Proteins (AREA)
Abstract
A kind of solid phase for being used to detect O-shaped FMDV antibody blocks ELISA kit, contain ELISA Plate, cleaning solution, sample diluting liquid, rabbit anti-serum, ELIAS secondary antibody, substrate A liquid, substrate B liquid, terminate liquid, positive control serum, negative control sera in the kit, wherein the ELISA Plate is coated with O-shaped FMDV gene engineering antigens.Kit of the present invention uses solid phase blocked method, with traditional indirect ELISA reagent kit ratio, reduces non-specific antibody interference, testing result is more accurate, and does not differentiate between sample Hosts.The how anti-progress kit production of albumen and serum that kit of the present invention is prepared using technique for gene engineering, simple and safe, cost is low, can be widely applied to O-shaped FMD immune effect of vaccine monitoring and epidemic monitoring.
Description
Technical field
It is more particularly to a kind of to can be used for detecting O-shaped FMDV antibody the present invention relates to a kind of kit of detection FMDV antibody
Solid phase block ELISA kit, belong to biological products detection technique field.
Background technology
Aftosa (foot-and-mouth disease, FMD) is by foot and mouth disease virus (foot-and-mouth
Disease virus, FMDV) caused by acute, highly contagious disease, artiodactyl is mainly encroached on, occasionally in people and its
Its animal.OIE (Office International des Epizooties, OIE) is classified as legal
The infectious disease declared, China is by its class animal epidemic of row one.FMDV has 7 serotypes, including O, A, C, Asia I in the whole world
Without cross immunity phenomenon between (Asia l), South Africa I (SAT 1), South Africa II (SAT 2) and South Africa III (SAT 3), serotype.
The current popular strains of China FMDV are mainly O-shaped and A types, wherein O-shaped FMDV genotype is numerous, it is popular extensive, seriously endanger poultry
Herd aquaculture development.
China takes FMD mandatory immunization strategy, and immune effect of vaccine turns into the key point of influence FMD prevention and control.Epidemic disease
The monitoring of seedling immune effect mainly has two methods, indirect hemagglutination assay (IHA) and EUSA
(ELISA).IHA is simple and easy to apply, but its result judges more subjective, and is influenceed larger by antigen hemagglutinating antigen, in the world no longer
Use.ELISA method operation automation is strong, reduces interference caused by subjective factors, it is adaptable to the antibody detection of a large amount of clinical samples.Often
Include VP1 structural proteins antibody indirect ELISA and LPB-ELISA with ELISA method.VP1 protein antibodies indirect ELISAs are
Serum specific antibody is captured using FMDV VP1 albumen as envelope antigen, serum antibody is then recognized by ELIAS secondary antibody, most
Signal amplification is carried out by substrate colour developing.Indirect ELISA is easy to operate, but this method has species specificity, clinically needs
For different hosts reagent preparation box, using cumbersome.In addition, indirect ELISA is influenceed larger by non-specific factors, and can only
Detect a kind of serum antibody, such as IgG.
LPB-ELISA carries out external neutralization test using totivirus inactivation antigen with serum antibody, then by double
The superfluous antigen of antibody sandwich capture, most carries out substrate colour developing through ELIAS secondary antibody afterwards and amplifies with signal.LPB-ELISA is
The standard method for the detection FMDV antibody that OIE recommends, is worldwide widely used.But this method also has shortcoming：(1) make
Totivirus inactivation antigen is used, is related to Virus culture, inactivation with concentrating and purifying, to working condition, producers' quality and produced
Range request is stricter, while there is bio-safety risk；(2) totivirus antigen prepares complex, is related to cell culture
And virus breeding, the production cycle is long, and yield is also restricted；(3) totivirus antigen production need to use P3 laboratories, exceed the speed limit from
The high end instrument facility such as scheming, is unfavorable for popularization and application.
Made constant progress with immunologic, subunit vaccine, polypeptide vaccine based on FMDV neutralizing epitopes etc. are new
Vaccine will progressively substitute traditional inactivated vaccine by higher purity and immune precision.At the same time, built for FMDV antibody
Immune effect monitoring of vertical safe, sensitive, the special detection method to vaccine is significant.
The content of the invention
ELISA kit is blocked it is an object of the invention to provide a kind of indirect solid phase for being used to detect O-shaped FMDV antibody,
The virus protein expressed by the use of technique for gene engineering can be detected as envelope antigen for O-shaped FMDV specific antibodies, and
Sample Hosts are not differentiated between.
The Cleaning Principle of kit of the present invention be sample moderate resistance FMDV antibody combined with envelope antigen cause rabbit anti-serum with
The binding capacity of antigen is reduced, and is formed " envelope antigen-rabbit anti-serum-enzyme labelled antibody " compound, is eventually adding substrate and is developed the color
Reaction, the colour developing depth is inversely proportional with antibody content in sample.
Contain ELISA Plate, cleaning solution, sample diluting liquid, rabbit anti-serum, ELIAS secondary antibody, substrate A in the kit of the present invention
Liquid, substrate B liquid, terminate liquid, positive control serum, negative control sera, wherein the ELISA Plate is coated with O-shaped FMDV genes work
Cheng Kangyuan.
Specifically, wherein the process that the ELISA Plate is coated with O-shaped FMDV gene engineering antigens is：Will be anti-with coating buffer solution
Original dilution, ELISA Plate is added with the amount in 100uL/ holes, and 4 DEG C of coating 12h are washed 3 times with cleaning solution；Add confining liquid, 200uL/
Hole, 4 DEG C of closing 12h；Deblocking liquid is got rid of, board-washing 3 times is placed in 37 DEG C of dryings, sealed with aluminum foil under vacuum.
Under preferable case, the carbonate buffer solution that the coating buffer solution is pH9.6.
Under preferable case, the confining liquid is the PBS containing 10% tryptone.
In a preferred embodiment of the invention, the ELIAS secondary antibody uses the goat anti-rabbit igg antibody that HRP is marked.
In one particular embodiment of the present invention, wherein the rabbit anti-serum is exempted from by O-shaped FMDV gene engineering antigens
It is prepared by epidemic disease rabbit.Serum antibody carries out preliminary purification through caprylic acid-ammonium and G-protein post method, then with O-shaped FMDV genes work
Cheng Kangyuan and unlabelled antigen carry out affinitive layer purification, eliminate non-specific antibody, and antibody purity is high.With monoclonal antibody phase
Simple, cost is prepared than, the serum antibody low, with multiple epitope binding sites, with envelope antigen binding ability more
By force.
The positive effect of the present invention is：
1st, envelope antigen used in kit of the present invention is gene engineering expression, is not related to Virus culture.Compared to tradition
LPB-ELISA kit, its production is good with biological safety during use.
2nd, kit of the present invention uses solid phase blocked method, with traditional indirect ELISA reagent kit ratio, reduces antigen non-specific
The interference of property antibody, testing result is more accurate.
3rd, kit of the present invention avoids the ELIAS secondary antibody using sample Hosts, with traditional indirect ELISA reagent kit
Than, do not limited by the Hosts of sample, it is easy to use available for the detection of many animals blood serum sample.
4th, the how anti-progress kit production of albumen and serum that kit of the present invention is prepared using technique for gene engineering, operation
Simple and safe, cost is low, is beneficial to popularization and application.
Embodiment
Blocked according to the solid phase for being used to detect O-shaped FMDV antibody of the present invention in ELISA kit, kit provided with washing
Liquid, sample diluting liquid, ELIAS secondary antibody, substrate A liquid, substrate B liquid, terminate liquid, shrouding film, in addition to the control of O-shaped FMDV yin and yang attributes
Serum, has been coated with the ELISA Plate of O-shaped FMDV antigen proteins, and the rabbit anti-serum that can be specifically bound with envelope antigen.Its
Middle rabbit anti-serum has very high binding activity with envelope antigen, is prepared by O-shaped FMDV antigen proteins immune rabbit.
Lower mask body introduce the source (or preparation process) of each reagent in kit of the present invention, the test philosophy of kit and
Test process.
The source of main raw material(s) in following embodiments is：HRP marks goat anti-rabbit igg and O-shaped FMDV genetic engineerings anti-
Original is purchased from the logical experiment material center difficult to understand of Luoyang one hundred.O-shaped FMDV LPB-ELISAs kit is purchased from Chinese Academy of Agricultural Sciences Lanzhou
Veterinary institute.Other reagents and material are derived from commercial sources.Described experimental method, unless otherwise noted, be
Normal experiment method.
First, the source of each reagent and preparation process in kit
The preparation of 1.1 ELISA Plates
(1) the coating process of ELISA Plate
The ELISA Plate be the O-shaped FMDV gene engineering antigens albumen expressed using technique for gene engineering as envelope antigen,
Antigen is diluted with pH9.6 carbonate buffer solution, ELISA Plate is added with the amount in 100uL/ holes, 4 DEG C of coating 12h, with washing
Liquid is washed to wash 3 times.Add confining liquid, 200uL/ holes, 4 DEG C of closing 12h.Deblocking liquid is got rid of, board-washing 3 times is placed in 37 DEG C of dryings, used
Aluminum foil under vacuum is sealed.
(2) determination of envelope antigen best effort concentration
The optimal coating concentration of antigen protein is determined using square formation titration.It is anti-with pH9.6 carbonate buffer solution dilution
Former albumen, concentration is respectively 1ug/mL, 0.5ug/mL, 0.25ug/mL, 0.125ug/mL, 0.0625ug/mL, 0.03125ug/
ML, laterally adds ELISA Plate, 4 DEG C of coating 12h.The tryptone for plus 10%, 200uL/ holes, 37 DEG C of closing 2h.Yin and yang attribute rabbit-anti
Serum does doubling dilution 1: 100,1: 200,1: 400,1: 800,1: 1600,1: 3200,1: 6400,1: 12800 simultaneously, and longitudinal direction adds
Enter ELISA Plate, composition square formation carries out indirect ELISA.Add terminate liquid 50uL per hole, determine OD450nm values.Selection positive OD values connect
Nearly 1.0, antigen coat concentration and antibody dilution when positive OD values/feminine gender OD values (P/N values) are maximum are best effort concentration.
The antigen best effort concentration of determination is 0.0625ug/mL.
(3) determination of confining liquid and off-period
With the most suitable working concentration coated elisa plate of fixed antigen, respectively with the PBS containing 3% gelatin, containing 10% degreasing
The PBS of milk powder, the PBS containing 10% tryptone are used as confining liquid.37 DEG C close off 30min, 1h, 2h and 3h.Plus substrate is aobvious
Color, reads OD450nm values.Selection positive OD values are used as most suitable bar close to confining liquid during 1.0, P/N values maximum and off-period
Part.PBS of the selection containing 10% tryptone is as optimal confining liquid, and off-period is 2h.
The preparation of 1.2 ELIAS secondary antibodies
Tris 2.42g, enzyme stabilizers 0.8g, sodium chloride 8.5g, red 2g, NBCS 100ml, Proclin
300 0.5ml, triton x-100 1.5ml, plus distilled water 850ml, are carefully added into hydrochloric acid 1.65ml, fully mix, and adjust pH
Value 7.0, is settled to 1000ml, adds the goat anti-rabbit igg antibody 0.2ml of HRP marks, stirs, with 0.22 μm of degerming filter
Device is filtered.It is aseptic subpackaged.
The preparation of 1.3 rabbit anti-serums
From the adult large ear rabbit of health, blood is taken to collect non-immune serum as control before immune.By O-shaped FMDV bases
Emulsified because engineering antigen and complete Freund's adjuvant are mixed, carry out the subcutaneous multi-point injection of rabbit back, every μ g of rabbit 500.Every 2 weeks
Booster immunization 1 time, uses incomplete Freund's adjuvant, every μ g of rabbit 250 during booster immunization.Three exempt from 14 days afterwards, and blood sampling is imitated
Valency is detected.Potency reaches 1: 104When, arteria carotis sacrificed by exsanguination animal separates serum.Serum uses caprylic acid-ammonium and G eggs
Bai Zhufa carries out preliminary purification, then carries out affinity chromatography with O-shaped FMDV gene engineering antigens and unlabelled antigen, to go unless special
Heterogenetic antibody.Serum puts -20 DEG C of preservations after purification.
The preparation of 1.4 negative control seras
From 5 week old sodium selenites, ELSIA kits are blocked to be accredited as O-shaped FMDV negative antibodies through O-shaped FMDV liquid phases
Pig, O-shaped FMDV antigen negatives pig is accredited as through PCR method.Arteria carotis sacrificed by exsanguination animal, separates serum, with octanoic acid-ammonium sulfate
The precipitation method and G post affinity chromatography purified blood serums.The serum of purifying is made into 10 times of dilutions with negative control sera dilution, fully
Mix, it is degerming with 0.22 μm of membrane filtration, as negative control sera, put -20 DEG C of preservations.
The preparation of 1.5 positive control serums
From 5 week old sodium selenites, O-shaped FMDV negative antibodies are accredited as through O-shaped FMDV LPB-ELISAs kit
Pig, O-shaped FMDV antigen negatives pig is accredited as through PCR method.O-shaped FMDV inactivated vaccines are immunized, with O-shaped FMDV LPB-ELISAs
Kit carries out titration.When potency reaches 1: 512, arteria carotis sacrificed by exsanguination animal, separating and purifying serum.Use positive control
The serum of purifying is made 10 times of dilutions by serum dilution, is fully mixed, degerming with 0.22 μm of membrane filtration, is used as positive control blood
Clearly, -20 DEG C of preservations are put.
The preparation of 1.6 sample diluting liquids
Sodium dihydrogen phosphate 1.28g, disodium hydrogen phosphate 0.41g, sodium chloride 20.09g, indicator 0.2g, NBCS
The 0.5ml of 50ml, Proclin 300, plus distilled water 950ml, are fully mixed, and are adjusted pH value 6.0, are settled to 1000ml.Then divide
Dress.
The preparation of 1.7 cleaning solutions
Potassium dihydrogen phosphate 4g, disodium hydrogen phosphate 58g, the 0.5ml of sodium chloride 160g, Proclin 300, Tween-20
10ml, plus distilled water are settled to 1000ml.Then dispense.
The preparation of 1.8 substrate A liquid and substrate B liquid
Substrate A liquid：Citric acid 2.10g, crystallizes sodium acetate 12.25g, urea peroxide 0.6g, plus distilled water 950ml, fully
Mix, adjust pH value 5.0, plus distilled water is settled to 1000ml.Packing.
Substrate B liquid：Citric acid 2.10g, EDTA 0.3g, TMB-HCl 0.6g, plus distilled water 950ml, are fully mixed, and are adjusted
PH value 3.0 is saved, plus distilled water is settled to 1000ml.Packing.
The preparation of 1.9 terminate liquids
Distilled water 850ml is taken, sulfuric acid 108.5ml is carefully added into, dissolving is complete, and 1000ml is settled to after cooling.Packing.
2nd, test process and principle
2.1 test process
Step 1：Testing sample is diluted, ELISA Plate, 37 DEG C of incubation 30min are added with the dosage in 100uL/ holes.Dry, board-washing
3 times, 3min/ times.
Step 2：Dilute rabbit anti-serum, additive capacity 100uL/ holes, 37 DEG C of incubation 30min.
Step 3：Dry, board-washing, plus ELIAS secondary antibody, 37 DEG C of incubation 30min.
Step 4：Dry, board-washing, add bottom each 50uL of liquid A, B, 37 DEG C of colour developing 10min.
Step 5：Add terminate liquid 50uL per hole, determine OD450nm values.
2.2 solid phases block the determination of ELISA optimum reaction conditionses
Carry out indirect ELISA with the most suitable working concentration of fixed antigen, using square formation titration determine rabbit anti-serum and
The most suitable working concentration of ELIAS secondary antibody.After the completion of closing, add gradient dilution rabbit anti-serum (1: 6000,1: 8000,1:
10000th, 1: 12000,16000,18000,20000,24000) and HRP ELIAS secondary antibodies (1: 1000,1: 2000,1: 4000,1:
8000th, 1: 16000,1: 32000,1: 64000,1: 128000), developed the color after the completion of 37 DEG C of incubations, read OD450nm values.Selection
Positive OD values are used as most suitable working concentration close to the rabbit anti-serum and HRP ELIAS secondary antibodies dilution factor during 1.0, P/N values maximum.It is determined that
Rabbit anti-serum best effort concentration is 16000 times of dilutions, and HRP ELIAS secondary antibodies are 8000 times of dilutions.
2.3 solid phases block the determination of ELISA criterion
100 parts are taken to identify positive blood serum sample through O-shaped FMDV LPB-ELISAs antibody assay kit, by 1: 32
Indirect ELISA detections are carried out after dilution, OD450 values are read.Regulation is with the average OD450 values of 100 parts of serum plus 3 times of marks
Quasi- error (SD) calculates inhibiting rate as the critical value of yin and yang attribute.As a result show, it is the positive, inhibiting rate that inhibiting rate, which is more than 20%,
It is feminine gender less than 20%.Positive serum controls inhibiting rate should be 30% ± 1, and negative serum control inhibiting rate must not be no higher than 7%
Then result of the test is invalid.
Inhibiting rate calculation formula：
2.4 solid phases block ELISA sensitivity tests
(1) sensitivity test of FMDV antibody positives control serum O-shaped to pig
The O-shaped FMDV positive control serums of pig are subjected to 2 times of gradient dilutions, the solid phase prepared with the present invention blocks kit to enter
Row is determined, and is determined sensitivity of the kit to positive control serum, be the results are shown in Table 1.Kit detects the positive of gradient dilution
Serum, detection potency reaches 1: 128 times (inhibiting rate is more than 20%).
Sensitivity test of the table 1 to the positive control serum of gradient dilution
| Extension rate | Inhibiting rate (%) | Result judgement |
| 1∶8 | 79.93 | + |
| 1∶16 | 78.02 | + |
| 1∶32 | 75.6 | + |
| 1∶64 | 69.03 | + |
| 1∶128 | 38.5 | + |
| 1∶256 | 14.25 | - |
| 1∶512 | 12.53 | - |
| Positive control | 29.75 | / |
| Negative control | 6.19 | / |
(2) to the sensitivity tests of known positive serum samples
120 parts of positives are obtained using the identification of O-shaped FMDV LPB-ELISAs kit.Use the solid phase of the present invention
ELISA kit is blocked to detect sample.ELISA kit 116 parts of the positive of detection of the present invention, has 4 parts not examine
Go out.As a result it is 96.7% to the sensitiveness of 120 parts of positives to show this kit.
2.5 solid phases block ELISA specific tests
(1) specific test of FMDV negative antibodies quality controlled serum O-shaped to pig
Block ELISA kit to detect 7 parts of O-shaped FMDV negative antibodies quality controlled serums with the solid phase of the present invention, the results are shown in Table
2, blocking ELISA kit of the invention and these antiviral antibody no cross reactions, particularly and A types FMDV and Asia I type
FMDV antibody is also without cross reaction.Wherein N1 is A type FMDV Positive Seras, and N2 is Asia I type FMDV antibody positive blood
Clearly, N3 is antibody against swine fever virus positive serum, and N4 is PRRS virus Positive Sera, and N5 is pig parvoviral antibody sun
Property serum, N6 be porcine pseudorabies virus Positive Sera, N7 be swine influenza virus Positive Sera.
The sensitivity test of positive serum samples known to 2 pairs of table
| Serum is numbered | Inhibiting rate (%) | Result judgement |
| N1 | 8.71 | - |
| N2 | 8.48 | - |
| N3 | 9.35 | - |
| N4 | 4.92 | - |
| N5 | 7.52 | - |
| N6 | 6.71 | - |
| N7 | 8.64 | - |
| Positive control | 30.38 | / |
| Negative control | 6.1 | / |
(2) to the specific test of known negative blood serum sample
120 parts of negative samples are obtained using the identification of O-shaped FMDV LPB-ELISAs kit.Use the liquid phase of the present invention
ELISA kit is blocked to detect sample.ELISA kit 115 parts of the negative sample of detection of the present invention, has 5 parts not examine
Go out.As a result it is 95.8% to the specificity of 120 parts of negative samples to show this kit.
2.6 solid phases block ELISA replica tests
Using the ELISA Plate of 3 different batches, carry out criticizing interior repetition experiment on one block of plate, carried out on different plates between criticizing
Repeat to test, determine OD450 values, calculate the coefficient of variation.Coefficient of variation ＜ 10%, illustrates that kit repeatability and stability are good.3
The ELISA Plate of individual different batches, in batch with batch between testing result it is consistent, the equal ＜ 6% of the coefficient of variation illustrates that kit is repeated very
It is good.
The coefficient of variation (CV) calculation formula：
3rd, solid phase blocks the assembling of ELISA kit
Each reagent is assembled into kit, takes each component to be sealed respectively by table 3, inner packing label, assembling examination is pasted
Agent box.
The kit component of table 3
The kit assembled is placed in 4 DEG C of preservations, interval determines its Sensitivity and Specificity for 2 months, to determine reagent
The storage life of box.4 DEG C of the kit of the present invention is kept in dark place, and storage life is 6 months.
Claims (4)
1. a kind of solid phase for being used to detect O-shaped FMDV antibody is blocked in ELISA kit, the kit containing ELISA Plate, washing
Liquid, sample diluting liquid, rabbit anti-serum, ELIAS secondary antibody, substrate A liquid, substrate B liquid, terminate liquid, positive control serum, negative control
Serum, wherein the ELISA Plate is coated with O-shaped FMDV gene engineering antigens；
The ELIAS secondary antibody uses the goat anti-rabbit igg antibody that HRP is marked；
The rabbit anti-serum is prepared by O-shaped FMDV gene engineering antigens immune rabbit, serum antibody through caprylic acid-ammonium and
G-protein post method carries out preliminary purification, then carries out affinitive layer purification with O-shaped FMDV gene engineering antigens and unlabelled antigen, goes
Except non-specific antibody.
2. the solid phase according to claim 1 for being used to detect O-shaped FMDV antibody blocks ELISA kit, wherein the enzyme
The process that target is coated with O-shaped FMDV gene engineering antigens is：With coating buffer solution by antigen diluent, added with the amount in 100 μ L/ holes
ELISA Plate, 4 DEG C of coating 12h, is washed 3 times with cleaning solution；Add confining liquid, 200 μ L/ holes, 4 DEG C of closing 12h；Deblocking liquid is got rid of,
Board-washing 3 times, is placed in 37 DEG C of dryings, is sealed with aluminum foil under vacuum.
3. the solid phase according to claim 2 for being used to detect O-shaped FMDV antibody blocks ELISA kit, the coating is delayed
Fliud flushing is pH9.6 carbonate buffer solution.
4. the solid phase according to claim 2 for being used to detect O-shaped FMDV antibody blocks ELISA kit, the confining liquid
For the PBS containing 10% tryptone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610212417.3A CN105675866B (en) | 2016-04-07 | 2016-04-07 | For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610212417.3A CN105675866B (en) | 2016-04-07 | 2016-04-07 | For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105675866A CN105675866A (en) | 2016-06-15 |
| CN105675866B true CN105675866B (en) | 2017-09-15 |
Family
ID=56309473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610212417.3A Active CN105675866B (en) | 2016-04-07 | 2016-04-07 | For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105675866B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109142727A (en) * | 2018-10-10 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of the visualization quick detection kit and its application of O-shaped antibodies against foot-and-mouth disease virus |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106226519A (en) * | 2016-07-18 | 2016-12-14 | 洛阳现代生物技术研究院有限公司 | A kind of O type antibodies against foot-and-mouth disease virus chemiluminescence detection kit |
| CN106501512A (en) * | 2016-11-22 | 2017-03-15 | 盐城拜明生物技术有限公司 | A kind of foot-and-mouth disease antibody detection kit, detection method and its application |
| CN109799342A (en) * | 2018-12-07 | 2019-05-24 | 中国农业科学院兰州兽医研究所 | A kind of O-shaped antibodies against foot-and-mouth disease virus competitive ELISA detection kit |
| CN110187105B (en) * | 2019-06-25 | 2022-03-22 | 艾军 | B-ELISA kit for detecting O-type antibody of foot-and-mouth disease virus of cattle and sheep and preparation method thereof |
| CN113495157A (en) * | 2020-03-20 | 2021-10-12 | 郑州达诺生物技术有限公司 | Enzyme conjugate diluent, total thyroxine quantitative detection kit and use method thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1765370A (en) * | 2004-10-26 | 2006-05-03 | 屈国胜 | Method for preparing anti-aftosa serum |
| CN102662062B (en) * | 2012-04-17 | 2015-05-27 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) |
| CN103884839B (en) * | 2012-12-19 | 2015-08-19 | 中国农业科学院兰州兽医研究所 | A kind of reverse LPB-ELISA method for quantitatively detecting FMDV Effective Antigens |
| CN103554234B (en) * | 2013-09-05 | 2016-01-27 | 广西壮族自治区动物疫病预防控制中心 | Based on the competitive ELISA method of foot and mouth disease A type VP1 albumen and monoclonal antibody thereof |
-
2016
- 2016-04-07 CN CN201610212417.3A patent/CN105675866B/en active Active
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109142727A (en) * | 2018-10-10 | 2019-01-04 | 中国农业科学院兰州兽医研究所 | A kind of the visualization quick detection kit and its application of O-shaped antibodies against foot-and-mouth disease virus |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105675866A (en) | 2016-06-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105675866B (en) | For detecting that the solid phase of O-shaped FMDV antibody blocks ELISA kit | |
| CN105807052B (en) | O-shaped FMDV antibody direct competive ELISA detection kit | |
| CN104407137B (en) | A kind of CSFV velogen strain and low virulent strain differentiate Test paper | |
| CN107894508A (en) | A kind of solid phase competitive ELISA kit and its application for the detection of Senecan antiviral antibody | |
| CN107741495A (en) | A kind of double crush syndrome kit and its application for Senecan virus antigen detection | |
| CN102305859B (en) | Activated toxic antibody kit for detecting porcine reproductive and respiratory syndrome virus | |
| WO2008003236A1 (en) | Method of jointly detecting hbv pres1 antigen and core antigen, test kit, solid substrate and virus lysis solution | |
| CN110927390A (en) | ELISA method and kit for detecting African swine fever CD2v protein antibody and application | |
| TWI809010B (en) | Determination method and assay kit for hepatitis B virus s antigen | |
| CN109187968B (en) | Bivalent gold-labeled test paper for detecting classical swine fever virus and porcine pseudorabies virus and preparation method thereof | |
| WO2013043125A1 (en) | Enterovirus 71 specific antibodies and uses thereof | |
| Yoon et al. | Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses | |
| CN106405093A (en) | Detection kit for detecting infectious bovine rhinotracheitis virus antibody | |
| CN104280551A (en) | Duck tembusu virus E-ELISA (E-enzyme-linked immuno sorbent assay) detection kit and preparation method thereof | |
| CN104569428A (en) | ELISA (enzyme-linked immunosorbent assay) kit for EV (enterovirus) 71 inactivated vaccine antigen | |
| CN112745387B (en) | Anti-porcine Seneca valley virus monoclonal antibody and application thereof | |
| CN109799342A (en) | A kind of O-shaped antibodies against foot-and-mouth disease virus competitive ELISA detection kit | |
| CN103558386B (en) | The method of ELISA detection fusion albumen rMBP NAP | |
| CN105136782A (en) | Immune magnetic micro-particle chemiluminescence method HCMV IE1 antigen detection kit | |
| CN102435732A (en) | Toxoplasma IgM antibody immunoblotting kit and preparation method thereof | |
| CN109613249A (en) | A kind of russian spring-summer encephalitis virus IgM antibody ELISA detection kit and preparation method thereof | |
| CN105567643B (en) | Hybridoma cell capable of secreting anti-EV 71 virus protein VP1 monoclonal antibody, monoclonal antibody and application | |
| CN111398594B (en) | Immunodiagnosis kit for specifically detecting yellow fever virus NS1 antigen | |
| CN114578047A (en) | Foot-and-mouth disease virus O-type and A-type antibody joint detection kit and preparation method and application thereof | |
| CN102288768A (en) | Toxoplasma gondii IgG (immunoglobulin G) antibody immunoblotting kit and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |