CN110161237A - A kind of foot and mouth disease virus cELISA antibody assay kit extending storage life - Google Patents
A kind of foot and mouth disease virus cELISA antibody assay kit extending storage life Download PDFInfo
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- CN110161237A CN110161237A CN201810342072.2A CN201810342072A CN110161237A CN 110161237 A CN110161237 A CN 110161237A CN 201810342072 A CN201810342072 A CN 201810342072A CN 110161237 A CN110161237 A CN 110161237A
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
- G01N2333/09—Foot-and-mouth disease virus
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Abstract
The present invention relates to a kind of by protecting solid-state foot-and-mouth disease virus antigen to extend the foot and mouth disease virus cELISA antibody assay kit of storage life; first prepare antigen protective agent: the PBS buffer solution for preparing 0.01M makees mother liquor, sequentially adds 10%BSA, 10% trehalose, 3% polysaccharides, 3% aloe polysaccharide, 0.01% thimerosal, 0.05% Tween-20；Through the membrane filtration degerming of the aperture 0.45nm after mixing well, can be used through steriling test qualification rear；Antigen protective agent is added into the foot-and-mouth disease virus antigen fixed plate prepared; aftosa Antigen Stability storage life in ELISA Plate can be extended to 12 months under the conditions of 4-6 DEG C; using the kit of this ELISA Plate preparation than one times of long shelf-life of current such kit; the cost for reducing pig farm purchase kit, also reduces the management intensity of veterinary work.
Description
Technical field
The invention belongs to biological diagnosis detection technique fields, and in particular to one kind passes through protection solid-state foot-and-mouth disease virus antigen
And extend the foot and mouth disease virus cELISA antibody assay kit of storage life.
Background technique
Aftosa is one of great animal epidemic, World Organization for Animal Health (OIE) be classified as A class animal epidemic it
It is first.Hostis Picornaviridae, Hostis, totally 7 serotype, a hypotype more than 70.The prevention of aftosa and
Control is global problem, the whole world every year because caused by the disease direct economic loss to tens billion of dollars.Annual China puts into number
Prevention and control and new industrial research of 1000000000 RMB for aftosa, however various regions aftosa epidemic situation still continuously emerges.Vaccine is exempted from
Epidemic disease is the main means of current China's prevention and control aftosa Epidemic outbreak of disease and sprawling, due to foot-and-mouth disease virus antigen immunogene
Property it is poor, vaccine quality is irregular, and susceptible animal needs multiple vaccine inoculation to can be only achieved ideal antibody level, therefore in time
Monitoring vaccine immunity after animal antibody level, evaluate immune effect and take corresponding counter-measure particularly important.
The Foot-and-mouth disease antibody detection method that OIE recommends is virus neutralization tests, liquid phase blocks enzyme-linked exempt from
Epidemic disease adsorption test (LPB-ELISA) and solid phase competition enzyme-linked immunosorbent adsorption test (cELISA) are (see OIE terrestrial animal diagnostic test
With vaccine handbook, the 7th edition version in 2012,166-169 pages).CELISA detection method is the specified international trade detection side OIE
Method, sensibility is identical compared with LPB-ELISA detection method, but specificity is higher.Foreign countries have commercialization aftosa at present
CELISA detection kit, the application of some of them cELISA detection kit is expression antigen, since expression antigen uses line
Shape amino acid sequence may be implemented to be directly fixed on ELISA Plate, but cannot cover totivirus, relatively narrow etc. there are antigen covering surface
The problem of aspect, therefore OIE is without being recommended to use.Another part aftosa cELISA kit is by being coated with rabbit-anti mouth hoof
Then epidemic disease antiviral antibody captures foot and mouth disease virus 146S antigen (complete virion antigen), is fixed on antigen on ELISA Plate,
Antibody test is used for after confining liquid closing.But foot-and-mouth disease virus antigen is easy to be denaturalized, pH8.0 or more, pH6.5 or less can be very
Fast inactivation, it is also extremely sensitive to temperature, it is relatively stable due to other reagents, so the stability of foot-and-mouth disease virus antigen is shadow
The main factor of the storage life of sound kit, and guarantor under the conditions of 4-6 DEG C of such aftosa cELISA kit at present
The phase of depositing is only 6 months, higher cost, and more demanding to animal doctor's tracing management on pig farm.Therefore, if aftosa can be protected
Stability of the viral antigen on ELISA Plate, so that it may extend storage life, be a Xiang Chong for the detection of foot-and-mouth disease antibody
The technological progress wanted.
Foot-and-mouth disease antigen Techniques of preserving (the application number of the inventions such as Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences's Wang Yong Lu
02124649.1) it is saved for liquid foot-and-mouth disease antigen, it is domestic without for the special of aftosa cELISA solid-state antigen so far
Door protective agent and corresponding reagent box.
Summary of the invention
To solve the above problems, the present invention proposes a kind of foot and mouth disease virus cELISA antibody test reagent for extending storage life
Box.
The foot and mouth disease virus cELISA antibody assay kit of extension storage life of the invention, be made by the steps and
At:
1) prepare antigen protective agent: the PBS buffer solution for preparing 0.01M makees mother liquor, sequentially adds the sea of 10%BSA, 10%
Algae sugar, 3% polysaccharides, 3% aloe polysaccharide, 0.01% thimerosal, 0.05% Tween-20；Through the hole 0.45nm after mixing well
Diameter membrane filtration degerming can be used through steriling test qualification rear；
2) it prepares foot-and-mouth disease virus antigen fixed plate: the coating buffer of rabbit-anti antibodies against foot-and-mouth disease virus pH9.6 being diluted, 50
The hole μ l/ is added in 96 hole elisa Plates, and 4 DEG C stand 16 hours；Coating buffer is outwelled, is washed 3 times with the PBST of 0.01M；Every hole adds
Enter 50 μ l of foot and mouth disease virus Antigen suspensions, 37 DEG C are reacted 1 hour；Liquid is outwelled, washs 3 times, as aftosa with 0.01MPBST
Viral antigen fixed plate；
3) the foot and mouth disease virus cELISA antibody assay kit that preparation extends storage life detects ELISA Plate: to step 2) institute
It obtains and closed protective agent obtained by step 1) is added in foot-and-mouth disease virus antigen fixed plate, 50 μ L closed protective agent are added in every hole, and 4 DEG C flat
It is steady to place 16 hours, closed protective agent, which is abandoned, with gently patting dry on blotting paper is placed in vent cabinet or other apparatus for drying room temperatures
It air-dries 1 hour, can be obtained the foot and mouth disease virus cELISA antibody assay kit detection ELISA Plate for extending storage life；
4) it is saved after reagent preparation box: 5 pieces of ELISA Plate of the detection obtained by step 3), with corresponding cavy foot-and-mouth disease virus resistant
600 μ L of serum, 300 μ L of foot and mouth disease virus positive control serum, 600 μ L of foot and mouth disease virus negative control sera, rabbit-anti cavy HRP
Mark 50 μ L of secondary antibody, dilution 60mL, TMB developing solution 30mL, 2 bags of the PBS pulvis of terminate liquid 30mL, 0.01M and spitting for 2mL
Temperature -20 collectively constitutes the foot and mouth disease virus cELISA antibody assay kit for extending storage life, is placed in 4 DEG C of preservations.
Further, the coating buffer is the carbonate buffer solution of 0.1M.
Preferably, the compositional purity requirement of the antigen protective agent are as follows: BSA >=98%, trehalose >=98%, polysaccharides
>=70%, aloe polysaccharide >=70%, thimerosal >=98%, Tween-20 >=99%.
The present invention utilizes the foot and mouth disease virus solid-state antigen protective agent of original creation, Neng Goubao by selection, proportion optimizing repeatedly
Retaining aphtovirus solid-state antigen maintains effective active up to 12 months or more on ELISA Plate, the mouth prepared using this ELISA Plate
Fever aphthous detection kit, storage life can extend to 12 months.In antigen protective agent formula of the invention, trehalose can be in cell
Surface can form unique protective film, be effectively protected protein molecule invariance inactivation, to maintain virion biology special
Sign；On the one hand aloe polysaccharide has stabilization, guarantee the stability of virion neoepitope Western, on the other hand containing there are many disappear
Except the ingredient of superoxide radical delays virion to degrade such as superoxide dismutase, catalase；Polysaccharides
Other than having stable protein, oxidation resistant function, also there is stronger hygroscopic effect, antigen and serum to be checked can be promoted anti-
Body, the combination for competing antibody；Thimerosal serves in this protective agent inhibits bacterium bacterium and mould；Tween-20 is surface-active
Agent, due to there is more one polyoxyethylene groups of hydrophilic radical in its molecule, therefore hydrophily is strong, has the function of renaturation antigen,
Recognition capability specific in antigen-antibody reaction can be improved；BSA (bovine serum albumin(BSA)), by improving Proteins In Aqueous Solutions
Concentration prevents the decomposition, denaturation and non-specific adsorption of enzyme, mitigates denaturation caused by surface tension and chemical factor.
Beneficial effects of the present invention are as follows:
1. extending the storage life of ELISA Plate or kit: the present invention can be by aftosa Antigen Stability storage life in ELISA Plate
12 months are extended under the conditions of 4-6 DEG C, using the preparation of this ELISA Plate kit than current such kit long shelf-life
One times, the cost of pig farm purchase kit is reduced, the management intensity of veterinary work is also reduced.
2. improving ELISA Plate sealing effect, more valid data are obtained: kit of the invention, since antigen protective agent has
Sealing effect can protect the stable components in 96 hole elisa Plates in all holes, when detecting can so ELISA Plate is closed well
Obtain more valid data.
3. method is simple, low in cost, practical: the present invention increases only on the basis of preparing conventional kit
One step of antigen protective agent is added, and the antigen protective agent is easily obtained using material, preparation, operating method are simple, right
Production technology, the requirement of facility are low, practical, are suitable for the demand of large-scale production.
4. facilitating detection, it is conducive to pig farm and prevents and treats aftosa: uses kit of the invention, it is easy to operate, it can rapid, high volume
Detection pig farm in aftosa morbidity and potential epidemic disease situation, being conducive to pig farm, to formulate aftosa in advance according to detection data anti-
Scheme is controlled, is got twice the result with half the effort.
Specific embodiment
Below by way of specific embodiment further illustrate technical solution of the present invention, but technical solution of the present invention not with
Embodiment is limited.
Embodiment 1:
A kind of foot and mouth disease virus cELISA antibody assay kit extending storage life, is made by the steps:
1) prepare antigen protective agent: the PBS buffer solution for preparing 0.01M makees mother liquor, sequentially adds the sea of 10%BSA, 10%
Algae sugar, 3% polysaccharides, 3% aloe polysaccharide, 0.01% thimerosal, 0.05% Tween-20；Through the hole 0.45nm after mixing well
Diameter membrane filtration degerming can be used through steriling test qualification rear；
2) it prepares foot-and-mouth disease virus antigen fixed plate: the coating buffer of rabbit-anti antibodies against foot-and-mouth disease virus pH9.6 being diluted, 50
The hole μ l/ is added in 96 hole elisa Plates, and 4 DEG C stand 16 hours；Coating buffer is outwelled, is washed 3 times with the PBST of 0.01M；Every hole adds
Enter 50 μ l of foot and mouth disease virus Antigen suspensions, 37 DEG C are reacted 1 hour；Liquid is outwelled, washs 3 times, as mouth hoof with 0.01M PBST
Epidemic disease viral antigen fixed plate；
3) the foot and mouth disease virus cELISA antibody assay kit that preparation extends storage life detects ELISA Plate: to step 2) institute
It obtains and antigen protective agent obtained by step 1) is added in foot-and-mouth disease virus antigen fixed plate, 50 μ L antigen protective agents are added in every hole, and 4 DEG C flat
It is steady to place 16 hours, antigen protective agent is abandoned, with gently patting dry on blotting paper, is placed in vent cabinet or other apparatus for drying room temperatures
It air-dries 1 hour, can be obtained the foot and mouth disease virus cELISA antibody assay kit detection ELISA Plate for extending storage life；
4) it is saved after reagent preparation box: 5 pieces of ELISA Plate of the detection obtained by step 3), with corresponding cavy foot-and-mouth disease virus resistant
600 μ L of serum, 300 μ L of foot and mouth disease virus positive control serum, 600 μ L of foot and mouth disease virus negative control sera, rabbit-anti cavy HRP
Mark 50 μ L of secondary antibody, dilution 60mL, TMB developing solution 30mL, 2 bags of the PBS pulvis of terminate liquid 30mL, 0.01M and spitting for 2mL
Temperature -20 collectively constitutes the foot and mouth disease virus cELISA antibody assay kit for extending storage life, is placed in 4 DEG C of preservations.
Embodiment 2: test sample
The detection ELISA Plate in the kit of 4 DEG C of preservations is taken out, is placed at room temperature for 20 minutes；Every hole be added 12 μ l to
Examine blood serum sample and foot and mouth disease virus positive control serum, foot and mouth disease virus negative control sera, 1 hole of blood serum sample/part to be checked,
2 holes of positive control serum/part, 4 holes of negative control sera/part；The anti-mouth of cavy is diluted by 1:100 with the dilution that kit provides
Aphtovirus serum (competition antibody), each 50 μ l cavy foot-and-mouth disease virus resistant serum dilution of Kong Zhongjia, concussion mixes 1 point
Clock；It sets and is incubated 60 minutes in 37 DEG C of incubators；Withdrawing plate outwells liquid, gently pats dry on blotting paper；It is provided with kit
Dilution marks secondary antibody by 1:1000 dilution rabbit-anti cavy HRP, and every 50 μ l of hole, 37 DEG C incubate 30 minutes, with PBS and Tween-20
The 0.01M PBST washing lotion of preparation board-washing 5 times, gently pats dry on blotting paper；Every hole adds 50 μ L one pack system TMB developing solutions to react
Liquid, Incubation in dark 8-10 minutes；Then every 50 μ L of Kong Zaijia terminate liquid terminates reaction.In 15min under microplate reader 450nm wavelength
Read OD value.
The kit for setting up unused antigen protective agent simultaneously compares.4 DEG C of preservations, monthly each 1 inspection of taking-up in 0-13 months
Item (8 hole) is surveyed to be detected.
As a result it calculates: being determined according to inhibiting rate as a result, inhibiting rate (PI)=(1- sample OD450/ negative control is averaged OD450)
× 100%, inhibiting rate is judged to the positive when being greater than or equal to 50%, is judged to feminine gender when inhibiting rate is less than 50%.
Antibodies against foot-and-mouth disease virus positive control sample and negative control sample testing result are shown in Table 1.Testing result is shown, is answered
It is showed over time now with antigen protective agent and not using the ELISA Plate negative control sample OD value of antigen protective agent
Drop trend (is shown in Table 1), at 13 months, drops to 0.865 using antigen protective agent, and not dropping to using antigen protective agent
0.245, antigen protecting effect is significant.Positive control sample testing result is shown, using ELISA Plate 1-12 of antigen protective agent
The moon, testing result was substantially steady, and PI value is remarkably decreased within the 13rd month, did not applied the 4th month PI of ELISA Plate of antigen protective agent
Value is remarkably decreased, and can not detect the positive within the 9th month.Therefore, antigen protective agent is significant to antigen protecting effect, can be effective
Extend in cELISA kit and detects the service life of ELISA Plate to 12 months.
Table 1 0~13 months application protective agents and not application protectant ELISA Plate testing result statistics
The foot and mouth disease virus cELISA antibody assay kit provided by the present invention for extending storage life is carried out above
It is discussed in detail.The principle of the present invention and embodiment are expounded herein by specific embodiment, it is described above to be
It is used to help understand method and its core concept of the invention.It should be pointed out that coming for those skilled in the art
It says, without departing from the principle of the present invention, can be with several improvements and modifications are made to the present invention, these improvement and modification
It also falls within the protection scope of the claims of the present invention.
Claims (3)
1. a kind of foot and mouth disease virus cELISA antibody assay kit for extending storage life, which is characterized in that as follows
It is prepared:
1) prepare antigen protective agent: the PBS buffer solution for preparing 0.01M makees mother liquor, sequentially add 10%BSA, 10% trehalose,
3% polysaccharides, 3% aloe polysaccharide, 0.01% thimerosal, 0.05% Tween-20；Through the aperture 0.45nm filter membrane after mixing well
Filtration sterilization can be used through steriling test qualification rear；
2) it prepares foot-and-mouth disease virus antigen fixed plate: the coating buffer of rabbit-anti antibodies against foot-and-mouth disease virus pH9.6 being diluted, 50 μ l/
Hole is added in 96 hole elisa Plates, and 4 DEG C stand 16 hours；Coating buffer is outwelled, is washed 3 times with the PBST of 0.01M；Mouth is added in every hole
50 μ l of aphtovirus Antigen suspensions, 37 DEG C are reacted 1 hour；Liquid is outwelled, washs 3 times, as hoof-and-mouth disease with 0.01M PBST
Malicious antigen fixed plate；
3) preparation extends the foot and mouth disease virus cELISA antibody test ELISA Plate of storage life: anti-to foot and mouth disease virus obtained by step 2)
Antigen protective agent obtained by step 1) is added in former fixed plate, 50 μ L antigen protective agents are added in every hole, and 4 DEG C are steadily placed 16 hours,
Antigen protective agent is abandoned, with gently being patted dry on blotting paper, vent cabinet is placed in or other apparatus for drying is air-dried at room temperature 1 hour, i.e.,
It can get the foot and mouth disease virus cELISA antibody test ELISA Plate for extending storage life；
4) it is saved after reagent preparation box: 5 pieces of ELISA Plate of the detection obtained by step 3), with corresponding cavy foot-and-mouth disease virus resistant serum
600 μ L, 300 μ L of foot and mouth disease virus positive control serum, 600 μ L of foot and mouth disease virus negative control sera, rabbit-anti cavy HRP label
50 μ L of secondary antibody, dilution 60mL, TMB developing solution 30mL, 2 bags of the PBS pulvis of terminate liquid 30mL, 0.01M and the Tween-20 of 2mL,
The foot and mouth disease virus cELISA antibody assay kit for extending storage life is collectively constituted, 4 DEG C of preservations are placed in.
2. extending the foot and mouth disease virus cELISA antibody assay kit of storage life according to claim 1, which is characterized in that
The coating buffer is the carbonate buffer solution of 0.1M.
3. extending the foot and mouth disease virus cELISA antibody assay kit of storage life according to claim 1, which is characterized in that
The compositional purity requirement of the antigen protective agent are as follows: BSA >=98%, trehalose >=98%, polysaccharides >=70%, aloe polysaccharide
>=70%, thimerosal >=98%, Tween-20 >=99%.
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| CN201810342072.2A CN110161237A (en) | 2018-04-17 | 2018-04-17 | A kind of foot and mouth disease virus cELISA antibody assay kit extending storage life |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112881710A (en) * | 2021-02-04 | 2021-06-01 | 中国农业科学院兰州兽医研究所 | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175852A (en) * | 2011-01-06 | 2011-09-07 | 云南省畜牧兽医科学院 | Detection method for solid phase competition ELISA (Enzyme Linked Immuno Sorbent Assay) of foot and mouth disease |
| CN107213117A (en) * | 2017-05-29 | 2017-09-29 | 钟术光 | A kind of preparation method of the composition of stable bioactive materials |
-
2018
- 2018-04-17 CN CN201810342072.2A patent/CN110161237A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102175852A (en) * | 2011-01-06 | 2011-09-07 | 云南省畜牧兽医科学院 | Detection method for solid phase competition ELISA (Enzyme Linked Immuno Sorbent Assay) of foot and mouth disease |
| CN107213117A (en) * | 2017-05-29 | 2017-09-29 | 钟术光 | A kind of preparation method of the composition of stable bioactive materials |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112881710A (en) * | 2021-02-04 | 2021-06-01 | 中国农业科学院兰州兽医研究所 | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
| CN112881710B (en) * | 2021-02-04 | 2022-04-22 | 中国农业科学院兰州兽医研究所 | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
| CN113267621A (en) * | 2021-05-14 | 2021-08-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
| CN113267621B (en) * | 2021-05-14 | 2021-12-17 | 北京金诺百泰生物技术有限公司 | Stabilizer for ELISA kit coated plate, preparation method of stabilizer, kit coated plate and kit |
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Application publication date: 20190823 |