CN110161238A - A kind of foot-and-mouth disease virus antigen protective agent - Google Patents
A kind of foot-and-mouth disease virus antigen protective agent Download PDFInfo
- Publication number
- CN110161238A CN110161238A CN201810342182.9A CN201810342182A CN110161238A CN 110161238 A CN110161238 A CN 110161238A CN 201810342182 A CN201810342182 A CN 201810342182A CN 110161238 A CN110161238 A CN 110161238A
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- China
- Prior art keywords
- foot
- mouth disease
- disease virus
- protective agent
- virus antigen
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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
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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
Abstract
The present invention relates to a kind of foot-and-mouth disease virus antigen protective agents, are formulated as follows: 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；After mixing well, through the membrane filtration degerming of the aperture 0.45nm；Through steriling test qualification, as foot-and-mouth disease virus antigen protective agent, it is placed in 4 DEG C or -20 DEG C saves backup.The present invention passes through selection, proportion optimizing repeatedly; form a kind of foot and mouth disease virus solid-state antigen protective agent; foot and mouth disease virus solid-state antigen can be protected to maintain effective active up to 12 months or more on ELISA Plate; the application effect of aftosa cELISA is improved, while improving ELISA Plate sealing effect, obtains more valid data; method is simple; it is low in cost, it is practical, it is suitable for the demand of large-scale production.
Description
Technical field
The invention belongs to biological diagnosis detection technique fields, and in particular to one kind is in solid phase competition enzyme-linked immunosorbent adsorption test
(cELISA) stability of the protection foot-and-mouth disease virus antigen in 96 hole elisa Plates in, and ELISA Plate carrier surface is closed, it prevents
The foot-and-mouth disease virus antigen protective agent of undesirable adsorption reaction.
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.
Summary of the invention
To solve the above problems, the present invention proposes a kind of stabilization for protecting foot-and-mouth disease virus antigen in 96 hole elisa Plates
Property, and ELISA Plate carrier surface is closed, prevent the foot-and-mouth disease virus antigen protective agent of undesirable adsorption reaction.
Foot-and-mouth disease virus antigen protective agent of the invention, is formulated as follows:
1) PBS buffer solution for preparing 0.01M makees mother liquor；
2) 10%BSA, 10% trehalose, 3% polysaccharides, 3% aloe are sequentially added in the step 1) mother liquor
Polysaccharide, 0.01% thimerosal, 0.05% Tween-20；
3) after mixing well step 2) acquired solution, through the membrane filtration degerming of the aperture 0.45nm；
4) by step 3) acquired solution through steriling test qualification, as foot-and-mouth disease virus antigen protective agent, be placed in 4 DEG C or-
20 DEG C save backup.
Further, foot-and-mouth disease virus antigen protective agent of the invention, when being applied to cELISA technology, to having fixed mouth
The foot-and-mouth disease virus antigen protective agent is added in 96 hole elisa Plates of fever aphthous antigen, 50 μ L protective agents are added in every hole, and 4 DEG C steady
It places 16 hours, abandons protective agent, ELISA Plate is gently patted dry on blotting paper, be placed in vent cabinet or other apparatus for drying room temperatures
It air-dries 1 hour, is sealed up for safekeeping using vacuum bag vacuum tightness spare.
Further, the foot-and-mouth disease virus antigen protective agent can also be used for protecting the blue tongue rims adsorbed by ELISA Plate or
Ah card's spot viral antigen.
Preferably, the purity requirement of the component are as follows: BSA >=98%, trehalose >=98%, polysaccharides >=70%, reed
Luxuriant growth polysaccharide >=70%, thimerosal >=98%, Tween-20 >=99%.
The present invention is formed a kind of foot and mouth disease virus solid-state antigen protective agent, can be protected by selection, proportion optimizing repeatedly
Foot and mouth disease virus solid-state antigen maintains effective active up to 12 months or more on ELISA Plate, improves the application effect of aftosa cELISA
Fruit.In formula, trehalose can form unique protective film in cell surface, be effectively protected the mistake of protein molecule invariance
It is living, to maintain virion biological characteristic；On the one hand aloe polysaccharide has stabilization, guarantee virion neoepitope Western
Stability, on the other hand containing there are many ingredients for eliminating superoxide radical, such as superoxide dismutase, catalase,
Virion is delayed to degrade；Polysaccharides also there is stronger moisture absorption to make other than having stable protein, oxidation resistant function
With the combination that can promote antigen and serum antibody to be checked, compete antibody；Thimerosal rises in this protective agent inhibits bacterium bacterium and mould
Bacterium effect；Tween-20 is surfactant, due to there is more one polyoxyethylene groups of hydrophilic radical in its molecule, thus it is hydrophilic
Property is strong, has the function of renaturation antigen, recognition capability specific in antigen-antibody reaction can be improved；BSA (bovine serum albumin
It is white), by improving the concentration of Proteins In Aqueous Solutions, prevent the decomposition, denaturation and non-specific adsorption of enzyme, mitigate surface tension and
Denaturation caused by 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, than one times of long shelf-life of current such kit, reduce pig farm purchase reagent
The cost of box also reduces the management intensity of veterinary work.
2. improving ELISA Plate sealing effect, obtain more valid data: foot-and-mouth disease virus antigen protective agent of the invention can
ELISA Plate sealing effect is significantly improved, the stable components in 96 hole elisa Plates in all holes are protected, can be obtained when detecting more
Valid data.
3. method is simple, low in cost, practical: foot-and-mouth disease virus antigen protective agent of the invention is easy to using material
It obtaining, preparation, operating method are simple, and the requirement to production technology, facility is low, and it is practical, it is suitable for the need of large-scale production
It asks.
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: foot-and-mouth disease virus antigen protective agent is prepared
A kind of foot-and-mouth disease virus antigen protective agent, is formulated as follows:
1) PBS buffer solution for preparing 0.01M makees mother liquor；
2) 10%BSA, 10% trehalose, 3% polysaccharides, 3% aloe are sequentially added in the step 1) mother liquor
Polysaccharide, 0.01% thimerosal, 0.05% Tween-20；
3) after mixing well step 2) acquired solution, through the membrane filtration degerming of the aperture 0.45nm；
4) by step 3) acquired solution through steriling test qualification, as foot-and-mouth disease virus antigen protective agent, be placed in 4 DEG C or-
20 DEG C save backup.
The purity requirement of the component are as follows: BSA >=98%, trehalose >=98%, polysaccharides >=70%, aloe polysaccharide >=
70%, thimerosal >=98%, Tween-20 >=99%.
Embodiment 2: closing ELISA Plate
The resulting foot-and-mouth disease virus antigen protective agent of embodiment 1 is applied to cELISA technology, to having fixed a mouthful hoof
The foot-and-mouth disease virus antigen protective agent is added in 96 hole elisa Plates of epidemic disease antigen, 50 μ L protective agents are added in every hole, and 4 DEG C steadily put
It sets 16 hours, abandons protective agent, ELISA Plate is gently patted dry on blotting paper, be placed in vent cabinet or other apparatus for drying room temperature wind
Dry 1 hour, sealed up for safekeeping using vacuum bag vacuum tightness, be placed in 4-6 DEG C it is spare.
The foot-and-mouth disease virus antigen protective agent can also be used for protecting the blue tongue rims adsorbed by ELISA Plate or Ah card's spot
Viral antigen.
Embodiment 3: test sample
The ELISA Plate for taking out 4 DEG C of preservations, is placed at room temperature for 20 minutes；The blood serum sample to be checked and aftosa of 12 μ l is added in every hole
Virus positive control serum, foot and mouth disease virus negative control sera, 1 hole of blood serum sample/part to be checked, 2 hole of positive control serum/
Part, 4 holes of negative control sera/part；The dilution provided with kit is (competing by 1:100 dilution cavy foot-and-mouth disease virus resistant serum
Strive antibody), each 50 μ l cavy foot-and-mouth disease virus resistant serum dilution of Kong Zhongjia, concussion mixes 1 minute；It sets in 37 DEG C of incubators
It incubates 60 minutes；Withdrawing plate outwells liquid, gently pats dry on blotting paper；The dilution provided with kit is dilute by 1:1000
Rabbit-anti cavy HRP label secondary antibody is released, every 50 μ l of hole, 37 DEG C incubate 30 minutes, are washed with 0.01MPBST prepared by PBS and Tween-20
It liquid board-washing 5 times, is gently patted dry on blotting paper；Every hole adds 50 μ L one pack system TMB developing solution reaction solutions, and Incubation in dark 8-10 divides
Clock；Then every 50 μ L of Kong Zaijia terminate liquid terminates reaction.OD value is read in 15min under microplate reader 450nm wavelength.
The ELISA Plate 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 at 13 months, drops to 0.865 using antigen protective agent, and does not drop to 0.245 using antigen protective agent,
Antigen protecting effect is significant.Positive control sample testing result is shown, using the ELISA Plate detection in 1-12 months of antigen protective agent
As a result substantially steady, PI value is remarkably decreased within the 13rd month, is not occurred using 4th month PI value of the ELISA Plate of antigen protective agent
It is remarkably decreased, the positive can not be detected within the 9th month.Therefore, antigen protective agent is significant to antigen protecting effect, can effectively extend
The service life of antigen ELISA Plate was to 12 months in cELISA.
Table 1 0~13 months application protective agents and not application protectant antigen ELISA Plate testing result statistics
A kind of foot-and-mouth disease virus antigen protective agent provided by the present invention is described in detail above.Herein by tool
Body embodiment is expounded the principle of the present invention and embodiment, it is described above be merely used to help understand it is of the invention
Method and its core concept.It should be pointed out that for those skilled in the art, not departing from the principle of the invention
Under the premise of, it can be with several improvements and modifications are made to the present invention, these improvement and modification also fall into the claims in the present invention
Protection scope in.
Claims (4)
1. a kind of foot-and-mouth disease virus antigen protective agent, which is characterized in that be formulated as follows:
1) PBS buffer solution for preparing 0.01M makees mother liquor；
2) sequentially added in the step 1) mother liquor 10%BSA, 10% trehalose, 3% polysaccharides, 3% aloe polysaccharide,
0.01% thimerosal, 0.05% Tween-20；
3) after mixing well step 2) acquired solution, through the membrane filtration degerming of the aperture 0.45nm；
4) step 3) acquired solution is placed in 4 DEG C or -20 DEG C through steriling test qualification, as foot-and-mouth disease virus antigen protective agent
It saves backup.
2. foot-and-mouth disease virus antigen protective agent according to claim 1, which is characterized in that when being applied to cELISA technology, to
It has fixed and the foot-and-mouth disease virus antigen protective agent is added in 96 hole elisa Plates of foot-and-mouth disease antigen, every hole is added 50 μ L and protects
Agent is protected, 4 DEG C are steadily placed 16 hours, abandon protective agent, ELISA Plate is gently patted dry on blotting paper, is placed in vent cabinet or other
Apparatus for drying is air-dried at room temperature 1 hour, is sealed up for safekeeping using vacuum bag vacuum tightness, be placed in 4-6 DEG C it is spare.
3. foot-and-mouth disease virus antigen protective agent according to claim 1, which is characterized in that the foot-and-mouth disease virus antigen protection
Agent can also be used for protecting the blue tongue rims adsorbed by ELISA Plate or Ah card's spot viral antigen.
4. foot-and-mouth disease virus antigen protective agent according to claim 1, which is characterized in that the purity requirement of the component are as follows:
BSA >=98%, trehalose >=98%, polysaccharides >=70%, aloe polysaccharide >=70%, thimerosal >=98%, Tween-20 >=
99%.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810342182.9A CN110161238A (en) | 2018-04-17 | 2018-04-17 | A kind of foot-and-mouth disease virus antigen protective agent |
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| CN201810342182.9A CN110161238A (en) | 2018-04-17 | 2018-04-17 | A kind of foot-and-mouth disease virus antigen protective agent |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112126628A (en) * | 2020-07-20 | 2020-12-25 | 天康生物股份有限公司 | Goat pox virus propagation method, goat pox live vaccine, preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112126628A (en) * | 2020-07-20 | 2020-12-25 | 天康生物股份有限公司 | Goat pox virus propagation method, goat pox live vaccine, preparation method and application thereof |
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Application publication date: 20190823 |