CN106771189A - A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine - Google Patents
A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine Download PDFInfo
- Publication number
- CN106771189A CN106771189A CN201611123598.9A CN201611123598A CN106771189A CN 106771189 A CN106771189 A CN 106771189A CN 201611123598 A CN201611123598 A CN 201611123598A CN 106771189 A CN106771189 A CN 106771189A
- Authority
- CN
- China
- Prior art keywords
- antigen
- oil
- checked
- dilution
- vaccine
- 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.)
- Granted
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)
- Urology & Nephrology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine, comprise the following steps：Aftosa synthetic peptide vaccine is demulsified, the antigen samples after demulsification are carried out into qualitative and quantitative detection using competitive ELISA method；The breaking method is：Oil-adjuvant vaccine is mixed with n-butanol, after being subsequently adding competitor mixing, centrifugation obtains final product antigen samples.The method competes antigen binding site by adding competitor with surfactant, and the antigen in oil-adjuvant vaccine is discharged into water phase, substantially increases the rate of recovery of antigen in water phase, is determined by sample is carried out purification process, shortens detection time；The present invention first first reacts antigen to be checked with the antibody of concentration known, and antibody is first neutralized completely with antigen；Then the solution after neutralization and immobilization are adsorbed the antigen-reactive in elisa plate to determine antigen concentration to be checked again, the slope of standard curve for obtaining can be more than conventional indirect competitive, so as to substantially increase detection sensitivity.
Description
Technical field
The present invention relates to aftosa synthetic peptide vaccine detection technique field, and in particular to a kind of the quick of oil-adjuvant vaccine is determined
Property quantitative detecting method.
Background technology
Regulation efficacy test must be tested using this animal in currently available vaccines, existing vaccines quality standard, because country carries out
100% reinforced immunological policy, it is difficult to selecting susceptible inspection uses animal, and animal attacks malicious high to Experimental Establishment requirement
(BSL3 grades of laboratory), time-consuming (more than one month), capital cost is big.If selecting impressibility using serum neutralization test
Thing, is technically difficult to exclude the non-susceptible animal with cellular immunity, and inspection data often occurs in practice in inspection does not advise
The problem of rule, influences the accuracy of inspection.Therefore aftosa vaccine perplexs always in particular for the quality testing of the vaccine of ox
Veterinary drug monitoring department and associated production enterprise.Therefore need to develop tested in vitro technology as early as possible, tried instead of existing animal
Test.
Inspection of the current country to vaccine is just progressively being transitioned into the detection to vaccine endoantigen, and currently more universal side
Method is that antigen is detected after vaccine is demulsified, and demulsification treatment is carried out by by vaccine, antigen is transferred in water phase, then right
It carries out follow-up detection and analysis.Due to complicated component in the oily adjuvant used in vaccine emulsion process, contain surface-active
The materials such as agent, immunopotentiator are present, and often contain in above-mentioned impurity and demulsifier, industry not in the water phase after demulsification
The method that above-mentioned impurity and demulsifier can be removed, and impurity and demulsifier etc. can also largely effect on detection process thereafter, make
Covered into signal or disturbed, forced down the intensity of antigen signals, in addition cannot effective detection to antigen therein, due to wherein miscellaneous
The being randomly assigned property of matter, causes the repeatability of its detection method not good, while increased instrument maintenance cost.
Aftosa synthetic peptide vaccine is a kind of protection synthesized by the amino acid sequence of native protein by manual method
Property small peptide.Existing method for quantitatively determining generally has the methods such as Lowry methods, BCA methods, HPLC.And in existing quantitative approach, due to
After vaccine demulsification, vaccine water-phase component is more complicated, and measuring samples generally need to take considerable time and can be only achieved after purification detection
It is required that, and many conventional method detection sensitivities are limited.
EUSA (ELISA) is quick together, convenient, special, the advantages of can quantify, in many fields
To extensive use.In conventional indirect competitive ELISA, antigen has antigen with the antibody of Finite Concentration while being added to solid phase adsorption
Elisa plate in reacted, antibody and free antigen, immobilised antigen binding rate probability are 50%, the knot for obtaining
The slope of standard curve that fruit is drawn is relatively low, and this causes that detection sensitivity is relatively low.Aftosa synthetic peptide vaccine can not well be reacted
Middle Effective Antigens content.
At present, there is no general accurate method for aftosa synthetic peptide vaccine antigenic content quantitative determination.It is badly in need of research and development
It is more quick always, effectively, the method for qualitative and quantitative detection of convenient oil-adjuvant vaccine.
The content of the invention
For defect of the prior art, the invention provides a kind of fast qualitative quantitative determination side of oil-adjuvant vaccine
Method.It is demulsified using special breaking method by by aftosa synthetic peptide vaccine, then to the water phase antigen samples after demulsification
Directly be at war with ELISA method, accurately can carry out qualitative and quantitative analysis to vaccine.
The purpose of the present invention is achieved through the following technical solutions：
The invention provides a kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine, comprise the following steps：By mouth hoof
Epidemic disease synthetic peptide vaccine is demulsified, and the antigen samples after demulsification are carried out into qualitative and quantitative detection using competitive ELISA method；
The breaking method is：Oil-adjuvant vaccine is mixed with n-butanol, after being subsequently adding competitor mixing, centrifugation, i.e.,
Obtain antigen samples.
Preferably, the competitor is at least one in amino acid and its derivative.
Preferably, the competitor is the one kind in lysine, arginine, phenylalanine, histidine and proline.
Preferably, the addition of the competitor is：1-40mg competitors are added in per 1ml oil-adjuvant vaccines, more preferably
1-20mg competitors.The excessive concentration of the competitor, the agent saturation that can constitute competition is separated out, and influences ultra-filtration process；Concentration is too low,
Can be constituted competition effect on driving birds is not good, it is impossible to the antigen of release detection enough.
Preferably, the oil-adjuvant vaccine and the volume ratio of n-butanol are 9:1-5:5.
It is highly preferred that the oil-adjuvant vaccine is 1 with the volume ratio of n-butanol:1.The volume of oil-adjuvant vaccine and n-butanol
It is identical, can preferably ensure that vaccine is demulsified completely.
Using described breaking method, because rate of recovery of antigen is high, the antigen samples for obtaining need not be further purified, you can
It is directly used in the quantitative and qualitative detection of antigen.
Preferably, the step of competitive ELISA method is used is as follows：
Antigen coat：The artificial synthesized foot-and-mouth disease antigen immobilization of concentration known is adsorbed onto on elisa plate；
Dilution antibody：The antibody of known potency is diluted, potency is 1 after antibody dilution:250000-1:1100000；
Antigen diluent：On serum-dilution plate, antigen samples to be checked are diluted with dilution, standard antigen is carried out not on year-on-year basis
Example dilution forms the standard antigen dilution of various concentrations；
The quantitative determination of antigen Specification Curve of Increasing and antigen samples to be checked：The antigen samples to be checked and difference that will have been diluted
The standard antigen dilution of concentration is reacted completely with the antibody for having diluted in plate is diluted respectively, is then reacted each completely
Reaction solution afterwards is added separately to continue to react in the elisa plate of immobilization antigen；It is each to add enzyme mark after reaction terminates
Thing, substrate react successively, then each addition terminate liquid terminates, and determines OD values, is drawn using the OD values of the standard antigen of various concentrations
Antigen standard curve, determined antigen sample is calculated during the OD values of antigen samples to be checked then are corresponded into the antigen standard curve
Product concentration, multiplied by with extension rate, divided by demulsification efficiency, as antigen actual content in determined antigen sample.
Preferably, in antigen coat step, the artificial synthesized foot-and-mouth disease antigen concentration is 0.5 μ g/mL-5 μ g/mL.
Preferably, it is described to react completely in the quantitative determination step of antigen Specification Curve of Increasing and antigen samples to be checked
Time is more than or equal to 60min, and reaction temperature is 37 DEG C.The reaction time can as far as possible ensure that reaction is complete.
Preferably, it is described each to react completely in the quantitative determination step of antigen Specification Curve of Increasing and antigen samples to be checked
Reaction solution afterwards is added separately to continue to react more than or equal to 30min in the elisa plate of immobilization antigen, and reaction temperature is 37
℃.If the reaction time is less than 30min, reaction can be caused not thorough, final OD values are relatively low, influence the accuracy of result.
Preferably, in the quantitative determination step of antigen Specification Curve of Increasing and antigen samples to be checked, the enzyme marker is
SPA-HRP, or be goat-anti pig biotin-IgG and Avidin-HRP.
It is highly preferred that the enzyme marker is goat-anti pig biotin-IgG and Avidin-HRP.Using goat-anti pig biotin-
IgG and Avidin-HRP due to the strong bonded of high-affinity between biotin-labeled pentylamine and multistage amplifies as enzyme marker
Effect, can make the test limit of antigen lower.
Preferably, the consumption of enzyme marker is 100 μ L after the dilution, and dilution ratio is 1:5000-1:10000.
Preferably, the substrate is：TMB solution.
Preferably, the terminate liquid is the H of 2mol/L2SO4Solution.
Principle of the invention is：Antigen binding site is competed with surfactant by adding competitor, so as to oil be helped
Antigen in vaccinating agent is discharged into water phase, so as to substantially increase the rate of recovery of antigen in water phase.To obtain after demulsification again
Water phase antigen samples use competitive ELISA method, antigen samples is first reacted in plate is diluted with the antibody of Finite Concentration, wait to resist
After antigen-antibody reaction is complete, reaction solution is added to during solid phase adsorption has elisa plate of antigen and is reacted, enzyme is added afterwards
Label is reacted, and is finally detected with substrate colour developing.It is higher that final testing result shows as antigen concentration, after colour developing
The OD values of detection are lower, inversely.Standard antigen is carried out into different proportion and dilutes the ELISA detection drafting standards that are at war with
Curve, detection sample is associated with standard curve so as to reach the requirement of qualitative, quantitative.
Prior art is compared, and the present invention has following beneficial effect：
1) present invention competes antigen binding site by adding competitor with surfactant, so as to by oil-adjuvant vaccine
Antigen discharge into water phase, compared with competitor is not added with, substantially increase the rate of recovery of antigen in water phase.
2) present invention uses competitive ELISA method, and first the antibody of antigen and Finite Concentration is first reacted in plate is diluted, and resists
Body is first neutralized completely with antigen to be checked, reaches the purpose of complete inhibition.In the elisa plate that subsequent immobilization is adsorbed with antigen,
Immobilization antigen is combined with antibody to be reduced therewith so that immobilization antigen, determined antigen and antibody combination unequal opportunities.Cause
This, the slope of standard curve for obtaining can be more than conventional indirect competitive, so as to greatly improve detection sensitivity.
2) after due to vaccine demulsification, water-phase component is more complicated.The method is simple, easy to operate, after demulsification aqueous sample without
It is measured by purification process need to being carried out, other detection methods that compare spend the time shorter.
3) requirements of the Competitive assays ELISA to reagent is considerably less, and either monoclonal antibody is still more anti-may be used to experiment, more
It is important that no matter checked object is the only one of which epitope such as macromolecular substances or polypeptide, small-molecule drug, small molecule hormone
Molecule can not can be detected using competitive ELISA using sandwich ELISA.
4) because antigen-antibody reaction has compared with high specific, when the interfering material in antigen-like material is difficult removal, or not
It is easy to get during to enough purifying antigens, detectable purpose is reached using competition law of the invention.It is dense after the dilution of simultaneous reactions antibody
Degree is relatively low, reduces the interference of non-specific responding and cross reaction.
5) present invention uses indirect method, and substantial amounts of catalytic reaction is can induce using minimal amount of enzyme, and generation is available for observation
Colour developing phenomenon so that antigen can be quantified under low concentration, and sensitivity is higher, and reproducible.
Brief description of the drawings
The detailed description made to non-limiting example with reference to the following drawings by reading, further feature of the invention,
Objects and advantages will become more apparent upon：
Fig. 1 is the antigen standard curve of embodiment 1；
Fig. 2 is the antigen standard curve of embodiment 2；
Fig. 3 is antigen concentration HPLC detection collection of illustrative plates in water phase after the method demulsification of comparative example 1.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that to the ordinary skill of this area
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Embodiment 1
A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine is present embodiments provided, specifically using following steps：
1) by commercially available Schweineseuche synthetic peptide vaccine (vaccine antigen concentration is 50 μ g/mL) as follows demulsification
Reason：
10ml vaccines to be checked are taken with n-butanol with volume ratio 1:1 mixing, adds 50mg histidines, and concussion is mixed, in 4 DEG C of bars
Under part, it is centrifuged 15 minutes with 3000r/min, lower floor's water phase is carefully extracted with 10ml syringes after centrifugation, obtains final product water phase antigen sample
Product.
The commercially available Schweineseuche synthetic peptide vaccine that the present embodiment is used is compareed with theoretical antigen concentration standard, to it
HPLC detection collection of illustrative plates samples go out peak position and are integrated, and as shown in table 1, antigenic content is integrating peak face in sample for its integration information
Long-pending form embodies, and its integrated peak areas is 1826453.To the present embodiment using antigen in water phase after Butanol+His demulsifications
The HPLC detection collection of illustrative plates samples of sample concentration go out peak position and are integrated, and its integration information is as shown in table 2, antigenic content in sample
Embodied in integrated peak areas form, its integrated peak areas is 1676683.The result for contrasting Tables 1 and 2 understands, both is integrated
After information contrast, its rate of recovery of antigen is 91.8%, that is, the efficiency that is demulsified is 91.8%.
Table 1
Table 2
2) competitive ELISA detection
2.1 antigen coats：By the 3 artificial synthesized foot-and-mouth disease antigens of μ g/mL, per the μ L of hole 100, on immobilization to elisa plate；
2.2 dilution antibody：The antibody of known potency is carried out into certain proportion dilution, antibody titer is 1 after dilution:
250000；
2.3 antigens (antigen to be checked is the water phase antigen samples that step 1 is obtained) to be checked and standard antigen dilution：In blood
It is thin to release on plate, antigen to be checked is pressed 1 with dilution:100 dilution, standard antigen respectively by dilution after concentration be 1000ng/mL,
500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 10ng/mL, 5ng/mL are diluted；
2.4 antigen-antibody reactions：In plate is diluted, add respectively in antigen to be checked and standard antigen that 100 μ L have been diluted
Enter antibody that equivalent diluted and mix, in being incubated 60min at 37 DEG C；
After 2.5 incubations terminate, each reaction solution in serum-dilution plate is drawn into 100 μ L respectively has antigen to immobilization
In elisa plate, in being incubated 30min at 37 DEG C；
After 2.6 are incubated and terminate, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ L in elisa plate
1:10000 SPA-HRP for having diluted, in being incubated 30min at 37 DEG C；
After 2.7 are incubated and terminate, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ L in elisa plate
TMB solution, in lucifuge colour developing 15min at 37 DEG C；
After 2.8 colour developings terminate, to adding 100 μ L 2mol/LH in elisa plate2SO4Solution terminating reaction, and in 450nm ripples
Lower measure OD values long；
2.9 according to the OD values for determining, by each standard antigen concentration using 10 be bottom logarithm as abscissa, the survey of each standard antigen
The OD450 for obtaining is ordinate, carries out linear regression, obtains the regression equation of standard curve for y=-0.3611x+1.4586, such as
Shown in Fig. 1；The OD values of antigen to be checked are brought into standard curve and calculates correspondence antigen concentration to be measured, antigen actual content=[(right
Answer antigen concentration × extension rate)/demulsification efficiency]/2 be sample in antigen actual content.Antigen is actual as in sample contains
Amount.In the present embodiment, it is 0.404 to measure the OD values of antigen to be checked, and the actual content for calculating antigen in sample is 45.30 μ g/
mL。
Note：After due to vaccine demulsification, oil phase is separated from the water, and actual antigen concentration increases 1 times in water phase, calculates vaccine reality
Border antigen concentration need to be divided by 2.
Embodiment 2
A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine is present embodiments provided, specifically using following steps：
1) by commercially available Schweineseuche synthetic peptide vaccine (vaccine antigen concentration is 50 μ g/mL) as follows demulsification
Reason：
10ml vaccines to be checked are taken with n-butanol with volume ratio 1:1 mixing, adds 10mg phenylalanines, and concussion is mixed, at 4 DEG C
Under the conditions of, it is centrifuged 15 minutes with 3000r/min, lower floor's water phase is carefully extracted with 10ml syringes after centrifugation, obtain final product water phase antigen
Sample.
Aftosa vaccine is demulsified using the method for the present embodiment, demulsification efficiency is 88.4%.
2) competitive ELISA detection
2.1 antigen coats：By the 3 artificial synthesized foot-and-mouth disease antigens of μ g/mL, per the μ L of hole 100, on immobilization to elisa plate；
2.2 dilution antibody：The antibody of known potency is carried out into certain proportion dilution, antibody titer is 1 after dilution:
1100000；
2.3 antigens (antigen to be checked is the water phase antigen samples that step 1 is obtained) to be checked and standard antigen dilution：In blood
It is thin to release on plate, antigen to be checked is pressed 1 with dilution:100 dilutions, it is known that standard antigen is diluted, and diluted concentration is respectively
1000ng/mL、500ng/mL、200ng/mL、100ng/mL、50ng/mL、10ng/mL、5ng/mL、1ng/mL；
2.4 antigen-antibody reactions：In plate is diluted, added in antigen to be checked and standard antigen that 100 μ L have been diluted etc.
The antibody that has diluted of amount is simultaneously mixed, in being incubated 60min at 37 DEG C；
After 2.5 incubations terminate, the reaction solution in serum-dilution plate is drawn into 100 μ L the elisa plate of antigen to immobilization
In, in being incubated 30min at 37 DEG C；
After 2.6 are incubated and terminate, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ L in elisa plate
1:The 10000 goat-anti pig biotin-IgG for having diluted, in being incubated 30min at 37 DEG C；
After 2.7 are incubated and terminate, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ L in elisa plate
1:5000 Avidin-the HRP for having diluted, in being incubated 30min at 37 DEG C；
After 2.8 are incubated and terminate, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ L in elisa plate
TMB solution, in lucifuge colour developing 15min at 37 DEG C；
After 2.9 colour developings terminate, to adding 100 μ L 2mol/LH in elisa plate2SO4Solution terminating reaction, and in 450nm ripples
Lower measure OD values long；
2.10 according to the OD values for determining, with standard antigen concentration using 10 be bottom logarithm as abscissa, OD450 sits for vertical
Mark, carries out linear regression, obtains the regression equation of standard curve for y=-0.4001x+1.5138, as shown in Figure 2；Will be to be checked anti-
Former OD values are brought into standard curve and calculate correspondence antigen concentration to be measured, antigen actual content=[(correspondence antigen concentration × dilution
Multiple)/demulsification efficiency]/2 be sample in antigen actual content.In the present embodiment, it is 0.338 to measure the OD values of antigen to be checked,
The actual content for calculating antigen in sample is 49.26 μ g/mL.
Note：After due to vaccine demulsification, oil phase is separated from the water, and actual antigen concentration increases 1 times in water phase, calculates vaccine reality
Border antigen concentration need to be divided by 2.
10 Duplicate Samples of the sample are carried out using the present embodiment method determining simultaneously, the fluctuation range of its result ±
In 0.80, illustrate that its repeatability is good.
Embodiment 3
A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine is present embodiments provided, specifically using following steps：
1) by the commercially available Schweineseuche synthetic peptide vaccine (vaccine antigen concentration is 50 μ g/mL) described in embodiment 1 according to
Following method demulsification treatment：
10ml vaccines to be checked are taken with n-butanol with volume ratio 1:1 mixing, often pipe addition 200mg proline, concussion is mixed,
Under the conditions of 4 DEG C, it is centrifuged 15 minutes with 3000r/min, lower floor's water phase is carefully extracted with 10ml syringes after centrifugation, is obtained final product water and mutually resist
Raw sample.
Aftosa vaccine is demulsified using the method for the present embodiment, demulsification efficiency is 95.6%.
2) competitive ELISA detection
The competitive ELISA method of use is same as Example 2.The actual content for calculating antigen in sample is 48.96 μ g/
mL。
Comparative example 1
Present embodiments provide a kind of fast qualitative quantitative determination side of oil-adjuvant vaccine (using the vaccine of embodiment 1)
Method, the method with embodiment 1 is essentially identical, the difference is that only：Competitor is added without in this comparative example.
The antigen actual content for using the method for this comparative example to measure in water phase is zero.The comparative example is demulsified using n-butanol
Antigen concentration HPLC detection collection of illustrative plates in water phase afterwards is as shown in figure 3, from this figure it can be seen that in antigen theory retention time simultaneously
Antigen is not detected, illustrates that the amount of antigen contained in sample is extremely low, rate of recovery of antigen is substantially zeroed, its demulsification efficiency is substantially zeroed.
Therefore subsequent detection requirement cannot be met.
Concrete application approach of the present invention is a lot, and the above is only the preferred embodiment of the present invention.It should be pointed out that more than
Embodiment is merely to illustrate the present invention, and the protection domain being not intended to limit the invention.For the common skill of the art
For art personnel, under the premise without departing from the principles of the invention, some improvement can also be made, these improvement also should be regarded as this hair
Bright protection domain.
Claims (10)
1. the fast qualitative quantitative detecting method of a kind of oil-adjuvant vaccine, it is characterised in that comprise the following steps：Aftosa is closed
Into peptide vaccine demulsification, the antigen samples after demulsification are carried out into qualitative and quantitative detection using competitive ELISA method；
The breaking method is：Oil-adjuvant vaccine is mixed with n-butanol, after being subsequently adding competitor mixing, centrifugation is obtained final product anti-
Raw sample.
2. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, it is characterised in that the competition
Agent is at least one in amino acid and its derivative.
3. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 2, it is characterised in that the competition
Agent is the one kind in lysine, arginine, histidine and proline.
4. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, it is characterised in that the competition
The addition of agent is：1-40mg competitors are added in per 1ml oil-adjuvant vaccines.
5. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, it is characterised in that the competition
The step of ELISA method is used is as follows：
Antigen coat：The artificial synthesized foot-and-mouth disease antigen immobilization of concentration known is adsorbed onto on elisa plate；
Dilution antibody：The antibody of known potency is diluted, potency is 1 after antibody dilution:250000-1:1100000；
Antigen diluent：On serum-dilution plate, antigen samples to be checked are diluted with dilution, it is dilute that standard antigen carries out different proportion
Release the standard antigen dilution to form various concentrations；
The quantitative determination of antigen Specification Curve of Increasing and antigen samples to be checked：The antigen samples to be checked and various concentrations that will have been diluted
Standard antigen dilution reacted completely in plate is diluted with the antibody for having diluted respectively, then will be each complete reacted
Reaction solution is added separately to continue to react in the elisa plate of immobilization antigen；After reaction terminates, enzyme marker, bottom are respectively added
Thing reacts successively, then each addition terminate liquid terminates, and determines OD values, and antigen mark is drawn using the OD values of the standard antigen of various concentrations
Directrix curve, then the OD values of antigen samples to be checked to be corresponded to calculate determined antigen sample in the antigen standard curve dense
Degree, multiplied by with extension rate, divided by demulsification efficiency, as antigen actual content in determined antigen sample.
6. the fast qualitative quantitative detecting method according to claim 5 oil-adjuvant vaccine, it is characterised in that antigen coat step
In, the artificial synthesized foot-and-mouth disease antigen concentration is 0.5 μ g/mL-5 μ g/mL.
7. the fast qualitative quantitative detecting method according to claim 5 oil-adjuvant vaccine, it is characterised in that antigen standard curve is painted
In the quantitative determination step of system and antigen samples to be checked, the time reacted completely is more than or equal to 60min, and reaction temperature is 37
℃。
8. the fast qualitative quantitative detecting method according to claim 5 oil-adjuvant vaccine, it is characterised in that antigen standard curve is painted
In the quantitative determination step of system and antigen samples to be checked, each reacted reaction solution completely is added separately to immobilization antigen
Elisa plate in continue react be more than or equal to 30min, reaction temperature be 37 DEG C.
9. the fast qualitative quantitative detecting method according to claim 5 oil-adjuvant vaccine, it is characterised in that antigen standard curve is painted
In the quantitative determination step of system and antigen samples to be checked, the enzyme marker is SPA-HRP, or is goat-anti pig biotin-IgG
With Avidin-HRP.
10. the competitive ELISA quantitative approach after aftosa synthetic peptide vaccine according to claim 1 is demulsified, its feature exists
In the consumption of enzyme marker is 100 μ L after the dilution, and dilution ratio is 1:5000-1:10000.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611123598.9A CN106771189B (en) | 2016-12-08 | 2016-12-08 | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611123598.9A CN106771189B (en) | 2016-12-08 | 2016-12-08 | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106771189A true CN106771189A (en) | 2017-05-31 |
| CN106771189B CN106771189B (en) | 2018-08-10 |
Family
ID=58877482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611123598.9A Active CN106771189B (en) | 2016-12-08 | 2016-12-08 | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106771189B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110554188A (en) * | 2019-10-23 | 2019-12-10 | 北京智飞绿竹生物制药有限公司 | Method for detecting content of adjuvant adsorption component vaccine |
| CN111273035A (en) * | 2020-03-06 | 2020-06-12 | 中国农业科学院兰州兽医研究所 | Chemiluminescence kit for quantitatively detecting non-structural protein residues in foot-and-mouth disease inactivated vaccine and detection method thereof |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0641853A2 (en) * | 1993-08-16 | 1995-03-08 | Exxon Chemical Patents Inc. | Method of demulsifying water-in-oil emulsions |
| WO2010114552A1 (en) * | 2009-04-03 | 2010-10-07 | Kroff Chemical Company, Inc. | Demulsification compositions, systems and methods for demulsifying and separating aqueous emulsions |
| CN102380232A (en) * | 2011-06-30 | 2012-03-21 | 金宇保灵生物药品有限公司 | Emulsion breaking method for aftosa oil emulsion inactivated vaccine |
| CN103076451A (en) * | 2013-01-17 | 2013-05-01 | 中国农业科学院兰州兽医研究所 | O type foot-and-mouth disease 146S antigen quantitative ELISA detection kit and method for using same |
| CN103088157A (en) * | 2013-01-11 | 2013-05-08 | 金宇保灵生物药品有限公司 | Method for identifying and quantifying serotype of antigens in bivalent vaccines and multi-valence vaccines of foot-and-mouth disease |
| CN103235139A (en) * | 2013-04-11 | 2013-08-07 | 肇庆大华农生物药品有限公司 | A rapid antigen detection method for inactivated oil emulsion vaccine against avian influenza finish products |
| CN104634891A (en) * | 2015-01-19 | 2015-05-20 | 中国科学院过程工程研究所 | Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S |
| CN105467138A (en) * | 2015-12-04 | 2016-04-06 | 中国农业科学院兰州兽医研究所 | Method for determining components and estimating anti-gen content of foot-and-mouth disease vaccine |
-
2016
- 2016-12-08 CN CN201611123598.9A patent/CN106771189B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0641853A2 (en) * | 1993-08-16 | 1995-03-08 | Exxon Chemical Patents Inc. | Method of demulsifying water-in-oil emulsions |
| WO2010114552A1 (en) * | 2009-04-03 | 2010-10-07 | Kroff Chemical Company, Inc. | Demulsification compositions, systems and methods for demulsifying and separating aqueous emulsions |
| CN102380232A (en) * | 2011-06-30 | 2012-03-21 | 金宇保灵生物药品有限公司 | Emulsion breaking method for aftosa oil emulsion inactivated vaccine |
| CN103088157A (en) * | 2013-01-11 | 2013-05-08 | 金宇保灵生物药品有限公司 | Method for identifying and quantifying serotype of antigens in bivalent vaccines and multi-valence vaccines of foot-and-mouth disease |
| CN103076451A (en) * | 2013-01-17 | 2013-05-01 | 中国农业科学院兰州兽医研究所 | O type foot-and-mouth disease 146S antigen quantitative ELISA detection kit and method for using same |
| CN103235139A (en) * | 2013-04-11 | 2013-08-07 | 肇庆大华农生物药品有限公司 | A rapid antigen detection method for inactivated oil emulsion vaccine against avian influenza finish products |
| CN104634891A (en) * | 2015-01-19 | 2015-05-20 | 中国科学院过程工程研究所 | Method for rapidly, accurately and repeatedly determining foot-and-mouth disease vaccine antigen 146S |
| CN105467138A (en) * | 2015-12-04 | 2016-04-06 | 中国农业科学院兰州兽医研究所 | Method for determining components and estimating anti-gen content of foot-and-mouth disease vaccine |
Non-Patent Citations (3)
| Title |
|---|
| ANDREA PENWELL等: "Development and validation of an HPLC-UV assay for separation and quantification of peptide antigens from a liposomal vaccine delivery platform", 《JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS》 * |
| 张怀宇: "Asia_型口蹄疫疫苗效力检验替代方法的初步研究", 《中国动物检疫》 * |
| 马丽等: "口蹄疫灭活疫苗蛋白质含量测定操作程序的确立", 《中国兽药杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110554188A (en) * | 2019-10-23 | 2019-12-10 | 北京智飞绿竹生物制药有限公司 | Method for detecting content of adjuvant adsorption component vaccine |
| CN111273035A (en) * | 2020-03-06 | 2020-06-12 | 中国农业科学院兰州兽医研究所 | Chemiluminescence kit for quantitatively detecting non-structural protein residues in foot-and-mouth disease inactivated vaccine and detection method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106771189B (en) | 2018-08-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100420947C (en) | Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor | |
| CN102928606B (en) | The Procalcitonin quick detection kit of multispecific antibody mark | |
| JP6756611B2 (en) | Competitive ligand binding assay to detect neutralizing antibodies | |
| CN102288765B (en) | Immunofluorescence chloramphenicol detecting method based on quantum dots and special kit | |
| CN103293299B (en) | Widen method and the test kit thereof of double-antibody sandwich immunodetection concentration range | |
| CN108152512A (en) | Heparin-binding protein detection kit and preparation method thereof | |
| CN111273017B (en) | Fluorescent immunochromatography kit for rapidly detecting novel coronaviruses | |
| CN107677808A (en) | A kind of immue quantitative detection reagent box of thyroglobulin and preparation method thereof and detection method | |
| US20240002959A1 (en) | Rna separation and related techniques for determining viruses such as coronaviruses | |
| CN106706924A (en) | Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine | |
| CN106771189B (en) | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine | |
| JP2020506395A5 (en) | ||
| CN113321715A (en) | Novel coronavirus antigen and detection use thereof | |
| CN107064492B (en) | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine | |
| EP3147371B1 (en) | Novel method for detecting detection object in sample, and detection kit using same | |
| JP2008275511A (en) | Method for measuring immunity of influenza virus antigen and device used for the same | |
| US20040241700A1 (en) | Calibrating microarrays | |
| CN106596875B (en) | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine | |
| CN105315166B (en) | The preparation method and its application in quantum dot immune fluorescent kit of salbutamol haptens and antigen | |
| CN103454418B (en) | A kind of Fasciola gigantica immune chromatography rapid detecting test paper strip and preparation method thereof | |
| CN106596876A (en) | Method for accurate qualitative and quantitative detection of oil adjuvant vaccine | |
| CN107436350B (en) | A kind of separation method carrying out each ingredient in mixture using ELISA Plate and monoclonal antibody | |
| CN112129933A (en) | Reagent, kit and method for resisting biotin interference in immunoassay system | |
| CN110055336A (en) | A kind of kit whether quick identification yak milk is adulterated | |
| WO2014122974A1 (en) | Vitamin d measurement method using chaotropic agent |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |