CN107064492B - 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
- CN107064492B CN107064492B CN201611123580.9A CN201611123580A CN107064492B CN 107064492 B CN107064492 B CN 107064492B CN 201611123580 A CN201611123580 A CN 201611123580A CN 107064492 B CN107064492 B CN 107064492B
- Authority
- CN
- China
- Prior art keywords
- antigen
- dilution
- oil
- acid
- checked
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The present invention provides a kind of fast qualitative quantitative detecting methods of oil-adjuvant vaccine, comprising the following steps: aftosa synthetic peptide vaccine is demulsified, the antigen samples after demulsification are carried out qualitative and quantitative detection using competitive ELISA method；The breaking method are as follows: aftosa synthetic vaccine is mixed with demulsifier, water phase antigen samples are obtained after layering；The demulsifier includes the mixture of organic solvent and acid, and the organic solvent is selected from one of acetonitrile, n-butanol, benzyl alcohol, butyl diglycol, ethyl alcohol, and the acid is selected from one of hydrochloric acid, acetic acid, formic acid, trifluoroacetic acid.Breaking method demulsification ability provided by the invention is stronger, measure that sample can without carrying out purification process, shortens detection time；The present invention carries out qualitative and quantitative detection antigen concentration using competitive ELISA method, and obtained slope of standard curve can be greater than conventional indirect competitive, to substantially increase detection sensitivity.
Description
Technical field
The present invention relates to oil-adjuvant vaccine detection technique fields, and in particular to a kind of fast qualitative of oil-adjuvant vaccine is quantitative
Detection method.
Background technique
Regulation efficacy test must be tested using this animal in currently available vaccines, existing vaccines quality standard, since country carries out
100% reinforced immunological policy is difficult to select susceptible inspection with animal, and to attack poison high to Experimental Establishment requirement for animal
(BSL3 grades of laboratories), time-consuming (one month or more), capital cost is big.If selecting impressibility using serum neutralization test
Object is technically difficult to the non-susceptible animal for excluding to have cellular immunity, and in inspection, inspection data is not advised frequent occurrence in practice
The problem of rule, influences the accuracy examined.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 it needs to develop tested in vitro technology as early as possible, be tried instead of existing animal
It tests.
Current country is just gradually being transitioned into the detection to vaccine endoantigen to inspections of vaccine, and currently more universal side
Method is to be detected after vaccine demulsification to antigen, by the way that vaccine is carried out demulsification processing, is transferred to antigen in water phase, then right
It carries out subsequent detection and analysis.Complicated component in the oil adjuvant as used in vaccine emulsion process, contains surface-active
Agent, the substances such as immunopotentiator exist, and often contain above-mentioned impurity and demulsifier in the water phase after demulsification, in industry not
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
It covers or interferes at signal, the intensity of antigen signals has been forced down, or even can not effectively detect antigen therein, due to wherein miscellaneous
The being randomly assigned property of matter causes the repeatability of its detection method bad, while increasing instrument maintenance cost.
Aftosa synthetic peptide vaccine is a kind of protection synthesized by manual method by the amino acid sequence of native protein
Property small peptide.Existing method for quantitatively determining usually has the methods of Lowry method, BCA method, HPLC.And in existing quantitative approach, due to
After vaccine demulsification, vaccine water-phase component is more complex, and measuring samples, which usually require to take considerable time after purification, can be only achieved detection
It is required that and much conventional method detection sensitivity is limited.
Enzyme-linked immunosorbent assay (ELISA) together quickly, conveniently, specifically, the advantages that can quantifying, in many fields
To extensive use.In conventional indirect competitive ELISA, the antibody of antigen and Finite Concentration is added to solid phase adsorption simultaneously antigen
Elisa plate in reacted, antibody and free antigen, immobilised antigen binding rate probability are 50%, obtained knot
The slope of standard curve that fruit is drawn is lower, this makes detection sensitivity lower.Aftosa synthetic peptide vaccine cannot be reacted well
Middle Effective Antigens content.
Currently, there is no general accurate method for aftosa synthetic peptide vaccine antigenic content quantitative detection.It is badly in need of research and development
Always more quickly, effectively, the method for qualitative and quantitative detection of convenient oil-adjuvant vaccine.
Summary of the invention
For the defects in the prior art, the present invention provides a kind of fast qualitative quantitative detection sides of oil-adjuvant vaccine
Method.By the way that aftosa synthetic peptide vaccine is demulsified using special breaking method, 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 what is be achieved through the following technical solutions: a kind of fast qualitative quantitative detection of oil-adjuvant vaccine
Method, comprising the following steps: aftosa synthetic peptide vaccine is demulsified, the antigen samples after demulsification are used into competitive ELISA method
Carry out qualitative and quantitative detection；
The breaking method are as follows: aftosa synthetic vaccine is mixed with demulsifier, water phase antigen samples are obtained after layering；It is described
Demulsifier includes the mixture of organic solvent and acid, and the organic solvent is selected from acetonitrile, n-butanol, benzyl alcohol, butyl diethyl two
One of alcohol, ethyl alcohol, the acid are selected from one of hydrochloric acid, acetic acid, formic acid, trifluoroacetic acid.Water intaking is mutually examined through liquid chromatogram
It surveys, rate of recovery of antigen is up to 90% or more.
Preferably, organic solvent is selected from one of acetonitrile, ethyl alcohol in demulsifier, and acid is in trifluoroacetic acid, hydrochloric acid
It is a kind of.
Preferably, 9:1~5:5 is mixed oil-adjuvant vaccine by volume with demulsifier.Demulsifier too high levels can increase
Add the subsequent processing time for being lyophilized or being concentrated to aqueous sample, it is too low, it can not be demulsified.
Preferably, the volume ratio of acid and organic solvent is (0.05~0.5) in the demulsifier: 100.
Using the breaking method, due to rate of recovery of antigen height, obtained antigen samples are without being further purified
It is directly used in the quantitative and qualitative detection of antigen.
Preferably, the step of competitive ELISA method uses is as follows:
Antigen coat: the artificial synthesized foot-and-mouth disease antigen immobilization of known concentration is adsorbed on elisa plate；
Dilution antibody: the antibody of known potency is diluted, and potency is 1:250000-1:1100000 after antibody dilution；
Antigen diluent: on serum dilution plate, antigen samples to be checked are diluted with dilution, standard antigen carries out not year-on-year
Example dilution forms the standard antigen dilution of various concentration；
The quantitative detection of antigen Specification Curve of Increasing and antigen samples to be checked: the antigen samples and difference to be checked that will have been diluted
The standard antigen dilution of concentration is reacted in dilution plate with the antibody diluted completely respectively, then by each complete reaction
Reaction solution afterwards be added separately to immobilization antigen elisa plate in the reaction was continued；After reaction, enzyme label is respectively added
Object, substrate successively react, then each terminate liquid that is added terminates, and measures OD value, is drawn using the OD value of the standard antigen of various concentration
Then the OD value of antigen samples to be checked is corresponded in the antigen standard curve and calculates to obtain determined antigen sample by antigen standard curve
Product concentration, multiplied by extension rate, divided by demulsification efficiency, being that antigen is practical in determined antigen sample divided by dilution effect contains
Amount.
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, described to react completely in the quantitative detection 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 guarantee fully reacting as far as possible.
Preferably, in the quantitative detection step of antigen Specification Curve of Increasing and antigen samples to be checked, each complete reaction
Reaction solution afterwards be added separately to immobilization antigen elisa plate in the reaction was continued be more than or equal to 30min, reaction temperature 37
℃.If the reaction time is less than 30min, it will lead to reaction and be not thorough, final OD value is relatively low, influences the accuracy of result.
Preferably, in the quantitative detection 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 is used as enzyme marker, due to the firm connection of high-affinity between biotin-labeled pentylamine and multistage amplification
Effect can be such that the detection of antigen limits lower.
Preferably, the dosage of enzyme marker is 100 μ L, dilution ratio 1:5000-1:10000 after the dilution.
Preferably, the substrate are as follows: TMB solution.
Preferably, the terminate liquid is the H of 2mol/L2SO4Solution.
The principle of the present invention is: acetonitrile has very strong dissolution, energy to emulsifying film as a kind of splendid organic solvent
It reaches and dissolves vaccine emulsifying film, to release antigen water phase therein, antigen is soluble in acetonitrile, and acetonitrile is miscible with water, can incite somebody to action
Antigen is brought into water phase, and the rate of recovery of antigen in water phase is substantially increased.Trifluoroacetic acid promotes demulsification similar to catalyst, accelerates
Emulsifying film cracks, and reduces the dosage of acetonitrile.The water phase antigen samples obtained after demulsification are used into competitive ELISA method again, are made
Antigen samples are first reacted in dilution plate with the antibody of Finite Concentration, and after antigen-antibody reaction is complete, reaction solution is added to
Solid phase adsorption have antigen elisa plate in reacted, later be added enzyme marker reacted, finally develop the color with substrate into
Row detection.Final testing result shows as that antigen concentration is higher, and the OD value detected after colour developing is lower, inversely.It will mark
Quasi- antigen carries out the different proportion dilution ELISA detection that is at war with and draws standard curve, test sample be associated with standard curve from
And reach the requirement of qualitative, quantitative.
The prior art is compared, the present invention have it is following the utility model has the advantages that
1) breaking method demulsification ability provided by the invention is stronger, can guarantee that antigen is distributed mainly in water phase, and water phase
Can be mutually kept completely separate with oily, can actual response go out the content and quality of Effective Antigens in vaccine, rate of recovery of antigen up to 90% with
On.
2) present invention uses competitive ELISA method, first first reacts antigen in dilution plate with the antibody of Finite Concentration, resists
Body and antigen elder generation to be checked complete neutralization, achieve the purpose that complete inhibition.In the elisa plate that subsequent immobilization is adsorbed with antigen,
Immobilization antigen is reduced therewith in conjunction with antibody, so that immobilization antigen, the determined antigen unequal opportunities in conjunction with antibody.Cause
This, obtained slope of standard curve can be greater than conventional indirect competitive, to greatly improve detection sensitivity.
3) after due to vaccine demulsification, water-phase component is more complex.This method is simple, easy to operate, after demulsification aqueous sample without
Need to carry out purification process can be measured, other detection methods that compare spend the time shorter.
4) since antigen-antibody reaction has compared with high specific, when the interfering substance in antigen-like material is not easy to remove, or not
When being easy to get to enough purifying antigens, detectable purpose is reached using competition law of the invention.It is dense after the dilution of simultaneous reactions antibody
It spends lower, reduces the interference of non-specific responding and cross reaction.
5) present invention uses indirect method, can induce a large amount of catalysis reaction using minimal amount of enzyme, generates for observation
Develop the color phenomenon, antigen is quantified under low concentration, sensitivity is higher, and reproducible.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other 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 test map in water phase after the method demulsification of comparative example 1.
Specific embodiment
The present invention is described in detail combined with specific embodiments below.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill of this field
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 scope.
Embodiment 1
1) selecting commercially available foot and mouth disease vaccine seedling (vaccine concentration is 50 μ g/mL), demulsification is handled as follows:
Vaccine to be checked is mixed with demulsifier, organic solvent selects acetonitrile in the demulsifier, and acid selects trifluoroacetic acid, second
The volume ratio of nitrile and trifluoroacetic acid is 100:0.05.8ml vaccine to be checked is taken to be placed in centrifuge tube, vaccine and demulsifier are with volume ratio
8:2 mixing, concussion mixes, and under the conditions of 4 DEG C, with 3000G centrifugation 15 minutes, carefully extracts lower layer with 10ml syringe after centrifugation
Water phase.After liquid-phase chromatographic analysis, compared with antigen concentration standard theoretical in vaccine, wherein theoretical antigen is dense in vaccine
The integration information that the HPLC test map sample of degree goes out peak position is as shown in table 1, using water after the demulsification of the present embodiment breaking method
In phase the HPLC test map sample of antigen samples concentration go out peak position integration information it is as shown in table 2.It analyzes, uses by contrast
This method rate of recovery of antigen is 91.7%, i.e., demulsification efficiency is 91.7%, and compared to existing method, rate of recovery of antigen mentions significantly
It is high.
Table 1
Table 2
Note:
Vaccine is emulsified by adjuvant and antigen, and the volume ratio of adjuvant and antigen is 1:1, therefore the water phase obtained after being demulsified is
The 50% of vaccine volume to be checked, it is miscible with water by demulsifier in this present embodiment, therefore its water phase after the method for the present invention is demulsified
Volume has more the volume of demulsifier, i.e. diluting effect compared with vaccine inner aqueous phase, therefore the more theoretical water phase of water phase (4ml) in the present embodiment
Increase 50% (2ml), the integral result for the water phase detection being demulsified in the present embodiment should calculate dilution effect, i.e. detection integral
As a result divided by 66.67%.
2) competitive ELISA detects
2.1 antigen coats: by the 3 artificial synthesized foot-and-mouth disease antigens of μ g/mL, every 100 μ L of hole, on immobilization to elisa plate；
2.2 dilution antibody: the antibody of known potency is subjected to 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 obtains) to be checked dilute with standard antigen: in blood
It is thin to release on plate, with dilution by antigen to be checked by 1:100 dilute, standard antigen press respectively 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 dilution plate, add respectively in antigen and standard antigen to be checked that 100 μ L have been diluted
Enter the antibody that equivalent has diluted and mixing, is incubated for 60min at 37 DEG C；
After 2.5 are incubated for, each reaction solution in serum dilution plate is drawn 100 μ L respectively has antigen to immobilization
In elisa plate, 30min is incubated at 37 DEG C；
After 2.6 are incubated for, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
The SPA-HRP that 1:10000 has diluted is incubated for 30min at 37 DEG C；
After 2.7 are incubated for, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
TMB solution is protected from light colour developing 15min at 37 DEG C；
After 2.8 colour developings, 100 μ L 2mol/LH are added into elisa plate2SO4Solution terminates reaction, and in 450nm wave
Long lower measurement OD value；
2.9 are used as abscissa, each standard antigen survey using 10 according to the OD value of measurement, by each standard antigen concentration for bottom logarithm
The OD450 obtained is ordinate, carries out linear regression, and the regression equation for obtaining standard curve is y=-0.357x+1.448, such as Fig. 1
It is shown；The OD value of antigen to be checked is brought into standard curve and calculates corresponding antigen concentration to be measured, antigen actual content=[(corresponding
Antigen concentration × extension rate)/(demulsification efficiency × dilution effect)]/it 2 is antigen actual content in sample.As in sample
Antigen actual content.In the present embodiment, the OD value for measuring antigen to be checked is 0.466, calculates to obtain the actual content of antigen in sample
For 45.99 μ g/mL.
Note: after vaccine demulsification, oil is mutually separated from the water, and practical antigen concentration increases 1 times in water phase, and it is real to calculate vaccine
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, following steps are specifically used:
1) selecting commercially available foot and mouth disease vaccine seedling (vaccine concentration is 50 μ g/mL), demulsification is handled as follows:
Vaccine to be checked is mixed with demulsifier, organic solvent selects acetonitrile in the demulsifier, and acid selects trifluoroacetic acid, second
The volume ratio of nitrile and trifluoroacetic acid is 100:0.05.5ml vaccine to be checked is taken to mix with demulsifier with volume ratio 5:5, concussion mixes,
Under the conditions of 4 DEG C, with 3000G centrifugation 15 minutes, water phase antigen samples are obtained.
It is demulsified using the method for the present embodiment to aftosa vaccine, the efficiency that must be demulsified is 90.3%, and dilution effect is
33.33%.
2) competitive ELISA detects
2.1 antigen coats: by the 3 artificial synthesized foot-and-mouth disease antigens of μ g/mL, every 100 μ L of hole, on immobilization to elisa plate；
2.2 dilution antibody: the antibody of known potency is subjected to 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 obtains) to be checked dilute with standard antigen: in blood
It is thin to release on plate, antigen to be checked is diluted with dilution by 1:100, 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 dilution plate, addition etc. in antigen and standard antigen to be checked that 100 μ L have been diluted
The antibody diluted and mixing are measured, is incubated for 60min at 37 DEG C；
After 2.5 are incubated for, the 100 μ L of reaction solution absorption in serum dilution plate are had to the elisa plate of antigen to immobilization
In, 30min is incubated at 37 DEG C；
After 2.6 are incubated for, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
The goat-anti pig biotin-IgG that 1:10000 has diluted, is incubated for 30min at 37 DEG C；
After 2.7 are incubated for, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
Avidin-the HRP that 1:5000 has diluted, is incubated for 30min at 37 DEG C；
After 2.8 are incubated for, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
TMB solution is protected from light colour developing 15min at 37 DEG C；
After 2.9 colour developings, 100 μ L 2mol/LH are added into elisa plate2SO4Solution terminates reaction, and in 450nm wave
Long lower measurement OD value；
2.10 are used as abscissa using 10 according to the OD value of measurement, with standard antigen concentration for bottom logarithm, and OD450 is vertical seat
Mark carries out linear regression, and the regression equation for obtaining standard curve is y=-0.403x+1.523, as shown in Figure 2；By antigen to be checked
OD value bring into standard curve and calculate corresponding antigen concentration to be measured, antigen actual content=[(corresponding antigen concentration × dilution times
Number)/(demulsification efficiency × dilution effect)]/it 2 is antigen actual content in sample.Antigen actual content as in sample.This reality
Apply in example, the OD value for measuring antigen to be checked is 0.528, calculate in sample the actual content of antigen be 49.02 μ g/mL.
Note: after vaccine demulsification, oil is mutually separated from the water, and practical antigen concentration increases 1 times in water phase, and it is real to calculate vaccine
Border antigen concentration need to be divided by 2.
10 Duplicate Samples of the sample are carried out while being measured using the present embodiment method, the fluctuation range of 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, following steps are specifically used:
1) selecting commercially available foot and mouth disease vaccine seedling (vaccine concentration is 50 μ g/mL), demulsification is handled as follows:
Vaccine to be checked is mixed with demulsifier, in the demulsifier organic solvent select ethyl alcohol, acid select hydrochloric acid, ethyl alcohol with
The volume ratio of hydrochloric acid is 100:0.33, and 7ml vaccine to be checked is taken to mix with demulsifier with volume ratio 7:3, and concussion mixes, in 4 DEG C of items
Under part, with 3000G centrifugation 15 minutes, water phase antigen samples are obtained.
It is demulsified using the method for the present embodiment to aftosa vaccine, demulsification efficiency is 93.5%, and dilution effect is
53.85%.
2) competitive ELISA detects
The competitive ELISA method of use is same as Example 2.Calculate in sample the actual content of antigen be 48.52 μ g/
mL。
Comparative example 1
Present embodiments provide a kind of qualitative and quantitative detection side of aftosa synthetic peptide vaccine (using the vaccine of embodiment 1)
Method, essentially identical with the method for embodiment 1, the difference is that only: this comparative example demulsifier uses n-butanol.
The method for using this comparative example measures the antigen actual content in water phase as zero.The comparative example is demulsified using n-butanol
Antigen concentration HPLC test map 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, the rate of recovery of antigen of this comparative example is substantially zeroed, and be demulsified efficiency
It is substantially zeroed.Therefore it is unable to satisfy subsequent detection requirement.
There are many concrete application approach of the present invention, the above is only a preferred embodiment of the present invention.More than it should be pointed out that
Embodiment is merely to illustrate the present invention, and the protection scope being not intended to restrict the invention.For the common skill of the art
For art personnel, without departing from the principle of the present invention, several improvement can also be made, these improvement also should be regarded as this hair
Bright protection scope.
Claims (7)
1. a kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine, which comprises the following steps: close aftosa
It is demulsified at peptide vaccine, the antigen samples after demulsification is subjected to qualitative and quantitative detection using competitive ELISA method；
The breaking method are as follows: aftosa synthetic vaccine is mixed with demulsifier, water phase antigen samples are obtained after layering；The demulsification
Agent includes the mixture of organic solvent and acid；In the demulsifier organic solvent select acetonitrile, acid select trifluoroacetic acid, acetonitrile with
The volume ratio of trifluoroacetic acid is 100:0.05；Alternatively, organic solvent selects ethyl alcohol in the demulsifier, acid selects hydrochloric acid, ethyl alcohol
Volume ratio with hydrochloric acid is 100:0.33；
9:1~5:5 is mixed oil-adjuvant vaccine by volume with demulsifier.
2. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 1, which is characterized in that the competition
The step of ELISA method uses is as follows:
Antigen coat: the artificial synthesized foot-and-mouth disease antigen immobilization of known concentration is adsorbed on elisa plate；
Dilution antibody: the antibody of known potency is diluted, and potency is 1:250000-1:1100000 after antibody dilution；
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 concentration；
The quantitative detection of antigen Specification Curve of Increasing and antigen samples to be checked: the antigen samples to be checked and various concentration that will have been diluted
Standard antigen dilution reacted completely in dilution plate with the antibody that has diluted respectively, then will be after each complete reaction
Reaction solution be added separately to immobilization antigen elisa plate in the reaction was continued；After reaction, enzyme marker, bottom is respectively added
Object successively reacts, then each terminate liquid that is added terminates, and measures OD value, draws antigen mark using the OD value of the standard antigen of various concentration
Directrix curve, then the OD value of antigen samples to be checked is corresponded in the antigen standard curve calculate determined antigen sample is dense
Degree, divided by demulsification efficiency, is antigen actual content in determined antigen sample divided by dilution effect multiplied by extension rate.
3. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 2, which is characterized in that antigen coat
In step, the artificial synthesized foot-and-mouth disease antigen concentration is 0.5 μ g/mL-5 μ g/mL.
4. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 2, which is characterized in that antigen standard
In the quantitative detection step of Drawing of Curve and antigen samples to be checked, the time reacted completely is more than or equal to 60min, reaction temperature
Degree is 37 DEG C.
5. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 2, which is characterized in that antigen standard
In the quantitative detection step of Drawing of Curve and antigen samples to be checked, the reaction solution after each complete reaction is added separately to solid phase
The reaction was continued in elisa plate of change antigen is more than or equal to 30min, and reaction temperature is 37 DEG C.
6. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 2, which is characterized in that antigen standard
In the quantitative detection step of Drawing of Curve and antigen samples to be checked, the enzyme marker is SPA-HRP, or is goat-anti pig biology
Element-IgG and Avidin-HRP.
7. the fast qualitative quantitative detecting method of oil-adjuvant vaccine according to claim 2, which is characterized in that the dilution
The dosage of enzyme marker is 100 μ L, dilution ratio 1:5000-1:10000 afterwards.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611123580.9A CN107064492B (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 |
|---|---|---|---|
| CN201611123580.9A CN107064492B (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 |
|---|---|
| CN107064492A CN107064492A (en) | 2017-08-18 |
| CN107064492B true CN107064492B (en) | 2019-04-23 |
Family
ID=59619668
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611123580.9A Active CN107064492B (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) | CN107064492B (en) |
Families Citing this family (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 |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0641853B1 (en) * | 1993-08-16 | 1998-10-21 | Nalco/Exxon Energy Chemicals L.P. | Method of demulsifying water-in-oil emulsions |
| CA2757568C (en) * | 2009-04-03 | 2016-08-30 | Kroff Chemical Company, Inc. | Demulsification compositions, systems and methods for demulsifying and separating aqueous emulsions |
| US10598666B2 (en) * | 2012-03-08 | 2020-03-24 | Glaxosmithkline Biologicals Sa | In vitro potency assay for protein-based meningococcal vaccines |
| CN103076451B (en) * | 2013-01-17 | 2016-01-20 | 中国农业科学院兰州兽医研究所 | A kind of O type aftosa 146S antigen quantify ELISA detection kit and using method thereof |
| CN105445462B (en) * | 2014-09-25 | 2017-04-12 | 金宇保灵生物药品有限公司 | Enzyme linked immunosorbent assay kit for specific quantitative detection of foot and mouth disease O type Guangxi strain antigen and application thereof |
-
2016
- 2016-12-08 CN CN201611123580.9A patent/CN107064492B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN107064492A (en) | 2017-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100420947C (en) | Method for quantitative determination of specific analyte with single trapping agent and reagent kit therefor | |
| Gomara et al. | Assessment of synthetic peptides for hepatitis A diagnosis using biosensor technology | |
| CN108333344A (en) | Highly sensitive chemical luminescence immune analysis reagent box and its preparation method and application | |
| US20220048025A1 (en) | Systems and methods to enhance consistency of assay performance | |
| CN106959372A (en) | Serum amyloid A protein and the two-in-one measure kit of C reactive proteins and preparation method | |
| CN107064492B (en) | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine | |
| CN106706924A (en) | Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine | |
| JP2020506395A5 (en) | ||
| CN106771189B (en) | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine | |
| Bang-Ce et al. | Simultaneous detection of sulfamethazine, streptomycin, and tylosin in milk by microplate-array based SMM–FIA | |
| Liu et al. | A supramolecular sensor array for selective immunoglobulin deficiency analysis | |
| KR20130090892A (en) | Co-coupling to control reactivity of reagents in immunoassays | |
| CN110596396A (en) | Method for detecting protein, test strip and kit | |
| US7846713B2 (en) | Calibrating microarrays | |
| CN101315379A (en) | Reagent kit for detecting Ractopamine and application thereof | |
| CN104122398A (en) | Multi-index parallel-detection protein chip detection kit, preparation method and detection method | |
| CN110596378A (en) | Multichannel universal chromatography method for detecting small molecules, test strip and kit | |
| Song et al. | Flow injection chemiluminescence immunosensor for the determination of clenbuterol by immobilizing coating-antigen on carboxylic resin beads | |
| CN106596875B (en) | A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine | |
| CN107436350B (en) | A kind of separation method carrying out each ingredient in mixture using ELISA Plate and monoclonal antibody | |
| Meriggioli | Use of immunoassays in neurological diagnosis and research | |
| CN106198958A (en) | Antisperm antibody chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN112415190A (en) | Time-resolved fluorescence immunochromatography method for detecting nicarbazin and application | |
| CN109030815A (en) | One kind is for detecting interactive protein-chip of liquid phase protein matter and its preparation method and application | |
| CN105315166A (en) | Preparation methods of salbutamol hapten and salbutamol antigen, and application thereof in quantum dot immunofluorescence kit |
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 |