CN106771189B - 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
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Abstract
The present invention provides a kind of fast qualitative quantitative detecting methods of oil-adjuvant vaccine, include the following steps：Aftosa synthetic peptide vaccine is demulsified, the antigen samples after demulsification are subjected to qualitative and quantitative detection using competitive ELISA method；The breaking method is：Oil-adjuvant vaccine is mixed with n-butanol, after competitor mixing then is added, is centrifuged to get antigen samples.This method competes antigen binding site by the way that competitor is added with surfactant, and the antigen in oil-adjuvant vaccine is discharged into water phase, the rate of recovery of antigen in water phase is substantially increased, and makes sample that can be measured without carrying out purification process, shortens detection time；The present invention first first reacts antigen to be checked with the antibody of known concentration, antibody and antigen elder generation complete neutralization；The solution after neutralization is then adsorbed on the antigen-reactive in elisa plate with immobilization again, to measure antigen concentration to be checked, obtained slope of standard curve can be more than conventional indirect competitive, to substantially increase detection sensitivity.
Description
Technical field
The present invention relates to aftosa synthetic peptide vaccine detection technique fields, and in particular to a kind of oil-adjuvant vaccine it is quick fixed
Property quantitative detecting method.
Background technology
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, it 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 exclude the non-susceptible animal with 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 to antigen after vaccine demulsification, by the way that vaccine is carried out demulsification processing, antigen is made to be transferred in water phase, then right
It carries out subsequent detection and analysis.Due to complicated component in the oil adjuvant used in vaccine emulsion process, contain 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 the amino acid sequence of native protein by manual method
Property small peptide.Existing method for quantitatively determining usually has the methods of Lowry methods, BCA methods, HPLC.And in existing quantitative approach, due to
After vaccine demulsification, vaccine water-phase component is more complex, and measuring samples usually require to take considerable time can be only achieved detection after purification
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 is 50% with free antigen, immobilised antigen binding rate probability, obtained knot
The slope of standard curve that fruit is drawn is relatively low, this so that detection sensitivity is relatively low.Aftosa synthetic peptide vaccine cannot be reacted well
Middle Effective Antigens content.
Currently, quantitatively detecting no general accurate method for aftosa synthetic peptide vaccine antigenic content.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.
Invention content
For the defects in the prior art, the present invention provides a kind of quantitative sides of detection of fast qualitative 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：
The present invention provides a kind of fast qualitative quantitative detecting methods of oil-adjuvant vaccine, include the following steps：By mouth hoof
Epidemic disease synthetic peptide vaccine is demulsified, and the antigen samples after demulsification are carried out qualitative and quantitative detection using competitive ELISA method；
The breaking method is：Oil-adjuvant vaccine is mixed with n-butanol, after competitor mixing then is added, centrifugation, i.e.,
Obtain antigen samples.
Preferably, the competitor is at least one of amino acid and its derivative.
Preferably, the competitor is 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 that can constitute competition saturation are precipitated, and influence ultra-filtration process；Concentration is too low,
It can constitute competition ineffective, the antigen of enough detections can not be discharged.
Preferably, the volume ratio of the oil-adjuvant vaccine and n-butanol is 9:1-5:5.
It is highly preferred that the volume ratio of the oil-adjuvant vaccine and n-butanol is 1:1.The volume of oil-adjuvant vaccine and n-butanol
It is identical, it can preferably ensure that vaccine is demulsified completely.
Using the breaking method, due to rate of recovery of antigen height, obtained antigen samples are without being further purified, you can
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 onto on elisa plate；
Dilute 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 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 with the antibody diluted in diluting plate completely respectively, then by each complete reaction
Reaction solution afterwards is added separately in elisa plate of immobilization antigen that the reaction was continued；After reaction, enzyme label is respectively added
Object, substrate react successively, then each terminate liquid that is added terminates, and measures OD values, is drawn using the OD values of the standard antigen of various concentration
Then the OD values of antigen samples to be checked are corresponded in the antigen standard curve and calculate to obtain determined antigen sample by 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, a concentration of 0.5 μ g/mL-5 μ g/mL of artificial synthesized foot-and-mouth disease antigen.
Preferably, described to react completely in antigen Specification Curve of Increasing and the quantitative detecting step of antigen samples to be checked
Time is more than or equal to 60min, and reaction temperature is 37 DEG C.The reaction time can ensure that the reaction was complete as far as possible.
Preferably, in antigen Specification Curve of Increasing and the quantitative detecting step of antigen samples to be checked, each complete reaction
Reaction solution afterwards is added separately in elisa plate of immobilization antigen that the reaction was continued and is more than or equal to 30min, reaction temperature 37
℃.If the reaction time is less than 30min, reaction can be caused to be not thorough, final OD values are relatively low, influence the accuracy of result.
Preferably, in antigen Specification Curve of Increasing and the quantitative detecting step of 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 after the dilution:5000-1:10000.
Preferably, the substrate is：TMB solution.
Preferably, the terminate liquid is the H of 2mol/L2SO4Solution.
The principle of the present invention is：Antigen binding site is competed with surfactant by the way that competitor is added, to help oil
Antigen in vaccinating agent is discharged into water phase, to substantially increase the rate of recovery of antigen in water phase.It will obtain after demulsification again
Water phase antigen samples use competitive ELISA method, so that antigen samples is first reacted in diluting plate with the antibody of Finite Concentration, wait resisting
After antigen-antibody reaction is complete, reaction solution is added to solid phase adsorption has in elisa plate of antigen and react, enzyme is added later
Marker 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 subjected to different proportion and dilutes the ELISA detection drafting standards that are at war with
Curve, detection sample are associated with to reach the requirement of qualitative, quantitative with standard curve.
The prior art is compared, and the present invention has following advantageous effect：
1) present invention competes antigen binding site by the way that competitor is added with surfactant, thus will be in oil-adjuvant vaccine
Antigen discharge into water phase, compared with being not added with competitor, substantially increase the rate of recovery of antigen in water phase.
2) present invention uses competitive ELISA method, first first reacts antigen in diluting plate with the antibody of Finite Concentration, resists
Body and antigen elder generation to be checked complete neutralization, achieve the purpose that complete inhibition.It is adsorbed in the elisa plate of antigen in subsequent immobilization,
Immobilization antigen is combined with antibody to be reduced therewith so that immobilization antigen, determined antigen and antibody combination unequal opportunities.Cause
This, obtained slope of standard curve can be more than conventional indirect competitive, to greatly improve detection sensitivity.
2) after being demulsified due to vaccine, 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.
3) requirements of the Competitive assays ELISA to reagent is considerably less, and either monoclonal antibody is still mostly anti-may be used to test, more
It is important that no matter checked object is that only there are one epitopes for macromolecular substances or polypeptide, small-molecule drug, small molecule hormone etc.
Molecule cannot use sandwich ELISA that can be detected using competitive ELISA.
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, competition law using the present invention reaches detectable purpose.It is dense after the dilution of simultaneous reactions antibody
It spends relatively low, 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 so that antigen can be quantified under low concentration, and sensitivity is higher, and reproducible.
Description of the drawings
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 maps in water phase after the method demulsification of comparative example 1.
Specific implementation mode
With reference to specific embodiment, the present invention is described in detail.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 domain.
Embodiment 1
A kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine is present embodiments provided, following steps are specifically used：
It 1) will be at commercially available Schweineseuche synthetic peptide vaccine (a concentration of 50 μ g/mL of vaccine antigen) as follows demulsification
Reason：
Take 10ml vaccines to be checked with n-butanol with volume ratio 1:1 mixing, is added 50mg histidines, mixing is shaken, in 4 DEG C of items
Under part, is centrifuged 15 minutes with 3000r/min, lower layer's water phase is carefully extracted with 10ml syringes to get water phase antigen sample after centrifugation
Product.
The commercially available Schweineseuche synthetic peptide vaccine that the present embodiment uses is compareed with theoretical antigen concentration standard, to it
HPLC test map samples go out peak position and are integrated, and integration information is as shown in table 1, and antigenic content is to integrate peak face in sample
Long-pending form embodies, integrated peak areas 1826453.To the present embodiment using antigen in water phase after Butanol+His demulsifications
The HPLC test map samples of sample concentration go out peak position and are integrated, and integration information is as shown in table 2, antigenic content in sample
It is embodied in the form of integrated peak areas, integrated peak areas 1676683.The result of Tables 1 and 2 is compared it is found that the two is integrated
After information comparison, rate of recovery of antigen 91.8%, that is, the efficiency that is demulsified is 91.8%.
Table 1
Table 2
2) competitive ELISA detects
2.1 antigen coat：By the 3 artificial synthesized foot-and-mouth disease antigens of μ g/mL, per 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, antigen to be checked is pressed 1 with dilution:100 dilution, standard antigen respectively press dilution after a concentration of 1000ng/mL,
500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL, 10ng/mL, 5ng/mL are diluted；
2.4 antigen-antibody reaction：In diluting 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, 60min is incubated at 37 DEG C；
After 2.5 are incubated, 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, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
1:10000 SPA-HRP diluted, 30min is incubated at 37 DEG C；
After 2.7 are incubated, 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 waves
Long lower measurement OD values；
2.9 are used as abscissa, each standard antigen survey using 10 according to the OD values 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=-0.3611x+1.4586, such as
Shown in Fig. 1；The OD values of antigen to be checked are brought into standard curve and calculate corresponding 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 practical as in sample contains
Amount.In the present embodiment, the OD values for measuring antigen to be checked are 0.404, calculate in sample the actual content of antigen be 45.30 μ g/
mL。
Note：After being demulsified due to vaccine, oil phase is 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 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：
It 1) will be at commercially available Schweineseuche synthetic peptide vaccine (a concentration of 50 μ g/mL of vaccine antigen) as follows demulsification
Reason：
Take 10ml vaccines to be checked with n-butanol with volume ratio 1:1 mixing, is added 10mg phenylalanines, mixing is shaken, at 4 DEG C
Under the conditions of, it is centrifuged 15 minutes with 3000r/min, lower layer's water phase is carefully extracted with 10ml syringes to get water phase antigen after centrifugation
Sample.
It is demulsified to aftosa vaccine using the method for the present embodiment, demulsification efficiency is 88.4%.
2) competitive ELISA detects
2.1 antigen coat：By the 3 artificial synthesized foot-and-mouth disease antigens of μ g/mL, per 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 dilution is pressed 1:100 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 reaction：In diluting plate, be added in antigen and standard antigen to be checked that 100 μ L have been diluted etc.
The antibody diluted and mixing are measured, 60min is incubated at 37 DEG C；
After 2.5 are incubated, the 100 μ L of reaction solution absorption in serum dilution plate are had to the elisa plate of antigen to immobilization
In, it is incubated 30min at 37 DEG C；
After 2.6 are incubated, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
1:The 10000 goat-anti pig biotin-IgG diluted, 30min is incubated at 37 DEG C；
After 2.7 are incubated, using PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, and 100 μ L are added into elisa plate
1:5000 Avidin-the HRP diluted, 30min is incubated at 37 DEG C；
After 2.8 are incubated, 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 waves
Long lower measurement OD values；
2.10 according to the OD values of measurement, and it is vertical seat to be used as abscissa, OD450 using 10 for bottom logarithm with standard antigen concentration
Mark carries out linear regression, and the regression equation for obtaining standard curve is y=-0.4001x+1.5138, as shown in Figure 2；It will be to be checked anti-
Former OD values, which are brought into standard curve, calculates corresponding antigen concentration to be measured, antigen actual content=[(corresponding antigen concentration × dilution
Multiple)/demulsification efficiency]/2 be sample in antigen actual content.In the present embodiment, the OD values for measuring antigen to be checked are 0.338,
Calculate in sample the actual content of antigen be 49.26 μ g/mL.
Note：After being demulsified due to vaccine, oil phase is 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 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) by commercially available Schweineseuche synthetic peptide vaccine (a concentration of 50 μ g/mL of vaccine antigen) described in embodiment 1 according to
Following method demulsification processing：
Take 10ml vaccines to be checked with n-butanol with volume ratio 1:1 mixing, often pipe addition 200mg proline, shakes mixing,
It under the conditions of 4 DEG C, is centrifuged 15 minutes with 3000r/min, carefully extract lower layer's water phase with 10ml syringes after centrifugation resists to get water phase
Raw sample.
It is demulsified to aftosa vaccine using the method for the present embodiment, demulsification efficiency is 95.6%.
2) competitive ELISA detects
The competitive ELISA method of use is same as Example 2.Calculate in sample the actual content of antigen be 48.96 μ g/
mL。
Comparative example 1
Present embodiments provide a kind of fast qualitative of oil-adjuvant vaccine (vaccine for using embodiment 1) the quantitatively side of detection
Method, it is essentially identical with the method for embodiment 1, it the difference is that only：Competitor is added without in this comparative example.
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 maps in water phase afterwards are 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, and demulsification efficiency is substantially zeroed.
Therefore it cannot be satisfied 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 domain 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 domain.
Claims (7)
1. a kind of fast qualitative quantitative detecting method of oil-adjuvant vaccine, which is characterized in that include the following steps：Aftosa is closed
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 is：Oil-adjuvant vaccine is mixed with n-butanol, after competitor mixing then is added, centrifugation is to get anti-
Raw sample；
The competitor is one kind in histidine, phenylalanine, proline；
The addition of the competitor is：1-40mg competitors are added in per 1ml oil-adjuvant vaccines.
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 onto on elisa plate；
Dilute 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 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 diluting plate with the antibody diluted respectively, then will be after each complete reaction
Reaction solution is added separately in elisa plate of immobilization antigen that the reaction was continued；After reaction, enzyme marker, bottom is respectively added
Object reacts successively, then each terminate liquid that is added terminates, and measures OD values, and antigen mark is drawn using the OD values of the standard antigen of various concentration
Directrix curve, then the OD values of antigen samples to be checked are corresponded in the antigen standard curve calculate determined antigen sample is dense
Degree, multiplied by with extension rate, divided by demulsification efficiency, as antigen actual content in determined antigen sample.
3. according to the fast qualitative quantitative detecting method of claim 2 oil-adjuvant vaccine, which is characterized in that antigen coat step
In, a concentration of 0.5 μ g/mL-5 μ g/mL of artificial synthesized foot-and-mouth disease antigen.
4. according to the fast qualitative quantitative detecting method of claim 2 oil-adjuvant vaccine, which is characterized in that antigen standard curve is painted
In the quantitative detecting step of system and antigen samples to be checked, the time reacted completely is more than or equal to 60min, reaction temperature 37
℃。
5. according to the fast qualitative quantitative detecting method of claim 2 oil-adjuvant vaccine, which is characterized in that antigen standard curve is painted
In the quantitative detecting step of system and antigen samples to be checked, the reaction solution after each complete reaction is added separately to immobilization antigen
Elisa plate in the reaction was continued be more than or equal to 30min, reaction temperature be 37 DEG C.
6. according to the fast qualitative quantitative detecting method of claim 2 oil-adjuvant vaccine, which is characterized in that antigen standard curve is painted
In the quantitative detecting 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.
7. the competitive ELISA quantitative approach after aftosa synthetic peptide vaccine demulsification according to claim 2, feature exist
In the dosage of enzyme marker is 100 μ L, dilution ratio 1 after the dilution:5000-1:10000.
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| 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 |
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