CN106706924A - Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine - Google Patents
Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine Download PDFInfo
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- 238000001514 detection method Methods 0.000 title claims abstract description 37
- 238000002965 ELISA Methods 0.000 title claims abstract description 28
- 229960005486 vaccine Drugs 0.000 title claims abstract description 27
- 230000002860 competitive effect Effects 0.000 title claims abstract description 23
- 239000002671 adjuvant Substances 0.000 title claims abstract description 18
- 239000000427 antigen Substances 0.000 claims abstract description 149
- 102000036639 antigens Human genes 0.000 claims abstract description 149
- 108091007433 antigens Proteins 0.000 claims abstract description 149
- 238000000034 method Methods 0.000 claims abstract description 39
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 9
- 239000000047 product Substances 0.000 claims abstract description 7
- 239000011265 semifinished product Substances 0.000 claims abstract description 5
- 238000007865 diluting Methods 0.000 claims abstract 3
- 238000010790 dilution Methods 0.000 claims description 34
- 239000012895 dilution Substances 0.000 claims description 34
- 238000006243 chemical reaction Methods 0.000 claims description 25
- 239000000243 solution Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 12
- 108090000790 Enzymes Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 10
- 239000003550 marker Substances 0.000 claims description 9
- 230000000890 antigenic effect Effects 0.000 claims description 6
- 229940023041 peptide vaccine Drugs 0.000 claims description 6
- 239000000758 substrate Substances 0.000 claims description 6
- 208000007212 Foot-and-Mouth Disease Diseases 0.000 claims description 5
- 241000710198 Foot-and-mouth disease virus Species 0.000 claims description 5
- 238000012360 testing method Methods 0.000 claims description 5
- 108010030694 avidin-horseradish peroxidase complex Proteins 0.000 claims description 4
- 239000003085 diluting agent Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000007787 solid Substances 0.000 claims 1
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 238000000746 purification Methods 0.000 abstract description 4
- 239000007790 solid phase Substances 0.000 abstract description 3
- 239000008346 aqueous phase Substances 0.000 abstract 1
- 239000011248 coating agent Substances 0.000 abstract 1
- 238000000576 coating method Methods 0.000 abstract 1
- 230000003472 neutralizing effect Effects 0.000 abstract 1
- 238000005406 washing Methods 0.000 description 14
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 239000006210 lotion Substances 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 5
- 239000012071 phase Substances 0.000 description 5
- 230000006872 improvement Effects 0.000 description 4
- 238000004364 calculation method Methods 0.000 description 3
- 238000012417 linear regression Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- DPBLXKKOBLCELK-UHFFFAOYSA-N pentan-1-amine Chemical compound CCCCCN DPBLXKKOBLCELK-UHFFFAOYSA-N 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012875 competitive assay Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000013100 final test Methods 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229940100684 pentylamine Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000003118 sandwich ELISA Methods 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Health & Medical Sciences (AREA)
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- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention provides a competitive ELISA qualitative and quantitative detection method of an oil adjuvant vaccine. The method comprises the following steps of coating an antigen, diluting an antibody, diluting the antigen, drawing an antigen standard curve and quantitatively detecting the to-be-detected antigen. The method comprises the specific steps: firstly enabling the to-be-detected antigen to react with the antibody with known concentration to completely neutralizing the antibody and the antigen; and then enabling the neutralized solution to react with the antigen adsorbed in an ELISA plate in a solid-phase way to determine the concentration of the to-be-detected antigen. The standard curve slope obtained by the invention is able to be greater than that obtained through an indirect competition method, thereby greatly improving the detection sensitivity; the detection method disclosed by the invention is extensive in detection, and capable of detecting the synthetic peptide antigen finished product, semi-finished product and vaccine antigen; and an aqueous-phase sample obtained after vaccine demulsification can be determined without performing the purification treatment; the spent time is short in comparison with other detection method; and the lowest detection limit of the antigen sample according to the invention can achieve 1ng/mL-1.5ng/mL.
Description
Technical field
The present invention relates to aftosa synthetic peptide vaccine detection technique field, and in particular to a kind of competition of oil-adjuvant vaccine
ELISA method for qualitative and quantitative detection.
Background technology
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.In existing quantitative approach mainly
For antigen after purification, if for antigen detection after vaccine demulsification, because vaccine water-phase component is more complicated, measuring samples are usual
Needs take considerable time and can be only achieved testing goal after purification, and many conventional method detection sensitivities are limited, it is impossible to reach
Detection is required.
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, for the detection of aftosa synthetic peptide antigen finished product, semi-finished product and vaccine antigen content quantitative without general standard
True method.It is more quick in order to find, effectively, convenient detection mode, the present inventor is by many experiments between routine
Connect and improved on the basis of competitive ELISA.
The content of the invention
For defect of the prior art, the invention provides a kind of competitive ELISA qualitative, quantitative inspection of oil-adjuvant vaccine
Survey method, is a kind of antigen quantitative detecting method.It is first that antigen is dense with limited by the improvement to indirect competitive ELISA method
The antibody of degree is first reacted in plate is diluted, and antibody is first neutralized completely with antigen.The ELISA of antigen is then being adsorbed with immobilization
Reacted in plate, the unequal opportunities for causing immobilization antigen, determined antigen to be combined with antibody.Finally, the standard for obtaining is bent
Line slope can be more than conventional indirect competitive ELISA, so as to greatly improve detection sensitivity.
The purpose of the present invention is achieved through the following technical solutions：
The invention provides a kind of competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine, comprise the following steps：
Antigen coat：The artificial synthesized foot-and-mouth disease antigen immobilizations of 0.5 μ g/mL-5 μ g/mL are adsorbed onto elisa plate
On；
Dilution antibody：The antibody of known potency is diluted, potency is 1 after antibody dilution:250000-1:1100000；
Antigen diluent：On serum-dilution plate, with dilution by antigen diluent to be checked, 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 to be checked：The antigen to be checked and the mark of various concentrations that will have been diluted
Quasi- antigenic dilution is reacted completely with the antibody for having diluted in plate is diluted respectively, then by each reacted reaction completely
Liquid be added separately to the elisa plate of immobilization antigen in continue react, reaction terminate after, respectively add enzyme marker, substrate according to
Secondary response, then each addition terminate liquid terminates, and determines OD values, and the logarithm value for using the standard antigen of various concentrations is abscissa, mark
The OD values of quasi- antigen are that ordinate draws antigen standard curve, then bring into be calculated in standard curve by the OD values of antigen to be checked and treat
Correspondence antigen concentration is surveyed, antigen actual content in testing sample is multiplied by with extension rate.
Preferably, the antigen lowest detection limit to be checked is 1ng/mL-1.5ng/mL.
Preferably, in the quantitative determination step of antigen Specification Curve of Increasing and antigen to be checked, the time reacted completely
More than or equal to 60min, reaction temperature is 37 DEG C.The reaction time can as far as possible ensure that reaction is complete.
Preferably, it is described each completely reacted in the quantitative determination step of antigen Specification Curve of Increasing and antigen to be checked
Reaction solution 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 DEG C.
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 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.
Preferably, the antigen to be checked is：Aftosa synthetic peptide vaccine through the water phase antigen antigenic synthetic peptide after demulsification into
One kind in product or semi-finished product.
Principle of the invention is：The antibody of antigen and Finite Concentration is first reacted in plate is diluted, antigen-antibody reaction is treated
After completely, reaction solution is added to during solid phase adsorption has elisa plate of antigen and is reacted, add enzyme marker to carry out afterwards
Reaction, is finally detected with substrate colour developing.Final testing result shows as the OD values that antigen concentration is higher, is detected after colour developing
It is lower, inversely.Standard antigen is carried out into different proportion and dilutes be at war with ELISA detection drafting standard curves, detection
Sample is associated with standard curve so as to reach quantitative requirement.
Prior art is compared, and the present invention has following beneficial effect：
1) this method first first reacts the antibody of antigen and Finite Concentration in plate is diluted, and antibody is first with antigen to be checked completely
Neutralize, reach the purpose of complete inhibition.In the elisa plate that subsequent immobilization is adsorbed with antigen, immobilization antigen and antibody knot
Conjunction is reduced therewith so that immobilization antigen, determined antigen and antibody combination unequal opportunities.Therefore, the standard curve for obtaining is oblique
Rate can be more than conventional indirect competitive, so as to greatly improve detection sensitivity.
2) 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.
3) 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, testing goal can be reached with competition law.Concentration is relatively low after the dilution of simultaneous reactions antibody, reduces
The interference of non-specific responding and cross reaction.
4) this method can to the quantitative determination of antigenic synthetic peptide finished product, semi-finished product and synthetic peptide vaccine antigen effective content,
Detection range is wide.After being demulsified with time vaccines, water-phase component is more complicated, and the method is simple, easy to operate, after demulsification aqueous sample without
Purification process need to be carried out and can reach quantitative determination purpose, other detection methods that compare spend the time shorter.
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 the antigen standard curve 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 competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine is present embodiments provided, specifically using following
Step：
1. antigen coat：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. antibody is diluted：The antibody of known potency is carried out into certain proportion dilution, antibody titer is 1 after dilution:250000；
3. (antigen to be checked is antigenic synthetic peptide finished product to antigen to be checked, and antigen to be checked measures it and resists using HPLC methods
Original content is 39.25 μ g/mL) diluted with standard antigen：On serum-dilution plate, antigen to be checked is pressed 1 with dilution:100 is dilute
Release, standard antigen respectively by dilution after concentration be 1000ng/mL, 500ng/mL, 200ng/mL, 100ng/mL, 50ng/mL,
10ng/mL, 5ng/mL are diluted；
4. antigen-antibody reaction：In plate is diluted, it is separately added into antigen to be checked and standard antigen that 100 μ L have been diluted
Antibody that equivalent has diluted simultaneously is mixed, in being incubated 60min at 37 DEG C；
5. it is incubated after terminating, 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；
6. it is incubated after terminating, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ in elisa plate
L1:10000 SPA-HRP for having diluted, in being incubated 30min at 37 DEG C；
7. it is incubated after terminating, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ in elisa plate
LTMB solution, in lucifuge colour developing 15min at 37 DEG C；
8. after colour developing terminates, to adding 100 μ L 2mol/LH in elisa plate2SO4Solution terminating reaction, and in 450nm ripples
Lower measure OD values long；
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=-0.3609x+1.4572, 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, multiplied by with extension rate, i.e.,
It is antigen actual content in sample.In the present embodiment, it is 0.525 to measure the OD values of antigen to be checked, calculates antigen in sample
Actual content is 38.02 μ g/mL.The result of calculation is basically identical with the result measured using HPLC methods.
Embodiment 2
A kind of competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine is present embodiments provided, specifically using following
Step：
1. antigen coat：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. antibody is diluted：The antibody of known potency is carried out into certain proportion dilution, antibody titer is 1 after dilution:
1100000；
3. (antigen to be checked is the aqueous sample after vaccine demulsification to antigen to be checked, and demulsification efficiency is 89.2%, purified
Afterwards, it is 148.83ng/mL to use HPLC methods to measure its antigen concentration) diluted with standard antigen：On serum-dilution plate, will treat
Inspection antigen presses 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；
4. antigen-antibody reaction：In plate is diluted, equivalent is added in antigen to be checked and standard antigen that 100 μ L have been diluted
The antibody that has diluted simultaneously is mixed, in being incubated 60min at 37 DEG C；
5. it is incubated after terminating, 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；
6. it is incubated after terminating, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ in elisa plate
L1:The 10000 goat-anti pig biotin-IgG for having diluted, in being incubated 30min at 37 DEG C；
7. it is incubated after terminating, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ in elisa plate
L1:5000 Avidin-the HRP for having diluted, in being incubated 30min at 37 DEG C；
8. it is incubated after terminating, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ in elisa plate
LTMB solution, in lucifuge colour developing 15min at 37 DEG C；
9. after colour developing terminates, to adding 100 μ L 2mol/LH in elisa plate2SO4Solution terminating reaction, and in 450nm ripples
Lower measure OD values long；
10. according to determine OD values, with standard antigen concentration using 10 be bottom logarithm as abscissa, OD450 is ordinate,
Linear regression is carried out, the regression equation of standard curve is obtained for y=-0.4021x+1.5186, as shown in Figure 2；By antigen to be checked
OD values bring into standard curve calculate it is to be measured correspondence antigen concentration, antigen actual content=[(correspondence antigen concentration × dilution again
Number)/demulsification efficiency]/2 be sample in antigen actual content.In the present embodiment, it is 1.350 to measure the OD values of antigen to be checked, meter
Calculate in sample the actual content of antigen be 147.43ng/mL.The result of calculation is basic with the result measured using HPLC methods
Unanimously.
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.
Comparative example 1
This comparative example provides a kind of competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine, comprises the following steps：
1. antigen coat：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. antibody is diluted：The antibody of known potency is carried out into certain proportion dilution, antibody titer is 1 after dilution:250000；
3. (antigen to be checked is antigenic synthetic peptide finished product to antigen to be checked, and antigen to be checked measures it and resists using HPLC methods
Original content is 39.25 μ g/mL) diluted with standard antigen：On serum-dilution plate, antigen to be checked is pressed 1 with dilution:100 is dilute
Release, known standard antigen is diluted, diluted concentration be respectively 1000ng/mL, 500ng/mL, 200ng/mL, 100ng/mL,
50ng/mL、10ng/mL、5ng/mL；The antigen to be checked is same sample with the antigen to be checked of embodiment 1；
4. antigen-antibody reaction：The antibody that has diluted of equivalent is added in the antigen to be checked and standard antigen that will dilute and is mixed
It is even, take 100 μ L and be added to during immobilization has an elisa plate of antigen, in being incubated 60min at 37 DEG C；
5. it is incubated after terminating, 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；
6. it is incubated after terminating, with PBST as washing lotion, board-washing 5 times simultaneously pats dry liquid in plate, to adding 100 μ L in elisa plate
Substrate solution, in lucifuge colour developing 15min at 37 DEG C；
7. after colour developing terminates, to adding 100 μ L 2mol/LH in elisa plate2SO4Solution terminating reaction, and in 450nm ripples
Lower measure OD values long；
8. according to determine OD values, with standard antigen concentration using 10 be bottom logarithm as abscissa, OD450 is ordinate,
Linear regression is carried out, the regression equation of standard curve is obtained for y=-0.2787+1.3962, as shown in Figure 3；By antigen to be checked
OD values are brought into standard curve and calculate correspondence antigen concentration to be measured, and antigen actual content in sample is multiplied by with extension rate.
In the present embodiment, it is 0.659 to measure the OD values of antigen to be checked, and the actual content for calculating antigen in sample is 44.67 μ g/mL.
The result of calculation has differed 5.42 μ g/mL with the result measured using HPLC methods.Therefore, comparative example 1 and comparative example 1
Result understands that the result determined using the method for the present invention is more accurate.
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 (9)
1. the competitive ELISA method for qualitative and quantitative detection of a kind of oil-adjuvant vaccine, it is characterised in that comprise the following steps：
Antigen coat：The artificial synthesized foot-and-mouth disease antigen immobilizations of 0.5 μ g/mL-5 μ g/mL are 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, with dilution by antigen diluent to be checked, standard antigen carries out different proportion dilution shape
Into the standard antigen dilution of various concentrations；
The quantitative determination of antigen Specification Curve of Increasing and antigen to be checked：The antigen to be checked and the standard of various concentrations that will have been diluted resist
Former dilution is reacted completely with the antibody for having diluted in plate is diluted respectively, then by each reacted reaction solution point completely
Continuation is reacted in not being added to the elisa plate of immobilization antigen, after reaction terminates, respectively adds enzyme marker, substrate anti-successively
Should, then each addition terminate liquid termination, OD values are determined, the logarithm value for using the standard antigen of various concentrations is abscissa, and standard resists
Former OD values are ordinate drafting antigen standard curve, then the OD values of antigen to be checked are brought into standard curve calculate it is to be measured right
Antigen concentration is answered, antigen actual content in testing sample is multiplied by with extension rate.
2. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that institute
The lowest detection limit for stating antigen to be checked is 1ng/mL-1.5ng/mL.
3. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 2, it is characterised in that anti-
In the quantitative determination step of primary standard Drawing of Curve and antigen to be checked, the time reacted completely is more than or equal to 60min, reaction
Temperature is 37 DEG C.
4. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that anti-
In the quantitative determination step of primary standard Drawing of Curve and antigen to be checked, each reacted reaction solution completely is added separately to solid
Continue to react in elisa plate of phaseization antigen and be more than or equal to 30min, reaction temperature is 37 DEG C.
5. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that anti-
In the quantitative determination step of primary standard Drawing of Curve and antigen to be checked, the enzyme marker is SPA-HRP, or for goat-anti pig gives birth to
Thing element-IgG and Avidin-HRP.
6. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that institute
The consumption of enzyme marker after diluting is stated for 100 μ L, dilution ratio is 1:5000-1:10000.
7. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that institute
Stating antigen to be checked is：Aftosa synthetic peptide vaccine is through in the water phase antigen after demulsification, antigenic synthetic peptide finished product or semi-finished product
Kind.
8. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that institute
Stating substrate is：TMB solution.
9. the competitive ELISA method for qualitative and quantitative detection of oil-adjuvant vaccine according to claim 1, it is characterised in that institute
State the H that terminate liquid is 2mol/L2SO4Solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611122914.0A CN106706924A (en) | 2016-12-08 | 2016-12-08 | Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine |
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|---|---|---|---|
| CN201611122914.0A CN106706924A (en) | 2016-12-08 | 2016-12-08 | Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine |
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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 quantitative detection of non-structural protein residues in inactivated foot-and-mouth disease vaccine and its detection method |
| CN112710852A (en) * | 2021-03-26 | 2021-04-27 | 上海美迪西生物医药股份有限公司 | GNP polypeptide detection kit and detection method |
| WO2022012673A1 (en) * | 2020-07-15 | 2022-01-20 | 南京岚煜生物科技有限公司 | Method for assigning antibody standard substance and determining minimum detection limit of antibody detection reagent |
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| WO2022012673A1 (en) * | 2020-07-15 | 2022-01-20 | 南京岚煜生物科技有限公司 | Method for assigning antibody standard substance and determining minimum detection limit of antibody detection reagent |
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| CN112710852A (en) * | 2021-03-26 | 2021-04-27 | 上海美迪西生物医药股份有限公司 | GNP polypeptide detection kit and detection method |
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