CN103091270B - A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application - Google Patents
A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application Download PDFInfo
- Publication number
- CN103091270B CN103091270B CN201310055748.7A CN201310055748A CN103091270B CN 103091270 B CN103091270 B CN 103091270B CN 201310055748 A CN201310055748 A CN 201310055748A CN 103091270 B CN103091270 B CN 103091270B
- Authority
- CN
- China
- Prior art keywords
- antigen
- asia
- mouth disease
- type foot
- type
- 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
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, comprise the following steps: 1) lay in Asia I type foot-and-mouth disease standard control antigen; 2) purifying quantitative Asia I type foot-and-mouth disease standard control antigen 1 46S; 3) Asia I type foot-and-mouth disease indirect sandwich ELISA method is set up; 4) drawing standard contrast antigen typical curve; 5) test sample 146S antigenic content is calculated; 6) detection sensitivity and specificity.And provide the kit of this detection method of application.Beneficial effect of the present invention is: a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method provided by the invention is when measuring the content of foot-and-mouth disease antigen, antigenic content can be calculated, somatotype can be carried out again to antigen, with kit prepared by its principle, responsive, special, simple to operate, time saving and energy saving, good stability, be suitable for batch detection, and can serotype be distinguished.
Description
Technical field
The present invention relates to a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application.
Background technology
The one that the Some Livestocks such as aftosa (foot and mouth disease, FMD) is pig, ox, sheep and other artiodactyl suffer from altogether is acute, hot, high degree in contact sexually transmitted disease, and susceptible animal reaches kind more than 70.Clinical symptoms is in mucous membrane of mouth, hoof and skin of breast generation blister rash.This sick route of transmission is many, speed is fast, once repeatedly worldwide outbreak of epidemic, causes huge politics, economic loss.OIE (OIE) is classified as must notifiable infectious disease, and China is also classified as first of a class zoonosis.
Foot and mouth disease virus (foot and mouth disease virus, FMDV) belongs to micro ribonucleic acid Viraceae Hostis, has seven serotypes: O type, A type, Asia I type, C type and South Africa 1,2,3 type.Cross protection is not had between each serotype.Present stage China's Major Epidemic O type, A type and Asia I type.
Vaccine inoculation is one of prevention, the effective means controlling aftosa.In China, inactivated foot-and-mouth disease vaccine uses the most extensive; For guaranteeing that it is safe and effective, strict control is carried out to its quality very necessary.At present, vaccine potency detection method general is in the world this animal protest test, or adopts PD
50determination method (Europe): namely use vaccine 50% animal protection dosage (PD
50) evaluate immune effect of vaccine, conventional vaccine at least need reach 3 PD
50, urgent vaccine inoculation at least need reach 6 PD
50; Or adopt (PGP) determination method (South America): namely protect percent (PGP) to evaluate immune effect of vaccine with vaccine, PGP reaches more than 75%, and this vaccine is qualified, and PGP is 62.5% ~ 68.8%, and need to carry out duplicate test, PGP is less than 62%, and this vaccine is defective.The animal of this animal protest test must be from without aftosa area, non-vaccine this animal immune without foot and mouth disease virus neutralizing antibody.Because China takes compulsory immunization means control the popular of aftosa and break out, so shaker test animal is quite difficult; In addition, the cost of this method is high, the cycle long, repeatability is poor, and needs high safe animal house, not easy to operate.
For this reason, researcher has carried out the research of the multiple method of inspection, attempts substituting this animal protest test.These researchs are mainly divided three classes: serology method of substitution, experimental animal method of substitution and vaccine antigen sizing technique.
It is evaluate vaccine immunity effect by detecting the antibody titer levels after vaccine immunity that aftosa serum effect detects alternative method, mainly comprises virus neutralization tests and LPB-ELISA test.Nineteen sixty-eight, Stellman etc. propose the statistical method analyzing vaccine potency with NAT; By 1976, Pay etc. established the correlation formula of the antigen dose of vaccine, NAT and protection ratio according to the relation of NAT and vaccine potency.Ma Junwu etc. detect vaccine immunity antibody by LPB-ELISA and attack malicious protection and analyze aftosa immune antiboidies such as determining ox, sheep, pig and attack the malicious relation protected.Vaccine immune sera antibody titer detects must with non-aftosa vaccine immune animal, and fundamentally cannot replace the use of this animal, the selection of animal still has difficulties.
Experimental animal method of substitution replaces this animal with experimental animal (as mouse, cavy etc.), carries out vaccine potency inspection.Research finds, carries out the potency test of ox inactivated foot-and-mouth disease vaccine, the PD checked with this Animal potency with cavy
50between have good correlativity (Eissner etc. 1976; Terpstra etc. 1976), also can substitute the ox Indirect evaluation effect of inactivated foot-and-mouth disease vaccine (Sutmoller etc. 1980 by mouse or BALB/c mouse; DusSantos etc. 2000).Substituting experimental animal is applicable to breeding scale, and homogeneity easily controls, and experimental animal and this animal attack the PD after poison
50in certain positive correlation, but substituting experimental animal has certain difference with the immune response of this animal, and uses test strain during substituting experimental animal artificial challenge virus, distinct in strain and route of infection with natural infection.
Foot and mouth disease virus is when sucrose density gradient centrifugation, different according to sedimentation coefficient, can be divided into complete virus particle (146S or 140S), hollow capsid (76S), virus infections related peptide (45S), 12 S protein subunits (12S).Research finds, complete virus particle (146S or 140S) is the principal ingredient that in inactivated foot-and-mouth disease vaccine, induced animal body produces protective immune response, and therefore in vaccine, the immune protection effectiveness of complete virus particle content and vaccine has obvious correlativity.The quantitative various methods of FMD vaccine antigen are are also researched and developed accordingly, mainly comprise sucrose density gradient centrifugation, sandwich ELISA method and size exclusion chromatography.
1971, Fayet etc. established the method quantitatively detecting aftosa complete virus particle (146S or 140S) in saccharose gradient with ultraviolet light; Subsequently, Barteling and Meloen(1974) and Doel etc. perfect further to it, and in nineteen eighty-two to OIE formal recommendation.In three more than ten years after this, the method is widely used by the aftosa vaccine researcher of countries in the world and supplier as the classical way of aftosa vaccine antigen quantify always.But this method also has its shortcoming: complicated operation, expensive equipment, repeatability poor, be not suitable for a large amount of sample detection, can't serotype be distinguished.
By comparison, the sandwich ELISA method that Van Maanen etc. (nineteen ninety) and Crowther etc. (1995) set up has more advantage: simple to operate, time saving and energy saving, good stability, be suitable for batch detection, and can distinguish serotype (detection bivalent seedling and multivalence seedling particularly important).They are using the neutralizing monoclonal antibody for VP1 linear epitope as capture antibody and detect antibody, establish the double crush syndrome that can detect 146S antigen, and not by the impact that 12 S protein subunits exist.But such monoclonal antibody is not easy to be separated to, limit its widespread use.
Size exclusion chromatography (SEC), is also called gel filtration chromatography, or molecular sieve chromatography, is a kind of liquid chromatography isolation technics.Be mainly used in the separation of macromolecular substances as protein.This technology is also used to the separation of FMD virion, quantitative (Spitteler etc., 2011), is first separated by foot-and-mouth disease antigen molecular sieve, then uses determination of ultra-violet (254nm), calculates the content of aftosa complete virus particle (140S).It is reported, very well, also comparatively the latter is simple in operation for the testing result of this method and the accordance of sucrose density gradient centrifugation; But it is the same with the latter, serotype can not be distinguished.
The exploration of this Animal potency of inactivated foot-and-mouth disease vaccine inspection alternative method faces both at home and abroad and urgently to be resolved hurrily problem, and effective alternative method should have good specificity, susceptibility and repeatability, and will be easy to standardization.
Summary of the invention
Object of the present invention is exactly for above-mentioned defect of the prior art, provides a kind of advantage both having maintained sucrose density gradient centrifugation, has again the Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method of the speciality of ELISA method.
To achieve these goals, technical scheme provided by the invention is: a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, comprises the following steps:
1) Asia I type foot-and-mouth disease standard control antigen is laid in;
2) purifying quantitative Asia I type foot-and-mouth disease standard control antigen 1 46S;
3) Asia I type foot-and-mouth disease indirect sandwich ELISA method is set up;
4) drawing standard contrast antigen typical curve;
5) test sample 146S antigenic content is calculated;
6) detection sensitivity and specificity.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, in described step 1), to Asia I type foot-and-mouth disease standard control antigen, a part, by 5 mL/bottle, puts-70 DEG C of deposits (only thawing once, for ELISA test); Another part, by 3 × 100 mL, puts-70 DEG C of deposits (only thaw once, quantitatively (divide three times) for 146S antigen purification).
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, described step 2) in, utilize sucrose density gradient method (45%, 35%, 25%, 15%), carry out purifying to Asia I type foot-and-mouth disease standard control antigen, measuring 146S antigenic content is 1.74 μ g/ml.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, in described step 3), the process setting up Asia I type foot-and-mouth disease indirect sandwich ELISA method is specific as follows:
A) bag is by elisa plate: be buffered liquid (pH 9.6) dilution aftosa Asia I type rabbit anti-serum (Lanzhou veterinary institute provides) with carbonate bag and add 1000 μ l carbonate bags to working concentration (1:1000) by 1 μ l aftosa Asia I type rabbit anti-serum and be buffered in liquid), mixing, every hole adds 50 μ l in bottom, to shake after 2-3 minute with shrouding film shrouding or puts ambient temperature overnight in wet box;
B) wash elisa plate: take shrouding film off, discard liquid in elisa plate, cleansing solution (0.01M pH 7.4 phosphate Tween buffer, 1 × PBST) is filled it up with in every hole, leave standstill and discard liquid after 30 seconds, repeat 4 times, pat dry;
C) application of sample: will contrast antigen and tested antigen from 1:2 with 1 × PBST, namely 50 μ l antigens add 50 μ l PBST, start to do 2 times of serial dilutions to 1:256, and every hole adds 50 μ l, 37 DEG C, incubation 1 hour, washs elisa plate 4 times with PBST, pats dry;
D) add two to resist: with guinea pig antiserum diluted aftosa Asia I type guinea pig antiserum to working concentration (1:1000, add in 1000 μ l guinea pig antiserum dilutions by 1 μ l aftosa Asia I type guinea pig antiserum), guinea pig antiserum dilution is for containing 10% normal calf serum, the PBST of 5% Healthy Rabbits serum, every hole adds 50 μ l, shrouding, 37 DEG C, incubation washed elisa plate 4 times with PBST after 1 hour, patted dry;
E) enzyme-added: to dilute rabbit anti-cavy-horseradish peroxidase bond to working concentration (1:500 with 1 × PBST, add in 1000 μ l PBST by 1 μ l rabbit anti-cavy-horseradish peroxidase bond), every hole adds 50 μ l, 37 DEG C of incubations washed elisa plate 4 times with PBST after 1 hour, patted dry;
F) develop the color: every hole adds 50 μ l substrate solutions, 37 DEG C of incubations 15 minutes, described substrate solution is 20mmol/L o-phenylenediamine OPD, 0.05M citrate phosphate buffer, 0.015%H
2o
2;
G) stop: every hole adds 50 μ l stop buffer cessation reactions again, described stop bath to be concentration be 1.25% H
2sO
4;
H) measure: under 490nm wavelength, read absorbance value.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, in described step 4), be with the concentration of standard control antigen for horizontal ordinate, OD value is ordinate, drawing standard curve.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, in described step 5), is find corresponding concentration with the OD value of sample by typical curve, then is multiplied by extension rate, obtain the actual concentrations of sample;
Or the linear regression equation of typical curve is calculated with the concentration of reference material and OD value, the OD value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, obtain the actual concentrations of sample.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, in described step 6), that Asia I type foot-and-mouth disease 146S antigen is made serial dilution, the minimum antigenic content of the linear regression equation of typical curve is calculated by the concentration of standard control antigen and OD value, during using foot-and-mouth disease antigen to be measured as tested antigen, detection reaction OD value is less than the sensitivity Detection value of standard control antigen.
Second object of the present invention there is provided a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit, and described Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit carries out detecting with above-mentioned Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method.
3rd object of the present invention there is provided a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit in Asia I type foot-and-mouth disease antigen quantify and the application in somatotype generation.
4th object of the present invention there is provided the application of a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit in aftosa vaccine quality evaluation and somatotype.
Asia I type foot-and-mouth disease 146S antigen quantify ELISA kit, ultimate principle is first carried out quantitatively (parallel 3 times) Asia I type foot-and-mouth disease antigen with sucrose density gradient centrifugation, be standard control antigen with it, ELISA detection is carried out to other sample, first use aftosa Asia I type rabbit anteserum bag by microwell plate, make insolubilized antibody, add antigen samples successively, aftosa Asia I type guinea pig serum, the anti-cavy IgG of horseradish peroxidase mark, form coated antibody-antigen-second antibody-hrp-antibody complex, add substrate solution colour developing again, acid adding stops, Asia I type foot-and-mouth disease 146S antigen concentration in the depth of color and sample is proportionate.Under 490nm wavelength, absorbance (OD value) is measured, by the concentration of Asia I type foot-and-mouth disease antigen in typical curve calculation sample by microplate reader.
Beneficial effect of the present invention is: a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method provided by the invention had both maintained the advantage (internationally recognized) of sucrose density gradient centrifugation, there is again the speciality of ELISA method, the indirect sandwich ELISA method of the antigen quantify that the present invention sets up, emphasis solves Asia I type foot-and-mouth disease antigen quantify and vaccine quality evaluation problem; The present invention is when measuring the content of foot-and-mouth disease antigen, antigenic content can be calculated, somatotype can be carried out again to antigen, with kit prepared by its principle, responsive, special, simple to operate, time saving and energy saving, good stability, be suitable for batch detection, and can serotype be distinguished, both can be used for half-finished on-line monitoring in Asia I type foot-and-mouth disease production of vaccine, production of vaccine technique can be optimized again, also can be used as aftosa finished product vaccine tested in vitro technology, the quality of assessment vaccine, substitutes the potency test of this animal.The present invention will produce in on-line monitoring, vaccine art optimization, vaccine quality assessment, vaccine potency inspection etc. at antigen and play a significant role.
experiment effect illustrates:
1, the application of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit in Asia I type foot-and-mouth disease antigen quantify and somatotype:
By Asia I type foot-and-mouth disease indirect sandwich ELISA method, mensuration is carried out four times to 5 parts of Asia I type foot-and-mouth disease virus 146s antigenic contents, result is stable, reproducible, survey 2 parts of antigens (O type and A type) of other type, result, all lower than its sensitivity range, shows that the specificity of this invention is good simultaneously.As shown in table 1 is that indirect ELISA is to 5 parts of Asia I type foot-and-mouth disease viral antigens and 2 parts of other type antigen 1 46s antigenic content measurement results.
2, the application of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit in aftosa vaccine quality evaluation and somatotype:
Carry out mensuration four times by Asia I type foot-and-mouth disease indirect sandwich ELISA method to 3 parts of Asia I type foot-and-mouth disease inactivated vaccine 146s antigenic contents, result is stable, reproducible; Measure other type inactivated foot-and-mouth disease vaccine (O type and A type) 146s antigenic content by Asia I type foot-and-mouth disease indirect sandwich ELISA method, result, all lower than its sensitivity range, shows that the specificity of this invention is good.As shown in table 2 is that indirect ELISA is to 3 parts of Asia I type foot-and-mouth disease inactivated vaccines and 4 parts of its alloytype aftosa vaccine 146s antigenic content measurement results.
Note: emulsion breaker (normal butyl alcohol) breakdown of emulsion first used by finished product vaccine, centrifugal (4 DEG C, 5000rpm 20min), sucking-off antigen part, then detect by ELISA method.
Accompanying drawing explanation
Fig. 1 is the operating process schematic diagram of a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method provided by the invention.
Embodiment
The source of present embodiment agents useful for same and material:
1, in this test, agents useful for same is provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences.
2, Asia I type foot-and-mouth disease standard control antigen and tested antigen samples all have the biological effective company of share in middle peasant Witter to provide.
3, Sigma company: the rabbit anti-igg of horseradish peroxidase-labeled, OPD, carbonate buffer solution capsule, citric acid phosphoric acid salt tablets, PEG6000.
4, elisa plate is that costar company produces, and antigen diluent plate is produced by Shenzhen Jin Can company.
5, experiment process as shown in Figure 1.
embodiment 1:
A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, comprises the following steps:
1) Asia I type foot-and-mouth disease standard control antigen is laid in:
To Asia I type foot-and-mouth disease standard control antigen, a part, by 5 mL/bottle, puts-70 DEG C of deposits (only thawing once, for ELISA test); Another part, by 3 × 100 mL, puts-70 DEG C of deposits (only thaw once, quantitatively (divide three times) for 146S antigen purification);
2) purifying quantitative Asia I type foot-and-mouth disease standard control antigen 1 46S:
Utilize sucrose density gradient method (45%, 35%, 25%, 15%), carry out purifying to Asia I type foot-and-mouth disease standard control antigen, measuring 146S antigenic content is 1.74 μ g/ml;
3) Asia I type foot-and-mouth disease indirect sandwich ELISA method is set up:
Specific as follows:
A) bag is by elisa plate: be buffered liquid (pH 9.6) dilution aftosa Asia I type rabbit anti-serum (Lanzhou veterinary institute provides) with carbonate bag and add 1000 μ l carbonate bags to working concentration (1:1000) by 1 μ l aftosa Asia I type rabbit anti-serum and be buffered in liquid), mixing, every hole adds 50 μ l in bottom, to shake after 2-3 minute with shrouding film shrouding or puts ambient temperature overnight in wet box;
B) wash elisa plate: take shrouding film off, discard liquid in elisa plate, cleansing solution (0.01M pH 7.4 phosphate Tween buffer, 1 × PBST) is filled it up with in every hole, leave standstill and discard liquid after 30 seconds, repeat 4 times, pat dry;
C) application of sample: will contrast antigen and tested antigen from 1:2 with 1 × PBST, namely 50 μ l antigens add 50 μ l PBST, start to do 2 times of serial dilutions to 1:256, and every hole adds 50 μ l, 37 DEG C, incubation 1 hour, washs elisa plate 4 times with PBST, pats dry;
D) add two to resist: with guinea pig antiserum diluted aftosa Asia I type guinea pig antiserum to working concentration (1:1000, add in 1000 μ l guinea pig antiserum dilutions by 1 μ l aftosa Asia I type guinea pig antiserum), guinea pig antiserum dilution is for containing 10% normal calf serum, the PBST of 5% Healthy Rabbits serum, every hole adds 50 μ l, shrouding, 37 DEG C, incubation washed elisa plate 4 times with PBST after 1 hour, patted dry;
E) enzyme-added: to dilute rabbit anti-cavy-horseradish peroxidase bond to working concentration (1:500 with 1 × PBST, add in 1000 μ l PBST by 1 μ l rabbit anti-cavy-horseradish peroxidase bond), every hole adds 50 μ l, 37 DEG C of incubations washed elisa plate 4 times with PBST after 1 hour, patted dry;
F) develop the color: every hole adds 50 μ l substrate solutions, 37 DEG C of incubations 15 minutes, described substrate solution is 20mmol/L o-phenylenediamine OPD, 0.05M citrate phosphate buffer, 0.015%H
2o
2;
G) stop: every hole adds 50 μ l stop buffer cessation reactions again, described stop bath to be concentration be 1.25% H
2sO
4;
H) measure: under 490nm wavelength, read absorbance value;
4) drawing standard contrast antigen typical curve:
With the concentration of standard control antigen for horizontal ordinate, OD value is ordinate, drawing standard curve;
5) test sample 146S antigenic content is calculated:
Be find corresponding concentration with the OD value of sample by typical curve, then be multiplied by extension rate, obtain the actual concentrations of sample;
Or the linear regression equation of typical curve is calculated with the concentration of reference material and OD value, the OD value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, obtain the actual concentrations of sample;
6) detection sensitivity and specificity:
That Asia I type foot-and-mouth disease 146S antigen is made serial dilution, the minimum antigenic content of the linear regression equation of typical curve is calculated by the concentration of standard control antigen and OD value, during using foot-and-mouth disease antigen to be measured as tested antigen, detection reaction OD value is less than the sensitivity Detection value of standard control antigen.
Last it is noted that the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment to invention has been detailed description, for a person skilled in the art, it still can be modified to the technical scheme described in foregoing embodiments, or carries out equivalent replacement to wherein portion of techniques feature.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (2)
1. an Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, is characterized in that, comprise the following steps:
1) lay in Asia I type foot-and-mouth disease standard control antigen: to Asia I type foot-and-mouth disease standard control antigen, a part presses 5mL/ bottle, and be placed in-70 DEG C of deposits, this part is only thawed once, tests for ELISA; Another part is by 3 × 100 mL, and be placed in-70 DEG C of deposits, this part is only thawed once, divide three times quantitative for 146S antigen purification;
2) purifying quantitative Asia I type foot-and-mouth disease standard control antigen 1 46S: be utilize sucrose density gradient method, gradient concentration is 45%, 35%, 25%, 15%, purifying Asia I type foot-and-mouth disease standard control antigen, the mean value recording 146S antigenic content for three times is 1.74 μ g/ml;
3) set up Asia I type foot-and-mouth disease indirect sandwich ELISA method, process is specific as follows:
A) bag is by elisa plate: be buffered liquid dilution aftosa Asia I type rabbit anti-serum to working concentration with the carbonate bag of pH 9.6, working concentration is 1:1000, adding 1000 μ l carbonate bags by 1 μ l aftosa Asia I type rabbit anti-serum is buffered in liquid, mixing, every hole adds 50 μ l in bottom, to shake after 2-3 minute with shrouding film shrouding or puts ambient temperature overnight in wet box;
B) wash elisa plate: take shrouding film off, discard liquid in elisa plate, cleansing solution is filled it up with in every hole, and cleansing solution is the phosphate Tween buffer of 0.01M, pH 7.4,1 × PBST, leave standstill and discard liquid after 30 seconds, repeat 4 times, pat dry;
C) application of sample: will contrast antigen and tested antigen from 1:2 with 1 × PBST, namely 50 μ l antigens add 50 μ l PBST, start to do 2 times of serial dilutions to 1:256, and every hole adds 50 μ l, 37 DEG C, incubation 1 hour, washs elisa plate 4 times with PBST, pats dry;
D) add two to resist: with guinea pig antiserum diluted aftosa Asia I type guinea pig antiserum to working concentration, working concentration is 1:1000, add in 1000 μ l guinea pig antiserum dilutions by 1 μ l aftosa Asia I type guinea pig antiserum, guinea pig antiserum dilution is for containing 10% normal calf serum, the PBST of 5% Healthy Rabbits serum, and every hole adds 50 μ l, shrouding, 37 DEG C, incubation washed elisa plate 4 times with PBST after 1 hour, patted dry;
E) enzyme-added: to dilute rabbit anti-cavy-horseradish peroxidase bond to working concentration with 1 × PBST, working concentration is 1:500, add in 1000 μ l PBST by 2 μ l rabbit anti-cavy-horseradish peroxidase bonds, every hole adds 50 μ l, 37 DEG C of incubations washed elisa plate 4 times with PBST after 1 hour, patted dry;
F) develop the color: every hole adds 50 μ l substrate solutions, 37 DEG C of incubations 15 minutes, described substrate solution is 20mmol/L o-phenylenediamine OPD, 0.05M citrate phosphate buffer, 0.015%H
2o
2;
G) stop: every hole adds 50 μ l stop buffer cessation reactions again, described stop buffer to be concentration be 1.25% H
2sO
4;
H) measure: under 490nm wavelength, read absorbance value;
4) drawing standard contrast antigen typical curve: be with the concentration of standard control antigen for horizontal ordinate, OD value is ordinate, drawing standard curve;
5) test sample 146S antigenic content is calculated: be find corresponding concentration with the OD value of sample by typical curve, then be multiplied by extension rate, obtain the actual concentrations of sample; Or the linear regression equation of typical curve is calculated with the concentration of reference material and OD value, the OD value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, obtain the actual concentrations of sample;
6) detection sensitivity and specificity: be that Asia I type foot-and-mouth disease 146S antigen is made serial dilution, the minimum antigenic content of the linear regression equation of typical curve is calculated by the concentration of standard control antigen and OD value, during using foot-and-mouth disease antigen to be measured as tested antigen, detection reaction OD value is less than the sensitivity Detection value of standard control antigen.
2. an Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit, it is characterized in that, described Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit detects with Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310055748.7A CN103091270B (en) | 2013-01-17 | 2013-01-17 | A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310055748.7A CN103091270B (en) | 2013-01-17 | 2013-01-17 | A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103091270A CN103091270A (en) | 2013-05-08 |
| CN103091270B true CN103091270B (en) | 2015-09-09 |
Family
ID=48204107
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310055748.7A Active CN103091270B (en) | 2013-01-17 | 2013-01-17 | A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103091270B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105467138B (en) * | 2015-12-04 | 2018-01-26 | 中国农业科学院兰州兽医研究所 | A kind of method for determining aftosa vaccine component and estimating antigenic content |
| CN109765365A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of detection kit of Asia1 type antibodies against foot-and-mouth disease virus |
| CN111239393A (en) * | 2020-03-06 | 2020-06-05 | 中国农业科学院兰州兽医研究所 | Method for detecting NSPs residues in foot-and-mouth disease inactivated antigen or inactivated vaccine |
| CN113189332A (en) * | 2021-04-19 | 2021-07-30 | 中国农业科学院兰州兽医研究所 | Foot-and-mouth disease virus liquid phase blocking BSA-ELISA antibody detection kit and application thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006138481A2 (en) * | 2005-06-15 | 2006-12-28 | The Trustees Of The University Of Pennsylvania | Detection of hiv-1 infection |
| CN1900115A (en) * | 2006-07-11 | 2007-01-24 | 中国农业科学院兰州兽医研究所 | Method for preparing monoclonal antibody resisting O-type foot and mouth disease virus and antibody and use |
| CN101082626A (en) * | 2007-06-14 | 2007-12-05 | 中国农业科学院兰州兽医研究所 | ELISA reagent kit for testing Asial type foot-and-mouth disease virus |
| CN101140281A (en) * | 2007-10-26 | 2008-03-12 | 云南省畜牧兽医科学研究所 | Foot-and-mouth disease 146S antigen quick-speed quantitative method |
| CN102517331A (en) * | 2011-12-26 | 2012-06-27 | 武汉中博生物股份有限公司 | 2 type subunit vaccine for porcine circovirus as well as preparation method and application thereof |
| CN102662062A (en) * | 2012-04-17 | 2012-09-12 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) |
-
2013
- 2013-01-17 CN CN201310055748.7A patent/CN103091270B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006138481A2 (en) * | 2005-06-15 | 2006-12-28 | The Trustees Of The University Of Pennsylvania | Detection of hiv-1 infection |
| CN1900115A (en) * | 2006-07-11 | 2007-01-24 | 中国农业科学院兰州兽医研究所 | Method for preparing monoclonal antibody resisting O-type foot and mouth disease virus and antibody and use |
| CN101082626A (en) * | 2007-06-14 | 2007-12-05 | 中国农业科学院兰州兽医研究所 | ELISA reagent kit for testing Asial type foot-and-mouth disease virus |
| CN101140281A (en) * | 2007-10-26 | 2008-03-12 | 云南省畜牧兽医科学研究所 | Foot-and-mouth disease 146S antigen quick-speed quantitative method |
| CN102517331A (en) * | 2011-12-26 | 2012-06-27 | 武汉中博生物股份有限公司 | 2 type subunit vaccine for porcine circovirus as well as preparation method and application thereof |
| CN102662062A (en) * | 2012-04-17 | 2012-09-12 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) |
Non-Patent Citations (2)
| Title |
|---|
| ELISA用于口蹄疫病毒146S抗原快速定量的研究;李乐等;《中国预防兽医学报》;20080430;第30卷(第4期);摘要部分，正文部分第2段，材料和方法1.2、1.3、1.4部分，结果2.1、2.2部分，图1 * |
| 用抗O型口蹄疫病毒的单克隆抗体建立双抗夹心ELISA方法;黄红亮等;《猪的重要传染病防治研究新成果》;20021231;正文306页最后一段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103091270A (en) | 2013-05-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103076451B (en) | A kind of O type aftosa 146S antigen quantify ELISA detection kit and using method thereof | |
| CN103091490B (en) | A kind of A type aftosa 146S antigen quantify ELISA detection method and kit thereof and application | |
| CN103884839B (en) | A kind of reverse LPB-ELISA method for quantitatively detecting FMDV Effective Antigens | |
| Thomas et al. | Dengue plaque reduction neutralization test (PRNT) in primary and secondary dengue virus infections: How alterations in assay conditions impact performance | |
| CN102662062B (en) | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) | |
| Hamblin et al. | A rapid enzyme-linked immunosorbent assay for the detection of foot-and-mouth disease virus in epithelial tissues | |
| Rainwater-Lovett et al. | Variation in dengue virus plaque reduction neutralization testing: systematic review and pooled analysis | |
| CN103091270B (en) | A kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method and kit thereof and application | |
| Maradei et al. | Updating of the correlation between lpELISA titers and protection from virus challenge for the assessment of the potency of polyvalent aphtovirus vaccines in Argentina | |
| CN101533016A (en) | Indirect ELISA reagent kit for detecting antibodies against rabbit hemorrhagic disease virus | |
| Jiang et al. | A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A | |
| CN105445462B (en) | Enzyme linked immunosorbent assay kit for specific quantitative detection of foot and mouth disease O type Guangxi strain antigen and application thereof | |
| Jaworski et al. | Validation of an r3AB1-FMDV-NSP ELISA to distinguish between cattle infected and vaccinated with foot-and-mouth disease virus | |
| Wang et al. | A neutralizing monoclonal antibody-based competitive ELISA for classical swine fever C-strain post–vaccination monitoring | |
| Robiolo et al. | Quantitative single serum-dilution liquid phase competitive blocking ELISA for the assessment of herd immunity and expected protection against foot-and-mouth disease virus in vaccinated cattle | |
| Yang et al. | Development of a competitive enzyme-linked immunosorbent assay for detection of antibodies against the 3B protein of foot-and-mouth disease virus | |
| Cassaniti et al. | Seroprevalence of SARS-CoV-2 in blood donors from the Lodi Red Zone and adjacent Lodi metropolitan and suburban area | |
| CN104569428B (en) | A kind of enterovirns type 71 killed vaccine antigen enzyme-linked immunologic detecting kit | |
| Wang et al. | Understanding the epidemiological characteristics of EV71 and CVA16 infection to aid the diagnosis and treatment of hand, foot, and mouth disease | |
| CN102062775A (en) | Immunodetection kit for detecting porcine reproductive and respiratory syndrome virus and application thereof | |
| CN105348386B (en) | The monoclonal antibody cocktail and detection kit of the O-shaped virus of resistant to foot and mouth disease | |
| Ran et al. | Development and validation of a competitive ELISA based on bacterium-original virus-like particles of serotype O foot-and-mouth disease virus for detecting serum antibodies | |
| Cerutti et al. | Large scale production and characterization of SARS‐CoV‐2 whole antigen for serological test development | |
| Guevarra Jr et al. | A synthetic peptide analog of in silico‐predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits | |
| CN112881710B (en) | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |