CN103091490B - A kind of A type aftosa 146S antigen quantify ELISA detection method and kit thereof and application - Google Patents
A kind of A type aftosa 146S antigen quantify ELISA detection method and kit thereof and application Download PDFInfo
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Abstract
The invention discloses a kind of A type aftosa 146S antigen quantify ELISA detection method, comprise the following steps: 1) lay in A type aftosa standard control antigen; 2) purifying quantitative A type aftosa standard control antigen 1 46S; 3) A type aftosa 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 A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection method and kit thereof and application.
Background technology
The one that the Some Livestocks such as aftosa (footandmouthdisease, 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 (footandmouthdiseasevirus, 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), 12S protein subunit (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, BartelingandMeloen(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 VanMaanen 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 12S protein subunit exists.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 A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection method, comprises the following steps:
1) A type aftosa standard control antigen is laid in;
2) purifying quantitative A type aftosa standard control antigen 1 46S;
3) A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection method, in described step 1), is to A type aftosa standard control antigen, and a part presses 5mL/ bottle, puts-70 DEG C of deposits (only thawing once, for ELISA test); Another part, by 3 × 100mL, puts-70 DEG C of deposits (only thaw once, quantitatively (divide three times) for 146S antigen purification).
Further, above-mentioned a kind of A type aftosa 146S antigen quantify ELISA detection method, described step 2) in, be utilize sucrose density gradient method (45%, 35%, 25%, 15%), carry out purifying to A type aftosa standard control antigen, measuring 146S antigenic content is 2.42 μ g/ml.
Further, above-mentioned a kind of A type aftosa 146S antigen quantify ELISA detection method, in described step 3), the process setting up A type aftosa indirect sandwich ELISA method is specific as follows:
A) bag is by elisa plate: be buffered liquid (pH9.6) with carbonate bag and dilute aftosa A type rabbit anti-serum (Lanzhou veterinary institute provides) and add 1000 μ l carbonate bags to working concentration (1:1000) by 1 μ l aftosa A 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 (0.01MpH7.4 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 lPBST, 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 A type guinea pig antiserum to working concentration (1:1000, add in 1000 μ l guinea pig antiserum dilutions by 1 μ l aftosa A 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 lPBST 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 A type aftosa 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 A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection method, in described step 6), that A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection kit, and described A type aftosa 146S antigen quantify ELISA detection kit carries out detecting with above-mentioned A type aftosa 146S antigen quantify ELISA detection method.
3rd object of the present invention there is provided a kind of A type aftosa 146S antigen quantify ELISA detection kit in the application of A type foot-and-mouth disease antigen quantitatively and in antigens genotyping.
4th object of the present invention there is provided the application of a kind of A type aftosa 146S antigen quantify ELISA detection kit in aftosa quality evaluation and antigens genotyping.
A type aftosa 146S antigen quantify ELISA kit, ultimate principle is first carried out quantitatively (parallel 3 times) A 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 A type rabbit anteserum bag by microwell plate, make insolubilized antibody, add antigen samples successively, aftosa A 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, A type aftosa 146S antigen concentration in the depth of color and sample is proportionate.Under 490nm wavelength, absorbance (OD value) is measured, by the concentration of A type foot-and-mouth disease antigen in typical curve calculation sample by microplate reader.
Beneficial effect of the present invention is: a kind of A type aftosa 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 antigen quantify that the present invention sets up, emphasis solve A type foot-and-mouth disease antigen quantitatively and vaccine potency check substitution 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 to antigen again, the kit prepared with 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 the production of A type aftosa vaccine, and production of vaccine technique can have been 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, A type aftosa 146S antigen quantify ELISA detection method is in the application of A type foot-and-mouth disease antigen quantitatively and in antigens genotyping:
By A type aftosa indirect sandwich ELISA method, three times are measured to 5 parts of A type foot and mouth disease virus 146s antigenic contents, result is stable, reproducible, detect the antigen (O type and Asia I type) of 2 parts of other types, 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 A type foot-and-mouth disease virus antigens and 2 parts of other type antigen 1 46s antigenic content measurement results.
2, the application of A type aftosa 146S antigen quantify ELISA detection method in aftosa quality evaluation and antigens genotyping:
Carry out mensuration three times by A type aftosa indirect sandwich ELISA method to 3 parts of A type inactivated foot-and-mouth disease vaccine 146s antigenic contents, result is stable, reproducible; Measure other type inactivated foot-and-mouth disease vaccine (O type and A type, each 2 parts) 146s antigenic content, 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 A type inactivated foot-and-mouth disease vaccines and 4 parts of other type antigen 1 46s antigenic content measurement results.
Note: emulsion breaker (normal butyl alcohol) breakdown of emulsion first used by finished product vaccine, centrifugal (4 DEG C, 5000rpm20min), sucking-off antigen part, then detect by ELISA method.
Accompanying drawing explanation
Fig. 1 is the operating process schematic diagram of a kind of A type aftosa 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, A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection method, comprises the following steps:
1) A type aftosa standard control antigen is laid in:
To A type aftosa standard control antigen, a part presses 5mL/ bottle, puts-70 DEG C of deposits (only thawing once, for ELISA test); Another part, by 3 × 100mL, puts-70 DEG C of deposits (only thaw once, quantitatively (divide three times) for 146S antigen purification);
2) purifying quantitative A type aftosa standard control antigen 1 46S:
Utilize sucrose density gradient method (45%, 35%, 25%, 15%), carry out purifying to A type aftosa standard control antigen, measuring 146S antigenic content is 1.74 μ g/ml;
3) A type aftosa indirect sandwich ELISA method is set up:
Specific as follows:
A) bag is by elisa plate: be buffered liquid (pH9.6) with carbonate bag and dilute aftosa A type rabbit anti-serum (Lanzhou veterinary institute provides) and add 1000 μ l carbonate bags to working concentration (1:1000) by 1 μ l aftosa A 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 (0.01MpH7.4 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 lPBST, 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 A type guinea pig antiserum to working concentration (1:1000, add in 1000 μ l guinea pig antiserum dilutions by 1 μ l aftosa A 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 lPBST 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 A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection method, is characterized in that, comprise the following steps:
1) A type aftosa standard control antigen is laid in; To A type aftosa 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 × 100mL, 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 A type aftosa standard control antigen 1 46S; Utilize sucrose density gradient method, gradient concentration is 45%, 35%, 25%, 15%, purifying A type aftosa standard control antigen, and the mean value recording 146S antigenic content for three times is 1.74 μ g/ml;
3) A type aftosa indirect sandwich ELISA method is set up; Process is specific as follows:
A) bag is by elisa plate: be buffered liquid dilution aftosa A type rabbit anti-serum to working concentration with the carbonate bag of pH9.6, working concentration is 1:1000, adding 1000 μ l carbonate bags by 1 μ l aftosa A 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, pH7.4,1 × 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 lPBST, 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 A 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 A 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 lPBST 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, be with the concentration of standard control antigen for horizontal ordinate, OD value is ordinate, drawing standard curve;
5) calculate test sample 146S antigenic content, 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 A type aftosa 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 A type aftosa 146S antigen quantify ELISA detection kit, is characterized in that, described A type aftosa 146S antigen quantify ELISA detection kit detects with A type aftosa 146S antigen quantify ELISA detection method according to claim 1.
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| 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 |
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