CN103091270A - Quantitative enzyme-linked immuno-sorbent assay (ELISA) detection method for Asia I-type foot-and-mouth disease antigens 146S as well as kit and application of quantitative ELISA detection method - Google Patents
Quantitative enzyme-linked immuno-sorbent assay (ELISA) detection method for Asia I-type foot-and-mouth disease antigens 146S as well as kit and application of quantitative ELISA detection method Download PDFInfo
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Abstract
The invention discloses a quantitative enzyme-linked immuno-sorbent assay (ELISA) detection method for Asia I-type foot-and-mouth disease antigens 146S. The method comprises the following steps of: (1) storing Asia I-type foot-and-mouth disease standard reference antigens; (2) purifying and quantifying the Asia I-type foot-and-mouth disease standard reference antigens 146S; (3) establishing an Asia I-type foot-and-mouth disease indirect sandwich ELISA method; (4) drawing a standard curve of the standard reference antigens; (5) calculating the content of the antigens 146S in a detected sample; and (6) detecting the sensitivity and specificity. The invention also provides a kit which adopts the detection method. The quantitative ELISA detection method for the Asia I-type foot-and-mouth disease antigens 146S and the kit have the beneficial effects that when the content of the foot-and-mouth disease antigens is measured by using the quantitative ELISA detection method, the content of the antigens can be calculated, and the antigens can be parted; and the kit prepared by using the principle of the quantitative ELISA detection method is sensitive, specific, simple to operate, good in stability and suitable for batch detection, saves time and labor, and can distinguish serotypes.
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 and application.
Background technology
Aftosa (foot and mouth disease, FMD) is a kind of acute, hot, the height contagious disease that the Some Livestock such as pig, ox, sheep and other artiodactyl suffer from altogether, 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 disease route of transmission is many, speed is fast, and once outbreak of epidemic worldwide repeatedly, caused huge politics, economic loss.OIE (OIE) classifies it as must notifiable infectious disease, and China also classifies it as first of a class zoonosis.
Foot and mouth disease virus (foot and mouth disease virus, FMDV) belongs to micro ribonucleic acid Viraceae Hostis, seven serotypes is arranged: O type, A type, Asia I type, C type and South Africa 1,2,3 types.There is no cross protection between each serotype.Present stage China's Major Epidemic O type, A type and Asia I type.
Vaccine inoculation is one of effective means of prevention, control aftosa.In China, inactivated foot-and-mouth disease vaccine uses the most extensive; Safe and effective for guaranteeing it, its quality is carried out strict control very necessary.At present, general vaccine potency detection method is this animal protest test in the world, or adopts PD
50Determination method (Europe): namely use vaccine 50% animal protection dosage (PD
50) estimate immune effect of vaccine, conventional vaccine need reach 3 PD at least
50, urgent vaccine inoculation need reach 6 PD at least
50Or adopt (PGP) determination method (South America): namely protect percent (PGP) to estimate immune effect of vaccine with vaccine, PGP reaches more than 75%, and this vaccine is qualified, and PGP is 62.5%～68.8%, need to carry out duplicate test, and PGP is less than 62%, and this vaccine is defective.The animal of this animal protest test must be from without aftosa area, without this animal of non-vaccine immunity of foot and mouth disease virus neutralizing antibody.Control the popular of aftosa and break out because China takes the compulsory immunization means, so shaker test animal difficult; In addition, the cost of this method is high, the cycle long, repeatability is relatively poor, and needs high safe animal house, and is not easy to operate.
For this reason, the researcher has carried out the research of the multiple method of inspection, attempts substituting this animal protest test.These researchs mainly are divided three classes: serology method of substitution, experimental animal method of substitution and vaccine antigen sizing technique.
It is to estimate vaccine immunity effect by the antibody titer level after the detection vaccine immunity that aftosa serum is learned effect detection alternative method, mainly comprises the test of virus neutralization tests and LPB-ELISA.Nineteen sixty-eight, Stellman etc. have proposed to analyze with NAT the statistical method of vaccine potency; By 1976, Pay etc. set up the correlation formula of antigen dose, NAT and the protection ratio of vaccine according to the relation of NAT and vaccine potency.Ma Junwu etc. have detected vaccine immunity antibody by LPB-ELISA and have attacked poison protection Analysis deterrmination the aftosa immune antiboidies such as ox, sheep, pig and attacked the malicious relation of protecting.The vaccine immunity serum antibody titer detects and must can't fundamentally replace the use of this animal with non-aftosa vaccine immune animal, and the selection of animal still has difficulties.
The experimental animal method of substitution is to replace this animal with experimental animal (as mouse, cavy etc.), carries out the vaccine potency check.Research is found, carries out the potency test of ox inactivated foot-and-mouth disease vaccine with cavy, with the PD of this animal efficacy test
50Between correlativity is arranged preferably (Eissner etc. 1976; Terpstra etc. 1976), also available mouse or BALB/c mouse substitute the ox Indirect evaluation effect of inactivated foot-and-mouth disease vaccine (Sutmoller etc. 1980; DusSantos etc. 2000).Substituting experimental animal is fit to breeding scale, and homogeneity is easily controlled, and experimental animal and this animal are attacked the PD after poison
50Be certain positive correlation, but substituting experimental animal with immune response this animal, certain difference arranged, and what use during substituting experimental animal artificial challenge virus is the test strain, distinct on strain and route of infection with natural infection.
Foot and mouth disease virus is when sucrose density gradient centrifugation, and is 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, the induced animal body produces protective immune response, so in vaccine, the immune protection effectiveness of complete virus particle content and vaccine has obvious correlativity.The quantitative the whole bag of tricks of FMD vaccine antigen is is also researched and developed accordingly, mainly comprises sucrose density gradient centrifugation, sandwich ELISA method and size exclusion chromatography method.
1971, Fayet etc. set up the method that quantitatively detects aftosa complete virus particle in saccharose gradient (146S or 140S) with ultraviolet light; Subsequently, Barteling and Meloen(1974) and Doel etc. further perfect to it, and in nineteen eighty-two to the OIE formal recommendation.In three more than ten years after this, the method is widely used by aftosa vaccine researcher and the supplier of countries in the world as the classical way of aftosa vaccine antigen quantify always.But this method also has its shortcoming: complicated operation, instrument are expensive, repeatability is relatively poor, be not suitable for a large amount of sample detection, can't distinguish serotype.
By comparison, the sandwich ELISA method that (1995) such as Van Maanen etc. (nineteen ninety) and Crowther are set up has more advantage: simple to operate, time saving and energy saving, good stability, be suitable for batch detection, and can distinguish serotype (bivalent seedling and multivalence seedling are particularly important detecting).They with for the neutralizing monoclonal antibody of VP1 linear epitope as capture antibody with detect antibody, set up the double antibodies sandwich ELISA that can detect 146S antigen, and the impact that not existed by 12 S protein subunits.But such monoclonal antibody is not easy to be separated to, and has limited its widespread use.
Size exclusion chromatography (SEC) is called again gel filtration chromatography, or molecular sieve chromatography, is a kind of liquid chromatography isolation technics.Be mainly used in the separation of macromolecular substances such as protein.This technology also is used to the separation of FMD virion, quantitative (Spitteler etc., 2011), first foot-and-mouth disease antigen is separated with molecular sieve, then uses determination of ultra-violet (254nm), calculates the content of aftosa complete virus particle (140S).It is reported, the testing result of this method and the accordance of sucrose density gradient centrifugation are fine, operate also simple than the latter; But it is the same with the latter, can not distinguish serotype.
The exploration of this animal of inactivated foot-and-mouth disease vaccine efficacy test alternative method is a problem that faces both at home and abroad and need to be resolved hurrily, and effectively alternative method should have good specificity, susceptibility and repeatability, and will be easy to standardization.
Summary of the invention
Purpose of the present invention is exactly for above-mentioned defective of the prior art, and a kind of advantage that had both kept sucrose density gradient centrifugation is provided, and 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) deposit Asia I type foot-and-mouth disease standard control antigen;
2) the quantitative Asia I type foot-and-mouth disease standard control of purifying antigen 1 46S;
3) set up the indirect sandwich ELISA method of Asia I type foot-and-mouth disease;
4) drawing standard contrast antigen typical curve;
5) calculate test sample 146S antigenic content;
6) detection sensitivity and specificity.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method is in described step 1), to Asia I type foot-and-mouth disease standard control antigen, a part is put-70 ℃ of deposits (only thaw once, be used for the ELISA test) by 5 mL/bottles; Another part is put-70 ℃ of deposits (only thaw once, be used for the 146S antigen purification and quantitatively (divide three times)) by 3 * 100 mL.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, described step 2) in, to utilize sucrose density gradient method (45%, 35%, 25%, 15%), Asia I type foot-and-mouth disease standard control antigen is carried out purifying, and measuring the 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 of setting up the indirect sandwich ELISA method of Asia I type foot-and-mouth disease is specific as follows:
A) coated elisa plate: be about to 1 μ l aftosa Asia I type rabbit anti-serum with coated damping fluid (pH 9.6) the dilution aftosa Asia I type rabbit anti-serum of carbonate (the Lanzhou veterinary institute provides) to working concentration (1:1000) and add in the coated damping fluid of 1000 μ l carbonate), mixing, every hole adds 50 μ l in the bottom, shakes after 2-3 minute with shrouding film shrouding or puts ambient temperature overnight in wet box;
B) washing elisa plate: take the shrouding film off, discard liquid in elisa plate, every hole fill it up with cleansing solution (0.01M pH 7.4 phosphate tween damping fluids, 1 * PBST), discard liquid after standing 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, begin to do 2 times of serial dilutions to 1:256, and every hole adds 50 μ l, and 37 ℃, incubation 1 hour with PBST washing elisa plate 4 times, pats dry;
D) adding two resists: use guinea pig antiserum diluted aftosa Asia I type guinea pig antiserum to working concentration (1:1000, being about to 1 μ l aftosa Asia I type guinea pig antiserum adds in 1000 μ l guinea pig antiserum dilutions), the guinea pig antiserum dilution is for containing 10% normal cow's serum, the PBST of 5% Healthy Rabbits serum, every hole adds 50 μ l, shrouding, 37 ℃, incubation washed elisa plate 4 times with PBST after 1 hour, patted dry;
E) enzyme-added: as to dilute the anti-cavy of rabbit-horseradish peroxidase bond to working concentration (1:500 with 1 * PBST, being about to the 1 anti-cavy of μ l rabbit-horseradish peroxidase bond adds in 1000 μ l PBST), every hole adds 50 μ l, 37 ℃ of incubations washed elisa plate 4 times with PBST after 1 hour, patted dry;
F) colour developing: every hole adds 50 μ l substrate solutions, 37 ℃ of incubations 15 minutes, and described substrate solution is 20mmol/L o-phenylenediamine OPD, 0.05M citric acid phosphoric acid salt buffer, 0.015%H
2O
2
G) stop: every hole adds 50 μ l stop buffer cessation reactions again, and described stop bath is that concentration is 1.25% H
2SO
4
H) measure: read absorbance value under the 490nm wavelength.
Further, above-mentioned a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, in described step 4), be concentration take standard control antigen as horizontal ordinate, the OD value is ordinate, the 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 to find corresponding concentration with the OD value of sample by typical curve, then multiply by extension rate, obtains the actual concentrations of sample;
Or the linear regression equation that calculates typical curve with concentration and the OD value of reference material, the OD value substitution equation with sample calculates sample concentration, then multiply by extension rate, obtains 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 that calculates typical curve with concentration and the OD value of standard control antigen, during as tested antigen, detection reaction OD value is less than the sensitivity Detection value of standard control antigen with foot-and-mouth disease antigen to be measured.
Second purpose of the present invention has been to provide 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 is to detect with above-mentioned Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method.
The 3rd purpose of the present invention has been to provide the application in generation at Asia I type foot-and-mouth disease antigen quantify and somatotype of a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit.
The 4th purpose of the present invention has been to provide 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 with sucrose density gradient centrifugation, Asia I type foot-and-mouth disease antigen to be carried out quantitatively (parallel 3 times), take it as standard control antigen, other sample is carried out ELISA to be detected, first with the coated microwell plate of aftosa Asia I type rabbit anteserum, make insolubilized antibody, add successively antigen samples, 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 again the substrate solution colour developing, acid adding stops, the depth of color and the Asia I type foot-and-mouth disease 146S antigen concentration in sample are proportionate.Measure absorbance (OD value) with microplate reader, by the concentration of Asia I type foot-and-mouth disease antigen in the typical curve calculation sample under the 490nm wavelength.
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 kept the advantage (internationally recognized) of sucrose density gradient centrifugation, the speciality that has again the ELISA method, the indirect sandwich ELISA method of the antigen quantify that the present invention sets up, emphasis has solved 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, can calculate antigenic content, can carry out somatotype to antigen again, kit with its principle preparation, responsive, special, simple to operate, time saving and energy saving, good stability, be suitable for batch detection, and can distinguish serotype, both can be used for half-finished on-line monitoring in the Asia I type foot-and-mouth disease production of vaccine, can optimize production of vaccine technique again, also can be used as the external inspection technology of aftosa finished product vaccine, the quality of assessment vaccine, the potency test of alternative this animal.The present invention will produce the aspects such as on-line monitoring, vaccine process optimization, vaccine quality assessment, vaccine potency check and play a significant role at antigen.
The experiment effect explanation:
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:
With the indirect sandwich ELISA method of Asia I type foot-and-mouth disease, 5 parts of Asia I type foot-and-mouth disease virus 146s antigenic contents are measured four times, result is stable, good reproducibility, survey simultaneously 2 parts of antigens (O type and A type) of other type, result shows that all lower than its sensitivity range the specificity of this invention is good.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:
With the indirect sandwich ELISA method of Asia I type foot-and-mouth disease, 3 parts of Asia I type foot-and-mouth disease inactivated vaccine 146s antigenic contents are measured four times, result is stable, good reproducibility; With the indirect sandwich ELISA method of Asia I type foot-and-mouth disease, other type inactivated foot-and-mouth disease vaccine (O type and A type) 146s antigenic content is measured, result shows that all lower than its sensitivity range 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.
Annotate: the finished product vaccine is first used emulsion breaker (normal butyl alcohol) breakdown of emulsion, and centrifugal (4 ℃, 5000rpm 20min), the sucking-off antigen part, then detect with the ELISA method.
Description of drawings
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 middle peasant Witter effective company of biological share to provide.
3, Sigma company: the anti-IgG of the rabbit 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 the 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) deposit Asia I type foot-and-mouth disease standard control antigen:
To Asia I type foot-and-mouth disease standard control antigen, a part is put-70 ℃ of deposits (only thaw once, be used for the ELISA test) by 5 mL/bottles; Another part is put-70 ℃ of deposits (only thaw once, be used for the 146S antigen purification and quantitatively (divide three times)) by 3 * 100 mL;
2) the quantitative Asia I type foot-and-mouth disease standard control of purifying antigen 1 46S:
Utilize sucrose density gradient method (45%, 35%, 25%, 15%), Asia I type foot-and-mouth disease standard control antigen is carried out purifying, measuring the 146S antigenic content is 1.74 μ g/ml;
3) set up the indirect sandwich ELISA method of Asia I type foot-and-mouth disease:
Specific as follows:
A) coated elisa plate: be about to 1 μ l aftosa Asia I type rabbit anti-serum with coated damping fluid (pH 9.6) the dilution aftosa Asia I type rabbit anti-serum of carbonate (the Lanzhou veterinary institute provides) to working concentration (1:1000) and add in the coated damping fluid of 1000 μ l carbonate), mixing, every hole adds 50 μ l in the bottom, shakes after 2-3 minute with shrouding film shrouding or puts ambient temperature overnight in wet box;
B) washing elisa plate: take the shrouding film off, discard liquid in elisa plate, every hole fill it up with cleansing solution (0.01M pH 7.4 phosphate tween damping fluids, 1 * PBST), discard liquid after standing 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, begin to do 2 times of serial dilutions to 1:256, and every hole adds 50 μ l, and 37 ℃, incubation 1 hour with PBST washing elisa plate 4 times, pats dry;
D) adding two resists: use guinea pig antiserum diluted aftosa Asia I type guinea pig antiserum to working concentration (1:1000, being about to 1 μ l aftosa Asia I type guinea pig antiserum adds in 1000 μ l guinea pig antiserum dilutions), the guinea pig antiserum dilution is for containing 10% normal cow's serum, the PBST of 5% Healthy Rabbits serum, every hole adds 50 μ l, shrouding, 37 ℃, incubation washed elisa plate 4 times with PBST after 1 hour, patted dry;
E) enzyme-added: as to dilute the anti-cavy of rabbit-horseradish peroxidase bond to working concentration (1:500 with 1 * PBST, being about to the 1 anti-cavy of μ l rabbit-horseradish peroxidase bond adds in 1000 μ l PBST), every hole adds 50 μ l, 37 ℃ of incubations washed elisa plate 4 times with PBST after 1 hour, patted dry;
F) colour developing: every hole adds 50 μ l substrate solutions, 37 ℃ of incubations 15 minutes, and described substrate solution is 20mmol/L o-phenylenediamine OPD, 0.05M citric acid phosphoric acid salt buffer, 0.015%H
2O
2
G) stop: every hole adds 50 μ l stop buffer cessation reactions again, and described stop bath is that concentration is 1.25% H
2SO
4
H) measure: read absorbance value under the 490nm wavelength;
4) drawing standard contrast antigen typical curve:
Take the concentration of standard control antigen as horizontal ordinate, the OD value is ordinate, the drawing standard curve;
5) calculate test sample 146S antigenic content:
Be to find corresponding concentration with the OD value of sample by typical curve, then multiply by extension rate, obtain the actual concentrations of sample;
Or the linear regression equation that calculates typical curve with concentration and the OD value of reference material, the OD value substitution equation with sample calculates sample concentration, then multiply by extension rate, obtains 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 that calculates typical curve with concentration and the OD value of standard control antigen, during as tested antigen, detection reaction OD value is less than the sensitivity Detection value of standard control antigen with foot-and-mouth disease antigen to be measured.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment, the present invention is had been described in detail, for a person skilled in the art, it still can be modified to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. an Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method, is characterized in that, comprises the following steps:
1) deposit Asia I type foot-and-mouth disease standard control antigen;
2) the quantitative Asia I type foot-and-mouth disease standard control of purifying antigen 1 46S;
3) set up the indirect sandwich ELISA method of Asia I type foot-and-mouth disease;
4) drawing standard contrast antigen typical curve;
5) calculate test sample 146S antigenic content;
6) detection sensitivity and specificity.
2. a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 1, it is characterized in that, in described step 1), to Asia I type foot-and-mouth disease standard control antigen, a part is pressed the 5mL/ bottle, be placed in-70 ℃ of deposits, this part is only thawed once, is used for the ELISA test; Another part is placed in-70 ℃ of deposits by 3 * 100 mL, and this part is only thawed once, is used for the 146S antigen purification quantitative minutes for three times.
3. a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 2, it is characterized in that, described step 2) in, to utilize the 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 that records the 146S antigenic content for three times is 1.74 μ g/ml.
4. a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 3, is characterized in that, in described step 3), the process of setting up the indirect sandwich ELISA method of Asia I type foot-and-mouth disease is specific as follows:
A) coated elisa plate: dilute aftosa Asia I type rabbit anti-serum to working concentration with the coated damping fluid of the carbonate of pH 9.6, working concentration is 1:1000, being about to 1 μ l aftosa Asia I type rabbit anti-serum adds in the coated damping fluid of 1000 μ l carbonate, mixing, every hole adds 50 μ l in the bottom, shakes after 2-3 minute with shrouding film shrouding or puts ambient temperature overnight in wet box;
B) washing elisa plate: take the shrouding film off, discard liquid in elisa plate, cleansing solution is filled it up with in every hole, and cleansing solution is 0.01M, the phosphate tween damping fluid of pH 7.4, and 1 * PBST discards liquid after standing 30 seconds, repeats 4 times, pats 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, begin to do 2 times of serial dilutions to 1:256, and every hole adds 50 μ l, and 37 ℃, incubation 1 hour with PBST washing elisa plate 4 times, pats dry;
D) adding two resists: use guinea pig antiserum diluted aftosa Asia I type guinea pig antiserum to working concentration, working concentration is 1:1000, being about to 1 μ l aftosa Asia I type guinea pig antiserum adds in 1000 μ l guinea pig antiserum dilutions, the guinea pig antiserum dilution is for containing 10% normal cow's serum, the PBST of 5% Healthy Rabbits serum, and every hole adds 50 μ l, shrouding, 37 ℃, incubation washed elisa plate 4 times with PBST after 1 hour, patted dry;
E) enzyme-added: as to dilute the anti-cavy of rabbit-horseradish peroxidase bond to working concentration with 1 * PBST, working concentration is 1:500, being about to the 1 anti-cavy of μ l rabbit-horseradish peroxidase bond adds in 1000 μ l PBST, every hole adds 50 μ l, 37 ℃ of incubations washed elisa plate 4 times with PBST after 1 hour, patted dry;
F) colour developing: every hole adds 50 μ l substrate solutions, 37 ℃ of incubations 15 minutes, and described substrate solution is 20mmol/L o-phenylenediamine OPD, 0.05M citric acid phosphoric acid salt buffer, 0.015%H
2O
2
G) stop: every hole adds 50 μ l stop buffer cessation reactions again, and described stop bath is that concentration is 1.25% H
2SO
4
H) measure: read absorbance value under the 490nm wavelength.
5. a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 4, is characterized in that, in described step 4), be concentration take standard control antigen as horizontal ordinate, the OD value is ordinate, the drawing standard curve.
6. a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 5, it is characterized in that, in described step 5), be to find corresponding concentration with the OD value of sample by typical curve, multiply by again extension rate, obtain the actual concentrations of sample;
Or the linear regression equation that calculates typical curve with concentration and the OD value of reference material, the OD value substitution equation with sample calculates sample concentration, then multiply by extension rate, obtains the actual concentrations of sample.
7. a kind of Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method according to claim 6, it is characterized in that, 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 that calculates typical curve with concentration and the OD value of standard control antigen, during as tested antigen, detection reaction OD value is less than the sensitivity Detection value of standard control antigen with foot-and-mouth disease antigen to be measured.
8. 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 the arbitrary described Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection method of claim 1-7.
9. the application of an 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.
10. the application of an Asia I type foot-and-mouth disease 146S antigen quantify ELISA detection kit in aftosa vaccine quality evaluation and somatotype.
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| 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 |
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| 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 |
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