CN106501512A - Foot-and-mouth disease antibody detection kit, detection method and application thereof - Google Patents
Foot-and-mouth disease antibody detection kit, detection method and application thereof Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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Abstract
The invention provides a foot-and-mouth disease antibody detection kit, which can solve the technical problems of low sensitivity and poor specificity of the existing detection kit. The kit comprises a coating plate, an antigen-antibody reaction plate, an antigen, a foot-and-mouth disease rabbit anti-working solution, a goat anti-rabbit IgG-HRP working solution, positive quality control serum, negative quality control serum, a sample diluent, a washing solution and a substrate, and is characterized in that: the coated plate is a streptavidin coated plate, and the antigen is a biotinylated foot-and-mouth disease virus antigen. The invention also provides a detection method of the detection kit and application thereof.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of foot-and-mouth disease antibody detection kit, detection method
And its application.
Background technology
Foot and mouth disease (FMD) is a kind of acute, the hot, highly contagious disease that the artiodactyls such as pig, cattle, sheep suffer from altogether,
Susceptible animal is up to kind more than 70.Clinical symptoms are, in oral mucosa, hoof and skin of breast, vesicle rash occurs.The sick route of transmission
Many, speed is fast, once multiple worldwide outbreak of epidemic, causes huge politics, economic loss.In consideration of it, world animal is defended
Raw tissue (OIE) is classified as first of A class infectious disease.At present, 2/3rds OIE member states prevalence FMD, moment threaten
Safety of livestock and Livestock Product Trade without FMD countries and regions.
The preventing and treating of foot and mouth disease typically realized by vaccinating, but due to animal species difference or individual variation, different
The effect of individual immunity is inconsistent.In order to detect immune effect, the detection of foot-and-mouth disease antibody need to be carried out.
Existing foot-and-mouth disease antibody detection kit detection sensitivity is relatively low, specificity is poor, it is impossible to meet high flux sample
This detection demand.
Content of the invention
For the problems referred to above, the invention provides a kind of foot-and-mouth disease antibody detection kit, which can solve existing detection examination
Agent box sensitivity is relatively low, the technical problem that specificity is poor.
Its technical scheme be such, its plate that includes being coated, antigen antibody reaction plate, antigen, foot and mouth disease rabbit-anti working solution,
Goat anti-rabbit igg-HRP working solutions, positive quality control serum, negative quality controlled serum, Sample dilution, washing liquid and substrate, its feature exist
In：The coated plate is Streptavidin coating plate, and the antigen is biotinylated foot-and-mouth disease virus antigen.
Further, Streptavidin of the Streptavidin coating plate by 2～20 μ g/mL, 100 μ l/ holes, coated shape
Into.
Further, the foot and mouth disease rabbit-anti working solution is containing 2～10 μ g/ml foot and mouth disease rabbit multi-resistance, 1wt% caseins
PBS, pH be 7.2～7.4.
Further, the goat anti-rabbit igg-HRP working solutions be containing 0.5～2.5 μ g/ml goat anti-rabbit igg-HRP,
The caseic PBSs of 1wt%, pH are 7.2～7.4.
Further, the negative quality controlled serum is the artiodactyl serum for being uninfected by foot and mouth disease viruses, and antibody test is imitated
Valency is less than 1：8；The positive quality control serum is that the foot and mouth disease multi-resistance of 20～50 μ g/mL of negative quality controlled serum interpolation is obtained, antibody
Detection potency is more than 1:720.
Further, the Sample dilution is that pH is 7.2～7.4 containing 1wt% caseic PBSs.
Further, the washing liquid is 20 times of concentration washing liquid, described 20 times of concentration washing liquid be containing 18wt%NaCl,
The Tris-HCl buffer of 1wt%Tween20 and 1wt%Proclin300, pH are 7.4～7.6.
Further, the substrate includes substrate A and substrate B；The substrate A is containing 1.26wt%N, N- dimethyl methyls
The Tris-HCl bufferings of amide, 5.85mmol/L luminols, 9.86mmol/L sodium tetraphenylborates and 1.06mg/L acetaminophen
Liquid, pH are 8.8～9.0；The substrate B is the Tris-HCl buffer containing 0.615mg/mL Dexol, and pH is 4.8～5.0.
Present invention also offers the detection method of above-mentioned foot-and-mouth disease antibody detection kit, comprises the following steps：
(1) in antigen antibody reaction plate, using Sample dilution to serum sample to be measured, positive quality control serum, feminine gender
Quality controlled serum carries out doubling dilution, 50 μ l/ holes, the test serum sample, positive quality control serum, the dilution of negative quality controlled serum
Scope is respectively (1：4～1：512)、(1：8～1：1024)、(1：2～1：4), antigen control arranges 4 holes, is separately added into 50 μ l
Sample dilution；
(2) biotinylated foot-and-mouth disease virus antigen working solution (2 μ g/ml) is added, 50 μ l/ holes, vibration, 37 DEG C incubate 60
Minute；
(3) reactant liquor is transferred in Streptavidin coating plate, 50 μ l/ holes, 37 DEG C incubate 30 minutes；
(4) board-washing, dries, and in Streptavidin coating plate adds foot and mouth disease rabbit-anti working solution, 50 μ l/ holes, 37 DEG C of temperature
Educate 30 minutes；
(5) board-washing, dries, the addition goat anti-rabbit igg-HRP working solutions in Streptavidin coating plate, 50 μ l/ holes, 37 DEG C
Incubate 30 minutes；
(6) board-washing, dries, and adds substrate A and substrate B, and 50 μ l/ holes determine relative light intensity (RLU)；
(7) judge the potency of test serum：4 holes of antigen control, discard highest and minimum RLU values, calculate remaining 2 hole
Average RLU values, with 55%～75% antigen control meansigma methodss as marginal value；Serum RLU values to be checked are the moon more than the hole of marginal value
Property hole, be positive hole less than or equal to the hole of marginal value；If marginal value is identical with the RLU in extension rate highest positive hole, to treat
Antibody titer of the highest extension rate in inspection seropositivity hole as the serum；If marginal value is between 2 dilution holes RLU,
Antibody titer takes the antilogarithm intermediate value of neighboring positive hole and negative hole extension rate.
Further, detection Recognized Standards are：Virus antigen control RLU values should in the range of 8000000 to 15000000,
Positive quality control serum antibody titer should be greater than 1:720, negative quality controlled serum antibody titer should be less than 1:8.
Present invention also offers application of the above-mentioned detection kit in foot-and-mouth disease antibody detection.
Experiment shows, the above-mentioned detection kit of the present invention is compared to existing foot-and-mouth disease antibody detection kit, sensitive
Degree is higher, specificity is preferable, disclosure satisfy that the detection demand of high flux sample.
Specific embodiment
Main agents explanation：
Foot-and-mouth disease virus antigen, is O-shaped, and purchased from Hangzhou Kitgen Biotechnology Co., Ltd., numbering is KT01.
Goat anti-rabbit igg-HRP, purchased from Hangzhou Kitgen Biotechnology Co., Ltd., numbering is HRP-GRI06.
Negative quality controlled serum, cultivates factory from hog, for being uninfected by the porcine blood serum sample of foot and mouth disease viruses.
Commercial ELISA Assay kit, is the O-shaped foot-and-mouth disease antibody detection kit of Pronics companies of Switzerland.
Interference sample S1～S3, cultivates factory from hog, is that injection strengthens bivalent foot-and-mouth disease vaccine (comprising O-shaped mouth hoof
Epidemic disease vaccine and Asia I type foot-and-mouth disease vaccine) porcine blood serum sample.
Sample to be tested 1～10, from hog plant, is the porcine blood serum sample for injecting O-shaped foot-and-mouth disease vaccine.
Biotinylated foot-and-mouth disease virus antigen, preparation process are as follows：
1. preparation of samples
1.1Buffer prepare
Buffer A：PB buffer, pH 7.4；
Buffer B：PBS, pH 6.0.
1.2 antigens are prepared
1ml foot-and-mouth disease virus antigens (200 μ g/mL) are taken, and desalination are carried out with buffer A and are changed liquid.
1.3NHS-LC-LC-Biotin solution is prepared
1mg NHS-LC-LC-Biotin are weighed, the dissolving of 200 μ l buffer As is added, it is 5mg/ml to make mass concentration.
2. labelling purification
2.1 labelling
The NHS-LC-LC-Biotin solution for taking 40 μ l is added in antigenic solution, is mixed, and reacts 1h, feed intake at 20 DEG C
It is 1 than (mass ratio)：1.
2.2 purification
After question response terminates, reaction solution is dialysed, elution buffer is buffer B, dialysed 3 times, each 3h, per
Secondary 1000ml；Dialysis environment is 4 DEG C, ensures dialysis speed using magnetic stirring apparatuss.
3. detect
Antigen concentration is determined using Coomassie Brilliant Blue.
The preparation of 3.1 protein standard substances
Take protein standard substance (BSA) to be dissolved completely in buffer B, make final concentration of 0.5mg/ml.
3.2 determination of protein concentration
A. standard substance are added in the standard sample wells of 96 orifice plates by 0,1,2,4,8,12,16,20 μ l, plus standard dilutions
20 μ l are supplied, equivalent to standard concentration respectively 0,0.025,0.05,0.1,0.2,0.3,0.4,0.5mg/ml.
Plus proper volume sample is in the sample well of 96 orifice plates b.；
C. each hole adds 200 μ l G250 dyeing liquors, room temperature to place 3～5min；
D. the absorbance of A595nm is determined with microplate reader；
E. according to standard curve and using sample volume calculate the protein concentration in sample.
Experimental arrangement is as shown in table 1.
Table 1：
Initial data is as shown in table 2.
Table 2：
The data deducted after blank value are as shown in table 3.
Table 3：
According to 3 data of table, protein quantification standard curve is measured for y=0.0013x+0.0962, R2=0.970, calculate institute
It is 117.5 μ g/ml to obtain biotinylated foot-and-mouth disease virus antigen concentration.
Foot and mouth disease rabbit multi-resistance,
1. under its preparation process：
Immunogen, the adjuvant of the O-shaped foot and mouth disease viruses and equivalent that take inactivation are mixed into stable emulsion, during preparation, inactivation
O-shaped foot and mouth disease viruses diluted by physiological saline solution；Immune animal, healthy Male New Zealand White Rabbit 2；Just exempt to use
Freund's complete adjuvant, two exempt from and use later incomplete Freund's adjuvant, and immunizing dose is 300 μ g/, and subcutaneous injection just exempts from 2 weeks
After carry out two and exempt from, two exempt from and interval is 2 weeks later.
2. rabbit immune effect monitoring：
Parameter：Foot-and-mouth disease antigen 100ng/ holes are coated, rabbit polyvalent antibody 1：100 dilutions, HRP-GRI06 1:5000 dilutions.
Monitoring Data is as shown in table 4.
Table 4：
Table 4 shows that No. 1 rabbit reached plateau at the 5th week, and No. 2 rabbits also reached plateau at the 6th week, can carry out
Serum collection.
3. final collection serum titre detection (4 exempt from after gather serum, the 7th week)：
Parameter：Foot-and-mouth disease antigen 100ng/ holes are coated, rabbit polyvalent antibody gradient dilution, HRP-GRI06 1:5000 dilutions make
With.
Testing result is as shown in table 5.
Table 5：
Table 5 shows：Two rabbit serum titers reach 1：100000, the multi-resistance can be used for the research and development of follow-up test kit.
Embodiment 1：
A kind of foot-and-mouth disease antibody detection kit, which includes：
Streptavidin coating plate, by the Streptavidin of 5 μ g/mL, 100 μ l/ holes, coated formation；
U-shaped 96 hole antigen antibody reaction plate；
Biotinylated foot-and-mouth disease virus antigen, working concentration are that (original concentration is 117.5 μ g/ml to 2 μ g/ml, using sample
Product diluted is obtained)；
Foot and mouth disease rabbit-anti working solution, is that pH is containing 4 μ g/ml foot and mouth disease rabbit multi-resistance, 1wt% caseic PBSs
7.2；
Goat anti-rabbit igg-HRP working solutions, are containing the caseic PBS bufferings of 1.0 μ g/ml goat anti-rabbit igg-HRP, 1wt%
Liquid, pH are 7.4；
Negative quality controlled serum, antibody test potency are less than 1：8；
Positive quality control serum, is that the foot and mouth disease rabbit multi-resistance of 30 μ g/mL of negative quality controlled serum interpolation is obtained, antibody test potency
It is more than 1:720；
Sample dilution, is that pH is 7.3 containing 1wt% caseic PBSs；
20 times of concentration washing liquid, is the Tris-HCl containing 18wt%NaCl, 1wt%Tween20 and 1wt%Proclin300
Buffer, pH are 7.6；
Substrate A, is containing 1.26wt%N, dinethylformamide, 5.85mmol/L luminols, tetra- benzene of 9.86mmol/L
The Tris-HCl buffer of boron sodium and 1.06mg/L acetaminophen, pH is 8.8；
Substrate B, is the Tris-HCl buffer containing 0.615mg/mL Dexol, and pH is 5.0.
Embodiment 2：
A kind of foot-and-mouth disease antibody detection kit, which includes：
Streptavidin coating plate, by the Streptavidin of 2 μ g/mL, 100 μ l/ holes, coated formation；
U-shaped 96 hole antigen antibody reaction plate；
Biotinylated foot-and-mouth disease virus antigen, working concentration are that (original concentration is 117.5 μ g/ml to 0.5 μ g/ml, uses
Sample diluting liquid dilution is obtained)；
Foot and mouth disease rabbit-anti working solution, is that pH is containing 2 μ g/ml foot and mouth disease rabbit multi-resistance, 1wt% caseic PBSs
7.4；
Goat anti-rabbit igg-HRP working solutions, are containing the caseic PBS bufferings of 0.5 μ g/ml goat anti-rabbit igg-HRP, 1wt%
Liquid, pH are 7.2；
Negative quality controlled serum, antibody test potency are less than 1：8；
Positive quality control serum, is foot and mouth disease rabbit multi-resistance that negative quality controlled serum adds 20 μ g/mL, and antibody test potency is more than
1:720；
Sample dilution, is that pH is 7.4 containing 1wt% caseic PBSs；
20 times of concentration washing liquid, is the Tris-HCl containing 18wt%NaCl, 1wt%Tween20 and 1wt%Proclin300
Buffer, pH are 7.5；
Substrate A, is containing 1.26wt%N, dinethylformamide, 5.85mmol/L luminols, tetra- benzene of 9.86mmol/L
The Tris-HCl buffer of boron sodium and 1.06mg/L acetaminophen, pH is 8.9；
Substrate B, is the Tris-HCl buffer containing 0.615mg/mL Dexol, and pH is 4.8.
Embodiment 3：
A kind of foot-and-mouth disease antibody detection kit, which includes：
Streptavidin coating plate, by the Streptavidin of 20 μ g/mL, 100 μ l/ holes, coated formation；
U-shaped 96 hole antigen antibody reaction plate；
Biotinylated foot-and-mouth disease virus antigen, working concentration are that (original concentration is 117.5 μ g/ml to 5 μ g/ml, using sample
Product diluted is obtained)；
Foot and mouth disease rabbit-anti working solution, is containing 10 μ g/ml foot and mouth disease rabbit multi-resistance, the caseic PBSs of 1wt%, pH
For 7.3；
Goat anti-rabbit igg-HRP working solutions, are containing the caseic PBS bufferings of 2.5 μ g/ml goat anti-rabbit igg-HRP, 1wt%
Liquid, pH are 7.3；
Negative quality controlled serum, antibody test potency are less than 1：8；
Positive quality control serum, is foot and mouth disease rabbit multi-resistance that negative quality controlled serum adds 50 μ g/mL, and antibody test potency is more than
1:720；
Sample dilution, is that pH is 7.2 containing 1wt% caseic PBSs；
20 times of concentration washing liquid, is the Tris-HCl containing 18wt%NaCl, 1wt%Tween20 and 1wt%Proclin300
Buffer, pH are 7.4；
Substrate A, is containing 1.26wt%N, dinethylformamide, 5.85mmol/L luminols, tetra- benzene of 9.86mmol/L
The Tris-HCl buffer of boron sodium and 1.06mg/L acetaminophen, pH is 9.0；
Substrate B, is the Tris-HCl buffer containing 0.615mg/mL Dexol, and pH is 4.9.
Embodiment 4：
The using method of the test kit of embodiment 1：
(1) in antigen antibody reaction plate, using Sample dilution to serum sample to be measured, positive quality control serum, feminine gender
Quality controlled serum carries out doubling dilution, 50 μ l/ holes, the test serum sample, positive quality control serum, the dilution of negative quality controlled serum
Scope is respectively (1：4～1：512)、(1：8～1：1024)、(1：2～1：4), antigen control arranges 4 holes, is separately added into 50 μ l
Sample dilution；
Sample layout and extension rate are as shown in table 6.
Table 6：
S1～S10 be test serum sample, S2～S10 extension rate distribution identical with S1.
(2) the biotinylated foot-and-mouth disease virus antigen of 2 μ g/ml, 50 μ l/ holes, shrouding, vibration, 37 DEG C of incubations 60 are added
Minute.
The extension rate added after foot-and-mouth disease virus antigen is as shown in table 7.
Table 7：
(3) reactant liquor is transferred in Streptavidin coating plate, 50 μ l/ holes, shrouding, 37 DEG C incubate 30 minutes；
(4) board-washing 5 times, dry, and in Streptavidin coating plate add foot and mouth disease rabbit-anti working solution, 50 μ l/ holes, envelope
Plate, 37 DEG C incubate 30 minutes；
(5) board-washing 5 times, dry, the addition goat anti-rabbit igg-HRP working solutions in Streptavidin coating plate, 50 μ l/ holes,
Shrouding, 37 DEG C incubate 30 minutes；
(6) board-washing 5 times, dry, and add substrate A and substrate B, and 50 μ l/ holes, using chemiluminescence immune assay in 5 minutes
Instrument determines relative light intensity (RLU)；
(7) result judgement：
A, detection Recognized Standards are：Virus antigen control RLU values should be in the range of 8000000 to 15000000, positive matter
Control serum antibody titer should be greater than 1:720, negative quality controlled serum antibody titer should be less than 1:8
B, the judgement of serum antibody titer：4 holes of antigen control, discard highest and minimum RLU values, calculate remaining 2 hole
Average RLU values, with 65% antigen control meansigma methodss as marginal value；Serum RLU values to be checked are negative hole more than the hole of marginal value, little
In the hole for being equal to marginal value be positive hole；If marginal value is identical with the RLU in extension rate highest positive hole, with serum to be checked sun
Antibody titer of the highest extension rate in property hole as the serum；If marginal value is between 2 dilution holes RLU, antibody titer
Take the antilogarithm intermediate value of neighboring positive hole and negative hole extension rate.
Antibody titer calculates control as shown in table 8.
Table 8
Additionally, the using method of the test kit of embodiment 2 adopts 75% antigen control meansigma methodss for marginal value；Embodiment 3
The using method of test kit adopt 55% antigen control meansigma methodss for marginal value.
Experimental example 1：Sensitivity experiment.
As described in Example 4,20 hole of parallel assay feminine gender quality controlled serum 1:Relative luminous intensity RLU during 4 dilution factor,
The standard deviation (SD) and RLU meansigma methodss (M) of RLU is calculated, (M-2SD) suppression ratio of corresponding RLU RLU average to antigen control is made
For weighing the index of test kit sensitivity.
Sensitivity experiment layout is as shown in table 9.
Table 9：
Sensitivity experiment result is as shown in table 10.
Table 10：
Table 10 shows, this test kit feminine gender quality controlled serum 1:Suppression ratio during 4 dilution factor is 7.49%, less than current techique
Index IC15The requirement of (suppression ratio 15%)；And the suppression ratio of commercial ELISA Assay kit is 25.37%, sensitivity relatively this test kit
Low.Experimental example 2：Specificity experiments.
Interference sample explanation：S1～S3 is three parts and comes from the individual serum sample of different pigs, antibody titer apparently higher than
Routine immunization porcine blood serum potency, while the matrix effect of serum and other interference factors are also more notable.
As described in Example 2, while determining the potency of 3 parts of interference samples, the different dilution factors of observation are strong with relative luminous
The plots changes of degree RLU, judge the specificity of test kit with this.
Specific test layout is as shown in table 11.
Table 11：
Specificity experiments result is as shown in table 12.
Table 12：
Table 12 shows that, with the rising of interference sample dilutions, the OD values of commercial ELISA Assay kit detection have significantly
The trend risen after falling before, and the test kit RLU value decline stages of the present invention are not obvious, capacity of resisting disturbance is significantly stronger than ELISA
Test kit, and serum titer gradation levels high compared with ELISA kit are detected, detection sensitivity is high.
Experimental example 3：Stability experiment.
The effect duration of commercial ELISA Assay kit is 6 months, take commercial ELISA Assay kit in exhaustion of effect 3 months and this
Invention test kit, while detecting yin and yang attribute quality controlled serum, judges the stability of test kit with this.
Stability test layout is as shown in table 13.
Table 13：
Stability experiment result is as shown in table 14.
Table 14：
Table 14 shows that, with the prolongation of test kit storage time, commercial ELISA Assay kit is with test kit of the present invention at 6
In the effect duration of the moon, yin and yang attribute quality controlled serum titer plateaus are detected, detection data is authentic and valid.
Additionally, after testing, embodiment 2,3 corresponding reagent boxes detection sample to be tested potency are same as Example 1, show this Shen
Mentioned reagent box and its detection method please has higher Stability and veracity.
Table 15：
The serum sample individual for coming from different pigs of sample to be tested 1～10.
Claims (10)
1. a kind of foot-and-mouth disease antibody detection kit, which includes be coated plate, antigen antibody reaction plate, antigen, foot and mouth disease rabbit-anti work
Make liquid, goat anti-rabbit igg-HRP working solutions, positive quality control serum, negative quality controlled serum, Sample dilution, washing liquid and substrate, which is special
Levy and be：The coated plate is Streptavidin coating plate, and the antigen is biotinylated foot-and-mouth disease virus antigen.
2. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：The Streptavidin bag
By plate by 2 ~ 20 μ g/mL Streptavidin, 100 μ l/ holes coated are formed.
3. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：The foot and mouth disease rabbit-anti work
It is that pH is 7.2 ~ 7.4 containing 2 ~ 10 μ g/ml foot and mouth disease rabbit multi-resistance, 1wt% caseic PBSs as liquid.
4. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：The goat anti-rabbit igg-
HRP working solutions are that pH is 7.2 ~ 7.4 containing 0.5 ~ 2.5 μ g/ml goat anti-rabbit igg-HRP, 1wt% caseic PBSs.
5. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：The negative quality controlled serum
For being uninfected by the artiodactyl serum of foot and mouth disease viruses, antibody test potency is less than 1：8；The positive quality control serum is negative matter
Control serum adds foot-and-mouth disease antibody and is obtained, and antibody test potency is more than 1:720.
6. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：The Sample dilution is
Containing the caseic PBSs of 1wt%, pH is 7.2 ~ 7.4.
7. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：, the washing liquid is 20 times
Concentration washing liquid, described 20 times of concentration washing liquid is the Tris- containing 18wt%NaCl, 1wt%Tween20 and 1wt%Proclin300
HCl buffer, pH are 7.4 ~ 7.6.
8. a kind of foot-and-mouth disease antibody detection kit according to claim 1, it is characterised in that：The substrate includes substrate
A and substrate B；The substrate A is containing 1.26wt%N, dinethylformamide, 5.85 mmol/L luminols, 9.86 mmol/L
Sodium tetraphenylborate and the Tris-HCl buffer of 1.06 mg/L acetaminophen, pH are 8.8 ~ 9.0；The substrate B be containing
The Tris-HCl buffer of 0.615 mg/mL Dexol, pH are 4.8 ~ 5.0.
9., such as the detection method of arbitrary described foot-and-mouth disease antibody detection kit in claim 1 ~ 8, comprise the following steps：
（1）In antigen antibody reaction plate, using Sample dilution to serum sample to be measured, positive quality control serum, negative Quality Control
Serum carries out doubling dilution, 50 μ l/ holes, the test serum sample, positive quality control serum, the dilution range of negative quality controlled serum
Respectively（1：4~1：512）、（ 1：8~1：1024）、（1：2~1：4）, 4 holes of antigen control setting are separately added into 50 μ l samples dilute
Release liquid；
（2）Biotinylated foot-and-mouth disease virus antigen working solution, 50 μ l/ holes is added to vibrate, 37 DEG C incubate 60 minutes；
（3）Reactant liquor is transferred in Streptavidin coating plate, 50 μ l/ holes, 37 DEG C incubate 30 minutes；
（4）Board-washing, dries, and in Streptavidin coating plate adds foot and mouth disease rabbit-anti working solution, 50 μ l/ holes, 37 DEG C of incubations 30
Minute；
（5）Board-washing, dries, and in Streptavidin coating plate adds goat anti-rabbit igg-HRP working solutions, 50 μ l/ holes, 37 DEG C of incubations
30 minutes；
（6）Board-washing, dries, and adds substrate A and substrate B, and 50 μ l/ holes determine relative light intensity（RLU）；
（7）Judge the potency of test serum：4 holes of antigen control, discard highest and minimum RLU values, calculate the average of remaining 2 hole
RLU values, with 55% ~ 75% antigen control meansigma methodss as marginal value；Serum RLU values to be checked are negative hole more than the hole of marginal value, little
In the hole for being equal to marginal value be positive hole；If marginal value is identical with the RLU in extension rate highest positive hole, with serum to be checked sun
Antibody titer of the highest extension rate in property hole as the serum；If marginal value is between 2 dilution holes RLU, antibody titer
Take the antilogarithm intermediate value of neighboring positive hole and negative hole extension rate.
10. such as application of arbitrary described foot-and-mouth disease antibody detection kit in foot-and-mouth disease antibody detection in claim 1 ~ 9.
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