CN108333352A - A kind of high-flux detection method of O-shaped foot-and-mouth disease antibody - Google Patents
A kind of high-flux detection method of O-shaped foot-and-mouth disease antibody Download PDFInfo
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Abstract
The technical issues of the present invention provides a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody, capable of solving to solve that Liquid-phase blocking ELISA method detection speed is slow, and detection flux is low, cannot be satisfied the fast high-flux detection demand under great amount of samples.The detection method, it uses the test serum relative light intensity RLU of chemiluminescence immunoassay determination of adsorption method sample to be tested and antigen control relative light intensity RLU, and inhibiting rate PI is calculated according to test serum relative light intensity RLU and antigen control relative light intensity RLU, it is characterised in that：Sample to be tested serum in the sample handling processes of chemiluminescence immunoassay absorption method, negative quality controlled serum and positive quality control serum carry out single dilutions, and the corresponding inhibiting rate PI of each potency serum counts the non-overlapping phenomenon in section under single dilutions multiple；Inhibiting rate PI by critical inhibiting rate corresponding with each potency serum compare the potency of determining test serum.
Description
Technical field
The present invention relates to technical field of biological, and in particular to a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody.
Background technology
Aftosa (foot-mouth disease, FMD) be the artiodactyl caused by foot and mouth disease virus (pig, ox, sheep,
Camel etc.) acute, the hot, contagious disease suffered from altogether, the disease is by World Organization for Animal Health (Office
International Des Epizooties, OIE) it is classified as first of A class infectious diseases, to the very harmful of Animal husbandry production.Disappear
It is the worldwide problem that national governments pay close attention to jointly to go out and control aftosa.
Serodiagnosis is one of the core technology in aftosa prevention, including Liquid-phase blocking ELISA, solid phase
The multinomial technologies such as competitive ELISA, nonstructural protein 3A BC antibody tests ELISA.Liquid-phase blocking ELISA is because of its good sensitivity
Property, specificity and stability are the aftosa serum diagnostic techniques being most widely used in the world.But this method is due to needing
Gradient dilution is carried out to sample, leads to it there are detection speeds slow, the low defect of detection flux cannot be satisfied under great amount of samples
The detection demand of fast high-flux.Lack the kit for carrying out high-throughput detection to foot-and-mouth disease antibody in existing market, mostly continues to use
Gradient dilution method carries out titration, and detection efficiency is low to have an immense impact on to aftosa epidemic monitoring.
Invention content
In view of the above-mentioned problems, the present invention provides a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody, can solve
Certainly Liquid-phase blocking ELISA method detection speed is slow, and detection flux is low, and the fast high-flux detection that cannot be satisfied under great amount of samples needs
The technical issues of asking.
Its technical solution is such, and a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody uses chemiluminescence to exempt from
The test serum relative light intensity RLU and antigen control relative light intensity RLU of epidemic disease determination of adsorption method sample to be tested, and according to be measured
Serum relative light intensity RLU and antigen control relative light intensity RLU calculate inhibiting rate PI, it is characterised in that：The chemiluminescence
It is dilute to carry out single for sample to be tested serum, negative quality controlled serum and positive quality control serum in the sample handling processes of immunoabsorption
It releases, the corresponding inhibiting rate PI of each potency serum counts the non-overlapping phenomenon in section under single dilutions multiple；The inhibiting rate PI passes through
Critical inhibiting rate corresponding with each potency serum compare the potency of determining test serum.
It is further, described that detection method includes the following steps：
(1) the sample to be tested serum after Sample dilution single dilutions, negative Quality Control blood are added in antigen-antibody reaction plate
The multiple of cleer and peaceful positive quality control serum, the single dilutions is 32 times；
(2) the biotinylated O-shaped viral antigen working solution of aftosa is added, while 4 antigen control holes are set, incubates, it is described
The antigen concentration of the biotinylated O-shaped viral antigen working solution of aftosa is 15 μ g/ml；
(3) it by the solution integral translation in reaction plate to the how anti-coating plate of rabbit, incubates, how anti-the coating of the rabbit coating plate be dense
Degree is 2 μ g/ml；
(4) board-washing, drying are added enzyme mark Streptavidin working solution to the how anti-plate of rabbit, incubate；
(5) substrate A and substrate B is added in board-washing, drying, measures relative light intensity RLU；
(6) judge, antigen control hole setting 4 is each, discards the peak and minimum of antigen control relative light intensity RLU, calculates
Remaining 2 hole average values are as antigen control RLU；
The corresponding critical inhibiting rate correspondence of each potency serum is as shown in the table
| Potency | Inhibiting rate |
| 1:512 or more | >=96.0% |
| 1:256 | 90.3% |
| 1:128 | 79.8% |
| 1:64 | 60.8% |
| 1:32 | 50.2% |
| 1:16 | 31.7% |
| 1:8 and less | ≤ 17.7% |
Further, the how anti-coating plate of the rabbit is by the O-shaped foot and mouth disease virus rabbit polyvalent antibodies of 2 μ g/ml, 100 holes μ l/, packet
It is formed.
Further, the antigen-antibody reaction plate is U-shaped 96 hole antigen-antibody reaction plate.
Further, the sample diluting liquid is the PBS buffer solution containing 0.02%Tween20,1% pig negative serum, pH
For 7.2-7.4.
Further, the O-shaped viral antigen working solution of the biotinylated aftosa is the biotinylation containing 15 μ g/ml
The PBS buffer solution of antigen, 0.02%Tween-20,1%BSA, pH 7.2-7.4.
Further, the enzyme mark Streptavidin working solution is containing 5 μ g/ml of enzyme conjugates, 0.5wt%2- chloroacetamides
PBS buffer solution, pH 7.0-7.2, enzyme HRP.
Further, the negative quality controlled serum is the artiodactyl serum for being uninfected by foot and mouth disease virus, and inhibiting rate is more than
80%；The positive quality control serum is that O-shaped foot and mouth disease virus storms malicious animal blood serum, and inhibiting rate is less than 20%.
Further, the substrate A is to contain 1.26wt%N, dinethylformamide, 5.85mmol/L luminols,
The Tris-HCl buffer solutions of 9.86mmol/L tetraphenylboron sodiums and 1.06mg/L paracetamol, pH 8.8-9.0；The substrate
B is the Tris-HCl buffer solutions of the sodium perborate containing 0.615mg/mL, pH 4.8-5.0.
Further, the washing lotion is 20 times of concentration washing lotion, described 20 times of concentration washing lotion be containing 18wt%NaCl,
The Tris-HCl buffer solutions of 1wt%Tween20 and 1wt%Proclin300, pH 7.4-7.6.
The determination of the detection method of the present invention, logical serum optimum diluting multiple not only makes sample to be tested serum, negative matter
Control serum and positive quality control serum carry out single dilutions can effectively promote detection speed without carrying out gradient dilution, and
And improve detection flux；The foundation of kit evaluation criterion passes through inhibiting rate PI critical inhibition corresponding with each potency serum
Rate carries out the potency that comparison judges test serum, and deterministic process is intuitively quick, can further promote detection speed, and then the two
It is comprehensive always meeting the detection demand of the fast high-flux under great amount of samples.Through examining, ground with Lanzhou animal doctor using the present invention
Study carefully O-shaped foot-and-mouth disease antibody Liquid-phase blocking ELISA detection kit (standard reagent box) and meanwhile detect 88 parts of serum, this reagent
Box detection time is only 3 hours, accelerates for 40% (2 hours) compared with standard reagent box, be greatly improved detection efficiency and
Save manpower and materials cost.
Description of the drawings
Fig. 1-1~Fig. 1-4 is under 8 times, 16 times, the 32 times and 64 times extension rates for determining experiment of serum diluting multiple
Box figure.
Fig. 2-1 is the box figure of the foundation experiment of agent box evaluation criterion；Fig. 2-2~Fig. 2-8 is building for agent box evaluation criterion
Vertical experiment corresponds to potency 1：512、1：256、1：128、1：64、1：32、1：16、1：8 ROC curve.
Fig. 3 is the present invention and standard reagent box coincidence rate contrast experiment.
Specific implementation mode
Main agents explanation：
O-shaped foot-and-mouth disease virus antigen is purchased from middle peasant Witter bio tech ltd；
O-shaped foot and mouth disease virus rabbit polyvalent antibody is purchased from the Hangzhou bio tech ltd Qi Tai；
Negative quality controlled serum and positive quality control serum are provided by middle peasant Witter bio tech ltd；
Commercial ELISA Assay kit is the O-shaped foot-and-mouth disease antibody Liquid-phase blocking ELISA detection kit (mark of Lanzhou veterinary institute
Quasi- kit)；
Totally 422 parts of serum sample derives from farm, for pig and the cow's serum sample of O-shaped aftosa vaccine is immunized；
The how anti-coating plate of rabbit is formed by the O-shaped foot and mouth disease virus rabbit polyvalent antibodies of 2 μ g/ml, 100 holes μ l/, coating；
Sample diluting liquid is the PBS buffer solution containing 0.02%Tween20,1% pig negative serum, pH 7.2-7.4；
The biotinylated O-shaped viral antigen working solution of aftosa is biotinylated antigen, 0.02%Tween- containing 15 μ g/ml
20, the PBS buffer solution of 1%BSA, pH 7.2-7.4；
Enzyme mark Streptavidin working solution is the PBS buffer solution containing 5 μ g/ml of enzyme conjugates, 0.5wt%2- chloroacetamides, and pH is
7.0-7.2, enzyme HRP；
Negative quality controlled serum is the artiodactyl serum for being uninfected by foot and mouth disease virus, and inhibiting rate is less than 20%；Positive quality control serum
Malicious animal blood serum is stormed for O-shaped foot and mouth disease virus, inhibiting rate is more than 80%；
Substrate A is to contain 1.26wt%N, dinethylformamide, 5.85mmol/L luminols, 9.86mmol/L tetraphenylboron sodiums and
The Tris-HCl buffer solutions of 1.06mg/L paracetamol, pH 8.8-9.0；Substrate B is sodium perborate containing 0.615mg/mL
Tris-HCl buffer solutions, pH 4.8-5.0；
The concentration washing lotion that washing lotion is 20 times, 20 times of concentration washing lotion is containing 18wt%NaCl, 1wt%Tween20 and 1wt%
The Tris-HCl buffer solutions of Proclin300, pH 7.4-7.6.
Embodiment 1
A kind of detection method of O-shaped foot-and-mouth disease antibody comprising following steps,
(1) use Sample dilution single dilute to 32 times of sample to be tested serum, negative quality controlled serum and positive quality control serum progress
It releases, antigen-antibody reaction plate is added with 50 holes μ l/；
(2) the biotinylated O-shaped viral antigen working solution of aftosa, 50 holes μ l/ are added, while 4 antigen control holes are set, add
Enter the antigen working solution in 100 holes μ l/, vibrate, 37 DEG C incubate 90 minutes；
(3) by the solution integral translation in reaction plate to the how anti-coating plate of rabbit, 50 holes μ l/, 37 DEG C incubate 40 minutes；
(4) enzyme mark Streptavidin working solution is added to the how anti-plate of rabbit in board-washing, drying, and 50 holes μ l/, 37 DEG C incubate 30 minutes；
(5) board-washing, drying are waited than mixed substrates A and substrate B, Substrate cocktail are added to the how anti-plate of rabbit, 100 holes μ l/ use
Chemiluminescence readings instrument measures relative light intensity RLU；
(6) yin and yang attribute of judgement test serum in 4 antigen control holes, is abandoned using antigen control inhibiting rate as criterion
Peak and minimum RLU values are removed, remaining 2 holes is calculated and is averaged RLU values as antigen control RLU values；
The corresponding critical inhibiting rate of each potency serum is as shown in table 1；
Table 1：The corresponding critical inhibiting rate of each potency serum
| Potency | Inhibiting rate |
| 1:512 or more | >=96.0% |
| 1:256 | 90.3% |
| 1:128 | 79.8% |
| 1:64 | 60.8% |
| 1:32 | 50.2% |
| 1:16 | 31.7% |
| 1:8 and less | ≤ 17.7% |
Inhibiting rate PI by critical inhibiting rate corresponding with each potency serum compare the potency of determining test serum.
Sample to be tested serum, antigen control, the layout of negative quality controlled serum and positive quality control serum are as shown in table 2.
Table 2：Laboratory reference is laid out
Embodiment 2
The determination of 2.1 rabbits mostly anti-best peridium concentration and antigen best effort concentration
Determine that rabbit resists best peridium concentration and antigen best effort concentration more using Checkerboard titration method.The how anti-peridium concentration of rabbit by
32 twice of serial dilution of μ g/ml to 1 μ g/ml are laterally coated with, and antigen concentration is by 40 twice of serial dilution of μ g/ml to 0.08 μ g/ml
Longitudinal sample-adding is finally considering the sufficiently large (RLU of signal value requirement>50000000) and antigen concentration is under corresponding peridium concentration
The singular point of variation selects peridium concentration for 2 μ g/ml, and corresponding antigen working concentration is 15 μ g/ml, and titration results are as shown in table 3.
Table 3：Titration results
The determination of 2.2 serum diluting multiples
(peridium concentration is 2 μ g/ml, and corresponding antigen working concentration is 15 μ g/ml), detection 24 are tested by above-mentioned determining condition
The Swine serum sample of part known background, serum titer are as shown in table 4.It is dilute to 8 times, 16 times, 32 times and 64 times of detection serum progress
Detection is released, the inhibiting rate of sample serum under each extension rate is calculated, using box figure to different potency serum under each dilution
Inhibiting rate is distinguished situation and is analyzed, and as shown in Fig. 1-1~Fig. 1-4, comments first box figure main part overlapping cases
Valence, it can be seen from the figure that, extension rate 1:When 8, potency is 1:Its box portion of 22 or more serum has apparent overlapping；It is dilute
It is 1 to release multiple:When 16, potency is 1:45 have the overlapping of part with upper box；Extension rate is 1:When 64, potency 1:22 and 1:
45 serum box portion is substantially overlapping；And it is 1 in extension rate:When 32, it can be seen that box portion has no weight between each potency
Folded situation, only in potency 1:45 and 1:There are crossing instances between 90；Secondly, bound is verified, bound is set using 95%
Letter section is determined, without the use of the maximum value that is shown in box figure and minimum as standard, 1:When 32 dilution, potency 1:
The number of photons upper limit of 90 detections is 2.09E+007, is less than potency 1:The number of photons lower limit (2.10E+007) of 45 detections；It is final true
Fixed 1:32 be best serum diluting multiple, i.e., when 32 times dilutions, each potency serum inhibiting rate is distinguished apparent, can be carried out to serum good
Good identification.
The corresponding inhibiting rate PI statistics non-overlapping phenomenon in section of each potency serum, which refers to, under single dilutions multiple detects serum
Single dilutions are carried out (preferably 32 times in embodiment) under a certain extension rate, (embodiment uses box through statistical analysis
Map analysis), the corresponding statistics section of each potency serum inhibiting rate distinguish it is apparent, it is non-overlapping.
Table 4：Virus monitory sample distribution table
| Potency | Number of samples |
| 1:4 | 2 |
| 1:11 | 2 |
| 1:22 | 3 |
| 1:45 | 4 |
| 1:90 | 6 |
| 1:180 | 3 |
| 1:360 | 4 |
The foundation of 2.3 doses of box evaluation criterions
Tested that (peridium concentration is 2 μ g/ml, and corresponding antigen working concentration is 15 μ g/ml, serum using above-mentioned determining condition
Extension rate is 32 times), 174 parts of known background serum are detected altogether.The inhibiting rate of testing result is analyzed using box figure,
As shown in Fig. 2-1, using ROC curve, the critical inhibiting rate value of each potency is determined, as shown in Fig. 2-2~Fig. 2-8.The result shows that
It is distinguished obviously between 174 parts of each potency of serum of detection, without overlapping cases, has confirmed 2.2 results.ROC curve figure simultaneously
It shows that area under the curve is 0.833~0.978, is all higher than 0.5, show that curve has very high accuracy to the judgement of critical value.
Finally, the critical inhibiting rate that each potency is determined by Youden index establishes the result criterion of the present invention with this, such as
Shown in table 5.
Table 5：The corresponding critical inhibiting rate correspondence of each potency serum
| Potency | Inhibiting rate |
| 1:512 or more | >=96.0% |
| 1:256 | 90.3% |
| 1:128 | 79.8% |
| 1:64 | 60.8% |
| 1:32 | 50.2% |
| 1:16 | 31.7% |
| 1:8 | 17.7% |
Embodiment 3
3.1 coincidence rates are tested
In order to be verified to kit performance, has chosen 244 parts of field serum and (refer to by farm, withdraw to the sources of students such as farm
Serum) be measured with the condition of above-mentioned determination, and simultaneously use Lanzhou veterinary institute O-shaped foot-and-mouth disease antibody liquid phase block
ELISA detection kit is detected as a comparison, and the results are shown in Figure 3, the results showed that, the two correlation is y=0.9435+
0.0831, correlation coefficient r 0.9551 belongs to highly relevant, illustrates that the present invention has good specificity and sensibility, detection knot
Fruit has very high confidence level.
Embodiment of the present invention is interpreted as illustrative, to be not intended to limit the present invention protection domain, for this field
For technical staff, under the premise of without departing substantially from spirit and scope of the present invention, this is still fallen within to all adjustment that the present invention makes
The protection domain of invention.
Claims (9)
1. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody, uses chemiluminescence immunoassay determination of adsorption method sample to be tested
Test serum relative light intensity RLU and antigen control relative light intensity RLU, and according to test serum relative light intensity RLU and
Antigen control relative light intensity RLU calculates inhibiting rate PI, it is characterised in that：The sample treatment of the chemiluminescence immunoassay absorption method
Sample to be tested serum, negative quality controlled serum and positive quality control serum carry out single dilutions in the process, are respectively imitated under single dilutions multiple
The corresponding inhibiting rate PI of valence serum counts the non-overlapping phenomenon in section；The inhibiting rate PI passes through corresponding critical with each potency serum
Inhibiting rate compare the potency of determining test serum.
2. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：The inspection
Survey method includes the following steps：
(1) the sample to be tested serum after Sample dilution single dilutions, negative Quality Control blood are added in antigen-antibody reaction plate
The multiple of cleer and peaceful positive quality control serum, the single dilutions is 32 times；
(2) the biotinylated O-shaped viral antigen working solution of aftosa is added, while 4 antigen control holes are set, incubates, it is described
The antigen concentration of the biotinylated O-shaped viral antigen working solution of aftosa is 15 μ g/ml；
(3) it by the solution integral translation in reaction plate to the how anti-coating plate of rabbit, incubates, how anti-the coating of the rabbit coating plate be dense
Degree is 2 μ g/ml；
(4) board-washing, drying are added enzyme mark Streptavidin working solution to the how anti-plate of rabbit, incubate；
(5) substrate A and substrate B is added in board-washing, drying, measures relative light intensity RLU；
(6) judge, antigen control hole setting 4 is each, discards the peak and minimum of antigen control relative light intensity RLU, calculates
Remaining 2 hole average values are as antigen control RLU；
Inhibiting rate
The corresponding critical inhibiting rate correspondence of each potency serum is as shown in the table
3. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：The rabbit
Mostly anti-coating plate is formed by the O-shaped foot and mouth disease virus rabbit polyvalent antibodies of 2 μ g/ml, 100 holes μ l/, coating.
4. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：The sample
Product dilution is the PBS buffer solution containing 0.02%Tween20,1% pig negative serum, pH 7.2-7.4.
5. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：The life
The O-shaped viral antigen working solution of aftosa of object element is the biotinylated antigen containing 15 μ g/ml, 0.02%Tween-20,1%
The PBS buffer solution of BSA, pH 7.2-7.4.
6. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：The enzyme
Mark Streptavidin working solution is the PBS buffer solution containing 5 μ g/ml of enzyme conjugates, 0.5wt%2- chloroacetamides, pH 7.0-
7.2, enzyme HRP.
7. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：Described the moon
Property quality controlled serum be the artiodactyl serum for being uninfected by foot and mouth disease virus, inhibiting rate be less than 20%；The positive quality control serum is O
The malicious animal blood serum of type foot and mouth disease virus storming, inhibiting rate are more than 80%.
8. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：The bottom
Object A is to contain 1.26wt%N, dinethylformamide, 5.85mmol/L luminols, 9.86mmol/L tetraphenylboron sodiums and
The Tris-HCl buffer solutions of 1.06mg/L paracetamol, pH 8.8-9.0；The substrate B is to cross boron containing 0.615mg/mL
The Tris-HCl buffer solutions of sour sodium, pH 4.8-5.0.
9. a kind of high-flux detection method of O-shaped foot-and-mouth disease antibody according to claim 1, it is characterised in that：It is described to wash
The concentration washing lotion that liquid is 20 times, described 20 times of concentration washing lotion is containing 18wt%NaCl, 1wt%Tween20 and 1wt%
The Tris-HCl buffer solutions of Proclin300, pH 7.4-7.6.
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Cited By (4)
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| CN110187105A (en) * | 2019-06-25 | 2019-08-30 | 艾军 | A kind of B-ELISA kit and preparation method detecting ox, the O-shaped antibody of sheep foot and mouth disease virus |
| CN110297091A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | A kind of multiple tube-type chemical luminescence detection kit of I type antibody of aftosa O, A, Asia |
| CN112881710A (en) * | 2021-02-04 | 2021-06-01 | 中国农业科学院兰州兽医研究所 | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
| CN113391061A (en) * | 2021-06-16 | 2021-09-14 | 王婷楠 | Application of animal immune antibody in preparation of medicine for detecting animal immunity |
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| CN110187105A (en) * | 2019-06-25 | 2019-08-30 | 艾军 | A kind of B-ELISA kit and preparation method detecting ox, the O-shaped antibody of sheep foot and mouth disease virus |
| CN110187105B (en) * | 2019-06-25 | 2022-03-22 | 艾军 | B-ELISA kit for detecting O-type antibody of foot-and-mouth disease virus of cattle and sheep and preparation method thereof |
| CN110297091A (en) * | 2019-07-17 | 2019-10-01 | 中国农业科学院兰州兽医研究所 | A kind of multiple tube-type chemical luminescence detection kit of I type antibody of aftosa O, A, Asia |
| CN110297091B (en) * | 2019-07-17 | 2020-12-01 | 中国农业科学院兰州兽医研究所 | Multi-tube chemiluminescence detection kit for foot-and-mouth disease O, A, Asia I-type antibody |
| CN112881710A (en) * | 2021-02-04 | 2021-06-01 | 中国农业科学院兰州兽医研究所 | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
| CN112881710B (en) * | 2021-02-04 | 2022-04-22 | 中国农业科学院兰州兽医研究所 | Inhibition ELISA method for detecting foot-and-mouth disease virus antibody and application thereof |
| CN113391061A (en) * | 2021-06-16 | 2021-09-14 | 王婷楠 | Application of animal immune antibody in preparation of medicine for detecting animal immunity |
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