CN106771134A - A kind of carp herpesvirusⅡtype sandwich ELISA detection kit and detection method - Google Patents
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit and detection method Download PDFInfo
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Abstract
The present invention is a kind of carp herpesvirusⅡtype sandwich ELISA detection kit, and the main composition of the kit is：Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution；The how anti-solution of enzyme mark ORF90；Dilution；Tmb substrate solution；Terminate liquid；Carp herpes virus type 2 positive control；Negative control；Coating buffer；96 hole elisa Plates.The invention further relates to a kind of detection method.Kit of the present invention and method can be used for the latent infection fish virus without clinical symptoms and detect, and can further apply the research of carp herpesvirusⅡtype Changing Pattern in fish body, so that for the viral prevention and control provide foundation.Other common virus of the inventive method and crucian, goldfish can be used for the disease diagnosis of domestic plant and entry and exit fish quarantine without nonspecific reaction.
Description
Technical field
The present invention relates to a kind of detection kit, particularly a kind of carp herpesvirusⅡtype sandwich ELISA detection reagent
Box, the method the invention further relates to be detected using the detection kit.
Background technology
In recent years, carp herpesvirusⅡtype（English name：Cyprinid herpesvirus II, abridge CyHV-2）Give
China crucian and goldfish aquaculture bring huge harm, and morbidity and mortality are high when epidemic disease breaks out, through not exclusively system
Meter, the economic loss caused by the disease in 2006 is up to 7.18 hundred million yuan.
In the prior art, diagnose most that intuitively method is that morbidity fish tissues are carried out after histological examination again through electronics to it
Microscope observes that herpesviral particle is confirmed.Conventional PCR or quantifying PCR method detect carp herpesviral both at home and abroad at present
There is latent infection in II type, herpesviral, the fish of appearance health may carry virus, and PCR method is able to detect that viral nucleic acid,
But cannot confirm in fish body with the presence or absence of communicable virion, it is therefore necessary to set up the immunology side of detection virus protein
Method.Therefore, the sandwich ELISA method for setting up detection carp herpesvirusⅡtype virus protein has for monitoring the latent infection of fish
Significance.
The content of the invention
The technical problems to be solved by the invention are directed to the deficiencies in the prior art, there is provided a kind of new carp herpesviral II
Type sandwich ELISA detection kit, can be used for enter and leave the border aquatic animal quarantine and the epidemic situation monitoring of domestic plant.
The technical problems to be solved by the invention are realized by following technical scheme.The present invention is a kind of carp blister
The type sandwich ELISA detection kit of exanthema virus II, is characterized in, the main composition of the kit is：
Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution, 1 100 μ L is that carp herpesvirusⅡtype ORF90 purified monoclonal antibodies are molten
In the solution that water is made, concentration is 10-20 mg/mL；
The how anti-solution of enzyme mark ORF90,1 100 μ L is to resist enzyme mark ORF90 to be dissolved in the solution that water is made more, and concentration is 10-20
mg/mL；
Dilution, 2 bottles, every bottle of 50 mL, solution is the phosphate-buffered of bovine serum albumin(BSA) that mass-volume concentration is 10 g/L
Liquid；
Tmb substrate solution, 1,250 μ L；It is the DMSO solution of TMB, its mass-volume concentration
It is 6 mg/mL；
Terminate liquid, concentration is 1 bottle of the sulfuric acid solution of 2 M, 20 mL；
Carp herpes virus type 2 positive control 1,500 μ L are the homogenate of goldfish assaypositive tissue；
Negative control 1,500 μ L are negative goldfish tissue homogenate；
Coating buffer, 1 bottle, 20 mL are the aqueous solution containing 0.015 M sodium carbonate and 0.035 M sodium acid carbonates；
96 hole elisa Plates, 1 piece.
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit of the present invention, its further preferred technology
Scheme is：In the kit, the w/v composition of described phosphate buffer is：
The g of sodium chloride 8.0
The g of potassium dihydrogen phosphate 0.2
The g of disodium hydrogen phosphate dodecahydrate 2.9
Potassium chloride 0.2g
The mL of Tween-80 0.5
The mL of distilled water 1000.
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit of the present invention, its further preferred technology
Scheme is：Also include in the kit：
Phosphate buffer PBST, 1 bottle of 50 mL, its mass volume ratio constitutes and is：
The g of sodium chloride 8.0
The g of potassium dihydrogen phosphate 0.2
The g of disodium hydrogen phosphate dodecahydrate 2.9
The g of potassium chloride 0.2
The mL of Tween-80 0.5
The mL of distilled water 1000.
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit of the present invention, its further preferred technology
Scheme is：The kit also includes：
Urea peroxide solution, 1 bottle of 100 mL, is mixed by urea peroxide with sodium acetate-citrate buffer solution；
Wherein, the preparation method of sodium acetate-citric acid solution is：Citric acid that 17 mL mass-volume concentrations are 94 g/L is molten
Liquid is added to during 500 mL mass-volume concentrations are the sodium acetate solution of 81 g/L, is well mixed and is obtained；
The preparation method of urea peroxide solution is as follows：140 mg urea peroxides are weighed, and adds it to 100 mL sodium acetates-citric acid
In cushioning liquid, it is well mixed and is obtained.
The technical problems to be solved by the invention can also further be realized by following technical scheme.The present invention is also
A kind of carp herpesvirusⅡtype sandwich ELISA detection method is disclosed, is characterized in, described in the method use above technical scheme
Kit detected that its step is as follows：
（1）By the carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution diluteds in kit to 10 μ g/mL, 100
μ L/ holes coated elisa plate, 4 DEG C overnight；
（2）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（3）Add dilution closing ELISA Plate, 200 μ L/ holes, 37 DEG C of reaction 2h；
（4）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（5）By detected sample, positive and negative control dilution according to 1:10 dilutions, 100 μ L/ holes are added in ELISA Plate,
25 DEG C of 0.5 h of reaction；
（6）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（7）The how anti-solution of enzyme mark ORF90 is pressed 1 with dilution:1000 dilutions, 100 μ L/ holes are added in ELISA Plate, and 25 DEG C anti-
Answer 0.5 h；
（8）Tmb substrate solution 1 is taken, adds 18 mL distilled waters and 2 mL urea peroxide solution to be configured to working solution, 100 μ
L/ holes add ELISA Plate, 25 DEG C of 15 min of reaction；
（9）Plus 50 μ L/ holes terminate liquids, determine optical density in 450 nm（OD）Value；
（10）Result judgement：P/N >=2.1, show that control is set up, wherein：P is positive control OD values, and N is negative control OD value；
When sample OD values/negative control OD value >=2.1, carp herpesvirusⅡtype is judged to positive；Sample OD values/negative control OD value ＜
When 2.1, carp herpesvirusⅡtype is judged to negative.
In kit of the present invention, resisting purchased from green grass or young crops described carp herpesvirusⅡtype ORF90 purified monoclonal antibodies and enzyme mark ORF90 more
Island riel Bioisystech Co., Ltd, ProductName is respectively：Carp herpesvirusⅡtype ORF90 purified monoclonal antibodies, ORF90 is more for enzyme mark
It is anti-.CompanyAddress：No. 63 Qingdao in Qingdao of Shandong province Haier road numeral science and technology center B-715, phone：0532-80999085.Carp
It is by prokaryotic expression carp herpes virus type 2 ORF90 albumen to resist herpesvirusⅡtype ORF90 purified monoclonal antibodies and enzyme mark ORF90 more
It is immunized prepared by animal afterwards, wherein monoclonal antibody is IgG1 types, and it is IgG2a types to resist more.
Compared with prior art, the invention provides a kind of new carp herpesvirusⅡtype sandwich ELISA detection kit,
Can be used to detect carp herpesvirusⅡtype.The present invention has also set up sandwich ELISA detection method, it is determined that sandwich ELISA side
The optimal coating concentration of method, the optimum reacting time of the best effort concentration, optimal off-period and antigen-antibody of labelled antibody,
For the detection of the virus provides effective method.The inventive method can be used for the latent infection fish virus inspection without clinical symptoms
Survey, and the research of carp herpesvirusⅡtype Changing Pattern in fish body can be further applied, so that for the viral prevention and control are carried
For foundation.Other common virus of the inventive method and crucian, goldfish can be used for domestic plant without nonspecific reaction
Disease diagnosis and entry and exit fish quarantine.
Specific embodiment
Concrete technical scheme of the invention described further below, is further understood that in order to those skilled in the art
The present invention, without constituting the limitation to its right.
Embodiment 1, a kind of carp herpesvirusⅡtype sandwich ELISA detection kit, the main composition of the kit is：
Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution, 1 100 μ L is that carp herpesvirusⅡtype ORF90 purified monoclonal antibodies are molten
In the solution that water is made, concentration is 10-20 mg/mL；
The how anti-solution of enzyme mark ORF90,1 100 μ L is to resist enzyme mark ORF90 to be dissolved in the solution that water is made more, and concentration is 10-20
mg/mL；
Dilution, 2 bottles, every bottle of 50 mL, solution is the phosphate-buffered of bovine serum albumin(BSA) that mass-volume concentration is 10 g/L
Liquid；
Tmb substrate solution, 1,250 μ L；It is the DMSO solution of TMB, its mass-volume concentration
It is 6 mg/mL；
Terminate liquid, concentration is 1 bottle of the sulfuric acid solution of 2 M, 20 mL；
Carp herpes virus type 2 positive control 1,500 μ L are the homogenate of goldfish assaypositive tissue；
Negative control 1,500 μ L are negative goldfish tissue homogenate；
Coating buffer, 1 bottle, 20 mL are the aqueous solution containing 0.015 M sodium carbonate and 0.035 M sodium acid carbonates；
96 hole elisa Plates, 1 piece.
Embodiment 2, in a kind of carp herpesvirusⅡtype sandwich ELISA detection kit described in embodiment 1：Described phosphorus
The w/v of phthalate buffer is constituted：
The g of sodium chloride 8.0
The g of potassium dihydrogen phosphate 0.2
The g of disodium hydrogen phosphate dodecahydrate 2.9
Potassium chloride 0.2g
The mL of Tween-80 0.5
The mL of distilled water 1000.
Embodiment 3, in a kind of carp herpesvirusⅡtype sandwich ELISA detection kit described in embodiment 1 or 2：The examination
Also include in agent box：
Phosphate buffer PBST, 1 bottle of 50 mL, its mass volume ratio constitutes and is：
The g of sodium chloride 8.0
The g of potassium dihydrogen phosphate 0.2
The g of disodium hydrogen phosphate dodecahydrate 2.9
The g of potassium chloride 0.2
The mL of Tween-80 0.5
The mL of distilled water 1000.
Embodiment 4, in a kind of carp herpesvirusⅡtype sandwich ELISA detection kit described in embodiment 1 or 2 or 3：Should
Kit also includes：
Urea peroxide solution, 1 bottle of 100 mL, is mixed by urea peroxide with sodium acetate-citrate buffer solution；
Wherein, the preparation method of sodium acetate-citric acid solution is：Citric acid that 17 mL mass-volume concentrations are 94 g/L is molten
Liquid is added to during 500 mL mass-volume concentrations are the sodium acetate solution of 81 g/L, is well mixed and is obtained；
The preparation method of urea peroxide solution is as follows：140 mg urea peroxides are weighed, and adds it to 100 mL sodium acetates-citric acid
In cushioning liquid, it is well mixed and is obtained.
A kind of carp herpesvirusⅡtype sandwich ELISA detection method of embodiment 5., the method using embodiment 1 or 2 or 3 or
Kit described in 4 is detected that its step is as follows：
（1）By the carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution diluteds in kit to 10 μ g/mL, 100
μ L/ holes coated elisa plate, 4 DEG C overnight；
（2）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（3）Add dilution closing ELISA Plate, 200 μ L/ holes, 37 DEG C of reaction 2h；
（4）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（5）By detected sample, positive and negative control dilution according to 1:10 dilutions, 100 μ L/ holes are added in ELISA Plate,
25 DEG C of 0.5 h of reaction；
（6）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（7）The how anti-solution of enzyme mark ORF90 is pressed 1 with dilution:1000 dilutions, 100 μ L/ holes are added in ELISA Plate, and 25 DEG C anti-
Answer 0.5 h；
（8）Tmb substrate solution 1 is taken, adds 18 mL distilled waters and 2 mL urea peroxide solution to be configured to working solution, 100 μ
L/ holes add ELISA Plate, 25 DEG C of 15 min of reaction；
（9）Plus 50 μ L/ holes terminate liquids, determine optical density in 450 nm（OD）Value；
（10）Result judgement：P/N >=2.1, show that control is set up, wherein：P is positive control OD values, and N is negative control OD value；
When sample OD values/negative control OD value >=2.1, carp herpesvirusⅡtype is judged to positive；Sample OD values/negative control OD value ＜
When 2.1, carp herpesvirusⅡtype is judged to negative.
A kind of carp herpesvirusⅡtype sandwich ELISA detection method experiment of embodiment 6.：
1.1 monoclonal antibodies-how anti-sandwich ELISA method is most preferably coated with concentration
Carp herpesvirusⅡtype ORF90 purified monoclonal antibodies（Call monoclonal antibody or ORF90 monoclonal antibodies in the following text）Solution is respectively with 5 μ g/mL, 10 μ g/
ML coated elisa plates, 4oC is overnight.Add dilution closing, 200 μ L/ holes, 37oC 2 h, PBST are washed three times.By enzyme mark
ORF90 resists more（Many anti-or ORF90 are called in the following text to resist more）From 1:250 start doubling dilution, and 100 μ L/ holes are added in ELISA Plate, room temperature
React 1 h.Enzyme-added labeling antibody：By the enzyme mark how anti-solution of ORF90 respectively according to 1:5000 and 1:10000 dilutions, 100 μ L/ holes,
Add in ELISA Plate, 25 DEG C are reacted 1 h.Plus tmb substrate, 25 DEG C of 10 min of effect.Plus 50 μ L/ holes terminate liquids, surveyed in 450 nm
Determine OD values.According to table 1, selection 10 μ g/mL coating concentration is best.
Table 1
The determination of 3.2 monoclonal antibodies-how anti-sandwich ELISA method labelled antibody working concentration
In the case of 10 μ g/mL monoclonal antibodies are coated, labelled antibody dilution is tested for 1000 times and 2500 times, detection knot
Really (table 2) display labelled antibody selects 1000 times of dilutions to be tested.
Table 2
3.3 monoclonal antibodies-how anti-sandwich ELISA method detection ORF90 albumen greatest dilutions
Detect that the concentration of ORF90 is 1.63 mg/mL with nucleic acid-protein detector.The μ g/mL of monoclonal antibody 10 that will be purified are coated with plate,
ORF90 albumen is from 1:250 start doubling dilution, and the minimum dilution factor of detection is 1:8000 (tables 3).
3 ORF of table 90 detects the greatest dilution of albumen
3.4 monoclonal antibodies-how anti-sandwich ELISA method optimal off-period and antigen-antibody optimum reacting time
The μ g/mL of ORF90 monoclonal antibodies 10 that will be purified are coated with plate, and 4 DEG C overnight, with the PBST closed plates containing 1% BSA, 150 μ L/
As a result hole, 37 DEG C of placements 0.5,1,2h show that 2h is optimal off-period（Table 4）.Coated antibody and antigen, detection antibody and
Antigen is respectively at room temperature（25 ℃）Reaction 0.5,1 and 1.5h, as a result show the optimum reacting time that 0.5 h is antigen-antibody（Table
5）.
The ORF90 monoclonal antibodies of table 4-how anti-sandwich ELISA optimal off-period
| Off-period | Positive homogenate | Negative control | P/N |
| 37℃0.5h | 0.264 | 0.181 | 1.461 |
| 37℃1h | 0.337 | 0.122 | 2.769 |
| 37℃2h | 0.452 | 0.129 | 3.517 |
The ORF90 monoclonal antibodies of table 5-how anti-sandwich ELISA antigen-antibody optimum reacting time
| Reaction time | Positive homogenate | Negative control | P/N |
| 0.5h | 1.005 | 0.292 | 3.441 |
| 1h | 0.848 | 0.651 | 1.303 |
| 1.5h | 1.140 | 0.554 | 2.058 |
3.5 monoclonal antibodies-how anti-sandwich ELISA method specific test
Detect that carp herpes virus type 2 tissue is homogenized and other several fishes simultaneously with the ORF90 monoclonal antibodies-how anti-sandwich ELISA set up
The virus-cell suspension of virus, including：SVCV (SVCV), popular Hematopoietic Necrosis (EHNV), infection
Property Hematopoietic Necrosis (IHNV), Koi herpesvirus (KHV), infectious pancreas necrosis virus（IPNV）, infectious salmon it is poor
Blood disease (ISAV), GCHV（GCHV）, as a result show that the method detection carp herpes virus type 2 is the positive, other diseases
Poison is all negative (table 6), illustrates specific preferable.
The specific test of the .CyHV-2 ORF90 monoclonal antibodies of table 6-how anti-sandwich ELISA
| Virus | OD values | P/N |
| SVC | 0.092 | 0.865 |
| EHNV | 0.099 | 0.924 |
| IHNV | 0.086 | 0.801 |
| KHV | 0.116 | 1.086 |
| IPNV | 0.103 | 0.961 |
| ISAV | 0.090 | 0.854 |
| GCHV | 0.111 | 1.041 |
| Kidney 1 | 0.306 | 2.868 |
| Kidney 2 | 0.334 | 3.126 |
| Negative control | 0.107 | |
| Blank | 0.060 |
3.6 sample detections
ORF90 monoclonal antibodies-how anti-the sandwich ELISA method for taking 40 parts of goldfish tissue homogenate this research foundation without clinical symptoms is examined
Survey, while extracting nucleic acid enters performing PCR detection, as a result show that this method can detect 18 parts of positive, and PCR method detection is positive
Property 15 parts of sample, show for the viruliferous fish of taking without clinical symptoms, the sandwich ELISA method detection that this research is set up is better than
PCR method, it is adaptable to the daily supervision of epidemic disease of enter and leave the border fish quarantine and plant.
The ORF90 monoclonal antibodies of table 7.-how anti-sandwich ELISA detection goldfish tissue homogenate testing result
| Sample number into spectrum | OD values | Sample number into spectrum | OD values | Sample number into spectrum | OD values | Sample number into spectrum | OD values | Sample number into spectrum | OD values |
| 1 | 2.609 | 9 | 8.079 | 17 | 1.164 | 25 | 1.897 | 33 | 1.193 |
| 2 | 1.751 | 10 | 1.461 | 18 | 1.496 | 26 | 2.935 | 34 | 3.289 |
| 3 | 3.077 | 11 | 2.953 | 19 | 3.446 | 27 | 1.123 | 35 | 2.276 |
| 4 | 3.012 | 12 | 1.279 | 20 | 1.735 | 28 | 1.575 | 36 | 1.541 |
| 5 | 2.903 | 13 | 2.093 | 21 | 2.990 | 29 | 2.356 | 37 | 1.035 |
| 6 | 2.366 | 14 | 4.224 | 22 | 1.238 | 30 | 1.173 | 38 | 1.401 |
| 7 | 1.271 | 15 | 2.279 | 23 | 1.625 | 31 | 2.175 | 39 | 2.784 |
| 8 | 1.578 | 16 | 1.847 | 24 | 1.900 | 32 | 1.290 | 40 | 1.161 |
The PCR of table 8 detection goldfish tissue homogenate results
| Sample number into spectrum | As a result | Sample number into spectrum | CT values | Sample number into spectrum | CT values | Sample number into spectrum | CT values | Sample number into spectrum | CT values |
| 1 | It is positive | 9 | It is positive | 17 | It is negative | 25 | It is negative | 33 | It is negative |
| 2 | It is negative | 10 | It is negative | 18 | It is negative | 26 | It is positive | 34 | It is negative |
| 3 | It is positive | 11 | It is positive | 19 | It is positive | 27 | It is negative | 35 | It is negative |
| 4 | It is positive | 12 | It is negative | 20 | It is negative | 28 | It is negative | 36 | It is negative |
| 5 | It is positive | 13 | It is positive | 21 | It is positive | 29 | It is positive | 37 | It is negative |
| 6 | It is positive | 14 | It is positive | 22 | It is negative | 30 | It is negative | 38 | It is negative |
| 7 | It is negative | 15 | It is positive | 23 | It is negative | 31 | It is positive | 39 | It is negative |
| 8 | It is negative | 16 | It is negative | 24 | It is negative | 32 | It is negative | 40 | It is negative |
3.7 reagent stabilities are tested
The antibody of preparation is put 20oC is preserved, and take out carries out sandwich ELISA assay detection purifying protein after six months, as a result
The display no significant change of OD values (table 9), illustrates that reagent stability is preferable.
The monoclonal antibody of table 9 coating-how anti-detection sandwich ELISA detection
Claims (4)
1. a kind of carp herpesvirusⅡtype sandwich ELISA detection kit, it is characterised in that the main composition of the kit is：
Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution, 1 100 μ L is that carp herpesvirusⅡtype ORF90 purified monoclonal antibodies are molten
In the solution that water is made, concentration is 10-20 mg/mL；
The how anti-solution of enzyme mark ORF90,1 100 μ L is to resist enzyme mark ORF90 to be dissolved in the solution that water is made more, and concentration is 10-20
mg/mL；
Dilution, 2 bottles, every bottle of 50 mL, solution is the phosphate-buffered of bovine serum albumin(BSA) that mass-volume concentration is 10 g/L
Liquid；
Tmb substrate solution, 1,250 μ L；It is the DMSO solution of TMB, its mass-volume concentration
It is 6 mg/mL；
Terminate liquid, concentration is 1 bottle of the sulfuric acid solution of 2 M, 20 mL；
Carp herpes virus type 2 positive control 1,500 μ L are the homogenate of goldfish assaypositive tissue；
Negative control 1,500 μ L are negative goldfish tissue homogenate；
Coating buffer, 1 bottle, 20 mL are the aqueous solution containing 0.015 M sodium carbonate and 0.035 M sodium acid carbonates；
96 hole elisa Plates, 1 piece.
2. a kind of carp herpesvirusⅡtype sandwich ELISA detection kit according to claim 1, it is characterised in that should
In kit, the w/v composition of described phosphate buffer is：
The g of sodium chloride 8.0
The g of potassium dihydrogen phosphate 0.2
The g of disodium hydrogen phosphate dodecahydrate 2.9
Potassium chloride 0.2g
The mL of Tween-80 0.5
The mL of distilled water 1000.
3. a kind of carp herpesvirusⅡtype sandwich ELISA detection kit according to claim 1, it is characterised in that should
Also include in kit：
Phosphate buffer PBST, 1 bottle of 50 mL, its mass volume ratio constitutes and is：
The g of sodium chloride 8.0
The g of potassium dihydrogen phosphate 0.2
The g of disodium hydrogen phosphate dodecahydrate 2.9
The g of potassium chloride 0.2
The mL of Tween-80 0.5
The mL of distilled water 1000.
4. a kind of carp herpesvirusⅡtype sandwich ELISA detection kit according to claim 1, it is characterised in that should
Kit also includes：
Urea peroxide solution, 1 bottle of 100 mL, is mixed by urea peroxide with sodium acetate-citrate buffer solution；
Wherein, the preparation method of sodium acetate-citric acid solution is：Citric acid that 17 mL mass-volume concentrations are 94 g/L is molten
Liquid is added to during 500 mL mass-volume concentrations are the sodium acetate solution of 81 g/L, is well mixed and is obtained；
The preparation method of urea peroxide solution is as follows：140 mg urea peroxides are weighed, and adds it to 100 mL sodium acetates-citric acid
Delay a kind of carp herpesvirusⅡtype sandwich ELISA detection method, it is characterised in that the method usage right requirement 1 or 2 or 3 or 4
Described kit is detected that its step is as follows：
（1）By the carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution diluteds in kit to 10 μ g/mL, 100
μ L/ holes coated elisa plate, 4 DEG C overnight；
（2）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（3）Add dilution closing ELISA Plate, 200 μ L/ holes, 37 DEG C of reaction 2h；
（4）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（5）By detected sample, positive and negative control dilution according to 1:10 dilutions, 100 μ L/ holes are added in ELISA Plate,
25 DEG C of 0.5 h of reaction；
（6）With phosphate buffer PBST board-washings, 250 μ L/ holes are washed three times；
（7）The how anti-solution of enzyme mark ORF90 is pressed 1 with dilution:1000 dilutions, 100 μ L/ holes are added in ELISA Plate, and 25 DEG C anti-
Answer 0.5 h；
（8）Tmb substrate solution 1 is taken, adds 18 mL distilled waters and 2 mL urea peroxide solution to be configured to working solution, 100 μ
L/ holes add ELISA Plate, 25 DEG C of 15 min of reaction；
（9）Plus 50 μ L/ holes terminate liquids, determine optical density in 450 nm（OD）Value；
（10）Result judgement：P/N >=2.1, show that control is set up, wherein：P is positive control OD values, and N is negative control OD value；
When sample OD values/negative control OD value >=2.1, carp herpesvirusⅡtype is judged to positive；Sample OD values/negative control OD value ＜
When 2.1, carp herpesvirusⅡtype is judged to negative.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109030821A (en) * | 2018-07-19 | 2018-12-18 | 北京农学院 | A kind of antibody, kit and its application method of lactobacillus plantarum bacteriocin |
| CN112279898A (en) * | 2018-11-05 | 2021-01-29 | 共鳞实业(深圳)有限公司 | Agent for preventing or treating fish infection CyHV-2 and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109030821A (en) * | 2018-07-19 | 2018-12-18 | 北京农学院 | A kind of antibody, kit and its application method of lactobacillus plantarum bacteriocin |
| CN109030821B (en) * | 2018-07-19 | 2021-07-20 | 北京农学院 | Antibody and kit of lactobacillus plantarum bacteriocin and use method of antibody and kit |
| CN112279898A (en) * | 2018-11-05 | 2021-01-29 | 共鳞实业(深圳)有限公司 | Agent for preventing or treating fish infection CyHV-2 and application thereof |
| CN112279898B (en) * | 2018-11-05 | 2022-06-21 | 共鳞实业(深圳)有限公司 | Agent for preventing or treating fish infection by CyHV-2 and application thereof |
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