CN106771134B - 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 PDF

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CN106771134B
CN106771134B CN201611096435.6A CN201611096435A CN106771134B CN 106771134 B CN106771134 B CN 106771134B CN 201611096435 A CN201611096435 A CN 201611096435A CN 106771134 B CN106771134 B CN 106771134B
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orf90
carp
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kit
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CN106771134A (en
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徐晔
段宏安
周毅
丁超
曹洁
王凤芝
程积进
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Lianyungang Entrance & Exist Inspection And Quarantine Administration People's
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens

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Abstract

The present invention is a kind of carp herpesvirusⅡtype sandwich ELISA detection kit, and main group of the kit becomes：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 methods.Kit and method of the present invention can be used for the latent infection fish virus detection of no clinical symptoms, and can further apply the research of carp herpesvirusⅡtype changing rule in fish body, to provide foundation for the prevention and control of the virus.The method of the present invention and crucian, goldfish other common virus without nonspecific reaction, can be used for disease diagnosis and the entry and exit fish quarantine of domestic farm.

Description

A kind of carp herpesvirusⅡtype sandwich ELISA detection kit and detection method
Technical field
The present invention relates to a kind of detection kit, especially a kind of carp herpesvirusⅡtype sandwich ELISA detection reagent Box, the invention further relates to the methods being detected using the detection kit.
Background technology
In recent years, carp herpesvirusⅡtype（English name：Cyprinid herpesvirus II, abridge CyHV-2）It gives China crucian and goldfish aquaculture bring huge harm, and morbidity and mortality are high when epidemic disease is broken out, through not exclusively uniting Meter, 2006 caused by the disease economic loss be up to 7.18 hundred million yuan.
In the prior art, it is diagnosed most intuitive method be to morbidity fish tissues carry out histological examination after again through electronics Microscope observes that herpesviral particle is confirmed.Common PCR or quantifying PCR method detect carp herpesviral both at home and abroad at present II type, there are latent infection, the fishes of appearance health may carry virus for herpesviral, and PCR method is able to detect that viral nucleic acid, But it can not confirm and whether there is communicable virion in fish body, it is therefore necessary to establish the immunology side of detection virus protein Method.Therefore, the sandwich ELISA method for establishing detection carp herpesvirusⅡtype virus protein has the latent infection for monitoring fish Significance.
Invention content
The technical problem to be solved by the present invention is in view of the deficiencies of the prior art, provide a kind of new carp herpesviral II Type sandwich ELISA detection kit, can be used for entering and leaving the border aquatic animal quarantine and the epidemic situation monitoring of domestic farm.
The technical problem to be solved by the present invention is to what is realized by technical solution below.The present invention is a kind of carp blister II type sandwich ELISA detection kit of exanthema virus, its main feature is that, main group of the kit becomes：
Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution, 1 100 μ L are that the ORF90 purifying of carp herpesvirusⅡtype is single It is anti-to be dissolved in solution made of water, a concentration of 10-20 mg/mL；
The how anti-solution of enzyme mark ORF90,1 100 μ L are dissolved in solution made of water, a concentration of 10- for enzyme mark ORF90 is mostly anti- 20 mg/mL；
Dilution, 2 bottles, every bottle of 50 mL, solution is the phosphate for the bovine serum albumin(BSA) that mass-volume concentration is 10 g/L Buffer solution；
Tmb substrate solution, 1,250 μ L；For the DMSO solution of TMB, mass-volume concentration
For 6 mg/mL；
Terminate liquid, 1 bottle of the sulfuric acid solution of a concentration of 2 M, 20 mL；
Carp herpes virus type 2 positive control 1,500 μ L are homogenized for goldfish assaypositive tissue；
Negative control 1,500 μ L, for negative goldfish tissue homogenate；
Coating buffer, 1 bottle, 20 mL, for the aqueous solution containing 0.015 M sodium carbonate and 0.035 M sodium bicarbonates；
96 hole elisa Plates, 1 piece.
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit of the present invention, further preferred technology Scheme is：In the kit, the w/v group of the phosphate buffer becomes：
8.0 g of sodium chloride
0.2 g of potassium dihydrogen phosphate
2.9 g of disodium hydrogen phosphate dodecahydrate
Potassium chloride 0.2g
0.5 mL of Tween-80
1000 mL of distilled water.
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit of the present invention, further preferred technology Scheme is：Further include in the kit：
Phosphate buffer PBST, 1 bottle of 50 mL, mass volume ratio group become：
8.0 g of sodium chloride
0.2 g of potassium dihydrogen phosphate
2.9 g of disodium hydrogen phosphate dodecahydrate
0.2 g of potassium chloride
0.5 mL of Tween-80
1000 mL of distilled water.
A kind of carp herpesvirusⅡtype sandwich ELISA detection kit of the present invention, further preferred technology Scheme is：The kit further includes：
Urea peroxide solution, 1 bottle of 100 mL, is mixed by urea peroxide and sodium acetate-citrate buffer solution；
Wherein, the preparation method of sodium acetate-citric acid solution is：By the lemon that 17 mL mass-volume concentrations are 94 g/L Acid solution is added in the sodium acetate solution that 500 mL mass-volume concentrations are 81 g/L, is uniformly mixed and is made；
The preparation method of urea peroxide solution is as follows：140 mg urea peroxides are weighed, and add it to 100 mL sodium acetates-lemon In lemon acid buffering solution, it is uniformly mixed and is made.
The technical problems to be solved by the invention can also be further realized by technical solution below.The present invention is also A kind of carp herpesvirusⅡtype sandwich ELISA detection method is disclosed, its main feature is that, this method uses described in above technical scheme Kit be detected, its step are as follows：
（1）By the carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution diluteds in kit to 10 μ g/mL, 100 holes μ L/ coated elisa plates, 4 DEG C overnight；
（2）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（3）Dilution is added and closes ELISA Plate, 200 holes μ L/, 37 DEG C of reaction 2h；
（4）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（5）By detected sample, the positive and negative control dilution according to 1:Enzyme mark is added in 10 dilutions, 100 holes μ L/ In plate, 25 DEG C of 0.5 h of reaction；
（6）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（7）Enzyme mark ORF90 how anti-solution dilution is pressed 1:1000 dilutions, 100 holes μ L/ are added in ELISA Plate, 25 DEG C reaction 0.5 h；
（8）Tmb substrate solution 1 is taken, 18 mL distilled waters are added and 2 mL urea peroxide solution are configured to working solution, ELISA Plate, 25 DEG C of 15 min of reaction are added in 100 holes μ L/；
（9）Add 50 holes μ L/ terminate liquids, optical density is measured 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, it is positive to be judged to carp herpesvirusⅡtype；Sample OD values/negative control OD When value ＜ 2.1, it is negative to be judged to carp herpesvirusⅡtype.
In kit of the present invention, the carp herpesvirusⅡtype ORF90 purified monoclonal antibodies and enzyme mark ORF90 are mostly anti-purchased from blueness Island riel Bioisystech Co., Ltd, ProductName are respectively：Carp herpesvirusⅡtype ORF90 purified monoclonal antibodies, enzyme mark ORF90 are more 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 that herpesvirusⅡtype ORF90 purified monoclonal antibodies and enzyme mark ORF90 resist more It is immunized prepared by animal afterwards, wherein monoclonal antibody is IgG1 types, resists for IgG2a types more.
Compared with prior art, the present invention provides a kind of new carp herpesvirusⅡtype sandwich ELISA detection kit, It can be used for detecting carp herpesvirusⅡtype.The present invention has also set up sandwich ELISA detection method, it is determined that sandwich ELISA side The best peridium concentration of method, the best effort concentration of labelled antibody, the optimum reacting time of best off-period and antigen-antibody, Effective method is provided for the detection of the virus.The method of the present invention can be used for the latent infection fish virus inspection of no clinical symptoms It surveys, and the research of carp herpesvirusⅡtype changing rule in fish body can be further applied, to be carried for the prevention and control of the virus For foundation.The method of the present invention and crucian, goldfish other common virus without nonspecific reaction, can be used for domestic farm Disease diagnosis and entry and exit fish quarantine.
Specific implementation mode
The specific technical solution of the present invention described further below, in order to which those skilled in the art is further understood that The present invention, without constituting the limitation to its right.
Embodiment 1, a kind of carp herpesvirusⅡtype sandwich ELISA detection kit, main group of the kit become：
Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution, 1 100 μ L are that the ORF90 purifying of carp herpesvirusⅡtype is single It is anti-to be dissolved in solution made of water, a concentration of 10-20 mg/mL；
The how anti-solution of enzyme mark ORF90,1 100 μ L are dissolved in solution made of water, a concentration of 10- for enzyme mark ORF90 is mostly anti- 20 mg/mL；
Dilution, 2 bottles, every bottle of 50 mL, solution is the phosphate for the bovine serum albumin(BSA) that mass-volume concentration is 10 g/L Buffer solution；
Tmb substrate solution, 1,250 μ L；For the DMSO solution of TMB, mass-volume concentration
For 6 mg/mL；
Terminate liquid, 1 bottle of the sulfuric acid solution of a concentration of 2 M, 20 mL；
Carp herpes virus type 2 positive control 1,500 μ L are homogenized for goldfish assaypositive tissue；
Negative control 1,500 μ L, for negative goldfish tissue homogenate；
Coating buffer, 1 bottle, 20 mL, for the aqueous solution containing 0.015 M sodium carbonate and 0.035 M sodium bicarbonates；
96 hole elisa Plates, 1 piece.
Embodiment 2, in a kind of carp herpesvirusⅡtype sandwich ELISA detection kit described in embodiment 1：The phosphorus The w/v group of phthalate buffer becomes：
8.0 g of sodium chloride
0.2 g of potassium dihydrogen phosphate
2.9 g of disodium hydrogen phosphate dodecahydrate
Potassium chloride 0.2g
0.5 mL of Tween-80
1000 mL of distilled water.
Embodiment 3, in a kind of carp herpesvirusⅡtype sandwich ELISA detection kit described in embodiment 1 or 2：The examination Further include in agent box：
Phosphate buffer PBST, 1 bottle of 50 mL, mass volume ratio group become：
8.0 g of sodium chloride
0.2 g of potassium dihydrogen phosphate
2.9 g of disodium hydrogen phosphate dodecahydrate
0.2 g of potassium chloride
0.5 mL of Tween-80
1000 mL of distilled water.
Embodiment 4, in a kind of carp herpesvirusⅡtype sandwich ELISA detection kit described in embodiment 1 or 2 or 3：It should Kit further includes：
Urea peroxide solution, 1 bottle of 100 mL, is mixed by urea peroxide and sodium acetate-citrate buffer solution；
Wherein, the preparation method of sodium acetate-citric acid solution is：By the lemon that 17 mL mass-volume concentrations are 94 g/L Acid solution is added in the sodium acetate solution that 500 mL mass-volume concentrations are 81 g/L, is uniformly mixed and is made；
The preparation method of urea peroxide solution is as follows：140 mg urea peroxides are weighed, and add it to 100 mL sodium acetates-lemon In lemon acid buffering solution, it is uniformly mixed and is made.
A kind of 5. carp herpesvirusⅡtype sandwich ELISA detection method of embodiment, this method using embodiment 1 or 2 or 3 or Kit described in 4 is detected, and its step are as follows：
（1）By the carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution diluteds in kit to 10 μ g/mL, 100 holes μ L/ coated elisa plates, 4 DEG C overnight；
（2）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（3）Dilution is added and closes ELISA Plate, 200 holes μ L/, 37 DEG C of reaction 2h；
（4）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（5）By detected sample, the positive and negative control dilution according to 1:Enzyme mark is added in 10 dilutions, 100 holes μ L/ In plate, 25 DEG C of 0.5 h of reaction；
（6）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（7）Enzyme mark ORF90 how anti-solution dilution is pressed 1:1000 dilutions, 100 holes μ L/ are added in ELISA Plate, 25 DEG C reaction 0.5 h；
（8）Tmb substrate solution 1 is taken, 18 mL distilled waters are added and 2 mL urea peroxide solution are configured to working solution, ELISA Plate, 25 DEG C of 15 min of reaction are added in 100 holes μ L/；
（9）Add 50 holes μ L/ terminate liquids, optical density is measured 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, it is positive to be judged to carp herpesvirusⅡtype；Sample OD values/negative control OD When value ＜ 2.1, it is negative to be judged to carp herpesvirusⅡtype.
A kind of 6. carp herpesvirusⅡtype sandwich ELISA detection method of embodiment is tested：
1.1 monoclonal antibodies-best the peridium concentration of how anti-sandwich ELISA method
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, and 10 μ g/mL coated elisa plates, 4oC is stayed overnight.Dilution is added to close, 200 holes μ L/, 37oC 2 h, PBST are washed three times.By enzyme mark ORF90 is mostly anti-（It is mostly anti-to call more anti-or ORF90 in the following text）From 1:250 start doubling dilution, and 100 holes μ L/ are added in ELISA Plate, room temperature React 1 h.Enzyme labeling antibody：By the how anti-solution of enzyme mark ORF90 respectively according to 1:5000 and 1:10000 dilutions, 100 holes μ L/, It is added in ELISA Plate, 25 DEG C of 1 h of reaction.Add tmb substrate, 25 DEG C of 10 min of effect.Add 50 holes μ L/ terminate liquids, is surveyed in 450 nm Determine OD values.According to table 1, select 10 μ g/mL peridium concentrations best.
Table 1
The determination of 3.2 monoclonal antibodies-how anti-sandwich ELISA method labelled antibody working concentration
It is coated in 10 μ g/mL monoclonal antibodies, labelled antibody dilution is tested for 1000 times and 2500 times, is examined It surveys result (table 2) and shows that labelled antibody selects 1000 times of dilutions to be tested.
Table 2
3.3 monoclonal antibodies-how anti-sandwich ELISA method detects ORF90 albumen greatest dilutions
A concentration of 1.63 mg/mL of ORF90 is detected with nucleic acid-protein detector.By the 10 μ g/mL coatings of monoclonal antibody of purifying Plate, ORF90 albumen is from 1:250 start doubling dilution, and the minimum dilution of detection is 1:8000 (tables 3).
The greatest dilution of table 3 ORF 90 detection albumen
3.4 monoclonal antibodies-how anti-sandwich ELISA method best off-period and antigen-antibody optimum reacting time
The 10 μ g/mL of ORF90 monoclonal antibodies of purifying are coated with plate, 4 DEG C overnight, with the PBST closed plates containing 1% BSA, 150 μ As a result the holes L/, 37 DEG C of placements 0.5,1,2h show that 2h is best off-period（Table 4）.Coated antibody and antigen, detection antibody With antigen respectively at room temperature（25 ℃）0.5,1 and 1.5h is reacted, as a result shows that 0.5 h is the optimum reacting time of antigen-antibody （Table 5）.
4 ORF90 monoclonal antibodies of table-how anti-sandwich ELISA best 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
5 ORF90 monoclonal antibodies of table-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 carp herpes virus type 2 tissue homogenate and other several simultaneously with ORF90 monoclonal antibodies-how anti-sandwich ELISA of foundation The virus-cell suspension of kind fish virus, including：Huichun viremia virus (SVCV), popular Hematopoietic Necrosis (EHNV), Infectious hematopoietic organ necrosis (IHNV), Koi herpesvirus (KHV), infectious pancreas necrosis virus（IPNV）, infectiousness salmon Fish anemia (ISAV), grass carp hemorrhagic disease virus（GCHV）, as a result show that this method detection carp herpes virus type 2 is the positive, Its virus is all negative (table 6), is illustrated specific preferable.
The specific test of 6 .CyHV-2 ORF90 monoclonal antibodies of table-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 control 0.060
3.6 sample detection
ORF90 monoclonal antibodies-how anti-sandwich ELISA the side for taking 40 parts of goldfish tissue homogenate without clinical symptoms to be established with this research Method detects, while extracting nucleic acid and carrying out PCR detections, as a result shows that this method can detect 18 parts of positive, and PCR method is examined Go out 15 parts of positive, shows the viruliferous fish of taking for no clinical symptoms, the sandwich ELISA method detection that this research is established Better than PCR method, the daily supervision of epidemic disease of be suitable for entering and leaving the border fish quarantine and farm.
7. ORF90 monoclonal antibodies of table-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
Table 8 PCR 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 takes out carry out sandwich ELISA assay detection purifying protein after six months, As a result it shows that OD values do not have significant change (table 9), illustrates that reagent stability is preferable.
9 monoclonal antibody of table coating-how anti-detection sandwich ELISA detection

Claims (2)

1. a kind of carp herpesvirusⅡtype sandwich ELISA detection kit, which is characterized in that the kit includes：
Carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution, 1 100 μ L are that carp herpesvirusⅡtype ORF90 purified monoclonal antibodies are molten The solution made of water, a concentration of 10-20 mg/mL；
The how anti-solution of enzyme mark ORF90,1 100 μ L are dissolved in solution made of water, a concentration of 10-20 for enzyme mark ORF90 is mostly anti- mg/mL；
Dilution, 2 bottles, every bottle of 50 mL, solution is the phosphate-buffered for the bovine serum albumin(BSA) that mass-volume concentration is 10 g/L Liquid；
Tmb substrate solution, 1,250 μ L；For the DMSO solution of TMB, mass-volume concentration
For 6 mg/mL；
Terminate liquid, 1 bottle of the sulfuric acid solution of a concentration of 2 M, 20 mL；
Carp herpes virus type 2 positive control 1,500 μ L are homogenized for goldfish assaypositive tissue；
Negative control 1,500 μ L, for negative goldfish tissue homogenate；
Coating buffer, 1 bottle, 20 mL, for the aqueous solution containing 0.015 M sodium carbonate and 0.035 M sodium bicarbonates；
96 hole elisa Plates, 1 piece；
The w/v group of the phosphate buffer becomes：
8.0 g of sodium chloride
0.2 g of potassium dihydrogen phosphate
2.9 g of disodium hydrogen phosphate dodecahydrate
Potassium chloride 0.2g
0.5 mL of Tween-80
1000 mL of distilled water；
Further include in the kit：
Phosphate buffer PBST, 1 bottle of 50 mL, mass volume ratio group become：
8.0 g of sodium chloride
0.2 g of potassium dihydrogen phosphate
2.9 g of disodium hydrogen phosphate dodecahydrate
0.2 g of potassium chloride
0.5 mL of Tween-80
1000 mL of distilled water；
The kit further includes：
Urea peroxide solution, 1 bottle of 100 mL, is mixed by urea peroxide and sodium acetate-citrate buffer solution；
Wherein, the preparation method of sodium acetate-citric acid solution is：17 mL mass-volume concentrations are molten for the citric acid of 94 g/L Liquid is added in the sodium acetate solution that 500 mL mass-volume concentrations are 81 g/L, is uniformly mixed and is made；
The preparation method of urea peroxide solution is as follows：140 mg urea peroxides are weighed, and add it to 100 mL sodium acetates-citric acid Buffer solution.
2. a kind of carp herpesvirusⅡtype sandwich ELISA detection method of non-diognose purpose, which is characterized in that this method right to use Profit requires the kit described in 1 to be detected, and its step are as follows：
（1）By the carp herpesvirusⅡtype ORF90 purified monoclonal antibody solution diluteds in kit to 10 μ g/mL, 100 The holes μ L/ coated elisa plate, 4 DEG C overnight；
（2）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（3）Dilution is added and closes ELISA Plate, 200 holes μ L/, 37 DEG C of reaction 2h；
（4）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（5）By detected sample, the positive and negative control dilution according to 1:10 dilutions, 100 holes μ L/ are added in ELISA Plate, 25 DEG C of 0.5 h of reaction；
（6）With phosphate buffer PBST board-washings, 250 holes μ L/ are washed three times；
（7）Enzyme mark ORF90 how anti-solution dilution is pressed 1:1000 dilutions, 100 holes μ L/ are added in ELISA Plate, and 25 DEG C anti- Answer 0.5 h；
（8）Tmb substrate solution 1 is taken, 18 mL distilled waters are added and 2 mL urea peroxide solution are configured to working solution, 100 μ ELISA Plate, 25 DEG C of 15 min of reaction are added in the holes L/；
（9）Add 50 holes μ L/ terminate liquids, optical density is measured 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, it is positive to be judged to carp herpesvirusⅡtype；Sample OD values/negative control OD value ＜ When 2.1, it is negative to be judged to carp herpesvirusⅡtype.
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