CN101215331A - Method for preparing tomato A virus Zhejiang separator monoclonal antibody and use thereof - Google Patents

Method for preparing tomato A virus Zhejiang separator monoclonal antibody and use thereof Download PDF

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CN101215331A
CN101215331A CNA2008100590751A CN200810059075A CN101215331A CN 101215331 A CN101215331 A CN 101215331A CN A2008100590751 A CNA2008100590751 A CN A2008100590751A CN 200810059075 A CN200810059075 A CN 200810059075A CN 101215331 A CN101215331 A CN 101215331A
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virus
monoclonal antibody
pva
elisa
zhejiang
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周雪平
吴建祥
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Zhejiang University ZJU
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Abstract

The invention discloses a preparation process of potato A virus zhejiang isolate monoclonal antibody and an application, which comprises merging purified potato A virus immune BALB/C mouse spleen cell and SP2/10 mouse myeloma cell, and obtaining which can stable generate and secrete anti-specify PVA monoclonal antibody (MAb) through screening and cloning, and then respectively preparing monoclonal antibody ascites. Monoclonal antibody builds up ACP-ELISA, DAS-ELISA and TAS-ELISA methods with high specify, sensibility and accuracy to PVA through utilizing the process. The process for preparing potato A virus isolate monoclonal antibody which is provided by the invention provides substance base and technology support for the fast detection, diagnosis and parting of PVA and molecular biology research, and builds up basis for preventing potato virus disease.

Description

The preparation method and its usage of tomato A virus Zhejiang separator monoclonal antibody
Technical field
Field under the present invention is a biological technical field, especially relates to a kind of preparation method and its usage of tomato A virus Zhejiang separator monoclonal antibody.
Background technology
(Pdtato virusA PVA) is Potyvirus (Potyvirus) member to marmor solani, and its virus particle is positive single stranded RNA, and wire is about 730 nanometers, about 15 nanometers of diameter.This virus knows that the host is a nightshade the sixth of the twelve Earthly Branches, as plants such as potato, tobaccos, is distributed widely in each potato planting country.The virus disease that is caused by PVA mainly occurs in Northern Europe, and potato planting is already endangered seriously, can cause the underproduction more than 40%, is that potato produces one of heavier virus of upward harm.This virus can be propagated with potato seed, and the field planting with the plantation of potato seed.We sent out the virus disease that the marmor solani disease is a kind of systemic infection of being caused by PVA in the Zhejiang Hangzhou suburb in 2003.Owing to have at least 7 kinds of aphids to propagate this virus in the mode of perishability, and these aphids are very general in China, and diffusion possibility that therefore should virus is very big, has higher popular risk, and the certain quarantine importance of tool.Thereby press for foundation fast, accurately, detection method simply and easily, be the diagnosis and the control of this virus disease, the production of virus-free seed potato provides technical support and material support.At present, the detection method of the PVA that has reported has electron microscopy observation and two kinds of many anti-serology detection methods that are combined into, but the electron microscopy observation method needs expensive electron microscope and can not popularize, many anti-serology detection methods that are combined into are because many anti-non-specific height that exists, accuracy and uniformity are poor, defectives such as output is limited and that these serological methods are existed is not enough, the specific antibody that the PVA monoclonal antibody specific method of the application's patent utilization development prepares anti-PVA has high specificity, uniformity is good, advantage such as can endlessly produce, strong thereby the ELISA serological method that uses this monoclonal antibody to set up has accuracy, characteristics such as easy stdn and extensive life.This method is used for the detection of PVA on field crops effectively, is the diagnosis and the control of this virus disease, the production service of virus-free seed potato.
Summary of the invention
The preparation method and its usage that the purpose of this invention is to provide a kind of tomato A virus Zhejiang separator monoclonal antibody.
Comprise the steps:
1) with the marmor solani immunity BALB/C mouse of purifying;
2) splenocyte and the SP2/0 rat bone marrow tumour cell of getting immunized mice merges under 50% polyoxyethylene glycol fusogen, and HAT screening culture medium screening hybridoma detects the positive hole of the anti-marmor solani of secretion with indirect ELISA method;
3) carry out cell clone with limiting dilution assay,, obtain to stablize the hybridoma of the anti-PVA monoclonal antibody specific of the justacrine that goes down to posterity through the indirect ELISA screening positive clone;
4) hybridoma is injected into BALB/C mouse abdominal cavity and prepares odd contradictive hydroperitoneum;
5) identify that with the described SA method of indirect ELI the preparation monoclonal antibody only has specific immune response with PVA virus, and with other plant virus without any immune response.
Described with the marmor solani immunity BALB/C mouse of purifying: the centrifugal 30-40 of 20000-30000g minute precipitation virus after precipitating viral 6-12 hour with 8-10%PEG, precipitation virus is extracted PVA with 20-45% sucrose discontinuous density gradient method, with extracting around the PVA virus immunity body weight 18-20g BALB/C female mice in age: 1mg/ml PVA virus 0.5-0.7ml mixes with equal-volume Fu Shi Freund's complete adjuvant, after fully emulsified, through every of back of the body subcutaneous abdomen multi-point injection 0.2-0.3ml, interval 3-4 week, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, for the second time abdominal injection 0.2-0.3ml is every, cross 3-4 after week the antigen with doubling dose carry out abdominal injection.
Described hybridoma is injected into BALB/C mouse abdominal cavity and prepares odd contradictive hydroperitoneum: get BALB/C mice about 8 ages in week, and abdominal injection 0.3-0.5ml pristane, pneumoretroperitoneum injected 5-10 * 10 in 7-10 days 5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3-5min of 3000-5000rpm collects supernatant liquor, is monoclonal antibody ascites.
Tomato A virus Zhejiang separator monoclonal antibody is used for field PVA and detects and import and export the PVA quarantine.
The described method that is used for field PVA detection is: ACP-ELISA, DAS-ELISA or TAS-ELISA.
The described method that is used to import and export the PVA quarantine is ACP-ELISA, DAS-ELISA or TAS-ELISA.
Advantage of the present invention is: the PVA virus-specific monoclonal antibody that 1) provides, utilization ACP-ELISA, DAS-ELISA and TAS-ELISA can high specials, accurately, sensitive detects PVA; 2) utilize the prepared monoclonal antibody of the present invention to detect PVA, do not need expensive electron microscopic mirror device; 3) utilize the prepared monoclonal antibody of the present invention, can be used for the detection of field crops PVA effectively.
Embodiment
The present invention has prepared the high specificity of PVA, the height of tiring, good stability, the mass-produced monoclonal antibody of energy, and set up with these monoclonal antibodies and to have detected fast diagnosis method--the ELISA that PVA has high degree of specificity, susceptibility and exactness, can be potato detoxicating evaluation and breeding for disease resistance strong technology and product support is provided, to the integrated control of this virus disease and increase production without increasing the work force direct theoretical foundation and technical guarantee are provided, simultaneously, can be applicable to import and export aspects such as check, quarantine and PVA investigation of epidemic situation analysis.
PVA MONOCLONAL ANTIBODIES SPECIFIC FOR method
1. immunogenic preparation
PVA is located away from the geographic potato of Zhejiang Hangzhou, precipitate viral 6-12 hour with 8-10%PEG after the centrifugal 30-40 of 20000-30000g minute precipitation virus, precipitation virus is extracted PVA with 20-45% sucrose discontinuous density gradient method.Virus purification liquid is observed particle shape through 2% phospho-wolframic acid (pH6.7) the rearmounted JEOL that dyes under the JEM-1200EX Electronic Speculum.
2. immune animal
With extracting around the PVA virus immunity body weight 18-20g BALB/C female mice in age: 1mg/ml extracts PVA virus 0.5-0.7ml to be mixed with equal-volume Fu Shi Freund's complete adjuvant, after fully emulsified, through every of back of the body subcutaneous abdomen multi-point injection 0.2-0.3ml, interval 3-4 week, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, for the second time abdominal injection 0.2-0.3ml is every, cross 3-4 after week the antigen with doubling dose carry out abdominal injection, extracting spleen cell merges after 3 days.
3. cytogamy
Get above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) in 5-10: 1 ratio, mixing in the RPMI-1640 of serum-free (Gibco) substratum, the centrifugal 5min of 1500rpm, remove substratum, with 50%PEG (polyoxyethylene glycol, Sigma) as fusogen, under 37 ℃ of following water-baths, add 0.5-0.7ml, make it merge 2min, RPMI-1640 substratum with serum-free stops the centrifugal 5min of fusion back 1500rpm, and precipitation suspends with HAT screening culture medium (Sigma), and branch installs to 96 holes and contains in the cell plate of feeder cell, 37 ℃, cultivate in the cell cultures vessel of 5%CO2.
4. the screening in hybridoma, positive hole and clone thereof
Cultivate after 5 days in the cell cultures vessel, change liquid once, changed liquid with the HT substratum on the 10th day with the HAT screening culture medium, by the time at the bottom of the fused cell coverage hole during 5%-50%, conventional indirect ELISA method screens positive hole, obtains more than 150 the positive hole that PVA is responded altogether, and positive rate is 28%.Select 6 cell holes that are strong positive reaction, carry out limiting dilution assay clone, obtain 1A2,3H9,7H2 and 8E12 totally 4 strains can secrete the hybridoma cell strain of the specific antibody of anti-PVA.Through subculture in vitro separately more than 6 months with repeatedly behind the cryopreservation resuscitation, 4 strain cell strains all can well be grown, and stably excreting antibody.After enlarged culturing, be used for ascites preparation and liquid nitrogen and preserve.
5. the specific detection of monoclonal antibody
With the marmor solani (PVA) of purifying, carnation mottle virus (CarMV), corn mosaic virus (SCMV), Brassica 2 et 4 (TuMV), Tomato mosaic virus (ToMV), tobacco mosaic virus (TMV) (TMV), cucumber mosaic virus (CMV), broad bean wilt virus 1 (BBWV-1), broad bean wilt virus 2 (BBWV-2), carnation ringspot virus (CaRSC), potato virus X (PVX) and marmor upsilon (PVY) 1ug/ml bag are by the ELISA Sptting plate, make negative control with corresponding strong leaf extract, use indirect elisa method, measure the specific reaction of 4 strain monoclonal antibodies respectively.Indirect ELISA method is specially above-mentioned virus and is diluted to behind the 1ug/mL 100ul/ hole bag by elisa plate with coating buffer respectively, 4 ℃ spend the night or 37 ℃ 2 hours, make it be adsorbed in the polystyrene plate hole; PBST washs skim-milk or 1-3%BSA or the 3-6% bovine serum sealing 30-60min that uses 1-10% after three times; Add monoclonal antibody 100ul/ hole, 37 ℃ 1-2 hour; PBST washs alkaline phosphatase lipase (AP) mark rabbit anti-mouse igg two anti-(Sigma company) the 100ul/ holes that add 10000 times of by specification dilutions after three times, 37 ℃ 1-2 hour, after the PBST washing four times, with the colour developing of PNPP substrate, after the 2mol/L sodium hydroxide termination reaction, read OD with microplate reader 405Value, with positive greater than 2.1 with negative OD value ratio.Found that 4 strain monoclonal antibodies have specific reaction to PVA, and all do not have specific reaction with CarMV, TMV, ToMV, CMV, SCMV, PVY, PVX, BBWV1, BBWV2, TuMV, other viruses of CaRSC.
6. monoclonal antibody ascites prepares and purifying
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5-10 * 10 in 7-10 days 5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3-5min of 3000-5000rpm collects supernatant liquor, is monoclonal antibody ascites.Get 1 times of volume ascites and add 2 times of volume 0.06M PH4.8 acetate buffer solution dilutions, add sad (30ul/ml ascites), the following edged of room temperature stirs, and clarifies 1 hour for 4 ℃, the centrifugal 20min of 12000rpm, collect supernatant, use 50% saturated ammonium sulphate immunoglobulin (Ig) again, placed 2 hours for 4 ℃, the centrifugal 20min of 3000-5000rpm, precipitation is dissolved with the PBS solution of 2 times of volumes, promptly obtains the ascites antibody of purifying after 4 ℃ of mobile dialysis 24 hours ,-70 ℃ of preservations.
7. the subgroup identification of monoclonal antibody and ascites titration
With the odd contradictive hydroperitoneum of purifying and the anti-BALB/C mice IgG of standard of Sigma company, IgG 1, IgG 2a, IgG 2b, IgG 3, IgM antibody is done two-way agar diffusion test, and the result is, and antibody type and the subclass of 3H9 and 8E12 are IgG1, and 1A2 and 7H2 are IgG3, the light chain of 4 strain monoclonal antibodies is the K chain.Detect odd contradictive hydroperitoneum with conventional indirect ELISA method and tire, the result tires all 10 for above-mentioned 4 strain ascites -6More than.
8.AP-the preparation of anti-PVA monoclonal antibody binding substances
In 0.5ml PBS (0.01mol/l, PH7.2), adding glutaraldehyde to final concentration is 0.2% with the monoclonal antibody mixing of 30mgAP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10mMTris-HCL (pH8.0,0.15M NaCl, 1MM MgCl2) in the reactant to 1ml, flow in 4C with above-mentioned Tris-HCL damping fluid then and dialysed 24 hours.The later product of dialysing is AP-monoclonal antibody binding substances, adds sterile glycerol to 50% (V/V) in the binding substances, and is standby in-20 ℃ of preservations.
9. the monoclonal antibody recognition site is analyzed
3ug/ml antigen (purification PVA) 100ul/ hole is in 96 hole elisa plates, and 4 ℃ of bags are spent the night; With PBST washing three times, 37 ℃ in 2%BSA 150ul/ hole was sealed 1 hour, added the another kind of monoclonal antibody 50ul of a kind of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃ 1 hour; Add nitrophenyl phosphate substrate 100ul/ hole after the PBST washing three times, 37 ℃ 20 minutes, OD405 measures absorption value.With odd contradictive hydroperitoneum the monoclonal antibody of same AP mark being suppressed is 100%, to the negative contrast of the inhibition of mark monoclonal antibody, calculates the inhibiting rate between each monoclonal antibody with known irrelevant odd contradictive hydroperitoneum.Be that inhibiting rate is (1-measured value OD/ negative control value OD) * 100.Inhibiting rate＞75% is for relevant, and＞50% is not exclusively relevant, and＜50% is uncorrelated, and＜25% for uncorrelated fully, and the result shows 4 incoherent antigen binding sites of the anti-PVA monoclonal antibody identification of 4 strains.
10. set up indirect antigen coated ELISA (ACP-ELISA) method with monoclonal antibody and detect virus
The operation steps of ACP-ELISA method
(1) the sick leaf sap 100ul/ hole with carbonate buffer solution (pH9.6) 30 times of dilutions adds elisa plate, the positive contrast of the sick leaf of PVA, and the negative contrast of corresponding strong leaf, 37 ℃ of 2h, or 4 ℃ spent the night;
(2) 30min is sealed with 5% skim-milk in PBST washing back;
(3) 100ul/ hole after 5000 times of dilutions of odd contradictive hydroperitoneum, 37 ℃ of 1h;
(4) PBST washing back adds the sheep anti-mouse igg binding substances (Sigma) of the AP mark of 5000 times of dilutions, 100ul/ hole, 37 ℃, 1h;
(5) add nitro phosphoric acid salt substrate 100ul/ hole, room temperature 30min with PBST washing back;
(6) detect by an unaided eye, it is positive that substrate colors becomes yellowish green hole, or survey the OD405 value with enzyme-linked immunosorbent assay instrument, with P/N＞2.1 as positive judging criterion.
ACP-ELISA method detection sensitivity detects
The odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, sick leaf is made doubling dilution from 1: 5 to 5120, purified virus (5mg/l) was made doubling dilution from 1: 1000 to 1: 512000, make negative control with corresponding dilution strong leaf sap respectively, carried out above-mentioned ACP-ELISA method and detected.The result shows that the ACP-ELISA method all is positive to the sick leaf sap of 1: 5～500 times of dilutions, promptly can reach 1: 500 to the sensitivity that detects the disease leaf, the ACP-ELISA method can reach 3ng/ml to the detection sensitivity of pure virus, and the viral absolute magnitude in every hole detects and is 0.3ng.Performance ACP-ELISA method has the susceptibility and the reliability of height.
11.PVA monoclonal antibody DAS-ELISA detection method
1) 5000 times of the strain monoclonal antibodies of anti-PVA dilution 100ul/ hole bag is by polystyrene board, and 37 ℃, 2-4h or 4 ℃ spend the night;
2) add the skim-milk of 1-10% or 1-3%BSA or 3-6% bovine serum sealing 200ul/ hole after the PBST washing three times in 37 ℃ of sealing 30-60min
3) add test sample 100ul/ hole.With the positive contrast of PVA, make negative control with corresponding healthy sample, 37 ℃ of 1-2h;
4) PBST washing back adds 10000 times of another monoclonal antibody binding substances of dilution alkaline phosphatase lipase (AP) mark 100ul/ holes, 37 ℃ of 1-2h
5) add the PNPP substrate in color development at room temperature 5-30min after the PBST washing, after the 2mol/L sodium hydroxide termination reaction, it is positive or survey the OD value of 405nm with 680 type enzyme-linked immunosorbent assay instruments that the visual inspection substrate colors becomes yellowish green hole, with P/N＞2.1 as positive judging criterion.
DAS-ELISA method detection sensitivity detects
Sick leaf is made doubling dilution from 1: 5 to 5120, and purified virus (5mg/l) was made doubling dilution from 1: 1000 to 1: 1024000, make negative control with corresponding dilution strong leaf sap respectively, carried out above-mentioned DAS-ELISA method and detected.The result shows that the DAS-ELISA method all is positive to the sick leaf sap of 1: 5～1000 times of dilutions, promptly can reach 1: 1000 to the sensitivity that detects the disease leaf, DAS-ELISA can reach 0.2ng/ml to the detection sensitivity of pure virus, and the viral absolute magnitude in every hole detects and is 0.02ng.Performance DAS-ELISA method has the susceptibility and the reliability of height.
12.PVA the TAS-ELISA detection method
12.1.TAS-ELISA the operating process of method:
1) rabbit anti-serum of anti-PVA (this institute makes by oneself) 100ul/ hole bag is by polystyrene board, and 37 ℃, 2-4h or 4 ℃ spend the night;
2) add the skim-milk of 1-10% or 1-3%BSA or 3-6% bovine serum sealing 200ul/ hole after the PBST washing three times in 37 ℃ of sealing 30-60min
3) add test sample 100ul/ hole.With the positive contrast of PVA, make negative control with corresponding healthy sample, 37 ℃ of 1-2h;
4) the odd contradictive hydroperitoneum 100ul/ hole of diluting with confining liquid after the washing, 37 ℃ of 1-2h
5) PBST washing back adds 10000 times of dilution alkaline phosphatase lipase (AP) mark rabbit anti-mouse igg two resistive connection compound (Sigma) 100ul/ holes, 37 ℃ of 1-2h
6) add the PNPP substrate in color development at room temperature 5-30min after the PBST washing, after the 2mol/L sodium hydroxide termination reaction, it is positive or survey the OD value of 405nm with 680 type enzyme-linked immunosorbent assay instruments that the visual inspection substrate colors becomes yellowish green hole, with P/N＞2.1 as positive judging criterion.
12.2.TAS-ELISA determining of detection method optimum condition:
Adopt the test of TAS-ELISA square formation to carry out, promptly laterally add the anti-PVA serum of rabbit (this institute makes by oneself) with wrap be cushioned liquid from 1: 100 to 1: 102400 doubling dilution; PVA 1ug/ml; Vertically add with the PBST damping fluid from 1: 5 to 1: 2048000 the doubling dilution odd contradictive hydroperitoneum; The rabbit anti-mouse igg two anti-specification sheets dilution of Sigma company, 1: 10000 times pressed of AP mark; Operate by the TAS-ELISA method flow.The result is 1: 6000 for the rabbit anti-serum optimal dilution of PVA; The optimal dilution of 1A2,3H9,7H2 and 8E12 was respectively 1: 8000, and 1: 10000,1: 5000,1: 10000.12.3.TAS-ELISA determining of method detection sensitivity
Under rabbit anti-serum and the suitableeest working concentration of odd contradictive hydroperitoneum, the PVA of 1ug/ml is carried out TAS-ELISA mensuration after containing the 1-3%BSAPBST doubling dilution, the result is: the sensitivity that TAS-ELISA detects 4 strain monoclonal antibodies all more than 0.01ng, illustrates that present method has good sensitivity.The application of PVA is detected in the field of 1 3. above-mentioned ELISA methods
Utilize the above-mentioned ELISA method of the PVA monoclonal antibody foundation of preparation that the field lily is detected.Gathering on the lily of Lishui of Zhejiang area is the sick sample of viral symptom, does 1: 30 times of dilution, carries out above-mentioned ELISA then and detects, and makes negative control with healthy tree juice, and the juice of inoculation PVA is made positive control.Detected result shows, the band of PVA poison rate is very high on the lily, 40% sample is positive, consistent with the electron microscopic observation detected result, this shows that the above-mentioned ELISA method of prepared monoclonal antibody and foundation can well be used for the detection of field PVA, simultaneously, clear and definite marmor solani is the main viral species of popular on the lily of field.

Claims (6)

1. the preparation method of a tomato A virus Zhejiang separator monoclonal antibody is characterized in that comprising the steps:
1) with the marmor solani immunity BALB/C mouse of purifying;
2) splenocyte and the SP2/0 rat bone marrow tumour cell of getting immunized mice merges under 50% polyoxyethylene glycol fusogen, and HAT screening culture medium screening hybridoma detects the positive hole of the anti-marmor solani of secretion with indirect ELISA method;
3) carry out cell clone with limiting dilution assay,, obtain to stablize the hybridoma of the anti-PVA monoclonal antibody specific of the justacrine that goes down to posterity through the indirect ELISA screening positive clone;
4) hybridoma is injected into BALB/C mouse abdominal cavity and prepares odd contradictive hydroperitoneum;
5) identify that with the described SA method of indirect ELI the preparation monoclonal antibody only has specific immune response with PVA virus, and with other plant virus without any immune response.
2. the preparation method of a kind of tomato A virus Zhejiang separator monoclonal antibody according to claim 1, it is characterized in that described: the centrifugal 30-40 of 20000-30000g minute precipitation virus after precipitating viral 6-12 hour with 8-10%PEG with the marmor solani immunity BALB/C mouse of purifying, precipitation virus is extracted PVA with 20-45% sucrose discontinuous density gradient method, with extracting around the PVA virus immunity body weight 18-20g BALB/C female mice in age: 1mg/ml PVA virus 0.5-0.7ml mixes with equal-volume Fu Shi Freund's complete adjuvant, after fully emulsified, through every of back of the body subcutaneous abdomen multi-point injection 0.2-0.3ml, interval 3-4 week, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, for the second time abdominal injection 0.2-0.3ml is every, cross 3-4 after week the antigen with doubling dose carry out abdominal injection.
3. the preparation method of a kind of tomato A virus Zhejiang separator monoclonal antibody according to claim 1, it is characterized in that described hybridoma is injected into BALB/C mouse abdominal cavity and prepares odd contradictive hydroperitoneum: get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane, pneumoretroperitoneum injected 5-10 * 10 in 7-10 days 5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3-5min of 3000-5000rpm collects supernatant liquor, is monoclonal antibody ascites.
4. the purposes by the tomato A virus Zhejiang separator monoclonal antibody of claim 1 preparation is characterized in that, is used for field PVA and detects and import and export the PVA quarantine.
5. the purposes of a kind of tomato A virus Zhejiang separator monoclonal antibody according to claim 4 is characterized in that the described method that is used for field PVA detection is: ACP-ELISA, DAS-ELISA or TAS-ELISA.
6. the purposes of a kind of tomato A virus Zhejiang separator monoclonal antibody according to claim 4 is characterized in that the described method that is used to import and export the PVA quarantine is ACP-ELISA, DAS-ELISA or TAS-ELISA.
CNA2008100590751A 2008-01-09 2008-01-09 Method for preparing tomato A virus Zhejiang separator monoclonal antibody and use thereof Pending CN101215331A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105543176A (en) * 2016-02-22 2016-05-04 浙江大学 Hybridoma cell strain secreting potato-virus-Y-resistant monoclonal antibodies and monoclonal antibody application thereof
CN109929812A (en) * 2019-02-15 2019-06-25 云南省农业科学院生物技术与种质资源研究所 Marmor solani strain and its construction method and application in a kind of Potato Cultivars cooperation 88

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105543176A (en) * 2016-02-22 2016-05-04 浙江大学 Hybridoma cell strain secreting potato-virus-Y-resistant monoclonal antibodies and monoclonal antibody application thereof
CN105543176B (en) * 2016-02-22 2019-01-01 浙江大学 Secrete resistant to PVY monoclonal antibody hybridoma cell strain and its monoclonal antibody application
CN109929812A (en) * 2019-02-15 2019-06-25 云南省农业科学院生物技术与种质资源研究所 Marmor solani strain and its construction method and application in a kind of Potato Cultivars cooperation 88

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