CN1424326A - Monocloned antibody for eight plant viruses and inspection thereof - Google Patents
Monocloned antibody for eight plant viruses and inspection thereof Download PDFInfo
- Publication number
- CN1424326A CN1424326A CN 02159875 CN02159875A CN1424326A CN 1424326 A CN1424326 A CN 1424326A CN 02159875 CN02159875 CN 02159875 CN 02159875 A CN02159875 A CN 02159875A CN 1424326 A CN1424326 A CN 1424326A
- Authority
- CN
- China
- Prior art keywords
- monoclonal antibody
- specific reaction
- virus
- hole
- tomv
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The 19 monoclonal antibody strains of 8 monoclonal antibody for 8 plant viruses including broad bean wilt virus, cucumber mosaic virus, tomato mosaic virus, cane mosaic virus, etc are disclosed. The high-specificity high-sensitivity ACP-ELISA and TAS-ELISA methods for detecting relative viruses are disclosed on the basis of said 8 antibodies.
Description
Technical field
The present invention relates to the monoclonal antibody and the detection method thereof of eight kind of plant viruses.
Background technology
Plant virus is infected the main diseases original of plant as a class, causes the disease of farm crop, fruit tree, flowers, herbage, medicinal plant in worldwide, causes the decline of yield and quality, even forms crushing harm, greatly influences peasant's income.The present invention can specificity, susceptibility ground detects eight kind of plant viruses, is the generation of breeding for disease resistance, disease and distribution, virus quarantine, the diagnosis of virus disease, the foundation and the evaluation of virus-elimination seedlings, and the research of the viroses of plant provides material and technical guarantee.
Broad bean wilt virus (BBWV) is at first obtained from the broad bean separation by Australian Stubbs in nineteen forty-seven, is the typical member that the Comoviridae fabavirus belongs to, and worldwide widely takes place.Since China is infected by BBWV on reported first peas such as Xi Zhongxing, successively in Xinjiang, ground such as Zhejiang, Beijing, Jiangsu find crop and the flowers that BBWV infects, comprise various plants such as pea, broad bean, soybean, tomato, spinach, celery, Root of Indigowoad, Myosotis sylvatica, the parasitic scope of BBWV is very wide, according to statistics such as Edwardson, it can infect and belong to 328 kind of plant that 44 sections 186 belong to.The different isolates of BBWV can be divided into two kinds of serotypes, i.e. serotype I and serotype II are at present by called after BBWV1 of ICTV and BBWV2.Many to the evaluation of BBWV according to the particle proterties of virus and the observation of the biological character behind the virus inoculation, and the biology of BBWV1 and BBWV2 and physicochemical property difference are little, thereby be difficult to determine that the BBWV that is reported is BBWV1 or BBWV2, because BBWV1 and BBWV2 differ greatly on serology, can carry out the evaluation of BBWV1 by serological technique.The monoclonal antibody of virus not only can be distinguished the different virus kind, and since each monoclonal antibody at antigenic determinant different and also can produce differential responses to the not homophyletic system of virus of the same race.
Cucumber mosaic virus (CMV) is the typical member of Bromoviridae Cucumovirus, is the universal plant virus population of being propagated in the perishability mode by aphid.Its host range is very wide, has found at least 800 kinds of hosts, be distribute the widest, host plant is maximum, one of plant virus of tool Economic Importance.It can cause floral leaf cucumber and other cucurbitaceous plants, and spinach is withered, the floral leaf of tomato fern leaf and other a variety of unifacial leaves or dicotyledons.In China, CMV is cause of disease or the main pathogen that causes tobacco mosaic disease, masaic of tomato, capsicum and pimento virus disease, Flower of India Canna's leaf disease, petunia disease, rubber virus disease.And over nearest 10 years, it is more widespread that CMV in China harm takes place, be that the present discovery of crop of main pathogen all is to infect based on CMV originally with other viruses, and on many crops, also show as to endanger and increase the weight of or new disease popular trend, rise and " disease newly " big generation by CMV and the compound disease that infects of other virus, production loss is increased the weight of.Since the first CMV separates report, more than 100 CMV strain system or isolate have been reported all over the world.Serology is to analyze the relatively different isolates and the favourable instrument of studying the disease epidemiology of CMV.
Tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) (TMV) belong to Tobamovirus together, and comparatively close serological relation is arranged.ToMV is considered to strain system among the TMV always for a long time, but both the amino acid of host range, serological relation and capsid protein form and aspect such as sequence variant.ToMV tells from TMV as single virus after 1971, thinks that now ToMV is a kind of virus nearest with the TMV relation among the Tobamovirus.Their host range is very wide, can infect many plants such as Solanaceae, Cruciferae, Gramineae, Chenopodiaceae, pulse family, and be a kind of universal virus.Occurring in nature or infect separately or virus is compound infects with CMV, PVX, PVY etc. seriously descends crop yield, quality.TMV and ToMV can't help amboceptor and propagate, and be the easiest in the friction inoculative transmission.Various farming operations are main paties of the two kinds of virus disseminatings in field.
Past, we mainly were experimental techniques such as ultra micro pathology difference by the two host behind the symptomatic reaction on some plant indicator, virus infection to the evaluation of ToMV and TMV, though can distinguish ToMV and TMV, but these methods or experimental period are long or need accurate instrument, and use can be distinguished the monoclonal antibody of ToMV and TMV, evaluation is carried out than being easier to, and good reproducibility.
Since nineteen twenty-one reported first Brassica 2 et 4 (TuMV), Chinese scholars has been carried out deep research to TuMv.TuMV is the most important pathogenic virus of crop in cruciferae virus disease, and TuMV belongs to the member of Potyvirus.Can be by the aphis propagation more than 40 kinds, its distribute various places all over the world (Shukla, 1989; Shattuck, 1989).Zhejiang Province is that the plantation of hot pickled mustard tube, leaf mustard, Wenzhou assorted cold dishes, potherb mustard economizes greatly, and earn foreign exchange to plant these Vegetables Exportation every year, produces huge economic benefit.But in recent years,, cause enormous economic loss because infecting of turnip mosaic virus becomes the serious underproduction of principal post agricultural-food such as our province hot pickled mustard tube, leaf mustard, potherb mustard.
Do not detect the TuMV diagnostic method at present effectively and rapidly, this enantiopathy breeding, virus-elimination seedlings and the integrated control of this virus disease brought very big difficulty, and these work are to prevent this pathogenetic unique method.Electron microscopic observation commonly used of the evaluation of TuMV at present and serological reaction, but electron microscopic observation can not popularize on the one hand, and expense is very high on the other hand, and efficient is very low; Shortcomings such as serum at present commonly used identifies and almost all use polyvalent antibody, and this polyvalent antibody exists specificity not high, and it is low to tire, instability and can not applying well.
Corn mosaic virus (SCMV) is to be reported the earliest in 1919 by Brandes, be the member that Potyvirus belongs to, each continent 83 countries have at present spread all over the world, host range is confined to gramineous crop, cause serious sugarcane, the mosaic disease of gramineous crops such as corn and weeds is the main pathogen of China's corn short mosaic disease.The corn short mosaic disease is a kind of serious worldwide disease, and it is wide to distribute in China, and harm is heavy, has become China corn producing region especially North China, the important corn disease virus disease in the Northwest.The serious chlorisis of diseased plant, short and small, the grave illness strain can not be tied fringe, brings about great losses to agriculture production.Serological technique is mainly used in the detection of corn short mosaic disease cause of disease, and as the enzyme linked immunological adsorption technology etc., this SD accuracy depends on the specificity relation between employed antiserum(antisera) and the detected virus.Often both contained specific antibody component between kind owing to infect the polyclonal antiserum of the Potyvirus genus virus of corn, contain the antibody component of guarding in belonging to again, thereby specialization is not strong, simultaneously, known have 4 kinds of Potyvirus to belong to viruses all can to infect corn and cause short mosaic disease, cause of disease situation more complicated makes and utilizes traditional polyclonal antiserum detection technique to be difficult to accurately identify that Potyvirus belongs to virus.
Potato virus X (PVX), marmor upsilon (PVY) are the main virus that causes solanaceous crops morbidities such as potato.Wherein marmor upsilon can be propagated with non-persistent mode and mechanical inoculation through aphid, and this viral host range is Solanaceae and some Amaranthaceaes, Chenopodiaceae, composite family and leguminous plants, and mechanical inoculation can infect 120 kind of plant.PVY causes that potato is mottled, vein is downright bad, necrotic plaque, dwarfing plants, the bent deformity of leaf roll, yellow, shrinkage, and symptom such as plant and stem tuber necrosis can cause the production loss of 10%-80%.PVX is mainly by the mechanical friction contact transmission.Its host range is wider, can infect 16 sections, 240 kind of plant, and Solanaceae is main host.PVX causes that the light-duty floral leaf of potato becomes latent disease, infect for many years and then cause heavy floral leaf or dwarfing, the production loss that causes is 10%-80%, causes mottled on tobacco and necrotic spot, on tomato, cause heavy floral leaf and dwarfing, as PVX with PVY is compound symptom aggravation when infecting.Potato is through vegetative propagation, and virus-elimination seedlings and potato seed are the effective ways that prevent virus harm from the source.
Tomato yellow leaf curl virus is that morbidity is economized seriously in Guangxi, China southern province, Yunnan etc., and harm is very serious on crops such as tobacco, tomato.Bean golden mosaic virus in such Tobamovirus geminivirus infection section belongs to by Bemisia tabaci biography poison.Because the aleyrodid amboceptor progressively increases in recent years, thereby virus also increases the weight of year by year, badly influenced the cultivation of tomato in provinces such as Guangxi.Because this viroid concentration in tissue is lower, thereby bring difficulty to serum pref.
Summary of the invention
The monoclonal antibody and the detection method thereof that the purpose of this invention is to provide eight kind of plant viruses.
The monoclonal antibody of the anti-BBWV of 4 strains is characterized in that, BBWV2 BIZU01, and 02 couple of BBWV2 has specific reaction, and does not have specific reaction with other plant virus; BBWV1 BIZU03, the 04th, BBWV1 there is specific reaction, and does not have specific reaction with other plant virus.
The monoclonal antibody of the anti-CMV of 2 strains is characterized in that, CMV BIZU05, and 06 couple of CMV has specific reaction, and does not have specific reaction with other plant virus.
The monoclonal antibody of the anti-ToMV of 4 strains, it is characterized in that, ToMV BIZU07,08 monoclonal antibody has specific reaction to ToMV, and do not have specific reaction with other plant virus, ToMV BIZU09,10 monoclonal antibodies have specific reaction to ToMV and TMV, and do not have specific reaction with other plant virus.
The monoclonal antibody of the anti-TuMV of 2 strains is characterized in that, TuMV BIZU11, and 12 couples of TuMV have specific reaction, and do not have specific reaction with other plant virus.
The monoclonal antibody of the anti-SCMV of 2 strains is characterized in that, SCMV BIZU13, and 14 couples of SCMV have specific reaction, and do not have specific reaction with other plant virus.
The monoclonal antibody of the anti-PVX of 2 strains is characterized in that, PVX BIZU15, and 16 pairs of PVX specific reactions, and do not have specific reaction with other plant virus.
The monoclonal antibody of the anti-PVY of 2 strains is characterized in that, PVY BIZU17, and 18 couples of PVY have specific reaction, and do not have specific reaction with other plant virus.
The monoclonal antibody of the anti-TYLCMV of 1 strain is characterized in that TYLCMV BIZU19 has specific reaction to TYLCMV, and does not have specific reaction with other plant virus.
Detect tri-antibody sandwich (TAS-ELISA) method of eight kind of plant viruses:
1) this institute of 5-50ug/ml make BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or TYLCMV by oneself rabbit anti-serum IgG 100ul/ hole bag by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night;
2) add 3%-6% skim-milk 120-150ul/ hole after PBST washs three times in 37 ℃ of sealing 30-60min
3) add the purified virus that dilutes with confining liquid, or 1: the sick leaf extract 100ul/ hole that 10-100 doubly dilutes.With BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or the positive contrast of the pure virus of TYLCMV, make negative control, 37 ℃ of 1-2h with corresponding strong leaf extract;
4) BBWV that doubly dilutes of washing rear enclosed liquid 5000-10000 or the odd contradictive hydroperitoneum 100ul/ hole of CMV or ToMV or TuMV or SCMV or PVX or PVY or TYLCMV virus, 37 ℃ of 1-2h
5) the AP mark sheep anti-mouse igg binding substances 100ul/ hole of the Sigma company that adding 5000-10000 doubly dilutes after the PBST washing, 37 ℃ of 1-2h
6) add nitro phosphoric acid salt substrate in room temperature or 37 ℃ of 10-35min after the PBST washing, the visual inspection substrate colors, it is positive or with the OD value of 550 type enzyme-linked immunosorbent assay instruments survey 405nm to become yellowish green hole, with P/N＞2.1 as positive judging criterion.
Detect the indirect antigen ELISA method of eight kind of plant viruses, i.e. the ACP-ELISA method:
1) the sick leaf sap 100ul/ hole of doubly diluting with carbonate buffer solution 10-50 adds elisa plate, with BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or the positive contrast of the sick leaf of TYLCMV, and the negative contrast of corresponding strong leaf, 37 ℃ of 2h, or 4 ℃ spent the night;
2) PBST washing back is sealed 30-60min with 3%-6% skim-milk 120-150ul/ hole in 37 ℃
3) the odd contradictive hydroperitoneum 5000-10000 of BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or TYLCMV virus doubly dilutes 100ul/ hole, back, 37 ℃ of 1-2h;
4) the sheep anti-mouse igg binding substances of the AP mark of the Sigma company that adding 5000-10000 doubly dilutes after the PBST washing, 100ul/ hole, 37 ℃, 1-2h;
5) add nitro phosphoric acid salt substrate 100ul/ hole, room temperature or 37 ℃ of 30-60min 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.
The present invention prepares 8 kinds of corresponding monoclonal antibody specifics with 8 kind of plant viruses, and has set up that the corresponding 8 kinds of TAS-ELISA with high degree of specificity and susceptibility detect viral method and 8 kinds of ACP-ELISA detect viral method.Use agglutination absorption and the specificity of monoclonal antibody and the amplification and the color reaction of ELIAS secondary antibody of polyvalent antibody with monoclonal antibody combination TAS-ELISA method, thereby have higher sensitivity and specificity, advantage such as color reaction is fast, naked eyes can be judged.Also have specificity and higher susceptibility with monoclonal antibody combination ACP-ELISA method,, and operation steps is simplified, use and conveniently favored by people in addition owing to save the polyvalent antibody bag by this step.These two kinds of methods are with a wide range of applications at the aspects such as evaluation of the distributing of the detection of plant virus, diagnosis, viral somatotype, virus, breeding for disease resistance, import and export quarantine, virus-elimination seedlings, thereby the present invention patent has very high using value.
Embodiment
The present invention utilizes the mono-clonal body technique, obtain the hybridoma cell strain of the specific antibody of secretion BBWV2 and BBWV1, and TAS-ELISA detection architecture and ACP-ELISA detection architecture have been set up, this detection architecture has higher sensitivity, each isolate of energy specific detection BBWV2, and can not react with the BBWV1 isolate, have very high specificity.Monoclonal antibody with BBWV1 can only have specific reaction with the isolate of BBWV1, and the isolate of BBWV2 is not reacted.Application the present invention detects the BBWV isolate of China different areas, and find that the BBWV of domestic distribution is BBWV2, and extensively be present in the farm crop, be one of main pathogen on the leguminous crop.But still can not whether exist in China by clear and definite BBWV1.The present invention helps to understand that BBWV takes place at home and strain system distributes, for identifying that fast, accurately BBWV provides technical foundation, and can be that nontoxic seedling is identified, the screening of resistant variety and and disease control strong evidence is provided.
The present invention preparation is that CMV has specific monoclonal antibody and detection method to homophyletic not, and the foundation of this virus detection, diagnosis, virus-elimination seedlings and detoxification flowers and the quarantine tool of virus disease are had very important significance.
Past, we mainly were experimental techniques such as ultra micro pathology difference by the two host behind the symptomatic reaction on some plant indicator, virus infection to the evaluation of ToMV and TMV, though can distinguish ToMV and TMV, but these methods or experimental period are long or need accurate instrument, and use can be distinguished monoclonal antibody and the detection method of ToMV and TMV, evaluation is carried out than being easier to, and good reproducibility.The generation of understanding the virus on the field crops that is established as of the acquisition of ToMV, TMV monoclonal antibody and detection system distributes and has created condition, so that provide scientific basis for the comprehensive regulation of crop breeding and virus disease.
We have prepared the high specificity of TuMV, the height of tiring, good stability, the mass-produced monoclonal antibody of energy, it is effective to set up TuMV, the rapid detection diagnostic method, can be the strong technology and the product support of seed seedling detoxification evaluation and breeding for disease resistance, 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 the import and export check of vegetables, seed seedling, aspects such as quarantine and TuMV investigation of epidemic situation analysis.
The present invention utilizes monoclonal antibody technique, obtains the Mab cell strain at No. 1 (ZJ1) a certain specific antigen determinant of SCMV Zhejiang isolate, and has set up the ACP-ELISA and the TAS-ELISA detection architecture of rapid sensitive.This detection architecture has very high detection sensitivity, and in reacting, the ELISA with maize rough dwarf virus poison and 6 kinds of Potyvirus genus viruses shows very strong specialization, thereby remedied the not strong limitation of polyclonal antibody specialization, it is more extensive that serological technique is used.
Using the present invention detects the corn short mosaic disease cause of disease of China 12 provinces and cities and identifies, determine that the corn short mosaic disease that China's wide geographic area takes place is caused by SCMV, and do not have the mixed infection with other 3 kinds of viruses, it is in full accord that detected result and RT-PCR and sequencing etc. detect qualification result.Therefore the present invention can be widely used in corn short mosaic disease cause of disease and identifies, and seed pollen is carried out on a large scale, fast, sensitivity, detection specifically, and can be the screening of resistant variety and evaluation and disease control strong evidence is provided.
The present invention has obtained at PVX, PVY monoclonal antibody, and sets up its ACP-ELISA and TAS-ELISA detection method, for good material and technical foundation have been laid in the foundation of the virus-elimination seedlings potato seed of the detection of PVX, PVY and potato.
The present invention utilizes monoclonal antibody technique, obtained monoclonal antibody at the tomato in China tomato yellow leaf curl China virus, and the ACP-ELISA and the TAS-ELISA detection method of rapid sensitive have been set up, this system has very high detection sensitivity, can be used for the detection of tomato yellow leaf curl virus, use this antibody we found on the tomato in Yunnan and tobacco should virus, take place popular and then control virus lays the foundation for further understanding this virus.
One. the preparation and the purifying of the preparation purifying of clonal antibody and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR () .BBWV MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.BBWV ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-BBWV 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-HCL (pH8.0,0.15M NaCl, 1MM MgCl2) in the reactant to 1ml, flow in 4 C 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.(2) preparation and the purifying of .CMV MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.CMV ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-CMV 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-HCL (pH8.0,0.15M NaCl, 1MM MgCl2) in the reactant to 1ml, flow in 4 C 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.(3) preparation and the purifying of .ToMV MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1ToMV ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-ToMV 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-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.(4) preparation and the purifying of .TuMV MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.TuMV ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-TuMV 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-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.(5) preparation and the purifying of .SCMV MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.SCMV ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-SCMV 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-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.(6) preparation and the purifying of .PVX MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.PVX ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-PVX 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-HCL (pH8.0,0.15M NaCl, 1MM MgCl2) in the reactant to 1ml, flow in 4 C 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.(7) preparation and the purifying of .PVY MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.PVY ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm 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 3000rpm, 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.2.AP-the preparation of anti-PVY 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-HCL (pH8.0,0.15M NaCl, 1MM MgCl2) in the reactant to 1ml, flow in 4 C 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.(8) preparation and the purifying of .TYLCMV MONOCLONAL ANTIBODIES SPECIFIC FOR purifying and AP-MONOCLONAL ANTIBODIES SPECIFIC FOR 1.TYLCMV ascites antibody
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5 * 10 in 7-10 days
5-10 * 10
5Individual hybridoma, the 7-10 days visible mouse web portions in injection back obviously expand, and take ascites, and the centrifugal 3min of 2000rpm collects supernatant liquor and is monoclonal antibody.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 3000rpm, 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.2.AP-the preparation of anti-TYLCMV 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 30mg AP and 1.0mg purifying, and mild stirring is 2 hours under room temperature.Add 10MM Tris-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.Two. IgG assay in the analysis on immunological characterization of monoclonal antibody () BBWV monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 9.56mg to No. 01 monoclonal antibody in every ml ascites, No. 02 is 2.69mg, No. 03 is 5.62mg, and No. 04 is 3.96mg (seeing Table 1).2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that 4 strain monoclonal antibodies are IgG
1(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.4 strain odd contradictive hydroperitoneums are tired and are 1 as a result; 640,000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed
3ug/ml antigen (purification BBWV) 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, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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,＞50% is not exclusively relevant,＜50% is uncorrelated,＜25% for uncorrelated fully, the result shows two incomplete antigen binding sites of the anti-BBWV2 monoclonal antibody identification of 2 strains, two incoherent antigen binding sites 5. of the anti-BBWV1 monoclonal antibody identification of 2 strains and different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with TMV, the ToMV, CMV, CTLV, RiMV, PVY, BBWV1, PVX, the TuMV that purify, with immunizing antigen BBWV1 or the positive contrast of BBWV2, make negative control with corresponding strong leaf extract, found that two strain monoclonal antibodies all only have specific reaction to BBWV2, other BBWV1, virus and negative control all there is not specific reaction, and the anti-BBWV1 monoclonal antibody of 2 strains only has specific reaction to BBWV1, and to BBWV2 and other virus reactionless (seeing Table 2).6. to the detection of the different isolates of BBWV
Get broad bean, capsicum, celery, soybean, the pea isolate of BBWV2, use above-mentioned indirect elisa method, measure the specific reaction of 2 strain BBWV2 monoclonal antibodies respectively.Found that 2 strain monoclonal antibodies all have specific reaction to different isolates such as the broad bean of BBWV2, capsicum, celery, soybean, peas, illustrate that this 2 strain monoclonal antibody has good broad spectrum to BBWV2.Get broad bean, capsicum, celery, soybean, the pea isolate of BBWV1, use above-mentioned indirect elisa method, measure the specific reaction of 2 strain BBWV1 monoclonal antibodies respectively.Found that, illustrate that 2 strain monoclonal antibodies all have specific reaction to different isolates such as the broad bean of BBWV1, capsicum, celery, soybean, peas, have good broad spectrum to BBWV1.7.BBWV2 the Western-blot of monoclonal antibody analyzes
The BBWV2 purified virus is carried out the SDS-PAGE electrophoresis, two bands of the bright dyeing visible big or small protein protomer in back of filial piety Maas, molecular weight is respectively 44.7KD and 21.9KD, another piece glue carries out electrotransfer, albumen is transferred on the nitrocellulose filter, and nitrocellulose filter carries out Westem-blot with two monoclonal antibodies respectively and analyzes.Analytical results shows that 2 strain monoclonal antibodies all are the specific antibodies at the big subunit of BBWV2 coat protein.(2) IgG assay in CMV monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 11.0mg to No. 05 monoclonal antibody in every ml ascites, is 3.21mg (seeing Table 1) No. 06.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that 2 strain monoclonal antibodies are respectively IgG
1And IgG
2b(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.2 strain odd contradictive hydroperitoneums are tired and are respectively 1: 1280 as a result, and 000 and 1: 640,000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed
3ug/ml antigen (purification CMV) 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, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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 two incomplete antigen binding sites of the anti-CMV monoclonal antibody identification of 2 strains.5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with TMV, the ToMV, CMV, CTLV, RiMV, PVY, BBWV1, PVX, the TuMV that purify, with the positive contrast of immune antigens c MV, make negative control with corresponding strong leaf extract, found that two strain monoclonal antibodies all only have specific reaction to CMV, other virus and negative control are not all had specific reaction (seeing Table 2).(3) IgG assay in ToMV monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 10.0mg to No. 07 monoclonal antibody in every ml ascites, No. 08 is 13.21mg, it is 3.20mg that No. 09 monoclonal antibody contains IgG, and it is 5.20mg (seeing Table 1) that No. 10 monoclonal antibodies contain IgG.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that 07,08,09, three strain monoclonal antibody is IgG
1, No. 10 strain monoclonal antibody IgGs
2a(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.4 strain odd contradictive hydroperitoneums are tired and are respectively 1: 2560000 as a result, and 1: 1280000,1: 800000,1: 1280000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed
3ug/ml antigen (purification ToMV) 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, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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,＞50% is not exclusively relevant,＜50% is uncorrelated,＜25% for uncorrelated fully, the result shows 2 incomplete related antigen binding sites of the anti-ToMV monoclonal antibody identification of 2 strains, the anti-ToMV of 2 strains and 2 incomplete related antigen binding sites of TMV monoclonal antibody identification, 2 complete uncorrelated antigen binding sites of the anti-ToMV monoclonal antibody identification of 07 and 09 2 strains, 2 complete uncorrelated antigen binding sites of 07 and 10 2 strain monoclonal antibodies identification, 2 complete uncorrelated antigen binding sites of 08 and 09 2 strain monoclonal antibodies identification, 2 complete uncorrelated antigen binding sites of 08 and 10 2 strain monoclonal antibodies identification.5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with TMV, the ToMV, CMV, CTLV, RiMV, PVY, BBWV1, PVX, the TuMV that purify, with the positive contrast of immunizing antigen ToMV, make negative control with corresponding strong leaf extract, found that 07,08 liang of strain monoclonal antibody all only has specific reaction to ToMV, other virus and negative control all there is not specific reaction, 09,10 liang of strain monoclonal antibodies all only have specific reaction to ToMV and TMV, and other virus and negative control are not all had specific reaction (seeing Table 2).(4) IgG assay in TuMV monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 3.10mg to No. 11 monoclonal antibodies in every ml ascites, is 2.45mg (seeing Table 1) No. 12.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that two strain monoclonal antibodies are IgG
1(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.2 strain odd contradictive hydroperitoneums are tired and are respectively 1: 640000 as a result, 1: 1280000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed
3ug/ml antigen (purification TuMV) 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, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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 2 incomplete related antigen binding sites of the anti-TuMV monoclonal antibody identification of 2 strains.5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with TMV, the ToMV, CMV, CTLV, RiMV, PVY, BBWV1, PVX, the TuMV that purify, with the positive contrast of immunizing antigen TuMV, make negative control with corresponding strong leaf extract, found that two strain monoclonal antibodies all only have specific reaction to TuMV, other virus and negative control are not all had specific reaction (seeing Table 2).(5) IgG assay in SCMV monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 2.10mg to No. 13 monoclonal antibodies in every ml ascites, is 6.78mg (seeing Table 1) No. 14.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that two strain monoclonal antibodies are IgG
1And IgG
2b(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.2 strain odd contradictive hydroperitoneums are tired and are respectively 1: 1280000 as a result, 1: 640000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed
3ug/ml antigen (purification SCMV) 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, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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 2 incomplete related antigen binding sites of the anti-SCMV monoclonal antibody identification of 2 strains.5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with TMV, the ToMV, CMV, CTLV, RiMV, PVY, BBWV1, PVX, the TuMV that purify, with the positive contrast of immunizing antigen SCMV, make negative control with corresponding strong leaf extract, found that two strain monoclonal antibodies all only have specific reaction to SCMV, other virus and negative control are not all had specific reaction (seeing Table 2).(6) IgG assay in PVX monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 13.0mg to No. 15 monoclonal antibodies in every ml ascites, is 9.21mg (seeing Table 1) No. 16.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that 2 strain monoclonal antibodies are IgG
1(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.2 strain odd contradictive hydroperitoneums are tired and are respectively 1: 320 as a result, and 000 and 1: 1280,000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed 3ug/ml antigen (purification PVX) 100ul/ hole in 96 hole elisa plates, and 4 ℃ of bags are spent the night; With PBST washing three times, 37 ℃ in 2%BSA 150ul/ hole, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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 2 incomplete related antigen binding sites of the anti-SCMV monoclonal antibody identification of 2 strains.5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with SCMV, the TMV, ToMV, CMV, CTLV, RiMV, PVY, BBWV1, the TuMV that purify, with the positive contrast of immunizing antigen PVX, make negative control with corresponding strong leaf extract, found that two strain monoclonal antibodies all only have specific reaction to PVX, other virus and negative control are not all had specific reaction (seeing Table 2).(7) IgG assay in PVY monoclonal antibody 1. ascites
After the monoclonal antibody of purifying is suitably diluted, measure the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and measure IgG content with the Lowry method, to contain IgG be 14.1.0mg to No. 17 monoclonal antibodies in every ml ascites, is 6.21mg (seeing Table 1) No. 18.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that 2 strain monoclonal antibodies are IgG
1(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.2 strain odd contradictive hydroperitoneums are tired and are respectively 1: 1280 as a result, and 000 and 1: 640,000 (seeing Table 1).4. the monoclonal antibody recognition site is analyzed 3ug/ml antigen (purification PVY) 100ul/ hole in 96 hole elisa plates, and 4 ℃ of bags are spent the night; With PBST washing three times, 37 ℃ in 2%BSA 150ul/ hole, add the monoclonal antibody 50ul of odd contradictive hydroperitoneum 50ul and AP mark, 37 ℃, sealed 1 hour; The monoclonal antibody 50ul that adds 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, OD
405Measure 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 2 incomplete related antigen binding sites of the anti-SCMV monoclonal antibody identification of 2 strains.5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with SCMV, the TMV, ToMV, CMV, CTLV, RiMV, PVY, BBWV1, the TuMV that purify, with the positive contrast of immunizing antigen PVY, make negative control with corresponding strong leaf extract, found that two strain monoclonal antibodies all only have specific reaction to PVY, other virus and negative control are not all had specific reaction (seeing Table 2).(8) IgG assay in TYLCMV monoclonal antibody 1. ascites
The monoclonal antibody of purifying is suitably after the dilution, measures the ultraviolet absorption value at its 260nm and 280nm wavelength place respectively with ultraviolet spectrophotometer, and with Lowry method mensuration IgG content, and to contain IgG be 21.10mg (seeing Table 1) to No. 19 monoclonal antibodies in every ml ascites.2. antibody type and subgroup identification
Adopt the sheep anti-mouse igg of U.S. Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgA, IgM standard antiserum(antisera) is done the ascites antibody of purifying to measure with agar double immunodiffusion method after the suitably dilution, observes precipitation line after 24 hours, found that 1 strain monoclonal antibody is IgG
1(seeing Table 1).3. ascites titration
Indirect ELISA method is adopted in the ascites titration, and pure virus antigen bag is 1ug/ml by concentration, and ascites is made doubling dilution.2 strain odd contradictive hydroperitoneums are tired and are respectively 1: 640 as a result, 000 (seeing Table 1).5. with different plant virus specific assay
Adopt indirect ELISA method, wrap by the ELISA Sptting plate with SCMV, the TMV, ToMV, CMV, CTLV, RiMV, PVY, BBWV1, the TuMV that purify, with the positive contrast of immunizing antigen TYLCMV, make negative control with corresponding strong leaf extract, found that monoclonal antibody has specific reaction to TYLCMV, other virus and negative control are not all had specific reaction (seeing Table 2).Three. the TAS-ELISA method of setting up with the anti-BBWV monoclonal antibody 1. usefulness monoclonal antibodies of method for detecting virus () of monoclonal antibody foundation detects operating process (1) 5ug/ml BBWV rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST adds 5% skim-milk 150ul/ hole after washing three times, adds the purified virus that dilute with confining liquid in 37 ℃ of sealing 30min (3), or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of BBWV, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of BBWV and different plant viruses.
Get broad bean, capsicum, celery, soybean, the pea isolate of BBWV2, and TMV, ToMV, CMV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that, 2 strain BBWV2 monoclonal antibodies all have specific reaction to different isolates such as the broad bean of BBWV2, capsicum, celery, soybean, peas, other viruses such as TMV, ToMV, CTLV, RiMV, CMV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to BBWV2.Get broad bean, capsicum, celery, soybean, the pea isolate of BBWV1, and TMV, ToMV, CMV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that, 2 strain BBWV1 monoclonal antibodies all have specific reaction to different isolates such as the broad bean of BBWV1, capsicum, celery, soybean, peas, other viruses such as TMV, ToMV, CTLV, RiMV, CMV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to BBWV1.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of BBWV is made doubling dilution from 1: 5 to 5 120, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-640 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 640; Detection sensitivity concentration to purified virus can reach 30.2ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 3ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.4 the TAS-ELISA method is to the detection application example of field sample
With the TAS-ELISA method of setting up the sick leaf samples of crop such as the broad bean in field, capsicum, celery, soybean, pea are detected.Measurement result shows: BBWV2 takes place very common in broad bean, pea, capsicum crop, and fragmentary generation is arranged in radish, eggplant, beans, soybean, Kidney bean.2. set up indirect antigen coated ELISA (ACP-ELISA) method with monoclonal antibody and detect the sick leaf sap 100ul/ hole adding elisa plate of the operation steps (1) of virus applications 2.1 ACP-ELISA methods with 30 times of dilutions of carbonate buffer solution (pH9.6), the positive contrast of the sick leaf of BBWV2, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of BBWV2 and different virus
Get broad bean, capsicum, celery, soybean, the pea isolate of BBWV2, and TMV, ToMV, CMV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that, 2 strain BBWV2 monoclonal antibodies all have specific reaction to different isolates such as the broad bean of BBWV2, capsicum, celery, soybean, peas, other viruses such as TMV, ToMV, CTLV, RiMV, CMV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to BBWV2.Get broad bean, capsicum, celery, soybean, the pea isolate of BBWV1, and TMV, ToMV, CMV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that, 2 strain BBWV1 monoclonal antibodies all have specific reaction to different isolates such as the broad bean of BBWV1, capsicum, celery, soybean, peas, other viruses such as TMV, ToMV, CTLV, RiMV, CMV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to BBWV1.2.3 ACP-ELISA method detection sensitivity detects
The odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, to making doubling dilution from 1: 5 to 5120, 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 ACP-ELISA method and detected.The result shows that the ACP-ELISA method all is positive to the sick leaf sap of 1: 5～200 times of dilutions, promptly can reach 1: 200 to the sensitivity that detects the disease leaf, the ACP-ELISA method can reach 10ng/ml to the detection sensitivity of pure virus, and the viral absolute magnitude in every hole detects and is 1ng.Performance ACP-ELISA method has the susceptibility and the reliability of height.2.4 detecting, the field of ACP-ELISA method uses
With the ACP-ELISA method of setting up the sick leaf samples of crop such as the broad bean in field, capsicum, celery, soybean, pea are detected.Measurement result shows: BBWV2 takes place very common in broad bean, pea, capsicum crop, and fragmentary generation is arranged in radish, eggplant, beans, soybean, Kidney bean.(2) the TAS-ELISA method of anti-CMV monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/ml CMV rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST washs and adds 5% skim-milk 150ul/ hole after three times in the purified virus of 37 ℃ of sealing 30min (3) adding with the confining liquid dilution, or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of CMV, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of CMV and different plant viruses.
Get CMVP1, CMVFnY, the CMVRB isolate of CMV, and TMV, ToMV, BBWV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain CMV monoclonal antibodies all have specific reaction to different isolates such as CMV, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to CMV.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of CMV 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-320 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 320; Detection sensitivity concentration to purified virus can reach 21.2ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 2.1ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.4 the TAS-ELISA method is to the detection application example of field sample
Utilize the monoclonal antibody of preparation and the TAS-ELISA system of foundation that field crops such as the geographic tobacco in suburbs, Hangzhou and Yunnan, tomato, capsicum, broad bean, cowpea, eggplant, potato, lettuce are detected.Tomato sample 7 examples, detecting to what CMV infected has 3 examples; Capsicum sample 7 examples are had 1 example by what CMV infected; Tobacco sample 26 examples (picking up from Yunnan), detecting to what CMV infected has 15 examples; Eggplant sample 4 examples have only 1 example to be infected by CMV; And on lettuce, broad bean, cowpea, do not detect CMV.The sample of tests positive is inoculated common cigarette again in the greenhouse, the result of its checking is consistent with the TAS-ELISA measurement result.This shows that prepared monoclonal anti physical efficiency well is used to detect field CMV.2.1 the operation steps of ACP-ELISA method (1) adds elisa plate with the sick leaf sap 100ul/ hole of carbonate buffer solution (pH9.6) 30 times of dilutions, the positive contrast of the sick leaf of CMV, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of CMV and different virus
Get CMVP1, CMVFnY, the CMVRB isolate of CMV, and TMV, ToMV, BBWV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned ACP-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain CMV monoclonal antibodies all have specific reaction to different isolates such as CMV, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to CMV.2.3 ACP-ELISA method detection sensitivity detects
The odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, to making doubling dilution from 1: 5 to 5120, 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 ACP-ELISA method and detected.The result shows that the ACP-ELISA method all is positive to the sick leaf sap of 1: 5～100 times of dilutions, promptly can reach 1: 100 to the sensitivity that detects the disease leaf, the ACP-ELISA method can reach 20ng/ml to the detection sensitivity of pure virus, and the viral absolute magnitude in every hole detects and is 2ng.Performance ACP-ELISA method has the susceptibility and the reliability of height.2.4 detecting, the field of ACP-ELISA method uses
Utilize the monoclonal antibody of preparation and the ACP-ELISA system of foundation that field crops such as the geographic tobacco in suburbs, Hangzhou and Yunnan, tomato, capsicum, broad bean, cowpea, eggplant, potato, lettuce are detected.Tomato sample 7 examples, detecting to what CMV infected has 3 examples; Capsicum sample 7 examples are had 1 example by what CMV infected; Tobacco sample 26 examples (picking up from Yunnan), detecting to what CMV infected has 15 examples; Eggplant sample 4 examples have only 1 example to be infected by CMV; And on lettuce, broad bean, cowpea, do not detect CMV.The sample of tests positive is inoculated common cigarette again in the greenhouse, the result of its checking is consistent with the ACP-ELISA measurement result.This shows that prepared monoclonal anti physical efficiency well is used to detect field CMV.(3) the TAS-ELISA method of anti-ToMV monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/ml ToMV rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST adds 5% skim-milk 150ul/ hole after washing three times, adds the purified virus that dilute with confining liquid in 37 ℃ of sealing 30min (3), or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of ToMV, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of ToMV and different plant viruses.
Get ToMVS 1, the ToMV-TL isolate of ToMV, and TMV, CMV, BBWV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of the anti-ToMV monoclonal antibody of 2 strains respectively.Found that 2 strain ToMV monoclonal antibodies all have specific reaction to different isolates such as ToMV, and other viruses such as TMV, CMV, CTLV, RiMV, BBWV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to ToMV.Get ToMVS1, ToMV-TL isolate and the TMV of ToMV, and CMV, BBWV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned TAS-ELISA method, measure the specific reaction of anti-ToMV of 2 strains and TMV monoclonal antibody respectively.Found that, 2 strain ToMV all have specific reaction with the TMV monoclonal antibody to different isolates such as ToMV and TMV, other viruses such as CMV, CTLV, RiMV, BBWV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to ToMV and TMV.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of ToMV is made doubling dilution from 1: 5 to 20480, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-10240 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 10240; Detection sensitivity concentration to purified virus can reach 5.2ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 0.52ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.4 the TAS-ELISA method is to the detection application example of field sample
Utilize the monoclonal antibody of preparation and the TAS-ELISA system of foundation that field crops such as the geographic tobacco in suburbs, Hangzhou and Yunnan, tomato, capsicum, broad bean, cowpea, eggplant, potato, lettuce are detected.Tomato sample 7 examples, detecting to what TMV infected has 1 example, 1 example that is infected by ToMV; Capsicum sample 7 examples are had 3 examples by what TMV infected, do not find that ToMV infects; Tobacco sample 26 examples (picking up from Yunnan), detecting to what TMV infected has 13 examples, does not also find that ToMV infects; Eggplant sample 4 examples have only 1 example to be infected by TMV; And on lettuce, broad bean, cowpea, do not detect TMV and ToMV.The sample of tests positive is inoculated common cigarette again in the greenhouse, the result of its checking is consistent with the TAS-ELISA measurement result.This shows that prepared monoclonal anti physical efficiency well is used to detect field ToMV and TMV.2.1 the operation steps of ACP-ELISA method (1) adds elisa plate with the sick leaf sap 100ul/ hole of carbonate buffer solution (pH9.6) 30 times of dilutions, the positive contrast of the sick leaf of ToMV, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of ToMV and different virus
Get ToMVS1, the ToMV-TL isolate of ToMV, and TMV, CMV, BBWV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned ACP-ELISA method, measure the specific reaction of the anti-ToMV monoclonal antibody of 2 strains respectively.Found that 2 strain ToMV monoclonal antibodies all have specific reaction to different isolates such as ToMV, and other viruses such as TMV, CMV, CTLV, RiMV, BBWV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to ToMV.Get ToMVS1, ToMV-TL isolate and the TMV of ToMV, and CMV, BBWV, CTLV, RiMV, PVX, PVY, TuMV, with above-mentioned ACP-ELISA method, measure the specific reaction of anti-ToMV of 2 strains and TMV monoclonal antibody respectively.Found that, 2 strain ToMV all have specific reaction with the TMV monoclonal antibody to different isolates such as ToMV and TMV, other viruses such as CMV, CTLV, RuMV, BBWV, TuMV, PVX, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to ToMV and TMV.2.3 ACP-ELISA method detection sensitivity detects
The sick leaf sap of ToMV is made doubling dilution from 1: 5 to 20480, 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.Measure with above-mentioned ACP-ELISA operating process.The result shows that ACP-ELISA is to 1: the sick leaf sap that 5-5120 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 5120; Detection sensitivity concentration to purified virus can reach 30.0ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 3.0ng.Show that the ACP-ELISA method of foundation has the susceptibility of height.2.4 detecting, the field of ACP-ELISA method uses
Utilize the monoclonal antibody of preparation and the ACP-ELISA system of foundation that field crops such as the geographic tobacco in suburbs, Hangzhou and Yunnan, tomato, capsicum, broad bean, cowpea, eggplant, potato, lettuce are detected.Tomato sample 7 examples, detecting to what TMV infected has 1 example, 1 example that is infected by ToMV; Capsicum sample 7 examples are had 3 examples by what TMV infected, do not find that ToMV infects; Tobacco sample 26 examples (picking up from Yunnan), detecting to what TMV infected has 13 examples, does not also find that ToMV infects; Eggplant sample 4 examples have only 1 example to be infected by TMV; And on lettuce, broad bean, cowpea, do not detect TMV and ToMV.The sample of tests positive is inoculated common cigarette again in the greenhouse, the result of its checking is consistent with the ACP-ELISA measurement result.This shows that prepared monoclonal anti physical efficiency well is used to detect field ToMV and TMV.(4) the TAS-ELISA method of anti-TuMV monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/ml TuMV rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST washs and adds 5% skim-milk 150ul/ hole after three times in the purified virus of 37 ℃ of sealing 30min (3) adding with the confining liquid dilution, or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of TuMV, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of TuMV and different plant viruses.
Get hot pickled mustard tube, leaf mustard, Wenzhou assorted cold dishes, potherb mustard, the green vegetables isolate of TuMV, and TMV, ToMV, BBWV, CTLV, RiMV, PVX, PVY, CMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain TuMV monoclonal antibodies all have specific reaction to different isolates such as TuMV, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, PVX, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to TuMV.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of TuMV is made doubling dilution from 1: 5 to 5 120, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-640 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 640; Detection sensitivity concentration to purified virus can reach 20.2ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 2.0ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.4 the TAS-ELISA method is to the detection application example of field sample
Utilize the monoclonal antibody of preparation and the TAS-ELISA system of foundation that field crops such as the geographic hot pickled mustard tube in Zhejiang and Yunnan, leaf mustard, Wenzhou assorted cold dishes, potherb mustard, green vegetables, tobacco, tomato, capsicum, broad bean, cowpea, eggplant, potato, lettuce are detected.Measurement result shows: TuMV takes place very common in crops such as hot pickled mustard tube, leaf mustard, Wenzhou assorted cold dishes, potherb mustard, green vegetables.2.1 the operation steps of ACP-ELISA method (1) adds elisa plate with the sick leaf sap 100ul/ hole of carbonate buffer solution (pH9.6) 30 times of dilutions, the positive contrast of the sick leaf of TuMV, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of TuMV and different virus
Get hot pickled mustard tube, leaf mustard, Wenzhou assorted cold dishes, potherb mustard, the green vegetables isolate of TuMV, and TMV, ToMV, BBWV, CTLV, RiMV, PVX, PVY, CMV, with above-mentioned ACP-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain TuMV monoclonal antibodies all have specific reaction to different isolates such as TuMV, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, PVX, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to TuMV.2.3 ACP-ELISA method detection sensitivity detects
The odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, to making doubling dilution from 1: 5 to 5120, 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 ACP-ELISA method and detected.The result shows that the ACP-ELISA method all is positive to the sick leaf sap of 1: 5～200 times of dilutions, promptly can reach 1: 200 to the sensitivity that detects the disease leaf, the ACP-ELISA method can reach 23.2ng/ml to the detection sensitivity of pure virus, and the viral absolute magnitude in every hole detects and is 2.3ng.Performance ACP-ELISA method has the susceptibility and the reliability of height.2.4 detecting, the field of ACP-ELISA method uses
Utilize the monoclonal antibody of preparation and the TAS-ELISA system of foundation that field crops such as the geographic hot pickled mustard tube in Zhejiang and Yunnan, leaf mustard, Wenzhou assorted cold dishes, potherb mustard, green vegetables, tobacco, tomato, capsicum, broad bean, cowpea, eggplant, potato, lettuce are detected.Measurement result shows: TuMV takes place very common in crops such as hot pickled mustard tube, leaf mustard, Wenzhou assorted cold dishes, potherb mustard, green vegetables.(5) the TAS-ELISA method of anti-SCMV monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/mlSCMV rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST adds 5% skim-milk 150ul/ hole after washing three times, adds the purified virus that dilute with confining liquid in 37 ℃ of sealing 30min (3), or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of SCMV, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of SCMV and different plant viruses.
Utilize two strain SCMV monoclonal antibodies to TMV, ToMV, BBWV, CTLV, RiMV, PVX, PVY, CMV, maize dwarf mosaic virus (MDMV), jowar mosaic virus (SrMV), Johnson grass mosaic virus (JGMV) and China Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi, Gansu, Sichuan, the TAS-ELISA that the 176 strain maize dwarf mosaic viruses in Yunnan 12 provinces and cities 15 places carry out, the result shows that these sick samples all contain SCMV, and there is not MDMV, SrMV or JGMV exist, and the maize dwarf mosaic virus that shows above-mentioned 12 provinces and cities is SCMV.TMV, ToMV, BBWV, CTLV, RiMV, PVX, other viruses of PVY, CMV all there is not specific reaction.1.5 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of SCMV is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-10240 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 10240; Detection sensitivity concentration to purified virus can reach 7.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 0.7ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.6 the TAS-ELISA method is to the detection application example of field sample
The TAS-ELISA that utilizes TAS-ELISA that the 176 strain maize dwarf mosaic viruses in maize dwarf mosaic virus (MDMV), jowar mosaic virus (SrMV), Johnson grass mosaic virus (JGMV) and China Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi, Gansu, Sichuan, Yunnan 12 provinces and cities 15 places are carried out, the result shows that these sick samples all contain SCMV, and there is not MDMV, SrMV or JGMV exist, and the maize dwarf mosaic virus that shows above-mentioned 12 provinces and cities is SCMV.2.1 the operation steps of ACP-ELISA method (1) adds elisa plate with the sick leaf sap 100ul/ hole of carbonate buffer solution (pH9.6) 30 times of dilutions, the positive contrast of the sick leaf of SCMV, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of SCMV and different virus
Utilize two strain SCMV monoclonal antibodies to TMV, ToMV, BBWV, CTLV, RiMV, PVX, PVY, CMV, maize dwarf mosaic virus (MDMV), jowar mosaic virus (SrMV), Johnson grass mosaic virus (JGMV) and China Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi, Gansu, Sichuan, the ACP-ELISA that the 176 strain maize dwarf mosaic viruses in Yunnan 12 provinces and cities 15 places carry out, the result shows that these sick samples all contain SCMV, and there is not MDMV, SrMV or JGMV exist, and the maize dwarf mosaic virus that shows above-mentioned 12 provinces and cities is SCMV.TMV, ToMV, BBWV, CTLV, RiMV, PVX, other viruses of PVY, CMV all there is not specific reaction.2.3 ACP-ELISA method detection sensitivity detects
The sick leaf sap of SCMV is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned ACP-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-5120 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 5120; Detection sensitivity concentration to purified virus can reach 12.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 1.2ng.Show that the ACP-ELISA method of foundation has the susceptibility of height.2.4 detecting, the field of ACP-ELISA method uses
The TAS-ELISA that utilizes TAS-ELISA that the 176 strain maize dwarf mosaic viruses in maize dwarf mosaic virus (MDMV), jowar mosaic virus (SrMV), Johnson grass mosaic virus (JGMV) and China Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi, Gansu, Sichuan, Yunnan 12 provinces and cities 15 places are carried out, the result shows that these sick samples all contain SCMV, and there is not MDMV, SrMV or JGMV exist, and the maize dwarf mosaic virus that shows above-mentioned 12 provinces and cities is SCMV.(6) the TAS-ELISA method of anti-PVX monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/ml PVX rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST washs and adds 5% skim-milk 150ul/ hole after three times in the purified virus of 37 ℃ of sealing 30min (3) adding with the confining liquid dilution, or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of PVX, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of PVX and different plant viruses.
Get the isolate of potato Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the tobacco of PVX, and TMV, ToMV, BBWV, CTLV, RiMV, TuMV, PVY, CMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain PVX monoclonal antibodies all have specific reaction to different isolates such as PVX, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, TuMV, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to PVX.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of PVX is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-320 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 320; Detection sensitivity concentration to purified virus can reach 22.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 2.2ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.7 the TAS-ELISA method is to the detection application example of field sample
Get the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the sick sample of Yunnan tobacco, utilize above-mentioned TAS-ELISA method to measure, found that PVX takes place very common in the crops such as the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and Yunnan tobacco.2.1 the operation steps of ACP-ELISA method (1) adds elisa plate with the sick leaf sap 100ul/ hole of carbonate buffer solution (pH9.6) 30 times of dilutions, the positive contrast of the sick leaf of PVX, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of PVX and different virus
Get the isolate of potato Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the tobacco of PVX, and TMV, ToMV, BBWV, CTLV, RiMV, TuMV, PVY, CMV, with above-mentioned ACP-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain PVX monoclonal antibodies all have specific reaction to different isolates such as PVX, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, TuMV, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to PVX.2.3 ACP-ELISA method detection sensitivity detects
The sick leaf sap of PVX is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned ACP-ELISA operating process.The result shows that ACP-ELISA is to 1: the sick leaf sap that 5-640 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 640; Detection sensitivity concentration to purified virus can reach 8.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 0.8ng.Show that the ACP-ELISA method of foundation has the susceptibility of height.2.4 detecting, the field of ACP-ELISA method uses
Get the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the sick sample of Yunnan tobacco, utilize above-mentioned ACP-ELISA method to measure, found that PVX takes place very common in the crops such as the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and Yunnan tobacco.(7) the TAS-ELISA method of anti-PVY monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/ml PVY rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST washs and adds 5% skim-milk 150ul/ hole after three times in the purified virus of 37 ℃ of sealing 30min (3) adding with the confining liquid dilution, or 1: 30 (W/V) times sick leaf extract 100ul/ hole of diluting.With the positive contrast of the pure virus of PVY, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, add nitro phosphoric acid salt substrate in color development at room temperature 30min after 37 ℃ of 1h (6) PBST washing, it is positive or survey the OD value of 405nm with 550 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.1.2 TAS-ELISA is to the specific detection reaction of different separation of PVY and different plant viruses.
Get the isolate of potato Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the tobacco of PVY, and TMV, ToMV, BBWV, CTLV, RiMV, TuMV, PVY, CMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain PVX monoclonal antibodies all have specific reaction to different isolates such as PVY, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, TuMV, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to PVY.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of PVY is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-320 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 320; Detection sensitivity concentration to purified virus can reach 10.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 1.0ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.8 the TAS-ELISA method is to the detection application example of field sample
Get the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the sick sample of Yunnan tobacco, utilize above-mentioned TAS-ELISA method to measure, found that PVY takes place very common in the crops such as the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and Yunnan tobacco.2.1 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 PVY, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of PVY and different virus
Get the isolate of potato Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the tobacco of PVY, and TMV, ToMV, BBWV, CTLV, RiMV, TuMV, PVY, CMV, with above-mentioned ACP-ELISA method, measure the specific reaction of 2 strain monoclonal antibodies respectively.Found that 2 strain PVY monoclonal antibodies all have specific reaction to different isolates such as PVY, and other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, TuMV, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to PVY.2.3 ACP-ELISA method detection sensitivity detects
The sick leaf sap of PVY is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned ACP-ELISA operating process.The result shows that ACP-ELISA is to 1: the sick leaf sap that 5-160 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 160; Detection sensitivity concentration to purified virus can reach 21.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 2.1ng.Show that the ACP-ELISA method of foundation has the susceptibility of height.2.4 detecting, the field of ACP-ELISA method uses
Get the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and the sick sample of Yunnan tobacco, utilize above-mentioned ACP-ELISA method to measure, found that PVY takes place very common in the crops such as the potato in Zhejiang, Jiangsu, Shanghai, Shandong, Henan, Hebei, Beijing, Shanxi, Shaanxi and Yunnan tobacco.(8) the TAS-ELISA method of anti-TYLCMV monoclonal antibody 1. usefulness monoclonal antibodies foundation detects operating process (1) 5ug/ml TYLCMV rabbit anti-serum IgG (this institute makes by oneself) the 100ul/ hole bag of viral application 1.1 TAS-ELISA by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night; (2) PBST adds 5% skim-milk 150ul/ hole after washing three times, in 37 ℃ of sealing 30min; (3) add the purified virus that dilutes with confining liquid, or sick leaf extract 100ul/ hole of 1: 30 (W/V) times dilution.With the positive contrast of the pure virus of TYLCMV, make negative control, 37 ℃ of 1h with corresponding strong leaf extract; (4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times of dilutions of washing rear enclosed liquid, 37 ℃ of 1h; (5) PBST washing back adds 7000 times of dilution AP mark sheep anti-mouse igg binding substances (Sigma) 100ul/ holes, 37 ℃ of 1h; (6) add nitro phosphoric acid salt substrate in color development at room temperature 30min after the PBST washing, it is positive or with the OD value of 550 type enzyme-linked immunosorbent assay instruments survey 405nm that the visual inspection substrate colors becomes yellowish green hole, with P/N＞2.1 as positive judging criterion.1.2 TAS-ELISA is to the specific detection reaction of different separation of TYLCMV and different plant viruses.
Get the isolate of the tobacco and the tomato in TYLCMV Guangxi, Yunnan, and TMV, ToMV, BBWV, CTLV, RiMV, TuMV, PVY, CMV, with above-mentioned TAS-ELISA method, measure the specific reaction of 1 strain monoclonal antibody respectively.Found that, 1 strain TYLCMV monoclonal antibody all has specific reaction to different isolates such as TYLCMV, other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, TuMV, PVY are not all had specific reaction, illustrate that this TAS-ELISA has excellent specificity to TYLCMV.1.3 TAS-ELISA method detection sensitivity is measured
The sick leaf sap of TYLCMV is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned TAS-ELISA operating process.The result shows that TAS-ELISA is to 1: the sick leaf sap that 5-320 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 320; Detection sensitivity concentration to purified virus can reach 10.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 1.0ng.Show that the TAS-ELISA method of foundation has the susceptibility of height.1.9 the TAS-ELISA method is to the detection application example of field sample
Get the sick sample of the tobacco and the tomato in TYLCMV Guangxi, Yunnan, utilize above-mentioned TAS-ELISA method to measure, found that, TYLCMV takes place very common in the crops such as the tobacco in Guangxi, Yunnan and tomato.2.1 the operation steps of ACP-ELISA method (1) adds elisa plate with the sick leaf sap 100ul/ hole of carbonate buffer solution (pH9.6) 30 times of dilutions, the positive contrast of the sick leaf of TYLCMV, 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.2.2 the ACP-ELISA method is to the specific assay of different isolates of TYLCMV and different virus
Get the isolate of the tobacco and the tomato in TYLCMV Guangxi, Yunnan, and TMV, ToMV, BBWV, CTLV, RiMV, TuMV, PVY, CMV, with above-mentioned ACP-ELISA method, measure the specific reaction of 1 strain monoclonal antibody respectively.Found that, 1 strain TYLCMV monoclonal antibody all has specific reaction to different isolates such as TYLCMV, other viruses such as TMV, ToMV, CTLV, RiMV, BBWV, CMV, TuMV, PVY are not all had specific reaction, illustrate that this ACP-ELISA has excellent specificity to TYLCMV.2.3 ACP-ELISA method detection sensitivity detects
The sick leaf sap of TYLCMV is made doubling dilution from 1: 5 to 40960, 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.Measure with above-mentioned ACP-ELISA operating process.The result shows that ACP-ELISA is to 1: the sick leaf sap that 5-640 doubly dilutes all is positive, and promptly the detection sensitivity to sick leaf reaches 1: 640; Detection sensitivity concentration to purified virus can reach 9.1ng/ml, and the viral absolute magnitude that each reacting hole can be detected is about 0.9ng.Show that the ACP-ELISA method of foundation has the susceptibility of height.2.4 detecting, the field of ACP-ELISA method uses
Get the sick sample of the tobacco and the tomato in TYLCMV Guangxi, Yunnan, utilize above-mentioned TAS-ELISA method to measure, found that, TYLCMV takes place very common in the crops such as the tobacco in Guangxi, Yunnan and tomato.
The immunological characteristic of table 1. monoclonal antibody
| Monoclonal antibody | Subclass | Ascites is tired | Odd contradictive hydroperitoneum IgG content (mg/ml) |
| ?01 | ?IgG1 | ?1∶640000 | ?9.56 |
| ?02 | ?IgG1 | ?1∶640000 | ?2.69 |
| ?03 | ?IgG1 | ?1∶640000 | ?5.62 |
| ?04 | ?IgG1 | ?1∶640000 | ?3.96 |
| ?05 | ?IgG1 | ?1∶1280000 | ?11.0 |
| ?06 | ?IgG1b | ?1∶640000 | ?3.21 |
| ?07 | ?IgG1 | ?1∶2560000 | ?10.00 |
| ?08 | ?IgG1 | ?1∶1280000 | ?13.21 |
| ?09 | ?IgG1 | ?1∶800000 | ?3.20 |
| ?10 | ?IgG2a | ?1∶1280000 | ?5.20 |
| ?11 | ?IgG1 | ?1∶640000 | ?3.10 |
| ?12 | ?IgG1 | ?1∶1280000 | ?2.45 |
| ?13 | ?IgG1 | ?1∶1280000 | ?2.10 |
| ?14 | ?IgG2b | ?1∶640000 | ?6.78 |
| ?15 | ?IgG1 | ?1∶320000 | ?13.00 |
| ?16 | ?IgG1 | ?1∶1280000 | ?9.21 |
| ?17 | ?IgG1 | ?1∶1280000 | ?14.10 |
| ?18 | ?IgG1 | ?1∶640000 | ?6.21 |
| ?19 | ?IgG1 | ?1∶640000 | ?21.10 |
The specific detection of table 2 monoclonal antibody
Annotate :+expression specific reaction, the no specific reaction of-expression
Claims (10)
1.4 the monoclonal antibody of the anti-BBWV of strain is characterized in that, BBWV2 BIZU01, and 02 couple of BBWV2 has specific reaction, and does not have specific reaction with other plant virus; BBWV1 BIZU03,04 couple of BBWV1 has specific reaction, and does not have specific reaction with other plant virus.
2.2 the monoclonal antibody of the anti-CMV of strain is characterized in that, CMV BIZU05, and 06 couple of CMV has specific reaction, and does not have specific reaction with other plant virus.
3.4 the monoclonal antibody of the anti-ToMV of strain, it is characterized in that, ToMV BIZU07,08 monoclonal antibody has specific reaction to ToMV, and do not have specific reaction with other plant virus, ToMV BIZU09,10 monoclonal antibodies have specific reaction to ToMV and TMV, and do not have specific reaction with other plant virus.
4.2 the monoclonal antibody of the anti-TuMV of strain is characterized in that, TuMV BIZU11, and 12 couples of TuMV have specific reaction, and do not have specific reaction with other plant virus.
5.2 the monoclonal antibody of the anti-SCMV of strain is characterized in that, SCMV BIZU13, and 14 couples of SCMV have specific reaction, and do not have specific reaction with other plant virus.
6.2 the monoclonal antibody of the anti-PVX of strain is characterized in that, PVX BIZU15, and 16 pairs of PVX specific reactions, and do not have specific reaction with other plant virus.
7.2 the monoclonal antibody of the anti-PVY of strain is characterized in that, PVY BIZU17, and 18 couples of PVY have specific reaction, and do not have specific reaction with other plant virus.
8.1 the monoclonal antibody of the anti-TYLCMV of strain is characterized in that, TYLCMV BIZU19 has specific reaction to TYLCMV, and does not have specific reaction with other plant virus.
9. detect tri-antibody sandwich ELISA (TAS-ELISA) method of eight kind of plant viruses, it is characterized in that its step is:
1) this institute of 5-50ug/ml make BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or TYLCMV by oneself rabbit anti-serum IgG 100ul/ hole bag by polystyrene board, 37 ℃, 2h or 4 ℃ spend the night;
2) PBST adds 3%-6% skim-milk 120-150ul/ hole after washing three times, 37 ℃ of sealing 30-60min;
3) add the purified virus that dilutes with confining liquid, or 1: the sick leaf extract 100ul/ hole that 10-100 doubly dilutes, with BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or the positive contrast of the pure virus of TYLCMV, make negative control, 37 ℃ of 1-2h with corresponding strong leaf extract;
4) BBWV that doubly dilutes of washing rear enclosed liquid 5000-10000 or the odd contradictive hydroperitoneum 100ul/ hole of CMV or ToMV or TuMV or SCMV or PVX or PVY or TYLCMV virus, 37 ℃ of 1-2h;
5) the AP mark sheep anti-mouse igg binding substances 100ul/ hole of the Sigma company that adding 5000-10000 doubly dilutes after the PBST washing, 37 ℃ of 1-2h;
6) add nitro phosphoric acid salt substrate in room temperature or 37 ℃ of 10-35min after the PBST washing, the visual inspection substrate colors, it is positive or with the OD value of 550 type enzyme-linked immunosorbent assay instruments survey 405nm to become yellowish green hole, with P/N＞2.1 as positive judging criterion.
10. detect the indirect antigen ELISA method of eight kind of plant viruses, it is characterized in that its step is:
1) the sick leaf sap 100ul/ hole of doubly diluting with carbonate buffer solution 10-50 adds elisa plate, with BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or the positive contrast of the sick leaf of TYLCMV, and the negative contrast of corresponding strong leaf, 37 ℃ of 2h, or 4 ℃ spent the night;
2) PBST washing back is sealed 30-60min with 3%-6% skim-milk 120-150ul/ hole in 37 ℃;
3) the odd contradictive hydroperitoneum 5000-10000 of BBWV or CMV or ToMV or TuMV or SCMV or PVX or PVY or TYLCMV virus doubly dilutes 100ul/ hole, back, 37 ℃ of 1-2h;
4) the sheep anti-mouse igg binding substances of the AP mark of the Sigma company that adding 5000-10000 doubly dilutes after the PBST washing, 100ul/ hole, 37 ℃, 1-2h;
5) add nitro phosphoric acid salt substrate 100ul/ hole, room temperature or 37 ℃ of 30-60min 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.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02159875 CN1424326A (en) | 2002-12-24 | 2002-12-24 | Monocloned antibody for eight plant viruses and inspection thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 02159875 CN1424326A (en) | 2002-12-24 | 2002-12-24 | Monocloned antibody for eight plant viruses and inspection thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1424326A true CN1424326A (en) | 2003-06-18 |
Family
ID=4753350
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 02159875 Pending CN1424326A (en) | 2002-12-24 | 2002-12-24 | Monocloned antibody for eight plant viruses and inspection thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1424326A (en) |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303424C (en) * | 2004-04-15 | 2007-03-07 | 云南省农业科学院 | CMV Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN1303422C (en) * | 2004-04-15 | 2007-03-07 | 云南省农业科学院 | PVX Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN1303423C (en) * | 2004-04-15 | 2007-03-07 | 云南省农业科学院 | PVY Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN102426234A (en) * | 2012-01-04 | 2012-04-25 | 潍坊科技学院 | Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV) |
| CN102702107A (en) * | 2012-06-27 | 2012-10-03 | 中国农业大学 | Amino acid derivative containing dinitro-trifluoromethylbenzene structure and preparation method and application of amino acid derivative |
| CN102928598A (en) * | 2012-10-30 | 2013-02-13 | 浙江大学 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN103540681A (en) * | 2013-07-02 | 2014-01-29 | 西南大学 | Method for detecting five plant viruses synchronously |
| CN105177187A (en) * | 2015-10-14 | 2015-12-23 | 浙江大学 | Preparing method and application of sample for detecting cucumber green mottle mosaic viruses carried by cucurbitaceae seeds |
| 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 |
| CN106497887A (en) * | 2016-09-23 | 2017-03-15 | 华南农业大学 | A kind of TMV substances virus colloidal gold Rapid detection test strip |
-
2002
- 2002-12-24 CN CN 02159875 patent/CN1424326A/en active Pending
Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303422C (en) * | 2004-04-15 | 2007-03-07 | 云南省农业科学院 | PVX Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN1303423C (en) * | 2004-04-15 | 2007-03-07 | 云南省农业科学院 | PVY Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN1303424C (en) * | 2004-04-15 | 2007-03-07 | 云南省农业科学院 | CMV Yunnan isolate TAS-ELISA test kit and its preparing method |
| CN102426234B (en) * | 2012-01-04 | 2014-06-25 | 潍坊科技学院 | Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV) |
| CN102426234A (en) * | 2012-01-04 | 2012-04-25 | 潍坊科技学院 | Method for rapid and accurate detection of tomato yellow leaf curl virus (TYLCV) |
| CN102702107A (en) * | 2012-06-27 | 2012-10-03 | 中国农业大学 | Amino acid derivative containing dinitro-trifluoromethylbenzene structure and preparation method and application of amino acid derivative |
| CN102702107B (en) * | 2012-06-27 | 2014-09-10 | 中国农业大学 | Amino acid derivative containing dinitro-trifluoromethylbenzene structure and preparation method and application of amino acid derivative |
| CN102937652A (en) * | 2012-10-30 | 2013-02-20 | 浙江大学 | Dot enzyme-linked immunosorbent assay (ELISA) method for detecting bemisia tabaci carrying tomato yellow leaf curl virus and application of method |
| CN102928598A (en) * | 2012-10-30 | 2013-02-13 | 浙江大学 | Dot-ELISA (dot Enzyme-Linked Immunosorbent Assay) method and tissue printing ELISA method for detecting presence of tomato yellow leaf curl virus in plant as well as reagent kit and application thereof |
| CN103540681A (en) * | 2013-07-02 | 2014-01-29 | 西南大学 | Method for detecting five plant viruses synchronously |
| CN103540681B (en) * | 2013-07-02 | 2015-04-22 | 西南大学 | Method for detecting five plant viruses synchronously |
| CN105177187A (en) * | 2015-10-14 | 2015-12-23 | 浙江大学 | Preparing method and application of sample for detecting cucumber green mottle mosaic viruses carried by cucurbitaceae seeds |
| CN105177187B (en) * | 2015-10-14 | 2018-08-14 | 浙江大学 | Detect sample preparation methods and purposes that Curcurbitaceae seed carries cucumber green mottle mosaic virus |
| 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 |
| CN106497887A (en) * | 2016-09-23 | 2017-03-15 | 华南农业大学 | A kind of TMV substances virus colloidal gold Rapid detection test strip |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1424326A (en) | Monocloned antibody for eight plant viruses and inspection thereof | |
| Wu et al. | Monoclonal antibody-based serological methods for maize chlorotic mottle virus detection in China | |
| CN104327186B (en) | Anti-bifenthrin monoclonal antibody and application thereof | |
| CN107937352A (en) | For detecting the colloidal gold immuno-chromatography test paper strip of PPR virus H protein antibodies | |
| CN110361542A (en) | The double-antibody sandwich elisa antigen detection kit of pig Delta coronavirus N protein | |
| CN107312088B (en) | Porcine epidemic diarrhea virus specificity SIgA ELISA detection kit and application thereof | |
| CN109187967A (en) | A kind of detection simultaneously distinguishes O-shaped, the duplex rapid detection card of A type foot and mouth disease virus and preparation method thereof | |
| Verma et al. | Immunological detection of bean common mosaic virus in French bean (Phaseolus vulgaris L.) leaves | |
| CN102559603B (en) | Hybridoma cell strain capable of secreting tomato yellow leaf curl virus monoclonal antibody and application of monoclonal antibody | |
| CN102533664B (en) | Hybridoma cell strain excreting monoclonal antibody (MAb) resisting rice blackstreaked dwarf virus (RBSDV) and application of MAb | |
| CN105543176A (en) | Hybridoma cell strain secreting potato-virus-Y-resistant monoclonal antibodies and monoclonal antibody application thereof | |
| Dheepa et al. | Transmission of Cucumber Mosaic Virus (CMV) infecting banana by aphid and mechanical methods | |
| WO2007013089A1 (en) | Development of diagnostic kit against the recombinant coat protein of prunus necrotic ringspot virus | |
| Reddy et al. | Comparison of neutralizing antigens of recent isolates of infectious bursal disease virus | |
| CN108165533B (en) | Secrete hybridoma cell strain and its monoclonal antibody application of water resistant rice stripe mosaic viral monoclonal antibodies | |
| CN104513810B (en) | Hybridoma cell strain secreting monoclonal antibody against cucumber mosaic virus and application of monoclonal antibody | |
| CN1680444A (en) | Monoclone antibody against virus of xiangshizhubanbo and its use | |
| CN113683685B (en) | anti-I group 4 type avian adenovirus monoclonal antibody, hybridoma cell strain and application of detection kit | |
| CN105567646B (en) | Secrete anti-corn mosaic virus monoclonal antibody hybridoma cell strain and its monoclonal antibody application | |
| CN105624121B (en) | Secrete anti-tomato black ring virus monoclonal antibody hybridoma cell strain and its monoclonal antibody application | |
| CN102559602B (en) | Monoclonal antibody hybridoma cell line for secreting southern rice black-streaked dwarf resistant viruses and monoclonal antibody application thereof | |
| CN114316037A (en) | Antibody m19 of O-type foot-and-mouth disease virus structural protein, preparation method and application | |
| CN104513811B (en) | Hybridoma cell strain secreting monoclonal antibody against watermelon mosaic virus and application of monoclonal antibody | |
| CN110498853B (en) | Monoclonal antibody for resisting duck growth retardation virus, hybridoma cell strain and application of monoclonal antibody | |
| CN101215331A (en) | Method for preparing tomato A virus Zhejiang separator monoclonal antibody and use thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |