CN104845937B

CN104845937B - Secrete anti-WYMV monoclonal antibody hybridoma cell strain and its monoclonal antibody application

Info

Publication number: CN104845937B
Application number: CN201510165442.6A
Authority: CN
Inventors: 周雪平; 吴建祥; 李娜
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2015-04-09
Filing date: 2015-04-09
Publication date: 2017-12-29
Anticipated expiration: 2035-04-09
Also published as: CN104845937A

Abstract

The invention discloses one kind to secrete anti-WYMV (WYMV) monoclonal antibody hybridoma cell strain and its application of monoclonal antibody.Virion with purification WYMV is antigen immune BALB/c mouse, through cell fusion, screening, clone, obtains 1 plant of hybridoma cell strain 4E11 that can secrete anti-WYMV monoclonal antibodies, its preserving number is CGMCC No.10415.Monoclonal antibody and the WYMV capsid proteins of cell line secretion react, and the odd contradictive hydroperitoneum ELISA potency of secretion is up to 10^‑7More than, Antibody types and subclass are IgG1, kappa.The monoclonal antibody of 4E11 secretions has specific reaction with WYMV, without being reacted with viruses such as Chinese wheat mosaic virus, barley yellow mosaic virus, marmor triticis.The hybridoma cell strain and its monoclonal antibody of secretion are the diagnosis of the wheat virus disease, detection and the foundation of science bridle system provide material support.

Description

Secrete anti-WYMV monoclonal antibody hybridoma cell strain and its monoclonal antibody application

Technical field

The present invention relates to biological technical field, more particularly to a kind of anti-WYMV monoclonal antibody hybridization of secretion The application of tumor cell strain and its monoclonal antibody.

Background technology

Wheat yellow mosaic (Wheat yellow mosaic disease), it is China's wheat also known as " Wheat spindle streak mosaic " A kind of important soil-borne disease virus disease in production.Wheat yellow mosaic is by WYMV（WYMV）It is caused.China The virosis is simply fragmentary before the 1960s occurs, and causes the disease to exist due to a large amount of plantations of susceptible variety the seventies China's wheat planting district occurs, is popular, and at present, the disease is distributed mainly on China the Changjiang river, the Yellow River middle and lower reaches and Basin of Huaihe River, including Sichuan, Shaanxi, Jiangsu, Zhejiang, Anhui, Hubei, Shandong and Henan etc. save, the wherein LX-river area He Ning town Yang Qiu in Jiangsu Province Mound area, Hubei Province northeast occur heavier along the area such as Dabie Mountains Region, Shaanxi Weihe basin Guanzhong area and Shandong Province. The Huangchuan of Henan in 1991 wheat yellow mosaic onset area has no harvest 3000 hectares up to 2.33 ten thousand hectares；2005 are only Jiangsu Province's hair Sick area is up to 4.636 ten thousand hectares；About 500,000 hectares of rough estimates whole nation onset area in 2011, current national onset area reach 2000000 hectares.General sick field underproduction 10%-30%, serious reaches 70%-80%, and some serious fields of falling ill even have no harvest.At present, The virosis has turned into the another important virus disease in China's Wheat Production after BYDV.

The virus by the more slime moulds of cereal (Polymyxa graminis) zoospore carry and propagate, and can be in soil Resting spore long-term survival, its sustainable survival more than ten years and keep its infection ability according to the literature.Electron microscope Observation shows that WYMV virions are long linear, and length is divided into two kinds of 275-300 nanometers and 575-600 nanometers, a diameter of 13-14 nanometers.Wheat infects yellow mosaic virus, did not showed symptom before the general winter, symptom just occurs to spring wheat period of seedling establishment.By Environment influences, and there is some difference for the symptom of various regions wheat yellow mosaic, but cardinal symptom is consistent.Early stage, in Newborn Leaves Occur light green or orange-yellow fusiformis or elliptical spot on piece, spot expands soon, mutually merges, turns into yellowish green alternate Floral leaf, mottled or random stripe, severe patient's young leaves shrinkage, are twisted into deformity；Fall ill the later stage, old leaf from floral leaf switch to necrosis, It is yellow withered, also occur the wire streak of chlorisis on stem sometimes；Susceptible wheat tillering is reduced, and root system development is bad, and severe one is substantially downgraded, Tiller atrophy is withered, or even whole strain is dead；The susceptible wheat plant plant type of Adult plant is loose, short and small, and fringe is short and small, seed insatiable hunger It is full.The just morbidity normal appearance point sheet infection center in field, then progressively extension is in blocks.Can be by virus infection, energy after wheat cultivation It is enough to detect virus in wheat root, but do not show symptom.The month in next year 2-3 just starts to show classical symptom in field.Often Year spring, when temperature is gone up to about 10 DEG C, the disease is put into onset peak period, and 4-15 DEG C is optimum temperature that disease shows disease.When When temperature is up to more than 20 DEG C, flower leaf paresthesia fades away, and young leaves shows as hidden disease, but bottom old leaf piece still has obvious disease Shape.

Inouye in 1969 determines that wheat yellow mosaic is propagated by the more slime moulds of cereal in soil.Cereal is more glutinous Bacterium is that a kind of low obligatory parasitism for belonging to the more slime mould category of Plasmodiophoromycetes found by Ledingham in nineteen thirty-nine is planted in grass family The eucaryote of thing.The bacterium can be propagated including WYMV in itself to host plant and non-hazardous A variety of cereal virus, such as barley yellow mosaic virus (Barley yellow mosaic virus, BaYMV), soil passes wheat floral leaf Viral (Soil-born wheat mosaic virus, SbWMV), Barley mild mosaic virus (Barley mild mosaic Virus, BaMMV), Chinese wheat mosaic virus (Chinese wheat mosaicvirus, CWMV) etc..The more slime moulds of cereal The most suitable soil moisture that wheat is infected in resting spore sprouting is 15 DEG C or so, and soil moisture is advantageous to greatly resting spore and sprouts and swim Zoospore infects.The more slime mould resting spores of cereal in Wheat Seedling, sick soil and invalid are sprouted into zoospore, infect Wheat seeding root system tissue, virus are bred and extended in intrusion root therewith.Sporosorus resistance is extremely strong caused by the bacterium, can With at least 20-25 of being survived in soil.

At present, effectively preventing measure there is no to wheat yellow mosaic in production.By years of researches and it was verified that Prevention and control wheat yellow mosaic should adhere to the control strategy of " promoting based on resistance to (anti-) sick kind, strengthen agricultural cultivation integrated management ".

Present invention is generally directed to the preparation of WYMV monoclonal antibody to organize work.The currently monitored WYMV body Be it is not perfect, or even Major Epidemic area do not carry out yet correlation predictive forecast work.It is used for detecting this viral side at present The methods of method is mainly field symptom observation, RT-PCR detections, electron microscopic observation.This several method all has its limitation, and Be not suitable for large batch of field sample detection.And serological method is suitable to field sample batch detection, but it is necessarily dependent upon spy The viral monoclonal antibody of the opposite sex.At present, WYMV Serology test is by preparing antiserum, utilizes ELISA or Western Blot carries out detection and research to virus.It is prepared for WYMV P1 and CP antiserum, Ke Yitong respectively to flourish etc. and Han Chenggui etc. Cross the serology such as ELISA or Western blot means detection virus；Shang Qiaoxia etc. establishes WYMV ELISA detecting systems.But These have no what it was applied in field sample on a large scale detection with the serological method of how anti-foundation due to its Shortcomings Report.Therefore, this patent is prepared for 1 plant of hybridoma that can secrete anti-WYMV monoclonal antibodies using hybridoma technology, and with its point Special, the sensitive monoclonal antibody secreted, which establishes, detects the viral serological method and detection kit, and is successfully applied to field The detection of WYMV, so as to the early monitoring for China's wheat yellow mosaic and early warning, scientific guidance prevention and control provide Material and technical support, ensure that the stable yields of China's wheat increases income.

The content of the invention

The purpose of the present invention is overcome the deficiencies in the prior art, there is provided one kind is secreted anti-WYMV monoclonal and resisted The hybridoma cell strain of body and its monoclonal antibody application.

Anti- WYMV monoclonal antibody hybridoma 4E11 is secreted, the specificity of anti-WYMV can be secreted Monoclonal antibody, China Committee for Culture Collection of Microorganisms's common micro-organisms center, ground are preserved on March 26th, 2015 Location：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, preserving number is CGMCC No.10415；

The monoclonal antibody ascites ELISA potency of anti-WYMV is up to 10^-7, Antibody types and subclass be IgG1, Kappa, the monoclonal antibody can have specific immunity association reaction with the capsid protein of WYMV, monoclonal antibody inspection The sensitivity for surveying disease leaf reaches 1:40960 times of dilutions；

The monoclonal antibody of anti-WYMV only has specific reaction with WYMV, without with China Wheat streak mosaic virus, barley yellow mosaic virus, marmor tritici, Barley mild mosaic virus, luteovirus GAV Strain, GPV strains and PAV strains, wheat dwarf virus, Brassica 2 et 4, TOMV, tobacco mosaic virus (TMV), Huang Melon mosaic virus, potato virus X, marmor upsilon, fractilinea oryzae, rice stripe virus, rice tingia dwarf virus Reaction；

Application of the monoclonal antibody of anti-WYMV on the Viral diagnosis is using monoclonal antibody as core The various immunological detection methods and immunological detecting kit established.

The present invention has the advantage that compared with prior art：1）It is special that the hybridoma cell strain of offer secretes anti-WYMV The property immunological method such as monoclonal antibody, dot-ELISA, ACP-ELISA and the TAS-ELISA established using the monoclonal antibody as core and The kit energy high special established with these methods, it is accurate, delicately detect WYMV；2）Utilize institute of the present invention The monoclonal antibody detection WYMV of preparation, it is not necessary to the equipment such as expensive electron microscope, PCR instrument；3）It is made using the present invention Standby monoclonal antibody, detection, the diagnosis of the WYMV being effectively used in field crops.

Brief description of the drawings

Fig. 1 is the sensitivity analysis of dot-ELISA methods detection WYMV；

Fig. 2 is the result of WYMV in dot-ELISA methods detection wheat sample.

Embodiment

Hybridoma cell strain provided by the invention can largely secrete anti-WYMV monoclonal antibodies, and its high specificity, sensitivity and effect Valency is high, stability is good.Detection WYMV high-throughout serological method and detection kit is established using the monoclonal antibody as core, and Field WYMV detection is can be applied to, so as to be China's WYMV early monitoring and early warning, scientific guidance prevention and control carry For material and technical support.

With reference to embodiment and accompanying drawing, the invention will be further described.

First, hybridoma acquisition and its preparation of monoclonal antibody

1. immunogene and the preparation for detecting antigen

WYMV method of purification is made with reference to Shirako ＆ Brakke (1984) and the method for week snow equality (1994) Improve, concrete operation step is as follows：

（1）By big mortar and tissue mixer device precooling；

（2）500g wheat disease leaves are weighed, pH 7.5 0.5M phosphate buffers (i.e. PB bufferings are added in every 100g disease leaves Liquid) 200ml (Na-EDTA containing 0.01M and 0.1% mercaptoethanol), use double-layer nylon filtered through gauze after being homogenized 2min, filtrate 6, 000rpm centrifugations 20min removes plant tissue residue；

（3）Gained supernatant adds Triton X-100, PEG (molecular weight 6,000) and NaCl respectively to end while stirring Solubility is 2.5%, 4% and 0.1M, and 4 DEG C are stirred more than 4h；

（4）11,000rpm centrifugations 15min must be precipitated；

（5）Precipitation is fully suspended with pH 7.5 0.5M PB (MgCl2 containing 0.01M and 0.5M ureas) 15mL, and 6,000rpm Supernatant is suctioned out after centrifugation 15min and is placed in centrifuge tube, precipitation is suspended with 5mL 0.5M PB again, centrifugation, is repeated 2 times；

（6）Merging supernatant, 33,000rpm ultracentrifugation 100min, gained precipitation is used 8 after 10mL 0.5M PB suspensions, 000rpm centrifuges 15min, and precipitation is suspended with 5mL 0.5M PB again, centrifugation, is repeated 2 times；

（7）Merge supernatant and be added to centrifuge tube added with the centrifuge tube of 30% sucrose cushions, 33,000rpm ultracentrifugations 100min；

（8）Gained precipitation pH 7.5 0.5M PB (MgCl2 containing 0.01M）Suspend, suspension is Virus purification Liquid；

（9）Virus purification liquid is through the 2 rearmounted JEOL JEM-1200EX electricity Microscopic observation grains of % phosphoric acid tungsten (pH 6.7) negative staining Sub- form.The virion of purifying can be used to immune and monoclonal antibody the preparation of mouse.

Immune animal

Six week old body weight 18-20g BALB/c female mices, i.e. WYMV disease are immunized with the WYMV of purification virion 100 μ L of virion mix with isometric Freund's complete adjuvant, after fully emulsified, through carrying on the back subcutaneous abdomen multi-point injection 0.2ml every, Interval 3 weeks, take with one exempt from equivalent viral antigen and isometric freund 's incomplete adjuvant it is fully emulsified after, second intraperitoneal injection 0.2ml every, it is injected intraperitoneally after spending 3 weeks with the antigen of doubling dose, extracting spleen cell carries out cell fusion after 3 days.

Cell fusion

Take above-mentioned immune mouse spleen cell and murine myeloma cell（SP2/0）By 8：1 ratio:In serum-free Mixed in RPMI-1640 culture mediums, 1,500rpm centrifugation 5min, culture medium is removed, with 50 % PEG（Sigma, molecular weight 1500）As fusion agent, 1ml is added under water-bath at 37 DEG C, it is merged 2min, with the RPMI-1640 culture mediums of serum-free 1,500rpm centrifuges 5min after terminating fusion, and precipitation is suspended with HAT culture mediums, is dispensed into the cell plates that feeder cells are contained in 96 holes In after 37 DEG C of 5% CO₂Cell culture incubator in cultivate.

Hybridoma, the screening in positive hole and its clone

After being cultivated 5 days in cell culture incubator, liquid is changed once with HAT culture mediums, changes liquid, Deng Daorong with HT culture mediums within the 10th day When closing cell covering bottom hole 5%-30%, using infect WYMV viruses wheat leaf blade and purified virus to detect antigen coat, with normal Indirect ELISA method screening antibodies positive hole is advised, obtains 98 positive holes altogether.Selection 7 is in the cell hole of strong positive reaction, is carried out Limiting dilution assay is cloned, and obtains the hybridoma cell strain 4E11 of 1 plant of specific monoclonal antibody that can secrete anti-WYMV.Through more than 6 months After subculture in vitro separately and multiple cryopreservation resuscitation, cell line can well grow, and stably excreting antibody.After expanding and cultivating, it is used for Ascites prepares and Liquid nitrogen storage.

The specific detection of monoclonal antibody

With infection of Chinese wheat streak mosaic virus (CWMV), barley yellow mosaic virus (BaYMV), marmor tritici (SbWMV), Barley mild mosaic virus (BaMMV), luteovirus GAV strains (BYDV GAV), GPV strains (BYDV ) and PAV strains (BYDV PAV), wheat dwarf virus (WDV), Brassica 2 et 4 (TuMV), TOMV GPV (ToMV), tobacco mosaic virus (TMV) (TMV), cucumber mosaic virus (CMV), potato virus X (PVX), marmor upsilon (PVY), Fractilinea oryzae (RDV), rice stripe virus (RSV), the sick leaf juice coating ELISA of rice tingia dwarf virus (RRSV) Plate, make negative control to be good for leaf extract accordingly, using WYMV as positive control, the special of monoclonal antibody is determined with ACP-ELISA methods Property reaction.ACP-ELISA method specific steps：The sick leaf liquid nitrogen grinding of above-mentioned various virus infection is into powder, by 1: 30 （w/v, g /mL）Add 100 μ l/ holes coating elisa plate after the grinding of ELISA coating buffers, 4 DEG C overnight or 37 DEG C of 2h make its absorption In elisa plate hole；PBST washings close 30-60min with 3% skimmed milk power afterwards three times；Add the μ of monoclonal antibody 100 suitably diluted L/ holes, 37 DEG C of 1-2h are incubated；PBST washings add alkaline phosphatase lipase (AP) the mark rabbit-anti mouse of 10000 times of dilution afterwards three times IgG secondary antibodies（Sigma companies）100 μ L/ holes, 37 DEG C of 1-2h；After PBST is washed four times, developed the color with PNPP substrates, 2mol/L hydrogen After sodium oxide molybdena terminating reaction, OD is read with ELIASA₄₀₅Value, with negative OD values ratio be more than 2.1 for the positive.As a result find, 4E11 monoclonal antibodies have specific reaction to WYMV, and pass wheat mosaic with Chinese wheat mosaic virus, barley yellow mosaic virus, soil Poison, Barley mild mosaic virus, luteovirus GAV strains, GPV strains and PAV strains, wheat dwarf virus, turnip Mosaic virus, TOMV, tobacco mosaic virus (TMV), cucumber mosaic virus, potato virus X, marmor upsilon, rice are short Virus, rice stripe virus, rice tingia dwarf virus and health plant contract without immune response.

Monoclonal antibody ascites prepares and purifying

8 week old or so BALB/C mice is taken, 0.3-0.5mL norphytanes are injected intraperitoneally（Sigma）, pneumoretroperitoneum injection in 7-10 days 5-10×10⁵Individual hybridoma, 7-10 days visible mouse web portions substantially expand after injection, take ascites, 3,000rpm centrifugations 3min, collect supernatant, as monoclonal antibody ascites.1 times of volume ascites is taken to add 2 times of M PH4.8 acetate buffers of volume 0.06 Liquid dilutes, and adds octanoic acid（30 μ L/ml ascites）, it is stirring while adding at room temperature, 4 DEG C of clarification 1h, 12,000rpm centrifugation 20min, collect Supernatant, then with 50% saturated ammonium sulphate immunoglobulin, 4 DEG C are placed 2 hours, 3,000rpm centrifugation 20min, are precipitated with 2 times The PBS solution dissolving of volume, the ascites antibody of purifying, -70 DEG C of preservations are obtained after 4 DEG C of flowings are dialysed 24 hours.

Subgroup identification and the titer of ascites measure of monoclonal antibody

By the odd contradictive hydroperitoneum of purifying and the anti-BALB/C mice IgG of standard of Sigma companies₁、 IgG_2a、IgG_2b、IgG₃、IgM Antibody makees double agar diffusion test, is as a result IgG1 for 4E11 monoclonal antibody subclass, kappa.It is coating with WYMV purified virus Antigen, doubling dilution ascites monoclonal antibody carry out indirect ELISA method detection odd contradictive hydroperitoneum potency, and testing result shows above-mentioned monoclonal antibody abdomen Water potency is 10^{- 7}More than.

2nd, virological immunology detection method and its detection kit

1. the foundation and its detection application of dot-ELISA methods

With liquid nitrogen grinding into powder after wheat leaf blade is weighed, by 1:10-30（w/v, g /mL）Add 0.01 mol/L PBS（pH7.4）After grind；The sick rpm of juice 5,000 centrifuges 3 min;3 μ L of supernatant points are taken to nitrocellulose filter（NC）On, Healthy and susceptible wheat leaf juice is set simultaneously respectively as negative and positive control；Drying at room temperature 10-20 min;NC films immerse Into PBST (the 0.01 mol/L PBS for containing 0.05% Tween-20) confining liquid containing 5% skimmed milk power, room temperature closes 30 min； NC films, which are put into the monoclonal antibody moderately diluted, is incubated at room temperature 30-60 min；Film is washed with PBST 3-4 times, every time 3 min；NC films are put into 30-60 min are incubated at room temperature in the AP enzymes mark sheep anti-mouse igg secondary antibody moderately diluted;PBST washes film 4-5 times, every time 3 min; 66 μ L NBT and 33 μ L BCIP substrates (Promega) are added to 10 ml substrate buffer solutions (0.1 mol/L Tris Cl, 0.1 Mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mix, film, which is put into substrate solution, to react, visual results.Treat the positive Control colour developing is obvious, and tap water rinse terminating reaction during negative no any colour developing, photographs to record result.

The most suitable working concentration of the dot-ELISA monoclonal antibodies and ELIAS secondary antibody that determine detection wheat disease leaf, examination are tested with square formation Test and show that the most suitable working concentration of 4E11 monoclonal antibodies and ELIAS secondary antibody is respectively 1:5000 and 1:8000 times of dilutions.With above-mentioned antibody Most suitable working concentration establishes the dot-ELISA methods of detection WYMV disease leaves.Sensitivity analysis shows, when wheat leaf blade is diluted to 1: 2,560 times（w/v, g/mL）When, the positive spots of purple are still presented in the dot-ELISA detections established with 4E11 monoclonal antibodies, i.e., its The sensitivity for detecting disease leaf reaches 1:2,560 times of dilutions（Fig. 1）.

2014 are picked up from Shandong with the dot-ELISA methods of foundation, Jiangsu wheat sample detects, as a result send out It is existing, there are 28 samples to produce purpuriferous positive spots in 47 samples（Fig. 2）.Positive is further analyzed with RT-PCR, knot Fruit shows that all dot-ELISA positives detect the specific PCR primers of WYMV, and PCR primer nucleic acid sequencing shows Positive infects WYMV.Illustrate that the dot-ELISA methods can accurately and reliably be used for the detection of WYMV in wheat samples.

ACP-ELISA methods detection virus is established by core of monoclonal antibody

The operating procedure of ACP-ELISA methods：

（1）After sick leaf is smashed with liquid nitrogen in mortar, with addition 0.05M carbonate buffer solutions（pH9.6）Homogenate, Supernatant is taken by 1 after 5000rpm centrifugations: 30（w/v, g /mL）The μ L/ holes of sick leaf sap 100 of dilution add elisa plate again, with It is positive control to infect WYMV wheat diseases leaf, and using healthy wheat as negative control, 37 DEG C are incubated 2h, or 4 DEG C are stayed overnight；

（2）PBST wash 3 times after with 3% skimmed milk power closing 30min；

（3）100 μ L/ holes after 5,000 times of dilution of odd contradictive hydroperitoneum, 37 DEG C of incubation 1h；

（4）PBST adds the alkaline phosphatase lipase of 8,000 times of dilutions after washing 3 times（AP）The sheep anti-mouse igg secondary antibody of mark （Sigma）, 100 μ L/ holes, 37 DEG C of incubation 1h；

（5）The μ L/ holes of nitro Phosphate substrate 100, room temperature 30min are added after being washed with PBST；

（6）Detecting by an unaided eye, substrate colors become the hole of yellow green as the positive, or with after 2M sodium hydroxide terminating reactions, OD405 values are surveyed with enzyme-linked immunosorbent assay instrument, with P/N>2.1 are used as positive criterion.

ACP-ELISA methods detection sensitivity detects：

Odd contradictive hydroperitoneum working concentration is 5,000 times of dilution, to sick leaf from 1:10 to 655,360 make doubling dilution, with corresponding The strong leaf sap of dilution factor makees negative control, carries out above-mentioned ACP-ELISA methods detection.As a result ACP-ELISA methods pair are shown 1:20-40, the sick leaf sap of 960 times of dilutions are positive, i.e., can reach 1 to the sensitivity for detecting sick leaf:40,960, table Bright ACP-ELISA methods have the sensitivity and reliability of height.

Detect WYMV TAS-ELISA detection methods

The operating process of TAS-ELISA methods：

1) 1:The anti-WYMV of 5,000 times of dilutions rabbit anti-serum（Prepared by the WYMV immune rabbits purified）100 μ L/ holes XPS is coated with, 37 DEG C, 2h or 4 DEG C is incubated overnight；

2) PBST washings add 1-10% skimmed milk power or 1-3% BSA or 3-6% cow's serum to close 200 μ l/ afterwards three times 30-60min is closed in 37 DEG C in hole;

3) the μ L/ holes of detection sample juice supernatant 100 are added.Using WYMV diseases leaf as positive control, using healthy wheat as feminine gender Control, 37 DEG C of 1-2h；

4) 5,000 times of the μ L of odd contradictive hydroperitoneum 100/hole, 37 DEG C of 1-2h are diluted after washing with confining liquid;

5) the AP mark rabbit anti-mouse igg secondary antibodies of 10,000 times of dilutions are added after PBST washings（Sigma）100 μ L/ holes, 37 DEG C 1-2h;

6) PNPP substrates are added to visually observe the hole that substrate colors become yellow green in color development at room temperature 30min after PBST washings For the positive, after 2mol/L sodium hydroxide terminating reactions, 405nm OD values are surveyed with 680 type enzyme-linked immunosorbent assay instruments, with P/N> 2.1 are used as positive criterion.

The determination of TAS-ELISA detection method optimum conditions：

Tested and carried out using TAS-ELISA square formations, i.e., laterally added respectively and use coating buffer solution from 1:100 to 1:102,400 The rabbit-anti WYMV serum of doubling dilution；Add WYMV disease leaf juices；Longitudinal direction adds respectively uses confining liquid from 1:5 to 1:2,048,000 times Than diluting odd contradictive hydroperitoneum；The rabbit anti-mouse igg secondary antibody of AP marks（Sigma companies）By 1:8,000 times of dilutions；By TAS-ELISA side Method flow is operated.As a result show that WYMV rabbit anti-serum and the optimal dilution of monoclonal antibody are respectively 1:10,000、1:5, 000。

The determination of TAS-ELISA method detection sensitivities：

Under the most suitable working concentration of rabbit anti-serum and odd contradictive hydroperitoneum, after WYMV diseases leaf juice is carried out into doubling dilution with PBS liquid TAS-ELISA measure is carried out, measurement result is：The sensitivity that TAS-ELISA detects sick leaf reaches 1::81,920 times of dilutions（w/ v, g/mL）, illustrate that this method has good sensitivity.

WYMV dot-ELISA detection kits

1）Kit main component：

The mL of 1 pipe of WYMV monoclonal antibodies 0.2

The mL of 1 pipe of AP mark sheep anti-mouse igg secondary antibodies 0.1

Each 1 bottle of NBT/BCIP substrates are respectively 2 mL and 1mL

Positive control（The leaf juice of wheat containing WYMV）The mL of 1 pipe 2

Negative control（Healthy wheat leaf juice）The mL of 1 pipe 2

Antibody diluent（10X）1 bottle of 80mL

Above reagent is stored at 4 DEG C

Nitrocellulose filter (NC) 10

2）Detect the operating procedure of wheat samples：

A. with liquid nitrogen grinding into powder after wheat leaf blade is weighed, by 1:10~30（w/v, g /mL）Add 0.01 mol/L PBS（pH7.4）After grind；

B. the sick rpm of juice 5,000 centrifuges 3 min;

C. 3 μ L of supernatant points are taken to NC, while set healthy and susceptible wheat leaf juice right respectively as the negative and positive According to drying at room temperature 10-20 min;

D. NC films are immersed in PBST (the 0.01 mol/L PBS for containing 0.05% Tween-20) closing containing 5% skimmed milk power Room temperature closes 30 min in liquid；

E. NC films are put into 1:30~60 min are incubated at room temperature in the monoclonal antibody of 5000 times of dilutions；

F. film is washed 3~4 times with PBST, every time 3 min；NC films are put into 1:The AP enzymes mark sheep anti-mouse igg of 8,000 dilutions 30~60 min are incubated at room temperature in secondary antibody;

G. PBST washes film 4~5 times, every time 3 min;66 μ L NBT and 33 μ L BCIP substrates are added to 10 ml substrates and delayed Fliud flushing (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mixes, and film is put into substrate Reacted in liquid, visual results；

H. treat that positive control colour developing is obvious（Purple）, and tap water rinse terminating reaction during negative no any colour developing, clap According to record result.

3）Preservation and the term of validity

It is kept in dark place in 2~8 DEG C, the term of validity 12 months.

4）Buffer formulation：

Phosphate buffer（PBS, 0.01 mol/L, pH7.4）：

NaCl 8 g

KCl 0.2 g

KH₂PO₄ 0.2 g

Na₂HPO₄•12H₂O 3g

The g of sodium azide 0.2

Add distilled water 950mL to adjust pH to 7.4 after dissolving, be settled to 1000 mL

ELISA cleaning solutions（0.01 mol/L PBST）：

Add 0.5 mL Tween-20 in 1000 mL 0.01mol/L PBS

ELISA confining liquids：

Skimmed milk power is added in 0.01 mol/L PBST to final concentration 5%（W/V）.

Claims

1. a kind of hybridoma cell strain 4E11 for secreting anti-WYMV monoclonal antibody, it is characterised in that can secrete anti- The monoclonal antibody specific of WYMV, hybridoma cell strain 4E11 are preserved in the micro- life of China on April 2nd, 2015 Thing culture presevation administration committee common micro-organisms center, preserving number are CGMCC No.10415.

2. a kind of monoclonal of the anti-WYMV of hybridoma cell strain 4E11 secretions as claimed in claim 1 resists Body, it is characterised in that described monoclonal antibody ascites ELISA potency is up to 10^-7, Antibody types and subclass are IgG1, kappa, The monoclonal antibody can have specific immunity association reaction with the capsid protein of WYMV, and the monoclonal antibody detects sick leaf Sensitivity reaches 1:40960 times of dilutions.

3. the monoclonal antibody of the anti-WYMV of hybridoma cell strain 4E11 secretions as claimed in claim 2, its Be characterised by that described monoclonal antibody can have specific reaction with WYMV, without with Wheat in China mosaic disease Poison, barley yellow mosaic virus, marmor tritici, Barley mild mosaic virus, luteovirus GAV strains, GPV strains System and PAV strains, wheat dwarf virus, Brassica 2 et 4, TOMV, tobacco mosaic virus (TMV), cucumber mosaic virus, Potato virus X, marmor upsilon, fractilinea oryzae, rice stripe virus, the reaction of rice tingia dwarf virus.

4. a kind of application of monoclonal antibody of anti-WYMV as claimed in claim 2 on the Viral diagnosis, It is characterized in that various exempt from applied to using the anti-WYMV monoclonal antibody described in claim 2 as what core was established Epidemiology detection method and immunological detecting kit.