CN104513812B

CN104513812B - Hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of monoclonal antibody

Info

Publication number: CN104513812B
Application number: CN201410629108.7A
Authority: CN
Inventors: 吴建祥; 于翠; 周雪平; 李娜
Original assignee: TECHNICAL CENTRAL FOR ANIMALS PLANTS AND FOOD INSPECTION AND QUARANTINE SHANGHAI ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU; Zhejiang University ZJU
Current assignee: TECHNICAL CENTRAL FOR ANIMALS PLANTS AND FOOD INSPECTION AND QUARANTINE SHANGHAI ENTRY-EXIT INSPECTION AND QUARANTINE BUREAU; Zhejiang University ZJU
Priority date: 2014-11-10
Filing date: 2014-11-10
Publication date: 2017-05-24
Anticipated expiration: 2034-11-10
Also published as: CN104513812A

Abstract

The invention discloses a hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of the monoclonal antibody. Iris yellow spot virus particles purified by a differential centrifugation method are used as an antigen to immunize BALB / c mice; through cell fusion, screening and cloning, one strain of hybridoma cell strain 12C10 capable of stable passage and secreting anti-Iris yellow spot virus monoclonal antibodies is obtained; and the strain has a preservation number CGMCC No.9336. The monoclonal antibody secreted from the hybridoma cell strain has ascites indirect ELISA titer of 10 ＜-7＞, and the antibody type and subtype are IgG1, kappa chain. The monoclonal antibody specifically and sensitively detect Iris yellow spot virus. The reaction with ZYMV. The monoclonal antibody secreted from 12C10 only has specific reaction with Iris yellow spot virus, but does not react with tomato spotted wilt virus, turnip mosaic virus and cucumber mosaic virus. The hybridoma cell strain 1C5 and monoclonal antibody secreted by the cell strain provide technical and material support for the diagnosis, inspection and quarantine, and scientific prevention and control of the virus.

Description

Secrete anti-iris macula lutea virus 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-iris macula lutea virus of secretion（IYSV）Monoclonal resists The application of body hybridoma cell strain and its monoclonal antibody.

Background technology

Iris macula lutea virus（Iris yellow spot virus, IYSV）It is bunyaviridae （Bunyaviridae）Tospovirus（Tospovirus）Virus.As plant virus is ground since the eighties in 20th century Study carefully the raising of means and developing rapidly for Protocols in Molecular Biology, various new virus are identified in Tospovirus category and name. IYSV is found according to the literature most earlier than 1992 on the iris of Holland, and the host range of IYSV is narrower, but can infect list Cotyledon plant.When iris is infected, initially there is chlorisis spot, be yellow necrotic plaque with disease development.According to cultivar Difference, is infected plant up to 50%~95%.In addition IYSV can also the vegetable crop such as natural infection onion, leek.With other Tospovirus viruses are the same, and IYSV is propagated also by thrips, the area being in a bad way, and usual thrips is also compared many.At present should Virus all has been reported that in Holland, Italy, Germany, the U.S., Japan, but the country's also report without occurrence injury.

In view of the complicated serological relation of Tospovirus category viruses and China are to the continuous of the import volumes such as iris, onion Increase, it is necessary to corresponding detection technique research is carried out to the virus, corresponding technological reserve is set up.

For iris macula lutea virus（IYSV）The preparation of monoclonal antibody is organized work.The currently monitored system is not perfect, Even Major Epidemic area does not carry out the work of correlation predictive forecast yet.It is used for detecting that the method for this virus is mainly used at present The methods such as RT-PCR detections, electron microscopic observation.This several method all has its limitation, and is 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 specific viral monoclonal antibody.It is domestic at present The outer specific monoclonal antibody do not developed for this virus.

With iris macula lutea virus（IYSV）It is anti-that purified virus particle is prepared for 1 plant of secretion for antigen by hybridoma technology The hybridoma 12C10 of the monoclonal antibody specific of IYSV, detects IYSV's with the monoclonal antibody that it is secreted as core is established High-throughout serological method and detection kit, and the detection of iris macula lutea virus is successfully applied to, so as to be China's iris The inspection and quarantine of macula lutea virus, scientific guidance prevention and control provide material and technical support.

The content of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, there is provided the anti-iris macula lutea viral monoclonal antibodies of one kind secretion Hybridoma cell strain and its monoclonal antibody application.

The hybridoma cell strain 12C10 of anti-iris macula lutea viral monoclonal antibodies is secreted, it can secrete anti-iris maculopathy The monoclonal antibody specific of poison, hybridoma cell strain 12C10 was preserved in Chinese microorganism strain preservation on July 3rd, 2014 Administration committee's common micro-organisms center, preserving number is CGMCC No.9336；

The ascites ELISA potency of the monoclonal antibody of anti-iris macula lutea virus 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 iris macula lutea virus, can detect biography Iris macula lutea virus in virus mediator thrips body；

The monoclonal antibody of anti-iris macula lutea virus is only capable of having specific reaction with iris macula lutea virus, without with tomato spot Wither virus, peanut ring spot virus, watermelon silver color mottle virus, Brassica 2 et 4, TOMV, tobacco mosaic virus (TMV), Cucumber mosaic virus, potato virus X, marmor upsilon, fractilinea oryzae, rice stripe virus, paddy rice tingia dwarf wilt The reaction of poison, WYMV, barley yellow mosaic virus, luteovirus GAV strains, GPV strains and PAV strains；

Application of the anti-iris macula lutea viral monoclonal antibodies in the Viral diagnosis, inspection and quarantine is to be with monoclonal antibody Various immunological detection methods and immunological reagent box that core is set up.

The present invention has the advantage that compared with prior art：1）The hybridoma cell strain of offer secretes anti-iris macula lutea Virus specific monoclonal antibody, with dot-ELISA, ACP-ELISA, DAS-ELISA and TAS- that the monoclonal antibody is set up as core The immunological methods such as ELISA and the kit energy high special set up with these methods, accurate, sensitive detection iris maculopathy Poison；2）Using the monoclonal antibody detection iris macula lutea virus prepared by the present invention, it is not necessary to expensive electron microscope, PCR The equipment such as instrument；3）Using the monoclonal antibody prepared by the present invention, kite in plant and its biography virus mediator thrips is effectively used for The detection of tail macula lutea virus, it is also possible to for the viral inspection and quarantine.

Brief description of the drawings

Fig. 1 is the sensitivity analysis of dot-ELISA methods detection iris macula lutea virus；

Fig. 2 is the result of iris macula lutea virosis in dot-ELISA methods detection tobacco.

Specific embodiment

The hybridoma cell strain 12C10 of anti-iris macula lutea viral monoclonal antibodies is secreted, it can secrete anti-iris maculopathy The monoclonal antibody specific of poison, hybridoma cell strain 12C10 was preserved in Chinese microorganism strain preservation on July 3rd, 2014 Administration committee's common micro-organisms center, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, postcode：100101, preserving number It is CGMCC No.9336, Classification And Nomenclature is：Secrete anti-iris macula lutea virus (IYSV) monoclonal antibody hybridoma；

The hybridoma cell strain that the present invention is provided can largely secrete anti-iris macula lutea virus monoclonal antibody, and its high specificity, effect Valency is high, good stability.The high-throughout serological method and detection kit of detection IYSV are established as core with the monoclonal antibody, and Can be applied to the detection and its inspection and quarantine of IYSV.

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

First, hybridoma acquisition and its preparation of monoclonal antibody

1. the preparation of immunogene and detection antigen

1) by big mortar and tissue mixer device precooling；

2) 500g disease leaves are weighed, 0.5M phosphate buffers (the i.e. PB buffer solutions of pH 7.5 are added in disease leaf per 100g） 200ml（Na-EDTA containing 0.01M and 0.1% mercaptoethanol）, double-layer nylon filtered through gauze, filtrate centrifugation are used after being homogenized 2 minutes （6000r/min, 20min）Remove plant tissue residue；

3) gained supernatant is added dropwise 2.5% Triton X-100,4% PEG while stirring（Molecular weight is 6000）With 0.1 Mol/L NaCl, 4 DEG C of stirring more than 4h；

4) it is centrifuged（11000r/min, 15min）Must precipitate；

5) the precipitation 0.5M PB (MgCl containing 0.01M of pH 7.5₂With 0.5M ureas) fully washing, centrifugation（6000r/ min, 15min）Supernatant being suctioned out afterwards and being placed in centrifuge tube, precipitation is washed again, centrifugation 3 times repeatedly；

6) supernatant, ultracentrifugation are merged（33000r/min, 100min）Gained precipitation is centrifuged after suspending（8000r/min, 15min）；

7) supernatant ultracentrifugation again is merged（33000r/min, 100min）, centrifugation bottom of the tube is added with 20%-30% sucrose Pad；

8) the gained precipitation 0.5M PB (MgCl containing 0.01M of pH 7.5₂）Suspend, suspension is Virus purification liquid；

2. animal is immunized

With the iris macula lutea virus of purification（IYSV）The immune four week old body weight 18-20g BALB/c females of virion Mouse.I.e. iris macula lutea is viral（IYSV）The μ L/ of virion 100 only mix with isometric Freund's complete adjuvant, it is fully emulsified Afterwards, through carrying on the back subcutaneous abdomen multi-point injection 0.2ml every, it is spaced 3 weeks, takes and exempt from equivalent amount of antigen with one and isometric Fu Shi is incomplete After adjuvant is fully emulsified, second intraperitoneal injection 0.2ml every carries out intraperitoneal injection, 3 after spending 3 weeks with the antigen of doubling dose Extracting spleen cell is merged after it.

3. cell fusion

Take above-mentioned immune mouse spleen cell and murine myeloma cell（SP2/0）By 10：1 ratio, in serum-free Mixed in RPMI-1640 culture mediums, 1500rpm centrifugation 5min remove culture medium, with 50 % PEG（Sigma, molecular weight 1500） As fusion agent, 1ml is added under water-bath at 37 DEG C, make its fusion 2min, terminate melting with the RPMI-1640 culture mediums of serum-free 1500rpm centrifugations 5min after conjunction, precipitation is suspended with HAT culture mediums, is dispensed into the cell plates that feeder cells are contained in 96 holes, 37 DEG C, 5 % CO₂Cell culture incubator in cultivate.

4. 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, change liquid, Deng Daorong with HT culture mediums within the 10th day When closing cell covering bottom hole 5%-50%, to infect the tobacco leaf and purified virus of IYSV viruses to detect antigen coat, with normal The positive hole of rule indirect ELISA method screening, obtains 72 positive holes altogether.10 cell holes in strong positive reaction of selection, are had Limit dilution method clone, obtaining 1 plant can secrete the hybridoma cell strain 12C10 of specific monoclonal antibody of anti-IYSV.Through 6 months with upper body After outer passage and multiple cryopreservation resuscitation, cell line can well grow, and stably excreting antibody.After through Amplification Culture, for abdomen Water is prepared and Liquid nitrogen storage.

5. the specific detection of monoclonal antibody

With infection tomato spotted wilf virus, peanut ring spot virus, watermelon silver color mottle virus, Brassica 2 et 4, tomato Mosaic virus, tobacco mosaic virus (TMV), cucumber mosaic virus, potato virus X, marmor upsilon, fractilinea oryzae, paddy rice bar Line virus, paddy rice tingia dwarf virus, WYMV, barley yellow mosaic virus, luteovirus GAV strains, GPV Strain and the sick leaf juice coating elisa plate of PAV strains reaction, make negative control, with iris macula lutea to be good for leaf extract accordingly Virus（IYSV）It is positive control, the specific reaction of monoclonal antibody is determined with ACP-ELISA methods.ACP-ELISA methods are specially The sick leaf liquid nitrogen grinding of virus infection is stated into powder, by 1: 30（w/v, g /mL）After adding the grinding of ELISA coating buffers 100ul/ holes are coated with elisa plate, and 4 DEG C overnight or 37 DEG C make it be adsorbed in elisa plate hole for 2 hours；PBST is used after washing three times 3% skimmed milk power or 1% BSA or 3% cow's serum closing 30-60min；Add the monoclonal antibody 100ul/ holes of appropriate dilution, 37 DEG C 1-2 hours；PBST washs three times and adds the alkaline phosphatase lipase (AP) of 10000 times of dilution to mark rabbit anti-mouse igg secondary antibody afterwards （Sigma companies）100ul/ holes, 37 DEG C 1-2 hours；After PBST washs four times, developed the color with PNPP substrates, 2mol/L hydroxides After sodium terminating reaction, OD is read with ELIASA₄₀₅Value, with negative OD values ratio more than 2.1 for the positive.Result discovery, 12C10 monoclonal antibodies have specific reaction to IYSV, and with tomato spotted wilf virus, peanut ring spot virus, watermelon silver color mottle virus, grassland Cyanines mosaic virus, TOMV, tobacco mosaic virus (TMV), cucumber mosaic virus, potato virus X, marmor upsilon, water Rice dwarf virus, rice stripe virus, paddy rice tingia dwarf virus, WYMV, barley yellow mosaic virus, barley are yellow Dwarf virus GAV strains, GPV strains and PAV strains and health plant are without immune response.

6. monoclonal antibody ascites is prepared and purified

Take 8 week old or so BALB/C mice, intraperitoneal injection 0.3-0.5ml norphytanes（Sigma）, pneumoretroperitoneum injection in 7-10 days 5-10×10⁵Individual hybridoma, 7-10 days visible mouse web portions substantially expand after injection, take ascites, 3000rpm centrifugations 3min, collects supernatant, as monoclonal antibody ascites.Take 1 times of volume ascites plus 2 times of M PH4.8 acetate buffers of volume 0.06 Liquid dilutes, plus octanoic acid（30ul/ml ascites）, stirring while adding at room temperature, 4 DEG C are clarified 1 hour, 12000rpm centrifugation 20min, are received Collection supernatant, then with 50% saturated ammonium sulphate immunoglobulin, 4 DEG C are placed 2 hours, 3000rpm centrifugation 20min, precipitation uses 2 The PBS solution dissolving of times volume, the ascites antibody of purifying, -70 DEG C of preservations are obtained after 24 hours in 4 DEG C of flowing dialysis.

7. the subgroup identification of monoclonal antibody and titer of ascites are determined

The odd contradictive hydroperitoneum and the anti-BALB/C mice IgG of standard of Sigma companies that will be purified₁、 IgG_2a、IgG_2b、IgG₃、IgM Antibody makees double agar diffusion test, as a result for 12C10 monoclonal antibodies subclass is IgG1, kappa.Detected with indirect ELISA method Odd contradictive hydroperitoneum potency, testing result shows above-mentioned odd contradictive hydroperitoneum potency 10^{- 7}。

2nd, virological immunology detection method and its detection kit

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

The foundation of IYSV methods and its field sample detection in 1.1 dot-ELISA detection irises

With liquid nitrogen grinding into powder after Iris Leaves are weighed, by 1:10-30（w/v, g /mL）Add 0.01 mol/L PBS（pH7.4）After grind；The rpm of sick juice 5000 is centrifuged 3 min;Take and check nitrocellulose filter on 3 μ l（NC）On, Healthy and susceptible iris leaf juice is set simultaneously respectively as negative and positive control；Drying at room temperature 10-20 min;NC films immerse Room temperature closes 30 min in PBST (the 0.01 mol/L PBS containing the 0.05% Tween-20) confining liquid containing 5% skimmed milk power； NC films are incubated at room temperature 30-60 min in being put into the monoclonal antibody of appropriateness dilution；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 of appropriateness dilution;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 reacts in being put into substrate solution, visual results.Treat the positive Control colour developing is obvious, and it is negative without any colour developing when tap water rinse terminating reaction, Taking Pictures recording result.

The dot-ELISA monoclonal antibodies and the most suitable working concentration of ELIAS secondary antibody for determining detection iris disease leaf, examination are tested with square formation Test and show that the most suitable working concentration of 12C10 monoclonal antibodies and ELIAS secondary antibody is respectively 1:5000 and 1:8000 times of dilutions.With above-mentioned antibody Most suitable working concentration set up detection IYSV disease leaf dot-ELISA methods.Sensitivity analysis shows, when Iris Leaves are diluted to 1:20480 times（w/v, g/mL）When, the dot-ELISA detections set up with 12C10 monoclonal antibodies are still presented the positive spots of purple, i.e., The sensitivity of the sick leaf of its detection reaches 1:20480 times of dilutions（Fig. 1）.

The suspicious taint IYSV samples intercepted and captured to Shanghai Entry-Exit Inspection and Quarantine bureau with the dot-ELISA methods set up are carried out Detection, as a result finds, has 14 samples to produce purpuriferous positive spots in 22 samples（Fig. 2）.Positive further uses RT- PCR is analyzed, and as a result shows that all of dot-ELISA positives detect the specific PCR primers of IYSV, PCR primer core Acid sequencing shows that positive infects IYSV.Illustrate that the dot-ELISA methods can accurately and reliably be used for IYSV in iris sample Detection and quarantine.

The foundation of IYSV methods in 1.2 dot-ELISA detection thrips bodies

Single head thrips is put into the centrifuge tube of the eppendorf of 0.5 mL, and 10 μ L PBS of addition (0.01mol/L, PH7.4), after mashing thrips with toothpick, 5000 rpm 3 min are centrifuged, take 3 μ L of supernatant points to NC films, film is dried, monoclonal antibody is incubated Educate, secondary antibody is incubated, development step is identical with IYSV methods in dot-ELISA detection irises, different simply secondary antibodies is appropriate dilute The sheep anti-mouse igg secondary antibody of the HRP marks released, chromogenic substrate is the chromogenic substrate of HRP, i.e. such as TMB sedimentation types chromogenic substrate.Together When set non-band poison and band poison thrips as feminine gender and positive control.

The most suitable working concentration of the dot-ELISA monoclonal antibodies and ELIAS secondary antibody that determine detection thrips is tested with square formation, table is tested The most suitable working concentration of bright 12C10 monoclonal antibodies and ELIAS secondary antibody is respectively 1:4000 and 1:6000 times of dilutions.With above-mentioned antibody most Suitable working concentration sets up the dot-ELISA methods of IYSV in detection thrips body, and testing result shows that the aphid for carrying IYSV is presented Blue spot, and nontoxic thrips does not have any chromogenic reaction, that is, the dot-ELISA methods set up can specifically detect thrips Internal IYSV.

2. the antigen coat ELISA for being set up as core with monoclonal antibody（ACP-ELISA）Method detection IYSV viruses

The operating procedure of ACP-ELISA methods：

（1）Use 0.05M carbonate buffer solutions（pH9.6）By 1: 30（w/v, g /mL）Times dilution sick leaf sap or press The supernatant 100ul/ holes that 100 thrips of ul carbonate buffer solutions every are mashed after adding add elisa plate, IYSV disease leaves or carrying IYSV thrips is positive control, and accordingly strong leaf or non-band poison thrips are negative control, 37 DEG C of 2h, or 4 DEG C overnight；

（2）After PBST washings 30min is closed with 5% skimmed milk power；

（3）100ul/ holes, 37 DEG C of 1h after 5000 times of dilutions of odd contradictive hydroperitoneum；

（4）5000 times of alkaline phosphatase lipase of dilution are added after PBST washings（AP）Or horseradish peroxidase（HRP）Mark The sheep anti-mouse igg secondary antibody of note（Sigma）, 100ul/ holes, 37 DEG C, 1h；

（5）Nitro Phosphate substrate or TMB substrate 100ul/ holes, room temperature 30min are added after being washed with PBST；

（6）Detect by an unaided eye, it is the positive that substrate colors become the hole of yellow green or blueness, or uses 2mol/L NaOH Or after sulfuric acid terminating reaction, the values of OD405 or 450 are surveyed with enzyme-linked immunosorbent assay instrument, with P/N>2.1 used as positive criterion.

ACP-ELISA methods detection sensitivity is detected

Odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, to sick leaf from 1：10 to 5120 make doubling dilution or single head thrips enters Row doubling dilution 20uL/ makees negative control with the strong leaf sap of corresponding dilution factor respectively to 3200uL/ heads, carries out above-mentioned ACP-ELISA methods are detected.Result shows ACP-ELISA methods to 1：10~40960 times of sick leaf saps for diluting and single head Ji Horse is diluted to 160uL/ and is positive, i.e. the sensitivity to the sick leaf of detection can reach 1：40960, the detection spirit to thrips Sensitivity reaches 160 uL/ heads, shows that ACP-ELISA methods have the sensitivity and reliability of height.

3. the TAS-ELISA detection methods of IYSV are detected

3.1. the operating process of TAS-ELISA methods：

1) rabbit anti-serum 1 of anti-IYSV:After 3000 times of dilutions（The immunogen immune rabbit of synthesis obtains）Wrap in 100ul/ holes By XPS, 37 DEG C, 2-4h or 4 DEG C, overnight；

2) PBST washs three times afterwards plus skimmed milk power or 1-3% BSA or 3-6% cow's serum the closing 200ul/ of 1-10% 30-60min is closed in 37 DEG C in hole；

3) detection sample 100ul/ holes are added.With IYSV disease leaf or carry IYSV thrips as positive control, with corresponding The thrips of healthy sample or non-band IYSV makees negative control, 37 DEG C of 1-2h；

4) 5000 times of odd contradictive hydroperitoneum 100ul/ holes, 37 DEG C of 1-2h are diluted after washing with confining liquid

5) 10000 times of AP or HRP mark rabbit anti-mouse igg secondary antibodies of dilution are added after PBST washings（Sigma）100ul/ holes, 37℃ 1-2h

6) add PNPP substrates or tmb substrate in color development at room temperature 5-30min after PBST washings, visually observe substrate colors and become The hole of yellow green or blueness is the positive, after 2mol/L NaOH or sulfuric acid terminating reaction, with 680 type enzyme-linked immunosorbent assay instruments The OD values of 405nm or 450nm are surveyed, with P/N>2.1 used as positive criterion.

The determination of 3.2TAS-ELISA detection method optimum conditions：

Carried out using the experiment of TAS-ELISA square formations, i.e., laterally added respectively with coating buffer solution from the ﹕ 102400 of 1 ﹕ 100 to 1 The rabbit-anti IYSV serum of doubling dilution；Add IYSV disease leaf juices or thrips homogenate；Longitudinal direction respectively plus with confining liquid from 1 ﹕ 5 to The doubling dilution odd contradictive hydroperitoneums of 1 ﹕ 2048000；The rabbit anti-mouse igg secondary antibody of AP or HRP marks presses the specification dilution of Sigma companies, 1 ﹕ 10000 times；Operated by TAS-ELISA method flows.Result is the rabbit anti-serum of IYSV and the optimal dilution point of monoclonal antibody Wei not 1 ﹕ 5000,1 ﹕ 8000.

3.3.TAS-ELISA the determination of method detection sensitivity

Under the most suitable working concentration of rabbit anti-serum and odd contradictive hydroperitoneum, by IYSV disease leaf juices or thrips homogenate PBS multiple proportions TAS-ELISA measure is carried out after dilution, as a result for：The sensitivity of TAS-ELISA detections sick leaf and thrips respectively reaches 1: 81920 times of dilutions（w/v, g /mL）With 160 uL/ heads, illustrate that this method has good sensitivity.

4. iris macula lutea virus dot-ELISA detection kits

1）Kit main component：

The ml of 1 pipe of IYSV monoclonal antibodies 0.2

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

The pipe 0.1ml of sheep anti-mouse igg secondary antibody 1 of HRP marks

1 bottle of 10ml of tmb substrate

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

Positive control 1（The leaf juice of iris containing IYSV）The ml of 1 pipe 2

Positive control 2（Band poison IYSV thrips）The ml of 1 pipe 2

Negative control 1（Healthy iris leaf juice）The ml of 1 pipe 2

Negative control 2（Healthy thrips homogenate）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 iris sample：

A. with liquid nitrogen grinding into powder after Iris Leaves are 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 5000 is centrifuged 3 min;

C. take and checked on NC on 3 μ l, while it is right respectively as the negative and positive to set healthy and susceptible iris leaf juice According to drying at room temperature 10-20 min;

D. NC films are immersed in PBST (the 0.01 mol/L PBS containing the 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 3000 times of monoclonal antibodies of dilution；

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 8000 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 delay Fliud flushing (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) is mixed, 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）Detect the operating procedure of thrips sample：

A. single head thrips is put into the centrifuge tube of the eppendorf of 0.5 mL, and 20 μ L PBS of addition (0.01mol/L, PH7.4), after mashing insect with toothpick, 5000 rpm 3 min are centrifuged, 3 μ L of supernatant points are taken to NC films, while setting band poison Malicious thrips is respectively as negative and positive control, drying at room temperature 10-20 min with non-band;

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

C. NC films are put into 1:30~60 min are incubated at room temperature in 3000 times of monoclonal antibodies of dilution；

D. film is washed 3~4 times with PBST, every time 3 min；NC films are put into 1:3000 times of HRP enzymes mark sheep anti mouses of dilution 30~60 min are incubated at room temperature in IgG secondary antibodies;

E. PBST washes film 4~5 times, every time 3 min;Film reacts in being put into tmb substrate liquid, visual results；

F. positive control colour developing is treated substantially (blueness), and tap water rinse terminating reaction during negative no any colour developing, clap According to record result.

4）Preserve and the term of validity

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

5）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

Plus adjust pH to 7.4 after the dissolving of distilled water 950, it is 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 to final concentration 5% is added in 0.01 mol/L PBST（W/V）.

Claims

1. a kind of hybridoma cell strain 12C10 for secreting anti-iris macula lutea viral monoclonal antibodies, it is characterised in that can secrete and resist The monoclonal antibody specific of iris macula lutea virus, hybridoma cell strain 12C10 was preserved in Chinese micro- life on July 3rd, 2014 Thing culture presevation administration committee common micro-organisms center, preserving number is CGMCC No.9336.

2. a kind of monoclonal antibody of the anti-iris macula lutea virus that hybridoma cell strain 12C10 as claimed in claim 1 secretes, It is characterized in that described monoclonal antibody ascites ELISA potency is up to 10^-7, Antibody types and subclass are IgG1, kappa, are somebody's turn to do Monoclonal antibody can have specific immunity association reaction with the capsid protein of iris macula lutea virus, can detect biography virus mediator thrips Internal iris macula lutea virus.

3. a kind of monoclonal antibody of the anti-iris macula lutea virus that hybridoma cell strain 12C10 as claimed in claim 2 secretes, It is characterized in that described monoclonal antibody can have specific reaction with iris macula lutea virus, without with tomato spotted wilf virus, flower Raw ring spot virus, watermelon silver color mottle virus, Brassica 2 et 4, TOMV, tobacco mosaic virus (TMV), cucumber mosaic virus Poison, potato virus X, marmor upsilon, fractilinea oryzae, rice stripe virus, paddy rice tingia dwarf virus, Wheat Yellow The reaction of mosaic virus, barley yellow mosaic virus, luteovirus GAV strains, GPV strains and PAV strains.

4. a kind of monoclonal antibody of anti-iris macula lutea virus as claimed in claim 2 is in the Viral diagnosis, inspection and quarantine Application, it is characterised in that set up various immunological detection methods and immunology detection reagent by core of the monoclonal antibody Box.