CN104513812A - Hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of monoclonal antibody - Google Patents
Hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of monoclonal antibody Download PDFInfo
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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
Technical field
The present invention relates to biological technical field, particularly relate to the application of a kind of secretion anti-iris macula lutea virus (IYSV) monoclonal antibody hybridoma cell strain and monoclonal antibody thereof.
Background technology
Iris macula lutea virus (Iris yellow spot virus, IYSV) is bunyaviridae (Bunyaviridae) Tospovirus (Tospovirus) virus.Along with the raising of plant virus research means and developing rapidly of Protocols in Molecular Biology since the eighties in 20th century, the interior multiple new virus of Tospovirus genus is identified and name.IYSV was found according to the literature early than 1992 on the iris of Holland, and the host range of IYSV is narrower, but can infect monocotyledons.When iris is subject to infecting, occurring chlorisis spot at first, is yellow necrotic plaque with PD.According to the difference of Cultivar, infected plant and can be reached 50% ~ 95%.In addition IYSV also can the vegetable crop such as natural infection onion, leek.With other Tospovirus virus, IYSV is also propagated by thrips, the area be in a bad way, and usual thrips is also many.This virus has report in Holland, Italy, Germany, the U.S., Japan at present, but the domestic report also not having occurrence injury.
The serological relation complicated in view of Tospovirus genus virus and China, to the continuous increase of the import volume such as iris, onion, are necessary to carry out corresponding detection technique research to this virus, set up corresponding tachnical storage.
Preparation for iris macula lutea virus (IYSV) monoclonal antibody is organized work.Current monitoring system is not perfect, and even Major Epidemic district does not carry out the work of correlation predictive forecast yet.The method being used for detecting this virus at present mainly adopts the methods such as RT-PCR detection, 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 for field sample batch detection, but specific viral monoclonal antibody must be depended on.Do not develop the specific monoclonal antibody for this virus at present both at home and abroad.
Be that antigen has prepared by hybridoma technology the hybridoma 12C10 that the monoclonal antibody specific of anti-IYSV is secreted in 1 strain with iris macula lutea virus (IYSV) purified virus particle, with high-throughout serological method and detection kit that the monoclonal antibody of its secretion detects IYSV for core establishes, and be successfully applied to the detection of iris macula lutea virus, thus be the inspection and quarantine of China's iris macula lutea virus, scientific guidance prevention and control provide material and technical support.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of hybridoma cell strain and monoclonal antibody application thereof of secreting anti-iris macula lutea viral monoclonal antibodies are provided.
Secrete the hybridoma cell strain 12C10 of anti-iris macula lutea viral monoclonal antibodies, it can secrete the monoclonal antibody specific of anti-iris macula lutea virus, hybridoma cell strain 12C10 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 3rd, 2014, and preserving number is CGMCC No.9336;
The ascites ELISA of the monoclonal antibody of anti-iris macula lutea virus tires and reaches 10
-7, Antibody types and subclass are IgG1, kappa, and the capsid protein of this monoclonal anti physical efficiency and iris macula lutea virus has specific immunity association reaction, can detect the iris macula lutea virus passed in virus mediator thrips body;
The monoclonal antibody of anti-iris macula lutea virus only can have specific reaction with iris macula lutea virus, and does not react with 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, rice stripe virus, paddy rice tingia dwarf virus, Wheat yellow mosaic virus, barley yellow mosaic virus, barly yellow dwarf virus GAV strain, GPV strain and PAV strain;
The application of anti-iris macula lutea viral monoclonal antibodies in this Viral diagnosis, inspection and quarantine be with monoclonal antibody be core set up various immunological detection method and immunological reagent box.
The beneficial effect that the present invention compared with prior art has: the hybridoma cell strain 1) provided secretes anti-iris macula lutea virus monoclonal antibody specific, immunological methods such as dot-ELISA, ACP-ELISA, DAS-ELISA and TAS-ELISA of setting up for core with this monoclonal antibody and viral with the test kit energy high special of these method establishment, accurate, sensitive detection iris macula lutea; 2) utilize the monoclonal antibody prepared by the present invention to detect iris macula lutea virus, do not need the expensive equipment such as electron microscope, PCR instrument; 3) utilize the monoclonal antibody prepared by the present invention, effectively can pass the detection of iris macula lutea virus in virus mediator thrips for plant and its, also may be used for the inspection and quarantine of this virus.
Accompanying drawing explanation
Fig. 1 is the sensitivity analysis that dot-ELISA method detects iris macula lutea virus;
Fig. 2 is the result that dot-ELISA method detects iris maculopathy viral disease in tobacco.
Embodiment
Secrete the hybridoma cell strain 12C10 of anti-iris macula lutea viral monoclonal antibodies, it can secrete the monoclonal antibody specific of anti-iris macula lutea virus, hybridoma cell strain 12C10 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 3rd, 2014, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, preserving number is CGMCC No.9336, and Classification And Nomenclature is: secrete anti-iris macula lutea virus (IYSV) monoclonal antibody hybridoma;
The ascites ELISA of the monoclonal antibody of anti-iris macula lutea virus tires and reaches 10
-7, Antibody types and subclass are IgG1, kappa, and the capsid protein of this monoclonal anti physical efficiency and iris macula lutea virus has specific immunity association reaction, can detect the iris macula lutea virus passed in virus mediator thrips body;
The monoclonal antibody of anti-iris macula lutea virus only can have specific reaction with iris macula lutea virus, and does not react with 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, rice stripe virus, paddy rice tingia dwarf virus, Wheat yellow mosaic virus, barley yellow mosaic virus, barly yellow dwarf virus GAV strain, GPV strain and PAV strain;
The application of anti-iris macula lutea viral monoclonal antibodies in this Viral diagnosis, inspection and quarantine be with monoclonal antibody be core set up various immunological detection method and immunological reagent box.
Hybridoma cell strain provided by the invention can secrete anti-iris macula lutea virus monoclonal antibody in a large number, and its high specificity, height of tiring, good stability.With high-throughout serological method and detection kit that this monoclonal antibody detects IYSV for core establishes, and can be applicable to detection and the inspection and quarantine thereof of IYSV.
Below in conjunction with embodiment and accompanying drawing, the invention will be further described.
One, the preparation of hybridoma acquisition and monoclonal antibody thereof
1. the preparation of immunogen and detectable antigens
1) by large mortar and the precooling of tissue mixer device;
2) the sick leaf of 500g is taken, 0.5M phosphate buffered saline buffer (the i.e. PB damping fluid) 200ml(of pH 7.5 is added containing 0.01M Na-EDTA and 0.1% mercaptoethanol) in the sick leaf of every 100g, homogenate used double-layer nylon filtered through gauze after 2 minutes, filtrate centrifugal (6000r/min, 20min) removes plant tissue residue;
3) gained supernatant liquor drips 2.5% Triton X-100 while stirring, and 4% PEG(molecular weight is 6000) and 0.1 mol/L NaCl, 4 DEG C are stirred more than 4h;
4) centrifugal (11000r/min, 15min) must precipitate;
5) precipitation uses the 0.5M PB of pH 7.5 (containing 0.01M MgCl
2with 0.5M urea) fully wash, centrifugal (6000r/min, 15min) afterwards sucking-off supernatant is placed in centrifuge tube, and precipitation is washed again, centrifugal 3 times repeatedly;
6) supernatant liquor is merged, after ultracentrifugation (33000r/min, 100min) gained precipitation suspends centrifugal (8000r/min, 15min);
7) merge supernatant liquor ultracentrifugation (33000r/min, 100min) again, bottom centrifuge tube, be added with 20%-30% sucrose cushions;
8) gained precipitation uses the 0.5M PB of pH 7.5 (containing 0.01M MgCl
2) suspend, suspension is Virus purification liquid;
2. immune animal
With virus particle immunity surrounding body weight 18-20g in the age BALB/c female mice of the iris macula lutea virus (IYSV) of purifying.Namely the virus particle 100 μ L/ of iris macula lutea virus (IYSV) only mixes with equal-volume Freund's complete adjuvant, after fully emulsified, only every through back of the body subcutaneous abdomen multi-point injection 0.2ml, 3 weeks, interval, get to exempt from one equivalent amount of antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, 0.2ml is only every for second time abdominal injection, and the antigen crossing 3 weeks rear doubling doses carries out abdominal injection, and after 3 days, extracting spleen cell merges.
3. cytogamy
Get above-mentioned immune mouse spleen cell and murine myeloma cell (SP2/0) ratio in 10:1, mix in the RPMI-1640 substratum of serum-free, the centrifugal 5min of 1500rpm, remove substratum, with 50 % PEG(Sigma, molecular weight 1500) as fusogen, 1ml is added under water-bath at 37 DEG C, it is made to merge 2min, stop merging the rear centrifugal 5min of 1500rpm with the RPMI-1640 substratum of serum-free, precipitation HAT substratum suspends, and is dispensed into 96 holes and contains in the cell plate of feeder cell, 37 DEG C, 5 % CO
2cell culture incubator in cultivate.
4. the screening in hybridoma, positive hole and clone thereof
Cultivate in cell culture incubator after 5 days, liquid is changed once with HAT substratum, within 10th day, change liquid with HT substratum, by the time at the bottom of fused cell coverage hole during 5%-50%, to infect the tobacco leaf of IYSV virus and purified virus for detectable antigens bag quilt, screen positive hole with conventional indirect ELISA method, obtain 72 positive holes altogether.Select 10 cell holes in strong positive reaction, carry out limiting dilution assay clone, obtain the hybridoma cell strain 12C10 that 1 strain can secrete the specific monoclonal antibody of anti-IYSV.Through more than 6 months subculture in vitro separately with repeatedly after cryopreservation resuscitation, cell strain all can well grow, and stably excreting antibody.After enlarged culturing, for ascites preparation 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, rice stripe virus, paddy rice tingia dwarf virus, Wheat yellow mosaic virus, barley yellow mosaic virus, barly yellow dwarf virus GAV strain, the sick leaf juice bag of GPV strain and the reaction of PAV strain is by elisa plate, negative control is made to be good for leaf extract accordingly, with iris macula lutea virus (IYSV) for positive control, the specific reaction of monoclonal antibody is measured by ACP-ELISA method.ACP-ELISA method is specially the sick leaf liquid nitrogen grinding powdered of above-mentioned virus infection, by 1:30(w/v, g/mL) add ELISA coating buffer grinding after 100ul/ hole bag by elisa plate, 4 DEG C spend the night or 37 DEG C within 2 hours, make it be adsorbed in elisa plate hole; PBST wash three times afterwards with 3% skim-milk or 1% BSA or 3% bovine serum close 30-60min; Add the monoclonal antibody 100ul/ hole of suitably dilution, 37 DEG C of 1-2 hour; PBST adds dilution 10000 times alkaline phosphatase lipase (AP) after washing three times marks anti-(Sigma company) the 100ul/ hole of rabbit anti-mouse igg two, 37 DEG C of 1-2 hour; After PBST washs four times, with the colour developing of PNPP substrate, after 2mol/L sodium hydroxide termination reaction, read OD by microplate reader
405value, to be greater than 2.1 for positive with negative OD value ratio.Found that, 12C10 monoclonal antibody has specific reaction to IYSV, and with 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, rice stripe virus, paddy rice tingia dwarf virus, Wheat yellow mosaic virus, barley yellow mosaic virus, barly yellow dwarf virus GAV strain, GPV strain and PAV strain and health plant all without immune response.
6. the preparation of monoclonal antibody ascites and purifying
Get BALB/C mice about 8 week age, abdominal injection 0.3-0.5ml pristane (Sigma), within 7-10 days, pneumoretroperitoneum injects 5-10 × 10
5individual hybridoma, after injection, 7-10 days visible mouse web portions obviously expand, and take ascites, the centrifugal 3min of 3000rpm, collect supernatant liquor, are monoclonal antibody ascites.Get 1 times of volume ascites and add 2 times of volume 0.06 M PH4.8 acetate buffer solution dilutions, add sad (30ul/ml ascites), the following edged of room temperature stirs, and clarifies 1 hour for 4 DEG C, the centrifugal 20min of 12000rpm, collect supernatant, then use 50% saturated ammonium sulphate immunoglobulin (Ig), place 2 hours for 4 DEG C, the centrifugal 20min of 3000rpm, precipitation is dissolved by the PBS solution of 2 times of volumes, namely obtains the ascites antibody of purifying ,-70 DEG C of preservations 4 DEG C of flowing dialysis after 24 hours.
7. the subgroup identification of monoclonal antibody and titer of ascites measure
By the anti-BALB/C mice IgG of standard of the odd contradictive hydroperitoneum of purifying and Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgM antibody makes double agar diffusion test, result is 12C10 monoclonal antibody subclass is IgG1, kappa.Detect odd contradictive hydroperitoneum with indirect ELISA method to tire, detected result shows that above-mentioned odd contradictive hydroperitoneum is tired 10
-7.
Two, virological immunology detection method and detection kit thereof
1. the foundation of dot-ELISA method and Fields detection application thereof
1.1 dot-ELISA detect foundation and the field sample detection thereof of IYSV method in iris
Liquid nitrogen grinding powdered is used, by 1:10-30(w/v, g/mL after being weighed by Iris Leaves) add 0.01 mol/L PBS(pH7.4) grind afterwards; Centrifugal 3 min of sick juice 5000 rpm; Get on 3 μ l and check on nitrocellulose filter (NC), healthy and susceptible iris leaf juice is set respectively as feminine gender and positive control simultaneously; Drying at room temperature 10-20 min; NC film is immersed in room temperature in PBST (the 0.01 mol/L PBS containing the 0.05% Tween-20) confining liquid containing 5% skim-milk and closes 30 min; NC film puts into the monoclonal antibody incubated at room 30-60 min of appropriateness dilution; Film is washed 3-4 time, each 3 min with PBST; NC film puts into the anti-incubated at room 30-60 min of AP enzyme labelling sheep anti-mouse igg two of appropriateness dilution; PBST washes film 4-5 time, each 3 min; 66 μ L NBT and 33 μ L BCIP substrate (Promega) join 10 ml substrate buffer solutions (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mixing, film is put into substrate solution and is reacted, visual results.Treat positive control colour developing obviously, and feminine gender is without any tap water rinse termination reaction during colour developing, Taking Pictures recording result.
Determine to detect the sick dot-ELISA monoclonal antibody of leaf of iris and the suitableeest working concentration of ELIAS secondary antibody with square formation test, test shows that the suitableeest working concentration of 12C10 monoclonal antibody and ELIAS secondary antibody is respectively 1:5000 and 1:8000 and doubly dilutes.The dot-ELISA method detecting the sick leaf of IYSV is set up with the suitableeest working concentration of above-mentioned antibody.Sensitivity analysis shows, when Iris Leaves be diluted to 1:20480 doubly (w/v, g/mL) time, the dot-ELISA set up with 12C10 monoclonal antibody detects the positive spots still presenting purple, and namely its sensitivity detecting disease leaf reaches 1:20480 and doubly dilutes (Fig. 1).
By the dot-ELISA method set up, the suspicious taint IYSV sample that Shanghai Entry-Exit Inspection and Quarantine bureau intercepts and captures is detected, found that in 22 samples, there are 14 purpuric positive spots of sample (Fig. 2).Positive is analyzed with RT-PCR further, and result shows that all dot-ELISA positive all detect the specific PCR primer of IYSV, and PCR primer nucleic acid sequencing shows that positive infects IYSV.Illustrate this dot-ELISA method can accurately, reliably for detection and the quarantine of IYSV in iris sample.
1.2 dot-ELISA detect the foundation of IYSV method in thrips body
Single head thrips puts into the centrifuge tube of the eppendorf of 0.5 mL, and add 10 μ L PBS (0.01mol/L, pH7.4), after mashing thrips with toothpick, centrifugal 3 min of 5000 rpm, getting on 3 μ L checks on NC film, it is identical that film is dry, monoclonal antibody is hatched, two anti-ly to hatch, development step and dot-ELISA detect IYSV method in iris, different just two sheep anti-mouse iggs two resisting the HRP for appropriateness dilution to mark resist, chromogenic substrate is the chromogenic substrate of HRP, namely as TMB sedimentation type chromogenic substrate etc.Establish the thrips of non-band poison and band poison as negative and positive control simultaneously.
Determine to detect the dot-ELISA monoclonal antibody of thrips and the suitableeest working concentration of ELIAS secondary antibody with square formation test, test shows that the suitableeest working concentration of 12C10 monoclonal antibody and ELIAS secondary antibody is respectively 1:4000 and 1:6000 and doubly dilutes.The dot-ELISA method detecting IYSV in thrips body is set up with the suitableeest working concentration of above-mentioned antibody, detected result shows that the aphid of carrying IYSV presents blue spot, and nontoxic thrips is without any color reaction, the dot-ELISA method namely set up can detect the IYSV in thrips body specifically.
2. be the antigen coated ELISA(ACP-ELISA that core is set up with monoclonal antibody) method detection IYSV virus
The operation steps of ACP-ELISA method:
(1) use 0.05M carbonate buffer solution (pH9.6) by 1:30(w/v, g/mL) the sick leaf sap of doubly dilution or the supernatant 100ul/ hole of mashing after adding by 100 ul carbonate buffer solutions every thrips add elisa plate, the sick leaf of IYSV or to carry IYSV thrips be positive control, corresponding strong leaf or non-band poison thrips are negative control, 37 DEG C of 2h, or 4 DEG C are spent the night;
(2) 30min is closed with 5% skim-milk after PBST washing;
(3) 100ul/ hole after odd contradictive hydroperitoneum 5000 times dilution, 37 DEG C of 1h;
(4) sheep anti-mouse igg two anti-(Sigma) that the alkaline phosphatase lipase (AP) of 5000 times of dilutions or horseradish peroxidase (HRP) mark is added after PBST washing, 100ul/ hole, 37 DEG C, 1h;
(5) nitro Phosphate substrate or TMB substrate 100ul/ hole is added, room temperature 30min with after PBST washing;
(6) detect by an unaided eye, substrate colors becomes the hole of yellow-green colour or blueness for positive, or with after 2mol/L sodium hydroxide or sulfuric acid termination reaction, survey OD405 or 450 value, using P/N> 2.1 as positive judging criterion with enzyme-linked immunosorbent assay instrument.
ACP-ELISA method detection sensitivity detects
Odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, from 1:10 to 5120, doubling dilution is done to sick leaf or single head thrips carries out doubling dilution 20uL/ head to 3200uL/ head, make negative control with corresponding dilution strong leaf sap respectively, carry out above-mentioned ACP-ELISA method and detect.Result shows that ACP-ELISA method is diluted to 160uL/ head to the sick leaf sap of 1:10 ~ 40960 times dilution and single head thrips and is all positive, namely 1:40960 can be reached to the sensitivity detecting sick leaf, 160 uL/ heads are reached to the detection sensitivity of thrips, shows that ACP-ELISA method has susceptibility and the reliability of height.
3. detect the TAS-ELISA detection method of IYSV
3.1. the operating process of TAS-ELISA method:
1) after the rabbit anti-serum 1:3000 of anti-IYSV doubly dilutes, (the immunogen immune rabbit of synthesis obtains) 100ul/ hole is wrapped by polystyrene board, and 37 DEG C, 2-4h or 4 DEG C, spends the night;
2) PBST adds the skim-milk of 1-10% or 1-3% BSA or 3-6% bovine serum and closes 200ul/ hole in 37 DEG C of closed 30-60min after washing three times;
3) detection sample 100ul/ hole is added.With the sick leaf of IYSV or carry the thrips of IYSV for positive control, make negative control with the thrips of corresponding healthy sample or non-band IYSV, 37 DEG C of 1-2h;
4) the odd contradictive hydroperitoneum 100ul/ hole of 5000 times is diluted after washing with confining liquid, 37 DEG C of 1-2h
5) AP or HRP adding 10000 times of dilutions after PBST washing marks anti-(Sigma) 100ul/ hole of rabbit anti-mouse igg two, 37 DEG C of 1-2h
6) PNPP substrate or tmb substrate is added in color development at room temperature 5-30min after PBST washing, visual inspection substrate colors becomes the hole of yellow-green colour or blueness for positive, after 2mol/L sodium hydroxide or sulfuric acid termination reaction, the OD value of 405nm or 450nm is surveyed, using P/N> 2.1 as positive judging criterion with 680 type enzyme-linked immunosorbent assay instruments.
The determination of 3.2TAS-ELISA detection method optimum condition:
Adopt the test of TAS-ELISA square formation to carry out, namely laterally add respectively and be buffered the rabbit anti-IYSV serum of liquid from 1 ﹕ 100 to 1 ﹕ 102400 doubling dilution with bag; Add the sick leaf juice of IYSV or thrips homogenate; Longitudinally add respectively with confining liquid from 1 ﹕ 5 to 1 ﹕ 2048000 doubling dilution odd contradictive hydroperitoneum; The rabbit anti-mouse igg two of AP or HRP mark is anti-presses the specification sheets dilution of Sigma company, 1 ﹕ 10000 times; Operate by TAS-ELISA method flow.Result is that the rabbit anti-serum of IYSV and the optimal dilution of monoclonal antibody are respectively 1 ﹕ 5000,1 ﹕ 8000.
3.3.TAS-ELISA the determination of method detection sensitivity
Under rabbit anti-serum and the suitableeest working concentration of odd contradictive hydroperitoneum, TAS-ELISA mensuration is carried out by after sick for IYSV leaf juice or thrips homogenate PBS doubling dilution, result is: the sensitivity that TAS-ELISA detects disease leaf and thrips reaches 1:81920 respectively and doubly dilutes (w/v, g/mL) and 160 uL/ heads, illustrate that present method has good sensitivity.
4. iris macula lutea virus dot-ELISA detection kit
1) test kit main component:
IYSV monoclonal antibody 1 pipe 0.2 ml
AP marks anti-1 pipe 0.1 ml of sheep anti-mouse igg two
The anti-1 pipe 0.1ml of sheep anti-mouse igg two of HRP mark
Tmb substrate 1 bottle of 10ml
Each 1 bottle of NBT/BCIP substrate is respectively 2 ml and 1ml
Positive control 1 (containing IYSV iris leaf juice) 1 pipe 2 ml
Positive control 2 (being with malicious IYSV thrips) 1 pipe 2 ml
Negative control 1(healthy iris leaf juice) 1 pipe 2 ml
The healthy thrips homogenate of negative control 2() 1 pipe 2 ml
Antibody diluent (10X) 1 bottle of 80ml
At above reagent is all stored in 4 DEG C
Nitrocellulose filter (NC) 10
2) operation steps of iris sample is detected:
A. liquid nitrogen grinding powdered is used after being weighed by Iris Leaves, by 1:10 ~ 30(w/v, g/mL) add 0.01 mol/L PBS(pH7.4) grind afterwards;
B. centrifugal 3 min of sick juice 5000 rpm;
C. get and 3 μ l check on NC, healthy and susceptible iris leaf juice is set respectively as feminine gender and positive control, drying at room temperature 10-20 min simultaneously;
D. during NC film is immersed in containing 5% skim-milk PBST (the 0.01 mol/L PBS containing 0.05% Tween-20) confining liquid, room temperature closes 30 min;
E. NC film puts into monoclonal antibody incubated at room 30 ~ 60 min that 1:3000 doubly dilutes;
F. film is washed 3 ~ 4 times with PBST, each 3 min; NC film puts into anti-incubated at room 30 ~ 60 min of AP enzyme labelling sheep anti-mouse igg two of 1:8000 dilution;
G. PBST washes film 4 ~ 5 times, each 3 min; 66 μ L NBT and 33 μ L BCIP substrates join 10 ml substrate buffer solutions (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mixing, and film is put into substrate solution and reacted, visual results;
H. treat positive control colour developing obviously (purple), and feminine gender is without any tap water rinse termination reaction during colour developing, Taking Pictures recording result.
3) operation steps of thrips sample is detected:
A. single head thrips puts into the centrifuge tube of the eppendorf of 0.5 mL; and add 20 μ L PBS (0.01mol/L; pH7.4); after mashing insect with toothpick, centrifugal 3 min of 5000 rpm; getting on 3 μ L checks on NC film; the thrips of band poison and non-band poison is set simultaneously respectively as negative and positive control, drying at room temperature 10-20 min;
B. during NC film is immersed in containing 5% skim-milk PBST (the 0.01 mol/L PBS containing 0.05% Tween-20) confining liquid, room temperature closes 30 min;
C. NC film puts into monoclonal antibody incubated at room 30 ~ 60 min that 1:3000 doubly dilutes;
D. film is washed 3 ~ 4 times with PBST, each 3 min; NC film puts into anti-incubated at room 30 ~ 60 min of HRP enzyme labelling sheep anti-mouse igg two that 1:3000 doubly dilutes;
E. PBST washes film 4 ~ 5 times, each 3 min; Film is put into tmb substrate liquid and is reacted, visual results;
F. treat positive control colour developing obviously (blueness), and feminine gender is without any tap water rinse termination reaction during colour developing, Taking Pictures recording result.
4) preservation and validity period
Keep in Dark Place in 2 ~ 8 DEG C, validity period 12 months.
5) buffer formulation:
Phosphate buffered saline buffer (PBS, 0.01 mol/L, pH7.4):
NaCl 8 g
KCl 0.2 g
KH
2PO
40.2 g
Na
2HPO
412H
2O 3g
Sodiumazide 0.2 g
Adding distil water 950 dissolves rear tune pH to 7.4, is settled to 1000 ml
ELISA washings (0.01 mol/L PBST):
0.5 ml Tween-20 is added in 1000 ml 0.01mol/L PBS
ELISA confining liquid:
Skim-milk is added to final concentration 5%(W/V) in 0.01 mol/L PBST.
Claims (4)
1. the hybridoma cell strain 12C10 of the anti-iris macula lutea viral monoclonal antibodies of secretion, it is characterized in that the monoclonal antibody specific secreting anti-iris macula lutea virus, hybridoma cell strain 12C10 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on July 3rd, 2014, and preserving number is CGMCC No.9336.
2. a monoclonal antibody for the anti-iris macula lutea virus of hybridoma cell strain 12C10 secretion as claimed in claim 1, is characterized in that described monoclonal antibody ascites ELISA tires and reaches 10
-7, Antibody types and subclass are IgG1, kappa, and the capsid protein of this monoclonal anti physical efficiency and iris macula lutea virus has specific immunity association reaction, can detect the iris macula lutea virus passed in virus mediator thrips body.
3. the monoclonal antibody of the anti-iris macula lutea virus of a hybridoma cell strain 12C10 secretion as claimed in claim 2, it is characterized in that described monoclonal anti physical efficiency and iris macula lutea virus have specific reaction, and not with 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, rice stripe virus, paddy rice tingia dwarf virus, Wheat yellow mosaic virus, barley yellow mosaic virus, barly yellow dwarf virus GAV strain, GPV strain and the reaction of PAV strain.
4. the application of monoclonal antibody in this Viral diagnosis, inspection and quarantine for anti-iris macula lutea virus as claimed in claim 2, it is characterized in that being applied to monoclonal antibody be core set up various immunological detection method and immunological detecting kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410629108.7A CN104513812B (en) | 2014-11-10 | 2014-11-10 | Hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of monoclonal antibody |
Applications Claiming Priority (1)
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|---|---|---|---|
| CN201410629108.7A CN104513812B (en) | 2014-11-10 | 2014-11-10 | Hybridoma cell strain secreting monoclonal antibody against Iris yellow spot virus and application of monoclonal antibody |
Publications (2)
| Publication Number | Publication Date |
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| CN104513812A true CN104513812A (en) | 2015-04-15 |
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| CN104991069A (en) * | 2015-07-17 | 2015-10-21 | 上海理工大学 | Fruit blotch immune colloidal gold detection test strip, and monoclonal antibody and hybridoma cell strain thereof |
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| CN104991069A (en) * | 2015-07-17 | 2015-10-21 | 上海理工大学 | Fruit blotch immune colloidal gold detection test strip, and monoclonal antibody and hybridoma cell strain thereof |
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