CN104513810B - Hybridoma cell strain secreting monoclonal antibody against cucumber mosaic virus and application of monoclonal antibody - Google Patents
Hybridoma cell strain secreting monoclonal antibody against cucumber mosaic virus and application of monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting monoclonal antibody against cucumber mosaic virus and application of the monoclonal antibody. Purified virus particles of cucumber mosaic virus (CMV) are used as an antigen to immunize BALB / c mice; through cell fusion, screening and cloning, one strain of hybridoma cell strain 21H5 capable of stable passage and secreting anti-CMV monoclonal antibodies is obtained; and the strain has a preservation number CGMCC No.9342. The monoclonal antibody secreted from the hybridoma cell strain has ascites indirect ELISA titer of 10＜-8＞, and the antibody type and subtype are IgG1, kappa chain. The monoclonal antibody has specificity reaction with CMV. The 21H5 antibody is used to establish a dot-ELISA virus method for detection of CMV in virus transmission medium aphid and in cucumber. The hybridoma cell strain 21H5, monoclonal antibody thereof and the establishment of the related serological detection method provide technical and material support for the diagnosis, detection, prediction and scientific prevention and control of the disease.
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of hybridization for secreting anti cucumber mosaic virus monoclonal antibody
The application of tumor cell strain and its monoclonal antibody.
Background technology
Cucumber mosaic virus（Cucumber mosaic virus, CMV）It is Bromoviridae (Bromovirus)
The representative member of Cucumovirus is single stranded positive-sense RNA (+ssRNA) virus.CMV can be infected including single, double son
Leaf plant, in 1000 interior various plants, is one of important crushing cause of disease of many crops and ornamental plant.CMV is certainly
Right boundary, mainly by host plant seed or propagating materials and entomochory, is that host known today is most, it is most wide most to be distributed
Has the plant virus of economic damage.From 1916 first report CMV be the cause of disease of cucumber mosaic virus since, domestic and foreign scholars
Harm of the virus on different hosts is reported in succession.CMV seriously suppresses the normal growth of many crops, such as causes cucumber leaf
The yellow of piece floral leaf, the thread deformity of tomato leaf, banana floral leaf（Centre rot）, the mottled deformity of Pepper Leaves and dieback etc., this gives the world
The production estimation of various countries causes serious economic loss.Cucumber virosis is to send out in China's open country and autumn greenhouse cultivation in recent years
The more serious disease of life.According to the literature, can cause the cucumber pathogenetic cause of disease of virus has various, wherein being drawn by CMV
The cucumber virosis for rising is more serious, and the influence to cucumber is very big, mainly causes fruit abnormal in addition to the growth of influence cucumber
Shape, loses commodity, or even cause total crop failure.
CMV virions are spherical, including 3 kinds of geneome RNAs (RNA1-3) and subgenomic RNA (RNA4), have
Also contain the 5th kind of RNA, i.e. satellite RNA.The size of RNA1, RNA2, RNA3 is respectively 3 390~3 410
Nt, 3 035~3 350 nt and 2 190~2 200 nt.A diameter of 35 nm of virion, lethal temperature is 60~70
DEG C, the poison phase is protected for 3~4 d.The host range of virus is very wide, and the single, double cotyledon of kind more than 1000 that can contaminate the category of 85 section 365 is planted
Thing, can be through 75 kinds of aphis propagations, mainly with melon aphid, and black peach aphid etc. passes poison, also by machinery, seed dispersal, but can not pass through soil
Earth is propagated.The most common symptom for causing is floral leaf, mottled, the downright bad, virus disease such as wither, wither, and virion is mainly divided
Cloth is in cytoplasm and vacuole.Compared with other viruses, CMV is maximum to the harmfulness of vegetable crop, and main harm is melon
Crop, huge economic loss is caused to China.The disease performance shape of this kind of virus has very big difference under different condition, is both cucumber
Mosaic virus also shows as different symptoms on different plants, hence sets up Cucumber Mosaic Virus detection architecture for tobacco leaf
The preventing and treating of virosis is significant.
In order to grasp cucumber mosaic virus（CMV）Disease strengthens China's cucumber mosaic virus in the morbidity hazard conditions of China
Viral disease early monitoring and early warning, scientific guidance prevention and control are badly in need of setting up the high-flux detection method of detection Cucumber Mosaic Virus, and
Currently without this high-throughout detection method, only entered with methods such as inefficient electron microscopic observation, RT-PCR method, nucleic acid electrophoresis
Row small sample is detected.The CMV viruses of present invention purification secrete the special of anti-CMV for antigen is prepared for 1 plant by hybridoma technology
Property monoclonal antibody hybridoma 21H5, with prepare monoclonal antibody for core can set up detect CMV high-throughout serology
Method and its kit, so that for the detection of China Cucumber Mosaic Virus, early monitoring and early warning, scientific guidance prevention and control provide thing
Matter 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 one kind secretion anti cucumber mosaic virus monoclonal antibody
Hybridoma cell strain and its monoclonal antibody application.
The hybridoma cell strain 21H5 of anti cucumber mosaic virus monoclonal antibody is secreted, it can secrete anti cucumber mosaic virus
Monoclonal antibody specific, hybridoma cell strain 21H5 is preserved in Chinese microorganism strain preservation pipe on July 3rd, 2014
Reason committee common micro-organisms center, preserving number is CGMCC No. 9342, and Classification And Nomenclature is：Secretion anti cucumber mosaic virus
(CMV) monoclonal antibody hybridoma.
The monoclonal antibody ascites indirect ELISA titer of anti cucumber mosaic virus is up to 10-8, Antibody types and subclass are
IgG1, kappa, the monoclonal antibody can have specific immunity association reaction with the capsid protein of cucumber mosaic virus, can examine
Measure the cucumber mosaic virus passed in virus mediator aphid body.
The monoclonal antibody of anti cucumber mosaic virus only has a specific immune response with cucumber mosaic virus, and with tomato
It is mosaic virus, odontoglossum ring spot virus, cucumber green mottle mosaic virus, Zucchini yellow mosaic virus, tobacco mosaic virus (TMV), melon
Chlorisis yellow virus, Bean common mosaic virus, rice black-streaked dwarf virus, Brassica 2 et 4, potato virus X, Ma Ling
Potato Y virus, marmor solani, potato virus S, rape mosaic disease poison, tomato spotted wilf virus, cymbidium mosaic virus are not sent out
Raw immune response.
Application of the anti cucumber mosaic virus monoclonal antibody in anti cucumber mosaic virus detection 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 21H5 of offer secretes anti-yellowing melon
The immunological methods such as mosaic virus monoclonal antibody specific, the dot-ELISA set up as core with the monoclonal antibody and ACP-ELISA
And set up with these methods kit energy high special, it is accurate, sensitively detect cucumber mosaic virus；2）Using institute of the present invention
The monoclonal antibody detection cucumber mosaic virus of preparation, it is not necessary to the equipment such as expensive electron microscope, PCR instrument；3）Using this
The prepared monoclonal antibody of invention, the inspection of the cucumber mosaic virus being effectively used in field crops and biography virus mediator aphid
Survey.
Brief description of the drawings
Fig. 1 is the sensitivity analysis that dot-ELISA methods detect CMV.
Specific embodiment
The hybridoma cell strain 21H5 of anti cucumber mosaic virus monoclonal antibody is secreted, it can secrete anti cucumber mosaic virus
Monoclonal antibody specific, hybridoma cell strain 21H5 is preserved in Chinese Academy of Sciences's microbe research on July 3rd, 2014
China Committee for Culture Collection of Microorganisms's common micro-organisms center of institute, address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number, postcode：100101, preserving number is CGMCC No. 9342,
The monoclonal antibody ascites indirect ELISA titer of anti cucumber mosaic virus is up to 10-8, Antibody types and subclass are
IgG1, kappa, the monoclonal antibody can have specific immunity association reaction with the capsid protein of cucumber mosaic virus, can examine
Measure the cucumber mosaic virus passed in virus mediator aphid body.
The monoclonal antibody of anti cucumber mosaic virus only has a specific immune response with cucumber mosaic virus, and with tomato
It is mosaic virus, odontoglossum ring spot virus, cucumber green mottle mosaic virus, Zucchini yellow mosaic virus, tobacco mosaic virus (TMV), melon
Chlorisis yellow virus, Bean common mosaic virus, rice black-streaked dwarf virus, Brassica 2 et 4, potato virus X, Ma Ling
Potato Y virus, marmor solani, potato virus S, rape mosaic disease poison, tomato spotted wilf virus, cymbidium mosaic virus are not sent out
Raw immune response.
Application of the anti cucumber mosaic virus monoclonal antibody on the Viral diagnosis is set up by core of monoclonal antibody
Various immunological detection methods and immunological reagent box.
The hybridoma cell strain 21H5 that the present invention is provided can largely secrete anti cucumber mosaic virus monoclonal antibody, and its secretion list
Anti- high specificity, potency are high, good stability.The high-throughout serological method of the detection CMV set up as core with the monoclonal antibody and
Its detection kit can be successfully applied to the detection of field CMV, thus for China's Cucumber Mosaic Virus early monitoring and early warning,
Detection and scientific guidance prevention and control provide material and technical support.
With reference to embodiment and accompanying drawing, the invention will be further described.
First, the acquisition of hybridoma and its preparation of monoclonal antibody
1. the preparation of immunogene and detection antigen
CMV virion is purified with following operating procedure：
1) by big mortar and tissue mixer device precooling；
2) 500g tobacco disease leaves are weighed, (i.e. PB delays the 0.5M phosphate buffers of addition pH 7.5 in every 100g diseases leaf
Fliud flushing）200ml（Na-EDTA containing 0.01M and 0.1% mercaptoethanol）, double-layer nylon filtered through gauze, filtrate are used after being homogenized 2 minutes
Centrifugation（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 cushions；
8) the gained precipitation 0.5M PB (MgCl containing 0.01M of pH 7.52）Suspend, suspension is Virus purification liquid；
9）Sample is purified with electron microscope observation, the cucumber mosaic virus particle of a large amount of high-purities is found.
2. animal is immunized
Four week old body weight 18-20g BALB/C female mices are immunized with CMV purified virus.That is CMV purified virus particle 35
μ L/ only mixs with isometric Freund's complete adjuvant, it is fully emulsified after, through carrying on the back subcutaneous abdomen multi-point injection 0.2ml every, be spaced 3
Week, take with one exempt from equivalent amount of antigen and isometric freund 's incomplete adjuvant it is fully emulsified after, second intraperitoneal injection 0.2ml is every
Only, intraperitoneal injection is carried out with the antigen of doubling dose after spending 3 weeks, extracting spleen cell is merged after 3 days.
3. cell fusion
Take above-mentioned immune mouse spleen cell and murine myeloma cell（SP2/0）By 10：1 ratio, in serum-free
RPMI-1640（Gibco）Mixed in culture medium, 1500rpm centrifugation 5min remove culture medium, with 50 % PEG（Sigma, molecule
Amount 1500）As fusion agent, 1ml is added under water-bath at 37 DEG C, make its fusion 2min, cultivated with the RPMI-1640 of serum-free
Base terminates 1500rpm centrifugation 5min after fusion, and precipitation is suspended with HAT culture mediums, is dispensed into the cell that feeder cells are contained in 96 holes
In plate, 37 DEG C, cultivated in the cell culture incubator of 5 % CO2.
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
It is antigen coat with the tobacco leaf and purified virus that infect CMV viruses, between routine when closing cell covering bottom hole 10%-30%
The positive hole of ELISA method screening secretion monoclonal antibody is connect, 210 positive holes are obtained altogether.15 cell holes in strong positive reaction of selection,
Limiting dilution assay clone is carried out, obtaining 1 plant can secrete the hybridoma cell strain 21H5 of specific monoclonal antibody of anti-CMV.Through 6 months with
After upper subculture in vitro separately and multiple cryopreservation resuscitation, cell line can well grow, and stably excreting antibody.After Amplification Culture, use
Prepared and Liquid nitrogen storage in ascites.
5. the specific detection of monoclonal antibody
With infection TOMV, odontoglossum ring spot virus, cucumber green mottle mosaic virus, small cucurbita pepo yellow mosaic disease
Poison, tobacco mosaic virus (TMV), melon chlorisis yellow virus, Bean common mosaic virus, rice black-streaked dwarf virus, Turnip mosaic virus
Poison, potato virus X, marmor upsilon, marmor solani, potato virus S, rape mosaic disease poison, tomato spotted wilf virus,
The sick leaf juice coating elisa plate of cymbidium mosaic virus, cucumber mosaic virus, negative control is made to be good for leaf extract accordingly, with
The sick leaf sap for infecting cucumber mosaic virus is positive control, and the specific reaction of monoclonal antibody is determined with ACP-ELISA methods.ACP-
ELISA method is specially：It is above-mentioned virus infection sick leaf liquid nitrogen grinding into powder, by 1: 30（w/v, g /mL）Add
ELISA coating buffers grinding after 100ul/ holes coating elisa plate, 4 DEG C overnight or 37 DEG C 2 hours, it is adsorbed in ELISA polyphenyl second
Alkene plate hole；PBST washs three times and closes 30-60min with the skimmed milk power or 1-3% BSA or 3-6% cow's serum of 1-10% afterwards；
Add the monoclonal antibody 100ul/ holes of appropriate dilution, 37 DEG C 1-2 hours；PBST washs three times and adds 10000 times of alkalescence of dilution afterwards
Phospholipase (AP) marks sheep anti-mouse igg secondary antibody（Sigma companies）100ul/ holes, 37 DEG C 1-2 hours, PBST wash four times after,
Developed the color with PNPP substrates, after 2mol/L NaOH terminating reactions, OD is read with ELIASA405Value, with negative OD values compare
Value is the positive more than 2.1.Result finds that 21H5 monoclonal antibodies have specific reaction to CMV, and with TOMV, tooth orchid ring spot
Virus, cucumber green mottle mosaic virus, Zucchini yellow mosaic virus, tobacco mosaic virus (TMV), melon chlorisis yellow virus, dish
Beans mosaic viruses, rice black-streaked dwarf virus, Brassica 2 et 4, potato virus X, marmor upsilon, potato A
Virus, potato virus S, rape mosaic disease poison, tomato spotted wilf virus, cymbidium mosaic virus and health plant are without specific anti-
Should.
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 volume 0.06M PH4.8 acetate buffers
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 type and 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、 IgG2a、IgG2b、IgG3、IgM
Antibody makees double agar diffusion test, as a result shows, 21H5 monoclonal antibodies subclass is IgG1, kappa.With conventional indirect ELISA side
Method detects odd contradictive hydroperitoneum potency, as a result for above-mentioned odd contradictive hydroperitoneum potency reaches 10- 8。
2nd, virological immunology detection method and its kit
1. antigen coat ELISA is set up by core of monoclonal antibody（ACP-ELISA）Method detection virus
The operating procedure of ACP-ELISA methods：
（1）Use 0.05M carbonate buffer solutions（pH9.6）By 1: 30（w/v, g /mL）The sick leaf sap for diluting again
100ul/ holes add elisa plate, with CMV disease leaf as positive control, be good for leaf be 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）10000 times of alkaline phosphatase lipase of dilution are added after PBST washings（AP）The sheep anti-mouse igg secondary antibody of mark
（Sigma）, 100ul/ holes, 37 DEG C, 1h；
（5）Nitro Phosphate substrate 100ul/ holes, room temperature 30min are added after being washed with PBST；
（6）Detect by an unaided eye, the hole that substrate colors become yellow green is the positive, or terminate anti-with 2mol/L NaOH
Ying Hou, surveys OD405, with P/N with enzyme-linked immunosorbent assay instrument>2.1 used as positive criterion.
Testing result finds the CMV that the above-mentioned ACP-ELISA methods that the monoclonal antibody is set up can be detected in plant sample well
Virus.
ACP-ELISA methods carry out sensitivity technique：
Odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, to sick leaf from 1：20 to 40960 make doubling dilution respectively with corresponding
The strong leaf sap of dilution factor makees negative control, carries out above-mentioned ACP-ELISA methods detection.Result shows ACP-ELISA methods pair
1：40960 times of dilutions（w/v, g/ml）The detection of sick leaf sap be still positive, i.e. the sensitivity to the sick leaf of detection can reach
1：40960, show that ACP-ELISA methods have the sensitivity and reliability of height.
2. the foundation of dot-ELISA methods and Fields detection application
The foundation of CMV methods and its field sample detection in 2.1 dot-ELISA detection plants
With liquid nitrogen grinding into powder after sick leaf is 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, while
Healthy and susceptible sick leaf juice is set respectively as negative and positive control；Drying at room temperature 10-20 min;NC films are immersed in containing 5%
Room temperature closes 30 min in PBST (the 0.01 mol/L PBS containing 0.05% Tween-20) confining liquid of skimmed milk power；NC films
It is put into incubation at room temperature 30-60 min in the monoclonal antibody of appropriateness dilution；Film is washed with PBST 3-4 times, every time 3 min；NC films are put into appropriateness
30-60 min are incubated at room temperature in the AP enzymes mark sheep anti-mouse igg secondary antibody of 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 the sick leaf of detection are tested with square formation, table is tested
The most suitable working concentration of bright 21H5 monoclonal antibodies and ELIAS secondary antibody is respectively 1:6000 and 1:8000 times of dilutions.With the most suitable of above-mentioned antibody
Working concentration sets up the dot-ELISA methods of detection CMV.Sensitivity analysis shows, when tobacco leaf is diluted to 1:20480 times
（w/v, g/mL）When, the dot-ELISA detections set up with 21H5 monoclonal antibodies are still presented the positive spots of purple, the i.e. sick leaf of its detection
Sensitivity reach 1:20480 times of dilutions（Fig. 1）.
With the dot-ELISA methods set up 2014 are picked up from the doubtful morbidity sample in Zhejiang field to detect, is as a result sent out
It is existing, there are 51 samples to produce purpuriferous positive spots in 123 detection samples, positive is further analyzed with PCR, as a result table
Bright all of dot-ELISA positives detect the specific PCR primers of CMV, and PCR primer nucleic acid sequencing shows positive sample
Product infect CMV.Illustrate that the dot-ELISA methods can accurately and reliably be used for the detection of cucumber mosaic virus in plant sample.
The foundation of CMV methods and its field sample detection in 2.2 dot-ELISA detection aphid bodies
Single Aphids are 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 aphid with toothpick, take and checked on NC films on 3 μ L, film is dried, monoclonal antibody is incubated, secondary antibody is incubated, colour developing step
Suddenly identical with CMV methods in dot-ELISA detection plants, different simply secondary antibodies is the sheep anti mouse of the HRP marks of appropriateness dilution
IgG secondary antibodies, chromogenic substrate is the chromogenic substrate of HRP, i.e. TMB chromogenic substrates.Non-band poison and the aphid with poison are set simultaneously as the moon
Property and positive control.
The most suitable working concentration of the dot-ELISA monoclonal antibodies and ELIAS secondary antibody that determine detection aphid is tested with square formation, table is tested
It is bright to prepare monoclonal antibody and the most suitable working concentration of ELIAS secondary antibody is respectively 1:4000 and 1:5000 times of dilutions.With the most suitable of above-mentioned antibody
Working concentration sets up the dot-ELISA methods of CMV in detection aphid body, and testing result shows that the aphid for carrying CMV is presented blue
Spot, and nontoxic aphid does not have any chromogenic reaction, that is, the dot-ELISA methods set up can be detected specifically in aphid body
CMV.
With set up dot-ELISA methods 2014 are picked up from Zhejiang Hangzhou artificial feeding obtain poison and nontoxic aphid enter
Row detection.Result finds have 82 to produce blue positive spots in 213 aphid detection samples, and nontoxic aphid does not have
Produce any blue spot.Positive is further analyzed with RT-PCR, as a result shows that all of dot-ELISA positives are equal
The specific PCR primers of CMV are detected, PCR primer nucleic acid sequencing shows that positive infects CMV.Illustrate the dot- ELISA
Method can accurately and reliably be used for the detection of CMV in aphid sample.
3 cucumber mosaic virus dot-ELISA detection kits
1）Kit main component：
The ml of 1 pipe of CMV monoclonal antibodies 0.2
The ml of 1 pipe of AP mark sheep anti-mouse igg secondary antibodies 0.1
The ml of 1 pipe of sheep anti-mouse igg secondary antibody 0.1 of HRP marks
Each 1 bottle of NBT/BCIP substrates are respectively 2 ml and 1ml
1 bottle of 10 ml of tmb substrate
Positive control 1（Leaf juice containing CMV）The ml of 1 pipe 2
Negative control 1（Health tobacco leaf juice）The ml of 1 pipe 2
Positive control 2（Band poison CMV aphid homogenates）The ml of 1 pipe 2
Negative control 2（Non- mosaic disease institute pesticide 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）The operating procedure of plant sample detection：
A. with liquid nitrogen grinding into powder after plant 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 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 tobacco 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 2000 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 two of 3000 dilutions
Incubation at room temperature 30-60 min in anti-;
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
Buffer solution (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 bottom
Reacted in thing 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 aphid sample：
A. Single Aphids are put into the centrifuge tube of the eppendorf of 0.5 mL, and add 10-50 μ L PBS
(0.01mol/L, pH7.4), after mashing aphid with toothpick, takes and checked on NC films on 3 μ L, while setting band poison and non-band poison
Aphid respectively as negative and positive control, drying at room temperature 10-20 min；
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 4000 times of monoclonal antibodies of dilution；
D. film is washed 3~4 times with PBST, every time 3 min；NC films are put into 1:5000 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 keeps 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 (4)
1. it is a kind of secrete anti cucumber mosaic virus monoclonal antibody hybridoma cell strain 21H5, it is characterised in that can secrete anti-yellowing
The monoclonal antibody specific of melon mosaic virus, hybridoma cell strain 21H5 was preserved in China Microbiological on July 3rd, 2014
Culture presevation administration committee common micro-organisms center, preserving number is CGMCC No.9342.
2. a kind of monoclonal antibody of the anti cucumber mosaic virus of hybridoma cell strain 21H5 as claimed in claim 1 secretion,
It is characterized in that described monoclonal antibody ascites indirect ELISA titer is up to 10-8, Antibody types and subclass are IgG1, kappa
Chain, monoclonal antibody can have specific immunity association reaction with the capsid protein of cucumber mosaic virus, can detect biography virus mediator
Cucumber mosaic virus in aphid body.
3. the monoclonal antibody of the anti cucumber mosaic virus of hybridoma cell strain 21H5 as claimed in claim 2 secretion, it is special
It is that described monoclonal antibody only has specific immune response with cucumber mosaic virus to levy, and blue with TOMV, tooth
Ring spot virus, cucumber green mottle mosaic virus, Zucchini yellow mosaic virus, tobacco mosaic virus (TMV), melon chlorisis yellows
Poison, Bean common mosaic virus, rice black-streaked dwarf virus, Brassica 2 et 4, potato virus X, marmor upsilon, horse
There is no immune response in bell potato A viruses, potato virus S, rape mosaic disease poison, tomato spotted wilf virus, cymbidium mosaic virus.
4. a kind of monoclonal antibody of anti cucumber mosaic virus as claimed in claim 2 cucumber mosaic virus detection on should
With, it is characterised in that set up various immunological detection methods and immunological detecting kit by core of the monoclonal antibody.
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| CN101748136A (en) * | 2010-01-15 | 2010-06-23 | 浙江大学 | Expression of cymbidium mosaic virus coat protein gene and preparation method for antibody of cymbidium mosaic virus coat protein gene |
| CN101852807A (en) * | 2010-05-14 | 2010-10-06 | 云南省农业科学院生物技术与种质资源研究所 | Sorghum mosaic virus tri-anti sandwich enzyme-linked immuno sorbent assay kit |
| CN102994455A (en) * | 2012-12-27 | 2013-03-27 | 北京世纪元亨动物防疫技术有限公司 | Monoclonal antibody and kit for cucumber green mottle mosaic viruses (CGMMVs) |
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| CN101748136A (en) * | 2010-01-15 | 2010-06-23 | 浙江大学 | Expression of cymbidium mosaic virus coat protein gene and preparation method for antibody of cymbidium mosaic virus coat protein gene |
| CN101852807A (en) * | 2010-05-14 | 2010-10-06 | 云南省农业科学院生物技术与种质资源研究所 | Sorghum mosaic virus tri-anti sandwich enzyme-linked immuno sorbent assay kit |
| CN102994455A (en) * | 2012-12-27 | 2013-03-27 | 北京世纪元亨动物防疫技术有限公司 | Monoclonal antibody and kit for cucumber green mottle mosaic viruses (CGMMVs) |
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