CN102533665B - Hybridoma cell strain secreting monoclonal antibody against rice ragged stunt virus and use of monoclonal antibody - Google Patents
Hybridoma cell strain secreting monoclonal antibody against rice ragged stunt virus and use of monoclonal antibody Download PDFInfo
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Abstract
The invention discloses a hybridoma cell strain secreting a monoclonal antibody against rice ragged stunt viruses (RRSV) and use of the monoclonal antibody. Purified RRSV coat protein is used as an antigen to immunize a BALB/C mouse, and thus a hybridoma cell strain 4H5 which can be passed down steadily and can secrete monoclonal antibody against RRSV is obtained, wherein the collection number of the hybridoma cell strain 4H5 is CGMCCNo.5536. The result of Westernblot detection indicates the monoclonal antibody secreted by the hybridoma cell strain 4H5 reacts with the RRSV coat protein specifically. The indirect enzyme-linked immuno sorbent assay(ELSA) titer of the hybridoma cell strain 4H5 monoclonal antibody ascitic fluid reaches 10<-7>, the type and subtype of the antibody is IgG1 and kappa chain. According to a dot-ELISA detection method established by using the 4H5 monoclonal antibody for detecting brown rice plant hopper and RRSV in rice, when single-head brown rice plant hopper is diluted to 800mu L and affected leaves is diluted according to a weight-volume (g/mL) ratio of 1:320, viruses can still be detected. The established dot-ELISA method can detect brown rice plant hopper and RRSV in rice accurately, specifically and sensitively. The preparation of the monoclonal antibody against RRSV and the establishment of the detection method thereof provide technical and material supports for diagnosing, predicting and forecasting and scientifically preventing and controlling rice virus diseases.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of the secrete hybridoma cell strain of water resistant rice tingia dwarf virus monoclonal antibody and the application of monoclonal antibody thereof.
Background technology
Paddy rice tingia dwarf virus (Rice ragged stunt virus, RRSV) is a kind of new virus of finding at eighties of last century end of the seventies, in South East Asia, the local generation in East Asia and some countries of South Asia, Rice Production is caused certain influence.Mainly occur at present the ground such as Guangxi, Fujian, Hunan, Jiangxi, Guizhou, Yunnan of China.Dark green the stunting of diseased plant performance, the leaf curling indentation is incised, and has elongated arteries and veins swollen at blade back and leaf sheath.The symptoms such as before tillering phase, morbidity can not be eared, and the later stage falls ill, and heading is incomplete, grain does not enrich.The general underproduction 10%～20% of morbidity field, the serious underproduction can reach 80%～100%, and being affects one of more serious virus disease of rice yield.RRSV is the prototypical member of Reoviridae Oryzavirus (Oryzavirus), causes the paddy rice tingia disease of stunting.The RRSV genome comprises 10 double-stranded RNA fragments (S1-S10), and the virus that the electrophoretogram of its morphological structure and genome double-stranded RNA and two other genus-Phytoreovirus of this section (Phytoreovirus) and Fijivirus belong to (Fifivirus) has larger difference.RRSV has double capsid, is icosahedral structure of virus, and the diameter of intact virus plastochondria is about 65nm, and core particle is 50nm approximately.Can be observed under Electronic Speculum in the viroplast (viro-plasm) that diameter 50～66nm or the virus particle of 40nm be distributed in susceptible rice leaf bast cell; With also can be observed diameter 40～45nm or 50～75nm, the two crystalline particles of class spherical junctions in the organ or tissue in the poisonous insect body, assemble or be arranged in dispersedly in cytoplasmic viroplast.The paddy rice sawtooth leaf disease of stunting is mainly to be caused by the RRSV virus that brown paddy plant hopper is propagated, and diseased plant juice, seed and soil all do not pass poison.Virus particle is spherical, and wider projection is arranged at the bottom.Its natural host has paddy rice, yard grass, Shortleaf Kyllinga Herb and barnyard grass, and the plant of manually infecting has the kinds more than 10 such as barley, wheat, oat, corn, sugarcane, barnyard grass.The amboceptor insect of RRSV is brown paddy plant hopper (Nilaparvatalugens), is a kind of migrating property insect, and it is propagated and to be the persistence mode, but transovarian transmission not.The nymph of brown paddy plant hopper casts off a skin but does not lose poison, and the distribution of virus in polypide is the highest with content in sialisterium.It is 3 hours (h) that the amboceptor minimum obtains the poison time, average 9 days (d).The shortest biography poison phase is 1h, and after inoculation, 10-36d shows symptom, passes malicious rate and is approximately 40%.Larva may be higher than the biography toxic effect rate of adult, female worm and male worm, and long wing type is identical with brachypterism type biography toxic effect rate.Still pass poison when amboceptor is casted off a skin, amboceptor passes poison and usually has the intermittent poison phase that passes, and can keep 1-4 week or pass throughout one's life poison.Four types of brown paddy plant hopper have similar biography poison characteristic.In order to grasp brown paddy plant hopper Natural Population band poison and rice pathogenesis situation, strengthen China paddy rice tingia stunt disease early monitoring and early warning, the scientific guidance prevention and control, be badly in need of setting up the high-throughout detection method that reaches RRSV in paddy rice in detection brown paddy plant hopper body, and there is no at present this high-throughout detection method, only can detect small sample with inefficient electron microscopic observation, RT-PCR method, nucleic acid electrophoresis etc.The present invention has prepared the monoclonal antibody specific of the 1 anti-RRSV of strain take the RRSV capsid protein (CP) of prokaryotic expression by hybridoma technology as antigen, monoclonal antibody take preparation has been set up the high-throughout serological method that detects RRSV as core, and be successfully applied to the detection of field RRSV, thereby be China paddy rice tingia stunt disease early monitoring and early warning, the scientific guidance prevention and control provide material and technical support.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, provide a kind of hybridoma cell strain and monoclonal antibody thereof of secreting water resistant rice tingia dwarf virus monoclonal antibody to use.
The hybridoma cell strain of secretion water resistant rice tingia dwarf virus monoclonal antibody, preserving number is CGMCC No.5536, it can secrete the monoclonal antibody of water resistant rice tingia dwarf virus.
The monoclonal antibody ascites indirect ELISA titer of water resistant rice tingia dwarf virus reaches 10
-7, Antibody types and subclass are IgG1, kappa chain, the coat protein subunit of this monoclonal anti physical efficiency and paddy rice tingia dwarf virus has the specific immunity association reaction.
The application of water resistant rice tingia dwarf virus monoclonal antibody on this virus detects is various immunological detection methods and the immunological detecting kit thereof of setting up take monoclonal antibody as core.
The beneficial effect that the present invention compared with prior art has: the water resistant rice tingia dwarf virus monoclonal antibody specific of the hybridoma cell strain that 1) provides secretion, the immunological method such as dot-ELISA, ACP-ELISA, DAS-ELISA and the TAS-ELISA that sets up take this monoclonal antibody as core and the test kit energy high special of setting up with these methods, accurate, sensitive detection paddy rice tingia dwarf virus; 2) utilize the prepared monoclonal antibody of the present invention to detect paddy rice tingia dwarf virus, do not need the equipment such as expensive electron microscope, PCR instrument; 3) utilize the prepared monoclonal antibody of the present invention, can effectively be used for the detection of the paddy rice tingia dwarf virus of field water rice crops and brown paddy plant hopper.
Description of drawings
Fig. 1 is the sensitivity analysis that the dot-ELISA method detects paddy rice RRSV.Susceptible rice leaf is respectively from 1:10, and doubling dilution is until 1:5120, and CK-is healthy paddy rice, and 1,2 is the repetition of same sample;
Fig. 2 is the result that the dot-ELISA method detects RRSV in the rice field sample, and for having infected the paddy rice of paddy rice tingia dwarf virus, CK-is healthy paddy rice to CK+ for the band poison;
Fig. 3 is the sensitivity analysis that the dot-ELISA method detects RRSV in the brown paddy plant hopper body;
Fig. 4 is the result that the dot-ELISA method detects RRSV in the brown paddy plant hopper body of field, and 1,2,3,4,5,6,7,8 represent respectively 8 field brown paddy plant hopper samples.
Embodiment
The hybridoma cell strain of secretion water resistant rice tingia dwarf virus monoclonal antibody, on November 28th, 2011, be preserved in China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center, preserving number is CGMCC No.5536, and it can secrete the monoclonal antibody of water resistant rice tingia dwarf virus.
The monoclonal antibody ascites indirect ELISA titer of water resistant rice tingia dwarf virus reaches 10
-7, Antibody types and subclass are IgG1, kappa chain, the coat protein subunit of this monoclonal anti physical efficiency and paddy rice tingia dwarf virus has the specific immunity association reaction.
The application of water resistant rice tingia dwarf virus monoclonal antibody on this virus detects is various immunological detection methods and the immunological detecting kit thereof of setting up take monoclonal antibody as core.
Hybridoma cell strain provided by the invention is secreted water resistant rice tingia dwarf virus monoclonal antibody in a large number, and its high specificity, the height of tiring, good stability.The high-throughout immunological method and the immunological detecting kit thereof that detect RRSV have been set up take this monoclonal antibody as core, can be successfully applied to the detection of field RRSV, thereby be China paddy rice tingia stunt disease early monitoring and early warning, the scientific guidance prevention and control provide material and technical support.
The invention will be further described below in conjunction with embodiment and accompanying drawing.
One, the preparation of hybridoma acquisition and monoclonal antibody thereof
1. the preparation of immunogen and detectable antigens
According to capsid protein gene (CP gene) sequence of the paddy rice tingia dwarf virus (RRSV) of reporting in GeneBank (number of logging in: EU523360) design Auele Specific Primer, RRSV-CP-F:
GGATCC CGAATCATCACTGAACAAGTATTTGG and RRSV-CP-R:
AAGCTT TCATACACCGGAACCGCTGG, underscore are respectively BamHI and HindIII restriction enzyme site.Pcr amplification obtains the CP gene that total length is about 1500bp, goal gene is inserted into pMD-18T carrier (RRSV-CP-pMD18T) upward and cuts screening by PCR and enzyme and obtains the pMD-18T-CP recon after reclaiming, sequencing shows that the CP gene and the RRSV CP gene order that are cloned into are identical.CP gene in recombinant plasmid pMD-18T-CP is connected on prokaryotic expression carrier PMAL-C2X after BamHI and HindIII double digestion, obtain recombinant expression vector pMAL-C2X-CP, cut with sequencing analysis through PCR, enzyme and show that the CP gene inserts prokaryotic expression carrier by correct reading frame, and base is not undergone mutation.Recombinant plasmid pMAL-C2X-CP imports respectively e. coli bl21 (DE3), picking list bacterium colony is cultivated in the LB nutrient solution, after expressing, 0.5mmol/L IPTG inducible protein finds that the RRSV-CP-pMAL-c2x with the MBP label can the correction size be the recombinant protein of 80KDa left and right, and utilize MBP pillar purifying, the maltose buffer solution elution has obtained the higher purifying protein of concentration.Take the recombinant protein of purifying as immunogen and detectable antigens.
2. immune animal
With recombinant protein RRSV-CP immunity surrounding body weight in the age 18-20g BALB/C female mice of expressing, the fusion rotein 0.5ml of the RRSV of 1mg/ml purifying mixes with equal-volume Fu Shi Freund's complete adjuvant, after fully emulsified, through every of back of the body subcutaneous abdomen multi-point injection 0.2ml, 3 weeks of interval, get with one exempt from equivalent antigen and isopyknic freund 's incomplete adjuvant fully emulsified after, abdominal injection 0.2ml is every for the second time, antigen with doubling dose after spending for 3 weeks 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) in the ratio of 5-10:1, RPMI-1640(Gibco at serum-free) mixing in substratum, the centrifugal 5min of 1500rpm, remove substratum, with 50 % PEG(Sigma, molecular weight 1500) as fusogen, add 1ml under 37 ℃ of lower water-baths, make it merge 2min, RPMI-1640 substratum with serum-free stops the rear centrifugal 5min of 1500rpm of fusion, and precipitation suspends with the HAT substratum, divides to install in the cell plate in 96 holes, 37 ℃, cultivate in the cell culture incubator of 5 % CO2.
4. the screening in hybridoma, positive hole and clone thereof
Cultivate after 5 days in cell culture incubator, change liquid once with the HAT substratum, changed liquid with the HT substratum on the 10th day, by the time at the bottom of the fused cell coverage hole during 5%-50%, take the RRSV-CP detectable antigens as envelope antigen, with the positive hole of conventional indirect ELISA method screening, obtain altogether 123 positive holes.Select 5 cell holes that are strong positive reaction, carry out the limiting dilution assay clone, obtain the hybridoma cell strain 4H5 that 1 strain can be secreted the specific monoclonal antibody of anti-RRSV.Through subculture in vitro separately more than 6 months with repeatedly after cryopreservation resuscitation, cell strain all can well be grown, and can stably excreting antibody.After enlarged culturing, be used for ascites preparation and liquid nitrogen and preserve.
5. the specific detection of monoclonal antibody
with infecting Brassica 2 et 4 (TuMV), Tomato mosaic virus (ToMV), tobacco mosaic virus (TMV) (TMV), cucumber mosaic virus (CMV), potato virus X (PVX) and marmor upsilon (PVY), fractilinea oryzae (RDV), rice stripe virus (RSV), southern rice black-streaked dwarf virus (SRBSDV), rice black-streaked dwarf virus (RBSDV), the sick leaf juice of paddy rice tingia dwarf virus (RRSV) is coated with the ELISA Sptting plate, make negative control with corresponding healthy leaf extract, with the positive contrast of paddy rice tingia dwarf virus, measure the specific reaction of monoclonal antibody with indirect elisa method.Indirect ELISA method is specially the sick leaf liquid nitrogen grinding powdered of above-mentioned virus infection, press 1:30(w/v, the coated elisa plate in 100ul/ hole after g/mL) adds the ELISA coating buffer to grind, 4 ℃ spend the night or 37 ℃ 2 hours, make it be adsorbed in the polystyrene plate hole; Skim-milk or 1-3% BSA or the 3-6% bovine serum sealing 30-60min of 1-10% used in the PBST washing for three times afterwards; Add monoclonal antibody 100ul/ hole, 37 ℃ 1-2 hour; PBST washs anti-(Sigma company) the 100ul/ hole of alkaline phosphatase lipase (AP) mark rabbit anti-mouse igg two that adds 10000 times of by specification dilutions after three times, 37 ℃ 1-2 hour, after PBST washing four times, develop the color with the PNPP substrate, after 2mol/L sodium hydroxide termination reaction, read OD with microplate reader
405Value, with positive greater than 2.1 with negative OD value ratio.Found that, the 4H5 monoclonal antibody has specific reaction to RRSV, and with TuMV, TMV, ToMV, CMV, PVY, PVX, RDV, RSV, other viruses of SRBSDV, RBSDV all without specific reaction.
6. monoclonal antibody ascites prepares and purifying
Get BALB/C mice about 8 ages in week, abdominal injection 0.3-0.5ml pristane (Sigma), pneumoretroperitoneum injected 5-10 * 10 in 7-10 days
5Individual hybridoma, after injection, 7-10 days visible mouse web portions obviously expand, and take ascites, and the centrifugal 3min of 3000rpm collects supernatant liquor, is monoclonal antibody ascites.Get 1 times of volume ascites and add 2 times of volume 0.06M PH4.8 acetate buffer solution dilutions, add sad (30ul/ml ascites), the following edged of room temperature stirs, clarified 1 hour for 4 ℃, the centrifugal 20min of 12000rpm collects supernatant, then uses 50% saturated ammonium sulphate immunoglobulin (Ig), placed 2 hours for 4 ℃, the centrifugal 20min of 3000rpm, precipitation is with the PBS solution dissolving of 2 times of volumes, under 4 ℃ of conditions, the dialysis of flowing namely obtains the ascites antibody of purifying ,-70 ℃ of preservations after 24 hours.
7. the subgroup identification of monoclonal antibody and titer of ascites are measured
The anti-BALB/C mice IgG of standard with the odd contradictive hydroperitoneum of purifying and Sigma company
1, IgG
2a, IgG
2b, IgG
3, IgM antibody makes double agar diffusion test, experimental result shows: 4H5 monoclonal antibody subclass is IgG1, kappa chain.Detect odd contradictive hydroperitoneum with conventional indirect ELISA method and tire, result is that above-mentioned odd contradictive hydroperitoneum is tired 10
-7
Two, immunological detection method and test kit thereof
1. set up indirect antigen coated ELISA(ACP-ELISA with monoclonal antibody) method detection virus
The operation steps of ACP-ELISA method:
(1) press 1:30(w/v with 0.05M carbonate buffer solution (pH9.6), g/mL) doubly dilution sick leaf sap or add by every brown paddy plant hopper of 150 ul carbonate buffer solutions after the supernatant 100ul/ hole of mashing add elisa plate, the sick leaf of RRSV or the positive contrast of brown paddy plant hopper of carrying RRSV, corresponding healthy leaf or the negative contrast of non-band poison brown paddy plant hopper, 37 ℃ of 2h, or 4 ℃ of coated spending the night;
(2) after the PBST washing, with 5% skim-milk sealing 30min;
(3) 100ul/ hole after 5000 times of dilutions of odd contradictive hydroperitoneum, 37 ℃ of 1h;
(4) add alkaline phosphatase lipase (AP) or the horseradish mistake of 5000 times of dilutions after the PBST washing
The sheep anti-mouse igg two of oxide compound enzyme (HRP) mark anti-(Sigma), 100ul/ hole, 37
℃，1h；
(5) with adding nitro phosphoric acid salt substrate or TMB substrate 100ul/ hole, room temperature 30min after PBST washing;
(6) detect by an unaided eye, substrate colors become yellow-green colour or blue hole positive, or use
After 2mol/L sodium hydroxide or sulfuric acid termination reaction, with enzyme-linked immunosorbent assay instrument survey OD405 or
450 values are with P/N〉2.1 as positive judging criterion.
ACP-ELISA method detection sensitivity detects
The odd contradictive hydroperitoneum working concentration is 5000 times of dilutions, sick leaf is made doubling dilution or the single head brown paddy plant hopper carries out doubling dilution 50uL/ head to the 12800uL/ head from 1:10 to 5120, make negative control with corresponding dilution healthy leaf sap respectively, carry out above-mentioned ACP-ELISA method and detect.Result shows that the ACP-ELISA method all is positive when the sick leaf sap of 1:10～320 times dilution and single head brown paddy plant hopper are diluted to 3200uL/, namely can reach 1:320 to the sensitivity that detects the disease leaf, detection sensitivity to brown paddy plant hopper reaches 1600 uL/ heads, shows that the ACP-ELISA method has susceptibility and the reliability of height.
2. detect the TAS-ELISA detection method of RRSV
2.1. the operating process of TAS-ELISA method:
1) the coated polystyrene board in the rabbit anti-serum of the anti-RRSV purification of Recombinant RRSV CP protein immunization rabbit of the expression (obtain) 100ul/ hole, 37 ℃, 2-4h or 4 ℃, coated spending the night;
2) add the skim-milk of 1-10% or 1-3% BSA or 3-6% bovine serum sealing 200ul/ hole after PBST washing three times, in 37 ℃ of sealing 30-60min;
3) add test sample 100ul/ hole.With the sick leaf of RRSV or the positive contrast of brown paddy plant hopper of carrying RRSV, make negative control, 37 ℃ of 1-2h with corresponding healthy sample or non-brown paddy plant hopper with RRSV;
4) dilute the odd contradictive hydroperitoneum 100ul/ hole of 5000 times, 37 ℃ of 1-2h after the washing with confining liquid;
5) add the AP of 10000 times of dilutions or HRP mark rabbit anti-mouse igg two to resist (Sigma) 100ul/ hole, 37 ℃ of 1-2h after the PBST washing;
6) add PNPP substrate or tmb substrate after the PBST washing in color development at room temperature 5-30min, the visual inspection substrate colors become yellow-green colour or blue hole positive, after 2mol/L sodium hydroxide or sulfuric acid termination reaction, survey the OD value of 405nm or 450nm with 680 type enzyme-linked immunosorbent assay instruments, with P/N〉2.1 as positive judging criterion.
2.2.TAS-ELISA determining of detection method optimum condition:
Adopt the test of TAS-ELISA square formation to carry out, namely at first laterally add respectively with the RRSV rabbit anti-serum of coated damping fluid from 1 ﹕ 100 to 1 ﹕ 102400 doubling dilutions; Second step adds the sick leaf juice (1:10) of RRSV or brown paddy plant hopper homogenate (150ul/ only); The 3rd step vertically added respectively with confining liquid from 1 ﹕ 5 to 1 ﹕ 2048000 doubling dilution odd contradictive hydroperitoneums; The 4th step added the rabbit anti-mouse igg two of AP mark or HRP note anti-by 10000 times of dilutions of Sigma specification sheets 1 ﹕ of company; The 5th step PNPP or tmb substrate colour developing (operating by the TAS-ELISA method flow).Result is that the rabbit anti-serum of brown paddy plant hopper and the optimal dilution of monoclonal antibody are respectively 1 ﹕ 5000,1 ﹕ 8000.
2.3.TAS-ELISA determining of method detection sensitivity
Under rabbit anti-serum and the suitableeest working concentration of odd contradictive hydroperitoneum, the sick leaf juice of RRSV or brown paddy plant hopper homogenate are carried out TAS-ELISA mensuration after with the PBS doubling dilution, result is: the sensitivity of the TAS-ELISA sick leaf of detection and brown paddy plant hopper reaches respectively 1:1250 and doubly dilutes (w/v, g/mL) He 1600 uL/ heads illustrate that present method has good sensitivity.
3. the foundation of dot-ELISA method and Fields detection are used
3.1 dot-ELISA detects foundation and the field sample detection thereof of RRSV method in paddy rice
With the rice leaf rear liquid nitrogen grinding powdered of using of weighing, press 1:10～30(w/v, g/mL) adds 0.01 mol/L PBS(pH7.4) after grind; Centrifugal 3 min of sick juice 5000 rpm; Get on 3 μ l and check on the NC film, health and susceptible Rice Leaf juice are set simultaneously respectively as feminine gender and positive control; Drying at room temperature 10-20 min; The NC film is immersed in room temperature sealing 30 min in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim-milk; The NC film is put into the monoclonal antibody incubated at room 30-60 min of appropriateness dilution; Wash film 3～4 times with PBST, each 3 min; The NC film is put into anti-incubated at room 30 ~ 60 min of AP enzyme labelling sheep anti-mouse igg two of appropriateness dilution; PBST washes film 4～5 times, each 3 min; 66 μ L NBT and 33 μ L BCIP substrates (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, the visual inspection result.Treat positive control colour developing obviously, and negative tap water rinsing termination reaction during without any colour developing, the Taking Pictures recording result.
Determine to detect the dot-ELISA monoclonal antibody of the sick leaf of paddy rice and the suitableeest working concentration of ELIAS secondary antibody with the square formation test, test shows that the suitableeest working concentration of 4H5 monoclonal antibody and ELIAS secondary antibody is respectively 1:5000 and 1:8000 doubly dilutes.The suitableeest working concentration with above-mentioned antibody is set up the dot-ELISA method that detects the sick leaf of RRSV.Sensitivity analysis shows, when rice leaf is diluted to 1:320 times (w/v, g/mL), still presents the positive spots of purple with the dot-ELISA detection of 4H5 monoclonal antibody foundation, i.e. its sensitivity that detects sick leaf reaches 1:320 and doubly dilutes (Fig. 1).
Detect picking up from the doubtful morbidity of the rice field paddy rice samples such as Foochow, Fujian, Libo, Guizhou, Jinhua, Zhejiang, Shidian County, Western Yunnan county in 2010,2011 with the dot-ELISA method of setting up, found that, 21 purpuric positive spots of sample (Fig. 2) are arranged in 50 paddy rice test sample, positive is further analyzed with RT-PCR, result shows that all dot-ELISA positive all detect the specific PCR product of RRSV, and PCR product nucleic acid sequencing shows that positive infects RRSV.Illustrate this dot-ELISA method can be accurately, be used for reliably the detection of paddy rice sample paddy rice tingia dwarf virus.
3.2 dot-ELISA detects foundation and the field sample detection thereof of RRSV method in the brown paddy plant hopper body
The single head brown paddy plant hopper is put into the centrifuge tube of the eppendorf of 1.5 mL, and add 100 μ L PBS (0.01mol/L, pH7.4), after mashing brown paddy plant hopper with toothpick, centrifugal 3 min of 5000 rpm, get on 3 μ L and check on the NC film, film is dry, monoclonal antibody is hatched, two anti-ly hatch, development step is identical with RRSV method in dot-ELISA detection paddy rice, different just two anti-sheep anti-mouse iggs two for the appropriate HRP mark that dilutes are anti-, chromogenic substrate is the chromogenic substrate of HRP, i.e. the TMB chromogenic substrate.Establish simultaneously the brown paddy plant hopper of non-band poison and band poison as feminine gender and positive control.
Determine to detect the dot-ELISA monoclonal antibody of brown paddy plant hopper and the suitableeest working concentration of ELIAS secondary antibody with the square formation test, test shows that the suitableeest working concentration of 4H5 monoclonal antibody and ELIAS secondary antibody is respectively 1:4000 and 1:5000 doubly dilutes.The suitableeest working concentration with above-mentioned antibody is set up the dot-ELISA method that detects RRSV in the brown paddy plant hopper body, detected result shows that the brown paddy plant hopper of carrying RRSV presents blue spot, and nontoxic brown paddy plant hopper is without any color reaction, and the dot-ELISA method of namely setting up can detect the RRSV in the brown paddy plant hopper body specifically.Sensitivity analysis shows, when the single head brown paddy plant hopper adds 800 μ L PBS, still presents the positive spots of blueness with the dot-ELISA detection of 4H5 monoclonal antibody foundation, i.e. its sensitivity that detects brown paddy plant hopper reaches 1:800 dilution (head/μ L) (Fig. 3).
With the dot-ELISA method of setting up, the brown paddy plant hopper with nontoxic that brown paddy plant hopper, the artificial feeding who picked up from the rice pathogenesis field such as Foochow, Fujian, Libo, Guizhou, Shidian County, Western Yunnan county in 2010,2011 obtains poison is detected.Found that, have 61 to produce blue positive spots in 110 brown paddy plant hopper test sample, and nontoxic brown paddy plant hopper does not produce any blue spot (Fig. 4).Positive is further analyzed with RT-PCR, and result shows that all dot-ELISA positive all detect the specific PCR product of RRSV, and PCR product nucleic acid sequencing shows that positive infects RRSV.Illustrate this dot-ELISA method can be accurately, be used for reliably the detection of brown paddy plant hopper sample paddy rice tingia dwarf virus.
3.3 paddy rice tingia dwarf virus dot-ELISA detection kit (detecting paddy rice and brown paddy plant hopper sample)
1) test kit main component:
RRSV monoclonal antibody 1 pipe 0.2 ml
Anti-1 pipe 0.1 ml of AP mark sheep anti-mouse igg two
Anti-1 pipe 0.1 ml of the sheep anti-mouse igg two of HRP mark
The NBT/BCIP substrate each 1 bottle be respectively 2 ml and 1ml
1 bottle of 10 ml of tmb substrate
Positive control 1 (containing RRSV rice leaf juice) 1 pipe 2 ml
Positive control 2 (being with malicious RRSV brown paddy plant hopper homogenate) 1 pipe 2ml
The healthy paddy rice leaf juice of negative control 1() 1 pipe 2 ml
The malicious brown paddy plant hopper homogenate of the non-band of negative control 2() 1 pipe 2 ml
1 bottle of 80ml of antibody diluent (10X)
Above reagent all is stored under 4 ℃
10 of nitrocellulose filters (NC)
2) detect the operation steps of paddy rice sample:
A. the rear liquid nitrogen grinding powdered of using of rice leaf being weighed is pressed 1:10～30(w/v, and g/mL) adds 0.01 mol/L PBS(pH7.4) after grind;
B. centrifugal 3 min of sick juice 5000 rpm;
C. get on 3 μ l and check on NC, health and susceptible Rice Leaf juice are set simultaneously respectively as feminine gender and positive control, drying at room temperature 10～20 min;
The d.NC film is immersed in room temperature sealing 30 min in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim-milk;
The e.NC film is put into monoclonal antibody incubated at room 30～60 min that 1:5000 doubly dilutes;
F. wash film 3～4 times with PBST, each 3 min; The NC film is put 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, the visual inspection result;
H. treat positive control colour developing obviously (purple), and negative tap water rinsing termination reaction during without any colour developing, the Taking Pictures recording result.
3) detect the operation steps of white backed planthopper sample:
A. the single head brown paddy plant hopper is put into the centrifuge tube of the eppendorf of 1.5 mL, and add 50～100 μ L PBS (0.01mol/L, pH7.4), after mashing brown paddy plant hopper with toothpick, centrifugal 3 min of 5000 rpm, get on 3 μ L and check on the NC film, the brown paddy plant hopper of band poison and non-band poison is set simultaneously respectively as feminine gender and positive control, drying at room temperature 10～20 min;
B. the NC film is immersed in room temperature sealing 30 min in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim-milk;
C. the NC film is put into monoclonal antibody incubated at room 30～60 min that 1:4000 doubly dilutes;
D. wash film 3～4 times with PBST, each 3 min; The NC film is put into anti-incubated at room 30～60 min of HRP enzyme labelling sheep anti-mouse igg two that 1:5000 doubly dilutes;
E. PBST washes film 4～5 times, each 3 min; Film is put into tmb substrate liquid and is reacted, the visual inspection result;
F. treat positive control colour developing obviously (blueness), and negative tap water rinsing termination reaction during without any colour developing, the Taking Pictures recording result.
4) preservation and validity period
Keep in Dark Place in 2～8 ℃, 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
4 0.2 g
Na
2HPO
4·12H
2O 3g
Sodiumazide 0.2 g
Transfer pH to 7.4 after adding distil water 950 dissolvings, be settled to 1000 ml
ELISA washings (0.01 mol/L PBST):
Add 0.5 ml Tween-20 in 1000 ml 0.01mol/L PBS
The ELISA confining liquid:
0.01 add skim-milk in mol/L PBST to final concentration 5%(W/V).
Claims (3)
1. hybridoma cell strain of secreting water resistant rice tingia dwarf virus monoclonal antibody, preserving number is CGMCC No.5536, it is characterized in that secreting the monoclonal antibody of water resistant rice tingia dwarf virus.
2. the monoclonal antibody of the water resistant rice tingia dwarf virus of a hybridoma cell strain secretion as claimed in claim 1, is characterized in that this monoclonal antibody ascites indirect ELISA titer reaches 10
-7, Antibody types and subclass are IgG1, kappa chain, the coat protein subunit of this monoclonal anti physical efficiency and paddy rice tingia dwarf virus has the specific immunity association reaction.
3. the application of water resistant rice tingia dwarf virus monoclonal antibody as claimed in claim 2 on this virus detects, is characterized in that various immunological detection methods and the immunological detecting kit thereof set up take monoclonal antibody as core.
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