CN1706866A - Rice stripe virus monoclonal antibody and rice stripe virus immunodetecting method - Google Patents

Abstract

The present invention discloses monoclonal antibody of rice stripe virus (RSV) and its immunodetecting method. Specific monoclonal antibody of RSV is used as tool in establishing immunodetecting method for RSV in rice and RSV propagating insect. The specific process includes: immunizing bal b/c mouse with purified RSV to prepare hybridoma cell, specifically screening hybridoma with positive culture supernatant to obtain four monoclonal antibody strains of specifically resisting RSV, and establishing direct/indirect ELISA detection method with these monoclonal antibodies. The establishment and application of the said method makes it possible to detect rice disease sample and insect with RSV conveniently, accurately and fast.

Description

The immunological detection method of rice stripe virus monoclonal antibody and rice stripe virus

Technical field

The present invention relates to a kind of monoclonal antibody of rice stripe virus and the application on agricultural thereof---the immunological detection method of rice stripe virus, belong to the microbiological technique field.

Background technology

The stripe disease that rice stripe virus (RSV) causes found that all there is generation the back in Korea, Ukraine and China in the Japanese Northeast early than 1897.China from 1962 since this virus is found in the Jiangsu and Zhejiang Provinces, the whole nation 16 provinces popular ^[2]This disease at present in Yunnan, Liaoning, Beijing, Henan, Shandong, Jiangsu, Shanghai are still very common, the particularly Golconda in Yunnan, Chuxiong, Kunming, Pekinese's doube bridge, the Yuanyang, Henan, ground such as the Jiangyan City in Jining, Shandong and the north, Jiangsu, Hongze take place more general.

Its major cause that continues to break out is: warm winter weather in recent years helps surviving the winter of infection insect small brown rice planthopper, and small brown rice planthopper poison source radix is accumulated for many years, adds that easy susceptible rice varieties plants in a large number.According to data analysis, the expert estimates that stripe disease in 2003 will be and lays particular stress on occurrence tendency that prevention work is very urgent.

And stripe disease is the viroses of plant of propagating with small brown rice planthopper, still pasts medical help in case morbidity is caused harm, so the control of this disease should serve as crucial i.e. " controlling the worm diseases prevention " with the control small brown rice planthopper.And the band poisonous insect of small brown rice planthopper amount is a stripe disease generation popular main affecting factors ^[3]Therefore, setting up a kind of rapid sensitive, to detect small brown rice planthopper be very necessary with the method for malicious rate for the popular and disease control of prediction stripe disease etc.Be used at present to detect small brown rice planthopper in the world and comprise ELISA, RT-PCR, Northern hybridization etc., but RT-PCR and Northern cost height are unsuitable for the mass detection sample with the method for malicious rate; And the domestic RSV monoclonal antibody that do not prepare as yet is used for the virus detection, detect relatively rapid sensitive and carry out ELISA with monoclonal antibody, and simple, be applicable to mass detection, the establishing of this method helps the testing staff of China basic unit and carries out early detection and early warning in time to the band poisonous insect in early days.

Summary of the invention

The purpose of this invention is to provide the rice stripe virus monoclonal antibody, a kind of this application of rice stripe virus monoclonal antibody on agricultural that utilize is provided simultaneously, be about to it and be used to detect the immunological detection method of rice stripe virus.

Rice stripe virus monoclonal antibody of the present invention has following feature:

1) by a kind of immunoglobulin (Ig) of hybridoma excretory;

2) this immunoglobulin (Ig) has the height uniformity;

3) this immunoglobulin (Ig) can combine with rice stripe virus (rice stripe virus) specificity;

In addition, this immunoglobulin (Ig) can be used for detecting insect, the intravital rice stripe virus of plant.

Described monoclonal antibody is to obtain by following method:

(1) with the RSV of differential centrifugation and the sucrose gradient centrifugation BAL B/C mouse in immune 8 ages in week, gets its splenocyte after three immunity, merge through 50%PEG, obtain hybridoma with murine myeloma cell;

(2) merge after 10 days and use the HT substratum instead, when the 10%-50% of each culture hole hybridoma coverage hole, adopt indirect ELISA method to detect hybridoma growth hole culture supernatant;

(3) selection has the hybridoma cell line of positive reaction, through 3 limiting dilution assays clones, can obtain the monoclonal antibody hybridoma cell strain.In 3 months through 40 succeeding transfer culture, cell well-grown, and continue stably excreting antibody.Mouse peritoneal is injected pristane, inject monoclonal antibody hybridoma cell again after about 7 days, gather BALB/c mouse ascites, every desirable approximately 8-10mL ascites of mouse after about 10 days.Recording tiring of each strain ascites with indirect ELISA is respectively: 1: 5120000 (3B9); 1: 640000 (2H2); 1: 80000 (2E5); 1: 320000 (2E4).

The step that detects the immunology Indirect Detecting Method of rice stripe virus by rice stripe virus monoclonal antibody of the present invention is:

1) wraps by sample: paddy rice sample 0.05M Na to be checked ₂CO ₃-NaHCO ₃Coating buffer does 1: the 20-200 dilution, and the insect sample does 1: 30-3000 dilution, every hole add 100ul 37 degree and spent night in 2-4 hour or 4;

2) sealing: 1-5% skim-milk-PBST 37 degree sealings 20-30 minute;

3) add the rice stripe virus monoclonal antibody, promptly add 1: the monoclonal antibody ascites of 5000-50000 dilution, every hole add 100ul 37 degree reactions 1-2 hour;

4) washing: use pH7.2,0.01M PBST washing ELISA Sptting plate 3-5 time, each three minutes;

5) add horseradish peroxidase-labeled sheep or rabbit anti-mouse igg, add the horseradish peroxidase-labeled sheep anti mouse of working concentration, every hole adds 100ul 37 degree reactions 1-2 hour;

6) washing: same step 4);

7) colour developing: add the suitable every hole of substrate and add 100ul colour developing 37 degree 20-30 minute,

8) termination reaction: add 2M H ₂SO ₄Every hole 50ul stops the ELISA color reaction;

9) read the OD value: the absorbance value or the visual inspection color reaction judged result that read the 490nm wavelength.

The immunology that detects rice stripe virus by rice stripe virus monoclonal antibody of the present invention is straight

The step that connects detection method is:

1) by sample with paddy rice sample 0.05M Na to be checked ₂CO ₃-NaHCO ₃Coating buffer does 1: the 20-200 dilution, the insect sample does 1: the 30-3000 dilution.Every hole adds 100ul 37 degree and spent night in 2-4 hour or 4;

2) sealing: 1-5% skim-milk-PBST 37 degree sealings 20-30 minute;

3) add the enzyme labelling rice stripe virus monoclonal antibody that enzyme labelling rice stripe virus monoclonal antibody is diluted at 1: 10000, every hole adds 100ul 37 degree reactions 1-2 hour;

4) washing: with indirect ELISA step 4;

5) colour developing: add the suitable every hole of substrate and add 100ul colour developing 37 degree 20-30 minute,

6) termination reaction: add 2M H ₂SO ₄Every hole 50ul stops the ELISA color reaction;

7) read the OD value: the absorbance value or the visual inspection color reaction judged result that read the 490nm wavelength.(how to judge and to illustrate)

The present invention mainly serves the quick and precisely detection of rice stripe virus paddy rice and band kissing bug, can be used for basic unit and sanitary authority and in time detect early warning, and prevent trouble before it happens as early as possible, reduce the paddy rice number of infected, in time prevent to be with kissing bug further to bite other healthy water rice plants, thereby reduce the output of local paddy rice, prevent to be with kissing bug to take virus to other area simultaneously by migrating, endanger other healthy paddy rice, and carry out the work of pesticide desinsection before sudden in that insect is circuitous, in case the band kissing bug had bitten the healthy water rice plants, to there be effective means to control the morbidity of these rice plants and the harm of virus again, as seen be important route of infection with biting of kissing bug, thereby the quantity of control band kissing bug is one of key of control virus harm, understanding timely and accurately to the band poison situation of the band of paddy rice poison situation and insect just seems extremely important thus, in time the kind of the kissing bug that ungirds, the length of time of insect, work such as the density of insect, in time carry out and detect forecast work and the medication of suiting the medicine to the illness, laxative is controlled worm work, the medication of suiting the medicine to the illness, in time desinsection is the current important measures that effectively reduce financial loss.Thereby the early stage accurately detection of strip virus just seemed extremely important.Also do not have a kind of detection method that basic unit's sanitary authority used and had the rice stripe virus of independent intellectual property right that is suitable in view of domestic, of can be described as current present status in China of the present invention in time fills up.

Embodiment

The monoclonal antibody development of virus is by the BAL B/C mouse with 8 ages in week of RSV immunity of differential centrifugation and sucrose gradient centrifugation, get its splenocyte after three immunity, merge through 50%PEG with murine myeloma cell, in 288 culture hole, have the hybridoma growth is arranged in 235 holes, fusion rate is 82%, merge and use the HT substratum instead after 10 days, when the 10%-50% of every hole hybridoma coverage hole, adopt indirect ELISA method to detect 235 hybridoma growth hole culture supernatant, wherein 54 hole performance positive reactions, positive rate is 23%.Select the hybridoma cell line of 9 tool strong positive reactions, through 3 limiting dilution assays clone, obtain 4 strain monoclonal antibody hybridoma cell strains (2E4,2E5,3B9,2H2) altogether, in 3 months through 40 succeeding transfer culture, the cell well-grown, and continue stably excreting antibody.Mouse peritoneal is injected pristane, inject monoclonal antibody hybridoma cell again after about 7 days, gather BALB/c mouse ascites, every desirable approximately 8-10mL ascites of mouse after about 10 days.Recording tiring of each strain ascites with indirect ELISA is respectively: 1: 5120000 (3B9); 1: 640000 (2H2); 1: 80000 (2E5); 1: 320000 (2E4).

Determining of detection sensitivity

Behind the purified virus of RSV and the sick leaf sap doubling dilution, carry out indirect ELISA and measure.To picking up from the sick leaf of the paddy rice in land for growing field crops, 4 strain monoclonal antibodies all have higher detection sensitivity.The sensitivity that 3B9,2H2 detect the disease leaf can reach 1: 10240, and the sensitivity that 2E4,2E5 detect the disease leaf can reach 1: 5120.They are faint to the reaction of healthy juice.The sensitivity that 3B9 detects the RSV purified virus reaches 1: 256000, and the sensitivity that 2H2 detects the RSV purified virus reaches 1: 64000 (Fig. 1).

Monoclonal antibody is to different plant virus specific assay

Carry out indirect ELISA with 4 strain monoclonal antibodies respectively with 5 kinds of different plant viruses and detect, do positive control with the sick leaf sap of RSV, healthy paddy rice juice is cooked negative control.The result show 4 strain monoclonal antibodies all only with the RSV reaction, all do not react with other viruses.Wherein 3B9,2H2 and RSV reaction is stronger, a little less than 2E4,2E5 and the RSV reaction.

The specificity of table 1 monoclonal antibody is identified

Table?1?Specifities?of?MAbs?tested?by?ELISA

??Vrius	????????????????????????????MAbs
					??3B9	??2H2	??2E4	??2E5
	??RSV
??TMV					??1.003	??0.996	??0.834	??0.805
	??CMV	??0.06	??0.061	??0.061					??0.066
??ToMV					??0.048	??0.049	??0.048	??0.049
	??BBWV1	??0.065	??0.055	??0.056					??0.068
??BBWV2					??0.050	??0.054	??0.054	??0.062
	??PVX	??0.054	??0.050	??0.051					??0.051
??Health					??0.054	??0.058	??0.065	??0.062
	??CK	??0.060	??0.053	??0.054					??0.055

Above-mentioned monoclonal antibody has the specificity immunology recognition reaction at rice stripe virus, and at other virus, as: viruses such as TMV, CMV, ToMV, BBWV1, BBWV2, PVX, normal paddy rice leaf, normal insect etc. do not have the immunology cross reaction, we utilize these monoclonal antibody characteristics to set up directly/the indirect ELISA immunological detection method, for rice stripe virus detects means and the method that science is provided, provide an important instrument to the harm of timely early warning, controlling rice stripe disease.

Western?blot

To the RSV Western-blot analysis revealed of purifying, 4 strain monoclonal antibodies can both combine with the coat protein subunit specificity of RSV 35kD

Utilize monoclonal antibody to detect the band poison rate of small brown rice planthopper

Utilize the monoclonal antibody of preparation to detect to picking up from the band poison rate of Jiangsu Province's part counties and cities small brown rice planthopper.Pick up from the small brown rice planthopper in morbidity rice field, ground such as Jianhu, Wujin, Chu Zhou, Hongze, Gaoyou, Jingjiang, Hai'an, Jiangyan City, Guanyun and choose 96 wantonly as detected object, corresponding band poisonous insect number is respectively 12,18,20,24,30,32,32,36,40; Corresponding band poison rate is respectively 12.5%, 18.75%, 20.83%, 25%, 31.25%, 33.33%, 33.33%, 37.5%, 41.5%.The band poison rate that this shows small brown rice planthopper is still very high, and our monoclonal antibody of preparation can perform well in the detection of big Tanaka small brown rice planthopper with malicious rate.

Indirect elisa method

Be RSV monoclonal antibody hybridoma 3B9, the 2H2 cell strain ascites of development, tire and working concentration through the doubling dilution survey, after the mensuration, determine that OD value 490 is between 2.0-1.0, the odd contradictive hydroperitoneum extent of dilution is between 10000-50000, so select 1: 10000 working concentration as antibody at that time.

After having determined the monoclonal antibody working concentration, further the weaker concn of working sample is determined after measured, and the working concentration of paddy rice sample is 1: 30 (bag is by elisa plate), and the testing concentration of small brown rice planthopper (insect) is: 1: 300 (bag is by elisa plate)

Embodiment 1

The step of indirect method is:

1) wraps by sample paddy rice sample 0.05M Na to be checked ₂CO ₃-NaHCO ₃Coating buffer is done dilution in 1: 30, and every hole adds 100ul 37 degree and spent night in 2 hours or 4;

2) sealing: 1% skim-milk-PBST 37 degree sealed 30 minutes;

3) add our unit and develop anti-rice stripe virus specific cell strain (3B9) monoclonal antibody, this monoclonal antibody ascites is done 50000 dilutions, every hole adds 100ul 37 degree reactions 1 hour;

4) washing: with pH 7.2,0.01M PBST washing ELISA Sptting plate 3 times, each three minutes;

5) add horseradish peroxidase-labeled mark sheep or rabbit anti-mouse igg, the horseradish peroxidase-labeled sheep anti mouse is that SIGMA company or other companies sell, and adds the horseradish peroxidase-labeled sheep anti mouse of working concentration, and every hole adds 100ul 37 degree reactions 1 hour;

6) washing: with step 4;

7) colour developing: add the every hole of substrate and add 100ul colour developing 37 degree 20 minutes,

The substrate prescription is: 20mg O-Phenylene Diamine (OPD)

25.7ml??0.2M?Na ₂HPO4.12H ₂O

24.3ml 0.1M citric acid

75ul????30％H ₂O ₂；

8) termination reaction: add 2M H ₂SO ₄Every hole 50ul stops the ELISA color reaction;

9) read the OD value: the absorbance value or the visual inspection color reaction judged result that read the 490nm wavelength.

Embodiment 2

Removing detected object is the insect sample, and the object that detects---insect sample is done outside the dilution in 1: 300, and other are operated all with embodiment 1

Direct ELISA method step:

Reaction is with behind the odd contradictive hydroperitoneum purifying, further carries out enzyme labelling, prepares enzyme mark monoclonal antibody, is applied to detect test.

Concrete ELISA reactions steps is as follows:

1, by sample with paddy rice sample 0.05M Na to be checked ₂CO ₃-NaHCO ₃Coating buffer is done dilution in 1: 30, and the insect sample is done dilution in 1: 300.Every hole adds 100ul 37 degree and spent night in 2 hours or 4.

2, sealing: 1% skim-milk-PBST 37 degree sealed 30 minutes

3, the enzyme labelling monoclonal antibody that adds the dilution in 1: 10000 of enzyme labelling monoclonal antibody, every hole add 100ul 37 degree reactions 1 hour.

4, washing: with indirect ELISA step 4

5, colour developing: add the every hole of substrate and add 100ul colour developing 37 degree 20 minutes,

The substrate prescription is: 20mg O-Phenylene Diamine (OPD)

25.7ml??0.2M?Na ₂HPO4.12H ₂O

24.3ml 0.1M citric acid

75ul????30％H ₂O ₂

6, termination reaction: add 2M H ₂SO ₄Every hole 50ul stops the ELISA color reaction,

7, read the OD value: the absorbance value or the visual inspection color reaction judged result that read the 490nm wavelength.

With obtaining specificity at the rice stripe virus monoclonal antibody, build between foundation/direct ELISA method, be convenient to sanitary authority and basic unit and in time measure the state of an illness of rice stripe disease in paddy rice, in time understanding insect is with malicious situation in time to carry out desinsection work, stop the band kissing bug further to endanger other healthy paddy rice of this area, reduce financial loss, paddy rice prevents to be with kissing bug to migrate simultaneously, virus is spread other areas, and carry out the circular epidemic situation and take place and the work of epidemic situation development prediction, forecast according to epidemic situation, in time carry out desinsection work, carry out and prevent virus intrusion preparation work.

Above-mentioned enforcement does not limit the present invention in any form.

Claims (4)

1, a kind of rice stripe virus monoclonal antibody, it has following properties:

1) by a kind of immunoglobulin (Ig) of hybridoma excretory;

2) this immunoglobulin (Ig) has the height uniformity;

3) this immunoglobulin (Ig) can combine with the rice stripe virus specificity.

2, detect the immunology Indirect Detecting Method of rice stripe virus according to the described rice stripe virus monoclonal antibody of claim 1, it is characterized in that step is:

1) wraps by sample: paddy rice sample 0.05M Na to be checked ₂CO ₃-NaHCO ₃Coating buffer does 1: the 20-200 dilution, and the insect sample does 1: 30-3000 dilution, every hole add 100ul 37 degree and spent night in 2-4 hour or 4;

2) sealing: 1-5% skim-milk-PBST 37 degree sealings 2--30 minute;

3) add the rice stripe virus monoclonal antibody, promptly add 1: the monoclonal antibody ascites of 5000-50000 dilution, every hole add 100ul 37 degree reactions 1-2 hour;

4) washing: use pH7.0-7.2,0.01M PBST washing ELISA Sptting plate 3-5 time, each three minutes;

5) add horseradish peroxidase-labeled sheep or rabbit anti-mouse igg, add the horseradish peroxidase-labeled sheep anti mouse of working concentration, every hole adds 100ul 37 degree reactions 1-2 hour;

6) washing: same step 4);

7) colour developing: add suitable substrate, every hole adds 100ul colour developing 37 degree 20-30 minute;

8) termination reaction: add 2M H ₂SO ₄Every hole 50ul stops the ELISA color reaction;

9) read the OD value: the absorbance value or the visual inspection color reaction judged result that read the 490nm wavelength.

3, according to the immunology Indirect Detecting Method of the described rice stripe virus of claim 2, it is characterized in that: chromogenic substrate is by 20mg O-Phenylene Diamine, 25.7ml 0.2M Na ₂HPO ₄.12H ₂O, 24.3ml 0.1M citric acid, 75ul 30%H ₂O ₂Formulated.

4, detect the immunology direct detecting method of rice stripe virus according to the described rice stripe virus monoclonal antibody of claim 1, it is characterized in that step is:

1) by sample with paddy rice sample 0.05M Na to be checked ₂CO ₃-NaHCO ₃Coating buffer does 1: the 20-200 dilution, and the insect sample does 1: the 30-3000 dilution; Every hole adds 100ul 37 degree and spent night in 2-4 hour or 4;

2) sealing: 1-5% skim-milk-PBST 37 degree sealings 20-30 minute;

3) add enzyme labelling rice stripe virus monoclonal antibody: the enzyme labelling rice stripe virus monoclonal antibody of dilution in 1: 10000, every hole add 100ul 37 degree reactions 1-2 hour;

4) washing: use pH7.0-7.2,0.01M PBST washing ELISA Sptting plate 3-5 time, each three minutes;

5) colour developing: add suitable substrate, every hole adds 100ul colour developing 37 degree 20-30 minute,

6) termination reaction: add 2M H ₂SO ₄Every hole 50ul stops the ELISA color reaction;

7) read the OD value: the absorbance value or the visual inspection color reaction judged result that read the 490nm wavelength.