CN109633152A - A kind of colloid gold immune test paper item and preparation method thereof of quick detection rice stripe virus - Google Patents
A kind of colloid gold immune test paper item and preparation method thereof of quick detection rice stripe virus Download PDFInfo
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- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The invention discloses a kind of colloid gold immune test paper items and preparation method thereof of quickly detection rice stripe virus (RSV).Anti- RSV monoclonal antibody is gold labeling antibody after colloid gold label, and it is coated on bonding pad made of polyester film, another monoclonal antibody of anti-RSV and sheep anti-mouse igg are respectively in connection in the detection line of nitrocellulose filter (T line) and nature controlling line (C line), then sample pad made of absorbent filter, RSV immune colloid gold pad, the nitrocellulose filter for being combined with RSV monoclonal antibody 11C1 and sheep anti-mouse igg and blotting paper are successively pasted on PVC offset plate respectively, test strips is made.Test strips of the invention can accurate, special, delicately detect the RSV in field rice and single head small brown rice planthopper sample.The test strips with the naked eye can directly observe testing result in 3-5min, be suitable for field large sample and detect, and provide technology and supporting substances to the diagnosis of stripe disease, early monitoring and early warning, breeding for disease resistance, epidemiological study and scientific prevention and cure.
Description
Technical field
Field belonging to the present invention is field of biotechnology, more particularly, to a kind of glue of quickly detection rice stripe virus
Body gold immunity test strip and preparation method thereof.
Background technique
It is distributed widely in Asia, especially in the stripe disease that the area outburst such as Japan, China is caused disaster is flown by ash
The rice stripe virus of louse-borne is (caused by (Rice stripe virus, RSV).The virus is directly led in rice seedling infection
It causes rice seedling withered, though the rice of forth infection is not withered, destroys the reproductive growth of rice, cause to ear abnormal, and with not plump
Based on paddy, rice yield is seriously affected.Therefore, which is the important virus disease for threatening China's Rice Production.Rice item
Line virus is the prototypical member of very thin Tobamovirus, by small brown rice planthopper with persistent fashion and transovarian transmission, can infect rice, wheat etc.
More than 20 kinds of gramineae plants.Rice stripe virus can be bred in small brown rice planthopper body, and small brown rice planthopper once obtains poison, i.e., lifelong band poison, height
Effect passes poison, and can transovarian transmission.And the Fast Practical detection technique of RSV is the viral diagnosis, resistant variety breeding, plant disease epidemic
Rule investigation, science bridle Establishing key.At present generally using field Symptom Observation, RT-PCR detection, Electronic Speculum observation
Carry out the checkout and diagnosis virus with dot-ELISA serological method.Field Symptom Observation accuracy is low, and virus infection rice early period
Equal plants do not have Symptoms, and RT-PCR and Electronic Speculum observation are required to precision instrument and professional operator, are only applicable to reality
Test the detection of room small sample.Only detection of the dot-ELISA serological method suitable for field batch samples at present, but it is consumed
When it is still longer, and colloid gold immune test paper item it is easy to operate, do not have to any instrument, complete sample detection in 3-5min, inspection
It surveys result to detect by an unaided eye judgement, is fully compatible for grass-roots unit and peasant's Field information.
Summary of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of colloidal gold of quickly detection rice stripe virus
Immunity test strip and preparation method thereof.
A kind of colloid gold immune test paper item of quick detection rice stripe virus, in which: immune colloid gold pad is using absorption
There is the polyester film of the anti-rice stripe virus monoclonal antibody of colloid gold label, combines water resistant rice on detection line, that is, T line of nitrocellulose filter
Strip virus monoclonal antibody is combined with sheep anti-mouse igg on nature controlling line, that is, C line.
The colloid gold immune test paper item, can it is accurate, special, delicately detect rice, wheat and the intracorporal rice of small brown rice planthopper
Strip virus, and its sensitivity for detecting rice disease leaf texture crude extract reaches 1:20,480 times of dilutions, unit are gram/mL, inspection
The sensitivity that survey single head takes malicious small brown rice planthopper reaches 1:5,120 times of dilutions, and unit is head/μ L.
The colloid gold immune test paper item, rice, wheat and the small brown rice planthopper sample of detection infection rice stripe virus are in Qiang Yang
Property as a result, and detect infection rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, rice tingia dwarf virus, rice
Dwarf virus, rice stripe mosaic virus, the rice of Rice Gall Dwarf In Guangdong Province and healthy rice and infection of Chinese wheat streak mosaic virus,
Wheat yellow mosaic virus, the wheat of wheat dwarf virus and healthy wheat, the small brown rice planthopper without RSV virus, carrying south rice are black
The white backed planthopper of dwarf virus carries rice stripe mosaic virus or Rice Gall Dwarf In Guangdong Province recilia dorsalis, carries rice tingia
Dwarf virus brown paddy plant hopper is negative result.
In addition, the present invention also provides a kind of preparation sides of the colloid gold immune test paper item of quickly detection rice stripe virus
Method comprising following steps:
1) prepared by colloidal gold solution: by 0.01% gold chloride ebuillition of heated of 50mL, 1% trisodium citrate of 0.55mL is added
After mix, then keep boiling 30min, gold chloride is reduced into colloid gold particle solution；
2) prepared by rice stripe virus immune colloid gold solution: colloidal gold solution described in 50mL is taken, with 0.1M K2CO3By its
PH value is adjusted to 7.8, and the anti-rice stripe virus monoclonal antibody of 2mL 1mg/mL is added while stirring, stirs 30min；It is added dropwise again
Polyethylene glycol, that is, PEG solution that 1mL 25M molecular weight is 20000, is stirred for 15min；4 DEG C of 20000rpm are centrifuged 15min, in abandoning
Clear liquid, the PBS buffer solution that 10mL pH 7.4 and PEG containing 0.4M 20000 is added suspend, and 4 DEG C of 20000rpm are centrifuged 15min, go
Supernatant, precipitating repeat with PBS buffer solution suspend, centrifugation, then with 5mL pH 7.4 and the PBS buffer solution containing 2%BSA dissolution sink
It forms sediment, after being filtered with 0.22 μm of sterilizing filter, filtrate is immune colloid gold solution, is saved backup in 4 DEG C；
3) by 0.01M pH 7.4PBS buffer of the above-mentioned rice stripe virus immune colloid gold solution containing 30% sucrose
8 times of dilution, is sprayed in polyester film label pad with metal spraying machine by the metal spraying amount of 2.3 μ L/cm, forms rice stripe virus and glue is immunized
Body gold pad；
4) the anti-rice stripe virus monoclonal antibody of 1.0mg/mL is used to the 0.01M pH 7.4PBS buffer dilution 6 containing 5% sucrose
Times, with Film-cutting machine point on nitrocellulose filter detection line, that is, T line；The sheep anti-mouse igg of 1.2mg/mL is used containing 2% sucrose
0.01M pH 7.4PBS buffer dilutes 5 times, with Film-cutting machine point on nitrocellulose filter nature controlling line, that is, C line, makes T line and C line
At a distance of 5mm；
5) sample pad made of absorbent filter, rice stripe virus immune colloid gold pad, point are had to the nitric acid of T line and C line
Cellulose membrane and blotting paper successively paste on PVC offset plate, are assembled into test strips after being then cut into the band of 60mm × 3mm.
Rice stripe virus monoclonal antibody in the above method can be prepared via a method which: from infection rice stripe virus
BALB/c mouse is immunized as antigen in purified virus particle in rice plants tissue, hybridizes through cell fusion, screening, cell clone
Tumor technology obtains anti-RSV monoclonal antibody.Certainly, the preparation method with rice stripe virus monoclonal antibody is reported in the prior art,
It can be prepared according to its record method, can prepare colloid gold immune test paper item, but there may be differences for effect.
The present invention has the advantages that 1) the invention patent develops into RSV's for the first time both at home and abroad compared with prior art
Colloid gold immune test paper item, and colloid gold immune test paper item can it is accurate, special, delicately detect rice, wheat and small brown rice planthopper
RSV in sample；2) RSV is detected using colloid gold immune test paper item of the present invention, does not need any instrument, and operate letter
It is single, quick, it is only necessary to which that the sample detection including sample treatment can be completed in 3-5min, and testing result is with the naked eye directly seen
It examines, is more suitable for the application of grass-roots unit and peasant field.
Detailed description of the invention
The structural schematic diagram of Fig. 1 rice stripe virus colloid gold immune test paper item.
In figure: 1: sample pad, 2: gold conjugation pad, 3: nitrocellulose (NC) film, 4: detection line (T line), 5: control
Line (C line), 6: blotting paper, 7: adhesive sticker, 8:PVC bottom plate.
Fig. 2 rice stripe virus colloid gold immune test paper item detects the specificity analysis knot of RSV in rice and small brown rice planthopper body
Fruit.
A:RSV, SRBSDV, RBSDV, RRSV, RGDV, RSMV, RDV and Rice respectively represent infection rice stripe disease in figure
Poison, southern rice black-streaked dwarf virus, rice black-streaked dwarf virus, rice tingia dwarf virus, Rice Gall Dwarf In Guangdong Province, rice item
Line mosaic virus, fractilinea oryzae rice and healthy rice；It is small that CWMV, WYMV, WDV and Wheat respectively represent infection of Chinese
Wheat mosaic virus, wheat yellow mosaic virus, the wheat of wheat dwarf virus and health wheat.B:SBPH, WBPH and BPH generation respectively
Table small brown rice planthopper, white backed planthopper and brown paddy plant hopper.
Fig. 3 rice stripe virus colloid gold immune test paper item detects the sensitivity point of RSV in field rice and small brown rice planthopper body
Analyse result.
A in figure: the sensitivity analysis result of the test strips infection RSV rice plants of foundation；B: the test strips detection of foundation
Carry the sensitivity analysis result of RSV single head small brown rice planthopper.
Specific embodiment
The object of the present invention is to provide a kind of colloid gold immune test paper item of quickly detection rice stripe virus and its preparations
Method.
As shown in Figure 1, quickly the colloid gold immune test paper item of detection rice stripe virus is combined by sample pad 1, colloidal gold
Pad 2, nitrocellulose (NC) film, detection line (T line) 4, control line (C line) 5, blotting paper 6, adhesive sticker 7 and PVC bottom plate 8 form.
Wherein, immune colloid gold pad is using the polyester film for being adsorbed with the anti-rice stripe virus monoclonal antibody of colloid gold label, nitrocellulose filter
Detection line, that is, T line on combine anti-rice stripe virus monoclonal antibody, be combined with sheep anti-mouse igg on nature controlling line, that is, C line.
The colloid gold immune test paper item of rice stripe virus of the invention can it is accurate, special, delicately detect rice, wheat
With the intracorporal rice stripe virus of small brown rice planthopper, and its sensitivity for detecting rice disease leaf texture crude extract can reach 1:20, and 480 times
Dilution, unit are gram/mL, and the sensitivity that detection single head takes malicious small brown rice planthopper can reach 1:5, and 120 times of dilutions, unit is head/μ L.
The rice, small of the colloid gold immune test paper item detection infection rice stripe virus of water rice stripe virus of the invention
Wheat and small brown rice planthopper sample are in strong positive as a result, and detecting infection rice black-streaked dwarf virus, southern rice black-streaked dwarf virus, water
Rice tingia dwarf virus, fractilinea oryzae, rice stripe mosaic virus, the rice of Rice Gall Dwarf In Guangdong Province and healthy rice and sense
Contaminate the wheat and healthy wheat, the ash without RSV virus of Chinese wheat mosaic virus, wheat yellow mosaic virus, wheat dwarf virus
Plant hopper, the white backed planthopper for carrying southern rice black-streaked dwarf virus carry rice stripe mosaic virus or Rice Gall Dwarf In Guangdong Province electricity
Light leafhopper carries rice tingia dwarf virus brown paddy plant hopper and is negative result.
The step of quickly detecting the preparation method of colloid gold immune test paper item of rice stripe virus:
1) preparation of rice stripe virus monoclonal antibody: the virus purified from the rice plants tissue of infection rice stripe virus
Particle is that BALB/c mouse is immunized in antigen, obtains anti-RSV monoclonal through cell fusion, screening, cell clone hybridoma technology and resists
Body；
2) prepared by colloidal gold solution: by 0.01% gold chloride ebuillition of heated of 50mL, 1% trisodium citrate of 0.55mL is added
After mix, then keep boiling 30min, gold chloride is reduced into colloid gold particle solution；
3) prepared by rice stripe virus immune colloid gold solution: 50mL colloidal gold solution is taken, with 0.1M K2CO3By its pH value
7.8 are adjusted to, the anti-rice stripe virus monoclonal antibody of 2mL 1mg/mL is added while stirring, stirs 30min；1mL is added dropwise again
Polyethylene glycol, that is, PEG solution that 25M molecular weight is 20,000, is stirred for 15min；4 DEG C of 20,000rpm are centrifuged 15min, abandon supernatant
Liquid, the PBS buffer solution that 7.4 PEG containing 0.4M of 10mL pH 20,000 is added suspend, and 4 DEG C of 20,000rpm are centrifuged 15min, go
Clearly, precipitating is suspended with PBS buffer solution, is centrifuged, and is repeated 1 time, and the PBS buffer solution dissolution with 5mL pH 7.4 containing 2%BSA is heavy
It forms sediment, after being filtered with 0.22 μm of sterilizing filter, filtrate is 4 DEG C of immune colloid gold solution and saves backup；
4) by 0.01M pH 7.4PBS buffer of the above-mentioned rice stripe virus immune colloid gold solution containing 30% sucrose
8 times of dilution, is sprayed in polyester film label pad with metal spraying machine by the metal spraying amount of 2.3 μ L/cm, forms rice stripe virus and glue is immunized
Body gold pad；
5) the anti-rice stripe virus monoclonal antibody of 1.0mg/mL is used to the 0.01M pH 7.4PBS buffer dilution 6 containing 5% sucrose
Times, with Film-cutting machine point on nitrocellulose filter detection line, that is, T line；The sheep anti-mouse igg of 1.2mg/mL is used containing 2% sucrose
0.01M pH 7.4PBS buffer dilutes 5 times, with Film-cutting machine point on nitrocellulose filter nature controlling line, that is, C line, makes T line and C line
At a distance of 5mm；
6) sample pad made of absorbent filter, rice stripe virus immune colloid gold pad, point are had to the nitric acid of T line and C line
Cellulose membrane and blotting paper successively paste on PVC offset plate, are assembled into test strips after being then cut into the band of 60mm × 3mm.
Test strips provided by the invention accurate, special, can be detected delicately in field rice, wheat and small brown rice planthopper sample
RSV, and it is easy to operate, quick, any instrument is not needed, the detection time for completing sample only needs 3-5min, and with the naked eye directly
Judging result is observed, grass-roots unit and peasant's Field information are highly suitable for, thus diagnosis, early stage for stripe disease
Monitoring and early warning, breeding for disease resistance, epidemiological study and scientific prevention and cure provide technology and supporting substances.
Below with reference to embodiment and attached drawing, the invention will be further described.
One, the preparation step of colloid gold immune test paper item
A kind of preparation method of the colloid gold immune test paper item of quick detection rice stripe virus mainly includes following step
It is rapid:
(1) preparation of RSV monoclonal antibody: the purification of RSV virus and RSV monoclonal antibody preparation method are referring to applicant laboratory
Deliver paper [preparation of rice stripe virus monoclonal antibody and detection are using Plant Pathology .2004,34 (4):
302-306] method obtain 2 plants of anti-RSV monoclonal antibodies (being denoted as 16E6 and 11C1 respectively) through cell fusion, screening, clone；
(2) prepared by colloidal gold solution: by 0.01% gold chloride ebuillition of heated of 50mL, 1% citric acid three of 0.55mL is added
It is mixed after sodium, then keeps boiling 30min, gold chloride is reduced into colloid gold particle solution；
(3) prepared by rice stripe virus immune colloid gold solution: 50mL colloidal gold solution is taken, with 0.1M K2CO3By its pH
Value is adjusted to 7.8, and the anti-rice stripe virus 16E6 monoclonal antibody of 2mL 1mg/mL is added while stirring, stirs 30min；It is added dropwise again
Polyethylene glycol, that is, PEG solution that 1mL 25M molecular weight is 20,000, is stirred for 15min；4 DEG C of 20,000rpm are centrifuged 15min, abandon
Supernatant, the PBS buffer solution that 7.4 PEG containing 0.4M of 10mL pH 20,000 is added suspend, and 4 DEG C of 20,000rpm are centrifuged 15min,
Supernatant is removed, precipitating is suspended with PBS buffer solution, is centrifuged, and after repeating 1 time, the PBS buffer solution with 5mL pH 7.4 containing 2%BSA is molten
Solution precipitating, after being filtered with 0.22 μm of sterilizing filter, filtrate is 4 DEG C of immune colloid gold solution and saves backup；
(4) the 0.01M pH 7.4PBS containing 30% sucrose is used to buffer above-mentioned rice stripe virus immune colloid gold solution
Liquid dilutes 8 times, is sprayed in polyester film label pad with metal spraying machine by the metal spraying amount of 2.3 μ L/cm, and it is immune to form rice stripe virus
Colloidal gold pad；
(5) by 0.01M pH 7.4PBS buffer of the anti-rice stripe virus 11C1 monoclonal antibody of 1.0mg/mL containing 5% sucrose
6 times of dilution, with Film-cutting machine point on nitrocellulose filter detection line, that is, T line；The sheep anti-mouse igg of 1.2mg/mL, which is used, contains 2% sucrose
0.01M pH 7.4PBS buffer dilute 5 times, with Film-cutting machine point on nitrocellulose filter nature controlling line, that is, C line, make T line and C
Line is at a distance of 5mm；
(6) sample pad made of absorbent filter, rice stripe virus immune colloid gold pad, point are had to the nitric acid of T line and C line
Cellulose membrane and blotting paper successively paste on PVC offset plate, are assembled into shown in Fig. 1 after being then cut into the band of 60mm × 3mm
Test strips.
Two, the step of rice stripe virus colloid gold immune test paper item detection RSV and its specificity and sensitivity analysis
1, the step of rice stripe virus colloid gold immune test paper item detection RSV:
1. sample preparation:
1) preparation of plant sample: old rice, wheat plant tissue weighing are placed in mortar with liquid nitrogen grinding into powder
End adds the ratio of 4mL that 0.01M PBS (pH 7.4) is added and continues grinding homogenate 1min in mortar afterwards in 0.1g plant tissue；It is tender
Rice, add the ratio of 4mL that 0.01M PBS (pH 7.4) mortar afterwards is added in 0.1g plant tissue after wheat plant tissue weighing
Middle grinding is homogenized 2min；Homogenate moves in 1.5mL eppendorf centrifuge tube, 5,000rpm centrifugation 3min, or stands 5min；
2) preparation of small brown rice planthopper sample: addition after a small brown rice planthopper is put into the eppendorf centrifuge tube of a 0.5ml
150-200 μ l 0.01M PBS mashes plant hopper in homogenate with toothpick butt end or 200 μ l pipette tips；
2. point sample: taking 1 colloid gold immune test paper item to lay flat, draw plant sample supernatant with suction pipe or small brown rice planthopper is homogenized
Drop 3 drips (about 150-300 μ L) in well；
3. visually observing judging result: visually observing judging result in 3-5min, i.e. red bar occurs simultaneously in T line and C line
The sample of band is the positive, and red stripes occurs in C line and T line red stripes sample does not occur for feminine gender.If C line and T line do not go out
Existing red stripes, or only red stripes occur in T line and illustrate test strips when there are not red stripes to C line and fail.
2, rice stripe virus colloid gold immune test paper item detects the specificity analysis of RSV in rice and small brown rice planthopper body
To infect the rice of RSV and the crude extract respectively positive and the negative control of healthy rice plants tissue, test paper is used
The wheat and small brown rice planthopper, infection rice black-streaked dwarf virus, southern rice black-streaked dwarf disease of item detection infection rice stripe virus
Poison, rice tingia dwarf virus, fractilinea oryzae, rice stripe mosaic virus, the rice of Rice Gall Dwarf In Guangdong Province and healthy water
Rice and infection of Chinese wheat streak mosaic virus, wheat yellow mosaic virus, the wheat of wheat dwarf virus and healthy wheat, without RSV disease
Small brown rice planthopper, the white backed planthopper, recilia dorsalis, brown paddy plant hopper of poison.Testing result shows, the colloid gold immune test paper of rice stripe virus
Rice, wheat and the small brown rice planthopper sample of item detection infection rice stripe virus are in strong positive as a result, and detecting infection rice secret note
Dwarf virus, southern rice black-streaked dwarf virus, rice tingia dwarf virus, fractilinea oryzae, rice stripe mosaic virus,
The rice of Rice Gall Dwarf In Guangdong Province and healthy rice and infection of Chinese wheat streak mosaic virus, wheat yellow mosaic virus, wheat dwarf wilt
Malicious wheat and healthy wheat, the white backed planthopper for carrying southern rice black-streaked dwarf virus, carry water at the small brown rice planthopper without RSV virus
Rice stripe mosaic virus or Rice Gall Dwarf In Guangdong Province recilia dorsalis, carry rice tingia dwarf virus brown paddy plant hopper be negative as a result,
As a result as shown in Figure 2.
3, the sensitivity analysis that rice stripe virus colloid gold immune test paper item detects RSV in rice and small brown rice planthopper body will be felt
The rice plants tissue crude extract supernatant for contaminating RSV presses 1:20-1:40,960 doubling dilution (w/v, g/mL), to correspond to dilution
Rice health plant crude extract supernatant is negative control；Or the small brown rice planthopper of RSV will be carried by 1:160-1:40,960 (head/μ L) times
Plant sample extracting solution supernatant ash is drawn with suction pipe and is flown using the nontoxic small brown rice planthopper for corresponding to dilution as negative control than dilution
Lice homogenate, 3 drop of drop is in test strips well, as the result is shown when rice disease leaf is diluted to 1:20,480 (w/v, g/mL), list
Headband poison small brown rice planthopper is diluted to 1:5, and when 120 μ L, test strips detection T line remains to the band for occurring red, the i.e. Test paper
Item reaches 1:20480 times of dilution (w/v, g/mL) for the detection sensitivity of rice diseased tissues, for the detection spirit with malicious small brown rice planthopper
Sensitivity reaches 1:5, and 120 μ L times dilute (head/mL), as a result as shown in figure 3, showing that test strip of the invention has the spirit of height
Quick property and reliability.
Claims (5)
1. a kind of colloid gold immune test paper item of quickly detection rice stripe virus, which is characterized in that immune colloid gold pad uses
It is adsorbed with the polyester film of the anti-rice stripe virus monoclonal antibody of colloid gold label, is combined on detection line, that is, T line of nitrocellulose filter anti-
Rice stripe virus monoclonal antibody is combined with sheep anti-mouse igg on nature controlling line, that is, C line.
2. the colloid gold immune test paper item of quickly detection rice stripe virus as described in claim 1, it is characterised in that can be quasi-
Really, specifically, delicately detect rice, wheat and the intracorporal rice stripe virus of small brown rice planthopper, and its to detect rice disease leaf texture thick
The sensitivity of extract reaches 1:20,480 times of dilutions, and unit is gram/mL, and the sensitivity that detection single head takes malicious small brown rice planthopper reaches 1:5,
120 times of dilutions, unit are head/μ L.
3. the colloid gold immune test paper item of quickly detection rice stripe virus as described in claim 1, it is characterised in that the examination
Rice, wheat and the small brown rice planthopper sample of paper slip detection infection rice stripe virus are in strong positive as a result, and detection infection rice is black
Dwarf virus, southern rice black-streaked dwarf virus, rice tingia dwarf virus, fractilinea oryzae, rice stripe mosaic disease
Poison, the rice of Rice Gall Dwarf In Guangdong Province and healthy rice and infection of Chinese wheat streak mosaic virus, wheat yellow mosaic virus, wheat stunt
The wheat of virus and healthy wheat, the white backed planthopper for carrying southern rice black-streaked dwarf virus, are taken the small brown rice planthopper without RSV virus
Band rice stripe mosaic virus or Rice Gall Dwarf In Guangdong Province recilia dorsalis carry rice tingia dwarf virus brown paddy plant hopper and are negative knot
Fruit.
4. a kind of preparation method of the colloid gold immune test paper item of quickly detection rice stripe virus, it is characterised in that including following
Step:
1) prepared by colloidal gold solution: 0.01% gold chloride ebuillition of heated of 50mL mixes after 1% trisodium citrate of 0.55mL is added
It is even, then boiling 30min is kept, gold chloride is reduced into colloid gold particle solution；
2) prepared by rice stripe virus immune colloid gold solution: colloidal gold solution described in 50mL is taken, with 0.1M K2CO3By its pH value
7.8 are adjusted to, the anti-rice stripe virus monoclonal antibody of 2mL 1mg/mL is added while stirring, stirs 30min；1mL is added dropwise again
Polyethylene glycol, that is, PEG solution that 25M molecular weight is 20000, is stirred for 15min；4 DEG C of 20000rpm are centrifuged 15min, abandon supernatant
Liquid, the PBS buffer solution that 10mL pH 7.4 and PEG containing 0.4M 20000 is added suspend, and 4 DEG C of 20000rpm are centrifuged 15min, go
Clearly, precipitating repeat with PBS buffer solution suspend, centrifugation, then with 5mL pH 7.4 and the PBS buffer solution containing 2%BSA dissolution precipitate,
After being filtered with 0.22 μm of sterilizing filter, filtrate is immune colloid gold solution, is saved backup in 4 DEG C；
3) use 7.4 PBS buffer solution of 0.01M pH containing 30% sucrose dilute above-mentioned rice stripe virus immune colloid gold solution
8 times are released, is sprayed in polyester film label pad with metal spraying machine by the metal spraying amount of 2.3 μ L/cm, rice stripe virus is formed and colloid is immunized
Gold pad；
4) 7.4 PBS buffer solution of 0.01M pH containing 5% sucrose is used to dilute 6 times the anti-rice stripe virus monoclonal antibody of 1.0mg/mL,
With Film-cutting machine point on nitrocellulose filter detection line, that is, T line；0.01M of the sheep anti-mouse igg of 1.2mg/mL containing 2% sucrose
7.4 PBS buffer solution of pH dilutes 5 times, with Film-cutting machine point on nitrocellulose filter nature controlling line, that is, C line, makes T line and C line apart
5mm；
5) sample pad made of absorbent filter, rice stripe virus immune colloid gold pad, point are had to the cellulose nitrate of T line and C line
Plain film and blotting paper successively paste on PVC offset plate, are assembled into test strips after being then cut into the band of 60mm × 3mm.
5. quickly detecting the preparation method of the colloid gold immune test paper item of rice stripe virus, feature as claimed in claim 4
It is, the rice stripe virus monoclonal antibody is prepared via a method which: from the rice plants tissue of infection rice stripe virus
BALB/c mouse is immunized as antigen in middle purified virus particle, is resisted through cell fusion, screening, cell clone hybridoma technology
RSV monoclonal antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN112014559A (en) * | 2020-08-14 | 2020-12-01 | 扬州大学 | Double-antibody sandwich enzyme-linked immunosorbent assay kit for rice stripe virus and preparation and detection methods thereof |
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