CN110133279A - A kind of joint inspection colloidal gold strip detecting transgenosis BT albumen and CP4-EPSPS albumen - Google Patents
A kind of joint inspection colloidal gold strip detecting transgenosis BT albumen and CP4-EPSPS albumen Download PDFInfo
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Abstract
A kind of joint inspection colloidal gold strip detecting transgenosis BT albumen and CP4-EPSPS albumen comprising bottom plate is fixed on the sample pad being sequentially connected on bottom plate, bonding pad, nitrocellulose filter and water absorption pad；Wherein, gold labeling antibody compound AuNPs-BTmAb1 and AuNPs-CP4mAb1 is coated on bonding pad；Nature controlling line and two detection lines are set on nitrocellulose filter.The joint inspection test strips can the quickly detection simultaneously of crops to the T-Cry1Ab of protein B containing transgenic pest-resistant, BT-Cry1Ac, BT-Cry1Ab/Ac fusion protein or herbicide resistant protein CP4-EPSPS, as a result accurate, with good specificity and higher sensitivity, reduce the dependence to instrument and professional operator, overcome single target test strips detection complex character crop, or detection single traits crop compound when limitation, have a good application prospect.
Description
Technical field
The invention belongs to transgene protein detection fields, and in particular to a kind of detection transgenosis BT albumen and CP4-EPSPS
The joint inspection colloidal gold strip of albumen.
Background technique
Cut-off 2017, the cultivated area of global genetically modified crops reaches 2.0 hundred million hectares, 1,700,000 compared to 1996
Hectare, the cultivated area of genetically modified crops increase as many as 100 times, and insect-resistant transgenic crops and Transgenic Resistant Herbicide Crops are
Most important two types in genetically modified crops, mainly by being inserted into Exogenous Bt gene in the genome of plant (from soil
Earth bacterium Bacillussubtilis) and Antiglyphosate gene CP4-epsps and glufosinate-resistant Bar gene/pat.
Under the so fast-developing situation of transgenosis plantation industry, the safety of genetically modified crops and products thereof is always
Focus concerned by people, especially transgenic product are to the health of people and animal, and influence to ecological environment is by extensive
Concern.Many countries carry out the product of transgenosis raw material production and force label measure, but genetically modified crops are illegally planted and flow
The case where going to supermarket also occasionally has generation, for the safe and effective management for realizing transgenic product, needs associate producer and inspection
It surveys mechanism and effective monitoring is carried out to the plantation of genetically modified crops.
Currently, transgenic product predominantly detects method and can be divided into two classes: one kind is the egg based on exogenous proteins target
White matter detection method；Another kind of is the nucleic acid detection method based on nucleic acid target.
Detection based on nucleic acid mainly has Standard PCR method, labelled by nested-PCR method, fluorescence quantitative PCR method etc., such method detection
Stability and sensitivity are higher, but this method needs accurate thermal cycler and a series of reagents, consumptive material, needs be equipped with
It is carried out in complete laboratory.
Immunological method based on Ag-Ab can be by detecting the albumen of exogenous gene expression come qualitative, quantitative
Transgenic product is detected, this kind of detection method mainly has Western Blot, ELISA method and immunity test strip method etc.；And
Western Blot and ELISA method need cumbersome operating procedure and longer incubation time, testing result that instrument is needed to sentence
It reads, the quick detection for being not particularly suited for the places such as field scene, storage uses；Immunity test strip method is easy to operate, testing result
Naked eyes do not need as it can be seen that detection 5~10min of needs using instrument etc., will quickly detect and rise in screening at the scene on a large scale
Important function.
With the continuous development of biotechnology transgenic technology, genetically modified crops are also sent out from single traits to complex character
Exhibition, by the end of 2017, the corn of complex character accounted for the 77% of transgenic corns, and the cotton of complex character accounts for 80%, and currently,
Colloidal gold strip for genetically modified crops detection is mostly the test format of single target, is not able to satisfy multiple characters transgenosis work
The demand of object and the compound analyte detection of single traits crop.
Summary of the invention
Aiming at the problems existing in the prior art, the purpose of the present invention is to provide a kind of detection transgenosis BT albumen and
The joint inspection colloidal gold strip of CP4-EPSPS albumen, for the detection of three kinds of target proteins, to albumen containing BT-Cry1Ab, BT-
The genetically modified crops of Cry1Ac albumen or BT-Cry1Ab/Ac fusion protein and CP4-EPSPS albumen quickly detect simultaneously,
The dependence to instrument and professional operator is reduced, overcomes single target test strips detection complex character crop, or detect several
The limitation when compound of single traits crop, has a good application prospect.
In order to achieve the above object, the invention provides the following technical scheme:
A kind of joint inspection colloidal gold strip detecting transgenosis BT albumen and CP4-EPSPS albumen comprising bottom plate, fixation
Sample pad, bonding pad, nitrocellulose filter and the water absorption pad being sequentially connected on bottom plate；
Wherein, gold labeling antibody compound AuNPs-BTmAb1 and AuNPs-CP4mAb1 are coated on bonding pad；It is described compound
Object AuNPs-BTmAb1 is colloidal gold AuNPs and the mouse monoclonal antibody BTmAb1 of anti-BT albumen is coupled and obtained, the BT
Albumen is BT-Cry1Ab albumen, BT-Cry1Ac albumen or BT-Cry1Ab/Ac fusion protein；Gold labeling antibody compound AuNPs-
CP4mAb1 is colloidal gold AuNPs and the mouse monoclonal antibody CP4mAb1 of anti-CP4-EPSPS albumen is coupled and obtained；
BT detection line and CP4-EPSPS detection line, BT inspection is arranged in parallel close to bonding pad end on the nitrocellulose filter
Survey line is made of monoclonal antibody BTmAb2, and CP4-EPSPS detection line is made of monoclonal antibody CP4mAb2；The nitric acid is fine
It ties up and nature controlling line is set on plain film, sheep anti-mouse igg antibody is coated on the nitrocellulose filter close to water absorption pad end, as Quality Control
Line.
Further, the monoclonal antibody CP4mAb1 is closed with after colloidal gold coupling with 3-5%BSA, monoclonal antibody
1-3%BSA is added in the dilution buffer of CP4mAb2；Monoclonal antibody BTmAb1 and colloidal gold coupling after with 1-3%BSA into
Row is closed, and adds 0.5-1%BSA in the dilution buffer of monoclonal antibody BTmAb2.
Also, in the gold labeling antibody compound AuNPs-BTmAb1, the mass body of monoclonal antibody BTmAb1 and colloidal gold
Product is than being 50-60 μ g:1mL；In gold labeling antibody compound AuNPs-CP4mAb1, the matter of monoclonal antibody CP4mAb1 and colloidal gold
Amount volume ratio is 60-72 μ g:1mL.
Preferably, coated monoclonal antibody BTmAb2 dosage distinguishes 1.5-2.0mg/mL, CP4- in the BT detection line
The dosage of coated monoclonal antibody CP4mAb2 is 1.5-2.0mg/mL in EPSPS detection line.
Again in the gold labeling antibody compound AuNPs-BTmAb1, the dosage of mouse monoclonal antibody BTmAb1 is 1.5-
2.0μg；In gold labeling antibody compound AuNPs-CP4mAb1, the dosage of mouse monoclonal antibody CP4mAb1 is 1.0-1.5 μ g；
The antibody concentration of dynamics is 0.5-1mg/mL on the nature controlling line.
Further, supporting back board is equipped with after the bottom plate of the colloidal gold strip.
Also, the colloidal gold strip further includes the shell being set in outside bottom plate；Sample-adding is provided on the shell of the shell
Hole and observation window, the well are set at the shell above the corresponding sample pad, and the observation window is set to the corresponding fibre
It ties up at the shell above plain film.
Preferably, in the sample pad, bonding pad, nitrocellulose filter and water absorption pad, 1-2mm is equipped between adjacent component
Overlay region.
Also, the sample pad, bonding pad are glass fibre, water absorption pad is cellulose fibre；Glue on the bonding pad
Body goldc grains diameter is 20-40nm；The nitrocellulose filter is CN95 film or CN140 film, and the aperture of the nitrocellulose filter is
8-15μm。
Joint inspection colloidal gold strip of the present invention detects albumen containing BT-Cry1Ab, BT- for detecting genetically modified crops
The complex character genetically modified crops of Cry1Ac albumen or BT-Cry1Ab/Ac fusion protein and CP4-EPSPS albumen are several single
The compound of character genetically modified crops.
BT-Cry1Ab/Ac in the present invention is fusion protein, and monoclonal antibody BTmAb1 and BTmAb2 are for BT-
The common antibody of Cry1Ab, BT-Cry1Ac albumen and BT-Cry1Ab/Ac fusion protein, inventor have screened gold labeling antibody or inspection
The suitable amounts of monoclonal antibody on survey line determine that the dosage of monoclonal antibody BTmAb1 is 1.5-2.0 μ g, monoclonal antibody
The dosage of CP4mAb1 is 1.0-1.5 μ g, and the antibody concentration of dynamics is 0.5-1mg/mL, if monoclonal antibody dosage mistake
It is more, then it will lead to different degrees of false positive, it is excessively few then detection sensitivity to be made to reduce.
Due to antibody BTmAb1, BTmAb2 and CP4-EPSPS albumen of BT albumen of the invention antibody CP4mAb1 and
CP4mAb2 has differences in affinity, and the affinity of antibody CP4mAb1 and CP4mAb2 are stronger, therefore the present invention is anti-to its gold mark
Body is closed by force, otherwise non-specific binding can occur with BT detection line antibody mAb2 in joint inspection, while need to be to BTmAb2
It is closed；To prevent the non-specific binding between BTmAb1 and CP4mAb2, closing effect is further strengthened to BTmAb1 needs
Fruit, therefore, the dosage of BSA have different degrees of increase.
In the present invention, antibody CP4mAb1 is closed by force after coupling with colloidal gold using 3-5%BSA, and antibody CP4mAb2's is dilute
It releases and adds 1-3%BSA in buffer, 1-3%BSA is closed after antibody BTmAb1 is coupled with colloidal gold, antibody BTmAb2's
0.5-1%BSA is added in dilution buffer, false positive caused by can avoid because of non-specific binding.
Testing principle and result interpretation process of the invention are as follows: if containing the BT-Cry1Ab in BT albumen to be measured in sample
Sample is added drop-wise in sample pad by albumen, BT-Cry1Ac albumen or BT-Cry1Ab/Ac fusion protein, when detection, in capillary
Under effect, sample is mobile by the direction of sample pad, bonding pad, nitrocellulose filter, water absorption pad, when flowing through bonding pad, makes first
Gold labeling antibody compound AuNPs-BTmAb1 redissolves on bonding pad, secondly, transgenosis specific B T albumen and AuNPs- in sample
BTmAb1 is combined, and forms immune complex, subsequent immune complex flows through nitrocellulose filter, when immune complex flow to Dan Ke
When the detection line that grand antibody BTmAb2 is fixed, the monoclonal antibody BTmAb2 of tested survey line is captured, and forms gold labeling antibody
The double-antibody sandwich mode of AuNPs-BTmAb1+BT albumen+BTmAb2, the colloidal gold being gathered in detection line shows naked eyes can
The red seen；Likewise, when there is CP4-EPSPS albumen to be measured in sample, when the combined pad of sample flow, turn first in sample
For gene C P4-EPSPS albumen in conjunction with gold labeling antibody compound AuNPs-CP4mAb1, subsequent compound flows through NC film, with detection
Fixed CP4mAb2 is combined on line, forms gold labeling antibody AuNPs-CP4mAb1+CP4-EPSPS albumen+CP4mAb2 double antibody folder
Heart mode, the colloidal gold being gathered in detection line show red, and extra gold labeling antibody and immune complex flow through nature controlling line
When, it is captured by the solid phase antibody of nature controlling line, shows red Quality Control lines in Quality Control line position；If there is no BT- in sample
Cry1Ab albumen, BT-Cry1Ac albumen or BT-Cry1Ab/Ac fusion protein and CP4-EPSPS proteantigen, then gold labeling antibody
It will not be accumulated in detection line, test strips only have a nature controlling line.
Thus, BT detection line and CP4-EPSPS detection line, but also display nature controlling line had not only been shown for double positive samples；It is described
BT protein positive sample only shows BT detection line and nature controlling line；CP4-EPSPS positive sample only show CP4-EPSPS detection line and
Nature controlling line；' negative ' specimens do not have detection line, only show nature controlling line；Other in addition to this is 4 kinds are as a result, regard as tying in vain
Fruit.
The present invention compares with prior art, has the following beneficial effects:
Joint inspection colloidal gold colloidal gold detection test paper strip of the invention can be used for transgenic pest-resistant BT-Cry1Ab, BT-Cry1Ac or BT-
The complex character crop and single traits crop of Cry1Ab/Ac fusion protein and antiweed CP4-EPSPS form compound
The quick detection of object, to the detection specificity of BT albumen and CP4-EPSPS albumen, sensitivity and to actual sample (soybean, jade
Rice, cotton and rape etc.) detection sensitivity be all remarkably higher than the prior art.
It can be to the transgenic pest-resistant BT albumen and antiweed CP4-EPSPS using colloidal gold strip of the invention
Albumen is used for quickly detecting, and testing result is accurate, has good specificity and higher sensitivity, detects CP4-EPSPS egg
White sensitivity can reach 5ng/mL, and the soybean RRS sensitivity for detecting the albumen containing CP4-EPSPS can be to 0.01%, to containing CP4-
The corn NK603 of EPSPS albumen, cotton MON88913, beet H7-1 sensitivity can be to 0.1%；Detect BT-Cry1Ab/Ac
Fusion protein sensitivity can reach 100ng/mL, and the sensitivity for detecting the soybean MON87701 of the albumen containing BT-Cry1Ac can arrive
The detection sensitivity of 1%, the corn MON810 of the albumen containing BT-Cry1Ab are 1%.
Existing PCR detection technique, which can be solved, using the present invention needs instrument, reagent, technical staff's operation etc. and ELISA
Method needs the problems such as longer incubation time, and the detection target type that can solve transgenosis immunoassay test strip is single shows
Shape is more able to satisfy the demand of the compound analyte detection in complex character genetically modified crops and containing single traits crop.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of immune colloidal gold detection test paper strip in the embodiment of the present invention.
Fig. 2 is the detection specific outcome of immune colloidal gold detection test paper strip in the embodiment of the present invention.
Fig. 3-4 is the detection albumen sensitivity results of immune colloidal gold detection test paper strip in the embodiment of the present invention, Fig. 3 unit
Ng/mL, Fig. 4 unit μ g/mL.
Fig. 5-8 be detection albumen containing CP4-EPSPS of immune colloidal gold detection test paper strip in the embodiment of the present invention corn,
The sensitivity results of soybean, cotton and beet；
In Fig. 5 the content of CP4-EPSPS soybean (RRS) be from left to right followed successively by 10%, 1%, 0.1%, 0.01%,
0.001%, blank control；In Fig. 6 the content of CP4-EPSPS corn (NK603) be from left to right followed successively by 5%, 0.5%,
0.1%, 0.05%, blank control；In Fig. 7 the content of CP4-EPSPS cotton (MON88913) be from left to right followed successively by 10%,
1%, 0.1%, 0.01%, blank control；In Fig. 8 the content of CP4-EPSPS beet (H7-1) be from left to right followed successively by 10%,
1%, 0.1%, 0.01%, blank control.
Fig. 9-10 is the detection actual sample corn of albumen containing BT of immune colloidal gold detection test paper strip in the embodiment of the present invention
And the sensitivity results of soybean, in Fig. 9 the content of BT-Cry1Ab corn (MON810) be from left to right followed successively by 10%, 5%,
1%, 0.5%, blank control, in Figure 10 the content of BT-Cry1Ac soybean (MON87701) be from left to right followed successively by 100%,
10%, 1%, 0.1%, blank control.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Trisodium citrate, Tween-20 are purchased from Shanghai Sheng Gong bioengineering Co., Ltd；BSA, gold chloride are purchased from the U.S.
Sigma company；Nitrocellulose filter (NC film) CN 95 is purchased from Germany Sartorius；The pairing of sheep anti-mouse igg antibody, BT albumen
Antibody BTmAb1 and BTmAb2, pairing the antibody CP4mAb1 and CP4mAb2 of CP4-EPSPS albumen are purchased from Canadian creation difficult to understand
Object company.
Nanodrop micro-spectrophotometer is purchased from U.S. Thermo company；Point sample instrument and micro computer cutting machine are purchased from Hangzhou
Han Gan scientific & technical corporation；Magnetic stirring apparatus is purchased from Shanghai Si Le instrument company.
The preparation and application of embodiment joint inspection test strips of the present invention
1. preparing gold labeling antibody compound
2mL colloidal gold solution (absorbance value under maximum absorption band wavelength is 1.0-1.2) is taken, brown cillin bottle is added to
In, potassium carbonate is added and adjusts pH to most just when 7.5-8.0, under magnetic stirring, is slowly added to suitable monoclonal antibody
BTmAb1 (or CP4mAb1), and continue to be stirred to react 40min.
The BSA capping 40min of different final concentrations is then added, mixed liquor is placed in centrifuge tube, 4 DEG C, l500rpm
It is centrifuged 15min, removes the free gold of sediment fraction；12000rpm centrifugation 30min removes supernatant, precipitating 0.2mL colloidal gold
Liquid is saved to be resuspended, as BT gold labeling antibody compound AuNPs-BTmAb1 (or CP4-EPSPS gold labeling antibody compound AuNPs-
CP4mAb1), it is kept in dark place for 4 DEG C.
2. assembling test strips
By sample cushion material (20cm × 30cm), in conjunction with cushion material (20cm × 30cm) and water suction cushion material (20cm ×
It 30cm) is cut, the sample pad (2cm × 30cm) and bonding pad (0.5cm × 30cm) after cutting out are separately immersed in sample pad
It is taken out in treatment fluid and bonding pad treatment fluid, after 10min and in drying at room temperature.
As shown in Figure 1, colloidal gold strip by bottom plate A, be fixed on the sample pad 1 being sequentially connected on bottom plate, bonding pad 2,
NC film 3 and water absorption pad 4, sample pad 1 and bonding pad 2 are glass fibre, and water absorption pad 4 (2cm × 30cm) is cellulose fibre.
By the BT gold labeling antibody compound AuNPs-BTmAb1 and CP4-EPSPS gold labeling antibody compound AuNPs- of preparation
CP4mAb1 is sprayed on bonding pad 2, is then dried at room temperature for；By monoclonal antibody BTmAb2/CP4mAb2 and 1mg/mL
Sheep anti-mouse igg antibody is transferred on NC film 3 (2.5cm × 30cm) according to the volume of 1 μ L/cm, is respectively formed BT detection line 31
(being T1 detection line after colour developing), CP4-EPSPS detection line 32 (being T2 detection line after colour developing) and nature controlling line 33 (are C after colour developing
Line), wherein in the BT gold labeling antibody compound AuNPs-BTmAb1, the dosage of mouse monoclonal antibody BTmAb1 is 1.5
μg；In gold labeling antibody compound AuNPs-CP4mAb1, the dosage of mouse monoclonal antibody CP4mAb1 is 1.0 μ g.
The NC film of antibody, bonding pad will be coated, sample pad and water absorption pad are successively attached on PVC bottom plate (6cm × 30cm),
Assembled test paper, the band of 3mm wide is cut into using automatic strip-cutting machine, is then sealed in equipped with desiccant by each overlapping 1-2mm
Polybag in, and save at room temperature.
3. specific detection
Detect soya seeds (MON87701, RRS), the corn seed of transgenosis respectively using transgenosis joint inspection test strips
(MON810, NK603), BT-Cry1Ab/Ac and CP4-EPSPS albumen.
By seed grind into powder, and distilled water progress water is added according to the ratio of 1:5 and mentions, acutely rocks 20s, stand and divide
Layer, takes supernatant to be detected, and determines to make after the specificity of test strips, the blank seed water of corresponding crop mention by testing result
For negative control, as a result referring to fig. 2, wherein C1 is blank soybean, and C2 is blank corn flour, C3 PBS, as negative right
According to；11 samples are 10% soybean RRS+10% soybean MON87701；12 be 10% soybean RRS；21 be 10% corn MON810+
10% soybean MON87701；22 be BT-11 corn；31 be BT-Cry1Ab/Ac+CP4-EPSPS albumen.
Fig. 2 can be seen that joint inspection test strips can detect the albumen containing BT soybean MON87701 (albumen containing Cry1Ac) and
Corn MON810 (albumen containing Cry1Ab) detects soybean RRS (albumen containing CP4-EPSPS) and corn NK603 containing CP4-EPSPS
(albumen containing CP4-EPSPS), it is anti-without intersecting with PAT albumen at test sample BT-11 (albumen containing CP4-EPSPS and PAT)
It answers, specificity is good.
4. sensitivity detects
Sensitivity determination: transgenosis BT-Cry1Ab/Ac fusion protein and CP4-EPSPS albumen are diluted to difference with PBS
Concentration gradient, to the diluted concentration of CP4-EPSPS albumen be respectively as follows: 1ng/mL, 5ng/mL, 10ng/mL, 50ng/mL,
100ng/mL, 500ng/mL, 1000ng/mL, 2500ng/mL are respectively as follows: 0.05 to the diluted concentration of BT-Cry1Ab/Ac albumen
μ g/mL, 0.1 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, 20 μ g/mL are used to measure the joint inspection colloidal gold of preparation
The sensitivity of immunity test strip, each sample are drawn the sample pad that 75 μ L are added drop-wise to test strips, are observed after 5-10min as a result, same
The PBS of volume is added drop-wise to sample pad as negative control.
By albumen according to detecting after different gradient dilutions, as a result referring to Fig. 3-4, joint inspection test strips are to CP4-
The detection sensitivity of EPSPS albumen is 5ng/mL (Fig. 3), and the detection sensitivity to BT-Cry1Ab/Ac albumen is 0.1 μ g/mL
(Fig. 4).
5. sample measures
Actual sample is detected first is that determine the practicability of test strips detection, second is that determine the detection spirit to actual sample
Sensitivity.
Gradient dilution is carried out after mentioning to seed sample water, using joint inspection test strips of the invention to albumen containing CP4-EPSPS
Genetically engineered soybean RRS (GTS-40-3-2) detected, as a result referring to Fig. 5-10.
As seen from Figure 5, test strips can detect the genetically engineered soybean that target protein content is 0.01%；Such as the institute of Fig. 6,7,8
Show, test strips are to the detection sensitivity of the corn NK603 of the albumen containing CP4-EPSPS, cotton MON88913, beet H7-1
0.1%, as shown in Figures 9 and 10, joint inspection test strips are to the MON810 corn of the albumen containing BT-Cry1Ab, the albumen containing BT-Cry1Ac
The detection sensitivity of MON87701 soybean is 1%, and sensitivity is higher.
By experimental verification, in immunity colloidal gold test paper strip of the invention, transgenosis BT-Cry1Ab/Ac and CP4-EPSPS
Monoclonal antibody mAb1 and mAb2 successfully match respectively, have it is good specificity and higher sensitivity, detect CP4-
The sensitivity of EPSPS albumen can reach 5ng/mL, and the sensitivity of detection BT-Cry1Ab/Ac albumen can reach 0.1 μ g/mL.
Joint inspection test strips of the invention can be used for detecting BT-Cry1Ab containing transgenosis, BT-Cry1Ac, BT-Cry1Ab/Ac
The soybean of fusion protein and CP4-EPSPS albumen, corn, cotton, beet, rape etc., the inspection to the protein soybeans containing CP4-EPSPS
Surveying sensitivity is 0.01%, and the detection sensitivity to protein maize containing CP4-EPSPS, cotton, beet is 0.1%；To containing BT-
The corn of Cry1Ab, BT-Cry1Ac albumen and the detection sensitivity of soybean are 1%, and anti-without intersecting with transgene protein PAT
It answers, has the characteristics that high sensitivity, high specificity, easy to detect, simple and quick.
Claims (10)
1. a kind of joint inspection colloidal gold strip for detecting transgenosis BT albumen and CP4-EPSPS albumen comprising bottom plate is fixed on
Sample pad, bonding pad, nitrocellulose filter and the water absorption pad being sequentially connected on bottom plate；
Wherein, gold labeling antibody compound AuNPs-BTmAb1 and gold labeling antibody compound AuNPs- are coated on bonding pad
CP4mAb1, the gold labeling antibody compound AuNPs-BTmAb1 are colloidal gold AuNPs and the mouse monoclonal of anti-BT albumen is anti-
Body BTmAb1 coupling obtains, and the BT albumen is that BT-Cry1Ab albumen, BT-Cry1Ac albumen or BT-Cry1Ab/Ac merge egg
It is white；Gold labeling antibody compound AuNPs-CP4mAb1 is colloidal gold AuNPs and the mouse monoclonal of anti-CP4-EPSPS albumen is anti-
Body CP4mAb1 coupling obtains；
BT detection line and CP4-EPSPS detection line, BT detection line is arranged in parallel close to bonding pad end on the nitrocellulose filter
It is made of monoclonal antibody BTmAb2, CP4-EPSPS detection line is made of monoclonal antibody CP4mAb2；The nitrocellulose
Nature controlling line is set on film, sheep anti-mouse igg antibody is coated on the nitrocellulose filter close to water absorption pad end, as nature controlling line.
2. the joint inspection colloidal gold strip of transgenosis BT albumen and CP4-EPSPS albumen is detected according to claim 1, it is special
Sign is that monoclonal antibody CP4mAb1 is closed after coupling with colloidal gold with 3-5%BSA, the dilution of monoclonal antibody CP4mAb2
1-3%BSA is added in buffer；Monoclonal antibody BTmAb1 is closed after coupling with colloidal gold with 1-3%BSA, monoclonal
0.5-1%BSA is added in the dilution buffer of antibody BTmAb2.
3. the joint inspection colloidal gold strip of detection transgenosis BT albumen and CP4-EPSPS albumen according to claim 1 or claim 2,
It is characterized in that, in the gold labeling antibody compound AuNPs-BTmAb1, the mass body of monoclonal antibody BTmAb1 and colloidal gold
Product is than being 50-60 μ g:1mL；In gold labeling antibody compound AuNPs-CP4mAb1, the matter of monoclonal antibody CP4mAb1 and colloidal gold
Amount volume ratio is 60-72 μ g:1mL.
4. the joint inspection colloidal gold strip of transgenosis BT albumen and CP4-EPSPS albumen is detected according to claim 1, it is special
Sign is that coated monoclonal antibody BTmAb2 dosage distinguishes 1.5-2.0mg/mL, CP4-EPSPS detection in the BT detection line
The dosage of coated monoclonal antibody CP4mAb2 is 1.5-2.0mg/mL on line.
5. the joint inspection colloidal gold strip of transgenosis BT albumen and CP4-EPSPS albumen is detected according to claim 1, it is special
Sign is, in the gold labeling antibody compound AuNPs-BTmAb1, the dosage of mouse monoclonal antibody BTmAb1 is 1.5-2.0
μg；In gold labeling antibody compound AuNPs-CP4mAb1, the dosage of mouse monoclonal antibody CP4mAb1 is 1.0-1.5 μ g；Institute
The antibody concentration for stating dynamics on nature controlling line is 0.5-1mg/mL.
6. the joint inspection colloidal gold strip of transgenosis BT albumen and CP4-EPSPS albumen is detected according to claim 1, it is special
Sign is, supporting back board is equipped with after the bottom plate of the colloidal gold strip.
7. the joint inspection colloidal gold strip of detection transgenosis BT albumen and CP4-EPSPS albumen according to claim 1 or 6,
It is characterized in that, the colloidal gold strip further includes the shell being set in outside bottom plate；Sample-adding is provided on the shell of the shell
Hole and observation window, the well are set at the shell above the corresponding sample pad, and the observation window is set to the corresponding fibre
It ties up at the shell above plain film.
8. the joint inspection colloidal gold strip of transgenosis BT albumen and CP4-EPSPS albumen is detected according to claim 1, it is special
Sign is, in the sample pad, bonding pad, nitrocellulose filter and water absorption pad, the overlapping of 1-2mm is equipped between adjacent component
Area.
9. the joint inspection colloidal gold strip of transgenosis BT albumen and CP4-EPSPS albumen is detected according to claim 1, it is special
Sign is that the sample pad, bonding pad are glass fibre, and water absorption pad is cellulose fibre；Colloidal gold on the bonding pad
Partial size is 20-40nm；The nitrocellulose filter is CN95 film or CN140 film, and the aperture of the nitrocellulose filter is 8-15 μ
m。
10. application of the joint inspection colloidal gold strip as described in claim 1 in detection genetically modified plants, detection contain transgenosis
The complex character of BT-Cry1Ab albumen, BT-Cry1Ac albumen or BT-Cry1Ab/Ac fusion protein and CP4-EPSPS albumen turns
Gene crops or the compound of several single traits genetically modified crops.
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| CN112526133A (en) * | 2020-11-25 | 2021-03-19 | 浙江新安化工集团股份有限公司 | Test strip, preparation method thereof and application thereof in transgenic protein AM79EPSPS detection |
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