CN208224281U - A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor - Google Patents
A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor Download PDFInfo
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- CN208224281U CN208224281U CN201820793751.7U CN201820793751U CN208224281U CN 208224281 U CN208224281 U CN 208224281U CN 201820793751 U CN201820793751 U CN 201820793751U CN 208224281 U CN208224281 U CN 208224281U
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- colloidal gold
- trypsin inhibitor
- quick measuring
- measuring card
- detecting
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Abstract
A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor comprising, offset plate adheres to the sample pad being sequentially connected on offset plate, gold-marking binding pad, reaction film and water absorption pad；Polyclonal antibody-the colloidal gold composite prepared by CpTI albumen is fixed in the gold-marking binding pad；Being coated in described reaction film one end can be with the specific antibody in conjunction with antigens c pTI, and as detection line, the specific antibody is the polyclonal antibody prepared by CpTI albumen；Two antiantibody goat anti-rabbit iggs are coated in the other end of reaction film, as nature controlling line.The utility model structure is simple, can effectively avoid the occurrence of cross reaction and pollution, high specificity, high sensitivity, and, easy to operate, detection process is not necessarily to specialized equipment and skilled technical ability, the field quick detection and safety evaluatio of CpTI suitable for transgenic product.
Description
Technical field
The utility model belongs to the detection field of Cowpea Trypsin Inhibitor, and in particular to a kind of detection cowpea tryptose
The colloidal gold immunochromatographimethod quick measuring card of enzyme inhibitor.
Background technique
Cowpea Trypsin Inhibitor (Cowpea Trypsin Inhibitor, CpTI) belongs to serine protease and inhibits
Agent.
After insect takes in protease inhibitors, stable compound is formed with the protease of insect gut, to make insect
Proteopepsis enzymatic activity be suppressed；Meanwhile the compound of protease and protease inhibitors is also possible to bear instead as one
The feed of feedback signal inhibition insect.Above-mentioned double effect will reduce pest to the effective rate of utilization of food proteins, meanwhile, also reduce
The intake of food eventually leads to insect due to lacking required nutrition (protein) and stops development, upset the normal of insect
Metabolism, it is slow or even dead to eventually lead to insect growth.
Hilder (1987) first reported CpTI channel genes tobacco, and obtaining has significant resistance to cigarette beetle
Transgenic plant.At home, Liu Chunming (1993) is separated cloned CpTI gene first, and obtain transgene tobacco, rice,
Cotton.Then, Hao Guixia (1999), Zhang Qixian (2001), Guo Sandui (1999) etc., which are obtained, turns CpTI gene Chinese white poplar, sweet
Indigo plant, tobacco, corn, wheat, pimento, cauliflower.
Currently, being examined for Cowpea Trypsin Inhibitor in genetically modified plants based on the specificity that immunology principle is established
Survey method has 3 kinds: enzyme linked immunosorbent assay (Enzyme Linked Immuno Sorbent Assay, ELISA), lateral flow
Type immunity test strip method (Lateral Flow strips) and Western hybridization assay.
ELISA method directly detects exogenous gene albumen, can quantitative detection, required sample is few, operation standard, detect it is sensitive,
It can be to a large amount of sample detections.But the restricted application of ELISA detection method, need technical professional and complexity
Instrument and equipment, it is time-consuming long, insecticidal proteins need to be also extracted completely from sample, the detection work being suitable under laboratory environment
Make, is not suitable for field screening and on-site test.
Western hybridization assay is the principle specifically bound on nitrocellulose filter using antibody and antigen, right
Foreign protein is detected.This method passes through PAGE electrophoresis first, by protein delivery to nitrocellulose filter, then carries out trace
It is immune, it is detected finally by the secondary antibody of label.The sensitivity of Western detection is very high, and detection limit is generally nanogram rank.
But Western detecting step is comparatively laborious, needs to carry out protein extraction, the processes such as PAGE electrophoresis and immune response, technology and
Equipment requirement is high, is not suitable for the detection of conventional a large amount of samples.
It is anti-to be also based on antigen for colloidal gold immunochromatographimethod detection method (Immune colloidal gold technique)
The sandwich formula technical principle of the specific binding of body, the antigen-antibody complex lateral movement of label, until with fixation
Another antibody on the surface combines colour developing, with ELISA method except that replacing polystyrene anti-with nitrocellulose filter
Answering plate is solid phase carrier.Required reagent is only included in entire detection device, not the requirement of instrument, whole operation process phase
To simple.Compared with nucleic acid and ELISA detection method, this method is fairly simple to the processing of sample.Destination protein to be checked is general
It is the water-solubility protein to antibody high special, therefore, sample does not need complicated purification process, just can be carried out detection.
Colloidal gold immunochromatographimethod detection method is a kind of qualitative checking method quickly, easy, test strips is placed on to be measured
In Sample extraction object, 5-10min can obtain a result, and detection process is not necessarily to specialized equipment and skilled technical ability, economical convenient, especially
Detection suitable for field and field sample.
Currently, existing immune colloidal gold technique is applied in the foreign protein of detection transgenic product, however it remains
Many problems, for example, poor specificity, sensitivity is not high, at high cost, therefore, needs to change existing immune colloidal gold technique
Into developing high specificity, high sensitivity, stability is good, the new method of reproducible detection transgenic product foreign protein.
Currently, not yet being developed both at home and abroad for the colloid gold immune that can detecte CpTI for turning CpTI gene pest-resistant plant
The rapid detection method of chromatography.
Utility model content
The purpose of this utility model is to provide a kind of colloidal gold immunochromatographimethod speed for detecting Cowpea Trypsin Inhibitor
Card is surveyed, detects Cowpea Trypsin Inhibitor, high specificity, high sensitivity with the quick measuring card, also, operates quick, easy, inspection
Survey process is not necessarily to specialized equipment and skilled technical ability, and the field quick detection of CpTI and safety are commented suitable for transgenic product
Valence.
In order to achieve the above object, the utility model provides the following technical solutions:
A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor comprising, offset plate adheres to glue
Sample pad, gold-marking binding pad, reaction film and the water absorption pad being sequentially connected on plate；
Polyclonal antibody-the colloidal gold composite prepared by CpTI albumen is fixed in the gold-marking binding pad；
The reaction film is equipped with detection line and nature controlling line, and being coated in reaction film one end can be with the spy in conjunction with antigens c pTI
Xenoantibody, as detection line, the specific antibody is the polyclonal antibody prepared by CpTI albumen；In the other end packet of reaction film
By two antiantibody goat anti-rabbit iggs, as nature controlling line.
Further, the quick measuring card further includes the shell being set in outside offset plate；Well is provided on the shell of the shell
And observation window, the well are set at the shell above the corresponding sample pad, the observation window is set to the corresponding reaction
At shell above film.
Further, the detection line and nature controlling line interval 1.0-1.5cm.
Also, being equipped with the overlay region of 1-2mm between the sample pad, gold-labelled pad, reaction film and water absorption pad.
The preferably described polyclonal antibody is prepared by the CpTI albumen containing 80 or 88 amino acid.
Preferably, the standard gold amount of the polyclonal antibody-colloidal gold composite is 10 μ g/mL.
Preferably, the offset plate is polyvinyl chloride plastic sheet.
Also, the reaction film is nitrocellulose filter.
Preferably, the aperture of the nitrocellulose filter is 0.1-0.22 microns, and the sample pad and water absorption pad are by aperture
Filter paper by 30-50 microns is made.
In the prior art, the CpTI albumen being made of 80 amino acid is studied, and inventor utilizes UniProt number
According to library, analysis has obtained the structural gene of 88 amino acid CpTI and the leader sequence of 19 amino acid, and 88 based on purifying
The CpTI albumen of amino acid prepares polyclonal antibody, and the utility model is prepared for a kind of glue for detecting Cowpea Trypsin Inhibitor
Body gold immunochromatography quick measuring card, this quick measuring card are more suitable CpTI albumen of the measurement in the different cowpea varieties and general at present
All over the existing albumen for carrying out artificial genetic modification to CpTI gene and expressing.
The quick measuring card of the utility model is divided into 4 functional areas: sample application zone, marker combined area, contrasting detection area and suction
Pool, after sample application zone is added in sample to be tested, under the capillarity of water absorption pad, sample is chromatographed forward, in conjunction with gold labeling antibody；
The phenomenon that double antibodies sandwich retains can be formed, a large amount of colloid gold particle is here in conjunction with detection antibody again when reaching detection line
Accumulation forms a red detection line, when extra gold labeling antibody continues to arrive forward nature controlling line again in conjunction with two antiantibodys,
Same retention forms a red nature controlling line.
The use of the utility model colloidal gold quick measuring card:
1) samples such as the blade of plant, stem, seed are ground, adds the extracting solution of 10ml (wherein containing 0.1mol/L
Tris-HCl, 0.2% ascorbic acid, 0.1% cysteine, 1mmol/L ethylenediamine tetra-acetic acid (EDTA), 1% polyvinyl pyrrole
Alkanone (PVP), pH7.0) standing is shaken up, take supernatant to be detected.
2) it detects: institute's pipette samples being added in the sample application zone of quick measuring card, act on 10min at room temperature, then observe result.
3) result judgement:
Positive: there are red stripes in nature controlling line and detection line position, illustrate in sample with the presence of CpTI.
Negative: detection line position does not occur red stripes, and red stripes occurs in Quality Control line position, illustrates in sample without CpTI
In the presence of.
Failure: it if detection line, Quality Control line position do not occur red stripes or only red stripes occurs in detection line, says
Bright examination quick measuring card failure.
Compared with prior art, the utility model has the following beneficial effects:
The utility model high specificity, high sensitivity, stability and reproducible, the detection to the specificity of CpTI albumen
It is limited to l μ g/mL, the detection CpTI albumen of specificity is remained to after placing 30 days at room temperature；The best standard gold amount of antibody is low, can be with
Significantly reduce quick measuring card cost of manufacture.
In the utility model, the interval of detection line and nature controlling line increases, and avoids the occurrence of cross reaction and pollution, preferably into
Row CpTI protein determination and quick measuring card performance test itself.
The utility model operating method is easy, quick, and detection time is short, is not necessarily to specialized equipment and skilled technical ability, economy is just
Victory instills sample to be tested in sample application zone, can be observed from observation area in 5-10 minutes as a result, especially suitable for field and now
The detection of field sample.
Detailed description of the invention
Fig. 1 is the structural schematic diagram of an embodiment of the present invention quick measuring card.
Fig. 2 an embodiment of the present invention stuck structure schematic diagram.
Fig. 3 be an embodiment of the present invention testing result schematic diagram wherein, A be testing result feminine gender schematic diagram；B
For testing result positive schematic diagram；C is the invalid schematic diagram of testing result.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment
Such as Fig. 1, a kind of colloidal gold immunochromatographimethod quick measuring card of detection Cowpea Trypsin Inhibitor of the utility model, packet
It includes offset plate 1, adhere to the sample pad 2 being sequentially connected on offset plate 1, gold-marking binding pad 3, nitrocellulose filter 4 and water absorption pad 5；Institute
It states and is fixed in gold-marking binding pad 3 by polyclonal antibody-colloidal gold composite of the CpTI albumen preparation of 88 amino acid；It is described according to
The overlapping of 1.0-2.0mm is equipped between secondary connected sample pad 2, gold conjugation pad 3, nitrocellulose filter 4 and water absorption pad 5
Area.
4 aperture of nitrocellulose filter is 0.1-0.22 microns, is equipped with detection line 41 and nature controlling line 42, and detection line 41 is wrapped
It can be to be prepared by the CpTI albumen with 88 amino acid with the specific antibody in conjunction with detection antigens c pTI, the specific antibody
Polyclonal antibody；Nature controlling line 42 is coated with two antiantibody goat anti-rabbit iggs, and the detection line 41 and nature controlling line 42 are spaced 1.0-
1.5cm。
Further, referring to fig. 2, shell 7 is additionally provided with outside the offset plate of the quick measuring card, shell 7 is equipped with well 71 and sees
Window 72 is examined, the well 71 is located above the corresponding sample pad, and the observation window 72 is located above nitrocellulose filter.
Also, the filter paper of the sample pad and water absorption pad by aperture by 30-50 microns is made.
The use of the present embodiment colloidal gold immunochromatographimethod quick measuring card:
Sample to be tested pre-treatment: the samples such as the blade, stem, seed of plant are ground, and add extracting solution (its of 10ml
In Tris-HCl containing 0.1mol/L, 0.2% ascorbic acid, 0.1% cysteine, 1mmol/L ethylenediamine tetra-acetic acid (EDTA),
1% polyvinylpyrrolidone (PVP), pH7.0) standing is shaken up, take supernatant to be detected.
Detection: samples taken is instilled in sample application zone, acts on 5-10min at room temperature, then the result from observation area.
Analysis of test results:
Negative: detection line does not occur red stripes, and nature controlling line goes out red stripes, illustrates exist in sample without CpTI, such as Fig. 3
Shown in middle A.
Positive: there are red stripes in detection line, nature controlling line, illustrate with the presence of CpTI in sample, as shown by B in fig. 3.
Failure: if only red stripes or detection line occurs in detection line, the non-outlet red stripes of nature controlling line illustrate speed
It is invalid to survey card, as shown in C in Fig. 3.
Claims (10)
1. a kind of colloidal gold immunochromatographimethod quick measuring card for detecting Cowpea Trypsin Inhibitor comprising, offset plate adheres to offset plate
On the sample pad, gold-marking binding pad, reaction film and the water absorption pad that are sequentially connected；It is characterized in that, fixed in the gold-marking binding pad
There is the polyclonal antibody-colloidal gold composite prepared by CpTI albumen；
The reaction film is equipped with detection line and nature controlling line, and being coated in reaction film one end can be special anti-in conjunction with antigens c pTI
Body, as detection line, the specific antibody is the polyclonal antibody prepared by CpTI albumen；Two are coated in the other end of reaction film
Antiantibody goat anti-rabbit igg, as nature controlling line.
2. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the quick measuring card further includes the shell being set in outside offset plate, and well and observation window are provided on the shell of the shell, described
Well is set at the shell above the corresponding sample pad, and the observation window is set to the shell above the corresponding reaction film
Place.
3. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the detection line and nature controlling line interval 1.0-1.5cm.
4. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In equipped with the overlay region of 1-2mm between the sample pad, gold-labelled pad, reaction film and water absorption pad.
5. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the polyclonal antibody is prepared by the CpTI albumen containing 80 or 88 amino acid.
6. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the standard gold amount of the polyclonal antibody-colloidal gold composite is 10 μ g/mL.
7. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the offset plate is polyvinyl chloride plastic sheet.
8. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the reaction film is nitrocellulose filter.
9. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 8, feature exists
In the aperture of the nitrocellulose filter is 0.1-0.22 microns.
10. detecting the colloidal gold immunochromatographimethod quick measuring card of Cowpea Trypsin Inhibitor according to claim 1, feature exists
In the filter paper of the sample pad and water absorption pad by aperture by 30-50 microns is made.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201820793751.7U CN208224281U (en) | 2018-05-25 | 2018-05-25 | A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor |
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| CN201820793751.7U CN208224281U (en) | 2018-05-25 | 2018-05-25 | A kind of colloidal gold immunochromatographimethod quick measuring card detecting Cowpea Trypsin Inhibitor |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110133292A (en) * | 2019-06-04 | 2019-08-16 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of herbicide resistant protein Bar |
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2018
- 2018-05-25 CN CN201820793751.7U patent/CN208224281U/en not_active Expired - Fee Related
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110133292A (en) * | 2019-06-04 | 2019-08-16 | 中国农业科学院生物技术研究所 | A kind of colloidal gold immunochromatographimethod quick measuring card of herbicide resistant protein Bar |
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| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20181211 |
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| CF01 | Termination of patent right due to non-payment of annual fee |