CN202522562U - Colloidal-gold-immunochromatographic-assay test paper for detecting tetrodotoxin - Google Patents
Colloidal-gold-immunochromatographic-assay test paper for detecting tetrodotoxin Download PDFInfo
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- CN202522562U CN202522562U CN2012200773612U CN201220077361U CN202522562U CN 202522562 U CN202522562 U CN 202522562U CN 2012200773612 U CN2012200773612 U CN 2012200773612U CN 201220077361 U CN201220077361 U CN 201220077361U CN 202522562 U CN202522562 U CN 202522562U
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Abstract
The utility model discloses colloidal-gold-immunochromatographic-assay test paper for detecting tetrodotoxin. The colloidal-gold-immunochromatographic-assay test paper is characterized by comprising a carrier board, a nitrocellulose membrane, a colloidal gold cushion, a sample adding cushion and an absorbing cushion, wherein the carrier board is positioned at the bottom layer; the nitrocellulose membrane, the colloidal gold cushion, the sample adding cushion and the absorbing cushion are stuck on the upper surface of the carrier board in sequence; the nitrocellulose membrane is positioned at the middle part of the carrier board; the colloidal gold cushion is arranged at one side of the nitrocellulose membrane and is partially overlapped with the nitrocellulose membrane; the sample adding cushion is arranged on the colloidal gold cushion and is partially overlapped with the colloidal gold cushion; the absorbing cushion is arranged at the other side of the nitrocellulose membrane and is partially overlapped with the nitrocellulose membrane; and the colloidal gold cushion is uniformly coated with a layer of rat-anti-tetrodotoxin monoclonal antibody labeled with colloidal gold. One end on the nitrocellulose membrane close to the sample adding cushion is provided with a detecting line coated with detecting antigens, and one end on the nitrocellulose membrane close to the absorbing cushion is provided with a quality control line coated with quality control antibodies. The colloidal-gold-immunochromatographic-assay test paper disclosed by the utility model has the advantages that the detection is fast and convenient, the operation is simple, and samples can be intuitively judged by the naked eyes without special treatment.
Description
Technical field
The utility model relates to a kind of biological detecting method, and concrete is a kind of colloidal gold immune chromatography test of fast detecting tetraodotoxin.
Background technology
Tetraodotoxin (tetrodotoxin; TIX) be the non-protide neurotoxin of the stronger micromolecule of a kind of toxicity; Its toxicity is more than 1250 times of sodium cyanide; Since its can with the combining of the sodium-ion channel acceptor high degree of specificity of neuron membrane, the blocking-up sodium-ion channel, thus suppress the conduction of nerve impulse.The globe fish meat flavour is delicious, nutritious; Its people such as China, Japan have edible custom, and usually cause eating by mistake poisoning owing to it contains fugutoxin, and the tetraodotoxin physicochemical property is more stable; Common cooking methods is difficult to its destruction; Tetraodotoxin poisoning disease time is fast, and mortality ratio is high, the saying that among the people have " defied death and eaten filefish ".
Detection method about tetraodotoxin mainly is divided at present: bioassay method, physics and chemistry detection method.Biological detection has mouse bioassay method and in vitro tissue method.The advantage of mouse detection method is simple to operate, but sensitivity is relatively poor, and specificity is also poor, can only whether contain toxin by judgement sample, and can not be qualitative whether be exactly tetraodotoxin.The physics and chemistry detection method has the glimmering detection method of ultraviolet etc., and fluorescence method and ultraviolet spectrophotometry process are simple, quick, but sample purity is had relatively high expectations.Instrument detecting methods such as the online detection method of GC/MS, efficient capillary zone electrophoresis method are relatively sensitiveer, but sample preparation is cumbersome, and used instrument is expensive, and check fee is high.
Immune colloidal gold technique is to encapsulate the collaurum ion through antibody (or antigen) to process immune colloid gold; React by the immune colloid gold antigen corresponding (or antibody) with it; Assemble the redness that demonstrates colloidal gold particle itself according to making colloidal gold particle; But therefore naked eyes intuitive judgment testing result, detection time is short.It is easy to use to process test strips, the specific apparatus that need not simple to operate, and sample need not special processing, can carry detection at any time at any time.
For the micromolecule detection of antigens, adopt competition inhibition method can obtain good effect.Because of micromolecule has only an antigen site, can not combine two antibody simultaneously, therefore can not adopt double antibody sandwich method to detect.It detects principle is the colloid gold particle labeled monoclonal antibody, and detection line encapsulates micromolecule antigen, and nature controlling line encapsulates anti-mouse IgG.During detection, the antigen to be checked in the sample at first combines with golden labeling antibody, when up arrival detects band; Saturated fully like antigen to be checked a certain amount of antibody; Then golden labeling antibody can not combine with the antigen that detects band, macroscopic red stripes can not occur, positive result.Like no antigen to be checked in the sample, then golden labeling antibody combines with the antigen that detects band, forms red stripes, negative result.Select for use this method to should be specifically noted that the antibody adsorbance and the envelope antigen amount of colloid gold particle.
Summary of the invention
The purpose of the utility model provides a kind of colloidal gold immune chromatography test that detects tetraodotoxin quickly and easily, and this test paper can realize whether containing tetraodotoxin in the fast detecting sample based on small amount of sample and test paper.The principle of work of this test paper is to utilize specific Ag-Ab to combine.
The described detection test paper of the utility model is made up of carrier board (1), nitrocellulose membrane (2), collaurum pad (3), application of sample pad (4) and absorption pad (5).Carrier board (1) is positioned at bottom; Nitrocellulose membrane (2), collaurum pad (3), application of sample pad (4), absorption pad (5) stick on the carrier board upper surface successively; Nitrocellulose membrane (2) is positioned at the carrier board middle part; Collaurum pad (3) is located at a side of nitrocellulose membrane (2) and overlaps with nitrocellulose membrane (2); Application of sample pad (4) is located at collaurum pad (3) and upward and with collaurum pad (3) overlaps, and absorption pad (5) is located at the opposite side of nitrocellulose membrane (2) and overlaps with nitrocellulose membrane (2).
Wherein carrier board (1) adopts the PVC plate, and collaurum pad (3) adopts glass fibre membrane, and application of sample pad (4) adopts spun glass, and absorption pad (5) adopts filter paper.Collaurum pad (3) is gone up and evenly is coated with the mouse-anti tetraodotoxin monoclonal antibody (is anti-) that one deck has colloid gold label.Nitrocellulose membrane (2) is provided with detection line T and nature controlling line C, and detection line T is coated with and detects antigen (tetraodotoxin), and detection line T is near application of sample pad one end; Nature controlling line C is coated with Quality Control antibody (anti-mouse IgG, two is anti-), and nature controlling line C is near absorption pad one end.
If contain tetraodotoxin in the sample; Under syphonic effect, tetraodotoxin will be penetrated on the collaurum pad through the application of sample pad, and with the collaurum pad on colloid gold label antibody (the anti-tetraodotoxin monoclonal antibody that contains; One is anti-); In conjunction with after, form bond 1, i.e. " collaurum-anti--antigen " bond; This bond 1 continues on nitrocellulose membrane, to move to the adsorptive pads place, and through detection line T place the time, the detection antigen (tetraodotoxin) that discord is embedded in this combines, so detection line T place does not develop the color; Bond 1 continues the migration to the adsorptive pads place, when arriving nature controlling line C place and the Quality Control antibody (anti-mouse IgG, two is anti-) that is embedded in this form bond 2, i.e. " collaurum-anti--antigen-two resist " bond; Bond 2 is because of producing aggregation shows collaurum at nature controlling line C place color.This kind situation is positive, and promptly detection line T does not develop the color, and nature controlling line C colour developing shows and contains tetraodotoxin in the sample.
If do not contain tetraodotoxin in the sample, under syphonic effect, the sample that does not contain tetraodotoxin will be penetrated on the collaurum pad through the application of sample pad; The colloid gold label antibody that contains on the collaurum pad (anti-tetraodotoxin monoclonal antibody, is anti-) is under the situation that is not attached to antigen (tetraodotoxin); On nitrocellulose membrane, move to the adsorptive pads place; Through detection line T place the time, will combine with the detection antigen (tetraodotoxin) that is embedded in this, form bond 1; I.e. " collaurum-anti--antigen " bond, bond 1 makes detection line T place the band that develops the color occur because of producing aggregation; Bond 1 continues the migration to the adsorptive pads place, when arriving nature controlling line C place and the Quality Control antibody (anti-mouse IgG, two is anti-) that is embedded in this form bond 2, i.e. " collaurum-anti--antigen-two resist " bond; Bond 2 is because of producing aggregation shows collaurum at nature controlling line C place color.This kind situation is negative, i.e. detection line T colour developing, and nature controlling line C colour developing shows not contain tetraodotoxin in the sample.
The utility model relates to the preparation method who detects test paper, and this method may further comprise the steps: the preparation of collaurum pad; The point sample of nitrocellulose membrane encapsulates; The assembling of colloidal gold immuno-chromatography test paper strip.
1, the preparation of collaurum pad.
Get collaurum (PH7.0-8.5) is sub-packed in 1.5ml with the 1ml/ pipe centrifuge tube; Every arm adds anti-tetraodotoxin monoclonal antibody 15-20mg respectively, places 5-10min behind the jog mixing, and every pipe adds and contains 10% calf serum (BSA) sealing damping fluid 100ml; The jog mixing; Place 5-10min, 4 ℃, the centrifugal 30-60min of 1000rpm abandon supernatant.Every arm adds phosphate buffer (PBS) 1ml that contains 1% BSA, and is the same centrifugal behind the mixing, abandons supernatant; Obtain the red loose deposits of grape, add the PBS damping fluid 500ml that contains 1% BSA, mixing; Evenly coat on the glass fibre membrane of wide 1cm, 37 ℃ of oven dry are prepared into the collaurum pad.
2, the point sample of nitrocellulose membrane encapsulates.
With the nitrocellulose membrane medium line is benchmark, apart from its left end 1cm place, detection line is set, and apart from 1cm place, its right side nature controlling line is being set.Point sample encapsulates tetraodotoxin on the detection line, and point sample encapsulates sheep anti-mouse igg (two is anti-) on the nature controlling line.The concentration that encapsulates of antibody is 1-4mg/ml, and the point sample amount is 1ml, forms the band of two wide about 1mm, and dry back is washed 2 times with PBS damping fluid (PH7.0), in 37 ℃ of oven dry with the PBS damping fluid sealing 24h that contains 1% BSA again.
3, the assembling of colloidal gold immuno-chromatography test paper strip
Carrier board is positioned at bottom, and nitrocellulose membrane, collaurum pad, application of sample pad, absorption pad are sticked on the carrier board upper surface successively, and its structure is as shown in Figure 1.Paste solid after, be cut into strip, obtain tetraodotoxin colloidal gold immunochromatographydetection detection test paper bar.
The utility model provides a kind of colloidal gold immuno-chromatography test paper strip that can be used for tetraodotoxin in the test sample, detect quick, convenient, the specific apparatus that need not simple to operate, sample need not special processing, but the intuitive judgment of testing result naked eyes.
Description of drawings
Fig. 1 is the positive side schematic view that practical colloid gold test paper structure is formed.
Wherein, 1 is carrier board; 2 are nitrocellulose membrane (T is a detection line, and C is a nature controlling line); 3. collaurum pad; 4 is the application of sample pad; 5 is absorption pad.
The side schematic view of bowing that Fig. 2 forms for the colloid gold test paper structure.
Wherein, 1 is carrier board; 2 is nitrocellulose membrane; 3 is the collaurum pad; 4 is the application of sample pad; 5 is absorption pad.
Fig. 3 is the experimental result pattern diagram.
Wherein, A is the synoptic diagram before the application of sample, and B is invalid test paper, the positive result of C, the negative result of D.T is a detection line, and C is a nature controlling line.
Embodiment
Following examples are used to explain the utility model, but the scope of the utility model is not limited only to this.
Embodiment 1.The preparation of tetraodotoxin colloidal gold immune chromatography test.
Material: the test strips structure is formed referring to accompanying drawing 1; Carrier board is the PVC plate; Absorption pad is a filter paper; Sample pad adopts glass fibre membrane, and the collaurum pad adopts glass fibre membrane.Collaurum (available from Huzhou Yingcheng Biological Technology Co., Ltd.); Anti-tetraodotoxin monoclonal antibody (one is anti-, available from Shanghai Ya Ji bio tech ltd), detection antigen is tetraodotoxin (available from U.S. Sigma company); Quality Control antibody is sheep anti-mouse igg (two is anti-, available from Beijing ancient cooking vessel state biotech company).
Method: the collaurum of getting pH 8.0 is managed the centrifuge tube that is sub-packed in 1.5ml with 1ml/, and every arm adds the anti-tetraodotoxin monoclonal antibody of 20mg respectively, places 5min behind the jog mixing, and antibody is combined with gold grain.Every pipe adds 10% BSA solution 100ml, and the jog mixing is placed 5min and sealed.Then in 4 ℃, centrifugal 30min (rotating speed 1000rpm), supernatant discarded.Every arm adds the PBS damping fluid 1ml of 1% BSA, and is centrifugal behind the mixing, abandons supernatant, obtains the red loose deposits of grape.The PBS damping fluid 500ml that adds 1% BSA evenly coats behind the mixing on the glass fibre membrane of wide 1cm, and 37 ℃ of oven dry are prepared into the collaurum pad.
Spacer segment 1cm place in nitrocellulose membrane, point sample detects antigen and Quality Control antibody respectively, and encapsulating concentration is 2mg/ml, and the point sample amount is 1ml.Form the band of two wide about 1mm, dry back is washed 2 times with PBS damping fluid (pH7.0) with the PBS damping fluid sealing 24h of 1%BSA again, and is subsequent use after 37 ℃ of oven dry.
Carrier board is positioned at bottom, and nitrocellulose membrane, collaurum pad, application of sample pad, absorption pad are sticked on the carrier board upper surface successively, and its structure is as shown in Figure 1.Paste solid after, be cut into strip, obtain tetraodotoxin colloidal gold immunochromatographydetection detection test paper bar.
Embodiment 2.The use of tetraodotoxin colloidal gold immune chromatography test.
Get the sample 20-40ml that contains (or not containing) tetraodotoxin with pipettor, it is dripped the application of sample pad place in the tetraodotoxin colloidal gold immuno-chromatography test paper strip, drip 100ml physiological saline, leave standstill and observed the test strips change color in 15-20 minute.
Only at nature controlling line C place the grape red stripes is arranged, then the result is positive, promptly contains tetraodotoxin; At detection line T and nature controlling line C the grape red stripes is arranged all, yin and yang attribute as a result then, promptly sample does not contain tetraodotoxin; Behind the application of sample, nature controlling line C is like no grape red stripes, and the result is invalid.
Claims (3)
1. a colloidal gold immune chromatography test that detects tetraodotoxin is characterized in that being made up of carrier board (1), nitrocellulose membrane (2), collaurum pad (3), application of sample pad (4) and absorption pad (5); Carrier board (1) is positioned at bottom; Nitrocellulose membrane (2), collaurum pad (3), application of sample pad (4), absorption pad (5) stick on the carrier board upper surface successively; Nitrocellulose membrane (2) is positioned at the carrier board middle part; Collaurum pad (3) is located at a side of nitrocellulose membrane (2) and overlaps with nitrocellulose membrane (2); Application of sample pad (4) is located at collaurum pad (3) and upward and with collaurum pad (3) overlaps, and absorption pad (5) is located at the opposite side of nitrocellulose membrane (2) and overlaps with nitrocellulose membrane (2).
2. colloidal gold immune chromatography test according to claim 1 is characterized in that described collaurum pad (3) upward is coated with the mouse-anti tetraodotoxin monoclonal antibody that one deck has colloid gold label uniformly.
3. colloidal gold immune chromatography test according to claim 1 is characterized in that last being provided with near application of sample pad one end of described nitrocellulose membrane (2) is coated with detection detection of antigens line, is provided with the nature controlling line that is coated with Quality Control antibody near absorption pad one end.
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| CN2012200773612U CN202522562U (en) | 2012-03-05 | 2012-03-05 | Colloidal-gold-immunochromatographic-assay test paper for detecting tetrodotoxin |
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| CN2012200773612U CN202522562U (en) | 2012-03-05 | 2012-03-05 | Colloidal-gold-immunochromatographic-assay test paper for detecting tetrodotoxin |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104142394A (en) * | 2013-05-10 | 2014-11-12 | 福建省水产研究所 | Making method of semi-quantitative tetrodotoxin detection card |
| CN106053819A (en) * | 2016-06-12 | 2016-10-26 | 深圳市计量质量检测研究院 | Tetrodotoxin immunochromatography detection card and application thereof |
| CN108931639A (en) * | 2017-05-25 | 2018-12-04 | 浙江清华长三角研究院萧山生物工程中心 | The chip and reagent of cortisol detection in a kind of saliva |
| CN110361545A (en) * | 2019-08-28 | 2019-10-22 | 重庆三峡医药高等专科学校 | A kind of colloidal gold strip detecting eosinophil source nerve toxin |
-
2012
- 2012-03-05 CN CN2012200773612U patent/CN202522562U/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104142394A (en) * | 2013-05-10 | 2014-11-12 | 福建省水产研究所 | Making method of semi-quantitative tetrodotoxin detection card |
| CN106053819A (en) * | 2016-06-12 | 2016-10-26 | 深圳市计量质量检测研究院 | Tetrodotoxin immunochromatography detection card and application thereof |
| CN108931639A (en) * | 2017-05-25 | 2018-12-04 | 浙江清华长三角研究院萧山生物工程中心 | The chip and reagent of cortisol detection in a kind of saliva |
| CN110361545A (en) * | 2019-08-28 | 2019-10-22 | 重庆三峡医药高等专科学校 | A kind of colloidal gold strip detecting eosinophil source nerve toxin |
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Granted publication date: 20121107 Termination date: 20130305 |