CN108226503A - Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof - Google Patents
Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof Download PDFInfo
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- CN108226503A CN108226503A CN201611157289.3A CN201611157289A CN108226503A CN 108226503 A CN108226503 A CN 108226503A CN 201611157289 A CN201611157289 A CN 201611157289A CN 108226503 A CN108226503 A CN 108226503A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/37—Assays involving biological materials from specific organisms or of a specific nature from fungi
- G01N2333/38—Assays involving biological materials from specific organisms or of a specific nature from fungi from Aspergillus
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Abstract
Immune colloid gold detection card of aflatoxin G 1 of the present invention and preparation method thereof, is related to animal-derived food detection of veterinary drugs in food technical field.Test strips in the detection card shell of the present invention, are made of PVC offset plates, sample pad, gold conjugation pad, coated film and water absorption pad；Colloidal gold film is the glass fibre element film of the monoclonal antibody containing aflatoxin G 1, and coated film is nitrocellulose filter, which is provided with T lines and C lines, and T lines are coated with aflatoxin G 1 protein conjugate, and C lines are coated with sheep anti-mouse igg antibody.The present invention is convenient, fast, result is accurate effective for quickly detecting aflatoxin G 1.
Description
Technical field
The present invention relates to Detecting Pesticide technical field in cereal foods, more particularly to the immune of aflatoxin G 1
Colloidal-gold detecting-card and preparation method thereof.
Background technology
Aflatoxin（AFT）It is the similar compound of a kind of chemical constitution, is the derivative of dihydrofuran cumarin.
Aflatoxin is mainly by aspergillus flavus（aspergillus flavus）Aspergillus parasiticus（a.parasiticus）Time generated
, there is the probability highest of aflatoxin in damp-heat area food and feed in raw metabolite.They are present in soil, dynamic plant
In object, various nuts, the grain oil products such as peanut, corn, rice, soybean, wheat, aspergillus flavus poison in 1993 are particularly easily polluted
Element is by the World Health Organization（WHO）Agency for Research on Cancer delimit as 1 class carcinogenic substance, be a kind of extremely strong extremely toxic substance of toxicity.
The harmfulness of aflatoxin is there is destruction to people and animal's liver tissue, and liver cancer even death can be caused when serious
The most common with aflatoxin G 1 in the food of natural contamination, toxicity and carcinogenicity are also most strong.G1 is most dangerous cause
Cancer object, often in corn, peanut, cotton seeds can be often detected in some dry fruits.They can generate glimmering under ultraviolet light irradiation
Light according to fluorescence color difference, is classified as B races and two major class of G races and its derivative.AFT has found that more than 20 plant.AFT exists
In soil, animals and plants, various nuts, oil and foodstuffs, animals and plants food etc. are mainly polluted；Such as peanut, corn, rice, wheat,
Beans, nut fruits, meat, milk and milk products, aquatic products etc. have aflatoxin contamination, be mycotoxicosis it is maximum,
To a kind of mycotoxin that human health risk is extremely prominent.
The prior art is mainly gas chromatography-mass spectrography, high performance liquid chromatography etc. for the detection of aflatoxin G 1
Detection method, but it is apparent the defects of these technologies：Equipment is expensive, it is complicated for operation, be difficult to promote.So establishing, one kind is simple, has
Effect, the assay method that base is suitble to use are extremely necessary.Showed the purpose of the present invention is development one kind is more suitable for enterprise
Field detecting and fast and simple, low-cost qualitative checking method.
Invention content
For case above, the purpose of the present invention is exactly to be provided a kind of yellow bent to overcome defect of the existing technology
Immune colloid gold detection card of mould toxin G1 and preparation method thereof can effectively solve quickly, easily detect aspergillus flavus poison
The problem of plain G1.
The aflatoxin G 1 colloidal-gold detecting-card of the present invention, the glue including being coated with monoclonal antibody colloid gold label object
Body gold bonding pad, the nitrocellulose filter for being coated with aflatoxin G 1-BSA and sheep anti-mouse igg, sample pad, water absorption pad, PVC
Offset plate and plastic mould composition, sample pad, bonding pad are adhered in one end of PVC offset plates successively, and nitrocellulose filter is pasted in centre,
The other end adheres to water absorption pad.
The preparation method of the aflatoxin G 1 colloidal-gold detecting-card of the present invention, is realized by following steps：
(1) prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added in, then adds in 1% gold chloride
(HAuCl4·3H2O) 5 mL is configured to 500 mL, 0.01% aqueous solution of chloraurate, heats after boiling in the situation of lasting stirring
1% trisodium citrate (Na of lower addition3C6H5O7·2H2O) solution 5-7 mL continue agitating and heating, when the color of solution becomes completely
During transparent aubergine, stop heating after maintaining 5 min, moisturizing to original volume is cooled to room temperature, and 2-8 DEG C saves backup；
(2) pretreatment of antibody：The aflatoxin G 1 antibody that will be marked is under the conditions of 1000 r/min, 4 DEG C, centrifugation 20
Min takes supernatant, and 1 mg/mL is diluted to 0.01 mol/L PBS；Or it is diluted to 1 mg/ml, mistake with 0.01 mol/L PBS
0.22 μm of filter membrane；
(3) preparation of colloid gold label object：40 mL of colloidal gold solution in step (1) is taken, is adjusted with 0.25 mol/L K2CO3
Colloidal gold solution pH to 8.5,250 r/min of magnetic stirrer are stirred, and the egg that 4 mL contain 0.3 mg antibody proteins is added dropwise
White solution reacts 10 min；4 mL, 10% BSA are added dropwise, continue to be stirred to react 10 min；Gold labeling antibody solution room temperature is low
Speed（1500 r/min）10 min are centrifuged, discard the precipitation formed by the gold particle agglomerated；Red 4 DEG C of supernatant solution, 12000
R/min centrifuges 20 min, abandons supernatant, collects precipitation, precipitation is settled to 1 mL with gold labeling antibody dilution, is prepared into Huang Qu
Mould toxin G1 monoclonal antibodies colloid gold label object；
(4) preparation of colloidal gold film：By step (3) aflatoxin G 1 protein conjugate monoclonal antibody colloid gold label object
It is sprayed on carrier glass cellulose membrane with the even concentration of 5 μ L/cm with film instrument is drawn, room temperature naturally dry or 37 DEG C of drying 3
The colloidal gold film of the monoclonal antibody colloid gold label object of protein conjugate containing aflatoxin G 1 is made in h；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, aflatoxin G 1 protein conjugate are diluted to 1 mg/mL, with draw
Film instrument is sprayed on the concentration of 1 μ L/cm on nitrocellulose filter successively, is prepared into coated film, and after 37 DEG C are coated with 2 h, room temperature is certainly
It so dries or 37 DEG C dries；
(6) sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, is less than 60% in air humidity
Under conditions of, room temperature naturally dry；
(7) assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent wool, group successively from top to bottom
Test strips are dressed up, cut into the strip of one fixed width, then test strips are mounted in the flat shelly-shaped detection card shell of strip.
In the step (5) coated detection line T be located at the lower section of nature controlling line C；
Sample pad treatment fluid in the step (6) is by 1 g Bovine serum albumins (BSA) and 0.8 g sodium chloride (NaCl), is used
The 0.01 mol/L PBS containing 0.5%TRITON-100 are settled to 100 mL；
The present invention can be effectively used for measuring aflatoxin G 1, and method is simple, convenient, fast, as a result accurately.
Description of the drawings
Fig. 1 is the structure chart of the immune colloid gold detection card of aflatoxin G 1 of the present invention：1. wells 2. are examined in figure
3. nature controlling line of survey line, 4. detection hole, 5. test strips 6. detect card shell.
Fig. 2 is the sectional structure chart of the test strips in the immune colloid gold detection of aflatoxin G 1 of the present invention blocks, in figure
7. 8. 10. nitrocellulose filter of gold conjugation pad 9.PVC offset plates of sample pad, 11. water absorption pad.
Specific embodiment
Embodiment 1
Fig. 1, embodiment shown in Fig. 2：9 be PVC offset plates in figure；7 be sample pad；8 be gold conjugation pad, which combines
Monoclonal antibody colloid gold label object has been coated on pad；10 be coated film, i.e. nitrocellulose filter, is wrapped on the nitrocellulose filter
By aflatoxin G 1-BSA and sheep anti-mouse igg；11 be water absorption pad, is made of water-absorbent material such as filter paper.
(sample end) adherency sample pad 7, bonding pad 8, sample pad 7 and bonding pad 8 is side by side on one end of PVC offset plates 9
Structure.
In the intermediate adhesion nitrocellulose filter 10 of PVC offset plates 9.Sheep anti-mouse igg is provided on nitrocellulose filter 10
Nature controlling line 3 and aflatoxin G 1-BSA detection lines 2.
Water absorption pad 11 is adhered in the other end of PVC offset plates 9.One end of nitrocellulose filter 10 slightly intersects with bonding pad 8, separately
One end slightly intersects with water absorption pad 11.The test strips 5 can be incorporated in detection card shell 6 made of plastic mould, and detection card is made,
Well 1 and detection hole 4 are equipped on the upper lid of detection card shell 6,7 face well 1 of sample pad, nitrocellulose filter 10 is just
To detection hole 4.
Embodiment 2
Prepared by aflatoxin G 1 colloidal-gold detecting-card, implemented by following steps：
It is prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added in, then adds in 1% gold chloride
(HAuCl4·3H2O) 5 mL is configured to 500 mL, 0.01% aqueous solution of chloraurate, heats after boiling in the situation of lasting stirring
1% trisodium citrate (Na of lower addition3C6H5O7·2H2O) solution 5-7 mL continue agitating and heating, when the color of solution becomes completely
During transparent aubergine, stop heating after maintaining 5 min, moisturizing to original volume is cooled to room temperature, and 2-8 DEG C saves backup；
The pretreatment of antibody：The aflatoxin G 1 antibody that will be marked is under the conditions of 1000 r/min, 4 DEG C, centrifugation 20
Min takes supernatant, and 1 mg/mL is diluted to 0.01 mol/L PBS；Or it is diluted to 1 mg/ml, mistake with 0.01 mol/L PBS
0.22 μm of filter membrane；
The preparation of colloid gold label object：40 mL of colloidal gold solution in step (1) is taken, glue is adjusted with 0.25 mol/L K2CO3
Body gold solution pH to 8.5,250 r/min of magnetic stirrer are stirred, and the albumen that 4 mL contain 0.3 mg antibody proteins is added dropwise
Solution reacts 10 min；4 mL, 10% BSA are added dropwise, continue to be stirred to react 10 min；Gold labeling antibody solution room temperature low speed
（1500 r/min）10 min are centrifuged, discard the precipitation formed by the gold particle agglomerated；Red 4 DEG C of supernatant solution, 12000 r/
Min centrifuges 20 min, abandons supernatant, collects precipitation, precipitation is settled to 1 mL with gold labeling antibody dilution, is prepared into aspergillus flavus
Toxin G1 monoclonal antibody colloid gold label objects；
The preparation of colloidal gold film：Step (3) aflatoxin G 1 protein conjugate monoclonal antibody colloid gold label object is used
It draws film instrument to be sprayed on carrier glass cellulose membrane with the even concentration of 5 μ L/cm, room temperature naturally dry or 37 DEG C of 3 h of drying,
The colloidal gold film of the monoclonal antibody colloid gold label object of protein conjugate containing aflatoxin G 1 is made；
It is coated with film preparation：Sheep anti-mouse igg antibody, aflatoxin G 1 protein conjugate are diluted to 1 mg/mL, with a stroke film instrument
It is sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively, is prepared into coated film, after 37 DEG C are coated with 2 h, room temperature is dried in the air naturally
Dry or 37 DEG C of drying；
Sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, is less than 60% item in air humidity
Under part, room temperature naturally dry；
Detect the assembling of card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent wool successively from top to bottom, are assembled into
Test strips cut into one fixed width strip, then test strips are mounted in the flat shelly-shaped detection card shell of strip.
Claims (2)
1. aflatoxin G 1 colloidal-gold detecting-card, it is characterised in that be prepared as steps described below：
(1) prepared by colloidal gold solution：1 L conical flasks 1 are taken, 495 mL of ultra-pure water is added in, then adds in 1% gold chloride
HAuCl4·3H2O) 5 mL is configured to 500 mL, 0.01% aqueous solution of chloraurate, heats after boiling in the case of lasting stirring
Add in 1% trisodium citrate Na3C6H5O7·2H2O solution 5-7 mL continue agitating and heating, when the color of solution becomes transparent completely
Aubergine when, stop heating after maintaining 5 min, moisturizing to original volume is cooled to room temperature, and 2-8 DEG C saves backup；
(2) pretreatment of antibody：The aflatoxin G 1 antibody that will be marked is under the conditions of 1000 r/min, 4 DEG C, centrifugation 20
Min takes supernatant, and 1 mg/mL is diluted to 0.01 mol/L PBS；Or it is diluted to 1 mg/ml, mistake with 0.01 mol/L PBS
0.22 μm of filter membrane；
(3) preparation of colloid gold label object：40 mL of colloidal gold solution in step (1) is taken, with 0.25 mol/L K2CO3It adjusts
Colloidal gold solution pH to 8.5,250 r/min of magnetic stirrer are stirred, and the egg that 4 mL contain 0.3 mg antibody proteins is added dropwise
White solution reacts 10 min；4 mL, 10% BSA are added dropwise, continue to be stirred to react 10 min；Gold labeling antibody solution room temperature
1500 r/min centrifuge 10 min, discard the precipitation formed by the gold particle agglomerated；Red 4 DEG C of supernatant solution, 12000 r/min
20 min are centrifuged, abandon supernatant, precipitation is collected, precipitation is settled to 1 mL with gold labeling antibody dilution, is prepared into aflatoxin
G1 monoclonal antibody colloid gold label objects；
(4) preparation of colloidal gold film：By step (3) aflatoxin G 1 protein conjugate monoclonal antibody colloid gold label object
It is sprayed on carrier glass cellulose membrane with the even concentration of 5 μ L/cm with film instrument is drawn, room temperature naturally dry or 37 DEG C of drying 3
The colloidal gold film of the monoclonal antibody colloid gold label object of protein conjugate containing aflatoxin G 1 is made in h；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, aflatoxin G 1 protein conjugate are diluted to 1 mg/mL, with draw
Film instrument is sprayed on the concentration of 1 μ L/cm on nitrocellulose filter successively, is prepared into coated film, and after 37 DEG C are coated with 2 h, room temperature is certainly
It so dries or 37 DEG C dries；
(6) sample pad pre-treatment：Sample pad treatment fluid is uniformly coated on glass fibre element film, is less than 60% in air humidity
Under conditions of, room temperature naturally dry；
(7) assembling of detection card：PVC offset plates paste sample pad, colloidal gold film, coated film and absorbent wool, group successively from top to bottom
Test strips are dressed up, cut into the strip of one fixed width, then test strips are mounted in the flat shelly-shaped detection card shell of strip.
2. aflatoxin G 1 colloidal-gold detecting-card according to claim 1, it is characterised in that wrapped in the step (5)
The detection line T of quilt is located at the lower section of nature controlling line C；
Sample pad treatment fluid in the step (6) is by 1 g Bovine serum albumins (BSA) and 0.8 g sodium chloride (NaCl), is used
The 0.01 mol/L PBS containing 0.5%TRITON-100 are settled to 100 mL.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611157289.3A CN108226503A (en) | 2016-12-15 | 2016-12-15 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611157289.3A CN108226503A (en) | 2016-12-15 | 2016-12-15 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
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| CN108226503A true CN108226503A (en) | 2018-06-29 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611157289.3A Pending CN108226503A (en) | 2016-12-15 | 2016-12-15 | Immune colloid gold detection card of aflatoxin G 1 and preparation method thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102087284A (en) * | 2010-12-29 | 2011-06-08 | 杭州南开日新生物技术有限公司 | Preparation method of reagent board for rapidly detecting aflatoxin B1 |
| CN103792358A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board |
| CN105510587A (en) * | 2014-10-16 | 2016-04-20 | 镇江先创生物科技有限公司 | Neomycin immuno-colloidal gold detection card and preparation method thereof |
| CN105572373A (en) * | 2014-10-17 | 2016-05-11 | 南京亿特生物科技有限公司 | Immune colloidal gold detecting card of carbendazin and preparation method of immune colloidal gold detecting card |
| CN105572371A (en) * | 2014-10-17 | 2016-05-11 | 南京亿特生物科技有限公司 | Immune colloidal gold detecting card of diclazuril and preparation method of immune colloidal gold detecting card |
-
2016
- 2016-12-15 CN CN201611157289.3A patent/CN108226503A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102087284A (en) * | 2010-12-29 | 2011-06-08 | 杭州南开日新生物技术有限公司 | Preparation method of reagent board for rapidly detecting aflatoxin B1 |
| CN103792358A (en) * | 2012-11-05 | 2014-05-14 | 江苏维赛科技生物发展有限公司 | Preparation and detection method of aflatoxin G1 colloidal gold immunochromatograohic assay detection board |
| CN105510587A (en) * | 2014-10-16 | 2016-04-20 | 镇江先创生物科技有限公司 | Neomycin immuno-colloidal gold detection card and preparation method thereof |
| CN105572373A (en) * | 2014-10-17 | 2016-05-11 | 南京亿特生物科技有限公司 | Immune colloidal gold detecting card of carbendazin and preparation method of immune colloidal gold detecting card |
| CN105572371A (en) * | 2014-10-17 | 2016-05-11 | 南京亿特生物科技有限公司 | Immune colloidal gold detecting card of diclazuril and preparation method of immune colloidal gold detecting card |
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Application publication date: 20180629 |
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