CN108226486A - Immune colloid gold detection card of Madumycin and preparation method thereof - Google Patents
Immune colloid gold detection card of Madumycin and preparation method thereof Download PDFInfo
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- CN108226486A CN108226486A CN201611158306.5A CN201611158306A CN108226486A CN 108226486 A CN108226486 A CN 108226486A CN 201611158306 A CN201611158306 A CN 201611158306A CN 108226486 A CN108226486 A CN 108226486A
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- madumycin
- gold
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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
- 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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- Investigating Or Analysing Biological Materials (AREA)
Abstract
Immune colloid gold detection card of Madumycin 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 Madumycin, and coated film is nitrocellulose filter, which is provided with T lines and C lines, and T lines are coated with Madumycin 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 Madumycin.
Description
Technical field
The present invention relates to Detecting Pesticide technical field in cereal foods, more particularly to the immune colloid of Madumycin
Gold detection card and preparation method thereof.
Background technology
Madumycin English name Maduramicin, also known as maduramicin are a kind of polyethers monovalence lists of newer type
Glucosides ion carrier antibiotic, this product anticoccidial spectrum extensively have coccidiostat activity to zygoblast and first generation schizont.This product energy
Effectively 6 kinds of common Eimeria species of control, also have good preventive effect to el pato's parasitosis.Mixed feeding, every 1000 kg feeds 5~10
G Madumycins are developed by American Cyanamid Company the 1980s, and China succeeds in developing and produces in enormous quantities within 1992, due to
Price is relatively low, has good preventive and therapeutic action to global-worm illness, Madumycin is made to be widely used in clinic.It but should
Medicine toxicity is larger, and safe range is narrow, and the dosis toxica of the very close chicken of dosage, Use out of range easily causes fowl to be poisoned.Poultry horse
Mycin poisoning of shutting out is generally acute process.Acute death（It is dead in 1~2 d）Animal generally will appear typical nosotoxicosis
Shape：East walk west alter, uneasy, the excited nervous symptoms such as hyperfunction or watery diarrhea, leg be soft, walking and astasia of standing.Severe patient
Two legs are benumbed, and lethargic sleep is until dead.Sub-chronic intoxication, which shows as subtracting the maximum phase of an eclipse to appetite, to give up absolutely, becomes thin rapidly, and color and luster is presented in excrement
Variation, from steel gray, light green, green to courage green, occurrence rate about 20% or so.Poultry lassitude squats that it is motionless to drive, very
To bipod paralysis, drive when gambrel land, two wings are flapped, powerless standing or even death.In clinical practice often because using
It is improper, poultry is caused to be poisoned, economic loss is larger.Ft%HWGE
The prior art is mainly the detection methods such as gas chromatography-mass spectrography, high performance liquid chromatography for the detection of Madumycin,
But the defects of these technologies, is apparent：Equipment is expensive, it is complicated for operation, be difficult to promote.So establish a kind of simple, effective, suitable base
The assay method that layer uses is extremely necessary.It is more suitable for enterprise the purpose of the present invention is developing one kind and carries out Site Detection and fast
Prompt easy, low-cost qualitative checking method.
Invention content
For case above, the purpose of the present invention is exactly to provide a kind of horse Du to overcome defect of the existing technology
Immune colloid gold detection card of mycin and preparation method thereof, can effectively solve quickly, easily detect asking for Madumycin
Topic.
The Madumycin colloidal-gold detecting-card of the present invention, the colloidal gold including being coated with monoclonal antibody colloid gold label object
Bonding pad, the nitrocellulose filter for being coated with Madumycin-BSA and sheep anti-mouse igg, sample pad, water absorption pad, PVC offset plates and modeling
Expect mold composition, adhere to sample pad, bonding pad successively in one end of PVC offset plates, nitrocellulose filter is pasted in centre, and the other end glues
Attached water absorption pad.
The preparation method of the Madumycin 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 Madumycin antibody that will be marked centrifuges 20 min under the conditions of 1000 r/min, 4 DEG C,
Supernatant is taken, 1 mg/mL is diluted to 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22
μm 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 horse Du
Mycin monoclonal antibody colloid gold label object；
(4) preparation of colloidal gold film：Step (3) Madumycin protein conjugate monoclonal antibody colloid gold label object is used and is drawn
Film instrument is sprayed on the even concentration of 5 μ L/cm on carrier glass cellulose membrane, room temperature naturally dry or 37 DEG C of 3 h of drying, system
Into the colloidal gold film of the monoclonal antibody colloid gold label object of protein conjugate containing Madumycin；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, Madumycin 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；
(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 Madumycin, 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 Madumycin of the present invention：1. well, 2. detection line in figure
3. 4. detection hole of nature controlling line, 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 Madumycin of the present invention blocks, 7. samples in figure
Product pad 8. 10. nitrocellulose filter of gold conjugation pad 9.PVC offset plates, 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 Madumycin-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 Madumycin-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 Madumycin 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 Madumycin antibody that will be marked centrifuges 20 min, takes under the conditions of 1000 r/min, 4 DEG C
Supernatant is diluted to 1 mg/mL with 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22 μ
M filter membranes；
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, and it is mould to be prepared into horse Du
Plain monoclonal antibody colloid gold label object；
The preparation of colloidal gold film：By step (3) Madumycin protein conjugate monoclonal antibody colloid gold label object stroke film
Instrument is sprayed on the even concentration of 5 μ L/cm on carrier glass cellulose membrane, and room temperature naturally dry or 37 DEG C of 3 h of drying are made
The colloidal gold film of the monoclonal antibody colloid gold label object of protein conjugate containing Madumycin；
It is coated with film preparation：Sheep anti-mouse igg antibody, Madumycin protein conjugate are diluted to 1 mg/mL, with stroke film instrument successively
Be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm, be prepared into coated film, after 37 DEG C of 2 h of coating, room temperature naturally dry or
37 DEG C of drying of person；
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. Madumycin 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 Madumycin antibody that will be marked centrifuges 20 min under the conditions of 1000 r/min, 4 DEG C,
Supernatant is taken, 1 mg/mL is diluted to 0.01 mol/L PBS；Or 1 mg/ml is diluted to 0.01 mol/L PBS, cross 0.22
μm 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 Madumycin list
Clonal antibody colloid gold label object；
(4) preparation of colloidal gold film：Step (3) Madumycin protein conjugate monoclonal antibody colloid gold label object is used and is drawn
Film instrument is sprayed on the even concentration of 5 μ L/cm on carrier glass cellulose membrane, room temperature naturally dry or 37 DEG C of 3 h of drying, system
Into the colloidal gold film of the monoclonal antibody colloid gold label object of protein conjugate containing Madumycin；
(5) it is coated with film preparation：Sheep anti-mouse igg antibody, Madumycin 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；
(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. Madumycin colloidal-gold detecting-card according to claim 1, it is characterised in that institute is coated in the step (5)
Detection line T 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 |
|---|---|---|---|
| CN201611158306.5A CN108226486A (en) | 2016-12-15 | 2016-12-15 | Immune colloid gold detection card of Madumycin and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611158306.5A CN108226486A (en) | 2016-12-15 | 2016-12-15 | Immune colloid gold detection card of Madumycin and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108226486A true CN108226486A (en) | 2018-06-29 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611158306.5A Pending CN108226486A (en) | 2016-12-15 | 2016-12-15 | Immune colloid gold detection card of Madumycin and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103353530A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for rapidly detecting maduramicin, and test card |
| CN103808932A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Maduramicin colloidal gold detection card |
| 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 |
| CN105572390A (en) * | 2014-10-13 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Immunocolloidal gold detection card for herbicide 2,4-D and preparation method thereof |
| CN105572372A (en) * | 2014-10-17 | 2016-05-11 | 南京亿特生物科技有限公司 | Immunity colloid gold detecting card of imidacloprid and preparing method of immunity colloid gold detecting card |
| CN105572392A (en) * | 2014-10-16 | 2016-05-11 | 丹阳亿太生物科技发展有限公司 | Immunity colloid gold detecting card of herbicide glyphosate and preparing method of immunity colloid 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 CN201611158306.5A patent/CN108226486A/en active Pending
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103808932A (en) * | 2012-11-06 | 2014-05-21 | 江苏维赛科技生物发展有限公司 | Maduramicin colloidal gold detection card |
| CN103353530A (en) * | 2012-12-12 | 2013-10-16 | 河南省农业科学院 | Test strip for rapidly detecting maduramicin, and test card |
| CN105572390A (en) * | 2014-10-13 | 2016-05-11 | 江苏维赛科技生物发展有限公司 | Immunocolloidal gold detection card for herbicide 2,4-D and preparation method thereof |
| 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 |
| CN105572392A (en) * | 2014-10-16 | 2016-05-11 | 丹阳亿太生物科技发展有限公司 | Immunity colloid gold detecting card of herbicide glyphosate and preparing method of immunity colloid gold detecting card |
| 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 |
| CN105572372A (en) * | 2014-10-17 | 2016-05-11 | 南京亿特生物科技有限公司 | Immunity colloid gold detecting card of imidacloprid and preparing method of immunity colloid 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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