CN104655835A - Immune colloidal gold detect card of chlorpromazine, and preparation method thereof - Google Patents

Immune colloidal gold detect card of chlorpromazine, and preparation method thereof Download PDF

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Publication number
CN104655835A
CN104655835A CN201310582213.5A CN201310582213A CN104655835A CN 104655835 A CN104655835 A CN 104655835A CN 201310582213 A CN201310582213 A CN 201310582213A CN 104655835 A CN104655835 A CN 104655835A
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chlorpromazine
gold
colloidal gold
solution
film
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洪霞
杜霞
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Nanjing Yite Biological Technology Co Ltd
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Nanjing Yite Biological Technology Co Ltd
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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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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
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  • Food Science & Technology (AREA)
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  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses an immune colloidal gold detect card of chlorpromazine, and a preparation method thereof, and belongs to the technical field of animal-derived food veterinary drug residue detection. A test strip of an outer housing of the immune colloidal gold detect card is composed of a PVC slab, a sample pad, a colloidal gold conjugate pad, a coating membrane, and an absorbent pad; a colloidal gold membrane is a glass cellulose membrane containing chlorpromazine monoclonal antibodies; the coating membrane is a nitrocellulose memebrane, and is provided with a T line and a C line; the T line contains chlorpromazine protein conjugates via coating; and the C line contains goat anti-mouse IgG antibodies via coating. The immune colloidal gold detect card is used for rapid detection of chlorpromazine effectively, and is convenient and rapid for using; and detection results are accurate.

Description

Immune colloid gold test card of chlorpromazine and preparation method thereof
Technical field
The present invention relates to animal-derived food detection of veterinary drugs in food technical field, immune colloid gold test card particularly relating to chlorpromazine and preparation method thereof.
Background technology
Chlorpromazine belongs to phenothiazines metabolin, this drug main will at liver metabolism, easy generation medicament residue, the metabolin that residual chlorpromazine and part have former medicine activity can cause Neuroleptic Leukocytopenia and agranulocytosis, thus cause the pathology of human liver, kidney, also can cause the complication etc. of eye.There is the raiser of some countries and regions to add chlorpromazine in consumption animal feeding, cause having in relevant animal food the residual of chlorpromazine, therefore regularly chlorpromazine is remained to the necessary means detecting the monitoring becoming many countries.
Prior art is for the detection mainly detection method such as high performance liquid chromatography-tandem mass method, high performance liquid chromatography of chlorpromazine, but the defect of these technology is obvious: apparatus expensive, complicated operation, be difficult to promote.So it is extremely necessary for setting up a kind of assay method that is simple, effective, that be applicable to basic unit's use.The object of the invention is a kind of enterprise that is more suitable for of development and carry out Site Detection and qualitative checking method fast and simple, with low cost.
Summary of the invention
For above situation, object of the present invention be exactly in order to overcome prior art exist defect and immune colloid gold test card that a kind of chlorpromazine is provided and preparation method thereof, effectively can solve the problem that can detect chlorpromazine fast, easily.
Chlorpromazine colloidal-gold detecting-card of the present invention, comprise and being formed by the nitrocellulose filter of chlorpromazine-BSA and sheep anti-mouse igg, sample pad, adsorptive pads, PVC offset plate and mould of plastics by the gold conjugation pad of monoclonal antibody colloid gold label thing, bag, sample pad, pad is adhered to successively in one end of PVC offset plate, nitrocellulose filter is pasted in centre, and the other end adheres to adsorptive pads.
The preparation method of chlorpromazine colloidal-gold detecting-card of the present invention is realized by following steps:
(1) colloidal gold solution preparation: get 1 L Erlenmeyer flask 1, add ultrapure water 495 mL, then add 1% gold chloride (HAuCl 43H 2o) 5 mL, are mixed with 500 mL 0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate (Na after boiling when Keep agitation 3c 6h 5o 72H 2o) solution 5-7 mL, continues agitating heating, and when the color of solution becomes transparent aubergine completely, stop heating after maintaining 5 min, moisturizing, to original volume, is cooled to room temperature, and 2-8 DEG C saves backup;
(2) pre-service of antibody: the chlorpromazine antibody that will mark is at 1000 r/min, and under 4 DEG C of conditions, centrifugal 20 min, get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Or be diluted to 1 mg/ml with 0.01 mol/L PBS, cross 0.22 μm of filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in step (1), colloidal gold solution pH to 8.5 is regulated with 0.25 mol/L K2CO3, magnetic stirrer 250 r/min stirs, dropwise add the protein solution of 4 mL containing 0.3 mg antibody protein, react 10 min; Dropwise add 4 mL 10% BSA, continue stirring reaction 10 min; Gold labeling antibody solution normal temperature low speed (1500 r/min) centrifugal 10 min, discard the precipitation formed by the gold grain condensed; Red supernatant solution 4 DEG C, centrifugal 20 min of 12000 r/min, abandon supernatant, collecting precipitation, and the golden labeling antibody dilution of precipitation is settled to 1 mL, are prepared into chlorpromazine monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: step (3) chlorpromazine protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with drawing film instrument with the even concentration of 5 μ L/cm, room temperature is dried or 37 DEG C of oven dry 3 h naturally, makes the colloidal gold film containing chlorpromazine protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, chlorpromazine protein conjugate are diluted to 1 mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, 37 DEG C of bags are by after 2 h, and room temperature is dried or 37 DEG C of oven dry naturally;
(6) sample pad pre-treatment: be uniformly coated on by sample pad treating fluid on glass fibre element film, under the condition of air humidity lower than 60%, room temperature is dried naturally;
(7) assembling of test card: PVC offset plate pastes sample pad, colloidal gold film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into the rectangular of one fixed width, then test strips is arranged in the test card shell of rectangular flat shelly.
In described step (5), the detection line T of institute's bag quilt is located at the below of nature controlling line C;
Sample pad treating fluid in described step (6) is by 1 g Bovine serum albumin (BSA) and 0.8 g sodium chloride (NaCl), is settled to 100 mL with the 0.01 mol/L PBS containing 0.5%TRITON-100;
The present invention can be effective to measure chlorpromazine, and method is simple, and convenient, fast, result is accurate.
Accompanying drawing explanation
Fig. 1 is the structural drawing of the immune colloid gold test card of chlorpromazine of the present invention: 1. well 2. detection line 3. nature controlling line 4. detect aperture 5. test strips 6. test card shells in figure.
Fig. 2 is the sectional structure chart of the test strips in the immune colloid gold test card of chlorpromazine of the present invention, 7. sample pad 8. gold conjugation pad 9.PVC offset plate 10. nitrocellulose filter 11. adsorptive pads in figure.
Embodiment
Embodiment 1
Embodiment shown in Fig. 1, Fig. 2: in figure, 9 is PVC offset plate; 7 is sample pad; 8 is gold conjugation pad, and on this gold conjugation pad, bag is by monoclonal antibody colloid gold label thing; 10 is coated film, i.e. nitrocellulose filter, and on this nitrocellulose filter, bag is by chlorpromazine-BSA and sheep anti-mouse igg; 11 is adsorptive pads, by absorbent material as filter paper is made.
On one end of PVC offset plate 9, (sample end) adheres to sample pad 7, pad 8, and sample pad 7 and pad 8 are side by side configuration.
At the intermediate adhesion nitrocellulose filter 10 of PVC offset plate 9.Nitrocellulose filter 10 is provided with sheep anti-mouse igg nature controlling line 3 and chlorpromazine-BSA detection line 2.
Adsorptive pads 11 is adhered at the other end of PVC offset plate 9.One end of nitrocellulose filter 10 slightly intersects with pad 8, and the other end slightly intersects with adsorptive pads 11.This test strips 5 can be incorporated with in the test card shell 6 that mould of plastics makes, and make test card, test card shell 6 covers and is provided with well 1 and detect aperture 4, sample pad 7 is just to well 1, and nitrocellulose filter 10 is just to detect aperture 4.
Embodiment 2
Prepared by chlorpromazine colloidal-gold detecting-card, be by following steps specific implementation:
Prepared by colloidal gold solution: get 1 L Erlenmeyer flask 1, add ultrapure water 495 mL, then add 1% gold chloride (HAuCl 43H 2o) 5 mL, are mixed with 500 mL 0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate (Na after boiling when Keep agitation 3c 6h 5o 72H 2o) solution 5-7 mL, continues agitating heating, and when the color of solution becomes transparent aubergine completely, stop heating after maintaining 5 min, moisturizing, to original volume, is cooled to room temperature, and 2-8 DEG C saves backup;
The pre-service of antibody: the chlorpromazine antibody that will mark is at 1000 r/min, and under 4 DEG C of conditions, centrifugal 20 min, get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Or be diluted to 1 mg/ml with 0.01 mol/L PBS, cross 0.22 μm of filter membrane;
The preparation of colloid gold label thing: get colloidal gold solution 40 mL in step (1), colloidal gold solution pH to 8.5 is regulated with 0.25 mol/L K2CO3, magnetic stirrer 250 r/min stirs, and dropwise adds the protein solution of 4 mL containing 0.3 mg antibody protein, reacts 10 min; Dropwise add 4 mL 10% BSA, continue stirring reaction 10 min; Gold labeling antibody solution normal temperature low speed (1500 r/min) centrifugal 10 min, discard the precipitation formed by the gold grain condensed; Red supernatant solution 4 DEG C, centrifugal 20 min of 12000 r/min, abandon supernatant, collecting precipitation, and the golden labeling antibody dilution of precipitation is settled to 1 mL, are prepared into chlorpromazine monoclonal antibody colloid gold label thing;
The preparation of colloidal gold film: step (3) chlorpromazine protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with drawing film instrument with the even concentration of 5 μ L/cm, room temperature is dried or 37 DEG C of oven dry 3 h naturally, makes the colloidal gold film containing chlorpromazine protein conjugate monoclonal antibody colloid gold label thing;
Prepared by coated film: sheep anti-mouse igg antibody, chlorpromazine protein conjugate are diluted to 1 mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, 37 DEG C of bags are by after 2 h, and room temperature is dried or 37 DEG C of oven dry naturally;
Sample pad pre-treatment: be uniformly coated on by sample pad treating fluid on glass fibre element film, under the condition of air humidity lower than 60%, room temperature is dried naturally;
The assembling of test card: PVC offset plate pastes sample pad, colloidal gold film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into one fixed width rectangular, then test strips is arranged in the test card shell of rectangular flat shelly.
Embodiment 3
Chlorpromazine minimum detectability amount is tested
Precise 0.500 g chlorpromazine antigen is dissolved in ultrapure water and is settled to 100 mL, is designated as A 1solution, gets A 1solution 1 mL adds ultrapure water in volumetric flask and is settled to 1000 mL, forms A 2solution, gets A 2solution 1 mL adds ultrapure water in volumetric flask and is settled to 1000 mL, forms A 3solution, then the concentration of A3 solution is 5 ng/mL (5ppb).Get A 3solution 1 mL is in 4 test tubes, and difference label 1 ~ 4, add respectively in test tube toward 9 mL, 4 mL, 1 mL, 0 mL ultrapure water, be made into chlorpromazine antigen solution concentration respectively: 0.5 ng/mL, 1 ng/mL, 2.5 ng/mL, 5 ng/mL; Separately get an empty test tube, add 5 mL ultrapure waters, be designated as No. 5 test tubes.Getting liquid in above-mentioned 1 ~ No. 5 test tube is added in chlorpromazine test card as sample drop, and 5 min observationss after application of sample, the colour developing of detection line 2 and nature controlling line 3 the results are shown in Table 1.
Table 1 chlorpromazine minimum detectability amount is tested
Through repeatedly checking, the recall rate of chlorpromazine reaches 99%, and minimum detectability amount is 5 ng/mL.

Claims (2)

1. chlorpromazine colloidal-gold detecting-card, is characterized in that preparing according to following step:
(1) colloidal gold solution preparation: get 1 L Erlenmeyer flask 1, add ultrapure water 495 mL, then add 1% gold chloride HAuCl 43H 2o) 5 mL, are mixed with 500 mL 0.01% aqueous solution of chloraurate, and heating adds 1% trisodium citrate Na after boiling when Keep agitation 3c 6h 5o 72H 2o solution 5-7 mL, continues agitating heating, and when the color of solution becomes transparent aubergine completely, stop heating after maintaining 5 min, moisturizing, to original volume, is cooled to room temperature, and 2-8 DEG C saves backup;
(2) pre-service of antibody: the chlorpromazine antibody that will mark is at 1000 r/min, and under 4 DEG C of conditions, centrifugal 20 min, get supernatant, are diluted to 1 mg/mL with 0.01 mol/L PBS; Or be diluted to 1 mg/ml with 0.01 mol/L PBS, cross 0.22 μm of filter membrane;
(3) preparation of colloid gold label thing: get colloidal gold solution 40 mL in step (1), with 0.25 mol/L K 2cO 3regulate colloidal gold solution pH to 8.5, magnetic stirrer 250 r/min stirs, and dropwise adds the protein solution of 4 mL containing 0.3 mg antibody protein, reacts 10 min; Dropwise add 4 mL 10% BSA, continue stirring reaction 10 min; Centrifugal 10 min of gold labeling antibody solution normal temperature 1500 r/min, discard the precipitation formed by the gold grain condensed; Red supernatant solution 4 DEG C, centrifugal 20 min of 12000 r/min, abandon supernatant, collecting precipitation, and the golden labeling antibody dilution of precipitation is settled to 1 mL, are prepared into chlorpromazine monoclonal antibody colloid gold label thing;
(4) preparation of colloidal gold film: step (3) chlorpromazine protein conjugate monoclonal antibody colloid gold label thing is sprayed on carrier glass cellulose membrane with drawing film instrument with the even concentration of 5 μ L/cm, room temperature is dried or 37 DEG C of oven dry 3 h naturally, makes the colloidal gold film containing chlorpromazine protein conjugate monoclonal antibody colloid gold label thing;
(5) coated film preparation: sheep anti-mouse igg antibody, chlorpromazine protein conjugate are diluted to 1 mg/mL, be sprayed on nitrocellulose filter with the concentration of 1 μ L/cm successively with drawing film instrument, be prepared into coated film, 37 DEG C of bags are by after 2 h, and room temperature is dried or 37 DEG C of oven dry naturally;
(6) sample pad pre-treatment: be uniformly coated on by sample pad treating fluid on glass fibre element film, under the condition of air humidity lower than 60%, room temperature is dried naturally;
(7) assembling of test card: PVC offset plate pastes sample pad, colloidal gold film, coated film and absorbent wool from top to bottom successively, is assembled into test strips, cuts into the rectangular of one fixed width, then test strips is arranged in the test card shell of rectangular flat shelly.
2. chlorpromazine colloidal-gold detecting-card according to claim 1, is characterized in that the detection line T of institute's bag quilt in described step (5) is located at the below of nature controlling line C;
Sample pad treating fluid in described step (6) is by 1 g Bovine serum albumin (BSA) and 0.8 g sodium chloride (NaCl), is settled to 100 mL with the 0.01 mol/L PBS containing 0.5%TRITON-100.
CN201310582213.5A 2013-11-20 2013-11-20 Immune colloidal gold detect card of chlorpromazine, and preparation method thereof Pending CN104655835A (en)

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Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106568957A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immune colloidal gold detection card of chlorothalonil, and preparation method thereof
CN106568947A (en) * 2015-10-13 2017-04-19 镇江亿特生物科技发展有限公司 Immune colloidal gold detection card of DDVP and preparation method thereof
CN106568959A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immunocolloidal gold test card of acetochlor and preparation method thereof
CN106568958A (en) * 2015-10-13 2017-04-19 丹阳亿太生物科技发展有限公司 Immunocolloidal-gold detection cassette for trifuralin and preparation method thereof
CN106568904A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immunity colloid gold detection card of paraquat and preparation method thereof
CN106568937A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immunity colloid gold detection card of benalaxyl and preparation method thereof
CN106568960A (en) * 2015-10-13 2017-04-19 丹阳亿太生物科技发展有限公司 Immunocolloidal gold test card of methomyl and preparation method thereof
CN106568946A (en) * 2015-10-13 2017-04-19 镇江亿特生物科技发展有限公司 immunogold detection card of propiconazole, and preparation method thereof
CN106596944A (en) * 2015-10-14 2017-04-26 江苏维赛科技生物发展有限公司 Tilmicosin phosphate immune colloidal gold detection card and preparation method thereof
CN108226505A (en) * 2016-12-15 2018-06-29 南京亿特生物科技有限公司 Immune colloid gold detection card of sodium sulfocyanate and preparation method thereof
CN109813886A (en) * 2017-11-20 2019-05-28 丹阳亿太生物科技发展有限公司 A kind of Rapid detection test strip detecting chlorpromazine medicament residue
CN109813891A (en) * 2017-11-18 2019-05-28 镇江亿特生物科技发展有限公司 A kind of time-resolved fluoroimmunoassay chromatography quantitative testing test paper item detecting chlorpromazine
CN109813900A (en) * 2017-11-21 2019-05-28 南京亿特生物科技有限公司 A kind of time-resolved fluoroimmunoassay chromatography quantitative testing test paper item detecting chloramphenicol
CN109813901A (en) * 2017-11-21 2019-05-28 南京亿特生物科技有限公司 A kind of immune colloid gold detection card and preparation method thereof detecting ethiprole

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CN101236202A (en) * 2008-02-06 2008-08-06 中国计量学院 Chlorpromazine-diazepam dual detection card and its detection method
CN101424688A (en) * 2008-11-20 2009-05-06 上海交通大学 Method for detecting chlorpromazine by colloidal gold immune chromatography test
CN102901819A (en) * 2012-09-27 2013-01-30 江苏维赛科技生物发展有限公司 Immune colloidal gold detection card for phenylethanolamine A and preparation method thereof

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Publication number Priority date Publication date Assignee Title
CN101236202A (en) * 2008-02-06 2008-08-06 中国计量学院 Chlorpromazine-diazepam dual detection card and its detection method
CN101424688A (en) * 2008-11-20 2009-05-06 上海交通大学 Method for detecting chlorpromazine by colloidal gold immune chromatography test
CN102901819A (en) * 2012-09-27 2013-01-30 江苏维赛科技生物发展有限公司 Immune colloidal gold detection card for phenylethanolamine A and preparation method thereof

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106568960A (en) * 2015-10-13 2017-04-19 丹阳亿太生物科技发展有限公司 Immunocolloidal gold test card of methomyl and preparation method thereof
CN106568957A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immune colloidal gold detection card of chlorothalonil, and preparation method thereof
CN106568959A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immunocolloidal gold test card of acetochlor and preparation method thereof
CN106568958A (en) * 2015-10-13 2017-04-19 丹阳亿太生物科技发展有限公司 Immunocolloidal-gold detection cassette for trifuralin and preparation method thereof
CN106568904A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immunity colloid gold detection card of paraquat and preparation method thereof
CN106568937A (en) * 2015-10-13 2017-04-19 江苏维赛科技生物发展有限公司 Immunity colloid gold detection card of benalaxyl and preparation method thereof
CN106568947A (en) * 2015-10-13 2017-04-19 镇江亿特生物科技发展有限公司 Immune colloidal gold detection card of DDVP and preparation method thereof
CN106568946A (en) * 2015-10-13 2017-04-19 镇江亿特生物科技发展有限公司 immunogold detection card of propiconazole, and preparation method thereof
CN106596944A (en) * 2015-10-14 2017-04-26 江苏维赛科技生物发展有限公司 Tilmicosin phosphate immune colloidal gold detection card and preparation method thereof
CN108226505A (en) * 2016-12-15 2018-06-29 南京亿特生物科技有限公司 Immune colloid gold detection card of sodium sulfocyanate and preparation method thereof
CN109813891A (en) * 2017-11-18 2019-05-28 镇江亿特生物科技发展有限公司 A kind of time-resolved fluoroimmunoassay chromatography quantitative testing test paper item detecting chlorpromazine
CN109813886A (en) * 2017-11-20 2019-05-28 丹阳亿太生物科技发展有限公司 A kind of Rapid detection test strip detecting chlorpromazine medicament residue
CN109813900A (en) * 2017-11-21 2019-05-28 南京亿特生物科技有限公司 A kind of time-resolved fluoroimmunoassay chromatography quantitative testing test paper item detecting chloramphenicol
CN109813901A (en) * 2017-11-21 2019-05-28 南京亿特生物科技有限公司 A kind of immune colloid gold detection card and preparation method thereof detecting ethiprole

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Application publication date: 20150527