CN102645532A - Device and method for detecting beta-agonists drug - Google Patents
Device and method for detecting beta-agonists drug Download PDFInfo
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- CN102645532A CN102645532A CN2012100015099A CN201210001509A CN102645532A CN 102645532 A CN102645532 A CN 102645532A CN 2012100015099 A CN2012100015099 A CN 2012100015099A CN 201210001509 A CN201210001509 A CN 201210001509A CN 102645532 A CN102645532 A CN 102645532A
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- 239000003814 drug Substances 0.000 title claims abstract description 56
- 229940079593 drug Drugs 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 14
- 229940125388 beta agonist Drugs 0.000 title abstract description 6
- 238000012360 testing method Methods 0.000 claims abstract description 50
- 238000001514 detection method Methods 0.000 claims abstract description 48
- 229940124748 beta 2 agonist Drugs 0.000 claims description 49
- 238000006243 chemical reaction Methods 0.000 claims description 33
- 238000003317 immunochromatography Methods 0.000 claims description 17
- 239000012528 membrane Substances 0.000 claims description 12
- 239000002131 composite material Substances 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 4
- 238000003908 quality control method Methods 0.000 claims description 4
- 239000004816 latex Substances 0.000 claims description 3
- 229920000126 latex Polymers 0.000 claims description 3
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- 239000002245 particle Substances 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 2
- 239000006249 magnetic particle Substances 0.000 claims 1
- 238000004587 chromatography analysis Methods 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000002360 preparation method Methods 0.000 description 14
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 8
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 6
- 239000000020 Nitrocellulose Substances 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 229920001220 nitrocellulos Polymers 0.000 description 6
- 238000010521 absorption reaction Methods 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- YJQZYXCXBBCEAQ-UHFFFAOYSA-N ractopamine Chemical compound C=1C=C(O)C=CC=1C(O)CNC(C)CCC1=CC=C(O)C=C1 YJQZYXCXBBCEAQ-UHFFFAOYSA-N 0.000 description 5
- 229940074095 ractopamine Drugs 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 4
- 239000001913 cellulose Substances 0.000 description 4
- 229960001399 clenbuterol hydrochloride Drugs 0.000 description 4
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 description 4
- 239000000084 colloidal system Substances 0.000 description 4
- 239000010931 gold Substances 0.000 description 4
- 229910052737 gold Inorganic materials 0.000 description 4
- 235000013372 meat Nutrition 0.000 description 4
- NDAUXUAQIAJITI-UHFFFAOYSA-N albuterol Chemical compound CC(C)(C)NCC(O)C1=CC=C(O)C(CO)=C1 NDAUXUAQIAJITI-UHFFFAOYSA-N 0.000 description 3
- 229960002052 salbutamol Drugs 0.000 description 3
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
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- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
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- 239000003651 drinking water Substances 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000009079 Bronchial Spasm Diseases 0.000 description 1
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- 229960001117 clenbuterol Drugs 0.000 description 1
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- 238000003255 drug test Methods 0.000 description 1
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- 229930195729 fatty acid Natural products 0.000 description 1
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- GOLXNESZZPUPJE-UHFFFAOYSA-N spiromesifen Chemical compound CC1=CC(C)=CC(C)=C1C(C(O1)=O)=C(OC(=O)CC(C)(C)C)C11CCCC1 GOLXNESZZPUPJE-UHFFFAOYSA-N 0.000 description 1
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- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of biological detection and discloses a detection device and a detection method of a large class of beta-agonists drugs by applying an immune chromatography technology. According to the invention, an antibody/receptor which is reacted with a large class of the beta-agonists drugs is used for preparing an immune chromatography test strip to realize the simultaneous detection of a large class of the beta-agonists drugs. Compared with the prior art, the detection on a large class of the beta-agonists drugs for one time can be realized, the detection cost is low and the method is convenient and fast.
Description
Technical field
The present invention relates to field of biological detection, relate in particular to and use detection method and the device thereof that immunochromatography technique detects beta-2-agonists class medicine.
Background technology
(β-agonists), be called beta-stimulants again is one type of synthetic medicine to beta-2-agonists, is mainly used in control people, animal bronchial astehma and bronchial spasm, pharmaceutically is being called the beta-adrenaline excitant.This type of medicine adds the medicine that can promote the lean meat growth in livestock and poultry feed, the potable water, suppress the fat meat growth to, is commonly called as clenbuterol hydrochloride.Because this type of medicine can make the eater occur nauseating, dizzy, toxicity symptoms such as muscle trembles, palpitaition easily in the accumulation of internal organ such as liver, lung, has had a strong impact on the healthy of consumer, even has threatened people's life security.States such as America and Europe made laws already and forbid on Production of Livestock and Poultry, using beta-2-agonists class medicine, and identical regulation was also made by China Ministry of Agriculture in 1997.However, receive ordering about of economic interests, illegal abuse beta-2-agonists class medicine phenomenon is still commonplace.2002; The Ministry of Agriculture, the Ministry of Public Health, State Food and Drug Administration announce once more; Prohibite and in feed and animal drinking water, add 7 kinds of clenbuterol hydrochlorides such as clenobuterol hydrochloride and Ractopamine; But in the operation of reality, only detect Clenizole Hydrochloride, Ractopamine, salbutamol etc. are not included conventional detection in for six kinds.So cause on the market phenomenon that a kind of clenbuterol hydrochloride remains incessant after repeated prohibition occurring, check clenobuterol hydrochloride severely like China, just the clenbuterol hydrochloride that occurs replacing with Ractopamine and salbutamol occur, to escape the detection of detection department.So realizing the detection of one type of beta-2-agonists class medicine is the strong approach of thoroughly forbidding the clenbuterol hydrochloride incident.
At present the detection method to beta-2-agonists class medicine mainly contains HPLC, HPLC/MS and GC/MS, but because analytical instrument is expensive, testing cost is high, and complex steps is consuming time longer, so and be not suitable for herding department, supervisory and management department and breed unit.
Immunochromatography technique is a kind of easy Fast Detection Technique of phase at the beginning of the eighties in last century, and the immuno-chromatographic test paper strip of being processed by this technology consists of the following components usually: base plate and stick on sample pad on the base plate, pad, cellulose membrane, absorption pad etc.During detection, sample drop is added on the sample pad, sample can move on chromatography strip through capillary action; In the process of migration; With the label generation specific reaction on the pad, produce immune complex, this immune complex continues migration; Combine with the corresponding antigens/antibody generation specificity of surveyed area on the cellulose membrane, form macroscopic detection band.This method is applied to the agricultural and veterinary chemicals residue detection based on immunochromatography and antigen-antibody reaction principle, can realize the residual fast quantitative analysis of medicine is highly suitable for the screening of batch samples.
But mostly the detection kit to beta-2-agonists class medicine is that the individual event of beta-2-agonists class medicine detects, and has only the small quantity of reagent box can realize the joint inspection of two kinds (clenobuterol hydrochloride and Ractopamines) or three kinds of beta-2-agonists class medicines (clenobuterol hydrochloride, Ractopamine and salbutamol) at present.Its method is a kind of beta-2-agonists class drug coupling high molecular weight protein of application and prepares monoclonal antibody or how anti-, with the corresponding beta-2-agonists class of preparation antibody test medicine.Like patent 02129704.X; Its with clenobuterol hydrochloride-carrier coupling protein as antigen; Immune mouse, the preparation monoclonal antibody is used the immunochromatographic method preparation and is detected the clenobuterol hydrochloride colloidal gold strip; Patent 200510126415.4, it is used with quadrat method and realizes in the sample detection to Ractopamine.Be a kind of beta-2-agonists class of a kind of antibody test medicine, detect when can't realize one type of medicine.
Summary of the invention
But the present invention provides the preparation and the method for application of the immuno-chromatographic test paper strip of one big type of beta-2-agonists class of a kind of one-time detection medicine.
1) to achieve these goals, a technical scheme of the present invention is:
A kind of pick-up unit is provided, comprises test strips and reaction cup two parts.Test strips comprises sample pad, immunochromatography composite membrane, the thieving paper that overlaps stickup on base plate and the base plate successively, wherein is coated with Quality Control district and detection zone on the immunochromatography composite membrane.Reaction cup contains label-beta-2-agonists class drug antibody/receptor complex.
Aforesaid label is characterized by the one type of material that can be used as signal tracer, like collaurum, electroselenium, latex particle, fluorescent grain, magnetic bead, and other any material that can be used as the signal mark.
Aforesaid test strips is characterized in that being made up of 1-3 sub-test strips, promptly detects one big type of beta-2-agonists class medicine with 1-3 sub-test strips.If be made up of 1 sub-test strips, then the detection zone of test strips is made up of the 1-3 sub-detection areas, can detect one big type of beta-2-agonists class medicine.
Aforesaid reaction cup is characterized by the buffer system that wherein contains label-beta-2-agonists class drug antibody/receptor complex and suitable biomolecular reaction, and the pH of this buffer system is between 1-13.
Aforesaid reaction cup is characterized in that content is the powder of freeze-drying, can also be liquid.
The present invention also provides a kind of detection method, it is characterized in that sample to be checked after pre-treatment, at first in reaction tank, with label-beta-2-agonists class drug antibody/receptor complex fully reaction takes place.
Detection method as stated; After it is characterized in that adding sample, the beta-2-agonists class drug antibody/acceptor in target substance in the sample and the reaction tank reacts 0-1h between 15-60 ℃, after reaction finishes; Test strip is inserted in the reaction cup interpretation testing result behind the reaction 0-1h.
Aforesaid detection method is characterized in that and can with the naked eye directly carry out interpretation as a result that also available corresponding instrument carries out interpretation.
2) another technical scheme of the present invention is:
A kind of immunochromatographydetecting detecting test strip is provided, and sample pad, label pad, immunochromatography composite membrane, thieving paper that this test strips is pasted by base plate and the last overlap joint of base plate wherein have Quality Control district and detection zone on the immunochromatography composite membrane.
Aforesaid test strips is characterized in that being made up of 1-3 sub-test strips, promptly detects one big type of beta-2-agonists class medicine with 1-3 sub-test strips.If be made up of 1 sub-test strips, then the detection zone of test strips is made up of the 1-3 sub-detection areas, can detect one big type of beta-2-agonists class medicine.
Aforesaid label pad is characterized by and is coated with label-beta-2-agonists class drug antibody/receptor complex.
Aforesaid label is characterized by the one type of material that can be used as signal tracer, like collaurum, electroselenium, latex particle, fluorescent grain, nanometer magnetic bead, and other any material that can be used as the signal mark.
Aforesaid detection zone is characterized by this zone and is made up of the 1-3 sub regions, is coated with beta-2-agonists class medicine-protein conjugate in these subregions, can detect one big type of beta-2-agonists class medicine.
The present invention also provides a kind of detection method, it is characterized in that test strip is inserted in the sample to be checked, between 15-60 ℃, reacts 0-1h, sentence read result.
Aforesaid detection method is characterized in that and can with the naked eye directly carry out interpretation as a result, can also carry out interpretation with corresponding instrument and equipment.
The antibodies/receptors of utilization of the present invention and the drug response of one big type of beta-2-agonists class prepares immuno-chromatographic test paper strip, detects when realizing one big type of beta-2-agonists class medicine.Compared with prior art, both save the detection cost, shortened detection time again.
Embodiment
Although content of the present invention is to combine this instance to describe, can not think restriction to patent of the present invention, scope of the present invention is limited appended claims.In addition, those skilled in the art carries out various changes or modification to the present invention in the appended claims restricted portion, and these changes or modified forms belong to protection scope of the present invention equally.
Real row 1 immuno-chromatography detection devices (test strips, reaction cup pattern)
1) preparation of test strips
The preparation of sample pad: cellulose membrane is cut into the band of 1.5 * 30cm specification, subsequent use;
The preparation of nitrocellulose filter: nitrocellulose filter is cut into the band of 2.5 * 30cm specification, dries at the antibody of the diverse location spraying beta-2-agonists class drug antibody/acceptor of NC film or two anti-(nature controlling line), beta-2-agonists class medicine parts (detection line), the room temperature of beta-2-agonists class drug antibody/acceptor;
The preparation of absorption pad: thieving paper is cut into the band of 3.5 * 30cm, subsequent use;
The assembling of test strips: absorption pad, nitrocellulose filter, sample pad are attached on the base plate successively, and are cut into the wide test strips of 5mm, 2-8 ℃ of kept dry.
2) preparation of reaction cup
According to the amount in 100 μ L/ holes, add the beta-2-agonists class drug antibody/acceptor of colloid gold label in the 96 hole microwell plates;
-80 ℃ of freezing 24h;
The beta-2-agonists class drug antibody/acceptor of the colloid gold label in the freeze drying hole;
Place dry environment to preserve the beta-2-agonists class drug antibody/acceptor of the colloid gold label after the freeze-drying.
3) detect urine sample
Get the 100ul urine sample and add reaction cup, behind 40 ℃ of abundant reaction 3min, test strips is inserted in the reaction cup, continue to read the result behind the reaction 5min.
4) beta-2-agonists is residual in the detection feed sample
Grind feed with mortar, claim the hydrochloric acid of the feed sample adding 10ml0.01M that 1g grinds, fully mixed 5 minutes; Regulate the pH value between the 6.5-8.0; The centrifugal 5min of 3500rpm migrates out supernatant; Directly get the 100ul supernatant and add reaction cup, behind 40 ℃ of abundant reaction 3min, test strips is inserted in the reaction cup, continue to read the result behind the reaction 5min.
Instance 2 immuno-chromatography detection devices (test strips pattern)
1) preparation of test strips
The preparation of sample pad: cellulose membrane is cut into the band of 1.5 * 30cm specification, subsequent use;
The preparation of pad: the beta-2-agonists class drug antibody/acceptor of colloid gold label is sprayed on the all-glass paper, and vacuum is drained, and is subsequent use.
The preparation of nitrocellulose filter: nitrocellulose filter is cut into the band of 2.5 * 30cm specification, and at the antibody of the diverse location spraying beta-2-agonists class drug antibody/acceptor of NC film, beta-2-agonists class medicine part (detection line), room temperature are dried;
The preparation of absorption pad: thieving paper is cut into the band of 3.5 * 30cm, subsequent use;
The assembling of test strips: absorption pad, label pad, nitrocellulose filter, sample pad are attached on the base plate successively, and are cut into the wide test strips of 5mm, 2-8 ℃ of kept dry.
2) detect beta-2-agonists in the meat
(1) liver of low-fat content, muscle, tissue
Sample and 25ml 50mM mixed in hydrochloric acid that 5g pulverizes, homogeneous 15min; Get the equal pledge of 6g, 10-15 ℃ of centrifugal 15min, 4000rpm or higher rotating speed; Migrate out supernatant to another centrifugal bottle, add 300 μ L 1M NaOH, mix 15min; Add 4ml 500mM potassium phosphate buffer (pH3.0), simple mixing is incorporated in 4 ℃ of preservations at least 1.5 hours or spends the night.10-15 ℃, the centrifugal 15min of 4000g.Separation of supernatant, balance are inserted test strips in the supernatant to room temperature, read the result behind the 5min.
(2) higher fatty acid meat tissue
Get the 5g sample in centrifuge tube, add 25ml 50mM Tris-HCl (pH 8.5), vibration 30min; Add 15ml heptane vibration 5min; The centrifugal 15min of 4000g; Remove top heptane layer and middle very thin fat deposit with pasteur pipet; Again with the vibration of 15ml heptane repeating step, centrifugal and suction degrease layer operation; In the meat liquid of homogeneous, add the hydrochloric acid of 0.5ml 6M, vibrate 1 hour (or shaken overnight); The centrifugal 15min of 4000g; Pipette supernatant in another centrifuge tube, add 300 μ l 1M NaOH, mix 15min; Add 4ml 500mM KH
2PO
4-damping fluid (pH3), simple mixing is incorporated in 4 ℃ and deposited at least 1.5 hours or spent the night; The centrifugal 15min of 4000g; Pipette supernatant, balance is to room temperature; Test strips is directly inserted in the supernatant, read the result behind the 5min.
Claims (9)
1. a beta-2-agonists class medicine immuno-chromatography detection device comprises test strip and reaction cup two parts, and this test strips comprises base plate, and overlaps sample pad, immunochromatography composite membrane, the thieving paper of pasting successively on the base plate.Wherein be coated with detection zone and Quality Control district on the immunochromatography composite membrane.Reaction cup contains label-beta-2-agonists class drug antibody/receptor complex and buffer system.
2. like right 1 described immuno-chromatography detection device, can also be the test paper slip, this test strips be by overlapping sample pad, pad, immunochromatography composite membrane, the thieving paper of pasting on base plate and the base plate successively.Wherein be coated with detection zone and Quality Control district on the immunochromatography composite membrane, be coated with label-beta-2-agonists class drug antibody/receptor complex on the pad.
3. the label described in claim 1 and 2 is characterized by the one type of material that can be used as the signal mark, like collaurum, electroselenium, latex particle, fluorescent grain, magnetic-particle, and other any material that can be used as the signal mark.
4. the test strips described in claim 1 and 2 is characterized in that being made up of 1-3 sub-test strips, promptly detects one big type of beta-2-agonists class medicine with 1-3 sub-test strips.If be made up of 1 sub-test strips, then the detection zone of test strips is made up of the 1-3 sub-detection areas, can detect one big type of beta-2-agonists class medicine.
5. reaction cup part as claimed in claim 1 and the described pad part of claim 2; It is characterized by the buffer system that wherein contains label-beta-2-agonists class drug antibody/receptor complex and suitable biomolecular reaction, the pH of this buffer system is between 1-13.
6. reaction cup part as claimed in claim 1 is characterized in that content is the powder of freeze-drying, can also be liquid.
7. immuno-chromatography detection device as claimed in claim 1; It is characterized in that its method that detects beta-2-agonists class medicine is after adding sample; Beta-2-agonists class drug antibody/acceptor in beta-2-agonists class medicine in the sample and the reaction tank reacts 0-1h between 15-60 ℃; Reaction is inserted test strips in the reaction cup after finishing, interpretation testing result behind reaction 0-1h between 15-60 ℃.
8. immuno-chromatography detection device as claimed in claim 2 is characterized in that its method that detects beta-2-agonists class medicine is that test strips is directly inserted sample to be checked, interpretation testing result behind reaction 0-1h between 15-60 ℃.
9. like the method for right 7 and the described detection beta-2-agonists of claim 8 class medicine, it is characterized in that testing result directly interpretation with the naked eye, also available corresponding instrument carries out interpretation.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102998446A (en) * | 2012-12-14 | 2013-03-27 | 深圳市易瑞生物技术有限公司 | Method for detecting multiple organic compounds with similar molecular structures by using immunochromatography technology in combination |
| CN103149352A (en) * | 2013-02-05 | 2013-06-12 | 江西中德生物工程有限公司 | Mabuterol colloidal gold test strip and preparation method thereof |
| CN107942052A (en) * | 2017-09-29 | 2018-04-20 | 杭州南开日新生物技术有限公司 | A kind of colloidal gold kit for detecting beta-agonist class medicine and preparation method thereof |
| WO2019109365A1 (en) * | 2017-12-06 | 2019-06-13 | 深圳市易瑞生物技术股份有限公司 | Immunochromatography detection device for high-flux agricultural veterinary drug residues |
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| CN102998446A (en) * | 2012-12-14 | 2013-03-27 | 深圳市易瑞生物技术有限公司 | Method for detecting multiple organic compounds with similar molecular structures by using immunochromatography technology in combination |
| CN103149352A (en) * | 2013-02-05 | 2013-06-12 | 江西中德生物工程有限公司 | Mabuterol colloidal gold test strip and preparation method thereof |
| CN107942052A (en) * | 2017-09-29 | 2018-04-20 | 杭州南开日新生物技术有限公司 | A kind of colloidal gold kit for detecting beta-agonist class medicine and preparation method thereof |
| WO2019109365A1 (en) * | 2017-12-06 | 2019-06-13 | 深圳市易瑞生物技术股份有限公司 | Immunochromatography detection device for high-flux agricultural veterinary drug residues |
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