CN102937647B - Lysergide detection kit and preparation method thereof - Google Patents
Lysergide detection kit and preparation method thereof Download PDFInfo
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- CN102937647B CN102937647B CN201210034598.7A CN201210034598A CN102937647B CN 102937647 B CN102937647 B CN 102937647B CN 201210034598 A CN201210034598 A CN 201210034598A CN 102937647 B CN102937647 B CN 102937647B
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- ergot
- ethylenediamine
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Abstract
A kind of detection kit for detecting ergot ethylenediamine, this kit comprises sample pad (1), colloidal gold pad (2), nitrocellulose filter (3), inhale sample pad (4) and PVC back up pad (5), PVC back up pad is stained with sample pad successively continuously, colloidal gold pad, nitrocellulose filter and suction sample pad, described colloidal gold pad is the ergot ethylenediamine monoclonal antibody polyester film of colloid gold label, described nitrocellulose filter wraps by ergot ethylenediamine-BSA coupled antigen successively as detection line (T line), sheep anti-mouse igg polyclonal antibody is as nature controlling line (C line).The present invention adopts colloidal gold immunochromatographimethod technology to prepare Lysergide detection kit, and for detecting the ergot ethylenediamine that may exist in sample, this kit preparation method is simple, have easy to use, be swift in response, the feature such as economical and practical.
Description
Technical field
The present invention relates to the determination techniques field of biology immunization method, particularly relate to a kind of a kind of Lysergide detection kit utilizing colloidal gold immunochromatographimethod fabrication techniques and preparation method thereof.
Background technology
Ergot ethylenediamine (LSD) is the psychedelic that the known efficacy of a drug is the strongest, extremely easily absorbed by the body.Photis, phonism and illusion can be produced after taking, occur the insane symptom panic-stricken, thought is dazed and confused, terribly suspicious, anxious, behavior is out of control and completely helpless.Can be caused disorienting, distinguish the ability of Distance geometry time simultaneously, thus causes health to be punished severely injured and dead.
At present about the detection method of ergot ethylenediamine has a lot, such as: the methods such as high performance liquid chromatography (HPLC), thin-layered chromatography (TLC), vapor-phase chromatography (GC), GC-MS(gas chromatography-mass spectrography) (GC-MS), but exist and need expensive instrument and equipment, high to the requirement of test material, need the restrictions such as purification processes.Therefore the detection method that research has an advantage such as quick, portable has realistic meaning.Invention describes and a kind ofly detect detection kit of ergot ethylenediamine and preparation method thereof fast with colloidal gold immunity chromatography, the method has simply, fast, without the need to any instrument and equipment, whether the feature such as economical and practical, be applicable to fast qualitative and detect in sample containing ergot ethylenediamine.
Summary of the invention
The object of the present invention is to provide a kind of portable, quick, be suitable for detection kit of Site Detection ergot ethylenediamine and preparation method thereof.
A kind of detection kit for detecting ergot ethylenediamine, comprise sample pad (1), colloidal gold pad (2), nitrocellulose filter (3), inhale sample pad (4) and PVC back up pad (5), in PVC back up pad, tight adhesion has sample pad, colloidal gold pad, nitrocellulose filter and suction sample pad successively.Described sample pad is glass fibre; Described colloidal gold pad is the ergot ethylenediamine monoclonal antibody polyester film of colloid gold label; Described nitrocellulose filter is coated with successively detection line (T line) and nature controlling line (C line), wherein the upper bag of detection line (T line) is by ergot ethylenediamine-BSA coupled antigen, and the upper bag of nature controlling line (C line) is by sheep anti-mouse igg polyclonal antibody; Described suction sample pad is thieving paper.
The present invention adopts nano-colloid technology for gold and antigen and antibody specific reaction, application Immune competition suppresses the principle of reaction to be prepared from, wrap the ergot ethylenediamine monoclonal antibody of the ergot ethylenediamine-BSA coupled antigen competition binding colloid gold label of quilt by detection line (T line) on the ergot ethylenediamine that contains in sample to be checked and nitrocellulose filter, whether judged in sample to be checked containing ergot ethylenediamine by the colour developing of T line.
The invention provides a kind of preparation method of Lysergide detection kit, comprise the following steps:
(1) ergot ethylenediamine coupled antigen is prepared
By ergot ethylenediamine and BSA coupling, synthesis ergot ethylenediamine-BSA coupled antigen, as ergot ethylenediamine coupled antigen.
(2) ergot ethylenediamine monoclonal antibody is prepared
Adopt ergot ethylenediamine-BSA coupled antigen to be immunogen immune BALB/C mice, by hybridoma technology, obtain the hybridoma cell strain secreting anti-ergot ethylenediamine monoclonal antibody; To induce a large amount of Dispersal risk of ascites method in body, use ProteinG post to carry out purifying, obtain ergot ethylenediamine monoclonal antibody.
(3) collaurum is prepared
Get 90ml purified water in the round-bottomed flask of cleaning, stir in Electromagnetic Heating cover and heat, after waiting for boiling, add the chlorogold solution of 4ml1%, continue to stir 1min, add rapidly the trisodium citrate of 6ml1%, solution color from pale yellow becomes black and finally becomes claret, continue heating 5min, take out cool to room temperature, normal temperature keeps in Dark Place.The size of the colloid gold particle made like this is 20-40nm.
(4) colloidal gold pad is prepared
Get the colloidal gold solution that grain size is 20-40nm, use 0.1mol/LK
2cO
3the pH value of colloidal gold solution is adjusted to 7.0-9.0, and room temperature places 10 minutes; In above-mentioned solution, add ergot ethylenediamine monoclonal antibody, make the concentration of ergot ethylenediamine monoclonal antibody be 20-80 μ g/ml collaurum, after mixing, room temperature places 30 minutes; The BSA solution adding 10% makes its concentration be 10-60 μ l/ml, and mix, room temperature places 10 minutes; 12000 leave the heart 30 minutes, and careful Aspirate supernatant, discards, the multiple solubilize of collaurum of remaining precipitation initial colloid gold volume; 12000 leave the heart 30 minutes, and careful Aspirate supernatant, discards, and the multiple solubilize of collaurum of remaining precipitation 30%-100% initial colloid gold volume, obtains ergot ethylenediamine monoclonal antibody-colloid gold label thing; Ergot ethylenediamine monoclonal antibody-colloid gold label thing is pressed 1mL and spread 40-70cm
2the ratio uniform of polyester film is layered on polyester film, then puts drying room, and temperature 38 DEG C, humidity to be less than under the condition of 30% dry 24 ± 2 hours, makes colloidal gold pad.
Above-mentioned collaurum redissolution liquid is the Tris containing 0.01-0.1%, the phosphate buffered solution of the 0.02MpH7.0-9.0 of the sucrose of 1.0-3.0%, the BSA of 0.1-1.0%.
(4) bag is by ergot ethylenediamine-BSA coupled antigen, sheep anti-mouse igg polyclonal antibody
Film instrument coating parameters 1 μ L/cm is drawn in setting, getting ergot ethylenediamine-BSA coupled antigen that concentration is 1.0-5.0mg/ml with microsyringe, get concentration is respectively 0.5-3.0mg/ml sheep anti-mouse igg polyclonal antibody, receives A, B pipe joint drawing film instrument in order.The PVC board posting nitrocellulose filter is placed on the to-and-fro movement platform of stroke film instrument, opens and draw a film instrument, nitrocellulose filter applies ergot ethylenediamine-BSA coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line).After line in the baking oven of temperature 38 DEG C dry 24 ± 2 hours, preserve, for subsequent use.
(5) process of sample pad
Glass fibre is soaked 20-40min in the phosphate buffered solution of 0.01MpH7.0-8.0, wherein contain 0.5-1.5%BSA, 0.5-1.0%Tweeen-20 in phosphate buffered solution, 38 DEG C of oven dry in drying baker, preserve, for subsequent use
(6) kit is assembled
PVC back up pad adheres to sample pad, colloidal gold pad, nitrocellulose filter and suction sample pad in order successively, and obtain described for detecting the test strips of ergot ethylenediamine, test strips can load in plastic clip, is assembled into test card.Wherein said sample pad is glass fibre, and inhaling sample pad is thieving paper.
The detection method of kit of the present invention is: by test sample balance to room temperature; Take out ergot ethylenediamine pick-up unit, horizontal positioned; In sample pad, add 2-3 drip sample, observe during 10-15 minute and record the colour developing situation of C, T line, judging testing result.
Kit of the present invention adopts colloidal gold immunochromatographimethod technology to measure ergot ethylenediamine, during detection, is added in by sample in the sample pad on kit, can observes directly immunoreactive result, complete sample detection.The present invention can be used for detecting in sample the ergot ethylenediamine that may exist, have easy to use, simple to operate, be swift in response, the feature such as economical and practical.
Accompanying drawing explanation
Fig. 1 Lysergide detection kit structural representation;
Reference numeral illustrates:
1: sample pad;
2: colloidal gold pad (polyester film of colloid gold label ergot ethylenediamine monoclonal antibody)
3: nitrocellulose filter (T: bag is by the detection line of ergot ethylenediamine-BSA coupled antigen; C: bag is by the nature controlling line of sheep anti-mouse igg polyclonal antibody);
4: inhale sample pad;
5:PVC back up pad;
The testing result schematic diagram of Fig. 2 kit of the present invention.
Be followed successively by C line line positive test symbol from left to right; T, C two line negative result; Invalid.
Embodiment:
Embodiment 1: the preparation of Lysergide detection kit
1. prepare ergot ethylenediamine coupled antigen
By ergot ethylenediamine and BSA coupling, synthesis ergot ethylenediamine-BSA coupled antigen, as ergot ethylenediamine coupled antigen.
2. prepare ergot ethylenediamine monoclonal antibody
Adopt ergot ethylenediamine-BSA coupled antigen to be immunogen immune BALB/C mice, by hybridoma technology, obtain the hybridoma cell strain secreting anti-ergot ethylenediamine monoclonal antibody; To induce a large amount of Dispersal risk of ascites method in body, use ProteinG post to carry out purifying, obtain ergot ethylenediamine monoclonal antibody.
3. prepare collaurum
Get 90ml purified water in the round-bottomed flask of cleaning, stir in Electromagnetic Heating cover and heat, after waiting for boiling, add the chlorogold solution of 4ml1%, continue to stir 1min, add rapidly the trisodium citrate of 6ml1%, solution color from pale yellow becomes black and finally becomes claret, continue heating 5min, take out cool to room temperature, normal temperature keeps in Dark Place.The size of the colloid gold particle made like this is 20-40nm.
4. prepare colloidal gold pad
Get the colloidal gold solution 5ml that grain size is 20-40nm, add the 0.1mol/LK of 0.15ml
2cO
3the pH value of colloidal gold solution is adjusted to 8.0, and room temperature places 10 minutes; In above-mentioned solution, add the ergot ethylenediamine monoclonal antibody that 52.6 μ l concentration are 3.8mg/ml, after mixing, room temperature places 30 minutes; Add the BSA solution of 0.15ml10%, mix, room temperature places 10 minutes; 12000 leave the heart 30 minutes, and careful Aspirate supernatant, discards, the multiple solubilize of collaurum of remaining precipitation initial colloid gold volume; 12000 leave the heart 30 minutes, and careful Aspirate supernatant, discards, and the multiple solubilize of collaurum of remaining precipitation 30%-100% initial colloid gold volume, obtains ergot ethylenediamine monoclonal antibody-colloid gold label thing; Ergot ethylenediamine monoclonal antibody-colloid gold label thing is pressed 1mL and spread 50cm
2the ratio uniform of polyester film is layered on polyester film, then puts drying room, and temperature 38 DEG C, humidity to be less than under the condition of 30% dry 24 hours, makes colloidal gold pad.
Above-mentioned collaurum redissolution liquid is the Tris containing 0.01%, the phosphate buffered solution of the 0.02MpH8.0 of the sucrose of 2.0%, the BSA of 0.5%.
(4) bag is by ergot ethylenediamine-BSA coupled antigen, sheep anti-mouse igg polyclonal antibody
A film instrument coating parameters 1 μ L/cm is drawn in setting, and getting ergot ethylenediamine-BSA coupled antigen that concentration is 2.5mg/ml with microsyringe, get concentration is respectively 1.0mg/ml sheep anti-mouse igg polyclonal antibody, receives A, B pipe joint drawing film instrument in order.The PVC board posting nitrocellulose filter is placed on the to-and-fro movement platform of stroke film instrument, opens and draw a film instrument, nitrocellulose filter applies ergot ethylenediamine-BSA coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line).After line in the baking oven of temperature 38 DEG C dry 24 hours, preserve, for subsequent use.
(5) process of sample pad
Glass fibre is soaked 30min in the phosphate buffered solution treating fluid of 50ml0.01MpH8.0, wherein contain 1.0%BSA, 0.5%Tweeen-2 in phosphate buffered solution, 38 DEG C of oven dry in drying baker, preserve, for subsequent use
(6) kit is assembled
PVC back up pad adheres to sample pad, colloidal gold pad, nitrocellulose filter and suction sample pad in order successively, and obtain described for detecting the test strips of ergot ethylenediamine, test strips can load in plastic clip, is assembled into test card.Wherein said sample pad is glass fibre, and inhaling sample pad is thieving paper.
Embodiment 2: the detection of Lysergide detection kit
1. detection method:
Take out Lysergide detection kit, horizontal positioned; Sample pad instills 3 samples, observes after 10 minutes and record the colour developing situation of C, T line, judging testing result.
2. result judges
Positive: T line does not develop the color, only C line colour developing, is judged to be positive findings;
Negative: T line, C line all develop the color, and are judged to be negative findings;
Invalid: C line does not develop the color, illustrate that maloperation or kit are rotten and damage.
When detecting sample, sample is because capillarity is to suction sample pad one end chromatography.If containing ergot ethylenediamine in sample, they will go up antibody combining site limited in the ergot ethylenediamine monoclonal antibody of the ergot ethylenediamine-BSA coupled antigen competition binding colloid gold label of bag quilt with detection line (T line), when the ergot ethylenediamine in sample reaches finite concentration, also completely saturated with the ergot ethylenediamine monoclonal antibody generation immune response of colloid gold label, now colloidal gold composite combines without the ergot ethylenediamine-BSA coupled antigen vacant site and detection line wrapping quilt, now T line does not develop the color, and this is positive findings.If not containing ergot ethylenediamine in sample, the colloid gold particle that marked ergot ethylenediamine monoclonal antibody will in company with after sample chromatography to T line position, immune association reaction is there is with ergot ethylenediamine-BSA coupled antigen T line wrapping quilt, colloid gold particle is piled up at T line position and is made T line present a macroscopic red stripes, and this is negative findings.No matter in sample whether containing ergot ethylenediamine, colloid gold label thing all can be coated on the sheep anti-mouse igg polyclonal antibody on nature controlling line (C line) and be combined and develop the color, the colour developing of C line has determined whether enough samples, the whether normal standard of chromatography process, simultaneously also as the inner quality standard of reagent.
Claims (5)
1. a Lysergide detection kit, it is characterized in that being made up of sample pad, colloidal gold pad, nitrocellulose filter, suction sample pad and PVC back up pad, sample pad, colloidal gold pad, nitrocellulose filter and suction sample pad are adhered tightly in PVC back up pad; Described sample pad is glass fibre; Described colloidal gold pad is the ergot ethylenediamine monoclonal antibody polyester film of colloid gold label; On described nitrocellulose filter, bag is by ergot ethylenediamine-BSA coupled antigen as detection line successively, and sheep anti-mouse igg polyclonal antibody is as nature controlling line; Described suction sample pad is thieving paper; The preparation method of described colloidal gold pad is: get the colloidal gold solution that grain size is 20-40nm, use 0.1mol/LK
2cO
3the pH value of colloidal gold solution is adjusted to 7.0-9.0, and room temperature places 10 minutes; In above-mentioned solution, add ergot ethylenediamine monoclonal antibody, make the concentration of ergot ethylenediamine monoclonal antibody be 20-80 μ g/ml collaurum, after mixing, room temperature places 30 minutes; The BSA solution adding 10% makes its concentration be 10-60 μ l/ml, and mix, room temperature places 10 minutes; 12000 leave the heart 30 minutes, and careful Aspirate supernatant, discards, the multiple solubilize of collaurum of remaining precipitation initial colloid gold volume; 12000 leave the heart 30 minutes, and careful Aspirate supernatant, discards, and the multiple solubilize of collaurum of remaining precipitation 30%-100% initial colloid gold volume, obtains ergot ethylenediamine monoclonal antibody-colloid gold label thing; Ergot ethylenediamine monoclonal antibody-colloid gold label thing is pressed 1mL and spread 40-70cm
2the ratio uniform of polyester film is layered on polyester film, then puts drying room, and temperature 38 DEG C, humidity to be less than under the condition of 30% dry 24 ± 2 hours, makes colloidal gold pad.
2. a Lysergide detection kit according to claim 1, is characterized in that described ergot ethylenediamine monoclonal antibody is obtained as immunogen immune BALB/C mice by ergot ethylenediamine-BSA coupled antigen.
3. a Lysergide detection kit according to claim 1, it is characterized in that the preparation method of described collaurum is: get 90ml purified water in the round-bottomed flask of cleaning, stir in Electromagnetic Heating cover and heat, after waiting for boiling, add the chlorogold solution of 4ml1%, continue to stir 1min, add rapidly the trisodium citrate of 6ml1%, solution color from pale yellow becomes black and finally becomes claret, continue heating 5min, take out cool to room temperature, normal temperature keeps in Dark Place, and the size of the colloid gold particle made like this is 20-40nm.
4. a Lysergide detection kit according to claim 1, is characterized in that described collaurum redissolution liquid is the Tris containing 0.01-0.1%, the phosphate buffered solution of the 0.02MpH7.0-9.0 of the sucrose of 1.0-3.0%, the BSA of 0.1-1.0%.
5. a Lysergide detection kit according to claim 1, it is characterized in that the method for coating of two lines on described nitrocellulose filter is: film instrument coating parameters 1 μ L/cm is drawn in setting, ergot ethylenediamine-BSA the coupled antigen that concentration is 1.0-5.0mg/ml is got respectively with microsyringe, getting concentration is 0.5-3.0mg/ml sheep anti-mouse igg polyclonal antibody, receive the A drawing film instrument in order, B pipe joint, the PVC board posting nitrocellulose filter is placed on the to-and-fro movement platform of stroke film instrument, open and draw film instrument, nitrocellulose filter applies ergot ethylenediamine-BSA coupled antigen as detection line, sheep anti-mouse igg polyclonal antibody is as nature controlling line, after line in the baking oven of temperature 38 DEG C dry 24 ± 2 hours, preserve, for subsequent use.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104076142A (en) * | 2014-03-05 | 2014-10-01 | 广东医学院附属医院 | Fluorescent microsphere lateral chromatographic detection strip for multiple joint inspection of trace target substances as well as preparation method and application thereof |
| CN105738582B (en) * | 2016-04-21 | 2017-11-21 | 天津科技大学 | Immuno-chromatographic test paper strip that is a kind of while detecting salbutamol and flumequine and preparation method thereof |
| CN107167593A (en) * | 2017-06-02 | 2017-09-15 | 亳州市新健康科技有限公司 | Illicit drugs inspection kit |
| CN114317449B (en) * | 2021-12-28 | 2023-08-15 | 无锡迪腾敏生物科技有限公司 | Ergot ethylenediamine antigen, ergot ethylenediamine monoclonal antibody, hybridoma cell strain and application |
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