CN102937647A - Lysergide detection kit and preparation method thereof - Google Patents
Lysergide detection kit and preparation method thereof Download PDFInfo
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- CN102937647A CN102937647A CN2012100345987A CN201210034598A CN102937647A CN 102937647 A CN102937647 A CN 102937647A CN 2012100345987 A CN2012100345987 A CN 2012100345987A CN 201210034598 A CN201210034598 A CN 201210034598A CN 102937647 A CN102937647 A CN 102937647A
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- ergot
- ethylenediamine
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Abstract
The invention relates to a detection kit for detecting lysergide. The kit comprises a sample pad (1), a colloid gold pad (2), a nitrocellulose membrane (3), a suction sample pad (4) and a PVC supporting plate (5), wherein the PVC supporting plate is successively adhered with the sample pad, the colloid gold pad, the nitrocellulose membrane and the suction sample; the colloid gold pad is a colloid gold marked lysergide monoclonal antibody polyester film; and the nitrocellulose membrane is successively coated by a lysergide-BBS coupled antigen as a detection line (T line), and a goat anti-mouse IgG fraction polyclonal antibody as a quality control line (C line). The lysergide detection kit is prepared by using a colloid gold immunochromatography assay, and is used for detecting lysergide which may exist in the detection sample. The kit is simple in preparation and convenient for use, reacts rapidly, and is 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 ergot ethylenediamine detection kit of utilizing the colloidal gold immunochromatographimethod fabrication techniques and preparation method thereof.
Background technology
Ergot ethylenediamine (LSD) is the strongest psychedelic of the known efficacy of a drug, very easily is absorption of human body.Can produce photis, phonism and illusion after taking, appearance is panic-stricken, thought is dazed and confused, terribly suspicious, anxious, behavior is out of control and complete helpless insane symptom.Can cause disorienting, distinguishing simultaneously the ability of distance and time, thereby it is injured and dead to cause health to be punished severely.
The detection method of at present relevant 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), need expensive instrument and equipment but exist, requirement to test material is high, needs the restrictions such as purification processes.Therefore the detection method of research with the advantage such as quick, portable has realistic meaning.The present invention has introduced a kind of detection kit with colloidal gold immunity chromatography fast detecting ergot ethylenediamine and preparation method thereof, the method has simply, fast, need not any instrument and equipment, the characteristics such as economical and practical are applicable to fast qualitative and detect in the sample whether contain the ergot ethylenediamine.
Summary of the invention
The object of the present invention is to provide a kind of portablely, quick, be suitable for detection kit of Site Detection ergot ethylenediamine and preparation method thereof.
A kind of detection kit for detection of the ergot ethylenediamine, comprise sample pad (1), collaurum pad (2), nitrocellulose filter (3), inhale sample pad (4) and PVC back up pad (5), tight adhesion has sample pad, collaurum pad, nitrocellulose filter and suction sample pad successively on the PVC back up pad.Described sample pad is glass fibre; The ergot ethylenediamine monoclonal antibody polyester film that described collaurum pad is colloid gold label; Be coated with successively detection line (T line) and nature controlling line (C line) on the described nitrocellulose filter, wherein be coated with ergot ethylenediamine-BSA coupled antigen on the detection line (T line), be coated with the sheep anti-mouse igg polyclonal antibody on the nature controlling line (C line); Described suction sample pad is thieving paper.
The present invention adopts nano-colloid technology for gold and antigen and antibody specific reaction, the principle of using the Immune competition inhibitory reaction is prepared from, by the ergot ethylenediamine monoclonal antibody of the coated ergot ethylenediamine of detection line (T line) on the ergot ethylenediamine that contains in the sample to be checked and the nitrocellulose filter-BSA coupled antigen competition association colloid gold mark, judge whether contain the ergot ethylenediamine in the sample to be checked by the colour developing of T line.
The invention provides a kind of preparation method of ergot ethylenediamine detection kit, may further comprise the steps:
(1) preparation ergot ethylenediamine coupled antigen
With ergot ethylenediamine and BSA coupling, synthetic ergot ethylenediamine-BSA coupled antigen is as ergot ethylenediamine coupled antigen.
(2) preparation ergot ethylenediamine monoclonal antibody
Ethylenediamine-the BSA coupled antigen is the immunogen immune BALB/C mice to adopt ergot, by hybridoma technology, obtains secreting the hybridoma cell strain of anti-ergot ethylenediamine monoclonal antibody; To induce a large amount of Dispersal risks of ascites method in the body, use Protein G post to carry out purifying, obtain ergot ethylenediamine monoclonal antibody.
(3) preparation collaurum
Get the 90ml purified water in the round-bottomed flask of cleaning, in the Electromagnetic Heating cover, stir and heating, after waiting for boiling, the chlorogold solution that adds 4ml 1% continues to stir 1min, adds rapidly the trisodium citrate of 6ml 1%, solution colour becomes claret at last by the pale yellow black that becomes, continue heating 5min, take out cool to room temperature, normal temperature keeps in Dark Place.The size of the colloid gold particle of making like this is 20-40nm.
(4) preparation collaurum pad
Get the colloidal gold solution that grain size is 20-40nm, use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7.0-9.0, and room temperature was placed 10 minutes; Add ergot ethylenediamine monoclonal antibody in mentioned solution, the concentration that makes ergot ethylenediamine monoclonal antibody is 20-80 μ g/ml collaurum, and after mixing, room temperature was placed 30 minutes; It is 10-60 μ l/ml that the BSA solution of adding 10% makes its concentration, mixes, and room temperature was placed 10 minutes; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining precipitation is dissolved with the collaurum redissolution liquid of initial collaurum volume; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining precipitation obtains ergot ethylenediamine monoclonal antibody-colloid gold label thing with the collaurum redissolution liquid dissolving of the initial collaurum volume of 30%-100%; Ergot ethylenediamine monoclonal antibody-colloid gold label thing is pressed 1mL spread 40-70cm
2The ratio uniform spreading of polyester film is put drying room again on polyester film, 38 ℃ of temperature, humidity was made the collaurum pad less than under 30% the condition dry 24 ± 2 hours.
Above-mentioned collaurum redissolution liquid is the Tris that contains 0.01-0.1%, the phosphate buffered solution of the 0.02M pH 7.0-9.0 of the sucrose of 1.0-3.0%, the BSA of 0.1-1.0%.
(4) coated ergot ethylenediamine-BSA coupled antigen, sheep anti-mouse igg polyclonal antibody
Set and draw film instrument coating parameters 1 μ L/cm, getting the ergot ethylenediamine that concentration is 1.0-5.0mg/ml-BSA coupled antigen, get concentration with microsyringe respectively is 0.5-3.0mg/ml sheep anti-mouse igg polyclonal antibody, receives in order A, the B pipe joint of drawing the film instrument.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, apply ergot ethylenediamine-BSA coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line) at nitrocellulose filter.After the line in the baking oven of 38 ℃ of temperature dry 24 ± 2 hours, preserve, for subsequent use.
(5) processing of sample pad
Glass fibre is soaked 20-40min in the phosphate buffered solution of 0.01M pH 7.0-8.0, wherein contain 0.5-1.5%BSA in the phosphate buffered solution, 0.5-1.0%Tweeen-20,38 ℃ of oven dry in drying baker are preserved, and are for subsequent use
(6) assembling kit
Adhere to successively in order sample pad, collaurum pad, nitrocellulose filter and suction sample pad on the PVC back up pad, obtain described test strips for detection of the ergot ethylenediamine, test strips can be packed in the plastic clip, is assembled into test card.Wherein said sample pad is glass fibre, and inhaling the sample pad is thieving paper.
The detection method of kit of the present invention is: with the test sample balance to room temperature; Take out ergot ethylenediamine pick-up unit, horizontal positioned; In sample pad, add 2-3 and drip sample, observe and record the colour developing situation of C, T line in the time of 10-15 minute, judge testing result.
Kit of the present invention adopts the colloidal gold immunochromatographimethod technology to measure the ergot ethylenediamine, during detection, sample is added on the sample pad on the kit, can observe directly immunoreactive result, finishes sample detection.The present invention can be used for detecting the ergot ethylenediamine that may exist in the sample, have easy to use, simple to operate, be swift in response, the characteristics such as economical and practical.
Description of drawings
Fig. 1 ergot ethylenediamine detection kit structural representation;
The reference numeral explanation:
1: sample pad;
2: collaurum pad (polyester film of colloid gold label ergot ethylenediamine monoclonal antibody)
3: nitrocellulose filter (T: the detection line that has been coated with ergot ethylenediamine-BSA coupled antigen; C: the nature controlling line that has been coated with the sheep anti-mouse igg polyclonal antibody);
4: inhale the sample pad;
The 5:PVC back up pad;
The testing result synoptic diagram of Fig. 2 kit of the present invention.
Be followed successively by from left to right line positive test symbol of C line; Two line negative result of T, C; Invalid.
Embodiment:
Embodiment 1: the preparation of ergot ethylenediamine detection kit
1. prepare ergot ethylenediamine coupled antigen
With ergot ethylenediamine and BSA coupling, synthetic ergot ethylenediamine-BSA coupled antigen is as ergot ethylenediamine coupled antigen.
2. prepare ergot ethylenediamine monoclonal antibody
Ethylenediamine-the BSA coupled antigen is the immunogen immune BALB/C mice to adopt ergot, by hybridoma technology, obtains secreting the hybridoma cell strain of anti-ergot ethylenediamine monoclonal antibody; To induce a large amount of Dispersal risks of ascites method in the body, use Protein G post to carry out purifying, obtain ergot ethylenediamine monoclonal antibody.
3. preparation collaurum
Get the 90ml purified water in the round-bottomed flask of cleaning, in the Electromagnetic Heating cover, stir and heating, after waiting for boiling, the chlorogold solution that adds 4ml 1% continues to stir 1min, adds rapidly the trisodium citrate of 6ml 1%, solution colour becomes claret at last by the pale yellow black that becomes, continue heating 5min, take out cool to room temperature, normal temperature keeps in Dark Place.The size of the colloid gold particle of making like this is 20-40nm.
4. prepare the collaurum pad
Getting grain size is the colloidal gold solution 5ml of 20-40nm, adds the 0.1mol/L K of 0.15ml
2CO
3The pH value of colloidal gold solution is transferred to 8.0, and room temperature was placed 10 minutes; Adding 52.6 μ l concentration in mentioned solution is the ergot ethylenediamine monoclonal antibody of 3.8mg/ml, and after mixing, room temperature was placed 30 minutes; Add the BSA solution of 0.15ml 10%, mix, room temperature was placed 10 minutes; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining precipitation is dissolved with the collaurum redissolution liquid of initial collaurum volume; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining precipitation obtains ergot ethylenediamine monoclonal antibody-colloid gold label thing with the collaurum redissolution liquid dissolving of the initial collaurum volume of 30%-100%; Ergot ethylenediamine monoclonal antibody-colloid gold label thing is pressed 1mL spread 50cm
2The ratio uniform spreading of polyester film is put drying room again on polyester film, 38 ℃ of temperature, humidity was made the collaurum pad less than under 30% the condition dry 24 hours.
Above-mentioned collaurum redissolution liquid is to contain 0.01% Tris, the phosphate buffered solution of the 0.02M pH 8.0 of 2.0% sucrose, 0.5% BSA.
(4) coated ergot ethylenediamine-BSA coupled antigen, sheep anti-mouse igg polyclonal antibody
Set and draw film instrument coating parameters 1 μ L/cm, getting the ergot ethylenediamine that concentration is 2.5mg/ml-BSA coupled antigen, get concentration with microsyringe respectively is 1.0mg/ml sheep anti-mouse igg polyclonal antibody, receives in order A, the B pipe joint of drawing the film instrument.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, apply ergot ethylenediamine-BSA coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line) at nitrocellulose filter.After the line in the baking oven of 38 ℃ of temperature dry 24 hours, preserve, for subsequent use.
(5) processing of sample pad
Glass fibre is soaked 30min in the phosphate buffered solution treating fluid of 50ml 0.01M pH 8.0, wherein contain 1.0%BSA in the phosphate buffered solution, 0.5%Tweeen-2,38 ℃ of oven dry in drying baker are preserved, and are for subsequent use
(6) assembling kit
Adhere to successively in order sample pad, collaurum pad, nitrocellulose filter and suction sample pad on the PVC back up pad, obtain described test strips for detection of the ergot ethylenediamine, test strips can be packed in the plastic clip, is assembled into test card.Wherein said sample pad is glass fibre, and inhaling the sample pad is thieving paper.
Embodiment 2: the detection of ergot ethylenediamine detection kit
1. detection method:
Take out ergot ethylenediamine detection kit, horizontal positioned; Splash into 3 samples in sample pad, observe and record the colour developing situation of C, T line after 10 minutes, judge testing result.
2. the result judges
Positive: the T line does not develop the color, and 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: the C line does not develop the color, and rotten damage of maloperation or kit is described.
During test sample, sample because of capillarity to inhaling sample pad one end chromatography.If contain the ergot ethylenediamine in the sample, they will and the ergot ethylenediamine monoclonal antibody of the upper coated ergot ethylenediamine of detection line (T line)-BSA coupled antigen competition association colloid gold mark on limited antibody combining site, when the ergot ethylenediamine in the sample reaches finite concentration, also fully saturated with the ergot ethylenediamine monoclonal antibody generation immune response of colloid gold label, this moment, colloidal gold composite was without ergot ethylenediamine coated on vacant site and the detection line-BSA coupled antigen combination, this moment, the T line did not develop the color this positive result.If do not contain the ergot ethylenediamine in the sample, mark the colloid gold particle of ergot ethylenediamine monoclonal antibody will be in company with the sample chromatography to the T line position, immune association reaction occurs with the ergot ethylenediamine that is coated with on the T line-BSA coupled antigen, colloid gold particle is piled up so that the T line presents a macroscopic red stripes at the T line position, this negative result.No matter whether contain the ergot ethylenediamine in the sample, the colloid gold label thing all can the sheep anti-mouse igg polyclonal antibody on being coated on nature controlling line (C line) be combined and be developed the color, the colour developing of C line is to have determined whether enough samples, whether chromatography process normal standard, simultaneously also as the inner quality standard of reagent.
Claims (6)
1. ergot ethylenediamine detection kit and preparation method thereof, it is characterized in that being comprised of sample pad, collaurum pad, nitrocellulose filter, suction sample pad and PVC back up pad, sample pad, collaurum pad, nitrocellulose filter and suction sample pad tight adhesion are on the PVC back up pad; Described sample pad is glass fibre; The ergot ethylenediamine monoclonal antibody polyester film that described collaurum pad is colloid gold label; Be coated with successively ergot ethylenediamine-BSA coupled antigen on the described nitrocellulose filter as detection line (T line), the sheep anti-mouse igg polyclonal antibody is as nature controlling line (C line); Described suction sample pad is thieving paper.
2. ergot ethylenediamine detection kit claimed in claim 1 and preparation method thereof is characterized in that described ergot ethylenediamine monoclonal antibody is obtained as the immunogen immune BALB/C mice by ergot ethylenediamine-BSA coupled antigen.
3. ergot ethylenediamine detection kit claimed in claim 1 and preparation method thereof, the preparation method who it is characterized in that described collaurum is: get the 90ml purified water in the round-bottomed flask of cleaning, in the Electromagnetic Heating cover, stir and heating, after waiting for boiling, the chlorogold solution that adds 4ml 1%, continue to stir 1min, the trisodium citrate that adds rapidly 6ml 1%, solution colour becomes claret at last by the pale yellow black that becomes, continue heating 5min, take out cool to room temperature, normal temperature keeps in Dark Place.The size of the colloid gold particle of making like this is 20-40nm.
4. ergot ethylenediamine detection kit claimed in claim 1 and preparation method thereof is characterized in that the preparation method of described collaurum pad is: get the colloidal gold solution that grain size is 20-40nm, use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to 7.0-9.0, and room temperature was placed 10 minutes; Add ergot ethylenediamine monoclonal antibody in mentioned solution, the concentration that makes ergot ethylenediamine monoclonal antibody is 20-80 μ g/ml collaurum, and after mixing, room temperature was placed 30 minutes; It is 10-60 μ l/ml that the BSA solution of adding 10% makes its concentration, mixes, and room temperature was placed 10 minutes; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining precipitation is dissolved with the collaurum redissolution liquid of initial collaurum volume; 12000 left the heart 30 minutes, carefully drew supernatant, discarded, and remaining precipitation obtains ergot ethylenediamine monoclonal antibody-colloid gold label thing with the collaurum redissolution liquid dissolving of the initial collaurum volume of 30%-100%; Ergot ethylenediamine monoclonal antibody-colloid gold label thing is pressed 1mL spread 40-70cm
2The ratio uniform spreading of polyester film is put drying room again on polyester film, 38 ℃ of temperature, humidity was made the collaurum pad less than under 30% the condition dry 24 ± 2 hours.
5. a claim 1 and ergot ethylenediamine detection kit claimed in claim 5 and preparation method thereof, it is characterized in that described collaurum redissolution liquid is the Tris that contains 0.01-0.1%, the phosphate buffered solution of the 0.02M pH 7.0-9.0 of the sucrose of 1.0-3.0%, the BSA of 0.1-1.0%.
6. ergot ethylenediamine detection kit claimed in claim 1 and preparation method thereof, the method for coating that it is characterized in that two lines on the described nitrocellulose filter is: set and draw film instrument coating parameters 1 μ L/cm, getting the ergot ethylenediamine that concentration is 1.0-5.0mg/ml-BSA coupled antigen, get concentration with microsyringe respectively is 0.5-3.0mg/ml sheep anti-mouse igg polyclonal antibody, receives in order A, the B pipe joint of drawing the film instrument.The PVC plate that posts nitrocellulose filter is placed on the to-and-fro movement platform of drawing the film instrument, open and draw the film instrument, apply ergot ethylenediamine-BSA coupled antigen (T line), sheep anti-mouse igg polyclonal antibody (C line) at nitrocellulose filter.After the line in the baking oven of 38 ℃ of temperature dry 24 ± 2 hours, preserve, for subsequent use.
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| CN201210034598.7A CN102937647B (en) | 2012-02-16 | 2012-02-16 | Lysergide detection kit and preparation method thereof |
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|---|---|---|---|
| CN201210034598.7A CN102937647B (en) | 2012-02-16 | 2012-02-16 | Lysergide detection kit and preparation method thereof |
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| CN102937647B CN102937647B (en) | 2015-11-25 |
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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 |
| CN105738582A (en) * | 2016-04-21 | 2016-07-06 | 天津科技大学 | Immunochromatography test strip capable of simultaneously detecting salbutamol and flumequine and preparation method of test strip |
| CN107167593A (en) * | 2017-06-02 | 2017-09-15 | 亳州市新健康科技有限公司 | Illicit drugs inspection kit |
| CN114317449A (en) * | 2021-12-28 | 2022-04-12 | 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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| Publication number | Priority date | Publication date | Assignee | Title |
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| 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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