CN102393464B - Preparation method of test strip for morphine detection and application thereof - Google Patents
Preparation method of test strip for morphine detection and application thereof Download PDFInfo
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- CN102393464B CN102393464B CN201110327560.4A CN201110327560A CN102393464B CN 102393464 B CN102393464 B CN 102393464B CN 201110327560 A CN201110327560 A CN 201110327560A CN 102393464 B CN102393464 B CN 102393464B
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Abstract
The invention discloses a preparation method of a test strip for morphine detection and an application thereof. The preparation method comprises the following steps of: preparing a colloidal gold labeling anti-morphine monoclonal antibody, and coating the antibody on a colloidal gold combining pad; preparing a conjugate of morphine and BSA; spraying the conjugate and goat anti-mouse IgG on a nitrocellulose film as a detection line and a control line respectively, and drying; and assembling the test strip. The test strip for morphine detection disclosed by the invention has strong specificity, high sensitivity and high accuracy, does not need special equipment and is suitable for field operation; and moreover, the test strip can be quickly known well by an operator without professional knowledge, is very suitable for the daily supervision of the supervision law-enforcing department, can meet the needs for supervising inspection of basic level, and is widely used for detecting foods and medicines.
Description
Technical field
The present invention relates to the preparation method for the test strips of morphine detection, also relate to the application of the test strips detecting for morphine.
Background technology
Morphine (Morphine) is a kind of opium alkaloid being refined by opium, is pure opioid receptor agonist, has powerful analgesic activity, therefore medically, morphine is narcotic analgeiscs.Such material just has floaty euphoria in using, and cannot concentrate on, and can produce dreamlike phenomenon, and excessive use causes acute poisoning, very easily produces tolerance and habituation.
At present, detect morphine in food and medicine, use detection method thin-layered chromatography (LC), vapor-phase chromatography (GC), the liquid phase chromatography (HPLC) of comparative maturity, Liquid Chromatography/Mass Spectrometry etc.Though the reinforcement that these methods are strong the supervision to food and medicine, but respectively there is advantage but there is obvious defect, the instrumental analysis such as liquid phase, gas phase, LC-MS because of instrument expensive, need specific laboratory, sense cycle is long, generally need the time of one day, need professional's operation; Thin layer chromatography trivial operations, and owing to often containing multiple spices in sample, experiment is caused to severe jamming, may obtain inaccurate result.
Summary of the invention
The object of the present invention is to provide the test strips preparation method and the application thereof that for morphine, detect.
The technical solution used in the present invention is:
The test strips preparation method who detects for morphine, comprises the steps:
1) prepare colloid gold label monoclonal antibodies against morphine, be coated on collaurum pad;
2) prepare the conjugate of morphine and BSA, itself and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and control line, dry;
3) test strips assembling: test strips is comprised of PVC template and PVC backboard, is stained with in order sample pad, collaurum pad, nitrocellulose filter and adsorptive pads on PVC backboard.
Preferably, step 3) nitrocellulose filter is sprayed with detection line, control line from sample pad successively to adsorptive pads direction.
Between the each several part of preferably, pasting on step 3) PVC backboard, have that 1～2mm's is overlapping.
The above-mentioned test strips detecting for morphine is in the application of food, medicine.
The invention has the beneficial effects as follows:
The test strips detecting for morphine of the present invention, specificity is strong, highly sensitive, accuracy is high, do not need Special Equipment, be suitable for execute-in-place, operating personnel do not have professional knowledge can grasp fast yet, are applicable to very much using in supervision law enforcement agency day-to-day supervision, the supervision and inspection demand that can meet basic unit, is widely used in the detection of food, medicine.
Accompanying drawing explanation
Fig. 1 is the test strips operation chart detecting for morphine;
Fig. 2 is the test strips result judgement figure detecting for morphine;
Fig. 3 is positive and negative sample testing result comparison diagram.
Embodiment
For ease of the present invention is further understood, now describe the present invention in conjunction with specific embodiments.The test strips preparation method who detects for morphine, comprises the steps:
1. prepare colloid gold label monoclonal antibodies against morphine
The big or small average out to 30nm of colloid gold particle adds 1% trisodium citrate 1ml in 100ml deionized water, boils the rear 1% gold chloride 1ml that adds rapidly, continues to boil 5min, cooling after, obtain colloidal gold solution, save backup at 4 ℃.
Get the colloidal gold solution 100ml having prepared, with 0.2mol/L sodium carbonate liquor, adjust pH to 8.0.Add while stirring monoclonal antibodies against morphine 1.5mg, stir 30min, more dropwise add 10ml 10%BSA, stir 15min.The centrifugal 15min of 12000G, abandons supernatant, adds 10mlpH7.4PBS damping fluid (containing 1%BSA), cleans altogether 3 times.Precipitation is dissolved containing the PBS damping fluid (pH7.4) of 1%BSA with 5ml, after filtering, obtain colloid gold label monoclonal antibodies against morphine with 0.2 μ m sterilizing filter, 4 ℃ save backup.
2. the conjugate of preparing morphine and BSA
Morphine is micromolecular compound, molecular weight is 285, there is hydrogenation and adjoin the silk fabric ring parent nucleus of smack one's lips phenanthrene, the light base of phenol of 3, the light base activity of alcohol of 6 are stronger, and itself non-immunogenicity adopts sodium monochloracetate method and bluffs and claps acid and join that method is synthetic bluffs that to clap phthalein morphine half cruel, under EDC catalysis, with macromolecular carrier protein-crosslinking, form 3,6 coupled complexes
Envelope antigen is the conjugate (MOP-BSA) of morphine and BSA, its synthetic method is: first 20mg morphine succinate, 15mgEDC and 30mgBSA are mixed and be dissolved in 1mlTris damping fluid, at 0～4 ℃, lucifuge stirs 1h, add again 5mgEDC, stirring reaction 12h at 0 ℃, after hold over night, with Tris damping fluid dialysis 3d, obtain the conjugate of morphine and BSA.
3. coated antibody and conjugate
By BioJetXYZ3000 type, select film instrument the conjugate of morphine and BSA and sheep anti-mouse igg are sprayed on to nitrocellulose filter (NC film) above, respectively as detection line (T) and control line (C), at 37 ℃ of oven drying 8h.In kind, the colloid gold label monoclonal antibodies against morphine preparing is coated on collaurum pad.
4. the test strips assembling detecting for morphine
The test strips detecting for morphine is comprised of a PVC backboard and a PVC template, is stained with in order sample pad, collaurum pad, nitrocellulose filter and adsorptive pads on PVC backboard, has that 1～2mm's is overlapping between each several part.On nitrocellulose filter, from sample pad, to adsorptive pads direction, be sprayed with successively detection line, control line.With band MARK adhesive tape, around backboard, cover on sample pad, collaurum pad and adsorptive pads.With cutting cutter, the plate posting is cut into the wide bar of 3mm.PVC template comprises well (S), detection zone, control zone, and it is placed on PVC backboard, and well (S) is corresponding to sample pad, and detection zone can show the detection line (T) of nitrocellulose filter, and control zone can show control line (C).The test strips of making is put into the aluminium foil bag sealed storage with drying agent.
the application of above-mentioned test strips in solid or semi-solid food products:
Approximately 2 spoonfuls of the about 5g(of sample thief) in 25mL sample hose, add water 10mL, powerful jolting 1 minute, draws supernatant liquid with disposable syringe, with 0.45 μ m filtering with microporous membrane.If sample solution is too dense, cannot filter, can add after equal-volume water dilutes and refilter, put after the well of above-mentioned test strips, judged result in 1～5 minute, as shown in Figure 1.
the application of above-mentioned test strips in soup for chafing dish:
Get soup for chafing dish standing, remove upper strata oil layer, with disposable syringe, draw lower aqueous layer 5mL in 25ml sample hose.With 0.45 μ m filtering with microporous membrane.If sample solution is too dense, cannot loading, refilter after adding the dilution of equal-volume water, put after the well of above-mentioned test strips judged result in 1～5 minute.
the application of above-mentioned test strips in capsule or tablet medicament:
Get 2 medicines (if medicine has capsule, need abandon capsule shell), add water 5ml, powerful jolting 1 minute, uses disposable syringe imbitition, with 0.45 μ m filtering with microporous membrane, puts after the well of above-mentioned test strips judged result in 1～5 minute.
the application of above-mentioned test strips in cough syrup:
Get about 5ml cough syrup, add the dilution of equal-volume water, use disposable syringe imbitition, with 0.45 μ m filtering with microporous membrane, put after the well of above-mentioned test strips judged result in 1～5 minute.
The result judgement of the above-mentioned test strips detecting for morphine:
1) negative (-): T line (p-wire, near well) than C line (control line) deeply or equally dark, represents in sample that morphine concentration is lower than detection limit, or containing morphine, shows not contain morphine;
2) positive (+): T line is more shallow than C line, or T line is without colour developing, represents in sample that morphine concentration is higher than detection limit; T line is higher than morphine concentration in the more shallow expression sample of C line, shows to contain morphine;
3) invalid: not occur Quality Control C line, show that the incorrect or test card of operating process lost efficacy.Can carry out result judgement with reference to Fig. 2.
Sample by two kinds of concentration of above-mentioned ELISA test strip: 1) negative sample; 2) in negative sample, adding morphine to concentration is 20ng/ml.Testing result as shown in Figure 3, the ELISA test strip on the left side be sample 2), contain 20ng/ml morphine, the ELISA test strip on the right be sample 1), positive, as figure shows, left side result is shown as the positive, the right is shown as feminine gender, result is consistent.Visible, the inventive method quick and precisely, and can detect the morphine of 20ng/ml.
2011, adopt liquid chromatography-tandem mass spectrometry method for determining to detect altogether chafing dish bottom flavorings and the condiment of the sampling observation of Liao86Pi Cong Guangdong Province prefectures and cities, confirm 6 batches and add morphine, by above-mentioned test strips, check this 86 batch sample, if there is positive findings, palpus retrial once, Bing Song further confirms in laboratory, result is as shown in table 1, and as shown in Table 1, result is not found false positive sample and false negative sample, ELISA test strip accuracy is 100%, false drop rate is 0, and loss is 0, and sensitivity and specificity are 100%.
2011, adopt liquid chromatography-tandem mass spectrometry method for determining to detect 21 batches of cough-relieving class Chinese patent drugs of random sampling observation from the market, confirm 4 batches and add morphine, with above-mentioned test strips check, result is not found false positive and false negative sample, ELISA test strip accuracy 100% yet.
Claims (1)
1. the test strips preparation method who detects for morphine, comprises the steps:
A) prepare colloid gold label monoclonal antibodies against morphine
1) the big or small average out to 30nm of colloid gold particle adds 1% trisodium citrate 1ml in 100ml deionized water, boils the rear 1% gold chloride 1ml that adds rapidly, continues to boil 5min, cooling after, obtain colloidal gold solution, save backup at 4 ℃;
2) get the colloidal gold solution 100ml having prepared, with 0.2mol/L sodium carbonate liquor, adjust pH to 8.0; Add while stirring monoclonal antibodies against morphine 1.5mg, stir 30min, more dropwise add 10ml 10%BSA, stir 15min; The centrifugal 15min of 12000 G, abandons supernatant, adds 10ml pH7.4 containing the PBS damping fluid of 1% BSA, cleans altogether 3 times;
3) precipitation is dissolved containing the pH7.4 PBS damping fluid of 1%BSA with 5ml, after filtering, obtain colloid gold label monoclonal antibodies against morphine with 0.2 μ m sterilizing filter, 4 ℃ save backup;
B) prepare the conjugate of morphine and BSA
First 20mg morphine succinate, 15mg EDC and 30mg BSA are mixed and be dissolved in 1ml Tris damping fluid, lucifuge stirs 1h at 0～4 ℃, then adds 5mgEDC, stirring reaction 12h at 0 ℃, after hold over night, with Tris damping fluid dialysis 3d, obtain the conjugate of morphine and BSA;
C) coated antibody and conjugate
By BioJetXYZ3000 type, select film instrument the conjugate of morphine and BSA and sheep anti-mouse igg are sprayed on nitrocellulose filter, respectively as detection line and control line, at 37 ℃ of oven drying 8h; In kind, the colloid gold label monoclonal antibodies against morphine preparing is coated on collaurum pad;
D) the test strips assembling detecting for morphine
The test strips detecting for morphine is comprised of a PVC backboard and a PVC template, is stained with in order sample pad, collaurum pad, nitrocellulose filter and adsorptive pads on PVC backboard, has that 1～2mm's is overlapping between each several part; On nitrocellulose filter, from sample pad, to adsorptive pads direction, be sprayed with successively detection line, control line; With band MARK adhesive tape, around backboard, cover on sample pad, collaurum pad and adsorptive pads; With cutting cutter, the plate posting is cut into the wide bar of 3mm; PVC template comprises well (S), detection zone, control zone, and it is placed on PVC backboard, and well (S) is corresponding to sample pad, and detection zone can show the detection line of nitrocellulose filter, and control zone can show control line.
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1018004A1 (en) * | 1998-07-22 | 2000-07-12 | Syntron Bioresearch, Inc. | Multiple analyte assay device |
| CN1259668A (en) * | 1998-11-10 | 2000-07-12 | 长沙市精神病医院 | Method and test paper of smei-quantitative determination of morphine by monoclone antibody immune chromatography |
| CN1307236A (en) * | 2000-02-02 | 2001-08-08 | 昆明广博科技有限公司 | Fast test paper strip for morphine, heroin and opium and its preparation |
| WO2001071353A1 (en) * | 2000-03-16 | 2001-09-27 | The University Of Wyoming | A practical method and apparatus for analyte detection with colloidal particles |
| CN1327158A (en) * | 2000-06-02 | 2001-12-19 | 北京大学 | Establishment of anti-morphine single anti-cell strain and method for producing double-side morphine test paper |
| CN1488941A (en) * | 2002-10-09 | 2004-04-14 | 上海市刑事科学技术研究所 | Test paper for detecting morphine |
| CN1517709A (en) * | 2003-01-14 | 2004-08-04 | 周中人 | Novel competition method for detecting antigen by immune chromatographic reaction on microporous membrane medium |
| CN101135685A (en) * | 2006-08-28 | 2008-03-05 | 王志洪 | Diacetyl morphine quick-speed testing apparatus |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20010026944A1 (en) * | 1999-04-21 | 2001-10-04 | Roy Chung | Immunoassay system |
-
2011
- 2011-10-25 CN CN201110327560.4A patent/CN102393464B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1018004A1 (en) * | 1998-07-22 | 2000-07-12 | Syntron Bioresearch, Inc. | Multiple analyte assay device |
| CN1259668A (en) * | 1998-11-10 | 2000-07-12 | 长沙市精神病医院 | Method and test paper of smei-quantitative determination of morphine by monoclone antibody immune chromatography |
| CN1307236A (en) * | 2000-02-02 | 2001-08-08 | 昆明广博科技有限公司 | Fast test paper strip for morphine, heroin and opium and its preparation |
| WO2001071353A1 (en) * | 2000-03-16 | 2001-09-27 | The University Of Wyoming | A practical method and apparatus for analyte detection with colloidal particles |
| CN1327158A (en) * | 2000-06-02 | 2001-12-19 | 北京大学 | Establishment of anti-morphine single anti-cell strain and method for producing double-side morphine test paper |
| CN1488941A (en) * | 2002-10-09 | 2004-04-14 | 上海市刑事科学技术研究所 | Test paper for detecting morphine |
| CN1517709A (en) * | 2003-01-14 | 2004-08-04 | 周中人 | Novel competition method for detecting antigen by immune chromatographic reaction on microporous membrane medium |
| CN101135685A (en) * | 2006-08-28 | 2008-03-05 | 王志洪 | Diacetyl morphine quick-speed testing apparatus |
Non-Patent Citations (5)
| Title |
|---|
| 任辉.免疫胶体金层析法快速检测食品中吗啡.《中国公共卫生》.2004,第20卷(第4期), |
| 免疫胶体金层析法快速检测食品中吗啡;任辉;《中国公共卫生》;20040430;第20卷(第4期);第488页右栏结果与分析部分 * |
| 吗啡胶体金免疫层析试纸条的研究与开发;赵海锋;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20090430;第33页第3.3.1、3.3.2节，第35页第3.3.5节，第38页第2段第2-4行，第41页第4.2.4.3节，第42页第4.2.8节，第43页图4-2 * |
| 吗啡胶体金免疫层析试纸条的研究与开发;赵海锋;《中国优秀硕士学位论文全文数据库医药卫生科技辑》;20090430;第33页第3.3.1、3.3.2节，第35页第3.3.5节，第38页第2段第2-4行，第41页第4.2.4.3节，第42页第4.2.8节，第43页图4-2，第43页第4.2.9节 * |
| 赵海锋.吗啡胶体金免疫层析试纸条的研究与开发.《中国优秀硕士学位论文全文数据库医药卫生科技辑》.2009, |
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