CN1547016A - Immune colloidal gold reagent for detecting sulfamethazine and preparation method thereof - Google Patents
Immune colloidal gold reagent for detecting sulfamethazine and preparation method thereof Download PDFInfo
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- CN1547016A CN1547016A CNA2003101094652A CN200310109465A CN1547016A CN 1547016 A CN1547016 A CN 1547016A CN A2003101094652 A CNA2003101094652 A CN A2003101094652A CN 200310109465 A CN200310109465 A CN 200310109465A CN 1547016 A CN1547016 A CN 1547016A
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- sulfamethazine
- preparation
- monoclonal antibody
- antibody specific
- colloid gold
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Abstract
The invention is a kind of immune colloidal gold reagent for measuring the sulfamethazole and the manufacturing method. The reagent includes a sample cushion, a colloidal gold combination cushion, aeotic acid cellulose film, a suction cushion and a PVC back lining, one end of the PVC back lining is adhered with sample cushion, colloidal gold combination cushion in order, the middle part is adhered with aeotic acid cellulose film, the other end is adhered with a suction cushion. The character lies in: the colloidal gold combination cushion is enclosed with a kind of sulfamethazole resisting specificity single clone antibody-colloidal gold label, the aeotic acid cellulose film is enclosed by sulfamethazole-BSA couplings and sheep (rabbit) antiplague IgG. The reagent has a strong specificity, high sensitivity, quick and simple, cheap, and convenient.
Description
Technical field
The present invention relates to a kind of immune colloid gold reagent that detects antibody, also relate to the preparation method of this reagent.
Background technology
The existing method that is used to detect sulfamethazine mainly contains high performance liquid chromatography, gas/matter coupling analytic approach (GC/MS), vapor-phase chromatography, thin-layered chromatography, spectroscopic methodology and spectral method, capillary electrophoresis and supercritical fluid chromatography.The defective of various chromatographys, gas/matter coupling analytic approach and capillary electrophoresis is: need the instrument and equipment of specialty auxiliary; The The pretreatment program is loaded down with trivial details, requires high; Complicated operating process, the time is long; Need the technician's operation through professional training, operating personnel will have abundant correlation experience; Operating personnel must understand the various disturbing factors that influence stratographic analysis, understand the relative merits of employed preprocess method, could obtain reliable analysis result; The instrument and equipment costliness that needs is difficult to popularize in city and medium-sized and small enterprises; The requirement height of instrument maintenance, the quality of maintenance is impact analysis result's accuracy directly; The testing cost height.The defective of spectroscopic methodology and spectral method is: need special instrument and equipment auxiliary, operating process is relatively complicated, and operating personnel also need through professional training.
Summary of the invention
The objective of the invention is in order to overcome the defective that current techniques exists in promoting the use of, providing a kind of does not need the auxiliary detectable of particular instrument equipment, and can reduce the detection cost effectively, alleviates the burden that needs to detect unit.The preparation method of this reagent is provided simultaneously.
Detect the immune colloid gold reagent and the preparation method of sulfamethazine, this reagent comprises sample pad, pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, pad successively, the middle nitrocellulose filter that adheres to, and the other end adheres to adsorptive pads.Bag is by anti-sulfamethazine monoclonal antibody specific--colloid gold label thing on the pad.Bag is by sulfamethazine-BSA conjugate and the anti-mouse IgG of sheep (rabbit) on the nitrocellulose filter.
The preparation method of this reagent may further comprise the steps: (1) preparation sulfamethazine-BSA conjugate: with sulfamethazine and BSA in 1: 5～50 (mol/mol) ratio mixing, make sulfamethazine and BSA form stable particle, form sulfamethazine-BSA conjugate by purifying; (2) the anti-sulfamethazine monoclonal antibody specific of preparation: with the repeatedly immune Balb/c mouse inbred lines of sulfamethazine-BSA conjugate, get mouse boosting cell and myeloma cell and form hybridoma, can get the positive hybridoma cell strain through the selective medium screening in external fusion.Mouse peritoneal obtains ascites or cell in vitro is cultivated modes such as collecting culture supernatant by hybridoma is injected, and prepares anti-sulfamethazine monoclonal antibody specific in a large number; (3) the anti-mouse IgG of preparation sheep (rabbit): with many immune mouses of sulfamethazine-BSA, extract antiserum immune goat or rabbit, get sheep anti-mouse igg or rabbit anti-mouse igg behind the purifying.(4) preparation collaurum: the colloid gold particle that gold chloride is reduced into 20nm～40nm with reductive agents such as trisodium citrates; (5) preparation monoclonal antibody colloid gold label: with collaurum and anti-sulfamethazine monoclonal antibody specific in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and anti-sulfamethazine monoclonal antibody specific form stable colloidal solid, by purifying, the concentrated anti-sulfamethazine monoclonal antibody specific-colloid gold label thing that forms; (6) will resist the sulfamethazine monoclonal antibody specific--the colloid gold label thing is coated on the collaurum pad, and sulfamethazine-BSA conjugate and the anti-mouse IgG of sheep (rabbit) are coated on the detection zone and the control zone of nitrocellulose filter, and is fully dry.
Good effect of the present invention is: cheap, production procedure is simple, and cost is low, and the expense of detection is than using other detection method all to want considerably cheaper; Detection speed is fast, and overall process only needs 15-30 minute, can realize that the oneself detects; Can on-the-spotly detect; Specificity is good, highly sensitive, good reproducibility; Easy and simple to handle, fast qualitative, the result is accurately, fast, and is easy and simple to handle, need not flushing process and standard control, can be in batches or single sample in time detect; Be easy to promote the use of, operating personnel need not professional training, and by specification gets final product complete operation.
Embodiment
PVC backing one end is adhered to sample pad, pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads.Bag is by anti-sulfamethazine monoclonal antibody specific--colloid gold label thing on the pad.Bag is by sulfamethazine-BSA conjugate and the anti-mouse IgG of sheep (rabbit) on the nitrocellulose filter.
Preparation according to the following steps: (1) preparation sulfamethazine-BSA conjugate: with sulfamethazine and BSA in 1: 5～50 (mol/mol) ratio mixing, make sulfamethazine and BSA form stable particle, form sulfamethazine-BSA conjugate by purifying; (2) the anti-sulfamethazine monoclonal antibody specific of preparation: with the repeatedly immune Balb/c mouse inbred lines of sulfamethazine-BSA conjugate, get mouse boosting cell and myeloma cell and form hybridoma, can get the positive hybridoma cell strain through the selective medium screening in external fusion.Mouse peritoneal obtains ascites or cell in vitro is cultivated modes such as collecting culture supernatant by hybridoma is injected, and prepares anti-sulfamethazine monoclonal antibody specific in a large number; (3) the anti-mouse IgG of preparation sheep (rabbit): with many immune mouses of sulfamethazine-BSA, extract antiserum immune goat or rabbit, get sheep anti-mouse igg or rabbit anti-mouse igg behind the purifying.(4) preparation collaurum: the particle that 100ml 0.01% chlorauride is reduced into the 40nm size with 0.9ml 1% trisodium citrate; (5) preparation monoclonal antibody colloid gold label: use 0.1mol/L K
2CO
3The pH value of colloidal gold solution is transferred to about 8.1, colloidal gold solution and monoclonal antibody are mixed in the ratio that adds the 1mg monoclonal antibody in the 100ml colloidal gold solution, make collaurum and antibody form stable colloidal gold composite, again by repeatedly centrifugal, abandon supernatant, cleaning, by purifying, the concentrated anti-sulfamethazine monoclonal antibody specific-colloid gold label thing that forms, refrigerate standby; (6) will resist the sulfamethazine monoclonal antibody specific with Biodot point film machine--the colloid gold label thing is coated on the collaurum pad, sulfamethazine-BSA conjugate and the anti-mouse IgG of sheep (rabbit) are coated on the detection zone and the control zone of nitrocellulose filter, fully dry.(7) nitrocellulose filter, collaurum pad, sample pad, adsorptive pads etc. are bonded on the PVC backing successively; (8) the PVC material that glues is cut into the reagent strip of certain width, promptly makes the immune colloid gold reagent that detects sulfamethazine.
Before detection, earlier sample and reagent strip (plate) are placed on placement a period of time (about 10 minutes) under the room temperature condition, make it restore to room temperature; Take out the detectable bar from aluminium foil bag, by the direction shown in the arrow under the MARK line reagent strip is immersed in the sample solution, liquid level must not surpass the MARK line, takes out after 5 seconds-8 seconds, lies on the operator's console; If agent plate: from aluminium foil bag, take out the detectable plate, lie on the operator's console, in well, drip 3 (about 120ul) sample solutions; Get final product judged result in 3 minutes-15 minutes, the result who judges after 30 minutes is invalid.The result judges: if there be " sulfamethazine " that will detect to exist in the sample, then band does not appear in the detection line place, and occurs red stripes on the nature controlling line, and this moment, the result was positive; If " sulfamethazine " that will not detect in the sample exists, then the detection line place has red stripes to occur, and red stripes also occurs on the nature controlling line simultaneously, and this moment, the result was negative.If there is not red stripes to occur on the nature controlling line, then this product is invalid.
Claims (2)
1, detects the immune colloid gold reagent and the preparation method of sulfamethazine, this reagent comprises sample pad, pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads, it is characterized in that bag is by anti-sulfamethazine monoclonal antibody specific-colloid gold label thing on the pad, bag is by sulfamethazine-BSA conjugate and the anti-mouse IgG of sheep (rabbit) on the nitrocellulose filter.
2, the immune colloid gold reagent of detection sulfamethazine according to claim 1 and preparation method, the preparation method who it is characterized in that this reagent may further comprise the steps: (1) preparation sulfamethazine-BSA conjugate: with sulfamethazine and BSA in 1: 5～50 (mol/mol) ratio mixing, make sulfamethazine and BSA form stable particle, form sulfamethazine-BSA conjugate by purifying; (2) the anti-sulfamethazine monoclonal antibody specific of preparation: with the repeatedly immune Balb/c mouse inbred lines of sulfamethazine-BSA conjugate, get mouse boosting cell and myeloma cell and form hybridoma, can get the positive hybridoma cell strain through the selective medium screening in external fusion.Mouse peritoneal obtains ascites or cell in vitro is cultivated modes such as collecting culture supernatant by hybridoma is injected, and prepares anti-sulfamethazine monoclonal antibody specific in a large number; (3) the anti-mouse IgG of preparation sheep (rabbit): with many immune mouses of sulfamethazine-BSA, extract antiserum immune goat or rabbit, get sheep anti-mouse igg or rabbit anti-mouse igg behind the purifying.(4) preparation collaurum: the colloid gold particle that gold chloride is reduced into 20nm～40nm with reductive agents such as trisodium citrates; (5) preparation monoclonal antibody colloid gold label: with collaurum and anti-sulfamethazine monoclonal antibody specific in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and anti-sulfamethazine monoclonal antibody specific form stable colloidal solid, by purifying, the concentrated anti-sulfamethazine monoclonal antibody specific-colloid gold label thing that forms; (6) will resist the sulfamethazine monoclonal antibody specific--the colloid gold label thing is coated on the collaurum pad, and sulfamethazine-BSA conjugate and the anti-mouse IgG of sheep (rabbit) are coated on the detection zone and the control zone of nitrocellulose filter, and is fully dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101094652A CN1547016A (en) | 2003-12-16 | 2003-12-16 | Immune colloidal gold reagent for detecting sulfamethazine and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101094652A CN1547016A (en) | 2003-12-16 | 2003-12-16 | Immune colloidal gold reagent for detecting sulfamethazine and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1547016A true CN1547016A (en) | 2004-11-17 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2003101094652A Pending CN1547016A (en) | 2003-12-16 | 2003-12-16 | Immune colloidal gold reagent for detecting sulfamethazine and preparation method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100396774C (en) * | 2006-07-17 | 2008-06-25 | 华中农业大学 | Immuno colloidal gold test paper strip for detecting sulfa drug residue |
| CN1888905B (en) * | 2006-06-23 | 2010-07-21 | 河南省动物免疫学重点实验室 | Test paper box for sulfa drug multi-residual conjoined detection |
| CN101949922A (en) * | 2010-08-11 | 2011-01-19 | 杭州南开日新生物技术有限公司 | Method for preparing reagent plate for detecting sulphadimidine |
| CN101995461A (en) * | 2009-08-27 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Sulfamethazine colloidal gold test card, production and using method thereof |
| CN101059512B (en) * | 2007-05-31 | 2012-02-15 | 北京望尔生物技术有限公司 | Immunoaffinity chromatography column and its uses in purifying sulpha drugs |
| CN103344758A (en) * | 2013-07-04 | 2013-10-09 | 广西壮族自治区兽医研究所 | Chemiluminescence enzyme immunoassay detection method and kit of sulfamethazine |
-
2003
- 2003-12-16 CN CNA2003101094652A patent/CN1547016A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1888905B (en) * | 2006-06-23 | 2010-07-21 | 河南省动物免疫学重点实验室 | Test paper box for sulfa drug multi-residual conjoined detection |
| CN100396774C (en) * | 2006-07-17 | 2008-06-25 | 华中农业大学 | Immuno colloidal gold test paper strip for detecting sulfa drug residue |
| CN101059512B (en) * | 2007-05-31 | 2012-02-15 | 北京望尔生物技术有限公司 | Immunoaffinity chromatography column and its uses in purifying sulpha drugs |
| CN101995461A (en) * | 2009-08-27 | 2011-03-30 | 深圳市三方圆生物科技有限公司 | Sulfamethazine colloidal gold test card, production and using method thereof |
| CN101995461B (en) * | 2009-08-27 | 2014-05-07 | 深圳市三方圆生物科技有限公司 | Sulfamethazine colloidal gold test card, production and using method thereof |
| CN101949922A (en) * | 2010-08-11 | 2011-01-19 | 杭州南开日新生物技术有限公司 | Method for preparing reagent plate for detecting sulphadimidine |
| CN103344758A (en) * | 2013-07-04 | 2013-10-09 | 广西壮族自治区兽医研究所 | Chemiluminescence enzyme immunoassay detection method and kit of sulfamethazine |
| CN103344758B (en) * | 2013-07-04 | 2016-03-23 | 广西壮族自治区兽医研究所 | Sulfadimidine chemiluminescence enzyme immunoassay detection method and kit |
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