CN1547019A - Immune colloidal gold reagent for detecting stilboestrol and preparation method thereof - Google Patents
Immune colloidal gold reagent for detecting stilboestrol and preparation method thereof Download PDFInfo
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- CN1547019A CN1547019A CNA2003101094686A CN200310109468A CN1547019A CN 1547019 A CN1547019 A CN 1547019A CN A2003101094686 A CNA2003101094686 A CN A2003101094686A CN 200310109468 A CN200310109468 A CN 200310109468A CN 1547019 A CN1547019 A CN 1547019A
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- China
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- diethylstilbestrol
- 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 ethene estraphenol 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 ethene estraphenol resisting specificity single clone antibody-colloidal gold label, the aeotic acid cellulose film is enclosed by ethene estraphenol-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 diethylstilbestrol mainly contains enzyme linked immunological absorption (EIA), radioimmunoassay (RIA), immune affinity chromatographic (IAC), gas/matter coupling analytic approach.The defective of enzyme linked immunological absorption (EIA) method is: need special instrument and equipment such as microplate reader to be used; The detecting operation personnel need pass through professional training; Operating process is relatively complicated, and it is long to detect required time; It is higher to detect required expense, can not realize that single part is detected.The defective of radioimmunoassay is: need the instrument and equipment of specialty auxiliary, and apparatus expensive; Operating personnel need pass through professional training; Complicated operating process requires high; Reagent needs cryopreservation; Because test relates to radioactive isotope, test is not very safe, so seldom adopt; Testing cost is higher; The detection required time is long.The defective of immune affinity chromatographic (IAC) is: need the special immune affinity chromatographic column of preparation; Operating personnel need pass through professional training; Complicated operating process requires high; The testing cost height; The detection required time is long; Be mainly used in sample preparation, application in retention analysis and research still are in the exploratory stage.The defective of gas/matter coupling analytic approach is: need the instrument and equipment of specialty auxiliary, and apparatus expensive; The The pretreatment program is loaded down with trivial details, requires high; The water and all kinds of solvents that use during to operation have strict requirement; 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; Need expensive instrument and equipment auxiliary, be difficult in city and medium-sized and small enterprises, popularize; The requirement height of instrument maintenance, the quality of maintenance is impact analysis result's accuracy directly; The testing cost height.
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 diethylstilbestrol, 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-diethylstilbestrol monoclonal antibody specific--colloid gold label thing on the pad.Bag is by diethylstilbestrol-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 diethylstilbestrol-BSA conjugate: with diethylstilbestrol and BSA in 1: 5～50 (mol/mol) ratio mixing, make diethylstilbestrol and BSA form stable particle, form diethylstilbestrol-BSA conjugate by purifying; (2) the anti-diethylstilbestrol monoclonal antibody specific of preparation: with the repeatedly immune Balb/c mouse inbred lines of diethylstilbestrol-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-diethylstilbestrol monoclonal antibody specific in a large number; (3) the anti-mouse IgG of preparation sheep (rabbit): with many immune mouses of diethylstilbestrol-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-diethylstilbestrol monoclonal antibody specific in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and anti-diethylstilbestrol monoclonal antibody specific form stable colloidal solid, by purifying, the concentrated anti-diethylstilbestrol monoclonal antibody specific-colloid gold label thing that forms; (6) will resist the diethylstilbestrol monoclonal antibody specific--the colloid gold label thing is coated on the collaurum pad, and diethylstilbestrol-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, collaurum pad successively, the middle nitrocellulose filter that adheres to, the other end adheres to adsorptive pads.Bag is by anti-diethylstilbestrol monoclonal antibody specific--colloid gold label thing on the collaurum pad.Bag is by diethylstilbestrol-BSA conjugate and the anti-mouse IgG of sheep (rabbit) on the nitrocellulose filter.
Preparation according to the following steps: (1) preparation diethylstilbestrol-BSA conjugate: in 1: 5～50 (mol/mol) ratio mixing, make diethylstilbestrol and BSA form stable particle diethylstilbestrol and BSA, by purifying formation diethylstilbestrol-BSA conjugate; (2) the anti-diethylstilbestrol monoclonal antibody specific of preparation: with the repeatedly immune Balb/c mouse inbred lines of diethylstilbestrol-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-diethylstilbestrol monoclonal antibody specific in a large number; (3) the anti-mouse IgG of preparation sheep (rabbit): with many immune mouses of diethylstilbestrol-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-diethylstilbestrol monoclonal antibody specific-colloid gold label thing that forms, refrigerate standby; (6) will resist the diethylstilbestrol monoclonal antibody specific with Biodot point film machine--the colloid gold label thing is coated on the collaurum pad, diethylstilbestrol-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 diethylstilbestrol.
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 " diethylstilbestrol " 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 " diethylstilbestrol " 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 diethylstilbestrol, this reagent comprises sample pad, collaurum pad, nitrocellulose filter, adsorptive pads and PVC backing, PVC backing one end adheres to sample pad, collaurum 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-diethylstilbestrol monoclonal antibody specific-colloid gold label thing on the collaurum pad, bag is by diethylstilbestrol-BSA conjugate and the anti-mouse IgG of sheep (rabbit) on the nitrocellulose filter.
2, the immune colloid gold reagent of detection diethylstilbestrol 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 diethylstilbestrol-BSA conjugate: with diethylstilbestrol and BSA in 1: 5～50 (mol/mol) ratio mixing, make diethylstilbestrol and BSA form stable particle, form diethylstilbestrol-BSA conjugate by purifying; (2) the anti-diethylstilbestrol monoclonal antibody specific of preparation: with the repeatedly immune Balb/c mouse inbred lines of diethylstilbestrol-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-diethylstilbestrol monoclonal antibody specific in a large number; (3) the anti-mouse IgG of preparation sheep (rabbit): with many immune mouses of diethylstilbestrol-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-diethylstilbestrol monoclonal antibody specific in 1: 0.005～0.015 (ml/mg) ratio mixing, make collaurum and anti-diethylstilbestrol monoclonal antibody specific form stable colloidal solid, by purifying, the concentrated anti-diethylstilbestrol monoclonal antibody specific-colloid gold label thing that forms; (6) will resist diethylstilbestrol monoclonal antibody specific-colloid gold label thing to be coated on the collaurum pad, diethylstilbestrol-BSA conjugate and the anti-mouse IgG of sheep (rabbit) will be coated on the detection zone and the control zone of nitrocellulose filter, fully dry.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101094686A CN1547019A (en) | 2003-12-16 | 2003-12-16 | Immune colloidal gold reagent for detecting stilboestrol and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2003101094686A CN1547019A (en) | 2003-12-16 | 2003-12-16 | Immune colloidal gold reagent for detecting stilboestrol and preparation method thereof |
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| Publication Number | Publication Date |
|---|---|
| CN1547019A true CN1547019A (en) | 2004-11-17 |
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| Application Number | Title | Priority Date | Filing Date |
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| CNA2003101094686A Pending CN1547019A (en) | 2003-12-16 | 2003-12-16 | Immune colloidal gold reagent for detecting stilboestrol and preparation method thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101865921A (en) * | 2010-06-11 | 2010-10-20 | 中国检验检疫科学研究院 | Method and a special kit for detecting bean pod mottle virus |
| CN102253224A (en) * | 2011-06-13 | 2011-11-23 | 清华大学深圳研究生院 | Immunochromatographic test paper for detecting diethylstilbestrol and preparation method thereof |
| CN102253222A (en) * | 2011-04-21 | 2011-11-23 | 中国农业科学院农业质量标准与检测技术研究所 | Method and special test paper for estrogen detection |
| CN101059511B (en) * | 2006-05-18 | 2012-09-05 | 李季 | ELISA reagent kit suitable for diethylstilbestrol residue analysis |
-
2003
- 2003-12-16 CN CNA2003101094686A patent/CN1547019A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101059511B (en) * | 2006-05-18 | 2012-09-05 | 李季 | ELISA reagent kit suitable for diethylstilbestrol residue analysis |
| CN101865921A (en) * | 2010-06-11 | 2010-10-20 | 中国检验检疫科学研究院 | Method and a special kit for detecting bean pod mottle virus |
| CN102253222A (en) * | 2011-04-21 | 2011-11-23 | 中国农业科学院农业质量标准与检测技术研究所 | Method and special test paper for estrogen detection |
| CN102253222B (en) * | 2011-04-21 | 2014-07-16 | 中国农业科学院农业质量标准与检测技术研究所 | Method and special test paper for estrogen detection |
| CN102253224A (en) * | 2011-06-13 | 2011-11-23 | 清华大学深圳研究生院 | Immunochromatographic test paper for detecting diethylstilbestrol and preparation method thereof |
| CN102253224B (en) * | 2011-06-13 | 2013-10-30 | 清华大学深圳研究生院 | Immunochromatographic test paper for detecting diethylstilbestrol and preparation method thereof |
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