CN106153907A - A kind of enzyme linked immunological colour reagent based on gold colloidal - Google Patents
A kind of enzyme linked immunological colour reagent based on gold colloidal Download PDFInfo
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- CN106153907A CN106153907A CN201510130222.XA CN201510130222A CN106153907A CN 106153907 A CN106153907 A CN 106153907A CN 201510130222 A CN201510130222 A CN 201510130222A CN 106153907 A CN106153907 A CN 106153907A
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- urea peroxide
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- linked immunological
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Abstract
The present invention provides a kind of novel enzyme linked immunological Color Appearance System, and this system reacts the principle producing red colloid gold based on gold chloride and peroxide redox.This system is by chlorauric acid solution and urea peroxide solution composition, it is not necessary to stop buffer；This system replaces poisonous and harmful or the developers of less stable such as the conventional o-phenylenediamine of enzyme linked immunological Color Appearance System, tetramethyl benzidine or nitrophenyl phosphate with gold chloride, improves the stability of test kit.
Description
Technical field
The present invention relates to check analysis technical field, in particular to the color appearance system reagent of a kind of Enzyme-Linked Immunospot.
Background technology
Enzyme-Linked Immunospot (elisa) is at present in the most widely used detection technique of medical test and field of detection of food safety, Enzyme-multiplied immune technique can detect little big to HIV (human immunodeficiency virus) antibody to clenbuterol hydrochloride residual, is applied to the every aspect of human health, field of food safety.
The Color Appearance System that euzymelinked immunosorbent assay (ELISA) is commonly used at present has two kinds, respectively horseradish peroxidase (HRP) system and alkali phosphatase (ALP) system.Horseradish peroxidase system is the substrate system with sulphuric acid as stop buffer with HRP for catalyst with tetramethyl Methanamide (TMB) and hydrogen peroxide；Alkaline phosphate ester enzyme system with ALP for catalyst with the p-nitrophenyl phosphate ester (AP) system as substrate, with NaOH as stop buffer.TMB is a kind of reducing substances, the most oxidized during storing, and commercial reagents box typically uses the buffer system of complexity to preserve, and during use, corkage can not be placed the most long.AP stability relatively TMB is stable, but ALP chromogenic reaction speed is very slow, it is generally required to more than 30min, compares and wastes time.
For solving the problem that TMB or AP exists as enzyme linked immunological developer, we use novel gold colloidal Color Appearance System, utilize gold chloride to produce red colloid gold solution with peroxy oxygen reduction reaction, and this solution has absorbance at 550nm.This system substrate component is single and stable, and the response time is short, it is not necessary to stop buffer, and sensitivity is suitable with existing Color Appearance System.
Technology contents
A kind of novel this system substrate component of enzyme linked immunological colour reagent based on gold colloidal is single and stable, and the response time is short, it is not necessary to stop buffer, and sensitivity is suitable with existing Color Appearance System.
The present invention is achieved through the following technical solutions:
A kind of novel enzyme linked immunological this system of Color Appearance System based on gold colloidal is by horseradish peroxidase, chlorauric acid solution and urea peroxide solution composition.
Described horseradish peroxidase is digit synbol and the horseradish peroxidase on monoclonal antibody or multi-resistance.
Described chlorauric acid solution is the AuCl of 0.01%～0.2%3Solution.
Described urea peroxide solution is the urea peroxide solution of 0.1%～5.0%.
Described gold chloride, its buffer is selected from phosphate, citrate, Tris hydrochloride buffer, and its pH is 7.0.
Described urea peroxide, its buffer is pH7.0 phosphate buffer.
The step applying elisa method of the present invention to develop the color is as follows:
Conventionally carry out elisa to operate to adding nitrite ion back.
It is sequentially added into chlorauric acid solution 50ul and urea peroxide solution 50ul.Mixing, room temperature colour developing 5min.
Lath after colour developing is put into microplate reader, and 550nm wavelength reads absorbance.
Data processing method is with conventional elisa.
The principle of present invention colour developing is: Au3+There is strong oxidizing property, urea peroxide can be aoxidized and produce gold simple substance, if urea peroxide is excessive, produce the colloidal gold solution of redness, otherwise, then produce blue gel gold solution.In color development system, HRP can be catalyzed urea peroxide decomposition simultaneously, if containing a large amount of HRP in system, the urea peroxide being catalyzed majority decomposes, thus produces blue gel gold solution, otherwise, then produce red colloid gold solution.
Advantage and good effect
Employing gold chloride is substrate, it is to avoid the shortcoming of conventional elisa test kit substrate poor stability, and this substrate stably can be placed the several years and not change.The method that developing time is more conventional simultaneously is short.Without stop buffer, decrease operating procedure, it is to avoid the etching chemicals such as strong acid and strong base.
Figure of description
Fig. 1 is the colour developing principle of this colour reagent.
Embodiment is illustrated
Embodiment 1 novel enzyme linked immunological Color Appearance System preparation of reagents method
Color Appearance System is made up of developer A and developer B.Developer A is 0.02% gold chloride, and developer B is 0.1% urea peroxide solution.
Developer A compound method is as follows: as a example by preparing 100ml, weighs gold chloride 0.02g and joins in 100ml pure water, and stirring and dissolving is subsequently adding 0.1ml Tween 80, stirs.Should be light yellow transparent solution.
Developer B compound method is as follows: as a example by preparing 100ml, weighs sodium acetate 2.8g, citric acid 0.3g and joins in 100ml pure water, and stirring and dissolving weighs 0.1g urea peroxide and joins in above-mentioned solution, stirring and dissolving.Should be colourless transparent solution.
The developer A prepared and developer B is placed in 2～8 DEG C of preservations in opaque plastics bottle.
Embodiment 2 Novel developing systematic difference method
Test material: developer A:0.02% chlorauric acid solution, developer B:0.1% urea peroxide solution, use HIV (human immunodeficiency virus) (1+2) antibody diagnosing reagent kit (euzymelinked immunosorbent assay (ELISA)) (Beijing Tso Biological Pharmaceutical Co produces, lot number: the 20140923) ELISA Plate of normal sale on market, enzyme labelled antibody.
Detection method is as follows:
1. sample-adding: pre-coating plates is fixed on grillage.Test sets negative control 2 hole, positive control 2 hole every time, is separately added into positive and negative comparison 100ul；If blank 1 hole, it is not added with sample.Remaining hole adds testing sample 100ul.
2. incubation: put 37 DEG C of incubations 60 minutes.
3. washing: discard liquid in hole, fills each hole by the washing liquid after dilution, stands the 30-60 second, discards washing liquid in hole, and button is dry.Repeat to wash 5 times.
The most enzyme-added: blank control wells is the most enzyme-added, remaining hole adds enzyme marker 100ul.
5. incubation: put 37 DEG C of incubations 30 minutes.
6. washing: discard enzyme marker in hole, repeat to wash 5 times (biconditional operation step 3).
7. colour developing: every hole adds developer A 50ul, adds developer B 50ul, vibration mixing, puts room temperature reaction 5 minutes.
8. select microplate reader to measure wavelength 550nm, use blank well zeroising, measure each hole OD value.Also may select 630nm as reference wavelength, measure with dual wavelength.
Claims (9)
1. an enzyme linked immunological kit colour reagent based on gold colloidal, described reagent comprises:
Gold chloride；
Urea peroxide；With
Make the solution that described gold chloride and urea peroxide dissolve.
During the detection of wherein said colour reagent, maximum absorbance is at 550nm.
2. gold chloride described in claim 1, its concentration range is 0.01%～0.2%.
3. urea peroxide described in claim 1, its concentration range is 0.1%～5%.
4. gold chloride described in claim 1, its buffer is selected from phosphate, citrate, Tris hydrochloride buffer.
5. buffer described in claim 4, its pH is 7.0.
6. urea peroxide described in claim 1, its buffer is pH7.0 phosphate buffer.
7. an enzyme linked immunological kit colour reagent based on gold colloidal, its detecting step includes: washes plate after adding detection sample, antibody and enzyme preparation in ELISA Plate micropore, is subsequently adding a certain amount of chlorauric acid solution and urea peroxide solution.Placement a period of time is placed on reading in 550nm wavelength instruments.
8. chlorauric acid solution described in claim 7 and urea peroxide solution, its volume ratio is 1: 1.
9. the reading duration described in claim 7 is 2-10min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510130222.XA CN106153907A (en) | 2015-03-25 | 2015-03-25 | A kind of enzyme linked immunological colour reagent based on gold colloidal |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510130222.XA CN106153907A (en) | 2015-03-25 | 2015-03-25 | A kind of enzyme linked immunological colour reagent based on gold colloidal |
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| CN106153907A true CN106153907A (en) | 2016-11-23 |
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| CN201510130222.XA Pending CN106153907A (en) | 2015-03-25 | 2015-03-25 | A kind of enzyme linked immunological colour reagent based on gold colloidal |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109490522A (en) * | 2018-12-04 | 2019-03-19 | 北京倍肯恒业科技发展股份有限公司 | A kind of nano colloid gold and the preparation method and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698505A (en) * | 2013-12-13 | 2014-04-02 | 山东博科生物产业有限公司 | Efficient stable TMB (Tetramethylbenzidine) color-developing liquor and preparation method thereof |
| CN104049083A (en) * | 2014-06-30 | 2014-09-17 | 江南大学 | Direct competitive enzyme immunoassay method based on nano-gold plasma excimer |
-
2015
- 2015-03-25 CN CN201510130222.XA patent/CN106153907A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103698505A (en) * | 2013-12-13 | 2014-04-02 | 山东博科生物产业有限公司 | Efficient stable TMB (Tetramethylbenzidine) color-developing liquor and preparation method thereof |
| CN104049083A (en) * | 2014-06-30 | 2014-09-17 | 江南大学 | Direct competitive enzyme immunoassay method based on nano-gold plasma excimer |
Non-Patent Citations (1)
| Title |
|---|
| ROBERTO DE LA RICA ET AL: "Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye", 《NATURE NANOTECHNOLOGY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109490522A (en) * | 2018-12-04 | 2019-03-19 | 北京倍肯恒业科技发展股份有限公司 | A kind of nano colloid gold and the preparation method and application thereof |
| CN109490522B (en) * | 2018-12-04 | 2022-03-11 | 北京倍肯恒业科技发展股份有限公司 | Nano colloidal gold and preparation method and application thereof |
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Application publication date: 20161123 |