CN104458717B - Sing-component enzyme substrate color development solution - Google Patents
Sing-component enzyme substrate color development solution Download PDFInfo
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- CN104458717B CN104458717B CN201410685230.6A CN201410685230A CN104458717B CN 104458717 B CN104458717 B CN 104458717B CN 201410685230 A CN201410685230 A CN 201410685230A CN 104458717 B CN104458717 B CN 104458717B
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- enzyme substrate
- color development
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- chromogenic enzyme
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Abstract
The invention discloses a single-component enzyme substrate color development solution. The color development solution per 1000 mL comprises the following components: 7-10 g of citric acid, 16-20 g of sodium hydrogen phosphate, 0.1-0.5 g of perboric acid soda, 20-30 ml of glycerol, 5-40 mg of enzyme substrates and 10-30 ml of alcohol. According to the single-component enzyme substrate color development solution, conventional hydrogen peroxide and urea peroxide are replaced with perboric acid soda which has a better oxidizing property compared with hydrogen peroxide and urea peroxide, so that the color development effect of the single-component enzyme substrate color development solution is improved; meanwhile, an operation step that a solution A and a solution B need to be mixed before use when the single-component enzyme substrate color development solution is used is eliminated, so that the operating process is simplified, the operation time is shortened, and the efficiency is improved; influence on the color development effect of a mixed color developing agent, caused by difference between the solution A and the solution B in different batches, is avoided; change of the concentration of effective color development components of the sing-component enzyme substrate color development solution is avoided, so that the sensitivity of the sing-component enzyme substrate color development solution is improved.
Description
Technical field
The present invention relates to chromogenic enzyme substrate liquid field, in particular it relates to a kind of one-component chromogenic enzyme substrate liquid.
Background technology
Zymolyte is TMB, for ELISA reactions.Substrate produces a kind of nattier blue end productses of solubility, can be 370
Or 635nm reads on spectrophotometer.The qualitatively and quantitatively analysis in its colour developing stage suitable for enzyme linked immunoassay.Its
The principle of inspection is：The reactions such as zymolyte, the hydrogen peroxide in the enzyme on be marked at antibody or antigen and nitrite ion, add and terminate
Solution is by colourless yellowing after liquid, in the yellow depth reaction ELISA systems antigen-antibody combine number, so as to judge certain
The number of antigen or antibody.Light absorption value is being estimated by microplate reader, the degree of immunoreation is being judged.
Existing commercially available enzyme linked immunological kit is mostly double-component using chromogenic enzyme substrate agent liquid, point A liquid and B liquid,
Needing first to mix before use, being not convenient enough there is shortcoming one using on, two is the presence of cost raising on packaging material
And the wasting of resources；Three is quality heterogeneity between the mixed process in use is easily caused batch；Four be go back existence and stability it is poor, preserve
Time is short, colour developing background is high, the shortcoming that sensitivity is low.
Chromogenic enzyme substrate liquid of prior art and preparation method thereof, obtains, the A liquid after being mixed by A liquid and B liquid equal-volume
The molar concentration of each component is：Citric acid 0.01-0.5mol/L, disodium hydrogen phosphate 0.01-0.5mol/L, hydrogen peroxidase 10 .01-
0.5mol/L, EDTA0.1-1mmol/L；The B liquid includes zymolyte, HCl and polyvinylpyrrolidone, and the zymolyte rubs
Your concentration is 0.1-1mmol/L, and the molar concentration of the HCl is 0.01-0.5mol/L, the quality of the polyvinylpyrrolidone
Fraction is 0.1-10%.
The content of the invention
The technical problem to be solved is to provide a kind of one-component chromogenic enzyme substrate liquid, to overcome existing nitrite ion
The low problem of troublesome poeration, sensitivity.
Additionally, present invention additionally comprises the application of above-mentioned one-component chromogenic enzyme substrate liquid.
The present invention the adopted technical scheme that solves the above problems is：A kind of one-component chromogenic enzyme substrate liquid, its feature exists
In the nitrite ion per 1000mL includes following components：Citric acid 7-10g, disodium hydrogen phosphate 16-20g, Dexol 0.1-
0.5g, glycerol 20-30mL, zymolyte 5-40mg, ethanol 10-30mL.
Existing chromogenic enzyme substrate liquid is typically all to be mixed to get by A liquid and B liquid, and the molar concentration of A liquid each components is：Lemon
Lemon acid 0.01-0.5mol/L, disodium hydrogen phosphate 0.01-0.5mol/L, hydrogen peroxidase 10 .01-0.5mol/L, EDTA0.1-
1mmol/L；The B liquid includes zymolyte, HCl and polyvinylpyrrolidone, and the molar concentration of the zymolyte is 0.1-
The molar concentration of 1mmol/L, the HCl is 0.01-0.5mol/L, and the mass fraction of the polyvinylpyrrolidone is 0.1-
10%, the shortcoming of existing chromogenic enzyme substrate agent is that two kinds of liquid of A, B needed equal-volume to mix before colour developing, and operation is not square
Just, time-consuming, it is less efficient, batch have differences between this, while cost is also high, and A liquid B liquid is during mix, HCl meetings
Volatilization, the concentration that can cause the effective ingredient for developing the color changes, thus can cause showing for mixed chromogenic enzyme substrate liquid
Sensitivity decrease during color；The present invention is one-component chromogenic enzyme substrate liquid, it is to avoid chromogenic enzyme substrate liquid is between use
Need the operating procedure for mixing A, B liquid, streamline operation reduces the operating time, improve efficiency, at the same avoid because
Difference between A, B liquid different batches causes the color developing effect of the developer for mixing；Also, be not in because caused by HCl volatilizations
The concentration of effective color composition of chromogenic enzyme substrate liquid changes, and then improves the sensitivity of chromogenic enzyme substrate liquid；This
Bright Dexol substitutes existing hydrogen peroxide, urea peroxide, it is demonstrated experimentally that compared with hydrogen peroxide, urea peroxide, crossing boron
Sour sodium has more preferable oxidisability.
Further, citric acid is 9-10g, disodium hydrogen phosphate 18-20g.
It is demonstrated experimentally that citric acid is 9-10g, the 1000mL one-component chromogenic enzyme substrate liquid of disodium hydrogen phosphate 18-20g has
More preferable stability.
Further, the quality settings of zymolyte are 10-30mg.Can be effective for 10-30mg by the quality settings of zymolyte
Raising color developing effect, improve colour developing sensitivity.
Further, Dexol is 0.4-0.5g.
It is demonstrated experimentally that Dexol has preferably colour developing effect for the 1000mL one-component chromogenic enzyme substrates liquid of 0.4-0.5g
Really.
A kind of such as right wants the application of one-component chromogenic enzyme substrate liquid, it is characterised in that by described one-component zymolyte
Nitrite ion is applied to enzyme linked immunological kit.
To sum up, the invention has the beneficial effects as follows：
1st, the present invention substitutes existing hydrogen peroxide, urea peroxide with Dexol, with hydrogen peroxide, urea peroxide phase
Than Dexol has more preferable oxidisability, and Dexol improves the color developing effect of one-component chromogenic enzyme substrate liquid.
2nd, the colour developing by the way that the one-component chromogenic enzyme substrate liquid in scope of the present invention is used in enzyme linked immunoassay
Stage, it is to avoid chromogenic enzyme substrate liquid needs between use the operating procedure for mixing A, B liquid, streamline operation to reduce
Operating time, efficiency is improve, while avoiding the colour developing of the developer for causing to mix because of difference between A, B liquid different batches
Effect.
3rd, the colour developing by the way that the one-component chromogenic enzyme substrate liquid in scope of the present invention is used in enzyme linked immunoassay
Stage, it is to avoid the concentration of effective color composition of chromogenic enzyme substrate liquid changes, and then improves chromogenic enzyme substrate liquid
Sensitivity.
Specific embodiment
With reference to embodiment, detailed description further, but embodiments of the present invention not limited to this are made to invention.
Embodiment 1：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 7g, phosphoric acid hydrogen two
Sodium 16g, Dexol 0.1g, glycerol 20mL, zymolyte 5mg, 10mL ethanol.
Embodiment 2：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 7g, phosphoric acid hydrogen two
Sodium 16g, Dexol 0.1g, glycerol 20mL, zymolyte 10mg, 10mL ethanol.
Embodiment 3：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 7g, phosphoric acid hydrogen two
Sodium 16g, Dexol 0.1g, glycerol 20mL, zymolyte 20mg, 10mL ethanol.
Embodiment 4：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 7g, phosphoric acid hydrogen two
Sodium 16g, Dexol 0.1g, glycerol 20mL, zymolyte 30mg, 10mL ethanol.
Embodiment 5：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 10g, phosphoric acid hydrogen
Disodium 20g, Dexol 0.5g, glycerol 30mL, zymolyte 40mg, 30mL ethanol.
Embodiment 6：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 9g, phosphoric acid hydrogen two
Sodium 18g, Dexol 0.2g, glycerol 20mL, zymolyte 30mg, 20mL ethanol.
Embodiment 7：
A kind of one-component chromogenic enzyme substrate liquid, the nitrite ion per 1000mL includes following components：Citric acid 9g, phosphoric acid hydrogen two
Sodium 18g, Dexol 0.4g, glycerol 20mL, zymolyte 30mg, 20mL ethanol.
Table 1
Table 2
Table 1 is the Performance comparision data of one-component nitrite ion；
Table 2 is that single Dexol compares with the absorbance of hydrogen peroxide, urea peroxide.
Developing time of the existing chromogenic enzyme substrate liquid in the colour developing stage of enzyme linked immunoassay is typically all 20min left
The right side, as shown in table 1, embodiments of the invention 1 to the developing time of embodiment 7 are below 20min, therefore the one-component enzyme of the present invention
Substrate nitrite ion has more preferable sensitivity, wherein, embodiment 6, embodiment 7, embodiment 2, embodiment 3, the colour developing of embodiment 4
Time is less than embodiment 1, embodiment 5, it was demonstrated that zymolyte is in 10-30mg with faster good sensitivity.
The absorbance of existing chromogenic enzyme substrate liquid is general all in 5.0 or so, as shown in table 1, the enforcement of this present invention
Example 1 to the absorbance of embodiment 7 is all higher than 5.0, therefore the one-component chromogenic enzyme substrate liquid of the present invention has preferably colour developing effect
Really；Wherein, embodiment 5, embodiment 6, embodiment 7 have higher absorbance, it was demonstrated that citric acid is 9-10g, phosphoric acid hydrogen two
The 1000mL one-component chromogenic enzyme substrate liquid of sodium 18-20g has more preferable stability；Wherein, embodiment 5, the extinction of embodiment 7
Angle value is higher than embodiment 6, it was demonstrated that Dexol has preferably aobvious for the 1000mL one-component chromogenic enzyme substrates liquid of 0.4-0.5g
Color effect, as shown in table 2, compared with hydrogen peroxide, urea peroxide, Dexol has more preferable oxidisability.
As described above, the present invention can be realized preferably.
Claims (5)
1. a kind of one-component chromogenic enzyme substrate liquid, it is characterised in that the nitrite ion per 1000mL includes following components：Citric acid 7-
10g, disodium hydrogen phosphate 16-20g, Dexol 0.1-0.5g, glycerol 20-30mL, zymolyte 5-40mg, ethanol 10-30mL.
2. a kind of one-component chromogenic enzyme substrate liquid according to claim 1, it is characterised in that the citric acid is 9-10g,
Disodium hydrogen phosphate 18-20g.
3. a kind of one-component chromogenic enzyme substrate liquid according to claim 1 and 2, it is characterised in that the matter of the zymolyte
Amount is set to 10-30mg.
4. a kind of one-component chromogenic enzyme substrate liquid according to claim 1 and 2, it is characterised in that the Dexol is
0.4-0.5g。
5. a kind of application of one-component chromogenic enzyme substrate liquid as claimed in claim 1 or 2, it is characterised in that by described list
Component chromogenic enzyme substrate liquid is applied to enzyme linked immunological kit.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410685230.6A CN104458717B (en) | 2014-11-25 | 2014-11-25 | Sing-component enzyme substrate color development solution |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410685230.6A CN104458717B (en) | 2014-11-25 | 2014-11-25 | Sing-component enzyme substrate color development solution |
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| CN104458717B true CN104458717B (en) | 2017-05-03 |
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| CN106872688A (en) * | 2017-03-02 | 2017-06-20 | 江苏华冠生物技术股份有限公司 | A kind of horseradish peroxidase stabilization substrate A B mixed liquors |
| CN110376376A (en) * | 2019-07-06 | 2019-10-25 | 郑州达诺生物技术有限公司 | A kind of two-in-one substrate of TMB |
Citations (4)
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| JPH08168394A (en) * | 1994-12-16 | 1996-07-02 | Nikken Food Kk | Monoclonal antibody to 4-hydroxy-2-nonenal modified protein, its production, hybrid cell producing the same and immonoassay kit for determining 4-hydroxy-2-nonenal |
| CN1150649A (en) * | 1996-05-24 | 1997-05-28 | 中国预防医学科学院流行病学微生物学研究所 | Enzyme linked immunosorbent assay kit for Leym disease |
| CN103063661A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Tetramethylbenzidine (TMB) coloration solution and preparation method thereof |
| CN103712982A (en) * | 2013-12-13 | 2014-04-09 | 山东博科生物产业有限公司 | High-sensitivity TMB (tetramethylbenzidine) color developing liquid and preparation method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080286812A1 (en) * | 2007-03-23 | 2008-11-20 | Joseph Thomas Ippoliti | Alcohol oxidase-based enzyme-linked immunosorbent assay |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08168394A (en) * | 1994-12-16 | 1996-07-02 | Nikken Food Kk | Monoclonal antibody to 4-hydroxy-2-nonenal modified protein, its production, hybrid cell producing the same and immonoassay kit for determining 4-hydroxy-2-nonenal |
| CN1150649A (en) * | 1996-05-24 | 1997-05-28 | 中国预防医学科学院流行病学微生物学研究所 | Enzyme linked immunosorbent assay kit for Leym disease |
| CN103063661A (en) * | 2012-12-21 | 2013-04-24 | 杭州茂天赛科技有限公司 | Tetramethylbenzidine (TMB) coloration solution and preparation method thereof |
| CN103712982A (en) * | 2013-12-13 | 2014-04-09 | 山东博科生物产业有限公司 | High-sensitivity TMB (tetramethylbenzidine) color developing liquid and preparation method thereof |
Non-Patent Citations (1)
| Title |
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| 市售单组分TMB显色液质量比较;潘庆军 等;《检验医学与临床》;20131231;第10卷(第23期);全文 * |
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