CN104459110B - A kind of one-component TMB nitrite ion - Google Patents
A kind of one-component TMB nitrite ion Download PDFInfo
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- CN104459110B CN104459110B CN201410685229.3A CN201410685229A CN104459110B CN 104459110 B CN104459110 B CN 104459110B CN 201410685229 A CN201410685229 A CN 201410685229A CN 104459110 B CN104459110 B CN 104459110B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses a kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 7-10g, sodium hydrogen phosphate 16-20g, sodium perborate 0.1-0.5g, glycerine 20-30ml, TMB5-40mg, 10-40mlDMSO.By the one-component TMB nitrite ion in scope of the present invention being used for the colour developing stage in enzyme linked immunoassay, avoid TMB nitrite ion to need A, B liquid to mix between use, decrease the running time, the concentration avoiding effective color composition of TMB nitrite ion changes, and then improves the sensitivity of TMB nitrite ion; The solvent of solvent for use TMB of the present invention is DMSO, compares with conventional solvent ethanol, and its speed of dissolving TMB is fast, and dissolve fully, the stability of solution after dissolving is better, improves color developing effect.
Description
Technical field
The present invention relates to TMB nitrite ion field, particularly, relate to a kind of one-component TMB nitrite ion.
Background technology
TMB is a kind of peroxidase substrate, reacts for ELISA.Substrate produces the nattier blue end products of a kind of solubility, can read in 370 or 635nm on spectrophotometer.It is applicable to the quantitative and qualitative analysis in the colour developing stage in enzyme linked immunoassay.The principle of its inspection is: when being marked at the reaction such as the enzyme on antibody or antigen and the TMB in nitrite ion, hydrogen peroxide, after adding stop buffer, solution is by colourless yellowing, in the yellow depth reaction ELISA system antigen-antibody combine number, thus judge certain antigen or antibody number.Estimate light absorption value by microplate reader, judge immunoreactive degree.
Existing commercially available enzyme linked immunological kit adopts TMB developer liquid to be mostly two component, and point A liquid and B liquid, need before use first to mix, and it is facilitate not that use exists shortcoming one, and two is on wrappage, there is cost improve and the wasting of resources; Three is quality heterogeneities between mixed process in using easily causes batch; Four is go back the shortcoming that existence and stability difference, holding time are short, colour developing background is high, sensitivity is low etc., and commercially available enzyme linked immunological kit also has the TMB nitrite ion adopting one-component, but whether good especially the stability of existing one-component TMB nitrite ion is.
TMB nitrite ion of prior art and preparation method thereof, obtain after being mixed by A liquid and B liquid equal-volume, the volumetric molar concentration of each component of described A liquid is: citric acid 0.01-0.5mol/L, sodium hydrogen phosphate 0.01-0.5mol/L, hydrogen peroxidase 10 .01-0.5mol/L, EDTA0.1-1mmol/L; Described B liquid comprises TMB, HCl and polyvinylpyrrolidone, and the volumetric molar concentration of described TMB is 0.1-1mmol/L, and the volumetric molar concentration of described HCl is 0.01-0.5mol/L, and the massfraction of described polyvinylpyrrolidone is 0.1-10%; The oxygenant of existing one-component TMB nitrite ion is urea peroxide.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of one-component TMB nitrite ion, to overcome the problem of existing nitrite ion troublesome poeration, poor stability.
In addition, the present invention also comprises the application of above-mentioned one-component TMB nitrite ion.
The present invention's adopted technical scheme that solves the problem is: a kind of one-component TMB nitrite ion, is characterized in that, the nitrite ion of every 1000mL comprises following component: citric acid 7-10g, sodium hydrogen phosphate 16-20g, sodium perborate 0.1-0.5g glycerine 20-30ml, TMB5-40mg, 10-40mlDMSO.
Existing TMB nitrite ion is all generally be mixed to get by A liquid and B liquid, and the volumetric molar concentration of each component of A liquid is: citric acid 0.01-0.5mol/L, sodium hydrogen phosphate 0.01-0.5mol/L, hydrogen peroxidase 10 .01-0.5mol/L, EDTA0.1-1mmol/L, described B liquid comprises TMB, HCl and polyvinylpyrrolidone, the volumetric molar concentration of described TMB is 0.1-1mmol/L, the volumetric molar concentration of described HCl is 0.01-0.5mol/L, the massfraction of described polyvinylpyrrolidone is 0.1-10%, the shortcoming of existing TMB developer is A, B two kinds of liquid needed equal-volume to mix before colour developing, inconvenient operation, time-consuming, efficiency is lower, criticize between this and there are differences, cost is also high simultaneously, and A liquid B liquid is in the process of mixing, HCl can volatilize, the concentration of the effective constituent developed the color can be caused to change, thus the sensitivity decrease in process color of mixed TMB nitrite ion can be caused, the present invention is one-component TMB nitrite ion, avoid the operation steps that TMB nitrite ion needs A, B liquid to mix between use, simplify the operation flow process, decreases the running time, improve efficiency, the difference between simultaneously avoiding because of A, B liquid different batches causes the color developing effect of the developer mixed, can not exist because the volatilize concentration of effective color composition of the TMB nitrite ion caused of HCl changes, and then improve the sensitivity of TMB nitrite ion, and, conventional solvent ethanol is replaced to DMSO by the present invention, DMSO is a kind of polar non-solute, there is good perviousness, can dissolve each other with organic solvent, water, therefore, DMSO can accelerate the dissolving of TMB, make it the more abundant of dissolving, and make the solution after dissolving more stable, the one-component TMB nitrite ion that improve good stability has better color developing effect, the present invention's sodium perborate substitutes existing hydrogen peroxide, urea peroxide, and experiment proves, compared with hydrogen peroxide, urea peroxide, sodium perborate has better oxidisability.
Further, DMSO is 20-30mL.The DMSO of 20-30mL has dissolution velocity faster, dissolving more abundant, and stability is better.
Further, citric acid is 9-10g, sodium hydrogen phosphate 18-20g.
Experiment proves, citric acid is 9-10g, and the TMB nitrite ion of sodium hydrogen phosphate 18-20g has better stability.
Further, the quality settings of TMB is 10-30mg.Be that 10-30mg effectively can improve color developing effect by the quality settings of TMB, improve the sensitivity of colour developing.
An application for one-component TMB nitrite ion, is applied to enzyme linked immunological kit by described one-component TMB nitrite ion.
To sum up, the invention has the beneficial effects as follows:
1, by the one-component TMB nitrite ion in scope of the present invention being used for the colour developing stage in enzyme linked immunoassay, avoid the operation steps that TMB nitrite ion needs A, B liquid to mix between use, decrease the running time, improve efficiency, the difference between simultaneously avoiding because of A, B liquid different batches causes the color developing effect of the developer mixed.
2, by the one-component TMB nitrite ion in scope of the present invention being used for the colour developing stage in enzyme linked immunoassay, the concentration avoiding effective color composition of TMB nitrite ion changes, and then improves the sensitivity of TMB nitrite ion.
3, the solvent that the present invention is used is DMSO, compares with conventional solvent ethanol, and its speed of dissolving TMB is fast, and dissolve fully, the stability of solution after dissolving is better, makes color developing effect better.
Embodiment
Below in conjunction with embodiment, to the detailed description further of invention do, but embodiments of the present invention are not limited thereto.
Embodiment 1:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 7g, sodium hydrogen phosphate 16g, sodium perborate 0.1g, glycerine 20ml, TMB5mg, 10mLDMSO.
Embodiment 2:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 7g, sodium hydrogen phosphate 16g, sodium perborate 0.1g, glycerine 20ml, TMB5mg, 20mLDMSO.
Embodiment 3:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 7g, sodium hydrogen phosphate 16g, sodium perborate 0.1g, glycerine 20ml, TMB5mg, 30mLDMSO.
Embodiment 4:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 7g, sodium hydrogen phosphate 16g, sodium perborate 0.1g, glycerine 20ml, TMB5mg, 40mLDMSO.
Embodiment 5:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 10g, sodium hydrogen phosphate 20g, sodium perborate 0.5g, glycerine 30ml, TMB10mg, 20mlDMSO.
Embodiment 6:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 10g, sodium hydrogen phosphate 20g, sodium perborate 0.5g, glycerine 30ml, TMB30mg, 30mlDMSO.
Embodiment 7:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 9g, sodium hydrogen phosphate 18g, sodium perborate 0.2g, glycerine 20ml, TMB20mg, 20mlDMSO.
Embodiment 8:
A kind of one-component TMB nitrite ion, the nitrite ion of every 1000mL comprises following component: citric acid 9g, sodium hydrogen phosphate 18g, sodium perborate 0.2g, glycerine 20ml, TMB30mg, 40mlDMSO.
Table 1
Table 2
Table 1 is the Performance comparision data of the one-component nitrite ion of good stability;
Table 2 is that independent sodium perborate compares with the absorbance of hydrogen peroxide, urea peroxide.
TMB nitrite ion, compared with the TMB nitrite ion of existing one-component, not only has two component to be set to one-component by the present invention, makes TMB nitrite ion in operation more for convenience; And alcohol solvents all for existing TMB nitrite ion is replaced to DMSO by the present invention, DMSO is a kind of polar non-solute, there is good perviousness, can dissolve each other with organic solvent, water, therefore, DMSO can accelerate the dissolving of TMB, makes it the more abundant of dissolving, and make the solution after dissolving more stable, make color developing effect better.
Existing TMB nitrite ion is all generally about 20min at the developing time in the colour developing stage of enzyme linked immunoassay, as shown in table 1, the developing time of embodiments of the invention 1 to embodiment 8 is all lower than 20min, therefore the one-component TMB nitrite ion of good stability of the present invention has better sensitivity, wherein, embodiment 5, embodiment 6, embodiment 7, embodiment 8 have the shorter time, when the content of TMB is 10-30mg in the one-component TMB nitrite ion of proof 1000mL good stability, there is better sensitivity.
Existing TMB nitrite ion absorbance general all about 5.0, as shown in table 1, these embodiments of the invention 1 to embodiment 8 absorbance be all greater than 5.0, therefore the one-component TMB nitrite ion of good stability of the present invention has better color developing effect; Wherein, embodiment 2, embodiment 3, embodiment 5, embodiment 6, embodiment 7 have better absorbance, when in the one-component TMB nitrite ion of proof 1000mL good stability, the content of DMSO is 20-30mL, there is better color developing effect, embodiment 7, compared with embodiment 2, embodiment 5, has higher absorbance, proves that citric acid is 9-10g, the one-component TMB nitrite ion of the good stability of sodium hydrogen phosphate 18-20g has better stability, has better color developing effect.
As shown in table 2, compared with hydrogen peroxide, urea peroxide, sodium perborate has higher absorbance, therefore it has better oxidisability.
As mentioned above, the present invention can be realized preferably.
Claims (5)
1. an one-component TMB nitrite ion, is characterized in that, the nitrite ion of every 1000mL comprises following component: citric acid 7-10g, sodium hydrogen phosphate 16-20g, sodium perborate 0.1-0.5g, glycerine 20-30ml, TMB5-40mg, 10-40mlDMSO.
2. a kind of one-component TMB nitrite ion according to claim 1, it is characterized in that, described DMSO is 20-30mL.
3. a kind of one-component TMB nitrite ion according to claim 1 and 2, it is characterized in that, described citric acid is 9-10g, sodium hydrogen phosphate 18-20g.
4. a kind of one-component TMB nitrite ion according to claim 1 and 2, is characterized in that, the quality settings of described TMB is 10-30mg.
5. an application for one-component TMB nitrite ion as claimed in claim 1 or 2, is characterized in that, described one-component TMB nitrite ion is applied to enzyme linked immunological kit.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105044327B (en) * | 2015-07-02 | 2016-09-14 | 深圳市卫光生物制品股份有限公司 | A kind of stop buffer of one-component TMB nitrite ion and preparation method thereof |
| CN105823871B (en) * | 2016-04-18 | 2017-11-07 | 苏州新赛美生物科技有限公司 | A kind of one-component enzyme linked immunological nitrite ion of efficient stable and preparation method thereof |
| CN109085342A (en) * | 2018-09-14 | 2018-12-25 | 武汉伊莱瑞特生物科技股份有限公司 | A kind of ELISA Sample dilution and preparation method improving detection sensitivity |
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