CN103645181A - Enzyme-linked immunosorbent assay chromogenic substrate solution - Google Patents
Enzyme-linked immunosorbent assay chromogenic substrate solution Download PDFInfo
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- CN103645181A CN103645181A CN201310676535.6A CN201310676535A CN103645181A CN 103645181 A CN103645181 A CN 103645181A CN 201310676535 A CN201310676535 A CN 201310676535A CN 103645181 A CN103645181 A CN 103645181A
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- nitrite ion
- chromogenic substrate
- enzyme
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Abstract
The invention relates to an enzyme-linked immunosorbent assay substrate solution. The substrate solution comprises a chromogenic solution (A) and a chromogenic solution (B). The substrate solution provided by the invention can be stably stored for two years at the temperature of 2 to 8 DEG C, the chromogenic effect can be kept for 1h, and the chromogenic strength is obviously improved; meanwhile, a background is not added, so that the detection sensitivity is improved, the aims of stability and high efficiency are achieved, and various requirements of an enzyme-linked immunosorbent assay kit on the chromogenic substrate solution are met.
Description
Technical field
The enzyme linked immunological substrate nitrite ion that the present invention relates to a kind of stability and high efficiency, belongs to clinical vitro detection technical field.
Background technology
Engvall in 1971 and Perlmann have delivered enzyme linked immunological
adsorbentmeasure (enzyme linked immunosorbent assay,
elisa), for the article of IgG quantitative measurement, make within 1966, to start to become for the enzyme labelled antibody technical development of antigen location the assay method of liquid sample micro substance.This method basic
principlebe: 1. make antigen or antibody be attached to certain surface of solid phase carriers, and keep its immunocompetence.2. make antigen or antibody and horseradish peroxidase (HRP) connect into enzyme-labelled antigen or antibody, this enzyme-labelled antigen or antibody had both retained its immunocompetence, retained again the activity of enzyme.When measuring, being examined sample (measuring antibody or antigen wherein) and enzyme-labelled antigen or antibody, by the antigen of different steps and surface of solid phase carriers or antibody, react.By the method for washing, the antigen antibody complex forming on solid phase carrier is separated with other materials, the enzyme amount being finally combined on solid phase carrier becomes certain ratio with the amount of tested substance in sample.Add after the substrate solution of horseradish peroxidase (HRP) reaction, substrate is become coloured product by enzymatic, and the amount of product is directly related with the amount of tested substance in sample.Because the catalysis frequency of enzyme is very high, thus iodine effect greatly, thus make assay method reach very high susceptibility.In this detection system, substrate nitrite ion as final decision the colour developing degree of last detection, the feature that desirable chromogenic substrate liquid should have that stability is high, the holding time is long, colour developing background is low, enzymatic colored intensity is high.
The oxidation reaction of HRP catalysis superoxide, the most representative superoxide is H
2o
2, its reaction equation is as follows: DH
2+ H
2o
2 
d+ H
2o, in above formula, DH
2for oxygen donator, H
2o
2for hydrogen acceptor.In ELISA, DH
2be generally leuco compound, after enzyme effect, become coloured product, to do colorimetric estimation.Conventional hydrogen donor has o-phenylenediamine (O-phenylenediamine, OPD), tetramethyl benzidine (3,3', 5,5'-tetramethylbenzidine, TMB) and ABTS [2,2'-azino-di-(3-ethylbenziazobine sulfonate-6)].
Product after OPD oxidation is orange red, with acid, stops, after enzyme reaction, at 492nm place, having the highest absorption peak, and highly sensitive, colorimetric is convenient, is the most frequently used substrate of HRP bond.Itself is insoluble in water OPD, and OPD2HCL is water-soluble.Once there is report OPD to cause mutation, during operation, should note.OPD is shown in that light is perishable, is mixed into after substrate application liquid more unstablely with hydrogen peroxide, must existing configuration is existing uses.In kit, OPD and H
2o
2generally be divided into two components, OPD can be made into a certain amount of pulvis or tablet form, contains foaming cosolvent in tablet, uses more convenient.Hydrogen peroxide is allocated in substrate buffer solution, has the concentrate of making easy preservation, distilled water diluting during use.In advanced ELISA kit, being directly made into containing protectant working concentration is 0.02% H
2o
2application liquid, can be used as substrate application liquid after only need adding OPD.
TMB common property thing after HRP effect is aobvious blue, visual with distinct contrast.TMB character is more stable, can wiring solution-forming reagent, only need and H
2o
2solution mixed application liquid, can directly make substrate and use.In addition, TMB has again the advantages such as non-carcinogenesis, and therefore in ELISA, application is increasingly extensive.HCL or H for enzyme reaction
2sO
4after termination, TMB product is yellow by blueness, can be in tintmeter quantitatively, and the suitableeest absorbing wavelength is 405nm.
Though ABTS is not as OPD and TMB sensitivity, blank value is extremely low, also by some kits, is adopted.
The character of various hydrogen donors is different, and that on market, common hydrogen donor is selected is TMB.But TMB is relatively insoluble in water, and character is unstable in solution state, is relatively difficult to deposit, and this has just caused conventional TMB nitrite ion poor stability on market, develop the color lasting not, and the problem such as colored intensity reduction after long-time placement.
Summary of the invention
Be directed to the problem that above conventional TMB chromogenic substrate liquid exists, the invention provides a kind of TMB nitrite ion formula and compound method of stability and high efficiency, assurance chromogenic substrate liquid can be stablized placement 2 years under 2～8 ℃ of conditions, and color developing effect can continue 1 hour, meets market kit.
TMB chromogenic substrate liquid of the present invention is comprised of two reagent component, and concrete component and concentration are as follows:
Nitrite ion A:
Sodium citrate 5-20g/L
Citric acid 2-10 g/L
Urea peroxide 0.5-1g/L
Sodium pyrophosphate 0.5-5g/L
Hydrogen peroxide 0.5-1mL/L
Brij30 0.2-1mL/L
N-Methylisothiazolone-HCl （MIT） 1-10g/L
Each component is dissolved in respectively in 1L distilled water have been prepared;
Nitrite ion B:
TMB 0.3-5 g/L
Citric acid 2-10 g/L
Disodium ethylene diamine tetraacetate 0.2-2 g/L
Glycerine 1-10 mL/L
Dimethyl sulfoxide (DMSO) 5-10 mL/L
First TMB is dissolved into after dimethyl sulfoxide (DMSO), then adds in 1L distilled water, other components add dissolving successively.
TMB chromogenic substrate liquid provided by the invention adds organic solvent to dissolve TMB, has strengthened the homogeneity of reagent component, and the nitrite ion of preparation is stable, has added in addition urea peroxide, sodium pyrophosphate in nitrite ion A, has effectively protected the stability of hydrogen peroxide.This compound method and component, having guaranteed that chromogenic substrate liquid can be stablized under 2～8 ℃ of conditions places 2 years, color developing effect can continue 1 hour, and the intensity of colour developing obviously strengthens, do not increase background simultaneously, improve the sensitivity detecting, reached the object of stability and high efficiency, met the requirements of enzyme linked immunological kit to chromogenic substrate liquid.
Accompanying drawing explanation
Fig. 1 is substrate solution of the present invention and the comparison diagram of the different detection times of conventional substrate solution to testing result.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated.
The preparation of embodiment 1:TMB chromogenic substrate liquid
Nitrite ion A:
Sodium citrate 20g/L
Citric acid 10 g/L
Urea peroxide 1g/L
Sodium pyrophosphate 5g/L
Hydrogen peroxide 1mL/L
Brij30 01mL/L
N-Methylisothiazolone-HCl （MIT） 10g/L
Each component is dissolved in respectively in 1L distilled water have been prepared;
Nitrite ion B:
TMB 5 g/L
Citric acid 10 g/L
Disodium ethylene diamine tetraacetate 2 g/L
Dimethyl sulfoxide (DMSO) 10 mL/L
First TMB is dissolved into after dimethyl sulfoxide (DMSO), then adds in 1L distilled water, other components add dissolving successively.
Utilize hepatitis C cAg detection kit (double antibodies sandwich ELISA) to carry out detection validation to chromogenic substrate liquid.Concrete operation step is: (1) sets well, 50 μ L series HCV calibration product is added on the coated plate of HCV kit, then adds the anti-HCV monoclonal antibody-HRP of 50 μ L enzyme conjugates, and 37 ℃ of reactions, after 1 hour, are washed plate 5 times, pat dry; (2) every hole adds nitrite ion A liquid and each 50uL of B liquid, 37 ℃ of colour developing 15min of lucifuge.(3) every hole adds each 50uL of stop buffer, reads absorbance (OD) value by microplate reader at wavelength 450nm/630nm place.
On contrast market, common chromogenic substrate liquid detects, and the OD value (λ=450nm/630nm) that obtains variable concentrations standard items gained is as shown in table 1 below:
Table 1 standard items testing result
| Standard items concentration (pg/mL) | |
Common chromogenic substrate liquid is measured |
| 0 | 0.078 | 0.096 |
| 10 | 0.249 | 0.218 |
| 100 | 1.021 | 0.946 |
| 1000 | 1.675 | 1.539 |
| 10000 | 2.110 | 1.987 |
| 100000 | 2.897 | 2.441 |
OD value while measuring 0 concentration is background intensity, measurement result by experiment, and the background of embodiment 1 is 0.078, and the background of common chromogenic substrate liquid is 0.096, the chromogenic substrate liquid background of the present invention's preparation is lower.The testing result of contrast 10pg/mL, signal to noise ratio (S/N ratio) (S/N) 0.249/0.078=3.12 that under same concentration, embodiment 1 detects, be greater than the signal to noise ratio (S/N ratio) 0.218/0.096=2.27 that common chromogenic substrate liquid is measured, therefore the colored intensity detecting is larger, is applied to the sensitivity that detects sample in kit and wants high.
Embodiment 2: preparation TMB chromogenic substrate liquid is as follows,
Nitrite ion A:
Sodium citrate 5g/L
Citric acid 2 g/L
Urea peroxide 0.5g/L
Sodium pyrophosphate 0.5g/L
Hydrogen peroxide 0.5mL/L
Brij30 0.2mL/L
N-Methylisothiazolone-HCl （MIT） 1g/L
Each component is dissolved in respectively in 1L distilled water have been prepared.
Nitrite ion B:
TMB 0.3g/L
Citric acid 2 g/L
Disodium ethylene diamine tetraacetate 0.2g/L
Glycerine 1mL/L
Dimethyl sulfoxide (DMSO) 5 mL/L
First TMB is dissolved into after dimethyl sulfoxide (DMSO), then adds in 1L distilled water, other components add dissolving successively.
The chromogenic substrate liquid of embodiment 2 preparations and common chromogenic substrate liquid simultaneous equal are placed in 37 ℃ of constant temperature ovens, place after 7 days, take out contrast simultaneously and verify, testing process is identical with embodiment 1.Testing result is as shown in table 2:
37 ℃ of placements of table 2 nitrite ion testing result after 7 days:
| Standard items concentration (pg/mL) | Embodiment 2 measures OD value | Common chromogenic substrate liquid is measured |
| 0 | 0.064 | 0.057 |
| 10 | 0.236 | 0.175 |
| 100 | 0.948 | 0.686 |
| 1000 | 1.524 | 1.038 |
| 10000 | 2.008 | 1.376 |
| 100000 | 2.697 | 1.948 |
Pass through Data Comparison, through 37 ℃ of 7 days constant temperature, place, gliding obviously appears in the OD value that common chromogenic substrate liquid detects, and the chromogenic substrate liquid of embodiment 2 preparations changes not quite, the chromogenic substrate liquid that embodiment 2 preparations have been described has stronger high-temperature resistance, has reduced the impact of hot weather transportation on kit.
Embodiment 3: preparation TMB chromogenic substrate liquid is as follows,
Nitrite ion A:
Sodium citrate 10g/L
Citric acid 5 g/L
Urea peroxide 0.7g/L
Sodium pyrophosphate 2g/L
Hydrogen peroxide 0.7mL/L
Brij30 0.5mL/L
N-Methylisothiazolone-HCl （MIT） 5g/L
Each component is dissolved in respectively in 1L distilled water have been prepared;
Nitrite ion B:
TMB 3g/L
Citric acid 5 g/L
Disodium ethylene diamine tetraacetate 1g/L
Dimethyl sulfoxide (DMSO) 7 mL/L
First TMB is dissolved into after dimethyl sulfoxide (DMSO), then adds in 1L distilled water, other components add dissolving successively.
Utilize embodiment 3 chromogenic substrate liquid and conventional chromogenic substrate liquid (substrate solution of DiaSorin South Africa (Pty) Ltd), 1000 pg/mL standard items are detected, testing process is identical with the flow process of embodiment 1, divide different time to testing result record, follow the tracks of detection along with the variation tendency of the colored intensity of the variation nitrite ion of time, concrete outcome as shown in Figure 1, by the result shown in Fig. 1, conventional chromogenic substrate liquid is along with the prolong intensity of colour of detection time is reducing, this is unfavorable for the application of kit, because each reacting hole of ELISA kit is not to add nitrite ion simultaneously, so chromogenic reaction time disunity of whole 96 orifice plates, if changing, colored intensity will cause testing result to occur deviation.The chromogenic substrate liquid of embodiment 3 preparations can keep almost not decay of nitrite ion in 30 minutes, had effectively guaranteed the accuracy of testing result.
The present invention is by optimizing conventional TMB chromogenic substrate liquid, prepared a kind of enzyme linked immunological chromogenic substrate liquid of stability and high efficiency, this substrate solution can be stablized placement 2 years under 2～8 ℃ of conditions, color developing effect can continue 1 hour, and the intensity of colour developing obviously strengthens, do not increase background simultaneously, improved the sensitivity detecting, reach the object of stability and high efficiency, met the requirements of enzyme linked immunological kit to chromogenic substrate liquid.
Claims (1)
1. an enzyme linked immunological chromogenic substrate liquid, is characterized in that, comprises nitrite ion A and nitrite ion B;
Component and the content of described nitrite ion A are as follows:
Sodium citrate 5-20g/L
Citric acid 2-10 g/L
Urea peroxide 0.5-1g/L
Sodium pyrophosphate 0.5-5g/L
Hydrogen peroxide 0.5-1mL/L
Brij30 0.2-1mL/L
N-Methylisothiazolone-HCl 1-10g/L
The preparation method of nitrite ion A: each component is dissolved in respectively in 1L distilled water have been prepared;
Component and the content of described nitrite ion B are as follows:
TMB 0.3-5 g/L
Citric acid 2-10 g/L
Disodium ethylene diamine tetraacetate 0.2-2 g/L
Glycerine 1-10 mL/L
Dimethyl sulfoxide (DMSO) 5-10 mL/L
The preparation method of nitrite ion B: first TMB is dissolved into after dimethyl sulfoxide (DMSO), then adds in 1L distilled water, other components add dissolving successively.
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Cited By (9)
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| CN103913566A (en) * | 2014-04-11 | 2014-07-09 | 苏州浩欧博生物医药有限公司 | ELISA (enzyme-linked immunosorbent assay) chromogenic substrate and preparation method thereof |
| CN104280557A (en) * | 2014-09-29 | 2015-01-14 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunoassay kit for detecting abamectin and preparation method and detection method of enzyme linked immunoassay kit |
| CN105116141A (en) * | 2015-07-02 | 2015-12-02 | 深圳市卫光生物制品股份有限公司 | Single-component TMB coloration liquid and preparation method thereof |
| CN105548173A (en) * | 2016-03-12 | 2016-05-04 | 北京易科拜德科技有限公司 | Adiponectin detection kit |
| 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 |
| CN107942054A (en) * | 2017-11-16 | 2018-04-20 | 北华大学 | A kind of arginyl butanedioic acid cracks enzyme reagent kit |
| CN111665241A (en) * | 2020-06-12 | 2020-09-15 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
| CN112280824A (en) * | 2020-09-28 | 2021-01-29 | 北京森康维特生物技术研究所 | Single-component TMB color developing solution, preparation method and kit thereof |
| CN117969817A (en) * | 2024-02-02 | 2024-05-03 | 禾旭(郑州)生物技术有限公司 | High-sensitivity and ultra-stable ELISA (enzyme-linked immunosorbent assay) bi-component chromogenic solution and stop solution |
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| CN103913566A (en) * | 2014-04-11 | 2014-07-09 | 苏州浩欧博生物医药有限公司 | ELISA (enzyme-linked immunosorbent assay) chromogenic substrate and preparation method thereof |
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| CN104280557A (en) * | 2014-09-29 | 2015-01-14 | 贵州勤邦食品安全科学技术有限公司 | Enzyme linked immunoassay kit for detecting abamectin and preparation method and detection method of enzyme linked immunoassay kit |
| CN105116141A (en) * | 2015-07-02 | 2015-12-02 | 深圳市卫光生物制品股份有限公司 | Single-component TMB coloration liquid and preparation method thereof |
| CN105548173A (en) * | 2016-03-12 | 2016-05-04 | 北京易科拜德科技有限公司 | Adiponectin detection kit |
| 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 |
| CN107942054A (en) * | 2017-11-16 | 2018-04-20 | 北华大学 | A kind of arginyl butanedioic acid cracks enzyme reagent kit |
| CN111665241A (en) * | 2020-06-12 | 2020-09-15 | 苏州良辰生物仪器试剂有限公司 | Tyrosine detection test strip and preparation method and application thereof |
| CN112280824A (en) * | 2020-09-28 | 2021-01-29 | 北京森康维特生物技术研究所 | Single-component TMB color developing solution, preparation method and kit thereof |
| CN112280824B (en) * | 2020-09-28 | 2022-07-19 | 北京森康维特生物技术研究所 | Single-component TMB color developing solution, preparation method and kit thereof |
| CN117969817A (en) * | 2024-02-02 | 2024-05-03 | 禾旭(郑州)生物技术有限公司 | High-sensitivity and ultra-stable ELISA (enzyme-linked immunosorbent assay) bi-component chromogenic solution and stop solution |
| CN117969817B (en) * | 2024-02-02 | 2024-11-26 | 禾旭(郑州)生物技术有限公司 | A highly sensitive, ultrastable enzyme-linked immunosorbent two-component colorimetric solution and stop solution |
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