CN102435598A - Stable HRP enzymatic enhanced chemiluminescent substrate solution - Google Patents
Stable HRP enzymatic enhanced chemiluminescent substrate solution Download PDFInfo
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- CN102435598A CN102435598A CN2011102623592A CN201110262359A CN102435598A CN 102435598 A CN102435598 A CN 102435598A CN 2011102623592 A CN2011102623592 A CN 2011102623592A CN 201110262359 A CN201110262359 A CN 201110262359A CN 102435598 A CN102435598 A CN 102435598A
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- 239000000758 substrate Substances 0.000 title claims abstract description 42
- 230000002255 enzymatic effect Effects 0.000 title abstract description 5
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000007800 oxidant agent Substances 0.000 claims abstract description 16
- 239000007788 liquid Substances 0.000 claims description 47
- 239000000126 substance Substances 0.000 claims description 25
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 238000004020 luminiscence type Methods 0.000 claims description 11
- 159000000000 sodium salts Chemical class 0.000 claims description 4
- 241001597008 Nomeidae Species 0.000 claims description 2
- 238000001514 detection method Methods 0.000 abstract description 7
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 abstract description 6
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 abstract description 5
- 239000012744 reinforcing agent Substances 0.000 abstract description 5
- 229960001922 sodium perborate Drugs 0.000 abstract description 5
- YKLJGMBLPUQQOI-UHFFFAOYSA-M sodium;oxidooxy(oxo)borane Chemical compound [Na+].[O-]OB=O YKLJGMBLPUQQOI-UHFFFAOYSA-M 0.000 abstract description 5
- 238000006911 enzymatic reaction Methods 0.000 abstract description 4
- 230000035484 reaction time Effects 0.000 abstract description 2
- 239000000891 luminescent agent Substances 0.000 abstract 3
- 239000000243 solution Substances 0.000 description 26
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 24
- 238000013016 damping Methods 0.000 description 16
- 239000012530 fluid Substances 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 11
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 7
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 7
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000004321 preservation Methods 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 6
- 235000010234 sodium benzoate Nutrition 0.000 description 6
- 239000004299 sodium benzoate Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- ZOPIBCDDKMAEII-UHFFFAOYSA-N 4-(1,2,4-triazol-1-yl)phenol Chemical compound C1=CC(O)=CC=C1N1N=CN=C1 ZOPIBCDDKMAEII-UHFFFAOYSA-N 0.000 description 4
- 238000003018 immunoassay Methods 0.000 description 4
- KQDJTBPASNJQFQ-UHFFFAOYSA-N 2-iodophenol Chemical compound OC1=CC=CC=C1I KQDJTBPASNJQFQ-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- -1 amino phthalylhydrazine Chemical compound 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000002038 chemiluminescence detection Methods 0.000 description 3
- 229960002163 hydrogen peroxide Drugs 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- RXNXLAHQOVLMIE-UHFFFAOYSA-N phenyl 10-methylacridin-10-ium-9-carboxylate Chemical compound C12=CC=CC=C2[N+](C)=C2C=CC=CC2=C1C(=O)OC1=CC=CC=C1 RXNXLAHQOVLMIE-UHFFFAOYSA-N 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003127 radioimmunoassay Methods 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002045 lasting effect Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Abstract
The invention discloses a stable HRP enzymatic enhanced chemiluminescent substrate solution. The stable enzymatic enhanced chemiluminescent substrate solution is composed of a luminescent agent solution A and an oxidizing agent solution B, which are mixed in volume ratios of 2:3-3:2. The luminescent agent solution A contains a luminescent agent: luminol or luminol derivatives, and a reinforcing agent: p-iodo-pheno, 4-(1, 2, 4-triazole-1-radical) phenol, etc., and the oxidizing agent solution B contains sodium perborate, hydrogen peroxide, etc. The stable HRP enzymatic enhanced chemiluminescent substrate solution of the invention is applied in an HRP enzymatic reaction with the characteristics of short reaction time and long platform period, can be stably preserved for 2 years at 4-8 EDG C, and is widely used in a kit using HRP as the detection object.
Description
Technical field
The present invention relates to the immunoassay field, more particularly, relating to a kind of is the chemical luminous substrate liquid of the detection architecture of enzymatic reaction with HRP.
Background technology
(chemiluminescence immunoassay is that chemiluminescence system is combined with immune system CLIA) to the chemical analysis immunization, and to detect antibody or antigen, it had both had immunoreactive specificity, had the high sensitivity of chemiluminescence reaction again.Its detailed process is with chemiluminescence reaction reagent (can be chemical illuminating reagent or catalyzer etc.) labelled antibody or antigen; Antibody behind the mark or antigen and determinand are measured determinand with the form that detects luminous intensity at last and are got content through a series of immune response and physics and chemistry step.It has overcome radiological hazard and the pollution problem of radioimmunoassay (RIA) because of using radioactive isotope to cause; Overcome required instrument complicacy in the fluoroimmunoassay (FIA), the shortcoming that background interference is big.With special advantages such as its high sensitivity, low instrument price, easy to use, safe, "dead" pollutions, extremely people's favor becomes an important directions of labelled immune.Wherein with acridinium ester (AE), alkaline phosphatase (ALP) and horseradish peroxidase (HRP) are main development trend for the chemiluminescence immune assay of label.
Luminol is one of chemiluminescence compound of understanding the earliest of people, and its luminescence mechanism and reaction conditions have much relations.Horseradish peroxidase can catalysis luminol-hydroperoxidation.Because luminol system chemiluminescence reaction has a conclusive oxidation step, horseradish peroxidase-labeled is on antigen or antibody the time, because the sterically hindered effect of protein macromolecule often makes oxidation step be suppressed; Luminol system chemiluminescence reaction need be carried out in strongly basic medium usually, and strong alkalescence can produce certain inhibiting effect to the activity of enzyme, and it is low therefore to utilize luminol-hydrogen peroxide-horseradish peroxidase directly to measure biomacromolecule sensitivity.
Until the eighties middle and later periods, people have found that successively some material (these materials are called as reinforcing agent) that can strengthen enzymatic luminol chemiluminescence system luminous intensity greatly just makes this technology promptly be applied to genetic analysis and field of immunodetection.In the presence of reinforcing agent, the luminous intensity of luminol can be enhanced 1000 times, and to make " background " that the time spent occurs separately luminous but also can reduce oxygenant and luminous agent etc. greatly.Compare with the system of original no reinforcing agent, the sensitivity that adds the luminol system of forming behind the reinforcing agent improves greatly, can fluorescent lifetime be extended to several hours simultaneously.Make could become lasting luminous that available sensitive film just can detect by detected instantaneous light emission with light-emitting appearance originally, these great improvement make this system be widely used in gene, immunoassay field.
Luminous substrate poor stability, luminous intensity on the market is low now, background is higher, and these have seriously limited the application that chemiluminescence immunoassay detects, and to these problems, we study its prescription.
Summary of the invention
The objective of the invention is to overcome the above-mentioned defective of conventional chemical luminous substrate on the market, obtain a kind of stable HRP enzyme-catalyzed chemical luminescence substrate liquid.
Technical scheme of the present invention is: a kind of stable HRP enzyme-catalyzed chemical luminescence substrate liquid; It is characterized in that; Mainly form, before use luminous agent solution A and oxidizing agent solution B are mixed with volume ratio 2: 3～3: 2 (optimum volume ratio is 1: 1) by luminous agent solution A and oxidizing agent solution B;
Said luminous solution A contains following component:
Said oxidizing agent solution B contains following component:
On the basis of such scheme, further optimize each component concentrations, confirm that optimal concentration is:
A liquid:
B liquid:
Said 4-(1-H-1,2,4-triazole) phenol (English name: 4-(1,2,4-Triazol-1-yl) phenol; CAS number is 68337-15-5; Structural formula
), molecular weight 161.16.
Said luminol derivant can be different luminol (the amino phthalylhydrazine of 4-), the amino hexyl of 4--N-ethyl different Shandong promise, luminol list sodium salt etc.; Said disodium EDTA, molecular formula C
10H
14N
2Na
2O
82 (H
2O), molecular weight is 372.23; Said sodium perborate molecular formula is NaBO
34H
2O, molecular weight are 153.88.
Know-why of the present invention is:
Above-mentioned reaction produces cold light, can be arrived by the chemiluminescence Equipment Inspection, and this light intensity becomes positive correlation with HRP (horseradish peroxidase) simultaneously, makes this detection system be widely used in the chemiluminescence detection with the HRP enzymatic reaction.
The invention has the beneficial effects as follows: the present invention is through after the performance test; Find that it has higher reaction stability, reaction reaches maximum emission intensity very soon, and a plateau is for a long time arranged afterwards; Compare with the luminous substrate liquid on the market, luminous intensity values is high and highly sensitive.The present invention is applied to the HRP enzymatic reaction and has the advantages that the reaction time is short, the detection platform phase is long, can stablize at 4-8 ℃ and preserve 2 years, and can be widely used in various is to detect in the kit of target with HRP.
Description of drawings
Fig. 1 is a HRP enzyme-catalyzed chemical luminescence kinetic curve.
Fig. 2 is the sensitivity gradient curve.
Fig. 3 be with market on the correlativity curve of like product contrast.
Embodiment
Below in conjunction with embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.
The preparation of embodiment 1 chemical luminous substrate liquid
The preparation of damping fluid: 0.1mol/L Tris-HCl damping fluid, pH8.4.0.01mol/L PBS damping fluid, pH7.4, concrete prescription are as follows:
A liquid (luminous agent solution):
B liquid (oxidizing agent solution):
The A liquid of chemical luminous substrate (luminous agent solution) 1000mL preparation can be adopted following method: earlier with 4-(1,2,4-triazole-1-yl) phenol 0.2417g, be dissolved in 10mL N, and in the dinethylformamide, mixing; Get luminol list sodium salt 0.1991g, to iodophenol 0.1100g, disodium EDTA 1.8610g, Sodium Benzoate 0.0144g, sodium chloride 0.1755g; Then 4-(1,2,4-triazole-1-yl) phenol solution and each material are added in the Tris-HCl damping fluid, and be settled to 1000ml with the Tris-HCl damping fluid, mixing is put in 4 ℃ of preservations.
B liquid (oxidizing agent solution) the 1000mL preparation of chemical luminous substrate can be adopted following method: the oxydol of getting 0.0567g30%; 0.769g sodium perborate, disodium EDTA 1.8610g, Sodium Benzoate 0.0144g; Add in the Tris-HCl damping fluid; And being settled to 1000ml with the Tris-HCl damping fluid, mixing is put in 4 ℃ of preservations.
Embodiment 2 enzyme-catalyzed chemical luminescence polymerization kinetics curves
Add 10 μ L HRP (10ng/mL) in the microwell plate respectively, every Kong Zhongzai adds the luminous substrate A liquid 50 μ L and the luminous substrate B liquid 50 μ L of preparation among the embodiment 1, detects luminous intensity (RLU) rapidly; Continue to detect 30 minutes, draw polymerization kinetics curves, the result sees accompanying drawing 1; As can beappreciated from fig. 1: the luminous substrate liquid of embodiment 1 preparation, the luminous intensity of reaction reaches maximal value about 3 minutes, and a long plateau is arranged afterwards; After detecting 30 minutes; Still keep very high luminous intensity, this has explained that luminous substrate liquid of the present invention has higher reaction stability, is applied to detection system and can effectively reduces error.
Embodiment 3 sensitivity studies
With the 1mg/mL HRP solution of newly preparing; Carry out doubling dilution with the PBS damping fluid; Select concentration from 100ng/mL to 0.001ng/mL, to detect; The detection step is: corresponding respectively 10 μ LPBS or the 10 μ L HRP (each detectable concentration) of adding in the microwell plate, in each hole, add luminous substrate A liquid 50 μ L and the luminous substrate B liquid 50 μ L that embodiment 1 prepares again, and place and detect RLU after 5 minutes.The parallel result who carries out detecting for three times representes with S/N, shown in table 1 and accompanying drawing 2.Can find out with Fig. 2 from table 1: the concentration of HRP becomes positive correlation with signal to noise ratio (S/N ratio); Along with reducing of HRP concentration; The luminous intensity signal to noise ratio (S/N ratio) is also in continuous decline, is ordinate with the logarithm of S/N, and the concentration system of HRP maps for horizontal ordinate; Each corresponding point has explained that the luminous substrate and the HRP of embodiment 1 preparation has stronger linear relationship almost point-blank.
The S/N value of table 1 variable concentrations HRP
Embodiment 4 THERMAL STABILITY
With 10 parts of luminous substrate liquid A liquid, the packing of B liquid equivalent of embodiment 1 preparation; Respectively get 5 parts respectively correspondence be positioned over 4 ℃, 37 ℃ and preserve down; Take out when placing 3 days, 5 days, 7 days, 10 days and 14 days and detect; The detection step is: corresponding respectively 10 μ L PBS or the 10 μ L HRP (concentration is 10ng/mL) of adding in the microwell plate, 4 ℃, 37 ℃ luminous substrate A liquid 50 μ L and luminous substrate B liquid 50 μ L that preserve down of corresponding adding in each hole place and detect RLU after 5 minutes again.The parallel result who carries out detecting for three times representes with S/N, and is as shown in table 2; Can find out from table 2: the luminous substrate liquid of embodiment 1 preparation has stability preferably, can infer this luminous substrate liquid in 4-8 ℃ of environment according to the equivalence experiment, can stablize and preserve 2 years.
Table 2 THERMAL STABILITY data
The correlativity of like product contrast on embodiment 5 and the market
The chemiluminescence detection substrate solution of chemical luminous substrate liquid described in the embodiment 1 and U.S. PIERCE company is compared.Prepare the carcinomebryonic antigen chemical luminescence reagent kit sold on the market; Use one of them detachable white luminous plate, be coated with antibody above, the enzyme conjugates solution of a correspondence; The standard items of six concentration, correspondence are respectively 5,10,20,40,80,160ng/mL.Concrete operation steps is following:
Step 2 is got rid of potpourri in the hole, washes plate machine distilled water and washes plate 5 times, does at the thieving paper arsis at last.
Step 3, the corresponding chemiluminescence detection substrate solution that adds mixed embodiment 1 chemical luminous substrate liquid of 100 μ L or U.S. PIERCE company in every hole, room temperature reaction detects luminous intensity RLU after 5 minutes.Testing result and related coefficient are shown in table 3 and accompanying drawing 3.
The chemical luminous substrate liquid testing result table of comparisons of table 3 and PIERCE company
Can find out with Fig. 3 from table 3: the luminous substrate liquid of embodiment 1 preparation has similar testing result with the like product on the market, and correlativity is higher, but luminous substrate liquid of the present invention connects measuring tool higher luminous intensity is arranged, and is more convenient for using.
In sum, chemical luminous substrate liquid of the present invention has the characteristic of efficient stable, is applicable to HRP to be the immune response that detects index.
Embodiment 6
A liquid (luminous agent solution):
B liquid (oxidizing agent solution):
The A liquid of chemical luminous substrate (luminous agent solution) 1000mL preparation can be adopted following method: earlier with 4-(1-H-1,2,4-triazole) phenol 0.1612g, be dissolved in 10mL N; In the dinethylformamide, mixing is got luminol list sodium salt 0.2987g, to iodophenol 0.1761g; Disodium EDTA 1.8610g, Sodium Benzoate 0.0144g, sodium chloride 0.1755g; Then 4-(1-H-1,2,4-triazole) phenol solution and each material are added in the Tris-HCl damping fluid; And being settled to 1000ml with the Tris-HCl damping fluid, mixing is put in 4 ℃ of preservations.
B liquid (oxidizing agent solution) the 1000mL preparation of chemical luminous substrate can be adopted following method: the oxydol of getting 0.0454g30%; 1.5388g sodium perborate, disodium EDTA 1.8610g, Sodium Benzoate 0.0144g; Add the Tris-HCl damping fluid; And being settled to 1000ml with the Tris-HCl damping fluid, mixing is put in 4 ℃ of preservations.
During use, A liquid is mixed with B liquid at 1: 1.
Embodiment 7
A liquid (luminous agent solution):
B liquid (oxidizing agent solution):
The A liquid of chemical luminous substrate (luminous agent solution) 1000mL preparation can be adopted following method: earlier with 4-(1-H-1,2,4-triazole) phenol 0.1934g, be dissolved in 10mL N; In the dinethylformamide, mixing is got different luminol 0.1952g, to iodophenol 0.0880g; Disodium EDTA 1.8610g, Sodium Benzoate 0.0144g, sodium chloride 0.1755g; Then 4-(1-H-1,2,4-triazole) phenol solution and each material are added in the Tris-HCl damping fluid; And being settled to 1000ml with the Tris-HCl damping fluid, mixing is put in 4 ℃ of preservations.
B liquid (oxidizing agent solution) the 1000mL preparation of chemical luminous substrate can be adopted following method: the oxydol of getting 0.1135g30%; 1.2310g sodium perborate, disodium EDTA 1.8610g, Sodium Benzoate 0.0144g; Add the Tris-HCl damping fluid; And being settled to 1000ml with the Tris-HCl damping fluid, mixing is put in 4 ℃ of preservations.
During use, A liquid is mixed with B liquid at 2: 3.
Claims (4)
1. a stable HRP enzyme-catalyzed chemical luminescence substrate liquid is characterized in that, mainly is made up of luminous agent solution A and oxidizing agent solution B, before use luminous agent solution A and oxidizing agent solution B is mixed with volume ratio in 2: 3～3: 2;
Said luminous solution A contains following component:
Said oxidizing agent solution B contains following component:
2. like right 1 described a kind of stable HRP enzyme-catalyzed chemical luminescence substrate liquid, it is characterized in that, before use luminous agent solution A and oxidizing agent solution B are mixed with volume ratio at 1: 1.
3. according to claim 1 or claim 2 a kind of stable HRP enzyme-catalyzed chemical luminescence substrate liquid is characterized in that,
Said luminous solution A contains following component:
Said oxidizing agent solution B contains following component:
4. according to claim 1 or claim 2 a kind of stable HRP enzyme-catalyzed chemical luminescence substrate liquid is characterized in that said luminol derivant is different luminol, 4-amino-ethyl-N-ethyl different Shandong promise or luminol list sodium salt.
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| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
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| CN103604918A (en) * | 2013-11-28 | 2014-02-26 | 中国科学院广州生物医药与健康研究院 | Luminescent substrate, use of luminescent substrate and detection kit containing luminescent substrate |
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| CN104049080A (en) * | 2014-06-27 | 2014-09-17 | 中国农业科学院农业质量标准与检测技术研究所 | Chemiluminescence sensibilization liquid and preparation method thereof |
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| CN105806830A (en) * | 2015-12-10 | 2016-07-27 | 北京联众泰克科技有限公司 | Stable HRP (Horseradish Peroxidase) enzyme-catalyzed chemiluminescence substrate solution as well as preparation method and application thereof |
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