A kind of HRP enzyme-catalyzed chemical luminescence substrate liquid of stabilizationTechnical field
The present invention relates to immunoassay field, in particular to a kind of change using HRP as the detection architecture of enzymatic reactionLearn luminous substrate liquid.
Background technology
Chemical analysis immunization (chemiluminescence immunoassay, CLIA) is by luminesceence analysis and is immunizedReaction is combined, and a kind of new detection trace antigen or the novel markings immuno analytical method of antibody set up, and it was bothSpecificity with immune response, the high sensitivity with chemiluminescence reaction.It overcomes radioimmunoassay (RIA) because makingWith radiological hazard caused by radio isotope and pollution problem；Overcome instrument needed for fluoroimmunoassay (FIA)Complexity, the big shortcoming of ambient interferences.With its high sensitivity, low instrument price, easy to use, safety, "dead" pollutionDeng the favor of unique advantage, extremely people, an important directions as labelled immune.Wherein with acridinium ester (AE), alkalescencePhosphatase (ALP) and the chemiluminescence immune assay that horseradish peroxidase (HRP) is label are Main Trends of The Development.
Luminol (Luminol), different luminol (Isoluminol) and its derivative are most early in making in chemoluminescence methodChemiluminescent substance.Luminol chemical name 3- amino phthalyl callositys, report its characteristics of luminescence by Albrecht earliest.This kind of material produces the intermediate of Alpha-hydroxy peroxide by aoxidizing, and the decomposition of the intermediate can release luminous energy, lightProperty it is relevant with the pH value of reaction system.The oxidation step of luminol class compound is relevant with solvent composition, in proton solventIn, it is necessary to enzyme come be catalyzed various oxygen derivative aoxidize luminol；In aprotic solvent, chemiluminescence only needs to oxygen and onePlant highly basic；And strong alkalescence can produce certain inhibitory action to the activity of enzyme, therefore utilize luminol-hydrogen peroxide-horseradishIt is low that peroxidase directly determines large biological molecule sensitivity.
Luminous substrate stability currently on the market generally is poor, background is higher；Luminous agent solution and oxidizing agent solution are mixedIt is extremely unstable after conjunction；Luminescent solution is colourless, is unfavorable for sample-adding, influences board-like chemiluminescence manual operations to influence the weight of experimentRenaturation, use cost is higher, and these seriously limit the application of chemiluminescence immunoassay detection, and for these problems, we are to itFormula is studied.
The content of the invention
It is an object of the invention to provide a kind of HRP enzyme-catalyzed chemical luminescence substrate liquid of stabilization, sent out with solving existing chemistryThe problems such as light substrate stability is poor, background is higher, result is inaccurate, cost is high.
The present invention uses following technical scheme：
A kind of HRP enzyme-catalyzed chemical luminescence substrate liquid of stabilization, including luminous agent solution and oxidizing agent solution, wherein, it is describedAgent solution light for the luminol containing final concentration of 0.5~10mmol/L or Derivative of Luminol or luminol sodium salt, 0.5~10mmol/L to iodophenol, 0.01~5mmol/L phenolphthalein, and pH be 8.5~10.5 buffer solution；
The oxidizing agent solution be the urea peroxide containing final concentration of 0.5~10mmol/L, mass concentration be 0.01~0.1% disodium ethylene diamine tetraacetate, volume ratio is 0.05~0.5% Tween-20, and its pH delays for 4.5~6.5 citric acidFliud flushing；
When in use, used after the luminous agent solution and the oxidizing agent solution being mixed.
HRP enzyme-catalyzed chemical luminescence substrate liquid as described above, it is preferable that the buffer solution of the luminous agent solution isCitrate buffer solution in 0.1mol/L~0.3mol/L boric acid-borate buffer solution, the oxidizing agent solution is final concentration of0.1mol/L~0.3mol/L.
HRP enzyme-catalyzed chemical luminescence substrate liquid as described above, it is preferable that the luminous agent solution is containing final concentration of1mmol/L luminol, 1~2mmol/L to iodophenol, 0.1~0.2mmol/L phenolphthalein, and 0.2mol/L boric acid-boronSand buffer solution, its pH is 9.0~10.0.
HRP enzyme-catalyzed chemical luminescence substrate liquid as described above, it is preferable that the oxidizing agent solution is containing final concentration of1mmol/L urea peroxide, mass concentration is 0.04% disodium ethylene diamine tetraacetate, and volume ratio is told for 0.1~0.25%Temperature -20,0.2mol/L citrate buffer solution, its pH is 5.0~6.0.
HRP enzyme-catalyzed chemical luminescence substrate liquid as described above, it is preferable that the oxidizing agent solution is also containing mass concentration0.01~0.1% ProClin 300.
HRP enzyme-catalyzed chemical luminescence substrate liquid as described above, it is preferable that the mass concentration of the ProClin 300 is0.05%.
HRP enzyme-catalyzed chemical luminescence substrate liquid as described above, it is preferable that before use, by the luminous agent solution and describedOxidizing agent solution by volume 1:Used after 1 mixing.
The HRP enzyme-catalyzed chemical luminescence substrate liquid that the present invention is provided, configuration is simple, and reinforcing agent, phenol are used as using to iodophenolPhthalein further improves the stability of luminescent solution as collaboration reinforcing agent, Tween-20 as stabilizer, obtains a kind of stabilization, heightSignal to noise ratio, highly sensitive, plateau length, aubergine HRP enzyme-catalyzed chemical luminescence substrate liquid.The HRP enzyme-catalyzed chemical luminescences1 day blank value is not raised after substrate solution mixing, signal value stabilization, and after performance test, it has higher reaction stability,Reaction can reach maximum emission intensity quickly, and have the plateau of a some time, the luminous substrate liquid with the market afterwardsContrasted, luminous intensity values are high and sensitivity is high.The present invention, which is applied to HRP enzymatic reactions, has that the reaction time is short, detection is flatThe characteristics of platform phase is long, 4-8 DEG C can stablize preserve 2 years, can be widely applied to it is various using HRP for detect target kit in.
The Chemoluminescent substrate cost of the present invention is low, and stability is good, and the mixed color of luminescent solution is aubergine,Relative to the easy-to-use discernable by eye luminescent solution dosage of Chemiluminescent plate of white, semi-automatic chemiluminescent behaviour by hand is particularly suitable for use inMake, reduce the operating mistake that manual operations is brought, phenolphthalein solution can be made to change colour additionally, due to pH changes, this luminescent solution can be according to faceIt is color identification reagent whether long bacterium, rotten, be prevented effectively from caused using failed reagent should not cost increase, and sample mistakeInspection rate.
Brief description of the drawings
Fig. 1 is fluorescent lifetime-two kinds of each period luminescent solution luminous value in one embodiment of the present invention relative to initialThe comparison diagram of the percentage of luminous value.
Fig. 2 is the comparison diagram of fluorescent lifetime-two kinds of luminescent solution signal to noise ratio of each period in one embodiment of the present invention.
Fig. 3 is used to detect gamma interferon concentration (LnX)-luminous value (LnY) for substrate solution in one embodiment of the present inventionCalibration graph.
Fig. 4 is the result figure in the embodiment of the present invention 4 by fluorescent lifetime-luminous value of each period drawing.
Fig. 5 be the embodiment of the present invention 4 in by fluorescent lifetime-two kinds of each period luminescent solution luminous value relative to initial luminousThe result figure of the per cents figure of value.
Embodiment
In the present invention using phenolphthalein as collaboration reinforcing agent, because phenolphthalein contains two phenolic hydroxyl groups；Tween-20 is as stableAgent, Tween-20 can ensure that oxidant slowly discharges.
Below with reference to the drawings and specific embodiments, the present invention will be described in detail, it will be appreciated that discussed belowThe technical scheme that is merely to illustrate and explain the present invention of embodiment, be not used to limitation of the invention.It is real belowIt is routinely experimental method to apply the experimental method in example unless otherwise instructed；Test reagent is commercially available purchase unless otherwise instructedProduct.
Embodiment 1
A kind of HRP enzyme-catalyzed chemical luminescence substrate liquid of stabilization, is respectively configured the luminous agent solutions of 1000mL and oxidizing agent solution,Wherein, the agent solution specific formula that lights is as follows：
Oxidizing agent solution specific formula is as follows：
In above-mentioned luminous agent solution, buffer solution used can use other conventional buffers systems, such as phosphate bufferDeng.
Before use, by the luminous agent solution and the oxidizing agent solution by volume 1:Used after 1 mixing.Above institute1 day blank value is not raised after the Chemoluminescent substrate mixing stated, signal value stabilization.
In order to ensure after mentioned reagent long-term storage, the stability of reagent can add mass concentration in oxidizing agent solutionFor 0.01~0.1% ProClin 300, this mass concentration has been effectively ensured the quality guarantee period of solution, has not also interfered with reagentReaction effect.
Embodiment 2
The present embodiment configuration Chemoluminescent substrate and oxidizing agent solution on the basis of implementing 1, specific formula are as follows：
Luminous agent solution in Chemoluminescent substrate：
Luminol 1mmol/L
Phenolphthalein 0.1mmol/L
To iodophenol 1.5mmol/L
0.2mol/L boric acid-borate buffer solution, with salt acid for adjusting pH value to 9.6.
Oxidizing agent solution in Chemoluminescent substrate：
0.2mol/L citrate buffer solutions, with salt acid for adjusting pH value to 5.0
By the luminous agent solution and oxidizing agent solution of above-mentioned configuration according to volume ratio be 1:After 1 mixing, luminous substrate is used asLiquid is determining 0.5 μ g/mL HRP with compareing the hypersensitive luminous substrate liquid of commercially available import ThermoFisher Pierce productionsStability (each period luminous value phase of signal to noise ratio (ratio of luminous value and background value) and 30 minutes luminous values in enzyme solutionsFor the percentage of initial luminescence values).
Specific steps：50 μ L concentration are added in luminescent screen for 0.5 μ g/mL HRP enzyme solutions, a hole are reserved not enzyme-added moltenLiquid respectively adds the luminous substrate liquid of above-mentioned preparation in the luminescent solution of commercially available import and the present invention each above as blank wellHole, is put into chemiluminescence detector reading, every 1 minute reading once.
By fluorescent lifetime-two kinds of each period luminescent solution luminous value relative to the per cents figure of initial luminescence values, paintMake result figure as shown in Figure 1；
Figure is drawn by fluorescent lifetime-two kinds of each period luminescent solution signal to noise ratio, as a result as shown in Figure 2.
It can be seen that in figure, the luminescent solution signal to noise ratio in the present embodiment is substantially better than import control luminescent solution, plateau is steadyIt is qualitative also slightly to compare luminescent solution better than import.
Embodiment 3
Using the Chemoluminescent substrate in embodiment 2, gamma interferon is detected applied to double-antibody method, wherein, antibodyThe antibody produced using Wuhan Hygiea Bioscience Co., Ltd..
Experimental procedure is as follows：
1st, it is loaded
(1) using calibration object dilution by gamma interferon calibration object reference material be diluted to 12.5pg/mL, 25pg/mL,50pg/mL, 100pg/mL, 200pg/mL, 400pg/mL totally 6 diluted concentrations, respectively take 50 μ L, are sequentially added into plate hole, put downRow does holes；The hole of blank control 1, blank control adds the μ L of calibration object dilution 50；
(2) the μ L of enzyme marking reagent 50 are added per hole；
2nd, incubate：It is gently mixed uniform, incubation 1 hour in 37 DEG C of constant incubator；
3rd, wash：The liquid in hole is got rid of, 300 μ L 1 × cleaning solution washing is added per hole, pats dry, is repeated 5 times；
4th, light：100 μ L luminous agent mixed liquors are added per hole；
5th, determine：In 20 minutes luminous value was read with chemical illumination immunity analysis instrument；
With the invention described above luminous substrate liquid gamma interferon concentration-luminous Value Data line of travel fitting：Ln(X)-Ln(Y), fit equation：Y=1.540x+3.584, wherein, coefficient R2=0.998.Substrate solution gamma interferon in the present embodimentConcentration (LnX)-luminous value (LnY) calibration graph, as shown in Figure 3.
As a result illustrate that reagent of the present invention is detected available for kit, good linearity.
Embodiment 4
On the basis of embodiment 2, luminous agent A1 (being free of phenolphthalein) is prepared, oxidant B1 (being free of Tween-20) is prepared, willLuminous agent, oxidant in embodiment 2 are labeled as luminous agent A, oxidant B.
Luminous agent A1 solution：
Luminol 1mmol/L
To iodophenol 1.5mmol/L
0.2mol/L boric acid-borate buffer solution, with salt acid for adjusting pH value to 9.6.
Oxidant B1 solution：
Urea peroxide 1mmol/L
Disodium ethylene diamine tetraacetate (EDTA) 0.04%
ProClin 300 0.05%
0.2mol/L citrate buffer solutions, with salt acid for adjusting pH value to 5.0
By luminous agent A and oxidant B, luminous agent A1 and oxidant B, luminous agent A and oxidant B1 difference 1:1 mixing, pointThe stabilization of the signal to noise ratio (ratio of luminous value and background value) and 30 minutes luminous values in 0.5 μ g/mL HRP enzyme solutions is not determinedProperty (percentage of each period luminous value relative to initial luminescence values).
Specific steps：50 μ L concentration are added in luminescent screen for 0.5 μ g/mL HRP enzyme solutions, a hole are reserved not enzyme-added moltenLiquid respectively adds the luminous substrate liquid of above-mentioned preparation in the luminescent solution of commercially available import and the present invention each above as blank wellHole, is put into chemiluminescence detector reading, every 1 minute reading once.
Drawn by fluorescent lifetime-luminous value of each period, drawn result figure such as Fig. 4；By fluorescent lifetime-each period twoLuminescent solution luminous value is planted relative to the per cents figure of initial luminescence values, drawn result figure such as Fig. 5.
Phenolphthalein is not added with as seen from Figure 4 as collaboration reinforcing agent, and luminescent solution luminous intensity is weaker.As seen from Figure 5, addStabilizer Tween-20, the luminous value stability of luminescent solution is significantly improved, and collaboration reinforcing agent equally has the work of stabilized illumination valueWith.