CN109799352A - It chemical illuminating reagent and its is applied in immune detection - Google Patents
It chemical illuminating reagent and its is applied in immune detection Download PDFInfo
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- CN109799352A CN109799352A CN201910084381.9A CN201910084381A CN109799352A CN 109799352 A CN109799352 A CN 109799352A CN 201910084381 A CN201910084381 A CN 201910084381A CN 109799352 A CN109799352 A CN 109799352A
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Abstract
It is applied the invention discloses chemical illuminating reagent and its in immune detection.Chemical illuminating reagent provided by the invention contains chlorogenic acid and chemiluminescent substrate, and the chemiluminescent substrate is luminol or luminol soluble-salt.The present invention passes through the chemiluminescence analysis of standard horseradish peroxidase enzyme catalytic luminol and hydrogen peroxide, it was demonstrated that the sensitivity of the chemical illuminating reagent detection horseradish peroxidase of the invention containing chlorogenic acid improves 100 times or more relative to the chemical illuminating reagent of the iodophenol containing 4-.Chemical illuminating reagent of the invention containing chlorogenic acid is used for protein immunoblot experiment, the detection sensitivity to target protein can be significantly improved.The present invention can be used for detecting trace of albumin in protein immunoblot.
Description
Technical field
It is applied the present invention relates to chemical illuminating reagent in biology and immunology and its in immune detection.
Background technique
In Biological Detection technology, immunology detection is in highly important status, utilizes antibody antigen reaction detection
Corresponding antigen-antibody concentration has the characteristics that high specific.However, classical characterized by antibody antigen directly reacts
The precipitation method, the sensitivity of agglutination detection antigen-antibody is low, accuracy is poor.Then researchers utilize peroxidase labelling the
Two antibody or streptavidin, by determined antigen in immune response or antibody signal conversion for convenience of the enzyme activity detected.Pass through
The amplification and good linear of enzyme reaction substantially increase the sensitivity and accuracy of detection.Horseradish peroxidase is a kind of steady
Qualitative peroxidase high, catalytic capability is strong.It is commonly used to label secondary antibody or Streptavidin, in being immunoreacted
Determined antigen or antibody signal are converted to the enzyme activity that can detecte.
The redox reaction of horseradish peroxidase enzyme catalytic can quantitative determine enzyme activity, common horseradish mistake
Oxide zymolyte is chromogenic substrate diaminobenzidine and tetramethyl benzidine.Horseradish peroxidase enzyme catalytic peroxidating hydrogen-oxygen
Change diaminobenzidine and generate the insoluble product of brown, suitable for protein immunoblot detection and Immunohistochemical detection；It is peppery
Root Catalyzed Synthesis By Peroxidase tetramethyl benzidine generates blue product and becomes stable yellow substance in acid condition, can be with
By absorbance Accurate Determining product formation and corresponding enzyme activity, it is suitable for MBP enzyme linked immuno-adsorbent assay.However this
The chromogenic substrate of a little horseradish peroxidases still has the critical defects such as detection sensitivity is low, quantitative linearity scope is narrow.Research
Persons have found the sensitivity and linear measurement range that horseradish peroxidase detection can be significantly improved using chemiluminescent substrate, then
Various chemiluminescent substrates are gradually developed.It is based particularly on the redox substrate of luminol and hydrogen peroxide, by adding
Enter the phenols such as 4- iodophenol chemiluminescence intensifier (Pure and Applied Chemistry, 1987,59 (5), 651-
654) enzyme activity of horseradish peroxidase, can be detected by chemiluminescence reaction well.
In protein immunoblot experiment, the albumen for needing to detect all is much trace of albumin.The increasing of existing market sale
The sensitivity of extensive chemical luminescent solution is not still able to satisfy the needs of protein immunoblot experiment.
Summary of the invention
The technical problem to be solved by the present invention is to how enhance horseradish peroxidase enzyme catalytic luminol and hydrogen peroxide
Chemiluminescence intensity in reaction process.
In order to solve the above-mentioned technical problems, the present invention provides chemical illuminating reagents.
Chemical illuminating reagent provided by the present invention is chemical luminous composite, and the chemical illuminating reagent contains chlorogenic acid
And chemiluminescent substrate, the chemiluminescent substrate are luminol or luminol soluble-salt.Chemical illuminating reagent of the invention
Alternatively referred to as containing the chemical illuminating reagent of chlorogenic acid.
In above-mentioned chemical illuminating reagent, the luminol soluble-salt can be luminol sodium salt.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent can be made of chlorogenic acid and the chemiluminescent substrate.
In the chemical illuminating reagent, the chlorogenic acid and the chemiluminescent substrate can be packed individually, can also be mixed.It should
In chemical illuminating reagent, the proportion of the chlorogenic acid and the chemiluminescent substrate can be 0.1-20.0mmol chlorogenic acid:
Chemiluminescent substrate described in 2.5mmol, 0.5-2.0mmol chlorogenic acid: chemiluminescent substrate or 1.0- described in 2.5mmol
2.0mmol chlorogenic acid: chemiluminescent substrate described in 2.5mmol.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent is also other needed for chemiluminescence reaction containing carrying out
Substance, such as trishydroxymethylaminomethane and/or hydrochloric acid and/or water.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent can be by chlorogenic acid, the chemiluminescent substrate and following
At least one material composition: trishydroxymethylaminomethane, hydrochloric acid and water.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent can also contain hydrogen peroxide.The chemical illuminating reagent
It can be made of chlorogenic acid, the chemiluminescent substrate and hydrogen peroxide.In the chemical illuminating reagent, hydrogen peroxide is individually packed,
Chlorogenic acid and the chemiluminescent substrate can be packed individually, can also be mixed.In the chemical illuminating reagent, green original
Sour, the described chemiluminescent substrate and the proportion of hydrogen peroxide can be 0.1-20.0mmol chlorogenic acid: chemiluminescence described in 2.5mmol
Substrate: 6mmol hydrogen peroxide, 0.5-2.0mmol chlorogenic acid: chemiluminescent substrate described in 2.5mmol: 6mmol hydrogen peroxide, or
1.0-2.0mmol chlorogenic acid: chemiluminescent substrate described in 2.5mmol: 6mmol hydrogen peroxide.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent is also other needed for chemiluminescence reaction containing carrying out
Substance, such as trishydroxymethylaminomethane and/or hydrochloric acid and/or water.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent can be by chlorogenic acid, the chemiluminescent substrate, peroxidating
Hydrogen and following at least one material compositions: trishydroxymethylaminomethane, hydrochloric acid and water.
In above-mentioned chemical illuminating reagent, the chemical illuminating reagent can be the reagent detected for chemiluminescence immunoassay.
In practical applications, the chemical illuminating reagent can be made of A liquid and B liquid, and A liquid can be to be made of solute and solvent
PH be 8.6 aqueous solution, solvent is water, solute and its concentration be respectively 0.1M trishydroxymethylaminomethane, 0.03M hydrochloric acid and
2.5mM luminol sodium salt, 0.1-20.0mM chlorogenic acid.B liquid can be the aqueous solution for being 8.6 by the pH that solute and solvent form, molten
Matter and its concentration are respectively 0.1M trishydroxymethylaminomethane, 0.03M hydrochloric acid and 6mM hydrogen peroxide, and solvent is water.A liquid and B liquid
It is used in mixed way in equal volume.
In above-mentioned chemical illuminating reagent, chemical illuminating reagent of the invention (chemical illuminating reagent containing chlorogenic acid) with contain 4-
The chemical illuminating reagent of iodophenol is compared, and the change during horseradish peroxidase enzyme catalytic luminol and hydroperoxidation is enhanced
Learn 114 times of luminous intensity.The change of chemical illuminating reagent (chemical illuminating reagent containing chlorogenic acid) and the iodophenol containing 4- of the invention
Luminescence reagent is learned to compare in composition, other than equimolar 4- iodophenol is replaced with equimolar chlorogenic acid, other components
And its content is all the same.
Following any applications also belong to protection scope of the present invention:
U0, chlorogenic acid or the chemical illuminating reagent detect (chemiluminescence in chemiluminescence detection or chemiluminescence immunoassay
Immunoassay, as protein immunoblot detect) in application；
U01, chlorogenic acid are preparing the application of application or chlorogenic acid as chemiluminescence intensifier in chemical illuminating reagent；
U1, chlorogenic acid and the chemiluminescent substrate are preparing the application in chemical illuminating reagent；
U2, chlorogenic acid, the chemiluminescent substrate and hydrogen peroxide are preparing the application in chemical illuminating reagent；
U3, chlorogenic acid, the chemiluminescent substrate and following at least one substances are preparing answering in chemical illuminating reagent
With: trishydroxymethylaminomethane and/or hydrochloric acid and/or water；
U4, chlorogenic acid, the chemiluminescent substrate, hydrogen peroxide and following at least one substances are in preparation chemiluminescence examination
Application in agent: trishydroxymethylaminomethane and/or hydrochloric acid and/or water；
U5, chlorogenic acid and hydrogen peroxide are preparing the application in chemical illuminating reagent；
U6, chlorogenic acid and trishydroxymethylaminomethane are preparing the application in chemical illuminating reagent；
U7, chlorogenic acid and hydrochloric acid are preparing the application in chemical illuminating reagent.
In above-mentioned application, the chemical illuminating reagent can be above-mentioned chemical illuminating reagent.The chemiluminescence intensifier can
For the reagent of the chemiluminescence intensity during enhancing horseradish peroxidase enzyme catalytic luminol and hydroperoxidation.
In above-mentioned application, chlorogenic acid enhances horseradish peroxidase enzyme catalytic luminol and peroxidating compared with 4- iodophenol
114 times of chemiluminescence intensity in hydrogen reaction process.
The present invention is screened by a large amount of luminol chemiluminescence humidification, and discovery chlorogenic acid has very strong chemistry hair
Photo-enhancement effect can greatly improve the detection sensitivity of protein immunoblot experiment.The present invention passes through standard horseradish peroxide
The chemiluminescence analysis of compound enzymatic luminol and hydrogen peroxide, it was demonstrated that the chemical illuminating reagent inspection of the invention containing chlorogenic acid
The sensitivity for surveying horseradish peroxidase improves 100 times or more relative to the chemical illuminating reagent of the iodophenol containing 4-.It will be of the invention
The chemical illuminating reagent containing chlorogenic acid be used for protein immunoblot experiment, the detection that can be significantly improved to target protein is sensitive
Degree.The present invention can be used for detecting trace of albumin in protein immunoblot.
Detailed description of the invention
Fig. 1 is that the enhanced chemiluminescence liquid of the iodophenol containing 4- and the enhanced chemiluminescence liquid containing chlorogenic acid are used for protein immunization
The result of trace detection.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Examples provided below can be used as the art ordinary skill
The guide that personnel are further improved, is not construed as limiting the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Material as used in the following examples
Material, reagent etc., are commercially available unless otherwise specified.
Reagent: trishydroxymethylaminomethane (article No.: T110600) is purchased from the limited public affairs of Shanghai Aladdin biochemical technology share
Department；Luminol sodium salt (article No.: V900354), chlorogenic acid (article No.: C3878), hydrogen peroxide (article No.: 88597) and 4- iodophenol
(article No.: I10201) is purchased from Sigma-Aldrich；Other chemical reagent are purchased from Beijing Chemical Plant.The training of DMEM cell
Base and fetal calf serum are supported, U.S. Corning company is purchased from.Protein electrophoresis related reagent has up to biotechnology purchased from Beijing one hundred is auspicious
Limit company.
Consumptive material: white 96 orifice plates (article No.: T110600) and common centrifuge tube and suction pipette head are purchased from U.S. Corning
Company.Polyvinylidene fluoride (PVDF) film is purchased from Merck KGaA company；Tissue Culture Flask, centrifuge tube and suction pipette head, are purchased from
Corning company, the U.S..
Instrument: Enspire Multimode Plate Reader is purchased from platinum Ai Ermo company, Germany.Protein electrophoresis phase
Equipment is closed, Bio Rad Laboratories is purchased from；4000 chemiluminescence imaging instrument of ImageQuant LAS is purchased from U.S. GE company.
Cell: RAW264.7 cell is purchased from Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences's cell centre.
Chemistry during embodiment 1, chlorogenic acid enhancing horseradish peroxidase enzyme catalytic luminol and hydroperoxidation
Luminous intensity
9 kinds of chemical illuminating reagents are present embodiments provided, this 9 kinds of chemical illuminating reagents are made of A liquid and B liquid.This 9 kinds
The B liquid phase of chemical illuminating reagent is same, and only A liquid is different.The B liquid of this 9 kinds of chemical illuminating reagents is made of solute and solvent
PH be 8.6 aqueous solution, solute and its concentration are respectively 0.1M trishydroxymethylaminomethane, 0.03M hydrochloric acid and 6mM peroxidating
Hydrogen, solvent are water.
This 9 kinds of chemical illuminating reagents be respectively 0mM chlorogenic acid chemical illuminating reagent, 0.1mM chlorogenic acid chemical illuminating reagent,
0.2mM chlorogenic acid chemical illuminating reagent, 0.5mM chlorogenic acid chemical illuminating reagent, 1.0mM chlorogenic acid chemical illuminating reagent, 2.0mM
Chlorogenic acid chemical illuminating reagent, 5.0mM chlorogenic acid chemical illuminating reagent, 10.0mM chlorogenic acid chemical illuminating reagent and 20.0mM are green
Ortho acid chemical illuminating reagent.
The A liquid of 0mM chlorogenic acid chemical illuminating reagent is 0mM solution of chlorogenic acid (control).0mM chlorogenic acid chemiluminescence examination
The A liquid of agent is the aqueous solution for being 8.6 by the pH that solute and solvent form, and solute and its concentration are respectively 0.1M trihydroxy methyl amino
Methane, 0.03M hydrochloric acid and 2.5mM luminol sodium salt, solvent are water.
The aqueous solution that the A liquid of 0.1mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 0.1mM chlorogenic acid chemical illuminating reagent is
0.1mM。
The aqueous solution that the A liquid of 0.2mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 0.2mM chlorogenic acid chemical illuminating reagent is
0.2mM。
The aqueous solution that the A liquid of 0.5mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 0.5mM chlorogenic acid chemical illuminating reagent is
0.5mM。
The aqueous solution that the A liquid of 1.0mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 1.0mM chlorogenic acid chemical illuminating reagent is
1.0mM。
The aqueous solution that the A liquid of 2.0mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 2.0mM chlorogenic acid chemical illuminating reagent is
2.0mM。
The aqueous solution that the A liquid of 5.0mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 5.0mM chlorogenic acid chemical illuminating reagent is
5.0mM。
The aqueous solution that the A liquid of 10.0mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 10.0mM chlorogenic acid chemical illuminating reagent
It is 10.0mM.
The aqueous solution that the A liquid of 20.0mM chlorogenic acid chemical illuminating reagent is 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is above-mentioned 0mM solution of chlorogenic acid.Chlorogenic acid content in the A liquid of 20.0mM chlorogenic acid chemical illuminating reagent
It is 20.0mM.
The above-mentioned 9 kinds of chemical illuminating reagents of horseradish peroxidase enzyme catalytic are tested respectively according to following chemiluminescence analysis method
It carries out the chemiluminescence intensity of chemiluminescence reaction: A liquid and B liquid is mixed in equal volume, be then drawn in white 96 orifice plates,
100 holes μ l/, are then added the horseradish peroxidase solution of 0.001U/ml, and 10 holes μ l/ are sufficiently mixed rear chamber room temperature reaction 5
Minute, microplate reader detects chemiluminescence intensity (relative light unit, Relative light unit, RLU).
Chemiluminescence analysis the result shows that horseradish peroxidase be catalyzed respectively 0.1mM chlorogenic acid chemical illuminating reagent,
0.2mM chlorogenic acid chemical illuminating reagent, 0.5mM chlorogenic acid chemical illuminating reagent, 1.0mM chlorogenic acid chemical illuminating reagent, 2.0mM
Chlorogenic acid chemical illuminating reagent, 5.0mM chlorogenic acid chemical illuminating reagent, 10.0mM chlorogenic acid chemical illuminating reagent and 20.0mM are green
The chemiluminescence intensity that ortho acid chemical illuminating reagent carries out chemiluminescence reaction is the green original of horseradish peroxidase enzyme catalytic 0mM respectively
Sour chemical illuminating reagent carries out 3660 times (183/0.05=3660) of the chemiluminescence intensity of chemiluminescence reaction, 7120 times
(356/0.05=7120), 9760 times (488/0.05=9760), 10760 times (538/0.05=10760), 10240 times (512/
0.05=10240), 8560 times (428/0.05=8560), 7060 times (353/0.05=7060) and 4460 times of (223/0.05=
4460).Horseradish peroxidase enzyme catalytic luminol and peroxidating can be significantly improved by illustrating that chlorogenic acid is added in chemical luminescence for liquid
The chemiluminescence intensity of hydrogen reaction, makes chemiluminescence intensity improve 3660-10760 times.Experiment is repeated 3 times, each each place
Manage 3 holes.
The chemiluminescence that the above-mentioned 9 kinds of chemical illuminating reagents of 1. horseradish peroxidase enzyme catalytic of table carry out chemiluminescence reaction is strong
Degree
Note: RLU value is indicated by mean+SD, utilizes One way ANOVA and Dunnett ' s post hoc
Test is for statistical analysis.* P < 0.05vs 0mM chlorogenic acid chemical illuminating reagent；#P < 0.05vs 1.0mM chlorogenic acid chemistry hair
Light reagent.
Embodiment 2 compares chlorogenic acid and 4- iodophenol using horseradish peroxidase standard items to luminol chemiluminescence
Humidification
3 kinds of chemical illuminating reagents are present embodiments provided, the enhancing of chemical luminescence for liquid, the iodophenol containing 4- is respectively compareed
Learn luminescent solution and the enhanced chemiluminescence liquid containing chlorogenic acid.This 3 kinds of chemical illuminating reagents are made of A liquid and B liquid.This 3 kinds changes
The B liquid phase for learning luminescence reagent is same, and only A liquid is different.What the B liquid of this 3 kinds of chemical illuminating reagents was made of solute and solvent
The aqueous solution that pH is 8.6, solute and its concentration are respectively 0.1M trishydroxymethylaminomethane, 0.03M hydrochloric acid and 6mM peroxidating
Hydrogen, solvent are water.
The A liquid for compareing chemical luminescence for liquid is the aqueous solution for being 8.6 by the pH that solute and solvent form, solute and its concentration point
Not Wei 0.1M trishydroxymethylaminomethane, 0.03M hydrochloric acid and 2.5mM luminol sodium salt, solvent be water.
The A liquid of the enhanced chemiluminescence liquid of the iodophenol containing 4- is the aqueous solution for being 8.6 by the pH that solute and solvent form, molten
Matter is 4- iodophenol, and solvent is the A liquid of above-mentioned control chemical luminescence for liquid, this contains the A liquid of the enhanced chemiluminescence liquid of 4- iodophenol
The content of middle 4- iodophenol is 1mM.(note: 1-5mM 4- iodobenzene phenol solution can play maximum chemical luminescence enhancement to luminol and make
With in order to compare its effect with chlorogenic acid, selection 1mM concentration 4- iodophenol prepares enhanced chemiluminescence liquid).
The A liquid of enhanced chemiluminescence liquid containing chlorogenic acid is the aqueous solution for being 8.6 by the pH that solute and solvent form, solute
For chlorogenic acid, solvent is the A liquid of above-mentioned control chemical luminescence for liquid.Green original in the A liquid of the enhanced chemiluminescence liquid containing chlorogenic acid
The content of acid is 1mM.
The above-mentioned 3 kinds of chemical illuminating reagents of horseradish peroxidase enzyme catalytic are tested respectively according to following chemiluminescence analysis method
It carries out the chemiluminescence intensity of chemiluminescence reaction: A liquid and B liquid is mixed in equal volume, be then drawn in white 96 orifice plates,
100 holes μ l/, are then added the horseradish peroxidase solution of 0.001U/ml, and 10 holes μ l/ are sufficiently mixed rear chamber room temperature reaction 5
Minute, microplate reader detects chemiluminescence intensity (relative light unit, Relative light unit, RLU).Experiment is repeated 3 times,
Each 3 holes of each processing.
Chemiluminescence analysis the result shows that horseradish peroxidase is catalyzed the enhanced chemiluminescence of the iodophenol containing 4- respectively
Liquid and the chemiluminescence intensity of the enhanced chemiluminescence liquid progress chemiluminescence reaction containing chlorogenic acid are horseradish peroxidase respectively
Enzymatic compare chemical luminescence for liquid carry out chemiluminescence reaction chemiluminescence intensity 1205 times (4.82/0.04=1205) and
13800 times (552/0.04=13800), enhanced chemiluminescence liquid of the horseradish peroxidase enzyme catalytic containing chlorogenic acid carries out chemical hair
The chemiluminescence intensity of light reaction is that the enhanced chemiluminescence liquid of horseradish peroxidase enzyme catalytic iodophenol containing 4- carries out chemical hair
115 times (552/4.82=115) (tables 2) of the chemiluminescence intensity of light reaction.Illustrate that chemistry is added in 4- iodophenol and chlorogenic acid
It can improve the chemiluminescence intensity of horseradish peroxidase enzyme catalytic luminol and hydroperoxidation in luminescent solution, and green original
The opposite 4- iodophenol of acid, the enhancement of chemiluminescence improve 100 times or more (tables 2).
The chemiluminescence that the above-mentioned 2 kinds of chemical illuminating reagents of 2. horseradish peroxidase enzyme catalytic of table carry out chemiluminescence reaction is strong
Degree
| Chemical illuminating reagent | RLU(×106) |
| Compare chemical luminescence for liquid | 0.04±0.01* |
| The enhanced chemiluminescence liquid of the iodophenol containing 4- | 4.82±0.09 |
| Enhanced chemiluminescence liquid containing chlorogenic acid | 552±15* |
Note: RLU value is indicated by mean+SD, utilizes Student ' s t test for statistical analysis.*P<
The enhanced chemiluminescence liquid of 0.05vs iodophenol containing 4-.
Embodiment 3 compares chlorogenic acid and 4- iodophenol using protein immunoblot experiment to luminol chemiluminescence enhancing
Effect
1,000,000 RAW264.7 cells for being in logarithmic growth phase are collected, are cracked using 4 DEG C of RIPA moderate strength lysate
Cell 30 minutes, 10000g was centrifuged 10 minutes collection albumen supernatants, and sample-loading buffer is then added and incubates in 98 DEG C of metal baths
It educates 10 minutes and prepares albuminate.Sample loading (5 parallel holes) is subjected to protein electrophoresis into 10% polyacrylamide gel,
Albumen electricity is gone on pvdf membrane after electrophoresis.Using 5% skimmed milk power TBS solution by film close 1 hour, then 4 DEG C with
Rat anti-mouse β-actin antibody incubation is stayed overnight, after the washing of TBST solution, the goat of room temperature and horseradish peroxidase-labeled
Anti- rat IgG is incubated for 2 hours, then after the washing of TBST solution, is utilized respectively the enhancing chemistry hair of the iodophenol containing 4- of embodiment 2
Light liquid and containing chlorogenic acid enhanced chemiluminescence liquid exposure.Wherein, by the A liquid and B liquid of two kinds of enhanced chemiluminescence liquid before exposure
Isometric mixing, is then drawn on pvdf membrane, selects auto exposure mode, saves exposure image and record time for exposure.
Protein immunoblot experiment is analyzed the result shows that the enhanced chemiluminescence liquid phase containing chlorogenic acid of embodiment 2 is for reality
Apply the iodophenol containing 4- of example 2 enhanced chemiluminescence liquid can more Sensitive Detection go out destination protein (β-actin), exposure band more
Slightly, the time for exposure is shorter (Fig. 1).Upper figure is to expose 5 using the enhanced chemiluminescence liquid of the iodophenol containing 4- of embodiment 2 in Fig. 1
Second protein immunoblot testing result, the following figure is the enhanced chemiluminescence liquid exposure containing chlorogenic acid for utilizing embodiment 2 in Fig. 1
0.5 second protein immunoblot testing result.
The present invention is had been described in detail above.To those skilled in the art, do not depart from spirit of the invention and
Range, and without carrying out under unnecessary experimental conditions, can synchronization parameters, concentration and under the conditions of, it is real in a wider range
Apply the present invention.Although The present invention gives particular embodiments, it is understood that, the present invention can be improved further.
In short, pressing the principle of the present invention, the application is intended to include any change, purposes or improvement of the present invention, including departing from this Shen
Please in the open scope, and the change carried out with routine techniques known in the art.By the range of following attached claims,
It can carry out the application of some essential characteristics.
Claims (10)
1. chemical illuminating reagent, it is characterised in that: the chemical illuminating reagent contains chlorogenic acid and chemiluminescent substrate, describedization
Learning luminous substrate is luminol or luminol soluble-salt.
2. chemical illuminating reagent according to claim 1, it is characterised in that: the chemical illuminating reagent is by chlorogenic acid and institute
State chemiluminescent substrate composition.
3. chemical illuminating reagent according to claim 1, it is characterised in that: the chemical illuminating reagent is by chlorogenic acid, institute
State chemiluminescent substrate and following at least one material compositions: trishydroxymethylaminomethane, hydrochloric acid and water.
4. chemical illuminating reagent according to claim 1, it is characterised in that: the chemical illuminating reagent also contains peroxidating
Hydrogen.
5. chemical illuminating reagent according to claim 1 or 4, it is characterised in that: the chemical illuminating reagent by chlorogenic acid,
The chemiluminescent substrate and hydrogen peroxide composition.
6. chemical illuminating reagent according to claim 1 or 4, it is characterised in that: the chemical illuminating reagent also contains three
Hydroxymethyl aminomethane and/or hydrochloric acid and/or water.
7. according to claim 1, chemical illuminating reagent described in 4 or 6, it is characterised in that: the chemical illuminating reagent is by green original
Sour, the described chemiluminescent substrate, hydrogen peroxide and following at least one material compositions: trishydroxymethylaminomethane, hydrochloric acid and water.
8. chemical illuminating reagent described in any claim is in chemiluminescence detection or chemistry in chlorogenic acid or claim 1-7
Application in electrochemiluminescent immunoassay detection.
9. chlorogenic acid is preparing the application of application or chlorogenic acid as chemiluminescence intensifier in chemical illuminating reagent.
10. following any applications:
U1, chlorogenic acid and chemiluminescent substrate are Rumi preparing the application in chemical illuminating reagent, the chemiluminescent substrate
Promise or luminol soluble-salt；
U2, chlorogenic acid, chemiluminescent substrate and hydrogen peroxide are preparing the application in chemical illuminating reagent, the chemiluminescence bottom
Object is luminol or luminol soluble-salt；
U3, chlorogenic acid, chemiluminescent substrate and C are Shandong preparing the application in chemical illuminating reagent, the chemiluminescent substrate
Minot or luminol soluble-salt, the C are trishydroxymethylaminomethane and/or hydrochloric acid and/or water；
U4, chlorogenic acid, chemiluminescent substrate, hydrogen peroxide and C are preparing the application in chemical illuminating reagent, the chemiluminescence
Substrate is luminol or luminol soluble-salt, and the C is trishydroxymethylaminomethane and/or hydrochloric acid and/or water；
U5, chlorogenic acid and hydrogen peroxide are preparing the application in chemical illuminating reagent；
U6, chlorogenic acid and trishydroxymethylaminomethane are preparing the application in chemical illuminating reagent；
U7, chlorogenic acid and hydrochloric acid are preparing the application in chemical illuminating reagent.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2023207221A1 (en) * | 2022-04-29 | 2023-11-02 | 广州中医药大学(广州中医药研究院) | Luminol derivative, method for preparing same, and use thereof |
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