CN113567679A - Hypersensitive ECL luminescent liquid reagent and using method thereof - Google Patents
Hypersensitive ECL luminescent liquid reagent and using method thereof Download PDFInfo
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- CN113567679A CN113567679A CN202110681650.7A CN202110681650A CN113567679A CN 113567679 A CN113567679 A CN 113567679A CN 202110681650 A CN202110681650 A CN 202110681650A CN 113567679 A CN113567679 A CN 113567679A
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 96
- 239000007788 liquid Substances 0.000 title claims abstract description 51
- 206010020751 Hypersensitivity Diseases 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 19
- NGSWKAQJJWESNS-UHFFFAOYSA-N 4-coumaric acid Chemical compound OC(=O)C=CC1=CC=C(O)C=C1 NGSWKAQJJWESNS-UHFFFAOYSA-N 0.000 claims abstract description 24
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000004020 luminiscence type Methods 0.000 claims abstract description 20
- 239000007790 solid phase Substances 0.000 claims abstract description 14
- FYFFGSSZFBZTAH-UHFFFAOYSA-N methylaminomethanetriol Chemical compound CNC(O)(O)O FYFFGSSZFBZTAH-UHFFFAOYSA-N 0.000 claims abstract description 13
- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000000243 solution Substances 0.000 claims description 31
- 208000026935 allergic disease Diseases 0.000 claims description 20
- 230000009610 hypersensitivity Effects 0.000 claims description 20
- 239000012046 mixed solvent Substances 0.000 claims description 10
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 10
- 108090000623 proteins and genes Proteins 0.000 abstract description 32
- 102000004169 proteins and genes Human genes 0.000 abstract description 31
- 238000011161 development Methods 0.000 abstract description 16
- 238000011534 incubation Methods 0.000 abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 6
- 235000018102 proteins Nutrition 0.000 description 19
- 239000007983 Tris buffer Substances 0.000 description 13
- 238000001262 western blot Methods 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000002998 immunogenetic effect Effects 0.000 description 1
- 230000036046 immunoreaction Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000010023 transfer printing Methods 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
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Abstract
The invention discloses a hypersensitive ECL luminescence liquid reagent and a using method thereof, wherein the hypersensitive ECL luminescence liquid reagent comprises reagent liquid A and reagent liquid B, the reagent liquid A comprises trihydroxymethyl aminomethane, luminol and p-hydroxycinnamic acid, the reagent liquid B comprises trihydroxymethyl aminomethane and hydrogen peroxide, when the hypersensitive ECL luminescence liquid reagent is used, the reagent liquid A and the reagent liquid B are mixed according to a proportion, the mass-to-volume ratio of the trihydroxymethyl aminomethane in the reagent A is 12g/L, the mass-to-volume ratio of the luminol is 100mg/L, and the mass-to-volume ratio of the p-hydroxycinnamic acid is 0.21 g/L; the mass volume ratio of the trihydroxymethyl aminomethane in the reagent B is 12g/L, and the volume ratio of the hydrogen peroxide is 750 mu L/L; the sensitivity is high, and a protein band with the protein content of nanogram grade can be displayed; incubation before luminescence and color development is not needed; the background of the chromogenic solid phase carrier is clean, other miscellaneous points do not exist, and the problem of protein chromogenic that some protein expression quantity is low or the binding capacity of an antibody and protein is weak is solved.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a hypersensitive ECL luminescence liquid reagent and a using method thereof.
Background
Western Blot experiment, i.e., Western Blot experiment, is a common experimental method in molecular biology, biochemistry and immunogenetics, and its basic principle is a method of staining a cell or biological tissue sample during gel electrophoresis treatment by a specific antibody, and obtaining information on the expression of a specific protein in the analyzed cell or tissue by analyzing the location and depth of staining. The method can be divided into 3 stages of electrophoresis, transfer printing and enzyme immunoassay, a protein sample separated by PAGE (polyacrylamide gel electrophoresis) is transferred to a solid phase carrier (such as a nitrocellulose membrane), the solid phase carrier adsorbs protein in a non-covalent bond form, the type and the biological activity of the polypeptide separated by electrophoresis can be kept unchanged, the protein or the polypeptide on the solid phase carrier is taken as an antigen, immunoreaction is carried out with a corresponding antibody, then the antibody reacts with a second antibody marked by enzyme, and the protein component expressed by a specific target gene separated by electrophoresis is detected by substrate coloration;
at present, the luminescence developing solution generally adopted in Western Blot experiment is ECL luminescence solution, however, for some proteins with less expression quantity, the sensitivity is relatively low or almost not used, so that some proteins with lower protein content can not be developed. The problem of color development for this class of proteins is not currently solved.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a hypersensitive ECL luminescent liquid reagent and a using method of the hypersensitive ECL luminescent liquid reagent, the color development speed is high, and a large amount of time can be saved; the sensitivity is high, and a protein band with the protein content of nanogram grade can be displayed; incubation before luminescence and color development is not needed; the chromogenic solid phase carrier has a cleaner background and no other miscellaneous points, solves the problem of protein chromogenic that the expression quantity of some proteins is low or the binding capacity of antibodies and the proteins is weak, and is used for solving the defects caused by the prior art.
In order to solve the technical problems, the invention provides the following technical scheme:
in a first aspect, the hypersensitivity ECL luminous liquid reagent comprises reagent liquid a and reagent liquid B, wherein the reagent liquid a comprises tris, luminol and p-hydroxycinnamic acid, and the reagent liquid B comprises tris and hydrogen peroxide, and when in use, the reagent liquid a and the reagent liquid B are mixed in proportion.
The hypersensitivity ECL luminous liquid reagent is characterized in that the reagent liquid A and the reagent liquid B are mixed according to the volume ratio of 1:1 when in use.
The hypersensitivity ECL luminescence liquid reagent is characterized in that the mass-to-volume ratio of the trihydroxymethyl aminomethane in the reagent A is 12g/L, the mass-to-volume ratio of the luminol is 100mg/L, and the mass-to-volume ratio of the p-hydroxycinnamic acid is 0.21 g/L.
The hypersensitivity ECL luminescence solution reagent is characterized in that the mass volume ratio of the trihydroxymethyl aminomethane in the reagent B is 12g/L, and the volume ratio of the hydrogen peroxide is 750 mu L/L.
The hypersensitivity ECL luminescent liquid reagent is based on a Western Blot hypersensitivity ECL luminescent liquid reagent, the hypersensitivity ECL luminescent liquid reagent can be used for quickly developing colors without incubating a solid phase carrier, the color development time of Western Blot protein can be shortened, meanwhile, after the hypersensitivity ECL luminescent liquid reagent is developed, the background of the solid phase carrier is clear, other snow spots or miscellaneous backgrounds do not exist, and the problem of protein color development that some protein expression quantity is low or the binding capacity of antibodies and the protein is weak is solved.
In a second aspect, a method for using the hypersensitivity ECL luminous liquid reagent comprises the following steps:
step 1, mixing reagent solution A and reagent solution B in proportion to prepare a mixed solvent;
and 2, loading the mixed solvent on the solid phase carrier without incubation.
The application method of the hypersensitivity ECL luminescent solution reagent comprises the step 1 of mixing the reagent solution A and the reagent solution B according to a ratio of 1:1 to prepare a mixed solvent.
The application method of the hypersensitivity ECL luminous liquid reagent comprises the steps that the reagent A comprises tris, luminol and p-hydroxycinnamic acid, and the reagent liquid B comprises tris and hydrogen peroxide.
The application method of the hypersensitivity ECL luminescence liquid reagent is characterized in that the mass-to-volume ratio of the trihydroxymethyl aminomethane in the reagent A is 12g/L, the mass-to-volume ratio of the luminol is 100mg/L, and the mass-to-volume ratio of the p-hydroxycinnamic acid is 0.21 g/L.
The application method of the hypersensitivity ECL luminescent liquid reagent is characterized in that the mass volume ratio of the tris in the reagent B is 12g/L, and the volume ratio of the hydrogen peroxide is 750 mu L/L.
The technical scheme provided by the hypersensitive ECL luminescence solution reagent and the using method thereof according to the invention has the following technical effects:
the color development speed is high, and a large amount of time can be saved; the sensitivity is high, and a protein band with the protein content of nanogram grade can be displayed; incubation before luminescence and color development is not needed; the background of the chromogenic solid phase carrier is clean, other miscellaneous points do not exist, and the problem of protein chromogenic that some protein expression quantity is low or the binding capacity of an antibody and protein is weak is solved.
Drawings
FIG. 1 is a color development comparison chart of the conventional luminescence solution reagent and the hypersensitive ECL luminescence solution reagent.
Detailed Description
In order to make the technical means, the characteristics, the purposes and the functions of the invention easy to understand, the invention is further described with reference to the specific drawings.
The invention provides a hypersensitive ECL luminescence liquid reagent, aiming at realizing higher color development speed and saving a large amount of time; the sensitivity is high, and a protein band with the protein content of nanogram grade can be displayed; incubation before luminescence and color development is not needed; the background of the chromogenic solid phase carrier is clean, other miscellaneous points do not exist, and the problem of protein chromogenic that some protein expression quantity is low or the binding capacity of an antibody and protein is weak is solved.
In the first embodiment, a reagent solution A and a reagent solution B are prepared, wherein the reagent solution A is prepared by mixing Tris (Tris-base) with a mass-volume ratio of 12g/L, luminol with a mass-volume ratio of 100mg/L and p-hydroxycinnamic acid with a mass-volume ratio of 0.21g/L to prepare a reagent solution A, and the reagent solution B is prepared by adding Tris (Tris-base) with a mass-volume ratio of 12g/L into hydrogen peroxide with a volume ratio of 750 muL/L to prepare a reagent solution B;
mixing the reagent solution A and the reagent solution B in proportion to prepare a mixed solvent;
the mixed solvent is loaded on a solid phase carrier to wait for color development without incubation, and as shown in figure 1, a color development image is obtained after the existing luminous liquid reagent and the hypersensitive ECL luminous liquid reagent are used.
In the second embodiment, a reagent solution A and a reagent solution B are prepared, wherein the reagent solution A is prepared by mixing Tris (Tris-base) with a mass-volume ratio of 12g/L, luminol with a mass-volume ratio of 100mg/L and p-hydroxycinnamic acid with a mass-volume ratio of 0.21g/L, and the reagent solution B is prepared by adding Tris (Tris-base) with a mass-volume ratio of 12g/L to hydrogen peroxide with a volume ratio of 750 μ L/L and mixing;
mixing the reagent solution A and the reagent solution B according to the proportion of 1:1 to prepare a mixed solvent;
loading the mixed solvent on a solid phase carrier for color development without incubation.
In conclusion, the hypersensitive ECL luminescence liquid reagent and the use method thereof have the advantages that the color development speed is high, and a large amount of time can be saved; the sensitivity is high, and a protein band with the protein content of nanogram grade can be displayed; incubation before luminescence and color development is not needed; the background of the chromogenic solid phase carrier is clean, other miscellaneous points do not exist, and the problem of protein chromogenic that some protein expression quantity is low or the binding capacity of an antibody and protein is weak is solved.
Specific embodiments of the invention have been described above. It is to be understood that the invention is not limited to the particular embodiments described above, in that devices and structures not described in detail are understood to be implemented in a manner common in the art; various changes or modifications may be made by one skilled in the art within the scope of the claims without departing from the spirit of the invention, and without affecting the spirit of the invention.
Claims (9)
1. The reagent is characterized by comprising reagent liquid A and reagent liquid B, wherein the reagent liquid A comprises tris (hydroxymethyl) aminomethane, luminol and p-hydroxycinnamic acid, and the reagent liquid B comprises tris (hydroxymethyl) aminomethane and hydrogen peroxide, and when the reagent is used, the reagent liquid A and the reagent liquid B are mixed in proportion.
2. The hypersensitivity ECL luminogenic solution reagent according to claim 1, wherein in use, the reagent solution A and the reagent solution B are mixed according to the volume of 1: 1.
3. The hypersensitivity ECL luminous liquid reagent as claimed in claim 1 or 2, wherein the mass volume ratio of said trihydroxymethyl aminomethane in said reagent A is 12g/L, the mass volume ratio of said luminol is 100mg/L, and the mass volume ratio of said p-hydroxycinnamic acid is 0.21 g/L.
4. The hypersensitivity ECL luminophore reagent according to claim 3, wherein said trihydroxymethyl aminomethane in said reagent B has a mass to volume ratio of 12g/L and said hydrogen peroxide has a volume ratio of 750 μ L/L.
5. A method for using a hypersensitive ECL luminescence solution reagent, which is characterized by comprising the following steps:
step 1, mixing reagent solution A and reagent solution B in proportion to prepare a mixed solvent;
and 2, loading the mixed solvent on a solid phase carrier.
6. The method for using the hypersensitivity ECL luminous liquid reagent as claimed in claim 5, wherein in step 1, the reagent liquid A and the reagent liquid B are mixed according to the ratio of 1:1 to prepare a mixed solvent.
7. The method for using the hypersensitivity ECL luminous liquid reagent according to the claim 5 or 6, wherein the reagent A comprises the trihydroxymethyl aminomethane, the luminol and the p-hydroxycinnamic acid, and the reagent B comprises the trihydroxymethyl aminomethane and the hydrogen peroxide.
8. The method for using the hypersensitivity ECL luminous liquid reagent as claimed in claim 7, wherein the mass volume ratio of the trihydroxymethyl aminomethane in the reagent A is 12g/L, the mass volume ratio of the luminol is 100mg/L, and the mass volume ratio of the p-hydroxycinnamic acid is 0.21 g/L.
9. The method for using the hypersensitivity ECL luminous liquid reagent as claimed in claim 8, wherein the mass volume ratio of the trihydroxymethyl aminomethane in the reagent B is 12g/L, and the volume ratio of the hydrogen peroxide is 750 μ L/L.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110681650.7A CN113567679A (en) | 2021-06-19 | 2021-06-19 | Hypersensitive ECL luminescent liquid reagent and using method thereof |
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| CN202110681650.7A CN113567679A (en) | 2021-06-19 | 2021-06-19 | Hypersensitive ECL luminescent liquid reagent and using method thereof |
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030073150A1 (en) * | 2001-05-09 | 2003-04-17 | Woerner Thomas M. | Stabilization of H2O2 under alkaline conditions for use in luminescence, fluorescence and colorimetric assays for enhanced detection of peroxidase type assays |
| CN107561261A (en) * | 2017-08-18 | 2018-01-09 | 江苏凯基生物技术股份有限公司 | A kind of enhanced chemical luminescence reagent box and preparation method thereof |
| CN110068568A (en) * | 2018-01-23 | 2019-07-30 | 深圳市泰锐生物科技有限公司 | A kind of chemiluminescence detection kit and its chemical luminescence detection method |
| CN110554183A (en) * | 2019-09-17 | 2019-12-10 | 沈阳万类生物科技有限公司 | Hypersensitive ECL chemiluminescent reagent |
| WO2020182318A1 (en) * | 2019-03-12 | 2020-09-17 | Symrise Ag | An antimicrobial mixture |
| CN112326636A (en) * | 2020-10-26 | 2021-02-05 | 通山县金瑞生物科技研发中心 | High-sensitivity ECL kit and preparation method thereof |
-
2021
- 2021-06-19 CN CN202110681650.7A patent/CN113567679A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030073150A1 (en) * | 2001-05-09 | 2003-04-17 | Woerner Thomas M. | Stabilization of H2O2 under alkaline conditions for use in luminescence, fluorescence and colorimetric assays for enhanced detection of peroxidase type assays |
| CN107561261A (en) * | 2017-08-18 | 2018-01-09 | 江苏凯基生物技术股份有限公司 | A kind of enhanced chemical luminescence reagent box and preparation method thereof |
| CN110068568A (en) * | 2018-01-23 | 2019-07-30 | 深圳市泰锐生物科技有限公司 | A kind of chemiluminescence detection kit and its chemical luminescence detection method |
| WO2020182318A1 (en) * | 2019-03-12 | 2020-09-17 | Symrise Ag | An antimicrobial mixture |
| CN110554183A (en) * | 2019-09-17 | 2019-12-10 | 沈阳万类生物科技有限公司 | Hypersensitive ECL chemiluminescent reagent |
| CN112326636A (en) * | 2020-10-26 | 2021-02-05 | 通山县金瑞生物科技研发中心 | High-sensitivity ECL kit and preparation method thereof |
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