CN114292217A - Bioactive cross-linking agent, biological cross-linked substance, kit and application - Google Patents
Bioactive cross-linking agent, biological cross-linked substance, kit and application Download PDFInfo
- Publication number
- CN114292217A CN114292217A CN202111671642.0A CN202111671642A CN114292217A CN 114292217 A CN114292217 A CN 114292217A CN 202111671642 A CN202111671642 A CN 202111671642A CN 114292217 A CN114292217 A CN 114292217A
- Authority
- CN
- China
- Prior art keywords
- cross
- group
- linking agent
- bioactive
- substance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a bioactive cross-linking agent, a biological cross-linking object, a kit and application, wherein the structural general formula of the cross-linking agent is SCN-Ph-N- (U- (W- (D-COOH)2n or 3n)a)bWherein N, a, b are positive integers, a is 1 or 2, b is 2 or 3, Ph is alkyl or alkyl containing a ring structure, and the radical of N is selected from: hexamethylenediamine, ethylenediamine, propylenediamine, butylenediamine, lysine, arginine, cyclohexanediamine, phenylenediamine, benzenetriamine and trimellitic acid, U is selected from acetoxy, propionyloxy, butanoyloxy, ethyl, propyl and butane, W is a polybasic group, and D has a structural general formula of- (CH)2)m-, m is a positive integer, m is more than or equal to 1 and less than or equal to 5. The cross-linking agent of the invention effectively improves the sensitivity, increases the signal intensity, and specifically adds two different functional groups so as toThe specificity of crosslinking is achieved.
Description
Technical Field
The invention relates to a biological detection technology, in particular to a bioactive cross-linking agent, a biological cross-linked substance, a kit and application.
Background
The cross-linking agent is a reagent which has the same or different active groups at two ends of a molecule and can be covalently combined with amino, sulfydryl, hydroxyl and the like on other molecules to generate a cross-linking effect, and can be divided into three types, namely homobifunctional reagent, heterobifunctional reagent and photoactivatable reagent. Currently, coupling methods for labeling by immunological techniques include both covalent crosslinking methods and physical adsorption methods. Among them, covalent crosslinking methods are the most. Covalent crosslinking methods are further classified into homocovalent crosslinking agents and heterocovalent crosslinking agents. The covalent cross-linking agent of the same type mainly comprises glutaraldehyde, glutaric acid, hexamethylene diamine, acetic anhydride, diglycidyl ether, methyl suberate and the like, and the cross-linking agent is easy to form self cross-linking in molecules or among molecules and has no direction selectivity. The heterotype cross-linking agent makes different active groups at two ends of a single molecule respectively cross-link with two different molecules, and self-cross-linking in molecules or among molecules can not be formed. The existing cross-linking agent is limited by the limitations of branched chain structure, space conformation, steric hindrance and the like, and the marking amplification capability is greatly limited.
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and should not be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person skilled in the art.
Disclosure of Invention
The invention aims to provide a bioactive cross-linking agent, a preparation method, a composition, a kit and application.
To achieve the above objects, embodiments of the present invention provide a bioactive cross-linking agent having the general structural formula SCN-Ph-N- (U- (W- (D-COOH)2n or 3n)a)b(the general formula means SCN-Ph-N- (U- (W- (D-COOH))2n)a)bOr SCN-Ph-N- (U- (W- (D-COOH)3n)a)b) Wherein N, a, b are positive integers, a is 1 or 2, b is 2 or 3, Ph is alkyl or alkyl containing a ring structure, and the radical of N is selected from: hexamethylenediamine, ethylenediamine, propylenediamine, butylenediamine, lysine, arginine, cyclohexanediamine, phenylenediamine, benzenetriamine and trimellitic acid, U is selected from acetoxy, propionyloxy, butanoyloxy, ethyl, propyl and butane, W is a polybasic group, and D has a structural general formula of- (CH)2)m-, m is a positive integer, m is more than or equal to 1 and less than or equal to 5.
In one or more embodiments of the invention, the crosslinker has the general structural formula SCN-Ph-N-(E-U-(W-(D-COOH)2n or 3n)a)bWherein N, a, b are positive integers, a is 1 or 2, b is 2 or 3, Ph is alkyl or alkyl containing a ring structure, and the radical of N is selected from: hexamethylenediamine, ethylenediamine, propylenediamine, butylenediamine, lysine, arginine, cyclohexanediamine, phenylenediamine, benzenetriamine and trimellitic acid, U is selected from acetoxy, propionyloxy, butanoyloxy, ethyl, propyl and butane, W is a polybasic group, and D has a structural general formula of- (CH)2)m-, m is a positive integer, m is not less than 1 and not more than 5, E is a carbon chain group or a heterochain group. W may be a tertiary amine or the like.
In one or more embodiments of the invention, the carbon chain group represented by group E also has a hydrophilic side chain or the heterochain group also has a hydrophilic side chain.
In one or more embodiments of the invention, the ratio of isothiocyanato to carboxyl groups in the crosslinking agent is 1: c, wherein c is a positive integer multiple of 2 or 3. Preferably, the ratio of isothiocyanato to carboxyl groups in the crosslinking agent is not less than 1: 10.
in one or more embodiments of the present invention, the crosslinking agent has the following structural formula:
this is a possible example of a crosslinking agent, not a limitation of the above formula, each of which can be substituted for a group, and which can be flexibly substituted.
In one or more embodiments of the invention, the biological cross-linker, comprises a backbone formed by a bioactive cross-linking agent as described above and an antigen or antibody linked to an isothiocyanato group, and/or a small molecule species linked to a carboxyl group.
In one or more embodiments of the invention, the small molecule substance comprises a fluorescent small molecule compound and/or a chemiluminescent substance and/or an electrochemiluminescent substance and/or a radioisotope and/or colloidal gold.
In one or more embodiments of the present invention, the small molecule substance includes at least one of fluorescein, acridine esters, colloidal gold, terpyridyl ruthenium, europium Eu derivatives, and radioactive isotopes.
In one or more embodiments of the invention, the labeling kit comprises a bioactive cross-linking agent as described above or a biological cross-linker as described above.
In one or more embodiments of the invention, the use of a biologically active cross-linking agent as described above or a biologically cross-linked material as described above or a labeling kit as described above for labeling a substance comprising an active amino group.
Aiming at the prior art, the invention provides a novel cross-linking agent, which has the advantage of heterotypic specific cross-linking, and can increase the signal intensity of a marker, thereby realizing the following steps of 1: n is advantageous. By bridging of the novel cross-linker, it is suggested that one antibody or antigen or other molecule may cross-link one or more cross-linker molecules, and that one cross-linker molecule may cross-link n marker molecules. Thus, a signal enhancement effect is achieved.
Detailed Description
The following detailed description is to be read in connection with specific embodiments, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Throughout the specification and claims, unless explicitly stated otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or component but not the exclusion of any other element or component.
In the following examples, the innovative aspects of the present invention will be described by taking as an example a crosslinking agent represented by the following formula, but the scope of the present invention is not limited thereto.
Crosslinking agent (noted as J, or SCN-J- (COOH)n)
The synthetic route of the cross-linking agent is as follows, and other possible cross-linking agents of the invention can be synthesized by referring to the route or other routes:
example 1: antigen (including hapten, complete antigen) or antibody, and is marked as P-NH2Crosslinking with chemiluminescent species, such as acridinium ester modifications (AE); the crosslinker is SCN-J- (COOH)n。
Acridine ester hexanediamine modifier (noted AE)
The reaction route of the biological cross-linker is as follows:
1.0mg of the antibody or antigen was dissolved in PBS (pH7.4), and the mixture was shaken and mixed. Adding 10.0mg of micromolecular cross-linking substance, shaking and mixing uniformly, and shaking and reacting at 4 ℃ overnight. The reaction solution was removed, dialyzed with a dialysis bag at 4 ℃ overnight.
After the dialysate was taken out, 10.0mg of acridine ester modifier (AE, formula shown in the specification) was added, 5.0mg of each of EDC and NHS was added, followed by shaking and mixing, and shaking reaction at 4 ℃ overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
And (4) taking out the dialyzate, dissolving and diluting the dialyzate by using a preservation solution, and fixing the volume to a proper concentration for later use.
Measuring the effect of the cross-linked marker through antigen-antibody immunoreaction, and comparing the effect with the marker after the conventional cross-linking process:
example 2: crosslinking of antigen (including hapten and complete antigen) or antibody with fluorescein substances, such as dansyl amide hexylamine modifier (D6), FITC modifier (F) and the like.
Dansyl amide hexylamine (noted as D6)
FITC hexamethylenediamine modifier (denoted as F)
The reaction route of the biological cross-linker is as follows:
1.0mg of the antibody or antigen was dissolved in PBS (pH7.4), and the mixture was shaken and mixed. Adding small molecule cross-linking substance (J, formula shown in the specification) 10.0mg, shaking, mixing, and shaking at 4 deg.C for reaction overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
After the dialysate was taken out, 10.0mg of FITC modifier (F, formula shown in the figure) was added, 5.0mg of each of EDC and NHS was added, followed by shaking and mixing, and shaking reaction at 4 ℃ overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
And (4) taking out the dialyzate, dissolving and diluting the dialyzate by using a preservation solution, and fixing the volume to a proper concentration for later use.
The effect of the crosslinked markers was determined by antigen-antibody immunoreaction, and the comparison with the markers after the conventional crosslinking process is shown in the following table:
example 3: crosslinking of antigen (including hapten and complete antigen) or antibody with electrochemiluminescence substance, such as terpyridyl ruthenium modifier (R6).
Terpyridyl ruthenium Ru (bpy)3 2+Hexamethylenediamine (HDA) modifier (R6)
The reaction route of the biological cross-linker is as follows:
1.0mg of the antibody or antigen was dissolved in PBS (pH7.4), and the mixture was shaken and mixed. Adding small molecule cross-linking substance (J, formula shown in the specification) 10.0mg, shaking, mixing, and shaking at 4 deg.C for reaction overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
Taking out the dialysate, adding 10.0mg of terpyridyl ruthenium modifier (R6, formula shown in the specification), adding 5.0mg of EDC and NHS respectively, shaking, mixing, and reacting at 4 deg.C overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
And (4) taking out the dialyzate, dissolving and diluting the dialyzate by using a preservation solution, and fixing the volume to a proper concentration for later use.
The effect of the crosslinked markers was determined by antigen-antibody immunoreaction, and the comparison with the markers after the conventional crosslinking process is shown in the following table:
example 4: crosslinking of antigens (including haptens, complete antigens) or antibodies with lanthanide metal species, such as europium metal modifications for time resolution (E6) and the like.
Eu(TTA)3L-Hexamethylenediamine (HDA) modifier (E6)
The reaction route of the biological cross-linker is as follows:
1.0mg of the antibody or antigen was dissolved in PBS (pH7.4), and the mixture was shaken and mixed. Adding small molecule cross-linking substance (J, formula shown in the specification) 10.0mg, shaking, mixing, and shaking at 4 deg.C for reaction overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
Taking out the dialysate, adding europium modifier (E6, formula shown in the specification) 10.0mg, adding EDC and NHS 5.0mg respectively, shaking, mixing, and shaking at 4 deg.C for reaction overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
And (4) taking out the dialyzate, dissolving and diluting the dialyzate by using a preservation solution, and fixing the volume to a proper concentration for later use.
The effect of the crosslinked markers was determined by antigen-antibody immunoreaction, and the comparison with the markers after the conventional crosslinking process is shown in the following table:
example 5: crosslinking of antigen (including hapten and complete antigen) or antibody with small molecule substance, such as pyridine iodide modifier (P0).
Propidium iodide PI12 (P0)
The reaction route of the biological cross-linker is as follows:
1.0mg of the antibody or antigen was dissolved in PBS (pH7.4), and the mixture was shaken and mixed. Adding small molecule cross-linking substance (J, formula shown in the specification) 10.0mg, shaking, mixing, and shaking at 4 deg.C for reaction overnight. After the reaction solution was taken out, the mixture was dialyzed with a dialysis bag at 4 ℃ overnight.
Taking out the dialysate, adding pyridine iodide modifier (P0, formula shown in the specification) 10.0mg, adding EDC and NHS 5.0mg respectively, shaking, mixing, and shaking at 4 deg.C for reaction overnight. The reaction solution was removed, dialyzed with a dialysis bag at 4 ℃ overnight.
And (4) taking out the dialyzate, dissolving and diluting the dialyzate by using a preservation solution, and fixing the volume to a proper concentration for later use.
The effect of the crosslinked markers was determined by antigen-antibody immunoreaction, and the comparison with the markers after the conventional crosslinking process is shown in the following table:
the foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (10)
1. A bioactive cross-linking agent has a general structural formula of SCN-Ph-N- (U- (W- (D-COOH)2n or 3n)a)bWherein N, a, b are positive integers, a is 1 or 2, b is 2 or 3, Ph is alkyl or alkyl containing a ring structure, and the radical of N is selected from: hexamethylenediamine, ethylenediamine, propylenediamine, butylenediamine, lysine, arginine, cyclohexanediamine, phenylenediamine, benzenetriamine and trimellitic acid, U is selected from acetoxy, propionyloxy, butanoyloxy, ethyl, propyl and butane, W is a polybasic group, and D has a structural general formula of- (CH)2)m-, m is a positive integer, m is more than or equal to 1 and less than or equal to 5.
2. The bioactive cross-linking agent of claim 1 having the general structural formula SCN-Ph-N- (E-U- (W- (D-COOH)2n or 3n)a)bWherein N, a, b are positive integers, a is 1 or 2, b is 2 or 3, Ph is alkyl or alkyl containing a ring structure, and the radical of N is selected from: hexamethylenediamine, ethylenediamine, propylenediamine, butylenediamine, lysine, arginine, cyclohexanediamine, phenylenediamine, benzenetriamine and trimellitic acid, U is selected from acetoxy, propionyloxy, butanoyloxy, ethyl, propyl and butane, W is a polybasic group, and D has a structural general formula of- (CH)2)m-, m is a positive integer, m is not less than 1 and not more than 5, E is a carbon chain group or a heterochain group.
3. The bioactive cross-linking agent of claim 2 wherein the carbon chain group represented by group E further has a hydrophilic side chain or the heterochain group further has a hydrophilic side chain.
4. A biologically active cross-linking agent according to any one of claims 1 to 3, wherein the ratio of isothiocyanato to carboxyl groups in the cross-linking agent is from 1: c, wherein c is a positive integer multiple of 2 or 3.
5. The bioactive cross-linking agent of claim 4 having the formula:
6. a biological cross-linker comprising a backbone formed from a biologically active cross-linker as claimed in any of claims 1 to 5 and an antigen or antibody linked to an isothiocyanato group, and/or a small molecule species linked to a carboxyl group.
7. The bioconjugate claimed in claim 6, wherein said small molecule substance comprises a fluorescent small molecule compound and/or a chemiluminescent substance and/or an electrochemiluminescent substance and/or a radioisotope and/or colloidal gold.
8. The crosslinked material according to claim 7, wherein the small-molecule substance comprises at least one of fluorescein group, acridine ester group, colloidal gold group, terpyridyl ruthenium group, europium group derivative and radioisotope.
9. A labelling kit comprising a biologically active cross-linking agent as claimed in any one of claims 1 to 5 or a biologically cross-linked material as claimed in any one of claims 6 to 8.
10. Use of a biologically active cross-linking agent according to any one of claims 1 to 5 or a biologically cross-linked material according to any one of claims 6 to 8 or a labelling kit according to claim 9 for labelling a substance containing a reactive amino group.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111671642.0A CN114292217A (en) | 2021-12-31 | 2021-12-31 | Bioactive cross-linking agent, biological cross-linked substance, kit and application |
| CN202211160408.6A CN116023312A (en) | 2021-12-31 | 2022-09-22 | Bioactive cross-linking agent, biological cross-linking substance, kit and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111671642.0A CN114292217A (en) | 2021-12-31 | 2021-12-31 | Bioactive cross-linking agent, biological cross-linked substance, kit and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114292217A true CN114292217A (en) | 2022-04-08 |
Family
ID=80975611
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111671642.0A Pending CN114292217A (en) | 2021-12-31 | 2021-12-31 | Bioactive cross-linking agent, biological cross-linked substance, kit and application |
| CN202211160408.6A Pending CN116023312A (en) | 2021-12-31 | 2022-09-22 | Bioactive cross-linking agent, biological cross-linking substance, kit and application |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202211160408.6A Pending CN116023312A (en) | 2021-12-31 | 2022-09-22 | Bioactive cross-linking agent, biological cross-linking substance, kit and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN114292217A (en) |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ES2054678T5 (en) * | 1986-08-18 | 1997-09-16 | Dow Chemical Co | STAR CONJUGATES. |
-
2021
- 2021-12-31 CN CN202111671642.0A patent/CN114292217A/en active Pending
-
2022
- 2022-09-22 CN CN202211160408.6A patent/CN116023312A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| 柳滢春 等: "《生物化学》", 30 June 2018 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116023312A (en) | 2023-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US4432907A (en) | Diamine acid fluorescent chelates | |
| JP4467869B2 (en) | Acridinium ester labels with hydrophilic modifiers | |
| DE69620902T2 (en) | FLUORESCENT AND LUMINESCENT MARKING COMPOSITIONS AND METHOD FOR USE THEREOF | |
| AU623352B2 (en) | Tridentate conjugate and method of use thereof | |
| US4082738A (en) | Cyanocobalamin derivatives | |
| EP0406473B1 (en) | Ion capture reagents and methods for performing binding assays | |
| US4668640A (en) | Fluorescence polarization immunoassay utilizing substituted carboxyfluoresceins | |
| CN102269762B (en) | Preparation method of conjugate and relative kit | |
| DE58907232D1 (en) | New digoxigenin derivatives and their use. | |
| JP4068137B2 (en) | Biotinylated chemiluminescent label, conjugate, assay and assay kit | |
| DE60113445T2 (en) | SUBSTANCES WITH BRANCHED LINKERMOLEKÜLEN | |
| JPH09504505A (en) | Pterin derivative and its production and use | |
| JPH04503865A (en) | Method for immobilizing cardiolipin, phosphatidylcholine and cholesterol on solid phase and immunoassay | |
| WO2023124154A1 (en) | Magnetic bead coating, preparation method therefor, and test kit | |
| CN114292217A (en) | Bioactive cross-linking agent, biological cross-linked substance, kit and application | |
| US5817527A (en) | Conjugation of ligand to immobilized protein in organic solvent | |
| CN109580934B (en) | Detection reagent and preparation method and application thereof | |
| JP2953501B2 (en) | Interference elimination reagent for measuring analytes using luminescent metal complexes | |
| WO1987004794A1 (en) | Latex agglutination using avidin/biotin system | |
| NZ199629A (en) | Fluorescent polarisation immunoassay and certain substituted carboxy fluoresceins | |
| US6670159B1 (en) | Preparing monomeric metal ion chelator containing diacetyl glycine group linked to proteinaceous molecule | |
| JPS5888660A (en) | Tyronine iodide immunogen composite body, antibody against said composite body and tyronine iodide derivative | |
| WO2001055722A1 (en) | Detection of biological target | |
| JPH03255959A (en) | Polymer | |
| CA1172560A (en) | Composition and method of immunofluorescent assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20220408 |
|
| WD01 | Invention patent application deemed withdrawn after publication |