CN116953222A - Nanometer polymerase labeled antibody and preparation method and application thereof - Google Patents
Nanometer polymerase labeled antibody and preparation method and application thereof Download PDFInfo
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- CN116953222A CN116953222A CN202310787737.1A CN202310787737A CN116953222A CN 116953222 A CN116953222 A CN 116953222A CN 202310787737 A CN202310787737 A CN 202310787737A CN 116953222 A CN116953222 A CN 116953222A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 102000004190 Enzymes Human genes 0.000 claims abstract description 76
- 108090000790 Enzymes Proteins 0.000 claims abstract description 76
- 239000000412 dendrimer Substances 0.000 claims abstract description 35
- 229920000736 dendritic polymer Polymers 0.000 claims abstract description 35
- 150000004676 glycans Chemical class 0.000 claims abstract description 32
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 32
- 239000005017 polysaccharide Substances 0.000 claims abstract description 32
- 229920000642 polymer Polymers 0.000 claims abstract description 15
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims description 21
- 239000011259 mixed solution Substances 0.000 claims description 18
- 229920000962 poly(amidoamine) Polymers 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 7
- 229920002307 Dextran Polymers 0.000 claims description 5
- 230000003213 activating effect Effects 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 5
- 229920002521 macromolecule Polymers 0.000 claims description 4
- 230000001590 oxidative effect Effects 0.000 claims description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 3
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 3
- 102000005936 beta-Galactosidase Human genes 0.000 claims description 3
- 108010005774 beta-Galactosidase Proteins 0.000 claims description 3
- 229920000499 poly(galactose) polymer Polymers 0.000 claims description 3
- 229920000157 polyfructose Polymers 0.000 claims description 3
- 102000003992 Peroxidases Human genes 0.000 claims description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 claims description 2
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 13
- 230000000694 effects Effects 0.000 abstract description 13
- 230000035945 sensitivity Effects 0.000 abstract description 9
- 238000001514 detection method Methods 0.000 abstract description 8
- 235000009754 Vitis X bourquina Nutrition 0.000 abstract description 7
- 235000012333 Vitis X labruscana Nutrition 0.000 abstract description 7
- 235000014787 Vitis vinifera Nutrition 0.000 abstract description 7
- 230000035699 permeability Effects 0.000 abstract description 4
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- 231100000419 toxicity Toxicity 0.000 abstract description 3
- 240000006365 Vitis vinifera Species 0.000 abstract 1
- LMDZBCPBFSXMTL-UHFFFAOYSA-N 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide Chemical compound CCN=C=NCCCN(C)C LMDZBCPBFSXMTL-UHFFFAOYSA-N 0.000 description 7
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 description 6
- 241000219095 Vitis Species 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 239000000427 antigen Substances 0.000 description 5
- 102000036639 antigens Human genes 0.000 description 5
- 108091007433 antigens Proteins 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
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- 239000000463 material Substances 0.000 description 3
- 230000035515 penetration Effects 0.000 description 3
- 229920001503 Glucan Polymers 0.000 description 2
- 125000003172 aldehyde group Chemical group 0.000 description 2
- 230000003197 catalytic effect Effects 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000002055 immunohistochemical effect Effects 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010029719 Nonspecific reaction Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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Abstract
The application provides a nano-polymerase-labeled antibody, a preparation method and application thereof, wherein the nano-polymerase-labeled antibody comprises dendritic polymer, enzyme, chain macromolecular polysaccharide and antibody, and the dendritic polymer is loaded with a plurality of enzymes to form grape-string-shaped nano-polymerase; the nano polymerase and the antibody are connected through the chain macromolecular polysaccharide to form the nano polymerase-labeled antibody, so that the enzyme content is increased, the grape string-shaped nano polymerase is formed, the nano polymerase and the antibody are coupled with the chain macromolecular polysaccharide, the problem of large steric hindrance caused by direct combination on the antibody is solved, the enzyme content is increased, the permeability of the nano polymerase-labeled antibody is improved, and the detection sensitivity is increased. The reagent EDC/NHS and sodium periodate are adopted to activate dendritic high molecular polymer and chain macromolecular polysaccharide, so that the toxicity of the reagent is reduced, the pollution to the environment is reduced, the self-crosslinking is avoided, the yield is improved, and the dye-patch effect of the prepared nano-polymerase-labeled antibody is excellent.
Description
Technical Field
The application relates to the technical field of medical detection, in particular to a nano polymerase labeled antibody and a preparation method and application thereof.
Background
Immunohistochemistry is a technique for locating, qualitatively or quantitatively detecting specific antigens or antibodies in tissues and cells by utilizing the specific binding principle between the antigens and the antibodies and a special labeling technique. The immunohistochemical technology has the characteristics of strong specificity, high sensitivity, accurate positioning, combination of morphology and function and the like, is favorable for deep research in the pathological field, plays an important role in modern pathological diagnosis, and is a gold standard for cancer diagnosis.
The immune grouping core technology is characterized in that the enzyme-labeled secondary antibody manufacturing process is adopted in the main stream of the market, and chain type multimeric enzyme-labeled secondary antibodies and grape string type multimeric enzyme-labeled secondary antibodies are adopted, so that compared with the enzyme-labeled secondary antibodies used in the traditional enzyme-linked immune reagent, the immune grouping core technology has obvious sensitivity and is greatly improved, and the immune grouping core technology is widely applied to domestic and foreign immune grouping reagents. Fig. 1 is a schematic diagram of a chain type secondary enzyme-labeled antibody and a grape string type secondary enzyme-labeled antibody in the prior art, the enzyme content in the chain type secondary enzyme-labeled antibody is low, the sensitivity is insufficient, the secondary enzyme-labeled antibody of the grape string type directly binds polymerase to the antibody, the permeability of polymer tissues is poor, and a secondary enzyme-labeled antibody is needed to solve the above problems.
Disclosure of Invention
In order to overcome the above-mentioned drawbacks in the prior art, the first aspect of the present application proposes a nano-polymerase-labeled antibody comprising a dendrimer, an enzyme, a chain-like macromolecular polysaccharide and an antibody, wherein the dendrimer is loaded with a plurality of enzymes to form a string-shaped nano-polymerase; the nano-polymerase is connected with the antibody through chain macromolecular polysaccharide to form the nano-polymerase labeled antibody.
According to the scheme, enzymes are loaded in a dendritic polymer material to form nano-polymerase, the dendritic polymer is of a three-dimensional structure, the surface of the dendritic polymer is provided with a plurality of functional groups, so that a large amount of enzymes can be loaded, the enzyme content is increased, and the grape cluster type nano-polymerase is formed; and then the nano-polymerase and the antibody are coupled with chain macromolecular polysaccharide, so that the problem of large steric hindrance caused by direct combination on the antibody is solved, meanwhile, the enzyme content is improved, the permeability of the nano-polymerase labeled antibody is improved, and the detection sensitivity is increased.
Furthermore, a plurality of nano-polymerases are connected in one chain macromolecular polysaccharide, so that the enzyme content in the enzyme-labeled secondary antibody is greatly improved, and the detection is more sensitive; meanwhile, a plurality of nanometer polymerases are coupled with a long-chain macromolecular polymer, so that steric hindrance generated by too tight connection is reduced, and the tissue penetration capacity of the polymer is enhanced.
Further, the dendrimer includes a PAMAM modified with a surface group.
Further, the chain macromolecular polysaccharide comprises at least one of dextran, polyfructose or polygalactose.
Further, the enzyme comprises at least one of peroxidase, alkaline phosphatase, or beta-galactosidase.
The second aspect of the present application provides a method for preparing a nano-polymerase labeled antibody, comprising the steps of: activating the dendritic polymer, adding the activated dendritic polymer into an enzyme solution, and obtaining a mixed solution A containing nano polymerase through centrifugation and purification; adding oxidized chain macromolecules into the mixed solution A for coupling reaction to obtain a mixed solution B of chain macromolecular polysaccharide coupled with nano polymerase; and adding the antibody into the mixed solution B to obtain a mixed solution containing the nano-polymerase labeled antibody.
The scheme provides a method for preparing a nano-polymerase-labeled antibody, which is characterized in that dendritic polymer compounds and chain macromolecular polysaccharides are treated without modifying enzymes and antibodies, so that the space structures and functions of the enzymes and the antibodies are not changed, and the activities and affinities of the enzymes and the antibodies are not influenced.
Further, the method further comprises the steps of: the dendritic polymer is added into a mixed solution of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), the groups on the surface of the dendritic polymer are activated, the activated dendritic polymer is obtained and coupled with enzyme, the enzyme space structure is not influenced, the enzyme molecular weight is obviously improved, the purification and separation are easy, and the industrialization is easy.
Further, the method further comprises the steps of: the chain macromolecular polysaccharide is added into the sodium periodate solution, the chain macromolecular polysaccharide is oxidized, oxidized chain macromolecules are obtained, the nano polymerase and the antibody are coupled, the space structures of the enzyme and the antibody are not affected obviously, the activity and the affinity are kept well, and the dye-sheet effect of the coupled nano polymerase-labeled antibody is excellent.
In a third aspect, the present application provides a kit comprising a nano-polymerase-labeled antibody according to the first aspect of the present application or a nano-polymerase-labeled antibody prepared by a method according to the second aspect of the present application.
According to the application, the enzyme is loaded in the dendritic polymer material to form the grape-string-shaped nano-polymerase, so that the enzyme content is improved, the nano-polymerase and the antibody are coupled through the chain-shaped macromolecular polysaccharide to form the nano-polymerase-labeled antibody, and a plurality of grape-string-shaped nano-polymerases are connected in one nano-polymerase-labeled antibody, so that the problem of steric hindrance caused by directly connecting the grape-string-shaped nano-polymerase to the antibody is solved, the tissue penetration capacity of the polymer is enhanced, the enzyme quantity in one antibody is increased, and higher sensitivity is brought in the detection process. The dendritic high molecular polymer and chain macromolecular polysaccharide are activated by adopting reagents EDC, NHS and sodium periodate, and the activated dendritic high molecular polymer and chain macromolecular polysaccharide are used for loading enzymes and coupling antibodies and nano-polymerase, so that the structures and functions of the enzymes and the antibodies are not changed, the activity and affinity of the modified enzymes and the modified antibodies are reduced, the toxicity of the reagents is reduced, the pollution to the environment is reduced, the self-crosslinking is avoided, the yield is improved, and the dye tablet of the nano-polymerase-labeled antibody is excellent. The nano-polymerase labeled antibody can be applied to a diagnosis kit, has high sensitivity, can detect trace antigens, and reduces the misdiagnosis probability of the kit.
Drawings
The drawings illustrate embodiments and together with the description serve to explain the principles of the application. The elements of the drawings are not necessarily to scale relative to each other. Like reference numerals designate corresponding similar parts.
FIG. 1 is a schematic diagram of a chain type enzyme-labeled secondary antibody and a grape string type enzyme-labeled secondary antibody in the prior art,
FIG. 2 is a schematic diagram of the structure of a nano-polymerase labeled antibody according to an embodiment of the present application,
FIG. 3 is a graph showing the effect of the secondary antibody staining of the inlet enzyme label in the prior art,
FIG. 4 is a graph showing the staining effect of a nano-polymerase labeled antibody according to another embodiment of the present application.
Reference numerals in the drawings: 101-nanometer polymerase, 102-antibody, 103-chain macromolecular polysaccharide.
Detailed Description
The following describes examples of the application to better understand the application, and many of the intended advantages of other embodiments and examples can be appreciated from the detailed description that follows. It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising" does not exclude the presence of additional identical elements in a process, method, article, or apparatus that comprises an element. Components of embodiments may be positioned in a number of different orientations, and so directional terminology, such as "top," "bottom," "left," "right," "up," "down," etc., is used with reference to the orientation of the figures to describe some embodiments. It is to be understood that the directional terminology is used for purposes of illustration and not limitation.
Example 1A nanopolymerase-labeled antibody
The nanometer polymerase labeling antibody comprises dendritic polymer, enzyme, chain macromolecular polysaccharide and antibody, wherein the dendritic polymer is preferably PAMAM with surface group modified; the enzyme comprises any one of oxidase, alkaline phosphatase or beta-galactosidase, and can be selected according to actual detection requirements; the chain macromolecular polysaccharide comprises any one of dextran, polyfructose or polygalactose.
Preferably, PAMAM is a generic term for a broad class of dendrimer polymers, having a molecular weight ranging from about 500 at generation 0 to about 120K at generation 7, and the surface modification comprises hydroxyl, carboxyl or amino groups, and the present application is directed to the modification of carboxyl groups on the PAMAM surface.
FIG. 2 is a schematic diagram of a nano-polymerase labeled antibody structure in an embodiment of the present application, wherein a dendrimer carries a plurality of enzymes to form a string of nano-polymerase 101; the nano-polymerase 101 is linked with the antibody 102 through the chain macromolecular polysaccharide 103 to form a nano-polymerase labeled antibody. Enzyme is loaded in a dendritic polymer material to form nano-polymerase, the dendritic polymer is of a three-dimensional structure, and the surface of the dendritic polymer is provided with a plurality of functional groups, so that a large amount of enzyme can be loaded, the enzyme content is increased, and the grape cluster type nano-polymerase is formed; and then the nano-polymerase and the antibody are coupled with chain macromolecular polysaccharide, so that the problem of large steric hindrance caused by direct combination on the antibody is solved, meanwhile, the enzyme content is improved, the permeability of the nano-polymerase labeled antibody is improved, and the detection sensitivity is increased.
Preferably, a plurality of nano-polymerases are connected in one chain macromolecular polysaccharide, so that the enzyme content in the enzyme-labeled secondary antibody is greatly improved, and the detection is more sensitive; meanwhile, a plurality of nano-polymerases are coupled with a long-chain macromolecular polymer, so that the steric hindrance generated by too tight connection is reduced, and the tissue penetration capacity of the polymer is enhanced; fig. 3 is a graph showing the staining effect of the second antibody of the inlet enzyme label in the prior art, fig. 4 is a graph showing the staining effect of the antibody of the nano polymerase label in another specific embodiment of the application, and the immunohistochemical experiment is carried out on the same tissue sample by using the second antibody of the inlet enzyme label and the antibody of the nano polymerase label of the application, compared with the second antibody of the inlet enzyme label, the staining effect of the antibody of the nano polymerase label in the embodiment is more obvious, meanwhile, clear staining background is maintained, positioning and qualitative research on antigens in tissues and cells are facilitated, the higher sensitivity enables the antibody to have good adaptability to different tissues, and qualitative and quantitative analysis can be carried out on the antibody even if only trace antigens exist.
Example 2 preparation method of a nanometer polymerase labeled antibody
A method for preparing a nano-polymerase labeled antibody, comprising the steps of: activating the dendritic polymer, adding the activated dendritic polymer into an enzyme solution, and obtaining a mixed solution A containing nano polymerase through centrifugation and purification; adding oxidized chain macromolecules into the mixed solution A for coupling reaction to obtain a mixed solution B of chain macromolecular polysaccharide coupled with nano polymerase; adding the antibody into the mixed solution B to obtain a mixed solution containing the nano polymerase labeled antibody; the uncoupled antibodies and long chain nanopolymerases were removed by column purification using Sephadex G-200 to obtain a nanopolymerase-only labeled antibody solution.
The embodiment provides a method for preparing a nano-polymerase labeled antibody, which is characterized in that a dendritic polymer compound and chain macromolecular polysaccharide are treated, enzymes and antibodies are not modified, the space structures and functions of the enzymes and the antibodies are not changed, and the activity and affinity of the enzymes and the antibodies are not influenced.
Preferably, under the condition of pH5.5-7.5, reagents EDC and NHS with the mole number of 5-100 times of that of the dendritic polymer PAMAM are added, the reagents EDC are firstly added for activation, then the reagents NHS are added, the two are cooperated for activating the surface carboxyl of the dendritic polymer, and the activated PAMAM is added into an enzyme solution for preparing the nano-polymerase mixed solution A through ultrafiltration tube centrifugation and purification. The reagent EDC and NHS are adopted to synergistically activate the carboxyl on the surface of the spherical high polymer PAMAM, the activated dendritic high polymer PAMAM is coupled with the enzyme, and the enzyme does not need to be treated, so that the space structure of the enzyme is not changed, the catalytic activity of the enzyme is not influenced, the molecular weight of the polymerase is obviously improved compared with that of the spherical high polymer and the enzyme, and the polymerase is easy to purify and separate and industrialization.
Preferably, the hydroxyl groups of the dextran are oxidized to aldehyde groups by oxidizing the dextran with sodium periodate in a molar amount of 5mmol/L to 200 mmol/L.
The nano-polymerase and the antibody are coupled to aldehyde groups of glucan, the nano-polymerase and the antibody can be coupled with glucan chains without processing the enzyme and the antibody, so that the space structures of the nano-polymerase and the antibody are not changed, the catalytic activity and the affinity of the nano-polymerase and the antibody are not changed, and compared with the existing enzyme-labeled secondary antibody, the conjugated nano-polymerase labeled antibody has no damage to the binding capacity of a substrate, so that the nano-polymerase labeled antibody has excellent dyeing effect.
Compared with the existing method for oxidizing the enzyme and the antibody by adopting a coupling reagent DVS, the method for oxidizing the chain macromolecular polysaccharide and the dendritic macromolecular polymer by adopting sodium periodate, reagents EDC and NHS respectively reduces the toxicity degree of the reagents to human bodies and the pollution to the environment, prevents the problems of low yield and difficult industrialization caused by self-crosslinking of the conjugate, and simultaneously avoids the damage to the space structures of the enzyme and the antibody, so that the activity of the enzyme and the antibody is reduced, the affinity of the enzyme and the antibody is reduced and the nonspecific reaction occurs.
The foregoing is a preferred embodiment of the present application, and it will be apparent to those skilled in the art that various modifications and variations can be made to the embodiment of the present application without departing from the spirit and scope of the application. In this manner, the application is also intended to cover such modifications and variations as come within the scope of the appended claims and their equivalents.
Claims (9)
1. The nano polymerase labeled antibody is characterized by comprising a dendritic polymer, an enzyme, chain macromolecular polysaccharide and an antibody, wherein the dendritic polymer is loaded with a plurality of enzymes to form grape-string-shaped nano polymerase; the nano-polymerase is connected with the antibody through the chain macromolecular polysaccharide to form the nano-polymerase labeled antibody.
2. The nanopolymer enzyme-labeled antibody of claim 1, wherein a plurality of nanopolymerases are linked in one of the chain macromolecular polysaccharides.
3. The nanopolymer-labeled antibody of claim 1, wherein the dendrimer polymer comprises a surface group modified PAMAM.
4. The nanopolymer enzyme-labeled antibody of claim 1, wherein the chain macromolecular polysaccharide comprises at least one of dextran, polyfructose, or polygalactose.
5. The nanopolymer enzyme-labeled antibody of claim 1, wherein the enzyme comprises at least one of peroxidase, alkaline phosphatase, or beta-galactosidase.
6. The preparation method of the nano-polymerase labeled antibody is characterized by comprising the following steps:
activating the dendritic polymer, adding the activated dendritic polymer into an enzyme solution, centrifuging and purifying to obtain a mixed solution A containing nano polymerase;
adding oxidized chain macromolecules into the mixed solution A for coupling reaction to obtain a mixed solution B of chain macromolecular polysaccharide coupled with the nano polymerase;
and adding the antibody into the mixed solution B to obtain a mixed solution containing the nano-polymerase labeled antibody.
7. The method for preparing the nano-polymerase labeled antibody according to claim 6, further comprising the steps of: and adding the dendritic polymer into a mixed solution of EDC and NHS, and activating groups on the surface of the dendritic polymer to obtain the activated dendritic polymer.
8. The method for preparing the nano-polymerase labeled antibody according to claim 6, further comprising the steps of: and adding the chain macromolecular polysaccharide into a sodium periodate solution, and oxidizing the chain macromolecular polysaccharide to obtain an oxidized chain macromolecular.
9. A kit comprising the nanopolymer-labeled antibody of any one of claims 1-5 or prepared by the method of any one of claims 6-8.
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| CN202310787737.1A CN116953222A (en) | 2023-06-30 | 2023-06-30 | Nanometer polymerase labeled antibody and preparation method and application thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202310787737.1A CN116953222A (en) | 2023-06-30 | 2023-06-30 | Nanometer polymerase labeled antibody and preparation method and application thereof |
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| CN202310787737.1A Pending CN116953222A (en) | 2023-06-30 | 2023-06-30 | Nanometer polymerase labeled antibody and preparation method and application thereof |
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