CN112326953A - Method for directionally labeling polybiotin by using antibody - Google Patents
Method for directionally labeling polybiotin by using antibody Download PDFInfo
- Publication number
- CN112326953A CN112326953A CN202011212275.3A CN202011212275A CN112326953A CN 112326953 A CN112326953 A CN 112326953A CN 202011212275 A CN202011212275 A CN 202011212275A CN 112326953 A CN112326953 A CN 112326953A
- Authority
- CN
- China
- Prior art keywords
- antibody
- biotin
- freeze
- reaction
- aldehyde
- 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
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000002372 labelling Methods 0.000 title claims abstract description 18
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 75
- 229960002685 biotin Drugs 0.000 claims abstract description 38
- 239000011616 biotin Substances 0.000 claims abstract description 38
- 235000020958 biotin Nutrition 0.000 claims abstract description 37
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 238000004108 freeze drying Methods 0.000 claims abstract description 17
- 229920000642 polymer Polymers 0.000 claims abstract description 15
- 239000003381 stabilizer Substances 0.000 claims abstract description 12
- 239000000126 substance Substances 0.000 claims abstract description 11
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 9
- 230000001590 oxidative effect Effects 0.000 claims abstract description 8
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 8
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 8
- 239000002981 blocking agent Substances 0.000 claims abstract description 7
- 239000002904 solvent Substances 0.000 claims abstract description 7
- 239000007800 oxidant agent Substances 0.000 claims abstract description 6
- 239000003223 protective agent Substances 0.000 claims abstract description 6
- 125000003172 aldehyde group Chemical group 0.000 claims abstract description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 5
- 238000004132 cross linking Methods 0.000 claims abstract description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 5
- 230000000903 blocking effect Effects 0.000 claims abstract description 4
- 238000004321 preservation Methods 0.000 claims abstract description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical group OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 18
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical group [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 claims description 18
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 12
- 229940098773 bovine serum albumin Drugs 0.000 claims description 12
- 150000001299 aldehydes Chemical class 0.000 claims description 9
- 239000000872 buffer Substances 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 5
- 229930006000 Sucrose Natural products 0.000 claims description 5
- 150000004676 glycans Polymers 0.000 claims description 5
- 150000004804 polysaccharides Polymers 0.000 claims description 5
- 239000005720 sucrose Substances 0.000 claims description 5
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 4
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 4
- 229920002521 macromolecule Polymers 0.000 claims description 4
- 229910000033 sodium borohydride Inorganic materials 0.000 claims description 4
- 239000012279 sodium borohydride Substances 0.000 claims description 4
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-L Carbonate Chemical compound [O-]C([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-L 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 claims description 3
- 230000008569 process Effects 0.000 claims description 2
- 241000894007 species Species 0.000 claims description 2
- 230000003321 amplification Effects 0.000 abstract description 10
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 10
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 abstract 2
- 238000005516 engineering process Methods 0.000 description 12
- 230000006287 biotinylation Effects 0.000 description 9
- 238000007413 biotinylation Methods 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 238000001514 detection method Methods 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 239000007853 buffer solution Substances 0.000 description 5
- 230000008878 coupling Effects 0.000 description 5
- 238000010168 coupling process Methods 0.000 description 5
- 238000005859 coupling reaction Methods 0.000 description 5
- 239000002808 molecular sieve Substances 0.000 description 5
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 5
- 230000007935 neutral effect Effects 0.000 description 4
- 230000035484 reaction time Effects 0.000 description 4
- YMXHPSHLTSZXKH-RVBZMBCESA-N (2,5-dioxopyrrolidin-1-yl) 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoate Chemical compound C([C@H]1[C@H]2NC(=O)N[C@H]2CS1)CCCC(=O)ON1C(=O)CCC1=O YMXHPSHLTSZXKH-RVBZMBCESA-N 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000008055 phosphate buffer solution Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000018071 Immunoglobulin Fc Fragments Human genes 0.000 description 2
- 108010091135 Immunoglobulin Fc Fragments Proteins 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 150000001615 biotins Chemical class 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000002274 desiccant Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012510 hollow fiber Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000007503 antigenic stimulation Effects 0.000 description 1
- 239000012496 blank sample Substances 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000001720 carbohydrates Chemical group 0.000 description 1
- 230000009920 chelation Effects 0.000 description 1
- 229940126678 chinese medicines Drugs 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004807 desolvation Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000007037 hydroformylation reaction Methods 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 239000004816 latex Substances 0.000 description 1
- 229920000126 latex Polymers 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000006249 magnetic particle Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
Abstract
The invention provides a method for directionally labeling polybiotin by an antibody, which comprises the following steps: reacting carboxyl on biotin or derivatives thereof with amino on the surface of the carrier, removing free biotin after the reaction is finished, and then adding a freeze-drying protective agent for freeze-drying treatment to obtain a biotin polymer; oxidizing adjacent hydroxyl on an Fc segment sugar chain of the antibody into aldehyde group by using an oxidant, removing non-protein substances after the blocking reaction of a blocking agent, and concentrating to obtain an aldehyde-based antibody; and dissolving the biotin polymer which is subjected to freeze drying preservation by using a double solvent, adding an aldehyde antibody to perform a cross-linking reaction for a period of time, and adding a stabilizer to obtain the biotinylated antibody. The method has strong universality and wide application range, and the obtained biotinylated antibody has strong affinity, high specificity and strong signal amplification capability.
Description
Technical Field
The application relates to the technical field of biology, in particular to a method for directionally labeling polybiotin by an antibody.
Background
Antibodies (antibodies) refer to proteins that are produced by the body as a result of antigenic stimulation and that are used by the immune system to identify proteins that neutralize foreign substances such as bacteria, viruses, etc. Igs can form a "Y" shaped structure, called an Ig monomer, which is the basic unit that constitutes an antibody. The antibody can be divided into different subtypes such as IgA, IgG, IgD, IgE, IgM, etc. according to its structural composition, and the antibody of each subtype has different functions and contents in vivo.
The overall structure of the antibody can be divided into the following three parts: first, heavy and light chains: the native Ig molecule contains four heterologous polypeptide chains, of which the two chains with the larger molecular weight are called heavy chains (H) and the two chains with the smaller molecular weight are called Light chains (L). Second, variable (Fab) and constant (Fc) regions: the regions of Ig light and heavy chains that vary widely near the N-terminal amino acid sequence are called variable regions (V), abbreviated as Fab fragments; the region near the C-terminus where the amino acid sequence is relatively stable is called the constant region (C), and is abbreviated as Fc region. Third, the hinge region: the hinge region (hinge region) is located between CH1 and CH2, and the two heavy chains of the hinge region are disulfide-linked to form the bivalent (multivalent) structure of the antibody.
Antibodies are widely applied to a plurality of fields such as medicine, diagnosis and the like at present, and according to different application scenes and purposes, natural antibodies need to be further modified or coupled with other substances. In the field of diagnostic reagents, the method is used for detecting different substances to be detected according to antigen-antibody specific reaction. The technology of coupling the antibody with corresponding markers and carriers is the basis for developing and applying diagnostic reagents. The application scenarios can be divided into the following categories: firstly, coupling the antibody with enzyme (alkaline phosphatase, horseradish peroxidase, etc.), and preparing enzyme-labeled antibody for luminescence of signal substance of diagnostic reagent. And secondly, coupling the antibody with a carrier, wherein the carrier comprises magnetic particles, latex microspheres and the like. And thirdly, coupling the antibody with small molecular substances, such as fluorescein, biotin, polypeptide substances and the like.
The Biotin-Avidin System (BAS) is a new type of amplification System for biological reactions developed in the late 70 s. BAS is widely used in various fields of medicine, and biotin-avidin system can be combined with various markers which have been successfully studied at present. The strong binding with high affinity between biotin and avidin and the multi-stage amplification effect make BAS immune labeling and related tracing analysis more sensitive. It has become a new technology widely used for qualitative and quantitative detection and positioning observation research of trace antigens and antibodies.
Disclosure of Invention
In view of the drawbacks and disadvantages of the prior art, the primary object of the present invention is to provide two aspects: firstly, realizing the directional labeling of biotin by an antibody; and secondly, the signal amplification capacity of the biotinylated antibody is improved. The scheme provided by the invention can realize the directional labeling of the biotin by the antibody without destroying the natural structure of the antibody and recombining and constructing the sequence and by artificially synthesizing a biotin polymer by utilizing the polysaccharide chain of the Fc segment of the natural antibody. The method has strong universality and wide application range, and the obtained biotinylated antibody has strong affinity, high specificity and strong signal amplification capability.
The invention provides a method for directionally labeling polybiotin by an antibody, which comprises the following steps: reacting carboxyl on biotin or derivatives thereof with amino on the surface of the carrier, removing free biotin after the reaction is finished, and then adding a freeze-drying protective agent for freeze-drying treatment to obtain a biotin polymer; oxidizing adjacent hydroxyl on an Fc segment sugar chain of the antibody into aldehyde group by using an oxidant, removing non-protein substances after the blocking reaction of a blocking agent, and concentrating to obtain an aldehyde-based antibody; and dissolving the biotin polymer which is subjected to freeze drying preservation by using a double solvent, adding an aldehyde antibody to perform a cross-linking reaction for a period of time, and adding a stabilizer to obtain the biotinylated antibody.
In the above method, wherein the biotin or a derivative thereof is N-hydroxysuccinimide ester.
In the above process, wherein the carrier is a macromolecular compound rich in primary amino groups.
In the above method, wherein the carrier is Bovine Serum Albumin (BSA).
In the above method, wherein the oxidizing agent is sodium periodate.
In the above method, the antibody used comprises monoclonal or polyclonal antibodies of different species, different subtypes, and different Fc fragment containing polysaccharide chains.
In the method, the freeze-drying protective agent is one or more of sucrose, trehalose and glucose with the mass percentage of 0.2% -3%.
In the above method, wherein the blocking agent is ethylene glycol.
In the above method, wherein the double solvent is an alkaline carbonate buffer or a borate buffer.
In the above method, wherein the stabilizer is sodium borohydride.
The signal amplification technology for directionally labeling the biotin by the antibody established by the invention can realize the technology for directionally labeling the biotin by the common antibody and has strong universality. The technology simultaneously comprises a directional labeling technology and a polymer signal amplification technology, and solves the problems in the prior art for preparing the biotinylated antibody. The biotinylated antibody prepared according to the invention can play a role in signal amplification due to the aggregation effect of biotin polymers. When the antibody is used for coating a diagnostic reagent, the dosage of the antibody is only 1/2 in the prior art when the same signal value is reached, the affinity of the antibody is greatly improved, and the product cost can be reduced most intuitively by reducing the dosage of the antibody. In addition, compared with a control group, the detection rate of a low-value sample and the correlation of a detection result of the biotinylated antibody prepared by the method are obviously improved, and the sensitivity and the specificity of the biotinylated antibody prepared by the method are superior to those of the conventional method. Plays an important role in improving the overall performance of the product.
Drawings
FIG. 1 shows the relationship between concentration values and signal values of a control group.
FIG. 2 shows the concentration values versus signal values for the experimental groups of the present invention.
FIG. 3 shows the correlation of experimental groups with reference reagents.
Fig. 4 shows the correlation of the control group with the reference reagent.
Detailed Description
The following examples are presented to enable those skilled in the art to more fully understand the present application and are not intended to limit the present application in any way.
As the variety of biotin derivatives is more abundant, the biotinylation technology of the antibody is mature day by day. The common technology is that the carboxyl on the surface of biotin is directly coupled with the amino on the surface of an antibody, and the method has the advantages of simple operation and high labeling efficiency. The directional labeling technology established according to the structure and characteristics of antibodies has been widely applied to various fields. The biotinylation of the antibody is realized by utilizing the direct reaction of biotin and derivatives thereof and amino acid residues on the surface of the antibody, although the technology is mature and the operation is simple, the defect is that the directional labeling of the antibody cannot be realized. If biotin binds to antibody active domain (Fab) amino acid residues, antibody affinity and specificity are impaired. In addition, methods of destroying the spatial structure of a bivalent antibody, synthesizing linear hexapeptide, protein A/G, metal chelation and the like have limitations, directional labeling of the antibody by utilizing an Fc segment sugar chain of the antibody has universality, but biotin and the antibody are low in coupling ratio, so that the signal amplification capability of the biotinylated antibody is poor.
The invention provides a signal amplification method for directionally labeling a poly-biotin by an antibody, which comprises the following specific scheme:
preparation of biotin multimers
The carboxyl on biotin or derivatives thereof is utilized to react with the amino on the surface of the carrier, free biotin is removed by molecular sieve chromatography after the reaction is finished, and then freeze-drying protective agent is added into the solution for freeze-drying treatment to obtain the biotin polymer.
The biotin used in the invention is a commercial biotin derivative: n-hydroxysuccinimide ester dissolved in neutral phosphate buffer solution is reacted with the carrier. The carriers used in the present invention are primary amino group-rich macromolecular compounds, such as Bovine Serum Albumin (BSA), polylysine, polymeric amino compounds and other macromolecular substances.
The carrier is prepared by reacting biotin N-hydroxysuccinimide ester with Bovine Serum Albumin (BSA) in a molar ratio of 10:1-20: 1; the reaction time is 1h-1.5 h; the reaction temperature was room temperature. The freeze-drying protective agent during freeze drying is one or more of sucrose, trehalose and glucose which are added into the solution in an amount of 0.2 to 3 percent, and the preferable amount is 1 percent.
Preparation of aldehyde-based antibody
Oxidizing the adjacent hydroxyl on the carbohydrate chain of the Fc segment of the antibody into aldehyde group by using sodium periodate (which can be other oxidizing agents for oxidizing the adjacent hydroxyl of the polysaccharide chain into aldehyde group), carrying out blocking reaction by using a blocking agent, removing non-protein substances by molecular sieve chromatography, and concentrating for later use. The antibody used in the present invention is a monoclonal antibody of murine origin, but the present invention is not limited thereto, and includes other monoclonal and polyclonal antibodies having an Fc fragment containing a polysaccharide chain. In some embodiments, the sodium periodate is reacted with the antibody at a molar ratio of 30:1 for a reaction time of 15min at room temperature. In some embodiments, the blocking agent used is ethylene glycol in a 2:1 molar ratio to sodium periodate and a reaction time of 15 min.
In some embodiments, the concentration mode of the present invention can be one or more of ultrafiltration and hollow fiber mode.
Antibody biotinylation
When the biotinylation of the antibody is marked, a double solvent is used for dissolving the biotin polymer which is stored by freeze drying, then the aldehyde-based antibody is added for a period of cross-linking reaction, and a stabilizing agent is added to obtain the biotinylation antibody. The biotinylated antibody was dialyzed against the buffer and the buffer was replaced. The double solvent is an alkaline carbonate buffer solution or a borate buffer solution. The molar ratio of the aldehyde antibody to the biotin polymer is 1:1, the reaction temperature is room temperature, the reaction time is 1.5h, and a stabilizer is added after the crosslinking reaction is finished. The stabilizer is sodium borohydride, and the molar ratio of the dosage of the stabilizer to the antibody is 100: 1. The preservation buffer solution is a neutral phosphate buffer solution. It should be understood that the above solutions or materials are exemplary only, and not limiting.
The buffer solution replacement mode provided by the invention is one or more of dialysis and hollow fiber washing and filtering.
Specific experimental procedures are described below to better understand the present invention.
Materials and methods
1. Material
1.1 reagents
Biotin N-hydroxysuccinimide ester, ethylene glycol, sodium periodate were purchased from Sigma; trehalose and sucrose are purchased from Chinese medicines; molecular sieve gels were purchased from GE corporation; monoclonal antibodies were purchased from Hytest; avidin beads were purchased from thermo;
1.2 instruments
The freeze drying agent is purchased from Fulong in Shanghai;
2. method of producing a composite material
2.1 preparation of Biotin multimers
1) Bovine Serum Albumin (BSA) and biotin N-hydroxysuccinimide ester (NHS-Bio) were dissolved at 1mg/ml with neutral phosphate buffer.
2) BSA and NHS-Bio were reacted at a molar ratio of 1:10 at room temperature for 30 min.
3) G25 molecular sieve filtration to remove free NHS-Bio.
4) Biotinylated BSA solution was added with 1% sucrose and lyophilized in a freeze-drying agent for storage.
2.2 hydroformylation of antibodies
1) Sodium periodate is dissolved to 1mg/ml by using an acidic citric acid buffer solution
2) The sodium periodate and the antibody are added into the antibody solution according to the molar ratio of 30:1 and reacted for 15min at room temperature.
3) Adding corresponding ethylene glycol according to the molar ratio of the ethylene glycol to the sodium periodate of 1:1, and reacting for 15min at room temperature.
4) G25 molecular sieve gel chromatography, removing non-protein substances, collecting aldehyde antibody, and concentrating.
2.3 antibody biotinylation
1) The biotin polymer was dissolved at 1mg/ml with a boric acid desolvation solution having a pH of 8.5.
2) The aldehyde-based antibody was added to the biotin polymer solution in an amount of 1:1 molar ratio of antibody to biotinylated polymer, and reacted at room temperature for 1 hour.
3) Dissolving sodium borohydride serving as a stabilizer into water to obtain a solution with the concentration of 1mg/ml, adding the corresponding stabilizer according to the molar ratio of the dosage of the stabilizer to the antibody of 100:1, and reacting for 30min at room temperature.
4) After the reaction is finished, dialyzing in neutral phosphate buffer solution overnight, and changing the solution for 2-3 times.
Third, experimental results
The control group biotinylation antibody is the prior general technology, biotin monomers are directly coupled with the antibody for reaction, and non-directional labeling is carried out to obtain the biotinylation antibody.
The experimental group is the biotinylated antibody prepared by the present patent.
3.1 affinity
The same gradient samples were measured by reacting avidin magnetic beads with biotinylated antibody at 0.15ug per single sample of test antibody, removing free unbound biotinylated antibody, and the results are shown in table 1 below and in figures 1 and 2:
TABLE 1
As can be seen from table 1, fig. 1 and fig. 2, the reactivity of the experimental group is about 2 times that of the control group under the same charging condition, which indicates that the affinity of the biotinylated antibody of the experimental group is stronger, and theoretically, the dosage of the antibody of the experimental group can be saved by more than one time under the condition of achieving the same reactivity. The experimental group is also more advantageous from the correlation coefficient of the gradient sample, which contributes to the improvement of the linear range.
3.2 blank limit
The blank limit reflects the background noise of the detection system to a certain extent, and the smaller the background noise of the detection system is, the lower the blank limit is, and the more advantageous the detection of a low-value sample is. Calculating according to a calculation formula according to a blank limit M +2 SD; m-mean value; SD is standard deviation;
when blank limit detection is carried out, firstly, the signal values of an experimental group and a control group are adjusted by taking the gradient sample as a standard, and the deviation of two groups of reactive signal values is not more than 10 percent as a standard, so that the final coating concentration of each group is obtained. Then, blank samples with the antigen amount to be detected being 0 were detected 20 times per group, and then result analysis was performed as shown in table 2 below.
TABLE 2
As can be seen from Table 2, the blank signal value of the experimental group is lower than that of the control group, which indicates that the biotinylated antibody prepared by the invention can reduce the non-specific adsorption of the reaction system and improve the sensitivity of the reaction system.
3.3 correlation
The control group and the experimental group are used for measuring the gradient positive samples respectively, and the correlation between the detection result of each group and the standard value of the positive sample is calculated, and the results are shown in the following table 3, fig. 3 and fig. 4.
TABLE 3
As can be seen from Table 3, FIG. 3 and FIG. 4, the correlation with the standard value of the positive sample was superior to that of the control group. In addition, one sample which is missed to be detected appears in the control group and can still be detected in the experimental group, which shows that the influence of the biotinylation antibody prepared by the invention on the specificity and sensitivity of the antibody is small, and the improvement of the product performance is facilitated.
Those skilled in the art will appreciate that the above embodiments are merely exemplary embodiments and that various changes, substitutions, and alterations can be made without departing from the spirit and scope of the application.
Claims (10)
1. A method for targeted labeling of polybiotin with an antibody, comprising:
reacting carboxyl on biotin or derivatives thereof with amino on the surface of the carrier, removing free biotin after the reaction is finished, and then adding a freeze-drying protective agent for freeze-drying treatment to obtain a biotin polymer;
oxidizing adjacent hydroxyl on an Fc segment sugar chain of the antibody into aldehyde group by using an oxidant, removing non-protein substances after the blocking reaction of a blocking agent, and concentrating to obtain an aldehyde-based antibody;
and dissolving the biotin polymer which is subjected to freeze drying preservation by using a double solvent, adding an aldehyde antibody to perform a cross-linking reaction for a period of time, and adding a stabilizer to obtain the biotinylated antibody.
2. The method of claim 1, wherein the biotin or derivative thereof is an N-hydroxysuccinimide ester.
3. The process of claim 1, wherein the carrier is a macromolecular compound rich in primary amino groups.
4. The method of claim 3, wherein the carrier is Bovine Serum Albumin (BSA).
5. The method of claim 1, wherein the oxidizing agent is sodium periodate.
6. The method of claim 1, wherein the antibodies used comprise monoclonal or polyclonal antibodies of different species, different subtypes, containing polysaccharide chains in the Fc region.
7. The method according to claim 1, wherein the lyoprotectant is one or more of sucrose, trehalose and glucose with a mass percentage of 0.2% -3%.
8. The method of claim 1, wherein the blocking agent is ethylene glycol.
9. The method of claim 1, wherein the double solvent is an alkaline carbonate buffer or a borate buffer.
10. The method of claim 1, wherein the stabilizer is sodium borohydride.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011212275.3A CN112326953A (en) | 2020-11-03 | 2020-11-03 | Method for directionally labeling polybiotin by using antibody |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011212275.3A CN112326953A (en) | 2020-11-03 | 2020-11-03 | Method for directionally labeling polybiotin by using antibody |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112326953A true CN112326953A (en) | 2021-02-05 |
Family
ID=74324647
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011212275.3A Pending CN112326953A (en) | 2020-11-03 | 2020-11-03 | Method for directionally labeling polybiotin by using antibody |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112326953A (en) |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010078376A2 (en) * | 2008-12-30 | 2010-07-08 | Ventana Medical Systems, Inc. | Fc-specific polymer-conjugated antibodies and their diagnostic use |
| CN101833000A (en) * | 2010-05-13 | 2010-09-15 | 北京海瑞祥天生物科技有限公司 | Efficient cascade amplification antibody testing method |
| CN103320428A (en) * | 2012-01-13 | 2013-09-25 | 大连大学 | Directional immobilization method of single-chain antibody by covalent bonds using carbohydrate chain labels |
| CN103792346A (en) * | 2014-02-14 | 2014-05-14 | 赫利森（厦门）生物科技有限公司 | Polymer chemiluminescent labeling reagent as well as preparation method and application of reagent |
| CN105209916A (en) * | 2013-03-14 | 2015-12-30 | 华盛顿大学商业中心 | Polymer dot compositions and related methods |
| CN106478824A (en) * | 2016-10-14 | 2017-03-08 | 潍坊医学院 | A kind of biotinylated antibody of accurate Fc site covalent coupling labelling |
| CN106501536A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | A kind of latex orientation coupling technology detection lipoprotein（a）Test kit |
| CN107764992A (en) * | 2017-10-16 | 2018-03-06 | 南京诺唯赞医疗科技有限公司 | The orientation coupling method of a kind of microballoon and antibody and application |
| CN107831306A (en) * | 2017-10-10 | 2018-03-23 | 武汉科前生物股份有限公司 | A kind of H7 subtype avian influenza virus double-antibody sandwich elisa kit and its detection method |
| CN109781978A (en) * | 2019-02-28 | 2019-05-21 | 湖南博奥瑞康生物科技有限公司 | A kind of polymer microsphere and preparation method thereof orienting the end coupled antibody Fc and its application |
| CN110568176A (en) * | 2019-09-09 | 2019-12-13 | 潍坊市康华生物技术有限公司 | method for directional coupling of antibody and magnetic bead |
| CN110850097A (en) * | 2019-11-07 | 2020-02-28 | 江苏宜偌维盛生物技术有限公司 | Calprotectin detection kit and detection method |
| CN111089965A (en) * | 2019-11-18 | 2020-05-01 | 宁波瑞源生物科技有限公司 | Antigen detection kit, preparation method thereof and antigen detection method |
-
2020
- 2020-11-03 CN CN202011212275.3A patent/CN112326953A/en active Pending
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010078376A2 (en) * | 2008-12-30 | 2010-07-08 | Ventana Medical Systems, Inc. | Fc-specific polymer-conjugated antibodies and their diagnostic use |
| CN101833000A (en) * | 2010-05-13 | 2010-09-15 | 北京海瑞祥天生物科技有限公司 | Efficient cascade amplification antibody testing method |
| CN103320428A (en) * | 2012-01-13 | 2013-09-25 | 大连大学 | Directional immobilization method of single-chain antibody by covalent bonds using carbohydrate chain labels |
| CN105209916A (en) * | 2013-03-14 | 2015-12-30 | 华盛顿大学商业中心 | Polymer dot compositions and related methods |
| CN103792346A (en) * | 2014-02-14 | 2014-05-14 | 赫利森（厦门）生物科技有限公司 | Polymer chemiluminescent labeling reagent as well as preparation method and application of reagent |
| CN106501536A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | A kind of latex orientation coupling technology detection lipoprotein（a）Test kit |
| CN106478824A (en) * | 2016-10-14 | 2017-03-08 | 潍坊医学院 | A kind of biotinylated antibody of accurate Fc site covalent coupling labelling |
| CN107831306A (en) * | 2017-10-10 | 2018-03-23 | 武汉科前生物股份有限公司 | A kind of H7 subtype avian influenza virus double-antibody sandwich elisa kit and its detection method |
| CN107764992A (en) * | 2017-10-16 | 2018-03-06 | 南京诺唯赞医疗科技有限公司 | The orientation coupling method of a kind of microballoon and antibody and application |
| CN109781978A (en) * | 2019-02-28 | 2019-05-21 | 湖南博奥瑞康生物科技有限公司 | A kind of polymer microsphere and preparation method thereof orienting the end coupled antibody Fc and its application |
| CN110568176A (en) * | 2019-09-09 | 2019-12-13 | 潍坊市康华生物技术有限公司 | method for directional coupling of antibody and magnetic bead |
| CN110850097A (en) * | 2019-11-07 | 2020-02-28 | 江苏宜偌维盛生物技术有限公司 | Calprotectin detection kit and detection method |
| CN111089965A (en) * | 2019-11-18 | 2020-05-01 | 宁波瑞源生物科技有限公司 | Antigen detection kit, preparation method thereof and antigen detection method |
Non-Patent Citations (1)
| Title |
|---|
| JERZY JANKUN: "Protein-based nanotechnology: Antibody conjugated with photosensitizer in targeted anticancer photoimmunotherapy", 《INTERNATIONAL JOURNAL OF ONCOLOGY》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU594651B2 (en) | Immunoassays for protein analytes, particularly HB A1c, involving sample denaturation | |
| DK160108C (en) | Method and equipment for direct or indirect detection of reaction between a specific binding agent and the corresponding acceptor substance | |
| CA1291028C (en) | Method for measuring antigens or antibodies in biological fluids using ligand labeled antigens or ligand labeled antibodies | |
| US4478744A (en) | Method of obtaining antibodies | |
| US4672045A (en) | Method for assaying antigen-antibody reactions and reagent thereof | |
| JPH07117536B2 (en) | Method for detecting specifically bindable substance in body fluid and ligand receptor | |
| JPH0926423A (en) | Synthetic calibrator for use in immunoassay containing body under test formed as composite for inactive carrier moleculeor partial alignment thereof | |
| CN110596404A (en) | IL-6 biotin-streptavidin immunochromatography detection card | |
| EP1780545A1 (en) | Probe complex | |
| US6262256B1 (en) | Article for tuesyl-activated dextran for solid-phase coupling | |
| JP2677753B2 (en) | Aggregation immunoassay | |
| EP0859793B1 (en) | Conjugation of ligand to immobilized protein in organic solvent | |
| KR20100132964A (en) | Method for detection of pneumococcus | |
| Bächi et al. | Staphylococcal protein A in immunoferritin techniques | |
| CN113049811A (en) | Nano magnetic bead coating material, preparation method thereof, detection reagent and detection kit | |
| CN112326953A (en) | Method for directionally labeling polybiotin by using antibody | |
| CN115951068A (en) | Kit for detecting TGF-beta 1, detection method and application | |
| US4460694A (en) | Bovine glycoproteins and use in diagnosing infectious mononucleosis | |
| JPH0232258A (en) | Method of measuring antibody factor in human body liquor and measurement of class specific antibody | |
| JP2004189665A (en) | Anti-c reactive protein antibody, biosensor using the same antibody, method for preparing the same antibody and method for measuring immunity by using the same antibody | |
| US5707878A (en) | Method for detecting blood component using conidiobolus hemagglutinin | |
| Rucklidge et al. | Preparation of biotinylated, affinity-purified antibodies for enzyme-linked immunoassays using blotting membrane as an antigen support | |
| CN114113614B (en) | Tannic acid immune network and clenbuterol hydrochloride Luo Shizhi detection method | |
| CN117706079A (en) | Method for directionally labeling biotin polymer by antibody | |
| JP2001519183A (en) | Monoclonal antibody against alpha2-macroglobulin and its use for glucan detection |
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 | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210205 |