CN116983437A - Nondestructive image marking method and product of alpha Olig2 and application thereof - Google Patents
Nondestructive image marking method and product of alpha Olig2 and application thereof Download PDFInfo
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- diethylenetriamine pentaacetic
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- 102000004169 proteins and genes Human genes 0.000 claims abstract description 35
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- 238000002372 labelling Methods 0.000 claims abstract description 27
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- 238000003384 imaging method Methods 0.000 claims abstract description 15
- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 claims description 48
- 239000000243 solution Substances 0.000 claims description 26
- 239000011259 mixed solution Substances 0.000 claims description 20
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 claims description 16
- 238000004108 freeze drying Methods 0.000 claims description 16
- 229960003330 pentetic acid Drugs 0.000 claims description 16
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 14
- 239000007864 aqueous solution Substances 0.000 claims description 13
- LEFGDNZNVQAADI-UHFFFAOYSA-N acetic acid N'-(2-aminoethyl)ethane-1,2-diamine gadolinium Chemical compound [Gd].CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O.NCCNCCN LEFGDNZNVQAADI-UHFFFAOYSA-N 0.000 claims description 11
- -1 gadolinium ions Chemical class 0.000 claims description 11
- 230000001105 regulatory effect Effects 0.000 claims description 11
- RPNUMPOLZDHAAY-UHFFFAOYSA-N Diethylenetriamine Chemical compound NCCNCCN RPNUMPOLZDHAAY-UHFFFAOYSA-N 0.000 claims description 10
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- 238000000502 dialysis Methods 0.000 claims description 10
- 238000002595 magnetic resonance imaging Methods 0.000 claims description 8
- 201000007983 brain glioma Diseases 0.000 claims description 7
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- 235000017557 sodium bicarbonate Nutrition 0.000 claims description 7
- PWGJDPKCLMLPJW-UHFFFAOYSA-N 1,8-diaminooctane Chemical compound NCCCCCCCCN PWGJDPKCLMLPJW-UHFFFAOYSA-N 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
- 238000000746 purification Methods 0.000 claims description 5
- 238000004090 dissolution Methods 0.000 claims description 4
- NAQMVNRVTILPCV-UHFFFAOYSA-N hexane-1,6-diamine Chemical compound NCCCCCCN NAQMVNRVTILPCV-UHFFFAOYSA-N 0.000 claims description 4
- 239000003550 marker Substances 0.000 claims description 4
- XHFLOLLMZOTPSM-UHFFFAOYSA-M sodium;hydrogen carbonate;hydrate Chemical compound [OH-].[Na+].OC(O)=O XHFLOLLMZOTPSM-UHFFFAOYSA-M 0.000 claims description 3
- 230000000536 complexating effect Effects 0.000 claims description 2
- 230000001066 destructive effect Effects 0.000 claims description 2
- QFTYSVGGYOXFRQ-UHFFFAOYSA-N dodecane-1,12-diamine Chemical compound NCCCCCCCCCCCCN QFTYSVGGYOXFRQ-UHFFFAOYSA-N 0.000 claims description 2
- 230000001276 controlling effect Effects 0.000 claims 4
- 239000000539 dimer Substances 0.000 claims 3
- 230000008685 targeting Effects 0.000 abstract description 11
- 230000008569 process Effects 0.000 abstract description 8
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- 238000013329 compounding Methods 0.000 abstract description 3
- 238000006471 dimerization reaction Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 2
- 125000000217 alkyl group Chemical group 0.000 abstract 1
- 238000012412 chemical coupling Methods 0.000 abstract 1
- 230000035484 reaction time Effects 0.000 description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
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- 208000032612 Glial tumor Diseases 0.000 description 7
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- RJOJUSXNYCILHH-UHFFFAOYSA-N gadolinium(3+) Chemical compound [Gd+3] RJOJUSXNYCILHH-UHFFFAOYSA-N 0.000 description 7
- 239000002904 solvent Substances 0.000 description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
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- 239000007787 solid Substances 0.000 description 6
- 239000012265 solid product Substances 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- PNYPSKHTTCTAMD-UHFFFAOYSA-K trichlorogadolinium;hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Cl-].[Gd+3] PNYPSKHTTCTAMD-UHFFFAOYSA-K 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000000108 ultra-filtration Methods 0.000 description 5
- 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 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 3
- 239000005695 Ammonium acetate Substances 0.000 description 3
- 229940043376 ammonium acetate Drugs 0.000 description 3
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- 239000002872 contrast media Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
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- 108010019160 Pancreatin Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
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- 229940055695 pancreatin Drugs 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- 230000004853 protein function Effects 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 238000002189 fluorescence spectrum Methods 0.000 description 1
- 238000012632 fluorescent imaging Methods 0.000 description 1
- UIWYJDYFSGRHKR-UHFFFAOYSA-N gadolinium atom Chemical compound [Gd] UIWYJDYFSGRHKR-UHFFFAOYSA-N 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
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- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- QAHWBDBQIHMPRN-UHFFFAOYSA-N octane-1,6-diamine Chemical compound CCC(N)CCCCCN QAHWBDBQIHMPRN-UHFFFAOYSA-N 0.000 description 1
- 238000010377 protein imaging Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0032—Methine dyes, e.g. cyanine dyes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0058—Antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/101—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals
- A61K49/103—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA
- A61K49/105—Organic compounds the carrier being a complex-forming compound able to form MRI-active complexes with paramagnetic metals the complex-forming compound being acyclic, e.g. DTPA the metal complex being Gd-DTPA
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- Health & Medical Sciences (AREA)
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- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Radiology & Medical Imaging (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a nondestructive image marking method and product of alpha Olig2 and application thereof, belonging to the technical field of protein image marking. According to the invention, fluorescent molecules Cy5 are marked into the structure of alpha Olig2 through chemical coupling, so that antibody protein alpha Olig2-Cy5 with a fluorescence imaging function is obtained; the dimerization Gd-DTPA and the alpha Olig2-Cy5 are compounded into an antibody probe with fluorescence/magnetic resonance double imaging function by utilizing hydrophobic action, wherein the introduction of alkyl chains in the molecular structure of the dimerization Gd-DTPA increases the binding action with the alpha Olig2-Cy 5. The antibody probe labeling method balances the limitations of reduced targeting binding capacity caused by protein structure change in the chemical labeling process and low image labeling efficiency in the physical compounding process, and furthest reserves the targeting binding capacity and the image labeling efficiency of antibody proteins. The antibody marking method is simple and easy to implement, and can be used as a novel method for nondestructive marking of antibody proteins.
Description
Technical Field
The invention relates to the technical field of protein image marking, in particular to a nondestructive image marking method and product of alpha Olig2 and application thereof.
Background
The imaging and marking technology of the protein has important significance in the aspects of cell, tissue and living body level imaging. Biotin labeling is the primary means of protein imaging labeling, i.e., the necessary fluorescent labeling of the target protein (antigen or antibody) for fluorescent imaging. The fluorescence imaging sensitivity is high, and the marker can be subjected to real-time contrast imaging through multichannel fluorescence signal detection, so that the method is more applied to imaging of cell layers or superficial tissues. Due to the low tissue penetration depth and interference of self-contained fluorescence of living organisms, fluorescence imaging tends to be poor at the living body level, especially for deep tissues. Magnetic resonance imaging is an important diagnostic means in clinic, and imaging has the characteristic of infinite penetration. However, magnetic resonance imaging contrast and sharpness tend to be poor. To achieve focused specific imaging and viewing of living tissue, it is often necessary to construct a probe-type contrast-enhancing contrast agent. The image mark of the antibody protein can be used as an effective mode to realize tissue specific contrast imaging, the efficient targeted contrast imaging can be realized by means of antigen-antibody combination, and the image signal change shown by the antibody protein is utilized to quantitatively analyze the specific protein in the specific tissue. Among them, the expression of the transcription factor Olig2 plays an important role in glioma invasion evolution, and can be used as a key transcription factor for elucidating the entry point of glioma invasion mechanism. Therefore, the noninvasive diagnosis of the invasion of the glioma can be realized by accurately judging the expression of the Olig2 protein in the glioma patient through an imaging means. However, in the technology, the antibody protein is used as an active macromolecule, and the influence of the change of the protein structure on the targeting function is required to be paid attention to in the labeling process, for example, the change of the protein structure in the chemical labeling process often leads to the reduction of the targeting binding capacity, and the image molecule and the target protein often cannot realize the protein with high efficiency through simple physical compounding, or the image molecule can destroy the structure and the targeting function of the protein while increasing the binding effect of the image molecule and the protein through an electrostatic adsorption mode. Therefore, development of a nondestructive antibody protein labeling method with simple preparation process and high image labeling efficiency for specific diagnosis of clinical diseases is urgently needed.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, in a first aspect of the present invention, there is provided a method for non-destructive image labelling of alpha Olig2 retaining the targeted binding capacity of an antibody protein, comprising the steps of:
(1) Introducing Cy5 into alpha Olig2 to obtain an alpha Olig2-Cy5 antibody probe;
(2) Reacting n-alkyl diamine with diethylenetriamine pentaacetic anhydride to obtain dimerized diethylenetriamine pentaacetic acid (di-DTPA);
(3) Complexing the dimerized diethylenetriamine pentaacetic acid (di-DTPA) with gadolinium ions to form dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA);
(4) And (3) the dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA) reacts with the alpha Olig2-Cy5 antibody probe to obtain alpha Olig2-Cy5@di-Gd-DTPA, and the nondestructive image marking of the alpha Olig2 is completed.
The traditional physical combination of the imaging molecule and the target protein usually has no specific acting force or is combined by an electrostatic adsorption mode, but the method often leads to the loss of the function of the protein. Aiming at the defects of the existing antibody protein labeling technology, the design idea of the invention is that a biotin labeling and physical compounding combined mode is utilized to chemically couple fluorescent molecules Cy5 into the structure of alpha Olig2, and simultaneously, T1 enhanced magnetic resonance imaging molecules dimerization Gd-DTPA and alpha Olig2-Cy5 are compounded into an antibody probe with fluorescence/magnetic resonance double imaging functions.
The di-Gd-DTPA and protein are combined through the hydrophobic structure in the di-Gd-DTPA molecule and the hydrophobic structure of the protein, the hydrophobic chain in the di-Gd-DTPA structure is utilized to increase the combination efficiency with the protein, the protein function can be preserved, the reaction condition is mild, the process ensures the retention of the targeting combination capability of the antibody protein after being marked, and meanwhile, the process can also endow the antibody protein with the image function, and can be used as a targeting contrast agent for diagnosing the invasiveness of glioma.
Preferably, the specific method of the step (1) is as follows:
dissolving alpha Olig2 and Sulfo-Cy5-NHS in a solvent, adjusting the pH of the obtained mixed solution, reacting, and purifying after the reaction is finished to obtain the alpha Olig2-Cy5 antibody probe.
Further preferably, the concentration of the αolig2 after dissolution is 0.05 to 0.2. Mu.M, and the molar ratio of the αolig2 to the Sulfo-Cy5-NHS is 1:1 to 10.
Still further, the molar ratio of the alpha Olig2 to the Sulfo-Cy5-NHS is 1:2 to 4.
The invention designs and adjusts the technological parameters, when the mole ratio of alpha Olig2 to Sulfo-Cy5-NHS is 1:1 to 10, better marking can be realized; and when the molar ratio is 1: 2-4, not only can the high-efficiency protein fluorescent marking be satisfied, but also the damage of protein functions caused by excessive marking can be prevented, and the best design effect is achieved.
Further preferably, the solvent is sodium bicarbonate aqueous solution, and the concentration thereof is 0.05-0.2 mol/L.
Further preferably, the pH of the mixed solution of the reaction is 7.5-9.5, the reaction temperature is room temperature, and the reaction time is 1-6 h.
Further preferably, the purification method is dialysis, lyophilization.
Preferably, the specific method of the step (2) is as follows:
dissolving n-alkyl diamine and diethylenetriamine pentaacetic anhydride (DTPAA) in a solvent, adjusting the pH of the obtained mixed solution, reacting, and purifying after the reaction is finished to obtain dimerized diethylenetriamine pentaacetic acid (di-DTPA).
Further preferably, the concentration of the n-alkyl diamine after dissolution is 0.05 to 0.2mM, and the molar ratio of the n-alkyl diamine to the diethylenetriamine pentaacetic anhydride is 1:2 to 5.
Further preferably, the n-alkyl diamine includes any one of 1, 6-hexamethylenediamine, 1, 8-octanediamine, and 1, 12-dodecanediamine.
Further preferably, the solvent is sodium bicarbonate aqueous solution, and the concentration thereof is 0.05-0.2 mol/L.
Further preferably, the pH of the mixed solution of the reaction is 7.5-9.5, the reaction temperature is room temperature, and the reaction time is 12-24 hours.
Further preferably, the purification method is dialysis, lyophilization.
Preferably, the specific method of the step (3) is as follows:
and (3) dissolving the dimerized diethylenetriamine pentaacetic acid (di-DTPA) in a solvent, adjusting the pH of the obtained mixed solution, then adding gadolinium ions for reaction, and purifying after the reaction is finished to obtain dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA).
Further preferably, the solvent is a trinna aqueous solution of citric acid, and the concentration of the solvent is 0.05-0.2 mol/L.
It is further preferred that the concentration of the dimerized diethylenetriamine pentaacetic acid is 0.05 to 0.2mM after the dimerized diethylenetriamine pentaacetic acid is dissolved in the aqueous solution of trisodium citrate.
Further preferably, in the reaction, the molar ratio of the dimerized diethylenetriamine pentaacetic acid to the gadolinium ions is 1:2 to 5.
Still further, the molar ratio of the dimerized diethylenetriamine pentaacetic acid to gadolinium ions is 1:2.5 to 3.5.
The molar ratio is 1: 2-5, the combined effect of the two can be optimized; and when the molar ratio is further optimized to 1:2.5 to 3.5, not only ensuring the full combination of Gd ions and di-DTPA, but also avoiding the waste of Gd ions caused by excessive raw materials.
Further preferably, the pH of the mixed solution of the reaction is 4.5-6.5, the reaction temperature is room temperature, and the reaction time is 6-24 hours.
Further preferably, the purification method is dialysis, lyophilization.
Further preferably, the di-Gd-DTPA comprises any one of Gd-6-Gd, gd-8-Gd and Gd-12-Gd, and sequentially corresponds to the following structural formula:
preferably, the specific method of the step (4) is as follows:
dissolving the dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA) and the alpha Olig2-Cy5 antibody probe in water for reaction, and purifying after the reaction is finished to obtain alpha Olig2-Cy5@di-Gd-DTPA, thereby completing the nondestructive image marking of the alpha Olig 2.
Further preferably, the concentration of the αolig2—Cy5 antibody probe after dissolution is 0.05 to 0.2. Mu.M, and the molar ratio of the αolig2—Cy5 antibody probe to the gadolinium dimerized diethylenetriamine pentaacetic acid is 1:1 to 10.
Still further, the molar ratio of the alpha Olig2-Cy5 antibody probe to the dimerized diethylenetriamine pentaacetic acid gadolinium is 1:6 to 10.
Further preferably, the reaction temperature is room temperature and the reaction time is 1 to 6 hours.
In a second aspect of the invention, an alpha Olig2-Cy5@di-Gd-DTPA antibody probe with excellent targeting binding capability and high image marking efficiency is provided, and the antibody probe is prepared by the method provided by the first aspect of the invention.
In a third aspect of the invention there is provided the use of an alpha Olig2-Cy5@di-Gd-DTPA antibody probe of the second aspect of the invention, in particular as a marker for fluorescence imaging and T1 magnetic resonance imaging of brain glioma.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention provides a nondestructive image marking method of alpha Olig2, which has simple process and high image marking efficiency, can keep the targeting binding capacity of antibody protein and endow the antibody protein with an image function.
The invention provides an alpha Olig2-Cy5@di-Gd-DTPA antibody probe which has strong targeting binding capacity and a fluorescence/magnetic resonance double imaging function.
The invention provides an application of an alpha Olig2-Cy5@di-Gd-DTPA antibody probe, which is used as a marker for fluorescence imaging and T1 magnetic resonance imaging of brain glioma and has a wide application prospect in specific diagnosis of clinical diseases.
Drawings
FIG. 1 is a graph and graph of the r1 relaxation rate measured for αOlig2-Cy5@Gd-8-Gd of example 1 in a magnetic field strength of 3.0T;
FIG. 2 is a graph and graph of the r1 relaxation rate measured for αOlig2-Cy5@Gd-8-Gd of example 1 in a 5.0T magnetic field strength;
FIG. 3 is a graph of flow cytometry data measured after incubation of example 1. Alpha. Olig2-Cy5@Gd-8-Gd with brain glioma cells for 1 h;
FIG. 4 is a graph showing the result of WB (Western blotting) after antigen-antibody reaction between the total protein extracted from the glioma cells and the αOlig2-Cy5@Gd-8-Gd of example 1.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
Wherein, alpha Olig2 related to the invention is purchased from Wuhansai Weibull biotechnology Co., ltd (product number: GB 11766), gadolinium ions are derived from gadolinium chloride hexahydrate (product number: 481618) provided by Beijing carboline technology Co., ltd, and diethylenetriamine pentaacetic acid is purchased from Shanghai Ala Di Biotechnology Co., ltd (product number: D106364). Mouse glioma cells (GL 261) were supplied by Shanghai Fuheng Biotechnology Inc. (cat# FH 1097).
Example 1
The nondestructive image marking method of alpha Olig2 comprises the following steps:
(1) Dispersing 0.1 mu M of alpha Olig2 and a set content of Sulfo-Cy5-NHS in 0.1M of sodium bicarbonate aqueous solution, regulating the pH of the obtained mixed solution to 8.5 through NaOH solution, rapidly stirring at room temperature for reaction set time, transferring the obtained reaction solution into a ultrafilter tube with the cut-off molecular weight of 10Kda, centrifuging, washing with deionized water for three times, and freeze-drying to obtain light blue solid, namely the alpha Olig2-Cy5 antibody probe;
(2) Dissolving 0.1mM 1, 8-octanediamine and a preset content of diethylenetriamine pentaacetic anhydride (DTPAA) in 0.1M sodium bicarbonate aqueous solution, regulating the pH of the obtained mixed solution to 8.5 by NaOH solution, rapidly stirring at room temperature for reaction for 24 hours, transferring the obtained reaction solution into dialysis with the molecular weight of 100-500, dialyzing in deionized water for three times, and freeze-drying the dialyzed reaction solution to obtain a white solid product which is recorded as 8-DTPA;
(3) Dissolving 0.1mM 8-DTPA and gadolinium chloride hexahydrate with a set content in 0.1M ammonium acetate aqueous solution, regulating the pH of the obtained mixed solution to 5.5 by hydrochloric acid, rapidly stirring at room temperature for reaction for a set time, transferring the obtained reaction solution into dialysis with a cut-off molecular weight of 500-1000, dialyzing in deionized water for three times, and freeze-drying the dialyzed reaction solution to obtain a white solid product which is marked as Gd-8-Gd;
(4) Dispersing 0.1 mu M alpha Olig2-Cy5 and Gd-8-Gd with a set content in water, rapidly stirring at room temperature for reaction for a set time, transferring the obtained reaction liquid into a ultrafiltration tube with a cut-off molecular weight of 10Kda, centrifuging, washing with deionized water for three times, and freeze-drying to obtain light blue solid which is marked as alpha Olig2-Cy5@Gd-8-Gd.
In the above step (1), the amount of Sulfo-Cy5-NHS (i.e., the ratio of the same to alpha Olig 2) and the reaction time were controlled, respectively, and the Cy5 labeling efficiency of alpha Olig2 was counted by fluorescence spectrum analysis to investigate the effect of the parameters on the labeling efficiency. The parameter settings and statistics are shown in Table 1-1.
Table 1-1: cy5 labeling efficiency of alpha Olig2-Cy5 at different molar ratios of alpha Olig2 to Sulfo-Cy5-NHS and different reaction times
| Name of the name | Sulfo-Cy5-NHS content (. Mu.M) | Reaction time (h) | Cy5 labeling efficiency (%) |
| 1-1 | 0.2 | 6 | 33 |
| 1-2 | 0.4 | 6 | 73 |
| 1-3 | 0.6 | 6 | 52 |
| 1-4 | 0.4 | 1 | 48 |
The fluorescence intensities of the αolig2-Cy5 antibody probes obtained at different molar ratios for αolig2 and Sulfo-Cy5-NHS were recorded and the results are shown in tables 1-2.
Table 1-2: alpha Olig2-Cy5 corresponding fluorescence intensities at different molar ratios of alpha Olig2 to Sulfo-Cy5-NHS and different reaction times (Ex=646nm, ex=664 nm)
In the above step (2), the amount of diethylenetriamine pentaacetic anhydride (DTPAA) (i.e., the ratio of DTPAA to 1, 8-octanediamine) was controlled, and the yield of 8-DTPA was counted to investigate the effect of the parameters on the yield of 8-DTPA. The parameter settings and statistics are shown in table 2.
Table 2: yields of 8-DTPA corresponding to 1, 8-octanediamine and DTPAA in different molar ratios
| Name of the name | DTPAA content (mM) | Yield of 8-DTPA (%) |
| 2-1 | 0.2 | 53 |
| 2-2 | 0.25 | 66 |
| 2-3 | 0.5 | 36 |
In the step (3), the dosage of gadolinium chloride hexahydrate (namely, the proportion of gadolinium ions and 8-DTPA) is controlled, and the yield of Gd-8-Gd is counted so as to study the influence of parameters on the Gd-8-Gd yield. The parameter settings and statistics are shown in table 3.
Table 3: gd-8-Gd yield of 8-DTPA and gadolinium ions corresponding to different molar ratios
| Name of the name | Gadolinium chloride hexahydrate content (mM) | Gd-8-Gd yield (%) |
| 3-1 | 0.2 | 61 |
| 3-2 | 0.25 | 78 |
| 3-3 | 0.35 | 46 |
In the step (4), the amount of Gd-8-Gd (i.e. the ratio of Gd-8-Gd to alpha Olig2-Cy 5) and the reaction time are controlled, and ICP-MASS analysis is adopted to count the Gd ion labeling efficiency of alpha Olig2 so as to study the influence of parameters on the labeling efficiency. The parameter settings and statistics are shown in table 4.
Table 4: gd ion marking efficiency corresponding to alpha Olig2-Cy5 and Gd-8-Gd under different molar ratios and reaction time
| Name of the name | Gd-8-Gd content (mu M) | Reaction time (h) | Gd ion marking efficiency (%) |
| 4-1 | 0.5 | 6 | 72 |
| 4-2 | 0.75 | 6 | 83 |
| 4-3 | 1 | 6 | 66 |
| 4-4 | 0.75 | 1 | 47 |
In summary, the optimal preparation process of alpha Olig2-Cy5@Gd-8-Gd is as follows: (1) the reaction molar ratio of alpha Olig2 to Sulfo-Cy5-NHS is 1:4, the fluorescence labeling efficiency of alpha Olig2-Cy5 is highest when the reaction time is 6 hours; (2) the molar ratio of 1, 8-octanediamine to DTPAA is 1: the yield of 8-DTPA is highest at 2.5; (3) similarly, the molar ratio of 8-DTPA to gadolinium ions is 1:2.5, the yield of Gd-8-Gd is highest; (4) And finally, when the molar ratio of the alpha Olig2-Cy5 obtained by the highest efficiency to the Gd-8-Gd obtained by the highest efficiency is 0.75 and the reaction time is 6 hours, obtaining the alpha Olig2-Cy5@Gd-8-Gd with the highest fluorescence and magnetic resonance marking efficiency.
Example 2
The nondestructive image marking method of alpha Olig2 comprises the following steps:
(1) Dispersing 0.1 mu M of alpha Olig2 and 0.4 mu M of Sulfo-Cy5-NHS in 0.1M of sodium bicarbonate water solution, regulating the pH of the obtained mixed solution to 8.5 through NaOH solution, rapidly stirring at room temperature for reaction for 6 hours, transferring the obtained reaction solution into a ultrafiltration tube with the cut-off molecular weight of 10Kda, centrifuging, washing with deionized water for three times, and freeze-drying to obtain light blue solid, namely the alpha Olig2-Cy5 antibody probe;
(2) Dissolving 0.1mM 1, 6-octanediamine and 2.5mM diethylenetriamine pentaacetic anhydride (DTPAA) in 0.1M sodium bicarbonate aqueous solution, regulating the pH of the obtained mixed solution to 8.5 by NaOH solution, rapidly stirring at room temperature for reaction for 24 hours, transferring the obtained reaction solution into dialysis with the molecular weight of 100-500, dialyzing in deionized water for three times, and freeze-drying the dialyzed reaction solution to obtain a white solid product which is denoted as 6-DTPA;
(3) Dissolving 0.1mM 6-DTPA and 2.5mM gadolinium chloride hexahydrate in 0.1M ammonium acetate aqueous solution, regulating the pH of the obtained mixed solution to 5.5 by hydrochloric acid, rapidly stirring at room temperature for reaction for 6 hours, transferring the obtained reaction solution into dialysis with the molecular weight of 500-1000, dialyzing in deionized water for three times, and freeze-drying the dialyzed reaction solution to obtain a white solid product which is marked as Gd-6-Gd;
(4) Dispersing 0.1 mu M alpha Olig2-Cy5 and Gd-6-Gd with a set content in water, rapidly stirring at room temperature for reaction for a set time, transferring the obtained reaction liquid into a ultrafiltration tube with a cut-off molecular weight of 10Kda, centrifuging, washing with deionized water for three times, and freeze-drying to obtain light blue solid which is marked as alpha Olig2-Cy5@Gd-6-Gd.
In the step (4), the amount of Gd-6-Gd (i.e. the ratio of Gd-6-Gd to alpha Olig2-Cy 5) and the reaction time are controlled, and ICP-MASS analysis is adopted to count the Gd ion labeling efficiency of alpha Olig2 so as to study the influence of parameters on the labeling efficiency. The parameter settings and statistics are shown in table 5.
Table 5: gd ion marking efficiency corresponding to alpha Olig2-Cy5 and Gd-6-Gd under different molar ratios and reaction time
| Name of the name | Gd-6-Gd content (mu M) | Reaction time (h) | Gd ion marking efficiency (%) |
| 5-1 | 0.5 | 6 | 53 |
| 5-2 | 0.75 | 6 | 68 |
| 5-3 | 1 | 6 | 43 |
| 5-4 | 0.75 | 1 | 36 |
In summary, when the molar ratio of alpha Olig2-Cy5 to Gd-6-Gd is 0.75 and the reaction time is 6 hours, the alpha Olig2-Cy5@Gd-6-Gd with highest gadolinium ion marking efficiency is obtained. Under the same preparation process conditions, the marking efficiency of Gd-6-Gd is lower than that of Gd-8-Gd.
Example 3
The nondestructive image marking method of alpha Olig2 comprises the following steps:
(1) Dispersing 0.1 mu M of alpha Olig2 and 0.4 mu M of Sulfo-Cy5-NHS in 0.1M of sodium bicarbonate water solution, regulating the pH of the obtained mixed solution to 8.5 through NaOH solution, rapidly stirring at room temperature for reaction for 6 hours, transferring the obtained reaction solution into a ultrafiltration tube with the cut-off molecular weight of 10Kda, centrifuging, washing with deionized water for three times, and freeze-drying to obtain light blue solid, namely the alpha Olig2-Cy5 antibody probe;
(2) Dissolving 0.1mM 1, 12-octanediamine and 2.5mM diethylenetriamine pentaacetic anhydride (DTPAA) in 0.1M sodium bicarbonate aqueous solution, regulating the pH of the obtained mixed solution to 8.5 by NaOH solution, rapidly stirring at room temperature for reaction for 24 hours, transferring the obtained reaction solution into dialysis with the molecular weight of 100-500, dialyzing in deionized water for three times, and freeze-drying the dialyzed reaction solution to obtain a white solid product which is denoted as 12-DTPA;
(3) Dissolving 0.1mM of 12-DTPA and 2.5mM of gadolinium chloride hexahydrate in 0.1M of ammonium acetate aqueous solution, regulating the pH of the obtained mixed solution to 5.5 by hydrochloric acid, rapidly stirring at room temperature for reaction for 6 hours, transferring the obtained reaction solution into dialysis with the molecular weight of 500-1000, dialyzing in deionized water for three times, and freeze-drying the dialyzed reaction solution to obtain a white solid product which is marked as Gd-12-Gd;
(4) Dispersing 0.1 mu M alpha Olig2-Cy5 and Gd-12-Gd with a set content in water, rapidly stirring at room temperature for reaction for a set time, transferring the obtained reaction liquid into a ultrafiltration tube with a cut-off molecular weight of 10Kda, centrifuging, washing with deionized water for three times, and freeze-drying to obtain light blue solid which is denoted as alpha Olig2-Cy5@Gd-12-Gd.
In the step (4), the amount of Gd-12-Gd (i.e. the ratio of Gd-12-Gd to alpha Olig2-Cy 5) and the reaction time are controlled, and ICP-MASS analysis is adopted to count the Gd ion labeling efficiency of alpha Olig2 so as to study the influence of parameters on the labeling efficiency. The parameter settings and statistics are shown in table 6.
Table 6: gd ion marking efficiency corresponding to alpha Olig2-Cy5 and Gd-12-Gd under different molar ratios and reaction time
| Name of the name | Gd-12-Gd containingQuantity (mu M) | Reaction time (h) | Gd ion marking efficiency (%) |
| 6-1 | 0.5 | 6 | 23 |
| 6-2 | 0.75 | 6 | 36 |
| 6-3 | 1 | 6 | 12 |
| 6-4 | 0.75 | 1 | 15 |
In summary, when the molar ratio of alpha Olig2-Cy5 to Gd-12-Gd is 0.75 and the reaction time is 6 hours, the alpha Olig2-Cy5@Gd-12-Gd with highest gadolinium ion marking efficiency is obtained. Under the same preparation process conditions, the marking efficiency of Gd-12-Gd is lower than that of Gd-6-Gd and Gd-8-Gd.
Test example 1
T1 magnetic resonance imaging and r1 relaxation rate determination of alpha Olig 2-Cy5@Gd-8-Gd:
200 mu L of alpha Olig2-Cy5@Gd-8-Gd and Gd-8-Gd containing 0.25, 0.5, 1.0 and 2.0mM Gd ions are respectively added into a 96-well plate, and the contrast is made by using a clinical contrast agent Gd-DTPA. The 96-well plate was placed in a 3.0T magnetic resonance imager for T1 MRI imaging and T1 mapping analysis. The T1 relaxation time was fitted to T1 mapping by mcsfdicomeyer software,the r1 relaxation rate is the gadolinium ion concentration (mM) and the inverse of the T1 relaxation time(s) -1 ) Is a linear equation slope value for (2). The test results are shown in figures 1 and 2, and after the alpha Olig2-Cy5 is compounded by Gd-8-Gd, the T1 relaxation rates of the alpha Olig2-Cy5 and the Gd-8-Gd are close to each other and are higher than those of the clinical Gd-DTPA. Likewise, the same results were measured in a 5.0T magnetic resonance imager.
Test example 2
Identification of targeting efficiency of alpha Olig2 after fluorescent and gadolinium ion labelling by flow cytometry:
1X 10 was added to each well of a 6-well plate 5 Brain glioma cells (GL 261) of individual mice were grown for 48h and then washed with pancreatin and PBS. Flow cytometric analysis was performed after incubation of cells collected from each well with αOlig2-Cy5@Gd-8-Gd for 1 h. Alpha IgG-Cy5@Gd-8-Gd prepared according to the optimal preparation process method of aOlig2-Cys@Gd-8-Gd is used as a contrast, and PBS is used as a blank contrast. The test result is shown in fig. 3, the alpha Olig2-Cy5@Gd-8-Gd can efficiently mark GL261, and the marking efficiency is obviously higher than that of alpha IgG-Cy5@Gd-8-Gd, which shows that alpha Olig2 has the capability of specifically marking brain glioma cells with high Olig2 protein expression after fluorescence and gadolinium ion marking.
Test example 3
Identifying the antigen-antibody binding efficiency of alpha Olig2 after fluorescent labeling and gadolinium ion labeling by using Western-blotting:
1X 10 was added to each well of a 6-well plate 5 Brain glioma cells (GL 261) of individual mice were grown for 48h and then washed with pancreatin and PBS. After protein (total protein) extraction, western-blotting is performed on cells collected from each well, wherein the primary antibody is incubated with unmodified and modified alpha Olig2 during the binding process, respectively. The test results are shown in fig. 4, and the alpha Olig2 still maintains the antigen-antibody binding capacity similar to that of the pure alpha Olig2 after fluorescence and gadolinium ion labeling, so that the binding of alpha Olig2-Cy5@Gd-8-Gd to GL261 cells is a specific label realized through antigen-antibody binding.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.
Claims (10)
1. A method for non-destructive imaging labeling of alpha Olig2, which retains the targeted binding capacity of an antibody protein, comprising the steps of:
(1) Introducing Cy5 into alpha Olig2 to obtain an alpha Olig2-Cy5 antibody probe;
(2) Reacting n-alkyl diamine with diethylenetriamine pentaacetic anhydride to obtain dimerized diethylenetriamine pentaacetic acid;
(3) Complexing the dimerized diethylenetriamine pentaacetic acid with gadolinium ions to form dimerized diethylenetriamine pentaacetic acid gadolinium;
(4) And the alpha Olig2-Cy5 antibody probe reacts with the dimerized diethylenetriamine pentaacetic acid gadolinium to obtain alpha Olig2-Cy5@di-Gd-DTPA, and the nondestructive image marking of the alpha Olig2 is completed.
2. The method according to claim 1, wherein the specific method of step (1) is as follows:
dissolving alpha Olig2 and Sulfo-Cy5-NHS in 0.05-0.2M sodium bicarbonate water solution, controlling the concentration of the alpha Olig2 to be 0.05-0.2 mu M after dissolving, and controlling the molar ratio of the alpha Olig2 to the Sulfo-Cy5-NHS to be 1:1 to 10; the pH of the obtained mixed solution is regulated to 7.5-9.5, the mixed solution reacts for 1-6 hours at room temperature, and after the reaction is finished, the alpha Olig2-Cy5 antibody probe is obtained through purification.
3. The method according to claim 1, wherein the specific method of step (2) is as follows:
dissolving n-alkyl diamine and diethylenetriamine pentaacetic anhydride in 0.05-0.2M sodium bicarbonate aqueous solution, controlling the concentration of the n-alkyl diamine to be 0.05-0.2 mM after dissolving, and controlling the molar ratio of the n-alkyl diamine to the diethylenetriamine pentaacetic anhydride to be 1:2 to 5; the pH value of the obtained mixed solution is regulated to 7.5-9.5, the mixed solution reacts for 12-24 hours at room temperature, and the dimeric diethylenetriamine pentaacetic acid is obtained after the reaction is finished and purified.
4. A method according to claim 3, characterized in that: the n-alkyl diamine comprises any one of 1, 6-hexamethylenediamine, 1, 8-octanediamine and 1, 12-dodecanediamine.
5. The method according to claim 1, wherein the specific method of step (3) is as follows:
dissolving the dimer diethylenetriamine pentaacetic acid in 0.05-0.2M of citric acid trinna aqueous solution to obtain a solution with the concentration of the dimer diethylenetriamine pentaacetic acid of 0.05-0.2 mM, adjusting the pH value of the obtained mixed solution to be 4.5-6.5, and then adding gadolinium ions, wherein the molar ratio of the dimer diethylenetriamine pentaacetic acid to the gadolinium ions is 1:2 to 5; and (3) reacting for 6-24 h at room temperature, and purifying after the reaction is finished to obtain the dimerized diethylenetriamine pentaacetic acid gadolinium.
6. The method of claim 5, wherein the dimerized diethylenetriamine pentaacetic acid gadolinium comprises any one of Gd-6-Gd, gd-8-Gd, gd-12-Gd, corresponding in sequence to the following structural formula:
7. the method according to claim 1, wherein the specific method of step (4) is as follows:
dissolving the alpha Olig2-Cy5 antibody probe and the dimerized diethylenetriamine pentaacetic acid gadolinium in water, wherein the concentration of the alpha Olig2-Cy5 antibody probe after dissolution is 0.05-0.2 mu M, and the molar ratio of the alpha Olig2-Cy5 antibody probe to the dimerized diethylenetriamine pentaacetic acid gadolinium is 1:1 to 10; and (3) reacting for 1-6 h at room temperature, purifying after the reaction is finished to obtain alpha Olig2-Cy5@di-Gd-DTPA, and finishing the nondestructive image marking of the alpha Olig 2.
8. The method according to any one of claims 2 to 7, wherein: the purification method is dialysis and freeze-drying.
9. An alpha Olig2-Cy5@di-Gd-DTPA antibody probe, which is characterized in that: marking obtainable by the method according to any one of claims 1 to 8.
10. Use of the αolig2-cy5@di-Gd-DTPA antibody probe as claimed in claim 9, wherein: the alpha Olig2-Cy5@di-Gd-DTPA antibody probe is used as a marker for fluorescence imaging and T1 magnetic resonance imaging of brain glioma.
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