A lossless image marking method and product of αOlig2 and its use

Abstract

The present invention discloses a non-destructive imaging labeling method and product of αOlig2 and its use, and belongs to the technical field of protein imaging labeling. The present invention labels the fluorescent molecule Cy5 into the structure of αOlig2 by chemical coupling to obtain an antibody protein αOlig2-Cy5 with fluorescence imaging function; utilizes hydrophobic interaction to compound dimerized Gd-DTPA and αOlig2-Cy5 into an antibody probe with fluorescence / magnetic resonance dual imaging function, wherein the introduction of alkyl chains in the molecular structure of dimerized Gd-DTPA increases the binding effect with αOlig2-Cy5. The antibody probe labeling method of the present invention balances the limitations of the weakened target binding ability caused by the change of protein structure in the chemical labeling process and the low image labeling efficiency in the physical compounding process, and retains the target binding ability and image labeling efficiency of the antibody protein to the greatest extent. The antibody labeling method of the present invention is simple and easy to implement, and can be used as a new method for non-destructive labeling of antibody proteins.

Description

Technical Field

[0001] The present invention relates to the technical field of protein image labeling, and in particular to a non-destructive image labeling method and product of αOlig2 and uses thereof. Background Art

[0002] Protein imaging labeling technology is of great significance in imaging at the cellular, tissue, and in vivo levels. Biotin labeling is the primary method for protein imaging labeling, which involves fluorescently labeling the target protein (antigen or antibody) for fluorescence imaging. Fluorescence imaging has high sensitivity and can perform real-time contrast imaging of the labeled substance through multi-channel fluorescence signal detection. It is particularly widely used in imaging of cellular or superficial tissues. However, due to the low tissue penetration depth and interference from the autofluorescence of living organisms, fluorescence imaging often has poor imaging effects at the in vivo level, especially in deep tissues. Magnetic resonance imaging is an important diagnostic tool in clinical practice, and its imaging has the characteristic of unlimited penetration. However, the contrast and clarity of magnetic resonance imaging are often poor. To achieve focused, specific imaging and observation of living tissues, it is often necessary to construct probe-type contrast-enhancing contrast agents. Antibody protein imaging labeling can be used as an effective method to achieve tissue-specific contrast imaging. It uses antigen-antibody binding to achieve efficient targeted contrast imaging, and uses the changes in the imaging signal exhibited by the antibody protein to quantitatively analyze specific proteins in specific tissues. Among them, the expression of transcription factor Olig2 plays an important role in the invasion and evolution of gliomas, and can be used as a key transcription factor to clarify the entry point of the glioma invasion mechanism. Therefore, accurately judging the expression of Olig2 protein in patients with brain gliomas through imaging means can achieve non-invasive diagnosis of the aggressiveness of brain gliomas. However, technically, antibody proteins are active macromolecules, and during their labeling process, it is necessary to pay attention to the impact of changes in protein structure on their targeting function. For example, changes in protein structure during chemical labeling often lead to a weakening of their targeting ability, and imaging molecules and target proteins often cannot achieve high efficiency through simple physical complexation, or imaging molecules increase their binding to proteins through electrostatic adsorption while often destroying the structure and targeting effect of the protein. Therefore, there is an urgent need to develop a non-destructive antibody protein labeling method with a simple preparation process and high imaging labeling efficiency for the specific diagnosis of clinical diseases. Summary of the Invention

[0003] In view of the above-mentioned defects of the prior art, in a first aspect of the present invention, a non-destructive imaging labeling method for αOlig2 that retains the targeting binding ability of the antibody protein is provided, comprising the following steps:

[0004] (1) Introducing Cy5 into αOlig2 to obtain an αOlig2-Cy5 antibody probe;

[0005] (2) reacting n-alkyldiamine and diethylenetriamine pentaacetic anhydride to obtain dimerized diethylenetriamine pentaacetic acid (di-DTPA);

[0006] (3) the dimerized diethylenetriamine pentaacetic acid (di-DTPA) is complexed with gadolinium ions to form dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA);

[0007] (4) The dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA) reacts with the αOlig2-Cy5 antibody probe to obtain αOlig2-Cy5@di-Gd-DTPA, thereby completing the non-destructive imaging labeling of αOlig2.

[0008] Traditional physical complexation of imaging molecules with target proteins often lacks specific interaction forces or relies on electrostatic adsorption, a method that often results in loss of protein function. To address the shortcomings of existing antibody-protein labeling technologies, the present invention utilizes a combination of biotin labeling and physical complexation to chemically couple the fluorescent molecule Cy5 to the αOlig2 structure. Simultaneously, the T1-enhanced magnetic resonance imaging molecule dimerization, Gd-DTPA, is complexed with αOlig2-Cy5 to create an antibody probe with dual fluorescence / magnetic resonance imaging capabilities.

[0009] The di-Gd-DTPA of the present invention binds to the protein via the hydrophobic structure in the di-Gd-DTPA molecule and the hydrophobic structure of the protein. The present invention utilizes the hydrophobic chain in the di-Gd-DTPA structure to increase the binding efficiency with the protein while preserving the protein function. The reaction conditions are mild, and the process ensures that the target binding ability of the antibody protein is retained after being labeled. At the same time, the antibody protein can be given imaging function, and can be used as a targeted contrast agent for diagnosing the aggressiveness of brain gliomas.

[0010] Preferably, the specific method of step (1) is as follows:

[0011] αOlig2 and Sulfo-Cy5-NHS are dissolved in a solvent, the pH of the resulting mixed solution is adjusted and reacted, and after the reaction is completed, the αOlig2-Cy5 antibody probe is obtained through purification.

[0012] Further preferably, the concentration of the αOlig2 after dissolution is 0.05-0.2 μM, and the molar ratio of the αOlig2 to the Sulfo-Cy5-NHS is 1:1-10.

[0013] Furthermore, the molar ratio of the αOlig2 to the Sulfo-Cy5-NHS is 1:2-4.

[0014] The present invention designs and adjusts the process parameters. When the molar ratio of αOlig2 to Sulfo-Cy5-NHS is 1:1-10, better labeling can be achieved; and when the molar ratio is 1:2-4, it can not only meet the requirements of efficient protein fluorescent labeling, but also prevent excessive labeling from causing damage to protein function, thereby achieving the best design effect.

[0015] More preferably, the solvent is an aqueous solution of sodium bicarbonate with a concentration of 0.05 to 0.2 mol / L.

[0016] More preferably, the pH of the mixed solution of the reaction is 7.5 to 9.5, the reaction temperature is room temperature, and the reaction time is 1 to 6 hours.

[0017] More preferably, the purification method is dialysis or freeze-drying.

[0018] Preferably, the specific method of step (2) is as follows:

[0019] n-Alkyl diamine and diethylenetriamine pentaacetic anhydride (DTPAA) are dissolved in a solvent, the pH of the obtained mixed solution is adjusted and a reaction is carried out. After the reaction is completed, the mixture is purified to obtain dimerized diethylenetriamine pentaacetic acid (di-DTPA).

[0020] More preferably, the concentration of the n-alkyldiamine after dissolution is 0.05-0.2 mM, and the molar ratio of the n-alkyldiamine to diethylenetriamine pentaacetic anhydride is 1:2-5.

[0021] More preferably, the n-alkyl diamine includes any one of 1,6-hexanediamine, 1,8-octanediamine, and 1,12-dodecanediamine.

[0022] More preferably, the solvent is an aqueous solution of sodium bicarbonate with a concentration of 0.05 to 0.2 mol / L.

[0023] More preferably, the pH of the mixed solution of the reaction is 7.5 to 9.5, the reaction temperature is room temperature, and the reaction time is 12 to 24 hours.

[0024] More preferably, the purification method is dialysis or freeze-drying.

[0025] Preferably, the specific method of step (3) is as follows:

[0026] The dimerized diethylenetriamine pentaacetic acid (di-DTPA) is dissolved in a solvent, the pH of the resulting mixed solution is adjusted, and then gadolinium ions are added and reacted. After the reaction is completed, the dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA) is obtained through purification.

[0027] More preferably, the solvent is a trisodium citrate aqueous solution with a concentration of 0.05 to 0.2 mol / L.

[0028] More preferably, after the dimerized diethylenetriamine pentaacetic acid is dissolved in the trisodium citrate aqueous solution, the concentration of the dimerized diethylenetriamine pentaacetic acid is 0.05 to 0.2 mM.

[0029] Further preferably, in the reaction, the molar ratio of the dimerized diethylenetriaminepentaacetic acid to the gadolinium ions is 1:2-5.

[0030] Furthermore, the molar ratio of the dimerized diethylenetriaminepentaacetic acid to gadolinium ions is 1:2.5-3.5.

[0031] Under the above conditions of a molar ratio of 1:2 to 5, the effect of the combination of the two can be optimized; and when the molar ratio is further optimized to 1:2.5 to 3.5, it can not only ensure the full combination of Gd ions and di-DTPA, but also avoid the waste of Gd ions caused by excessive raw materials.

[0032] More preferably, the pH of the mixed solution of the reaction is 4.5 to 6.5, the reaction temperature is room temperature, and the reaction time is 6 to 24 hours.

[0033] More preferably, the purification method is dialysis or freeze-drying.

[0034] Further preferably, the di-Gd-DTPA includes any one of Gd-6-Gd, Gd-8-Gd, and Gd-12-Gd, which correspond to the following structural formulas:

[0035]

[0036]

[0037] Preferably, the specific method of step (4) is as follows:

[0038] The dimerized diethylenetriamine pentaacetic acid gadolinium (di-Gd-DTPA) and the αOlig2-Cy5 antibody probe are dissolved in water and reacted. After the reaction is completed, the mixture is purified to obtain αOlig2-Cy5@di-Gd-DTPA, thereby completing the non-destructive imaging labeling of αOlig2.

[0039] Further preferably, the concentration of the αOlig2-Cy5 antibody probe after dissolution is 0.05-0.2 μM, and the molar ratio of the αOlig2-Cy5 antibody probe to the dimerized diethylenetriamine pentaacetic acid gadolinium is 1:1-10.

[0040] Furthermore, the molar ratio of the αOlig2-Cy5 antibody probe to the dimerized diethylenetriamine pentaacetic acid gadolinium is 1:6-10.

[0041] More preferably, the reaction temperature is room temperature, and the reaction time is 1 to 6 hours.

[0042] In the second aspect of the present invention, an αOlig2-Cy5@di-Gd-DTPA antibody probe with excellent antibody protein targeting binding ability and high image labeling efficiency is provided. The antibody probe is prepared by the method provided by the first aspect of the present invention.

[0043] In the third aspect of the present invention, an application of the αOlig2-Cy5@di-Gd-DTPA antibody probe according to the second aspect of the present invention is provided, specifically as a marker for fluorescence imaging and T1 magnetic resonance imaging of brain gliomas.

[0044] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0045] The present invention provides a non-destructive imaging labeling method for αOlig2. The method has a simple process and high imaging labeling efficiency, can retain the targeting binding ability of the antibody protein and impart the antibody protein with imaging function.

[0046] The present invention provides an αOlig2-Cy5@di-Gd-DTPA antibody probe, which has strong targeting and binding ability and has fluorescence / magnetic resonance dual imaging functions.

[0047] The present invention provides an application of an αOlig2-Cy5@di-Gd-DTPA antibody probe, which is used as a marker for fluorescence imaging and T1 magnetic resonance imaging of brain gliomas, and has broad application prospects in the specific diagnosis of clinical diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 The r1 relaxation rate curve and numerical diagram of αOlig2-Cy5@Gd-8-Gd of Example 1 measured in a magnetic field strength of 3.0 T;

[0049] Figure 2 The r1 relaxation rate curve and numerical diagram of αOlig2-Cy5@Gd-8-Gd of Example 1 measured in a magnetic field strength of 5.0 T;

[0050] Figure 3 Figure 1 is a flow cytometry data graph obtained after αOlig2-Cy5@Gd-8-Gd in Example 1 was incubated with glioma cells for 1 hour;

[0051] Figure 4This is a WB (Western-blotting) result diagram after the antigen-antibody reaction between αOlig2-Cy5@Gd-8-Gd in Example 1 and the total protein extracted from brain glioma cells. DETAILED DESCRIPTION

[0052] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0053] The αOlig2 involved in this invention was purchased from Wuhan Saiweier Biotechnology Co., Ltd. (Cat. No. GB11766), gadolinium ions were obtained from gadolinium chloride hexahydrate (Cat. No. 481618) provided by Beijing Bailingwei Technology Co., Ltd., and diethylenetriamine pentaacetic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Cat. No. D106364). Mouse glioma cells (GL261) were provided by Shanghai Fuheng Biotechnology Co., Ltd. (Cat. No. FH1097).

[0054] Example 1

[0055] αOlig2's lossless image labeling method:

[0056] (1) 0.1 μM αOlig2 and a set amount of Sulfo-Cy5-NHS were dispersed in a 0.1 M sodium bicarbonate aqueous solution. The pH of the resulting mixed solution was adjusted to 8.5 with a NaOH solution, and the mixture was rapidly stirred at room temperature for a set time. The resulting reaction solution was transferred to an ultrafiltration tube with a cutoff molecular weight of 10 kDa, centrifuged, washed three times with deionized water, and freeze-dried to obtain a light blue solid, i.e., the αOlig2-Cy5 antibody probe.

[0057] (2) 0.1 mM 1,8-octanediamine and a set amount of diethylenetriamine pentaacetic anhydride (DTPAA) were dissolved in a 0.1 M sodium bicarbonate aqueous solution. The pH of the resulting mixed solution was adjusted to 8.5 with a NaOH solution, and the mixture was rapidly stirred at room temperature for 24 h. The resulting reaction solution was dialyzed against a molecular weight cutoff of 100 to 500 and dialyzed three times in deionized water. The dialyzed reaction solution was freeze-dried to obtain a white solid product, which was recorded as 8-DTPA.

[0058] (3) 0.1 mM 8-DTPA and a set amount of gadolinium chloride hexahydrate were dissolved in a 0.1 M aqueous solution of ammonium acetate. The pH of the resulting mixed solution was adjusted to 5.5 with hydrochloric acid, and the mixture was rapidly stirred at room temperature for a set time. The resulting reaction solution was dialyzed against a molecular weight cutoff of 500-1000 and dialyzed three times in deionized water. The dialyzed reaction solution was freeze-dried to obtain a white solid product, which was designated as Gd-8-Gd.

[0059] (4) 0.1 μM αOlig2-Cy5 and a set amount of Gd-8-Gd were dispersed in water and reacted with rapid stirring at room temperature for a set time. The resulting reaction solution was transferred to an ultrafiltration tube with a cutoff molecular weight of 10 kDa. After centrifugation and washing three times with deionized water, it was freeze-dried to obtain a light blue solid, which was recorded as αOlig2-Cy5@Gd-8-Gd.

[0060] In step (1) above, the amount of Sulfo-Cy5-NHS (i.e., the ratio of Sulfo-Cy5-NHS to αOlig2) and the reaction time were controlled, and fluorescence spectroscopy was used to statistically analyze the Cy5 labeling efficiency of αOlig2 to study the effect of these parameters on labeling efficiency. The parameter settings and statistical results are shown in Table 1-1.

[0061] Table 1-1: Cy5 labeling efficiency of αOlig2-Cy5 at different αOlig2 to Sulfo-Cy5-NHS molar ratios and different reaction times

[0062] name Sulfo-Cy5-NHS content (μM) 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

[0063] The fluorescence intensity of the αOlig2-Cy5 antibody probe obtained at different molar ratios of αOlig2 and Sulfo-Cy5-NHS was recorded. The results are shown in Table 1-2.

[0064] Table 1-2: Fluorescence intensity of αOlig2-Cy5 at different αOlig2 to Sulfo-Cy5-NHS molar ratios and different reaction times (Ex = 646 nm, Ex = 664 nm)

[0065]

[0066] In step (2) above, the amount of diethylenetriamine pentaacetic anhydride (DTPAA) was controlled (i.e., the ratio of diethylenetriamine pentaacetic anhydride to 1,8-octanediamine) and the yield of 8-DTPA was statistically analyzed to study the effect of these parameters on the yield of 8-DTPA. The parameter settings and statistical results are shown in Table 2.

[0067] Table 2: 8-DTPA yields at different molar ratios of 1,8-octanediamine and DTPAA

[0068] name DTPAA content (mM) 8-DTPA yield (%) 2-1 0.2 53 2-2 0.25 66 2-3 0.5 36

[0069] In step (3), the amount of gadolinium chloride hexahydrate (i.e., the ratio of gadolinium ions to 8-DTPA) was controlled, and the yield of Gd-8-Gd was statistically analyzed to investigate the effect of these parameters on the yield of Gd-8-Gd. The parameter settings and statistical results are shown in Table 3.

[0070] Table 3: Gd-8-Gd yields at different molar ratios of 8-DTPA and gadolinium ions

[0071] 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

[0072] In step (4) above, the amount of Gd-8-Gd (i.e., the ratio of Gd-8-Gd to αOlig2-Cy5) and reaction time were controlled, and ICP-MASS analysis was used to statistically analyze the Gd ion labeling efficiency of αOlig2 to study the effect of these parameters on labeling efficiency. The parameter settings and statistical results are shown in Table 4.

[0073] Table 4: Gd ion labeling efficiency at different molar ratios of αOlig2-Cy5 and Gd-8-Gd and reaction times

[0074] name Gd-8-Gd content (μM) Reaction time (h) Gd ion labeling efficiency (%) 4-1 0.5 6 72 4-2 0.75 6 83 4-3 1 6 66 4-4 0.75 1 47

[0075] In summary, the optimal preparation process of αOlig2-Cy5@Gd-8-Gd is as follows: (1) The reaction molar ratio of αOlig2 and Sulfo-Cy5-NHS is 1:4, and the reaction time is 6 h, the fluorescence labeling efficiency of αOlig2-Cy5 is the highest; (2) The yield of 8-DTPA is the highest when the molar ratio of 1,8-octanediamine and DTPAA is 1:2.5; (3) Similarly, the yield of Gd-8-Gd is the highest when the molar ratio of 8-DTPA and gadolinium ion is 1:2.5; (4) Finally, when the molar ratio of the αOlig2-Cy5 obtained with the highest efficiency and the Gd-8-Gd obtained with the highest efficiency is 0.75 and the reaction time is 6 h, the αOlig2-Cy5@Gd-8-Gd with the highest fluorescence and magnetic resonance labeling efficiency is obtained.

[0076] Example 2

[0077] αOlig2's lossless image labeling method:

[0078] (1) 0.1 μM αOlig2 and 0.4 μM Sulfo-Cy5-NHS were dispersed in a 0.1 M sodium bicarbonate aqueous solution. The pH of the resulting mixed solution was adjusted to 8.5 with a NaOH solution, and the mixture was rapidly stirred at room temperature for 6 h. The resulting reaction solution was transferred to an ultrafiltration tube with a cutoff molecular weight of 10 kDa, centrifuged, washed three times with deionized water, and freeze-dried to obtain a light blue solid, i.e., the αOlig2-Cy5 antibody probe.

[0079] (2) 0.1 mM 1,6-octanediamine and 2.5 mM diethylenetriamine pentaacetic anhydride (DTPAA) were dissolved in 0.1 M sodium bicarbonate aqueous solution. The pH of the resulting mixed solution was adjusted to 8.5 with NaOH solution, and the mixture was rapidly stirred at room temperature for 24 h. The resulting reaction solution was dialyzed against a molecular weight cutoff of 100-500 and dialyzed three times in deionized water. The dialyzed reaction solution was freeze-dried to obtain a white solid product, which was recorded as 6-DTPA.

[0080] (3) 0.1 mM 6-DTPA and 2.5 mM gadolinium chloride hexahydrate were dissolved in a 0.1 M aqueous solution of ammonium acetate. The pH of the resulting mixed solution was adjusted to 5.5 with hydrochloric acid and then rapidly stirred at room temperature for 6 h. The resulting reaction solution was dialyzed against a molecular weight cutoff of 500-1000 and dialyzed three times in deionized water. The dialyzed reaction solution was freeze-dried to obtain a white solid product, designated as Gd-6-Gd.

[0081] (4) 0.1 μM αOlig2-Cy5 and a set amount of Gd-6-Gd were dispersed in water and reacted with rapid stirring at room temperature for a set time. The resulting reaction solution was transferred to an ultrafiltration tube with a cutoff molecular weight of 10 kDa. After centrifugation and washing three times with deionized water, it was freeze-dried to obtain a light blue solid, which was recorded as αOlig2-Cy5@Gd-6-Gd.

[0082] In step (4) above, the amount of Gd-6-Gd (i.e., the ratio of Gd-6-Gd to αOlig2-Cy5) and reaction time were controlled, and ICP-MASS analysis was used to statistically analyze the Gd ion labeling efficiency of αOlig2 to study the effect of these parameters on labeling efficiency. The parameter settings and statistical results are shown in Table 5.

[0083] Table 5: Gd ion labeling efficiency at different molar ratios of αOlig2-Cy5 and Gd-6-Gd and reaction times

[0084] name Gd-6-Gd content (μM) Reaction time (h) Gd ion labeling efficiency (%) 5-1 0.5 6 53 5-2 0.75 6 68 5-3 1 6 43 5-4 0.75 1 36

[0085] In summary, a molar ratio of αOlig2-Cy5 to Gd-6-Gd of 0.75 and a reaction time of 6 hours yielded the highest gadolinium ion labeling efficiency, αOlig2-Cy5@Gd-6-Gd. Under the same preparation conditions, the labeling efficiency of Gd-6-Gd was lower than that of Gd-8-Gd.

[0086] Example 3

[0087] αOlig2's lossless image labeling method:

[0088] (1) 0.1 μM αOlig2 and 0.4 μM Sulfo-Cy5-NHS were dispersed in a 0.1 M sodium bicarbonate aqueous solution. The pH of the resulting mixed solution was adjusted to 8.5 with a NaOH solution, and the mixture was rapidly stirred at room temperature for 6 h. The resulting reaction solution was transferred to an ultrafiltration tube with a cutoff molecular weight of 10 kDa, centrifuged, washed three times with deionized water, and freeze-dried to obtain a light blue solid, i.e., the αOlig2-Cy5 antibody probe.

[0089] (2) 0.1 mM 1,12-octanediamine and 2.5 mM diethylenetriamine pentaacetic anhydride (DTPAA) were dissolved in 0.1 M sodium bicarbonate aqueous solution. The pH of the resulting mixed solution was adjusted to 8.5 with NaOH solution, and the mixture was rapidly stirred at room temperature for 24 h. The resulting reaction solution was dialyzed against a molecular weight cutoff of 100-500 and dialyzed three times in deionized water. The dialyzed reaction solution was freeze-dried to obtain a white solid product, which was recorded as 12-DTPA.

[0090] (3) 0.1 mM 12-DTPA and 2.5 mM gadolinium chloride hexahydrate were dissolved in a 0.1 M aqueous solution of ammonium acetate. The pH of the resulting mixed solution was adjusted to 5.5 with hydrochloric acid and then rapidly stirred at room temperature for 6 h. The resulting reaction solution was dialyzed against a molecular weight cutoff of 500-1000 and dialyzed three times in deionized water. The dialyzed reaction solution was freeze-dried to obtain a white solid product, designated as Gd-12-Gd.

[0091] (4) 0.1 μM αOlig2-Cy5 and a set amount of Gd-12-Gd were dispersed in water and reacted with rapid stirring at room temperature for a set time. The resulting reaction solution was transferred to an ultrafiltration tube with a cutoff molecular weight of 10 kDa. After centrifugation and washing three times with deionized water, it was freeze-dried to obtain a light blue solid, which was recorded as αOlig2-Cy5@Gd-12-Gd.

[0092] In step (4) above, the amount of Gd-12-Gd (i.e., its ratio to αOlig2-Cy5) and reaction time were controlled, and ICP-MASS analysis was used to statistically analyze the Gd ion labeling efficiency of αOlig2 to study the effect of these parameters on labeling efficiency. The parameter settings and statistical results are shown in Table 6.

[0093] Table 6: Gd ion labeling efficiency at different molar ratios of αOlig2-Cy5 and Gd-12-Gd and reaction times

[0094] name Gd-12-Gd content (μM) Reaction time (h) Gd ion labeling efficiency (%) 6-1 0.5 6 23 6-2 0.75 6 36 6-3 1 6 12 6-4 0.75 1 15

[0095] In summary, a molar ratio of αOlig2-Cy5 to Gd-12-Gd of 0.75 and a reaction time of 6 hours yielded the highest gadolinium ion labeling efficiency, αOlig2-Cy5@Gd-12-Gd. Under the same preparation conditions, the labeling efficiency of Gd-12-Gd was lower than that of Gd-6-Gd and Gd-8-Gd.

[0096] Test Example 1

[0097] T1 MRI and r1 relaxivity determination of αOlig2-Cy5@Gd-8-Gd:

[0098] 200 μL of αOlig2-Cy5@Gd-8-Gd and Gd-8-Gd containing 0.25, 0.5, 1.0, and 2.0 mM Gd ion content was added to a 96-well plate, respectively, and the clinical contrast agent Gd-DTPA was used as a comparison. The 96-well plate was placed on a 3.0T magnetic resonance imaging system for T1MRI imaging and T1 mapping analysis. T1 relaxation time was obtained by fitting T1 mapping using McsfDicomViewer software. The r1 relaxation rate is the reciprocal of the gadolinium ion concentration (mM) and the T1 relaxation time (s -1 ) is the slope value of the linear equation. The test results are as follows Figure 1 and 2 As shown in the figure, after αOlig2-Cy5 was complexed with Gd-8-Gd, the T1 relaxation rates of both were similar and higher than those of the clinically used Gd-DTPA, as measured by a 3.0T MRI. Similarly, the same results were obtained using a 5.0T MRI.

[0099] Test Example 2

[0100] Flow cytometry was used to identify the targeting efficiency of αOlig2 after fluorescent labeling and gadolinium ion labeling:

[0101] 1×10 5 Mouse glioma cells (GL261) were cultured for 48 hours, digested with trypsin, and washed with PBS. The cells collected from each well were incubated with αOlig2-Cy5@Gd-8-Gd for 1 hour and then analyzed by flow cytometry. αIgG-Cy5@Gd-8-Gd prepared according to the optimal preparation process of aOlig2-Cys@Gd-8-Gd was used as a control, and PBS was used as a blank control. The test results are shown in Figure 2. Figure 3 As shown in the results, αOlig2-Cy5@Gd-8-Gd can efficiently label GL261, and its labeling efficiency is significantly higher than that of αIgG-Cy5@Gd-8-Gd, indicating that αOlig2 has the ability to specifically label brain glioma cells that highly express Olig2 protein after being labeled with fluorescence and gadolinium ions.

[0102] Test Example 3

[0103] Western blotting was used to identify the antigen-antibody binding efficiency of αOlig2 after fluorescent labeling and gadolinium ion labeling:

[0104] 1×10 5 Mouse glioma cells (GL261) were cultured for 48 hours and then digested with trypsin and washed with PBS. The cells collected from each well were extracted with protein (total protein) and then subjected to Western blotting. The primary antibody was incubated with unmodified and modified αOlig2 during the binding process. The test results are shown in Figure 2. Figure 4 As shown in the figure, after being labeled with fluorescence and gadolinium ions, αOlig2 still maintains antigen-antibody binding ability similar to that of simple αOlig2, further indicating that the binding of αOlig2-Cy5@Gd-8-Gd to GL261 cells is a specific labeling achieved through antigen-antibody binding.

[0105] The above describes in detail the preferred embodiments of the present invention. It should be understood that those skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solutions that can be derived by those skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A non-destructive imaging labeling method for αOlig2 that retains the targeting binding ability of the antibody protein, characterized in that: The steps include: (1) Dissolve αOlig2 and Sulfo-Cy5-NHS in a 0.05-0.2 M sodium bicarbonate aqueous solution. After dissolution, the concentration of αOlig2 is 0.05-0.2 μM, and the molar ratio of αOlig2 to Sulfo-Cy5-NHS is controlled to be 1:1-10. Adjust the pH of the resulting mixed solution to 7.5-9.5, and react at room temperature for 1-6 hours. After the reaction is completed, purify and obtain an αOlig2-Cy5 antibody probe. (2) reacting n-alkyldiamine and diethylenetriamine pentaacetic anhydride to obtain dimerized diethylenetriamine pentaacetic acid; (3) The dimerized diethylenetriamine pentaacetic acid is complexed with gadolinium ions to form dimerized diethylenetriamine pentaacetic acid gadolinium; (4) The αOlig2-Cy5 antibody probe reacts with the dimerized diethylenetriamine pentaacetic acid gadolinium to obtain αOlig2-Cy5@di-Gd-DTPA, thereby completing the non-destructive imaging labeling of αOlig2.

2. The method according to claim 1, characterized in that The specific method of step (2) is as follows: n-Alkyldiamine and diethylenetriamine pentaacetic anhydride are dissolved in a 0.05-0.2M sodium bicarbonate aqueous solution, wherein the concentration of n-alkyldiamine after dissolution is 0.05-0.2mM, and the molar ratio of n-alkyldiamine to diethylenetriamine pentaacetic anhydride is controlled to be 1:2-5; the pH of the resulting mixed solution is adjusted to 7.5-9.5, and the mixture is reacted at room temperature for 12-24 hours. After the reaction is completed, the mixture is purified to obtain dimerized diethylenetriamine pentaacetic acid.

3. The method according to claim 2, wherein: The n-alkyl diamine includes any one of 1,6-hexanediamine, 1,8-octanediamine, and 1,12-dodecanediamine.

4. The method according to claim 1, wherein The specific method of step (3) is as follows: The dimerized diethylenetriamine pentaacetic acid is dissolved in a 0.05-0.2M aqueous solution of trisodium citrate to obtain a solution with a dimerized diethylenetriamine pentaacetic acid concentration of 0.05-0.2mM. The pH of the obtained mixed solution is adjusted to 4.5-6.5, and then gadolinium ions are added with a molar ratio of dimerized diethylenetriamine pentaacetic acid to gadolinium ions of 1:2-5. The reaction is carried out at room temperature for 6-24 hours. After the reaction is completed, the mixture is purified to obtain dimerized diethylenetriamine pentaacetic acid gadolinium.

5. The method according to claim 4, wherein The dimerized diethylenetriamine pentaacetic acid gadolinium includes any one of Gd-6-Gd, Gd-8-Gd, and Gd-12-Gd, which correspond to the following structural formulas: 、 、 。 6. The method according to claim 1, characterized in that The specific method of step (4) is as follows: The αOlig2-Cy5 antibody probe and the dimerized diethylenetriamine pentaacetic acid gadolinium are dissolved in water, wherein the concentration of the αOlig2-Cy5 antibody probe after dissolution is 0.05-0.2 μM, and the molar ratio of the αOlig2-Cy5 antibody probe to the dimerized diethylenetriamine pentaacetic acid gadolinium is 1:1-10; the reaction is carried out at room temperature for 1-6 hours, and after the reaction is completed, the αOlig2-Cy5@di-Gd-DTPA is obtained after purification, thereby completing the lossless imaging labeling of αOlig2.

7. The method according to any one of claims 2 to 6, characterized in that: The purification method is dialysis and freeze-drying.

8. An αOlig2-Cy5@di-Gd-DTPA antibody probe, characterized by: The method is described in any one of claims 1 to 7.

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