Synthesis method and application of radioactive probe 68Ga-NOTA-mAbKv 1.3

Abstract

The invention relates to the field of rheumatoid arthritis nuclide imaging, and discloses a synthesis method and application of a radioactive probe 68Ga-NOT A-mAbKv 1.3. The synthesis method comprises the following steps: firstly, producing a mAbKv 1.3 monoclonal antibody, performing immunohistochemical staining, and producing the mAbKv 1.3 monoclonal antibody, namely inoculating a hybridoma cell AbM-F14FTSSCLS0167-KLH # 15 into the abdominal cavity of a mouse to obtain ascites containing the monoclonal antibody; the mAbKv 1.3 monoclonal antibody is purified from the ascites by using an affinity chromatography method; the method comprises the following steps: firstly, synthesizing a mA bKv 1.3 antibody, then synthesizing a radioactive probe 68Ga-NOTA-mAbKv 1.3, and sequentially connecting the mA bKv 1.3 antibody with NOTA, purifying the NOTA-mAbKv 1.3, chelating the NOTA-mAbKv 1.3 with 68Ga, determining the labeling rate of the 68Ga-NOTA-mAbKv 1.3, purifying the 68Ga-NOTA-mAbKv 1.3, and determining the radiochemical purity of the 68Ga-NOTA-mAbKv 1.3, so as to synthesize the radioactive probe 68Ga-NOTA-mAbKv 1.3. According to the invention, the problem that the inflammatory activity of immune cells at the rheumatoid arthritis diseased region is revealed in a living body state due to lack of appropriate targets and probes in clinical application at present is solved.

Description

Technical Field

[0001] The present invention relates to the field of radionuclide imaging of rheumatoid arthritis, and in particular to a radioactive probe 68 Ga-NOTA-mAb Kv 1.3 Synthesis methods and applications. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic inflammatory joint disease that affects approximately 1 in 200 adults worldwide. A diagnosis of RA often results in significant disability, premature job loss, and reduced life expectancy, placing a heavy burden on individuals and society. To improve outcomes for patients with arthritis, early diagnosis and timely initiation of treatment are key, along with the development of new therapeutic strategies to precisely control inflammation and prevent the ensuing damage.

[0003] Currently, there is no universally accepted gold standard for the diagnosis of rheumatoid arthritis (RA). Patients usually present with typical clinical symptoms such as joint pain, swelling, morning stiffness, and abnormal laboratory tests (such as rheumatoid factor and anti-citrullinated peptide antibodies), but these symptoms may not be obvious in the early stages of the disease. The main pathological feature of RA is synovial inflammation, which is manifested by the infiltration of innate and adaptive immune cells, including T lymphocytes, B lymphocytes, monocytes, and dendritic cells. Synovial fibroblasts accumulate and exhibit an invasive and inflammatory phenotype, which ultimately leads to joint destruction by promoting chondrocyte breakdown and osteoclastogenesis. The prominent features of inflammatory diseases are the exacerbation of autoimmune reactions and synovial infiltration mediated by inflammatory cells, which usually occur before the onset of clinical symptoms and signs. In addition, the presence of inflammatory cells is also an important indicator of disease remission, which emphasizes the need for regular reassessment during treatment. Molecular imaging, as a non-invasive technology, can provide detailed molecular information about the disease before the appearance of morphological lesions, thereby enabling the quantification of joint inflammation in vivo. This is of great value in diagnosing, grading and monitoring joint inflammation, especially when evaluating the effectiveness of treatment. 99m Tc-bisphosphonate SPECT and 18 F-FDG PET has been used to detect early signs of RA, but its lack of specificity for the inflammatory process limits its application. Therefore, it is critical to identify specific markers in inflamed tissues and develop tracer molecules that can target these markers with high specificity and efficiency.

[0004] Voltage-gated potassium channel 1.3 (Kv1.3) is a channel protein that can sense voltage changes and mediate potassium ion outflow across the membrane. The channel protein is encoded by the KCNA3 gene and is a homotetramer formed by non-covalent binding of subunits of approximately 500 amino acids, including four groups of protein subunits composed of six α-helices that span the membrane six times to form a single-pore channel. The gating of Kv1.3 is controlled by the membrane potential, and changes in membrane potential can be sensed by the "voltage sensor" region containing positive charges. When the cell is activated and the membrane potential is higher than -40mv, the channel opens, allowing potassium ions to flow across the membrane, increasing the negative value of the membrane potential, and increasing the transmembrane potential difference to form a relative hyperpolarized potential. Following the potential difference, calcium ions flow into the cell, resulting in an increase in intracellular calcium ion concentration. Calcium ions are important second messengers that can activate subsequent signals and further activate cells. Kv1.3 was found to be overexpressed in the proliferation and cytokine production of various inflammatory-related immune cells. In particular, when effector memory T cells are activated, the expression of Kv1.3 increases significantly, and the number of channels increases from 200-400 to about 1500-2000. In addition, the activation of macrophages, B lymphocytes, and dendritic cells also leads to the upregulation of Kv1.3, and these cells co-migrate and aggregate with T lymphocytes during infiltration, thereby initiating a chronic inflammatory process. Studies have shown that in the synovial fluid of RA patients, the expression level of Kv1.3 is high in effector memory T cells, and Kv1.3 channel inhibitors ShK-186 and PEG-HsTX1[R14A] can preferentially inhibit these cells. Therefore, the Kv1.3 channel is considered to be a promising target for the diagnosis and monitoring of RA.

[0005] The applicant previously developed a mouse anti-Kv1.3 channel monoclonal antibody (mAb) based on the extracellular cyclic peptide E314 of the human Kv1.3 channel. Kv1.3 ), has been granted a national invention patent (patent number: ZL201610325161.7). This monoclonal antibody has the characteristics of strong affinity and high specificity, and can specifically recognize human and mouse Kv1.3 channel proteins. In vivo experiments show that it is highly safe and does not affect blood counts, biochemical indicators and organ functions after intravenous injection. Based on the above research foundation, the applicant will mAb Kv1.3 Connect with NOTA and then with nuclide 68 Ga chelation, and the radioactive probe was obtained after purification 68 Ga-NOTA-mAb Kv1.3By injecting the probe in vivo, PET / CT imaging can clearly show the site of inflammation and the degree of inflammatory damage in the mouse joints in vivo, guiding clinical diagnosis and treatment as well as prognosis, to solve the problem of lack of suitable targets and probes in the clinic, and reveal the inflammatory activity of immune cells in rheumatoid arthritis diseased joints in vivo. Summary of the invention

[0006] The present invention is intended to provide a radioactive probe 68 Ga-NOTA-mAb Kv 1.3 The synthesis method and application of this novel novel molecule solve the problem of lack of suitable targets and probes in the clinic to reveal the inflammatory activity of immune cells in rheumatoid arthritis lesions in vivo.

[0007] In order to achieve the above object, the present invention provides the following method:

[0008] A radioactive probe provided by the present invention 68 Ga-NOTA-mAb Kv 1.3 The synthesis method is:

[0009] S1.mAb Kv 1.3 Monoclonal antibody production and immunohistochemical staining

[0010] mAbs Kv1.3 The production of monoclonal antibodies is carried out by inoculating hybridoma cells AbM-F14FTSSCLS0167-KLH#15 into the peritoneal cavity of mice to obtain ascites containing monoclonal antibodies, and then purifying mAbs from the ascites by affinity chromatography. Kv 1.3 Monoclonal antibodies;

[0011] S2. Radioactive probe 68 Ga-NOTA-mAb Kv 1.3 Synthesis

[0012] mAb Kv 1.3 Antibody-NOTA conjugation, NOTA-mAb Kv 1.3 Purification, NOTA-mAb Kv 1.3 Chelation 68 Ga, 68 Ga-NOTA-mAb Kv 1.3 Labeling rate determination, 68 Ga-NOTA-mAb Kv 1.3 purification, 68 Ga-NOTA-mAb Kv 1.3 Radiochemical purity determination to achieve synthetic radioactive probes 68 Ga-NOTA-mAb Kv 1.3 .

[0013] Preferably, the mAb Kv 1.3 Step of connecting the antibody with NOTA includes: taking protein sample mAb Kv 1.3 , adding it into Na 2 CO 3 buffer solution. The dosage ratio of the protein sample mAb Kv 1.3 and the Na 2 CO 3 buffer solution is 5:1. Adjust the pH of the mixed solution to 9.0 - 10.0 to obtain a protein sample solution; Weigh p-SCN-Bn-NOTA in a light-shielded environment, prepare a DMSO solution through solid DMSO, dissolve the p-SCN-Bn-NOTA in the DMSO solution to a concentration of 100 μg / μL to obtain a p-SCN-Bn-NOTA solution, where the volume of added solid DMSO needs to be less than 5% of the total volume; Quickly mix the protein sample solution and the p-SCN-Bn-NOTA solution, place it in a constant-temperature metal heater, react at 37°C for 2 h at a constant rotation speed of 400 r / min to complete the step of connecting the mAb Kv 1.3 antibody with NOTA, and obtain NOTA-mAb Kv 1.3 reactant.

[0014] Preferably, the step of purifying the NOTA-mAb Kv 1.3 includes: Open the upper lid of the PD-10 column and cut off the lower outlet, place the PD-10 column vertically to let the storage solution in the column drain out, and discard the liquid; Fill the upper chamber of the PD-10 column with PBS until the liquid drains out, repeat 3 - 4 times, and discard the liquid each time; Place the PD-10 column on a bracket, add the NOTA-mAb Kv1.3 reactant into the PD-10 column. After the NOTA-mAb Kv1.3 reactant completely enters the PD-10 column and there is no liquid flowing out, fill the PD-10 column with PBS; Connect an EP tube to the effluent, add PBS each time, and use an enzyme-linked immunosorbent assay instrument to measure the protein concentration of each tube of solution respectively to complete the purification process of NOTA-mAb Kv 1.3 and obtain a purified NOTA-mAb Kv 1.3 solution; Aliquot the purified NOTA-mAb Kv 1.3 solution, store it at -20°C, and use it within 3 months to avoid frequent freezing and thawing.

[0015] Preferably, the step of chelating Kv 1.3 Ga by the NOTA-mAb 68 includes: Use HCl to wash 68 Ge / 68 Ga generator to obtain a solution containing 68The eluate of Ga was collected using an EP tube containing 68 the eluate of Ga; the eluate containing 68 Ga was mixed with a sodium acetate solution, the pH was adjusted to 4.0 - 4.5, and then the purified NOTA-mAb Kv 1.3 solution was added thereto to obtain a first mixture; the first mixture was placed in a constant-temperature metal heater and reacted at 37 °C for 10 - 15 min at a constant rotation speed of 400 r / min to obtain 68 Ga-NOTA-mAb Kv 1.3 reactant, completing the process of chelating Kv 1.3 Ga with NOTA-mAb 68 .

[0016] Preferably, the step of measuring the labeling rate of 68 Ga-NOTA-mAb Kv 1.3 includes: analyzing using a size exclusion chromatography column, high-performance liquid chromatography, and a radioactive detector, with 10x PBS as the mobile phase and a flow rate of 1 mL / min; taking 740 - 1110 KBq of the 68 Ga-NOTA-mAb Kv 1.3 reactant after the reaction for on-machine detection, and calculating the radioactive labeling rate based on the peak area.

[0017] Preferably, the step of purifying 68 Ga-NOTA-mAb Kv 1.3 includes: opening the upper lid of the PD-10 column and cutting off the lower outlet, placing the PD-10 column vertically to drain the storage liquid in the column, and discarding the liquid; filling the upper chamber of the PD-10 column with PBS until the liquid drains out, repeating 3 - 4 times, and discarding all the liquids; placing the PD-10 column in a ventilated lead cabinet, adding the 68 Ga-NOTA-mAb Kv 1.3 reactant to the PD-10 column, waiting for the 68 Ga-NOTA-mAb Kv 1.3 reactant to completely enter the PD-10 column and no liquid is flowing out, then filling the PD-10 column with PBS; collecting the effluent with an EP tube and measuring the radioactivity of each tube; collecting the liquid with the highest radioactivity, which is the radioactive probe 68 Ga-NOTA-mAb Kv 1.3 .

[0018] Preferably, the 68 Ga-NOTA-mAb Kv 1.3The steps of radiochemical purity determination include: using size exclusion chromatography column, high performance liquid chromatography and radioactivity detector for analysis, using 10x PBS as mobile phase, flow rate of 1mL / min; taking 740-1110KBq radioactive probe 68 Ga-NOTA-mAb Kv 1.3 The radioactive labeling rate was calculated based on the peak area.

[0019] A radioactive probe provided by the present invention 68 Ga-NOTA-mAb Kv 1.3 The application of the synthetic method is:

[0020] Includes radioactive probe 68 Ga-NOTA-mAb Kv 1.3 PET / CT imaging and radioactive probes in a mouse model of rheumatoid arthritis 68 Ga-NOTA-mAb Kv 1.3 PET / CT Monitoring During Treatment in a Mouse Rheumatoid Arthritis Model:

[0021] The radioactive probe 68 Ga-NOTA-mAb Kv 1.3 PET / CT imaging in the mouse rheumatoid arthritis model was achieved by establishing a unilateral arthritis mouse model, radioactive probe injection, anesthesia and imaging;

[0022] The radioactive probe 68 Ga-NOTA-mAb Kv 1.3 PET / CT monitoring during the treatment of mouse rheumatoid arthritis model is to establish a bilateral arthritis mouse model, give the rheumatoid arthritis treatment drug elamod to the experimental group mice, and perform radioactive probe injection, anesthesia and imaging on the experimental and control group mice during the treatment to achieve PET / CT monitoring.

[0023] The beneficial effects of the present invention are as follows: according to existing literature reports, by tracing a single immune cell (such as T lymphocytes or macrophages) to evaluate the inflammation of rheumatoid arthritis lesions, the present invention 68 Ga-NOTA-mAb Kv 1.3The probe is the world's first inflammatory cell tracing probe targeting the Kv1.3 channel. It can reflect the common inflammatory activities of various immune cells (lymphocytes, monocytes / macrophages, neutrophils, etc.) in the inflamed joints, and can more objectively and comprehensively evaluate the degree, area and location of inflammatory damage in the lesion area. Existing detection technologies used in clinical evaluation of joint inflammation, such as 18F-FDG probe PET imaging, are used for imaging of rheumatoid arthritis, but cannot detect the area and degree of inflammatory activity in the lesion area. 68 Ga-NOTA-mAb Kv 1.3 The probe is used to image the inflammatory tissue in the lesion area, which is complementary to the existing technology; the mAb developed by the present invention Kv 1.3 It has the effect of inhibiting pro-inflammatory immune cells and promoting the polarization of anti-inflammatory immune cells, and can effectively inhibit the activity of inflammatory cells in the lesion site; the invention of this scheme 68 Ga-NOTA-mAb Kv 1.3 mAb-based probes Kv 1.3 It is synthesized and has not only the therapeutic effect of the antibody, but also the imaging and tracing function. It is a new type of probe that integrates treatment and imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn according to the actual scale.

[0025] Figure 1 H&E staining and Safranin O staining of inflamed joints and normal joint sections 2-3 days after the mouse arthritis model of the present invention is completed, and mAb is used to Kv 1.3 Shows immunofluorescence staining of Kv1.3, CD3, and CD68;

[0026] Figure 2 The radioactive probe of the present invention 68 Ga-NOTA-mAb Kv 1.3 After synthesis, the samples were incubated with PBS, and the instantaneous HPLC analysis chart group was performed immediately after purification by PD-10 column, 60 min after purification, 120 min after purification, and 240 min after purification;

[0027] Figure 3 The radioactive probe was injected into the tail vein 2-3 days after the mouse arthritis model of the present invention was established. 68 Ga-NOTA-mAb Kv 1.3Afterwards, PET / CT scans were performed in the transverse and coronal planes at 15, 30, 60, 120, and 240 minutes respectively.

[0028] Figure 4 The statistical analysis graph of radioactive uptake in the joints of the inflamed joints and normal joints of the present invention after PET / CT scanning 15min, 30min, 60min, 120min and 240min after the arthritis model is completed 2-3 days after the modeling is completed;

[0029] Figure 5 The bilateral arthritis mouse model of the present invention was treated with rheumatoid arthritis therapeutic drugs. The treatment group and the control group were injected with radioactive probes into the tail vein on days 0, 3, 6 and 10. 68 Ga-NOTA-mAb Kv 1.3 After 120 minutes, a coronal PET / CT scan was performed;

[0030] Figure 6 This is a statistical analysis chart of the change trend of arthritis scores of mice in the treatment group and the control group from the beginning of modeling to the end of treatment in the bilateral arthritis mouse model of the present invention;

[0031] Figure 7 The bilateral arthritis mouse model of the present invention was treated with rheumatoid arthritis therapeutic drugs. The treatment group and the control group were injected with radioactive probes into the tail vein on days 0, 3, 6 and 10. 68 Ga-NOTA-mAb Kv 1.3 Statistical analysis of the radioactive uptake values ​​of the joints 120 minutes after surgery;

[0032] Figure 8 This is a statistical analysis diagram of the correlation analysis between the arthritis score of the present invention and radioactive uptake in the joints. DETAILED DESCRIPTION

[0033] In order to make the technical personnel in the technical field better understand the scheme of the present invention, the technical scheme in the embodiment of the present invention will be clearly and completely described below in combination with the drawings in the embodiment of the present invention. Obviously, the described embodiment is only a part of the embodiment of the present invention, not all the embodiments. Based on the embodiment of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] The terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish different objects rather than to describe a specific order. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, device, product or end including a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or units inherent to these processes, methods, products or ends.

[0035] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0036] Currently, there is no universally accepted gold standard for the diagnosis of rheumatoid arthritis (RA). Patients usually present with typical clinical symptoms such as joint pain, swelling, morning stiffness, and abnormal laboratory tests (such as rheumatoid factor and anti-citrullinated peptide antibodies), but these symptoms may not be obvious in the early stages of the disease. The main pathological feature of RA is synovial inflammation, which is manifested by the infiltration of innate and adaptive immune cells, including T lymphocytes, B lymphocytes, monocytes, and dendritic cells. Synovial fibroblasts accumulate and exhibit an invasive and inflammatory phenotype, which ultimately leads to joint destruction by promoting chondrocyte breakdown and osteoclastogenesis. The prominent features of inflammatory diseases are the exacerbation of autoimmune reactions and synovial infiltration mediated by inflammatory cells, which usually occur before the onset of clinical symptoms and signs. In addition, the presence of inflammatory cells is also an important indicator of disease remission, which emphasizes the need for regular reassessment during treatment. Molecular imaging, as a non-invasive technology, can provide detailed molecular information about the disease before the appearance of morphological lesions, thereby enabling the quantification of joint inflammation in vivo. This is of great value in diagnosing, grading and monitoring joint inflammation, especially when evaluating the effectiveness of treatment. 99m Tc-bisphosphonate SPECT and 18 F-FDG PET has been used to detect early signs of RA, but its lack of specificity for the inflammatory process limits its application. Therefore, it is critical to identify specific markers in inflamed tissues and develop tracer molecules that can target these markers with high specificity and efficiency.

[0037] Voltage-gated potassium channel 1.3 (Kv1.3) is a channel protein that can sense voltage changes and mediate potassium ion outflow across the membrane. The channel protein is encoded by the KCNA3 gene and is a homotetramer formed by non-covalent binding of subunits of approximately 500 amino acids, including four groups of protein subunits composed of six α-helices that span the membrane six times to form a single-pore channel. The gating of Kv1.3 is controlled by the membrane potential, and changes in membrane potential can be sensed by the "voltage sensor" region containing positive charges. When the cell is activated and the membrane potential is higher than -40mv, the channel opens, allowing potassium ions to flow across the membrane, increasing the negative value of the membrane potential, and increasing the transmembrane potential difference to form a relative hyperpolarized potential. Following the potential difference, calcium ions flow into the cell, resulting in an increase in intracellular calcium ion concentration. Calcium ions are important second messengers that can activate subsequent signals and further activate cells. Kv1.3 was found to be overexpressed in the proliferation and cytokine production of various inflammatory-related immune cells. In particular, when effector memory T cells are activated, the expression of Kv1.3 increases significantly, and the number of channels increases from 200-400 to about 1500-2000. In addition, the activation of macrophages, B lymphocytes, and dendritic cells also leads to the upregulation of Kv1.3, and these cells co-migrate and aggregate with T lymphocytes during infiltration, thereby initiating a chronic inflammatory process. Studies have shown that in the synovial fluid of RA patients, the expression level of Kv1.3 is high in effector memory T cells, and Kv1.3 channel inhibitors ShK-186 and PEG-HsTX1[R14A] can preferentially inhibit these cells. Therefore, the Kv1.3 channel is considered to be a promising target for the diagnosis and monitoring of RA.

[0038] The applicant previously developed a mouse anti-Kv1.3 channel monoclonal antibody (mAb) based on the extracellular cyclic peptide E314 of the human Kv1.3 channel. Kv1.3 ), has been granted a national invention patent (patent number: ZL201610325161.7). This monoclonal antibody has the characteristics of strong affinity and high specificity, and can specifically recognize human and mouse Kv1.3 channel proteins. In vivo experiments show that it is highly safe and does not affect blood counts, biochemical indicators and organ functions after intravenous injection. Based on the above research foundation, the applicant will mAb Kv1.3 Connect with NOTA and then with nuclide 68 Ga chelation, and the radioactive probe was obtained after purification 68 Ga-NOTA-mAb Kv1.3By injecting the probe in vivo, PET / CT imaging can clearly show the site of inflammation and the degree of inflammatory damage in the mouse joints in vivo, guiding clinical diagnosis and treatment as well as prognosis, to solve the problem of lack of suitable targets and probes in the clinic, and reveal the inflammatory activity of immune cells in rheumatoid arthritis diseased joints in vivo.

[0039] The present invention is intended to provide a radioactive probe 68 Ga-NOTA-mAb Kv 1.3 The synthesis method and application of this novel novel molecule solve the problem of lack of suitable targets and probes in the clinic to reveal the inflammatory activity of immune cells in rheumatoid arthritis lesions in vivo.

[0040] like Figure 1-8 As shown, a specific embodiment of the present invention provides a radioactive probe 68 Ga-NOTA-mAb Kv 1.3 A synthetic method, such as Figure 1 As shown, the following steps are included:

[0041] S1.mAb Kv 1.3 Monoclonal antibody production and immunohistochemical staining

[0042] mAbs Kv1.3 The production of monoclonal antibodies is carried out by inoculating hybridoma cells AbM-F14FTSSCLS0167-KLH#15 into the peritoneal cavity of mice to obtain ascites containing monoclonal antibodies, and then purifying mAbs from the ascites by affinity chromatography. Kv 1.3 Monoclonal antibodies.

[0043] S2. Radioactive probe 68 Ga-NOTA-mAb Kv 1.3 Synthesis

[0044] mAb Kv 1.3 Antibody-NOTA conjugation, NOTA-mAb Kv 1.3 Purification, NOTA-mAb Kv 1.3 Chelation 68 Ga, 68 Ga-NOTA-mAb Kv 1.3 Labeling rate determination, 68 Ga-NOTA-mAb Kv 1.3 purification, 68 Ga-NOTA-mAb Kv 1.3 Radiochemical purity determination to achieve synthetic radioactive probes 68 Ga-NOTA-mAb Kv 1.3 .

[0045] In the present embodiment, mAb Kv 1.3The steps of connecting the antibody to NOTA include: taking a protein sample mAb Kv1.3 , added to Na 2 CO 3 In buffer, the protein sample mAb Kv 1.3 and the Na 2 CO 3 The amount ratio of the buffer solution is 5:1, and the pH of the mixed solution is adjusted to 9.0-10.0 to obtain a protein sample solution; p-SCN-Bn-NOTA is weighed in a light-proof environment, a DMSO solution is prepared by solid DMSO, and the p-SCN-Bn-NOTA is dissolved in the DMSO solution to a concentration of 100 μg / μL to obtain a p-SCN-Bn-NOTA solution, wherein the volume of the solid DMSO added needs to be less than 5% of the total volume; the protein sample solution and the p-SCN-Bn-NOTA solution are quickly mixed, and the mixture is placed in a constant temperature metal heater, and the reaction is carried out at a constant speed of 400 r / min at 37° C. for 2 hours to complete the mAb Kv 1.3 Steps for linking antibodies to NOTA to obtain NOTA-mAb Kv 1.3 Reactants;

[0046] NOTA-mAb Kv 1.3 The purification steps include: opening the upper cover of the PD-10 column and cutting off the lower outlet, placing the PD-10 column vertically to drain the preservation solution in the column and discard the solution; filling the upper chamber of the PD-10 column with PBS to drain the solution, repeating 3-4 times and discarding the solution; placing the PD-10 column on a bracket and placing the NOTA-mAb Kv1.3 The reactants were added to the PD-10 column and the NOTA-mAb Kv1.3 After the reactants completely enter the PD-10 column and no liquid flows out, the PD-10 column is filled with PBS; the outflow liquid is connected with an EP tube, and PBS is added each time. The protein concentration of each tube of solution is measured using an ELISA instrument to complete the NOTA-mAb Kv 1.3 Purification process to obtain purified NOTA-mAb Kv 1.3 Solution; the purified NOTA-mAb Kv 1.3 The solution is packaged and stored at -20°C. It should be used within 3 months and avoid frequent freezing and thawing.

[0047] NOTA-mAb Kv 1.3 Chelation 68 The steps of Ga include: eluting with HCl 68 Ge / 68 Ga generator, obtained containing 68 The Ga-containing eluate was collected using an EP tube. 68Ga eluent; the 68 The eluate of Ga was mixed with sodium acetate solution, the pH was adjusted to 4.0-4.5, and then the purified NOTA-mAb was added thereto. Kv1.3 The first mixed solution was placed in a constant temperature metal heater, and reacted at a constant speed of 400r / min and 37°C for 10-15min to obtain 68 Ga-NOTA-mAb Kv 1.3 Reactants, completed NOTA-mAb Kv1.3 Chelation 68 Ga process;

[0048] 68 Ga-NOTA-mAb Kv 1.3 The steps of labeling rate determination include: using size exclusion chromatography column, high performance liquid chromatography and radioactive detector for analysis, using 10x PBS as mobile phase, the flow rate is 1mL / min; taking 740-1110KBq of the reaction 68 Ga-NOTA-mAb Kv 1.3 The reactants were tested on the machine, and the radioactive labeling rate was calculated based on the peak area;

[0049] 68 Ga-NOTA-mAb Kv 1.3 The purification step comprises: opening the upper cover of the PD-10 column and cutting off the lower outlet, placing the PD-10 column vertically to allow the preservation solution in the column to drain out and discard the liquid; filling the upper chamber of the PD-10 column with PBS to allow the liquid to drain out, repeating 3-4 times and discarding the liquid; placing the PD-10 column in a ventilated lead cabinet, 68 Ga-NOTA-mAb Kv 1.3 The reactants were added to the PD-10 column and 68 Ga-NOTA-mAb Kv 1.3 After the reactants completely enter the PD-10 column and no liquid flows out, fill the PD-10 column with PBS; connect the outflow liquid with an EP tube and measure the radioactivity of each tube; collect the liquid with the highest radioactivity, which is the radioactive probe 68 Ga-NOTA-mAb Kv 1.3 ;

[0050] 68 Ga-NOTA-mAb Kv 1.3 The steps of radiochemical purity determination include: using size exclusion chromatography column, high performance liquid chromatography and radioactivity detector for analysis, using 10x PBS as mobile phase, flow rate of 1mL / min; taking 740-1110KBq radioactive probe 68 Ga-NOTA-mAbKv 1.3 The radioactive labeling rate was calculated based on the peak area.

[0051] A radioactive probe provided by the present invention 68 Ga-NOTA-mAb Kv 1.3 The application of the synthetic method is:

[0052] Includes radioactive probe 68 Ga-NOTA-mAb Kv 1.3 PET / CT imaging and radioactive probes in a mouse model of rheumatoid arthritis 68 Ga-NOTA-mAb Kv 1.3 PET / CT Monitoring During Treatment in a Mouse Rheumatoid Arthritis Model:

[0053] The radioactive probe 68 Ga-NOTA-mAb Kv 1.3 PET / CT imaging in the mouse rheumatoid arthritis model was achieved by establishing a unilateral arthritis mouse model, radioactive probe injection, anesthesia and imaging;

[0054] The radioactive probe 68 Ga-NOTA-mAb Kv 1.3 PET / CT monitoring during the treatment of mouse rheumatoid arthritis model is to establish a bilateral arthritis mouse model, give the rheumatoid arthritis treatment drug elamod to the experimental group mice, and perform radioactive probe injection, anesthesia and imaging on the experimental and control group mice during the treatment to achieve PET / CT monitoring. Specific embodiment:

[0056] mAbs Kv 1.3 Monoclonal antibody production and immunohistochemical staining

[0057] mAbs Kv 1.3 The production of monoclonal antibodies is based on the method disclosed in the existing patent "Monoclonal Antibodies Against Extracellular Cyclic Peptide Segments of Voltage-gated Potassium Channel 1.3 and Applications" (Publication No.: CN105968199A), that is, hybridoma cell line AbM-F14FTSSCLS0167-KLH#15 is inoculated into the peritoneal cavity of mice to obtain ascites containing monoclonal antibodies, and monoclonal antibodies mAbs are purified from the ascites by affinity chromatography. Kv 1.3 .

[0058] The mAb obtained Kv1.3 Immunohistochemical staining of arthritic mouse joints was performed to observe the expression of the antibody in inflamed joints and normal joints. The results showed that the expression of Kv1.3 channels in inflamed joints was significantly higher than that in normal joints. Figure 1 shown.

[0059] Figure 1 Immunofluorescence staining of Kv1.3, CD3 and CD68 in inflamed joints and normal joints 2-3 days after the completion of unilateral rheumatoid arthritis modeling in mice. mAb Kv1.3 , anti-mouse CD3 and CD68 antibodies were used as primary antibodies for Kv1.3, CD3 and CD68, respectively. Red fluorescence showed the expression of Kv1.3, green fluorescence showed the expression of CD3 and CD68, and blue fluorescence DAPI was the cell nucleus. The scale bar is 200μm. Staining showed that the expression of Kv1.3, CD3 and CD68 in inflammatory joint lesions was significantly higher than that in normal joints, indicating increased inflammatory cell infiltration.

[0060] Radioactive probe 68 Ga-NOTA-mAb Kv 1.3 Synthesis

[0061] mAb Kv1.3 Antibody-NOTA conjugation, NOTA-mAb Kv1.3 Purification, NOTA-mAb Kv1.3 Chelation 68 Ga, 68 Ga-NOTA-mAb Kv 1.3 Labeling rate determination, 68 Ga-NOTA-mAb Kv 1.3 purification, 68 Ga-NOTA-mAb Kv 1.3 The synthesis was carried out by radiochemical purity determination.

[0062] 1. mAb Kv1.3 Antibodies linked to NOTA

[0063] (1) Take 500 μL (about 25 nmol) of protein sample mAb Kv 1.3 , add 100 μL of 0.1 M Na 2 CO 3 Buffer solution, adjust the pH to 9.0-10.0;

[0064] (2) Weigh 0.4 mg of p-SCN-Bn-NOTA in a light-proof environment and dissolve it in 4 μL of DMSO solution to a concentration of 100 μg / μL;

[0065] (3) quickly mixing the above protein sample solution and p-SCN-Bn-NOTA solution, placing them in a constant temperature metal heater, and reacting them at a constant speed of 400 r / min and 37° C. for 2 h;

[0066] (4) The volume of DMSO added needs to be less than 5% of the total volume; if the volume of DMSO is too large, the volume of DMSO added to dissolve the p-SCN-Bn-NOTA powder can be appropriately reduced to increase the concentration.

[0067] 2. NOTA-mAb Kv1.3 purification

[0068] (1) Open the cap at the top of the PD-10 column and cut off the outlet at the bottom. Place the PD-10 column vertically to allow the preservation solution in the column to drain out and discard the liquid.

[0069] (2) Fill the upper chamber of the PD-10 column with PBS until the liquid flows out; repeat 4 times and discard the outflow liquid;

[0070] (3) Place the PD-10 column on the support and place the aforementioned synthesized NOTA-mAb Kv1.3 The reactants were added to the PD-10 column. After the sample completely entered the column and no liquid flowed out, the column was filled up to 2.5 mL with PBS (PBS volume = 2.5 mL - sample volume);

[0071] (4) Use a numbered 1.5 mL EP tube to collect the outflow liquid, add 500 μL PBS each time, connect 8 tubes in total, and measure the protein concentration of each tube of solution using an ELISA reader;

[0072] (5) The product was packaged and stored at -20°C. It was used within 3 months and was avoided from frequent freezing and thawing.

[0073] 3. NOTA-mAb Kv1.3 Chelation 68 Ga

[0074] (1) Elute the 68Ge / 68Ga generator with 4 mL of 0.1 M HCl and collect the 68Ge / 68Ga containing 1.0 mL, 1.0 mL, 1.0 mL, and 1.0 mL of EP tubes in the order of 1.5 mL. 68 The eluent for Ga is selected 68 The tube with the highest Ga activity is used for subsequent labeling;

[0075] (2) 68 The Ga solution was mixed with 250 μL of 0.5 M sodium acetate solution, the pH was adjusted to 4.0-4.5, and 10-15 nmol of NOTA-mAb was added to the above solution. Kv 1.3 ;

[0076] (3) Place the mixture in a constant temperature metal heater and react at a constant speed of 400 r / min and 37°C for 10-15 min to obtain 68 Ga-NOTA-mAb Kv 1.3 .

[0077] Four, 68 Ga-NOTA-mAb Kv1.3 Labeling rate determination

[0078] (1) Analyze using a size exclusion chromatography column, high performance liquid chromatography, and a radioactivity detector, using 10x PBS as the mobile phase at a flow rate of 1 mL / min;

[0079] (2) Take 740-1110 KBq (20 μCi) of the reacted sample and test it on the machine. Calculate the radioactive labeling rate based on the peak area.

[0080] five, 68 Ga-NOTA-mAb Kv1.3 purification

[0081] Purification of 68Ga-NOTA-mAb using PD-10 desalting chromatography column (referred to as PD-10 column) Kv1.3 .

[0082] (1) Preparation: Open the upper cap of the PD-10 column and cut off the lower outlet. Place the PD-10 column vertically to allow the preservation solution in the column to drain out and discard the liquid.

[0083] Balance: Fill the upper chamber of the PD-10 column with PBS until the liquid flows out; repeat 4 times and discard the outflow liquid (about 20-25 mL of PBS is used in total);

[0084] (2) Sample addition: Place the PD-10 column in a ventilated lead cabinet; 68 The Ga-NOTA-mAbKv1.3 reactant was added to the PD-10 column. After the sample completely entered the column and no liquid flowed out, it was filled up to 2.5 mL with PBS (PBS volume = 2.5 mL - sample volume);

[0085] (3) Elution: Use a numbered 1.5 mL EP tube to collect the outflow liquid, add 500 μL PBS each time, connect 6 tubes in total, and measure the radioactivity of each tube. Collect the liquid with the highest radioactivity, which is the purified labeled antibody. The product appears in the second or third tube.

[0086] six, 68 Ga-NOTA-mAb Kv1.3 Radiochemical purity determination, e.g. Figure 2 shown.

[0087] Figure 2 Radioactive probe 68 Ga-NOTA-mAb Kv1.3After synthesis, the samples were incubated with PBS and analyzed by HPLC at 0 min, 60 min, 120 min, and 240 min after purification by PD-10 column. The experimental results showed that the radioactive probe 68 Ga-NOTA-mAb Kv1.3 Good in vitro stability.

[0088] (1) Analyze using a size exclusion chromatography column, high performance liquid chromatography, and a radioactivity detector, using 10x PBS as the mobile phase at a flow rate of 1 mL / min;

[0089] (2) Take 740-1110 KBq (20 μCi) of the purified sample and test it on the machine. Calculate the radioactive labeling rate based on the peak area.

[0090] 68 Ga-NOTA-mAb Kv1.3 Probe PET / CT imaging in a unilateral arthritis mouse model, such as Figure 3 , Figure 4 shown.

[0091] Figure 3 The radioactive probe was injected into the tail vein 2-3 days after the mouse arthritis model of the present invention was established. 68 Ga-NOTA-mAb Kv 1.3 Afterwards, PET / CT scans were performed in the transverse and coronal planes at 15, 30, 60, 120 and 240 minutes respectively. As time went by, the radioactivity in the inflamed joints gradually accumulated, while no obvious radioactivity was observed in the contralateral normal joints.

[0092] Figure 4 This is a statistical analysis chart of the radioactive uptake in the joints of the inflamed joints and normal joints 2-3 days after the completion of the arthritis model, 15min, 30min, 60min, 120min and 240min after PET / CT scanning. There is a significant difference between the two at each time point, ****P<0.0001.

[0093] SPECT / CT imaging was achieved through the establishment of a unilateral arthritis model in mice, injection of radioactive probes, anesthesia and imaging

[0094] (I) Establishment of unilateral arthritis model in mice

[0095] After one week of adaptive feeding of 8-week-old DBA / 1 mice, type II collagen was dissolved in 3.3 mL of 0.1 M glacial acetic acid at a concentration of 3 mg / mL. 3.3 mL of complete Freund's adjuvant or incomplete Freund's adjuvant was added to the collagen three times for emulsification. The entire emulsification process was carried out on ice, and finally the collagen was emulsified until there was no oil scattered when the collagen was dropped on the water, forming an oil-in-water state. For the first immunization, 3 μL of type II collagen-complete Freund's adjuvant emulsified collagen was injected into the footpad of one lower limb of the mouse. The second booster immunization was carried out 21 days after the first immunization, and 3 μL of type II collagen-incomplete Freund's adjuvant emulsified collagen was injected into the mouse footpad.

[0096] (II) Radioactive Probe Injection

[0097] Each mouse was injected with about 3.7-7.4MBq (100-200μCi) via tail vein injection. 68 Ga-NOTA-mAb Kv1.3 (About 150 μL PBS). Record the time of drug extraction and drug activity, the time of drug injection, the activity and time of residual drug, and the weight of the mouse.

[0098] (III) Anesthesia and imaging

[0099] (1) Anesthetize mice with isoflurane gas anesthesia using a small animal gas anesthesia machine. Before imaging, place the animal in an anesthesia induction box. After anesthesia, place the animal in the PET / CT animal cabin and keep it in a prone position. Anesthesia lasts for about 15-20 minutes during PET / CT imaging. The isoflurane flow rate is set to 1.5 LPM (liters / minute).

[0100] (2) The mice were put on the machine to collect images 15, 30, 60, 120 and 240 minutes after the injection of the imaging agent. Using a small animal PET / CT imaging machine, the CT positioning phase was first acquired, and the scanning range of subsequent CT and PET was set to the whole body of the mouse. First, a CT spiral scan was performed. The CT scanning parameters were as follows: the scanning protocol was CT_Conventional_Scan, the voltage was 50KV, the current was 0.5mA, the step angle was 1.00°, and the single frame acquisition time was 2000ms. The CT reconstruction parameters were as follows: the reconstruction protocol was CT_FDK_Recon, the image matrix was 512×512, and the filter was soft.

[0101] (3) After the CT scan is completed, the PET scan is automatically performed. The PET scan parameters are as follows: the radionuclide setting and energy preset are 68 Ga. PET reconstruction parameters were as follows: algorithm type was OSEM+PSF, image matrix was 320x320x200, median filter was selected, number of iterations was 2, and 11 subsets were used.

[0102] (4) After the image acquisition is completed, the Inveon Research Workplace, an image post-processing workstation, is used for image processing. The PET / CT fusion image is obtained, and regions of interest (ROIs) on the fusion image are delineated, including joint regions, blood pools, and muscle tissues. Anatomical localization of the above regions is performed based on the CT images. After the delineation is completed, radioactive counts are obtained. The quantitative unit of the CT image is the CT value, i.e., the Hounsfield unit (Hu), and the PET image uses the percentage of injected dose per gram-tissue (%ID / g) as the quantitative unit for the radioactive counts of the image and the ROI.

[0103] 68 Ga-NOTA-mAb Kv 1.3 PET / CT monitoring of the probe during the treatment of the murine rheumatoid arthritis model is as Figure 5-8 shown.

[0104] Figure 5 This is a coronal PET / CT scan imaging map of the treatment group and the control group of the bilateral arthritis murine model of the present invention at 120 min after intravenous injection of the radioactive probe 68 Ga-NOTA-mAb Kv 1.3 on days 0, 3, 6, and 10 after treatment with the rheumatoid arthritis treatment drug; as the treatment progresses, the radioactive imaging of the joints of the mice in the treatment group gradually fades, while that of the mice in the control group remains basically unchanged.

[0105] Figure 6 This is a statistical analysis map of the changing trends of the arthritis scores of the treatment group and the control group of the bilateral arthritis murine model of the present invention from the start of model establishment to the end of treatment; after the start of model establishment, the arthritis score values of the mice increase and reach the highest value after the enhancement injection. After the start of treatment, the arthritis scores of the mice in the treatment group show a downward trend, while those of the mice in the control group remain basically stable. At the end of treatment, there is a difference between the two, *P<0.1.

[0106] Figure 7 This is a statistical analysis map of the radioactive uptake values of the joints of the treatment group and the control group of the bilateral arthritis murine model at 120 min after intravenous injection of the radioactive probe 68 Ga-NOTA-mAb Kv 1.3 on days 0, 3, 6, and 10 after treatment with the rheumatoid arthritis treatment drug; as the treatment progresses, the radioactive uptake values of the joints of the mice in the treatment group gradually decrease, while those of the mice in the control group remain basically unchanged. At the end of treatment, there is a significant difference between the two groups, **P<0.01.

[0107] Figure 8This is a statistical analysis diagram of the correlation analysis between the arthritis score of the present invention and the radioactive uptake in the joints; there is a significant correlation between the two, wherein r=0.8789, ****P<0.0001.

[0108] (1) A bilateral lower limb arthritis mouse model was constructed according to the above modeling aspects. After the modeling began, each mouse was regularly scored for arthritis according to the following criteria: grade 0, normal (no swelling); grade 1, mild but clear redness and swelling of the ankle or wrist, or obvious redness and swelling limited to one or more fingers; grade 2, moderate redness and swelling of the ankle or wrist; grade 3, severe redness and swelling of the entire paw including the fingers; grade 4, the most inflamed limb, involving multiple joints. The score for each limb is 0-4 points.

[0109] (2) The mice were randomly divided into a treatment group and a control group. Each mouse in the treatment group was given 30 mg / kg of ilamod solution by oral gavage every day, and each mouse in the control group was given an equal volume of PBS solution.

[0110] (3) On days 0, 3, 6, and 10 after the start of treatment, the mice in the treatment group and the control group were imaged 240 minutes after the tail vein injection of the radioactive drug according to the imaging parameters set above.

[0111] (4) After image acquisition, image processing is performed according to the above method.

[0112] The present invention is based on the Kv1.3 channel extracellular cyclic peptide E314, which has been authorized as a patent "Monoclonal antibody against voltage-gated potassium channel 1.3 extracellular cyclic peptide and its application" (publication number: CN105968199A). Kv1.3 Connect with NOTA and then with Nuclide 68 Ga chelation, and the radioactive probe was obtained after purification 68 Ga-NOTA-mAb Kv1.3 By injecting the probe in vivo, PET / CT imaging can clearly show the location of joint lesions and the degree of inflammatory damage in rheumatoid arthritis mice in vivo, guiding clinical diagnosis, treatment and prognosis.

[0113] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical schemes or characteristics in the scheme is not described in detail here; it should be pointed out that for those skilled in the art, several modifications and improvements can be made without departing from the scheme of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the present invention and the practicality of the patent. The protection scope required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.

Claims

1. A radioactive probe 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that The method comprises: S1.mAb Kv1.3 Monoclonal antibody production and immunohistochemical staining mAbs Kv1.3 The production of monoclonal antibodies is carried out by inoculating hybridoma cells AbM-F14FTSSCLS0167-KLH#15 into the peritoneal cavity of mice to obtain ascites containing monoclonal antibodies, and then purifying mAbs from the ascites by affinity chromatography. Kv1.3 Monoclonal antibodies; S2. Radioactive probe 68 Ga-NOTA-mAb Kv1.3 Synthesis mAb Kv1.3 Antibody-NOTA conjugation, NOTA-mAb Kv1.3 Purification, NOTA-mAb Kv1.3 Chelation 68 Ga, 68 Ga-NOTA-mAb Kv1.3 Labeling rate determination, 68 Ga-NOTA-mAb Kv1.3 purification, 68 Ga-NOTA-mAb Kv1.3 Radiochemical purity determination to achieve synthetic radioactive probes 68 Ga-NOTA-mAb Kv1.3 .

2. A radioactive probe according to claim 1 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that The mAb Kv1.3 The steps of connecting the antibody to NOTA include: Take protein sample mAb Kv1.3 , added to Na2CO3 buffer, the protein sample mAb Kv1.3 The ratio of the amount of the Na2CO3 buffer solution to that of the aqueous solution is 5:1, and the pH of the mixed solution is adjusted to 9.0-10.0 to obtain a protein sample solution; Weigh p-SCN-Bn-NOTA in a light-proof environment, prepare a DMSO solution using solid DMSO, dissolve the p-SCN-Bn-NOTA in the DMSO solution to a concentration of 100 μg / μL, and obtain a p-SCN-Bn-NOTA solution, wherein the volume of solid DMSO added needs to be less than 5% of the total volume; The protein sample solution and the p-SCN-Bn-NOTA solution were quickly mixed and placed in a constant temperature metal heater at a constant speed of 400 r / min at 37° C. for 2 h to complete the mAb Kv1.3 Steps for linking antibodies to NOTA to obtain NOTA-mAb Kv1.3 Reactants.

3. A radioactive probe according to claim 2 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that The NOTA-mAb Kv1.3 The purification steps include: Open the upper cover of the PD-10 column, cut off the lower outlet, place the PD-10 column vertically, drain the preservation solution in the column, and discard the solution; Fill the upper chamber of the PD-10 column with PBS to drain out the liquid, repeat 3-4 times, and discard the liquid; The PD-10 column is placed on the support, and the NOTA-mAb Kv1.3 The reactants were added to the PD-10 column and the NOTA-mAb Kv1.3 After the reactants completely enter the PD-10 column and no liquid flows out, the PD-10 column is filled up with PBS; Use EP tube to connect the outflow liquid, add PBS each time, and use microplate reader to measure the protein concentration of each tube solution to complete NOTA-mAb Kv1.3 Purification process to obtain purified NOTA-mAb Kv1.3 Solution; The purified NOTA-mAb Kv1.3 The solution was aliquoted and stored at -20°C. It was used within 3 months and avoided frequent freezing and thawing.

4. A radioactive probe according to claim 3 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that The NOTA-mAb Kv1.3 Chelation 68 The steps of Ga include: Use HCl elution 68 Ge / 68 Ga generator, obtained containing 68 The Ga-containing eluate was collected using an EP tube. 68 Ga eluent; The said 68 The eluate of Ga was mixed with sodium acetate solution, the pH was adjusted to 4.0-4.5, and then the purified NOTA-mAb was added thereto. Kv1.3 solution to obtain a first mixed solution; The first mixed solution was placed in a constant temperature metal heater and reacted at a constant speed of 400 r / min and 37°C for 10-15 min to obtain 68 Ga-NOTA-mAb Kv1.3 Reactants, completed NOTA-mAb Kv1.3 Chelation 68 Ga process.

5. A radioactive probe according to claim 4 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that Said 68 Ga-NOTA-mAb Kv1.3 The steps of labeling rate determination include: The analysis was performed using a size exclusion chromatography column, high performance liquid chromatography, and a radioactivity detector, with 10x PBS as the mobile phase and a flow rate of 1 mL / min; Take 740-1110KBq after reaction 68 Ga-NOTA-mAb Kv1.3 The reactants were tested on the machine and the radioactive labeling rate was calculated based on the peak area.

6. A radioactive probe according to claim 4 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that Said 68 Ga-NOTA-mAb Kv1.3 The purification steps include: Open the upper cover of the PD-10 column, cut off the lower outlet, place the PD-10 column vertically, drain the preservation solution in the column, and discard the solution; Fill the upper chamber of the PD-10 column with PBS to drain out the liquid, repeat 3-4 times, and discard the liquid; The PD-10 column was placed in a ventilated lead cabinet. 68 Ga-NOTA-mAb Kv1.3 The reactants were added to the PD-10 column and 68 Ga-NOTA-mAb Kv1.3 After the reactants completely enter the PD-10 column and no liquid flows out, the PD-10 column is filled up with PBS; Use EP tube to connect the outflow liquid and measure the radioactivity of each tube; collect the liquid with the highest radioactivity, which is the radioactive probe 68 Ga-NOTA-mAb Kv1.3 .

7. A radioactive probe according to claim 6 68 Ga-NOTA-mAb Kv1.3 The synthesis method is characterized in that Said 68 Ga-NOTA-mAb Kv1.3 The steps for radiochemical purity determination include: The analysis was performed using a size exclusion chromatography column, high performance liquid chromatography, and a radioactivity detector, with 10x PBS as the mobile phase and a flow rate of 1 mL / min; Take 740-1110KBq radioactive probe 68 Ga-NOTA-mAb Kv1.3 The radioactive labeling rate was calculated based on the peak area.

8. A radioactive probe 68 Ga-NOTA-mAb Kv1.3 The application of the synthesis method is characterized in that: Includes radioactive probe 68 Ga-NOTA-mAb Kv1.3 PET / CT imaging and radioactive probes in a mouse model of rheumatoid arthritis 68 Ga-NOTA-mAb Kv1.3 PET / CT Monitoring During Treatment in a Mouse Rheumatoid Arthritis Model: The radioactive probe 68 Ga-NOTA-mAb Kv1.3 PET / CT imaging in the mouse rheumatoid arthritis model was achieved by establishing a unilateral arthritis mouse model, radioactive probe injection, anesthesia and imaging; The radioactive probe 68 Ga-NOTA-mAb Kv1.3 PET / CT monitoring during the treatment of mouse rheumatoid arthritis model is to establish a bilateral arthritis mouse model, give the rheumatoid arthritis treatment drug elamod to the experimental group mice, and perform radioactive probe injection, anesthesia and imaging on the experimental and control group mice during the treatment to achieve PET / CT monitoring.

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