KR101699238B1 - Cell seperating method using phase difference and cell conductance controlling composition thereof - Google Patents
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Abstract
본 발명은 세포 임피던스 차이를 이용한 세포 분별 방법 및 이를 위한 세포 임피던스 조절용 조성물에 관한 것으로서, 더욱 상세하게는 세포 표면에 존재하는 단백질과 결합하는 항체, 및 상기 항체에 결합하는 전도성 입자를 포함하는 세포 전도성 조절용 조성물을 사용하여 특정 주파수에서 세포의 위상을 측정함으로써 세포를 분별하는 것을 특징으로 하는 세포 임피던스 차이를 이용한 세포 분별 방법 및 이를 위한 세포 임피던스 조절용 조성물에 관한 것이다. The present invention relates to a cell fractionation method using a cell impedance difference and a composition for controlling cell impedance therefor, and more particularly, to a cell fractionation method using a cytotoxic substance comprising an antibody binding to a protein existing on a cell surface, The present invention relates to a cell fractionation method using cell impedance difference and a composition for controlling cell impedance thereof, characterized by separating cells by measuring the phase of a cell at a specific frequency using a regulatory composition.
Description
본 발명은 주파수 위상차를 이용한 세포 분별 방법 및 이를 위한 세포 전도성 조절용 조성물에 관한 것으로서, 더욱 상세하게는 세포에 세포 표면에 존재하는 단백질과 결합하는 항체 등에 세포의 전도성을 조절할 수 있는 전도성 입자를 결합시켜 특정 주파수에 대한 응답이 다른 위상을 나타내도록 위상차를 유도한 후, 이를 이용하여 세포를 분별하는 방법에 관한 것이다.
The present invention relates to a cell fractionation method using a frequency phase difference and a composition for controlling cell conduction therefrom, and more particularly, to a cell fractionation method using a cell, To a method for discriminating a cell using a phase difference after inducing a response to a specific frequency to exhibit a different phase.
사람의 혈액(blood)에는 다양한 세포(cell)들과 단백질들이 포함되어 있다. 혈액 내의 특정 단백질들의 농도 변화를 통해, 그 사람의 건강 상태가 파악되기도 한다. 혈액 내 세포들로는 적혈구(red blood cell), 백혈구(white blood cell) 및 혈소판(platelet)이 주종을 이루고 있으며, 전체 세포들 부피의 약 55 ％ 정도를 차지하고 있다.Human blood contains various cells and proteins. Through changes in the concentration of certain proteins in the blood, the person's health status can be grasped. The cells in the blood are red blood cells, white blood cells and platelets, which account for about 55% of the total cell volume.
세포는 크게 세포막(plasma membrane), 핵(nucleus), 세포질(cytoplasm)로 나뉘며 세포의 종류 및 상태에 따라서 세포의 각부분이 기계적인 변형성(deformability)과 전기적인 유전율(permittivity)에 있어서 차이를 보이게 된다. 변형성은 탄성(elasticity)과 점성(viscosity)에 관계하며 물질이 손상되지 않고 변경하는 정도를 나타낸다. 그리고 유전율은 외부의 전기장에 물질이 반응하는 정도를 나타낸다. 세포의 경우 이러한 변형성과 유전률에 관계하는 물리량을 측정하게 되면 정상세포와 암세포간의 차이, 암세포의 전이단계 판별과 같은 세포의 생리학적 특성을 얻을 수 있다.Cells are largely divided into plasma membrane, nucleus, and cytoplasm. Depending on the type and condition of the cells, each part of the cell shows a difference in mechanical deformability and electrical permittivity do. Deformation affects the elasticity and viscosity and indicates the degree to which the material changes without being damaged. And the permittivity indicates the degree to which a substance reacts to an external electric field. In the case of the cells, physiological characteristics such as the difference between normal cells and cancer cells, and the stage of cancer cell metastasis can be obtained by measuring physical quantities related to the deformability and the dielectric constant.
암(cancer) 환자의 경우, 암의 진행 정도 또는 전이성의 유무에 따라, 고형 암으로부터 혈액 중으로 떨어져 나와 혈액 속을 떠다니게 되는 암 세포들이 존재하게 된다. 이러한 암 세포를 혈중 암 세포라 하며, 이 암 세포들의 평균적인 크기는 16 ~ 25 μm 정도의 범위인 것으로 알려져 있다.In the case of cancer patients, depending on the progress of the cancer or the presence or absence of the metastasis, there are cancer cells that fall into the blood from the solid cancer and float in the blood. These cancer cells are called blood cancer cells, and the average size of these cancer cells is known to be in the range of about 16 to 25 μm.
현재 혈액 내 암 세포를 분별, 분리 검출하기 위해서 사용되고 있는 방법들에는 비표지(label-free) 방식과 표지(label) 방식으로 나뉠 수 있다. 비표지 방식으로는 밀도 차이를 이용한 분리, 유전체전기영동(DiElectroPhoretic : DEP) 방법, 크기에 따른 관성력(inertial force)의 차이를 이용한 방법, 크기를 이용한 필터(filter) 형태의 분리 방법 등이 있으며, 표지 방식에는 대부분이 자기면역(autoimmunity) 표지 방식으로 상피 세포(epithelial cell) 특이적 항체(antibody)가 고정된 자석 입자를 이용한 분리 방법들이 사용되고 있다.Methods currently used to differentiate and detect cancer cells in blood can be divided into label-free and label-based methods. Non-labeling methods include separation using density difference, Dielectrophoretic (DEP) method, method using difference of inertial force according to size, and filter type separation method using size. In the labeling method, separation methods using magnet particles in which epithelial cell-specific antibodies are immobilized by autoimmunity labeling are mostly used.
이러한 방법으로 분리된 세포는 형광염색이나 RT-PCR 과 같은 방법으로 암세포를 구별하는 과정을 거치게 된다. 형광염색을 이용한 방법은 숙련자가 일일이 암세포를 선별하기 때문에 시간이 오래 걸리고 주관적인 판단에 의해 오차 범위가 커지게 된다. 또한 RT-PCR을 이용한 분자 분석 방법은 불특정 유전자의 증폭으로 인한 false-positive 결과가 나타나거나 특정 단백질의 발현 한계에 의하여 false-negative 결과가 나타날 수 있다는 문제점을 가지고 있다.Cells isolated by this method are subjected to a process such as fluorescent staining or RT-PCR to distinguish cancer cells. The method using fluorescence staining takes a long time because the expert selects each cancer cell, and the error range becomes larger due to subjective judgment. In addition, the molecular analysis method using RT-PCR has a problem that a false-positive result due to the amplification of an unspecific gene or a false-negative result due to the expression limit of a specific protein may occur.
flow cytometry를 이용하여 분리된 세포를 분별하는 방법은 실시간으로 지나가는 세포를 측정함으로써 측정 시간을 줄일 수 있다는 장점이 있다. Flow cytometry 방법은 빛을 이용하거나 전기적 방법을 이용하는 두 가지 방법으로 나눌 수 있는데 전기적 방법을 이용하여 세포를 구별하는 기준은 일반적으로 혈중 암세포는 혈액세포보다 크기가 크다는 점을 바탕으로 하여 전기적 측정 신호의 크기를 이용하여 암세포를 구별할 수 있다. 하지만 암세포는 이종(heterogeneous) 형질을 가지고 있기 때문에 크기만을 이용하여 암세포를 구별한다는 것은 한계가 있다.The method of separating the separated cells using flow cytometry has the advantage that the measurement time can be reduced by measuring the passing cells in real time. Flow cytometry can be divided into two methods using light or electrical methods. Based on the fact that blood cells are generally larger than hematocytes, the criteria for distinguishing cells using an electrical method are as follows: The size of cancer cells can be distinguished. However, since cancer cells have heterogeneous traits, it is limited to distinguish cancer cells using only their size.
또한, 세포분석의 경우 미세유체 채널에 세포를 흘려주어 전극사이로 세포가 지나가도록 한 후 세포의 전기임피던스를 측정하여 세포의 특성을 검출하는 방법이 한 예로 사용되었다. 그러나, 종래 방법의 경우 세포 자체의 임피던스 차이만을 검출했을 뿐, 별도의 조성물을 이용하여 세포의 임피던스를 조절한 후 이를 이용하여 세포를 분별하는 방법에 대해서는 연구되지 않았다.
In addition, in the case of cell analysis, a method of flowing the cells through the microfluidic channel to allow cells to pass between the electrodes, and then measuring the electrical impedance of the cells to detect the characteristics of the cells was used as an example. However, in the case of the conventional method, only the impedance difference of the cells themselves was detected, and no method of controlling the impedance of the cells using a separate composition and using them to separate the cells was studied.
본 발명은 상기와 같은 종래 세포 분별 방식 기술의 문제점을 해결하기 위하여 오직 전기적 신호만을 이용하여 주파수 위상차를 이용한 세포 분별 방법을 제공하는 것을 목적으로 한다. It is an object of the present invention to provide a cell sorting method using a frequency phase difference using only an electrical signal to solve the problems of the conventional cell sorting technique.
본 발명은 또한 본 발명에 의한 세포 분별 방법을 위한 세포 임피던스 조절용 조성물을 제공하는 것을 목적으로 한다.
It is another object of the present invention to provide a composition for controlling cell impedance for a cell fractionation method according to the present invention.
일 구현예에 따르면, 본 발명은 According to one embodiment,
(A) 분별을 원하는 세포를 포함하는 시료를 준비하는 단계; (A) preparing a sample containing cells to be fractionated;
(B) 상기 시료 속에 존재하는 세포 표면에 존재하는 단백질과 항체를 결합시키는 단계; (B) binding an antibody with a protein existing on the cell surface existing in the sample;
(C) 상기 항체 표면에 세포 전도성 조절을 위한 전도성 입자를 결합시키는 단계; 및(C) binding conductive particles for cell conduction control to the surface of the antibody; And
(D) 상기 전도성 입자가 결합된 세포의 위상을 측정하는 단계; 를 포함하는 상기 구현예에 따른 세포 분별용 조성물을 이용한 세포 분별 방법을 제공하고자 한다.
(D) measuring the phase of the cell to which the conductive particles are bound; The present invention provides a cell fractionation method using the cell fraction composition according to the above embodiment.
도 1은 종래 출력신호의 크기(amplitude) 변화를 이용한 세포 분별 방법의 일 예를 나타낸다. FIG. 1 shows an example of a cell sorting method using a change in amplitude of a conventional output signal.
도 1을 참고하면, 인가전극 1번에 out of phase로 전압을 인가하고 인가전극 2번에는 in phase로 전압을 인가할 때, 세포가 존재하지 않으면 두 감지전압의 합은 0 이기 때문에 출력신호도 0 V으로 측정된다. 그러나, 인가전극 1번과 감지전극 사이에 세포가 지나가게 되면, 일반적으로 세포는 일반적으로 dielectric 물질이므로 세포가 가지는 저항이 주변의 미디어보다 크기 때문에, 전류는 대부분 세포 주변의 미디어로 흐르게 되며, 세포에 의하여 인가전극 1번과 감지전극 사이의 저항이 인가전극 2번과 감지전극 사이의 저항에 비해 증가하게 된다. 따라서 두 전극 사이의 균형이 깨어지고 두 전극 사이에 발생하는 전압 차만큼 출력신호가 나타나고, 이때 출력신호는 세포의 부피에 비례하게 된다. Referring to FIG. 1, when a voltage is applied to the applied
암세포가 일반세포보다 부피가 크기 때문에 출력신호의 크기(amplitude)를 이용하여 두 세포를 구별할 수 있으나, 암세포는 크기가 매우 다양하기 때문에 단순히 크기만 가지고 세포를 구별하는 데에는 한계가 있다. Because cancer cells are bulkier than normal cells, we can distinguish between two cells using the amplitude of the output signal. However, since cancer cells are very large in size, there is a limitation in simply distinguishing cells from each other.
따라서 본 발명자들은 단순 전기적 출력 신호의 크기(amplitude)뿐만 아니라 본 발명에 의한 전도성 조절용 조성물에 의해 출력의 위상 변화(phase 변화)를 동시에 측정함으로써 암세포를 더욱 정확하게 분별할 수 있도록 하였다. Therefore, the present inventors have made it possible to more accurately discriminate cancer cells by measuring not only the amplitude of a simple electrical output signal but also the phase change (phase change) of the output by the composition for controlling conductivity according to the present invention.
도 2는 본 발명에 따른 전도성 입자가 결합된 세포의 위상차를 이용한 세포 분별 방법의 일 예를 나타낸다.FIG. 2 shows an example of a cell fractionation method using the phase difference of the cells to which the conductive particles according to the present invention are bound.
도 2를 참고하면, 전도성 물질이 결합된 암세포가 전극 사이를 지나가게 되면, 암세포 표면에 결합된 전도성 물질은 주변의 용매보다 전도도가 높기 때문에 전류가 쉽게 흐르게 된다. 따라서, 두 전극 사이의 저항값이 오히려 작아지는 효과가 나타나게 되고, 이에 따라 위상은 저항이 작아진 인가전극 1번의 위상을 따라가게 되어 결과적으로 out-of-phase의 출력신호가 나타나게 된다. 따라서 일반세포와는 위상차가 나게 되고 이러한 위상변화를 측정함으로써 세포를 정확하게 분별할 수 있는 것이다. Referring to FIG. 2, when a cancer cell to which a conductive substance is bound passes between the electrodes, the conductive substance bonded to the cancer cell surface has a higher conductivity than the surrounding solvent, so that the current easily flows. Accordingly, the resistance value between the two electrodes is reduced, so that the phase follows the phase of the applied
세포 임피던스는 세포의 종류에 따른 전기적 특성, 기질에 대한 부착능력등의 차이로 인해 달라질 수 있는 것으로 주파수의 영향을 또한 받는다. 저주파수의전류가 인가되는 경우 이중층 지질막으로 구성된 세포막을 쉽게 투과하지 못하고 미세한 간격을 갖는 세포-전극 및 세포-세포간 접합부를 통해 흐르게 되는데 이는 세포외질의 저항으로 나타낼 수 있다. 반면, 고주파수의 교류전류는 지질세포막을 통과할 수 있게 되고 해당 주파수 영역대에서 세포막 임피던스의 분산(dispersion)이 관측되며 따라서 세포막의 전기 임피던스 특성은 저주파수 성분과 고주파수 성분을 포함하고 있다.
Cell impedance is also affected by frequency, which can vary due to differences in electrical properties, attachment to substrate, etc., depending on the cell type. When a low-frequency current is applied, a cell membrane composed of a bilayer lipid membrane can not easily permeate and flows through a cell-electrode and a cell-cell junction having a minute gap. On the other hand, high-frequency alternating currents can pass through the lipid membrane and the dispersion of the membrane membrane impedance in the corresponding frequency region is observed, thus the electrical impedance characteristics of the membrane include the low-frequency component and the high-frequency component.
(A) 분별을 원하는 세포를 포함하는 시료를 준비하는 단계(A) preparing a sample containing cells to be separated
본 명세서에서 사용된 용어 “시료”는 표적 물질, 예를 들면 단백질을 함유하거나 함유하고 있는 것으로 추정되어 분석이 이루어질 조성물을 의미한다. 상기 시료는 조직, 혈액, 골수액, 림프액, 타액, 누액, 뇨, 점막액 또는 양수를 포함할 수 있으나, 이에 한정되는 것은 아니다. As used herein, the term " sample " refers to a composition to be analyzed which is presumed to contain or contain a target material, e.g., protein. The sample may include, but is not limited to, tissue, blood, bone marrow fluid, lymph fluid, saliva, fluid, urine, mucous membrane fluid or amniotic fluid.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 세포는 암, 류마티스성 관절염, 전신 홍반성 루푸스, 제1형 당뇨병, 다발성 경화증, 항호중구 세포질 항체-연관성 혈관염을 포함하는 자가면역질환, 또는 결핵(Tuberculosis), 리스테리아증(Listeriosis), 레지오넬라증 (Legionnaires’disease), 칸디다증 (candidiasis), 또는 전염성 단핵구증(infectious mononucleosis)을 포함하는 미생물 감염과 관련된 세포인 것을 특징으로 한다. In the cell fractionation method using the composition for cell fraction according to the present invention, the cell may be an autoimmune disease including cancer, rheumatoid arthritis, systemic lupus erythematosus,
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 암은 난소암, 유방암, 대장암, 전립선암, 흑색종, 호지킨 림프종, 비호지킨 림프종을 포함하는 림프종, 백혈병 (급성 골수성 백혈병, 만성 골수성 백혈병, 급성 림프구성 백혈병, 만성 림프구성 백혈병을 포함하는 백혈병, 위암, 신장세포암종, 대장암, 결장암, 폐암, 뇌암, 자궁 경부암 또는 식도암인 것을 특징으로 한다. In the cell fractionation method using the cell fractioning composition according to the present invention, the cancer is selected from the group consisting of ovarian cancer, breast cancer, colon cancer, prostate cancer, melanoma, Hodgkin's lymphoma, lymphoma including non-Hodgkin's lymphoma, leukemia , Chronic myelogenous leukemia, acute lymphocytic leukemia, leukemia including chronic lymphocytic leukemia, stomach cancer, renal cell carcinoma, colon cancer, colon cancer, lung cancer, brain cancer, cervical cancer or esophageal cancer.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 세포 표면에 존재하는 단백질은 암과 연관된 것을 특징으로 한다. In the cell fractionation method using the cell fraction composition according to the present invention, the protein present on the cell surface is characterized by being associated with cancer.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 세포 표면에 존재하는 단백질은 A33, ABL2, ADAM 10, AFP, ALA, ALIX, ALPL, ApoJ/CLU, ASCA, ASPH(A-IO), ASPH(DOIP), AURKB, B7H3, B7H3, B7H4, BCNP, BDNF, CA125(MUC16), CA-19-9, C-Bir, CD10, CD151, CD24, CD41, CD44, CD46, CD59(MEM-43), CD63, CD63, CD66eCEA, CD81, CD81, CD9, CD9, CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3(ErbB3), HSP70, HSPB1, hVEGFR2, iC3b, IL-1B, IL6R, IL6Unc, IL7Ralpha/CD127, IL8, INSIG-2, Integrin, KLK2, LAMN, Mammoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Mucl, MUC17, MUC2, Ncam, NDUFB7, NGAL, NK-2R(C-21), NT5E (CD73), p53, PBP, PCSA, PCSA, PDGFRB, PIMl, PRL, PSA, PSA, PSMA, PSMA, RAGE, RANK, ReglV, RUNX2, S100-A4, seprase/FAP, SERPINB3, SIM2(C-15), SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, TFF3, TGM2, TIMP-1, TMEM211, Trail-R2, Trail-R4, TrKB(poly), Trop2, TsglOl, TWEAK, UNC93A, VEGFA 또는 wnt-5a(C-16)인 것을 특징으로 한다.
In the cell fractionation method using the cell sorting composition according to the present invention, the protein present on the cell surface is A33, ABL2, ADAM10, AFP, ALA, ALIX, ALPL, ApoJ / CLU, ASCA, ASPH ), ASPH (DOIP), AURKB, B7H3, B7H3, B7H4, BCNP, BDNF, CA125 (MUC16), CA-19-9, C-A, CD10, CD151, CD24, CD41, CD44, CD46, 43, CD63, CD63, CD66eCEA, CD81, CD81, CD9, CD9, CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2 IL-1B, IL6R, IL6Unc, IL7Ralpha / CD127, IL-6, IL-8, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3 (ErbB3), HSP70, HSPB1, hVEGFR2, IL-8, INSIG-2, Integrin, KLK2, LAMN, Mammoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Mucl, MUC17, MUC2, Ncam, NDUFB7, NGAL, (SEQ ID NO: 21), NT5E (CD73), p53, PBP, PCSA, PCSA, PDGFRB, PIMI, PRL, PSA, PSA, PSMA, PSA, RAGE, RANK, ReglV, RUNX2, S100-A4, seprase / FAP, SERPINB3, SIM2 C-15), SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, TFF3, TGM2, TIMP-1, TMEM211, Trail -R2, Trail-R4, TrKB (poly), Trop2, TsglOl, TWEAK, UNC93A, VEGFA or wnt-5a (C-16).
(B) 세포 표면에 존재하는 단백질과 항체를 결합시키는 단계(B) a step of binding the antibody and the protein present on the cell surface
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 항체는 키메라 항체, 인간화 항체, 항체 단편 또한 항체 모방체를 포함하는 것을 특징으로 한다. In the cell fractionation method using the composition for cell fraction according to the present invention, the antibody is characterized in that the chimeric antibody, the humanized antibody, and the antibody fragment also include an antibody mimetic.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 항체 단편은 scFv, BITE, TandAb, Immunobody, Flexibody, Nanobody, Triomab, Troybody, Pepbody, Vaccibody, SMIP, Fab(fragment antigen binding), mAb2, UniBody, Fv (fragment variable), dAB, scFv-Fc, Diabody, Tetrabody, Minibody, scFab(single chain Fab), 또는 Fcab을 포함하는 것을 특징으로 한다. In the cell fractionation method using the cell sorting composition according to the present invention, the antibody fragment may be selected from scFv, BITE, TandAb, Immunobody, Flexibody, Nanobody, Triomab, Troybody, Pepbody, Vaccibody, SMIP, Fab , UniBody, Fv (fragment variable), dAB, scFv-Fc, Diabody, Tetrabody, Minibody, scFab (single chain Fab), or Fcab.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 항체 모방체는 DARPin, Tetranectin, Affibody, Transbody, Anticalin, AdNectin, Affilin, Microbody, Peptide aptamer, Phylomer, Stradobody, Avimer, Maxibodiy, Evibody, 또는 Fynomer을 포함하는 것을 특징으로 한다.
In the cell fractionation method using the cell sorting composition according to the present invention, the antibody mimetic may be DARPin, Tetranectin, Affibody, Transbody, Anticalin, AdNectin, Affilin, Microbody, Peptide aptamer, Phylomer, Stradobody, Avimer, Maxibodiy, Evibody, Or a Fynomer.
(C) 항체 표면에 전도성 입자를 결합시키는 단계(C) binding the conductive particles to the surface of the antibody
선택적으로, 본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 전도성 입자는 실험 단계를 단순화 시키기 위하여, 세포 표면에 존재하는 단백질과 항체를 결합시키는 단계 이전에 항체에 사전에 부착될 수도 있다. Alternatively, in the cell fractionation method using the cell fractioning composition according to the present invention, in order to simplify the experimental step, the conductive particles may be pre-attached to the antibody prior to the step of binding the antibody and the protein existing on the cell surface have.
도 3에 본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 사용되는 전도성 입자가 세포 표면 단백질에 결합된 상태를 나타내었다.
FIG. 3 shows the state where the conductive particles used in the cell fractionation method using the cell fractioning composition of the present invention are bound to the cell surface protein.
(D) 전도성 입자가 (D) conductive particles 결합된Combined 세포의 위상을 측정하는 단계 Measuring the phase of the cell
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 세포의 위상을 측정하는 단계에서는 1 KHz 이상의 주파수에서 측정하는 것을 특징으로 한다. 보편적으로 정도의 차이는 있겠으나, 인가되는 전류의 주파수가 너무 낮거나 높은 경우 세포존재시와 부재시 총임피던스의 차이가 감소하는 경향이 있다. 따라서, 적정범위의 주파수를 갖는 교류를 인가하는 것이 바람직하다.In the cell fractionation method using the cell fraction composition according to the present invention, the phase is measured at a frequency of 1 KHz or more in the step of measuring the phase of the cell. Although there is a general difference in the degree of difference, when the frequency of the applied current is too low or high, there is a tendency that the difference in total impedance between the presence and absence of the cell decreases. Therefore, it is preferable to apply an alternating current having an appropriate range of frequencies.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 세포의 위상을 측정하기 위해 인가하는 주파수는 분별하고자 하는 조성물의 전도도에 따라 변화시킬 수 있다. 본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서,상기 세포의 위상을 측정하는 단계에서 적용하는 주파수는 세포 분별용 조성물의 전도도에 따라 조절하는 것이 바람직하다. In the cell fractionation method using the cell fractioning composition according to the present invention, the frequency applied for measuring the phase of a cell can be changed according to the conductivity of the composition to be fractionated. In the cell fractionation method using the cell fraction composition according to the present invention, the frequency applied in the step of measuring the phase of the cell is preferably adjusted according to the conductivity of the cell fraction composition.
본 발명에 의한 세포 분별용 조성물을 이용한 세포 분별 방법에 있어서, 상기 전도성 입자가 결합된 세포의 전기 전도도는 10 mS/m 이상인 것을 특징으로 한다.
In the cell fractionation method using the cell fraction composition according to the present invention, the electric conductivity of the cell to which the conductive particles are bound is 10 mS / m or more.
일 구현 예에 따르면, 본 발명은According to one embodiment,
세포 표면에 존재하는 단백질에 특이적으로 결합하는 항체; 및 An antibody that specifically binds to a protein existing on the cell surface; And
상기 항체와 결합된 전도성 입자; 를 포함하는 세포 분별용 조성물을 제공하고자 한다. Conductive particles bound to the antibody; And a method for cell sorting.
본 발명에 따른 세포 분별용 조성물에 의하여 전도성 입자가 세포 표면의 항체와 결합된 예시적인 구현예를 도 3에 나타내었다.
An exemplary embodiment in which conductive particles are bound to an antibody on a cell surface by the composition for cell sorting according to the present invention is shown in Fig.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 세포는 암, 류마티스성 관절염, 전신 홍반성 루푸스, 제1형 당뇨병, 다발성 경화증, 항호중구 세포질 항체-연관성 혈관염을 포함하는 자가면역질환, 또는 결핵(Tuberculosis), 리스테리아증(Listeriosis), 레지오넬라증 (Legionnaires’disease), 칸디다증 (candidiasis), 또는 전염성 단핵구증(infectious mononucleosis)을 포함하는 미생물 감염과 관련된 세포인 것을 특징으로 한다. In the composition for cell division according to the present invention, the cell may be an autoimmune disease including cancer, rheumatoid arthritis, systemic lupus erythematosus,
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 암은 난소암, 유방암, 대장암, 전립선암, 흑색종, 호지킨 림프종, 비호지킨 림프종을 포함하는 림프종, 백혈병 (급성 골수성 백혈병, 만성 골수성 백혈병, 급성 림프구성 백혈병, 만성 림프구성 백혈병을 포함하는 백혈병, 위암, 신장세포암종, 대장암, 결장암, 폐암, 뇌암, 자궁 경부암 또는 식도암인 것을 특징으로 한다. In the composition for cell division according to the present invention, the cancer is selected from the group consisting of ovarian cancer, breast cancer, colon cancer, prostate cancer, melanoma, Hodgkin's lymphoma, lymphoma including non-Hodgkin's lymphoma, leukemia (acute myelogenous leukemia, Acute lymphoblastic leukemia, leukemia including chronic lymphocytic leukemia, gastric cancer, renal cell carcinoma, colon cancer, colon cancer, lung cancer, brain cancer, cervical cancer or esophageal cancer.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 세포 표면에 존재하는 단백질은 암과 연관된 것을 특징으로 한다. In the composition for cell separation according to the present invention, the protein present on the surface of the cell is characterized by being associated with cancer.
본 명세서에서 사용된 용어 “단백질”은 아미노산의 중합체쇄를 의미하는 것으로, “폴리펩티드”란 용어와 혼용될 수 있다. 상기 폴리펩티드는 천연 또는 합성 단백질, 단백질 단편 및 단백질 서열의 폴리펩티드 유사체를 포함한다.
As used herein, the term " protein " refers to a polymeric chain of amino acids and may be interchangeable with the term " polypeptide ". Such polypeptides include natural or synthetic proteins, protein fragments and polypeptide analogs of protein sequences.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 세포 표면에 존재하는 단백질은 A33, ABL2, ADAM 10, AFP, ALA, ALIX, ALPL, ApoJ/CLU, ASCA, ASPH(A-IO), ASPH(DOIP), AURKB, B7H3, B7H3, B7H4, BCNP, BDNF, CA125(MUC16), CA-19-9, C-Bir, CD10, CD151, CD24, CD41, CD44, CD46, CD59(MEM-43), CD63, CD63, CD66eCEA, CD81, CD81, CD9, CD9, CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3(ErbB3), HSP70, HSPB1, hVEGFR2, iC3b, IL-1B, IL6R, IL6Unc, IL7Ralpha/CD127, IL8, INSIG-2, Integrin, KLK2, LAMN, Mammoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Mucl, MUC17, MUC2, Ncam, NDUFB7, NGAL, NK-2R(C-21), NT5E (CD73), p53, PBP, PCSA, PCSA, PDGFRB, PIMl, PRL, PSA, PSA, PSMA, PSMA, RAGE, RANK, ReglV, RUNX2, S100-A4, seprase/FAP, SERPINB3, SIM2(C-15), SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, TFF3, TGM2, TIMP-1, TMEM211, Trail-R2, Trail-R4, TrKB(poly), Trop2, TsglOl, TWEAK, UNC93A, VEGFA 또는 wnt-5a(C-16)인 것을 특징으로 한다. In the composition for cell division according to the present invention, the protein present on the surface of the cell is A33, ABL2, ADAM10, AFP, ALA, ALIX, ALP, ApoJ / CLU, ASCA, ASPH (A- ), AURKB, B7H3, B7H3, B7H4, BCNP, BDNF, CA125 (MUC16), CA-19-9, C-A, CD10, CD151, CD24, CD41, CD44, CD46, CD59 CD63, CD66eCEA, CD81, CD81, CD9, CD9, CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, IL-6, IL7Ralpha / CD127, IL8, INSIG-2 (SEQ ID NO: 2), FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3 (ErbB3), HSP70, HSPB1, hVEGFR2, , Integrin, KLK2, LAMN, Mammoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Mucl, MUC17, MUC2, Ncam, NDUFB7, NGAL, NK- RAGE, RANK, ReglV, RUNX2, S100-A4, seprase / FAP, SERPINB3, SIM2 (C-15), P53, PBP, PCSA, PCSA, PDGFRB, PIM1, PRL, PSA, PSA, PSMA, PSMA, R4, TrKB (poly), Trop2, TMP2, TIMP-1, TMEM211, Trail-R2, SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, TFF3, TsglOl, TWEAK, UNC93A, VEGFA or wnt-5a (C-16).
본 명세서에서 사용된 용어 “항체”는 다클론 항체 및 단클론 항체를 모두 포함하며, 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태 뿐만 아니라 항체 분자의 단편도 포함한다. 항체 분자의 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 의미하며 단일 사슬(single-chain) Fv(scFv), Fab, F(ab'), F(ab')2 또는 단일 도메인(single domain)을 포함할 수 있으나, 이에 한정되는 것은 아니다. The term " antibody " as used herein includes both polyclonal and monoclonal antibodies, as well as complete forms with two full-length light chains and two full-length heavy chains, as well as fragments of antibody molecules. A fragment of an antibody molecule means a fragment having at least an antigen-binding function and is a single-chain Fv (scFv), Fab, F (ab ') 2, F (ab') 2, But is not limited thereto.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 항체는 키메라 항체, 인간화 항체, 항체 단편 또한 항체 모방체를 포함하는 것을 특징으로 한다. In the cell-dividing composition according to the present invention, the antibody is characterized in that the chimeric antibody, the humanized antibody, and the antibody fragment also include an antibody mimetic.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 항체 단편은 scFv, BITE, TandAb, Immunobody, Flexibody, Nanobody, Triomab, Troybody, Pepbody, Vaccibody, SMIP, Fab(fragment antigen binding), mAb2, UniBody, Fv (fragment variable), dAB, scFv-Fc, Diabody, Tetrabody, Minibody, scFab(single chain Fab), 또는 Fcab을 포함하는 것을 특징으로 한다. In the composition for cell sorting according to the present invention, the antibody fragment may be selected from the group consisting of scFv, BITE, TandAb, Immunobody, Flexibody, Nanobody, Triomab, Troybody, Pepbody, Vaccibody, SMIP, Fragment antigen binding, mAb2, UniBody, Fv fragment variable, dAB, scFv-Fc, Diabody, Tetrabody, Minibody, scFab (single chain Fab), or Fcab.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 항체 모방체는 DARPin, Tetranectin, Affibody, Transbody, Anticalin, AdNectin, Affilin, Microbody, Peptide aptamer, Phylomer, Stradobody, Avimer, Maxibodiy, Evibody, 또는 Fynomer을 포함하는 것을 특징으로 한다.
In the composition for cell sorting according to the present invention, the antibody mimetic includes DARPin, Tetranectin, Affibody, Transbody, Anticalin, AdNectin, Affilin, Microbody, Peptide aptamer, Phylomer, Stradobody, Avimer, Maxibodiy, Evibody, .
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 전도성 입자는 전기 전도도가 10 mS/m 이상인 것을 특징으로 한다. In the composition for cell separation according to the present invention, the conductive particles have an electrical conductivity of 10 mS / m or more.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 플레이트 형상의 전도성 입자는 본 발명자들이 2014년 6월 18일에 출원한 “미세 입자 분별용 플레이트 및 이의 제조 방법”에 의하여 제조할 수 있다. In the cell-dividing composition according to the present invention, the plate-shaped conductive particles can be produced by the inventors of the present invention on June 18, 2014, " a plate for fine particle fractionation and a method for producing the same. &Quot;
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 전도성 입자는 흑연, 금, 은, 크롬, 구리, 및 니켈을 포함하는 금속 또는 전도성 고분자인 것을 특징으로 한다. In the cell-dividing composition according to the present invention, the conductive particles are a metal or a conductive polymer including graphite, gold, silver, chromium, copper, and nickel.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 전도성 입자는 크기가 10 nm 내지 10 ㎛ 인 것을 특징으로 한다. In the cell-dividing composition according to the present invention, the conductive particles are characterized by a size of 10 nm to 10 탆.
본 발명에 의한 세포 분별용 조성물에 있어서, 상기 전도성 입자는 플레이트 형상 또는 입자 형상인 것을 특징으로 한다.
In the composition for cell separation according to the present invention, the conductive particles are characterized by being in the form of a plate or a particle.
본 발명에 의한 세포 분별용 조성물은 세포 표면 특이 단백질과 결합하는 항체 및 상기 항체에 결합하는 전도성 입자에 의하여 세포의 전도도를 높이게 되고 이와 같이 전도성 입자가 부착된 세포는 고주파수에서 위상 변화를 나타내므로 용이하게 분별할 수 있게 된다.
The composition for cell sorting according to the present invention enhances the conductivity of cells by an antibody that binds to a cell surface specific protein and a conductive particle that binds to the antibody. Since the cell having the conductive particles attached thereto exhibits a phase change at a high frequency, .
도 1은 본 발명에 따른 전도성 입자가 결합된 세포의 위상차를 이용한 세포 분별 방법의 일 예를 나타낸다.
도 2는 출력신호의 amplitude 변화를 이용한 세포 분별 방법의 일 예를 나타낸다.
도 3은 본 발명에 따른 세포 분별용 조성물이 세포에 결합된 예시적인 구현예를 나타낸다.
도 4는 본 발명의 일 실시예에 의한 전도성 입자가 부착된 세포의 형상을 나타낸다.
도 5는 본 발명의 일 실시예에 의한 백혈구, 대장암 세포, 그래핀 입자를 이용한 주파수에 따른 위상변화 측정 결과를 나타낸다.
도 6은 본 발명의 일 실시예에 의한 백혈구세포, 대장암세포 및 그래핀이 결합된 대장암 세포에 전압을 인가한 경우 500 kHz에서 응답 크기 및 10 MHz에서 응답 위상차를 측정하여 하나의 scatter plot으로 나타낸 결과이다. FIG. 1 shows an example of a cell sorting method using the phase difference of cells to which the conductive particles according to the present invention are bound.
2 shows an example of a cell sorting method using an amplitude change of an output signal.
Figure 3 shows an exemplary embodiment in which the cell sorting composition according to the present invention is bound to cells.
FIG. 4 shows the shape of cells with conductive particles according to an embodiment of the present invention.
FIG. 5 shows measurement results of phase shift according to frequency using white blood cells, colorectal cancer cells, and graphene particles according to an embodiment of the present invention.
FIG. 6 is a graph showing the response magnitudes at 500 kHz and the response phase difference at 10 MHz when voltage is applied to leukocyte cells, colon cancer cells, and graphene-conjugated colorectal cancer cells according to an embodiment of the present invention. Respectively.
이하에서는 본 발명을 실시예에 의하여 더욱 상세히 설명한다. 그러나, 본 발명이 이하의 실시예에 의하여 한정되는 것은 아니다.
Hereinafter, the present invention will be described in more detail by way of examples. However, the present invention is not limited by the following examples.
실시예Example 1: 세포 표면에 발현되는 1: expressed on the cell surface EpCAMEpCAM 단백질에 전도성 입자로서 As conductive particles to proteins 그래핀Grapina 부착 Attach
그래핀은 입자의 크기는 5 ㎛ 이하이고 두께는 50 nm 미만인 제품을 구매하여 사용하였으며, 플라즈마로 처리하여 10개 층 내지 100개 층을 포함하도록 박리(exfoliated) 되었다. The graphene was purchased and used as a product having a particle size of 5 μm or less and a thickness of less than 50 nm, and was treated with plasma to exfoliate to include 10 to 100 layers.
그래핀 표면을 dextran 으로 코팅하기 위해 Dextran 20 mg과 제조된 그래핀 10 mg을 0.5 mol/L 의 농도의 NaOH 수용액에 혼합하였다. 용액을 1시간 동안 온도를 상온으로 유지하면서 초음파로 분쇄하고 3시간 동안 정치한 후 3회 세정하였다. To coat the graphene surface with dextran, 20 mg of Dextran and 10 mg of the prepared graphene were mixed in a 0.5 mol / L NaOH aqueous solution. The solution was sonicated for 1 hour while maintaining the temperature at room temperature, allowed to stand for 3 hours, and then washed three times.
대장암 세포를 준비하고 대장암 세포 표면에 발현되는 EpCAM 단백질과 반응하는 항체 (STEM CELL TECH.)를 주입한 후 그 다음 dextran이 코팅된 그래핀을 주입한 후 각각 4℃ 에서 30분 반응시켰다. Colon cancer cells were prepared and injected with an antibody (STEM CELL TECH.) Reacting with EpCAM protein expressed on the surface of colorectal cancer cells. Then, dextran-coated graphene was injected and reacted at 4 ° C for 30 minutes.
이와 같이 그래핀이 표면에 결합된 세포의 SEM 사진을 측정하고 그 결과를 도 4에 나타내었다. 도 4에서 dextran이 코팅된 그래핀이 세포 표면에 부착된 것을 확인할 수 있다.
SEM photographs of cells bound to the surface of graphenes were measured, and the results are shown in FIG. 4, it can be confirmed that dextran-coated graphene adheres to the cell surface.
실시예Example 2: 세포 구별 효율 측정 2: Measurement of cell differentiation efficiency
일반 정상 혈액 세포 종류의 하나인 백혈구,암세포로서 대장암 세포, 및 그래핀 입자를 준비하여 주파수에 따른 위상을 측정하였다. 이때 측정 한계에 의하여 10 MHz 주파수까지 위상을 측정하였고 위상차가 가장 많이 나타나는 10 MHz 주파수를 위상 측정 주파수로 설정하였다.
White blood cells, which are one of the normal normal blood cell types, cancer cells as cancer cells, and graphene particles were prepared and their phases were measured according to frequency. At this time, the phase was measured up to the 10 MHz frequency by the measurement limit, and the 10 MHz frequency with the highest phase difference was set as the phase measurement frequency.
도 5로부터 알 수 있는 바와 같이, 백혈구와 그래핀이 부착되지 않은 대장암 세포는 주파수에 따른 위상이 유사하지만, 그래핀 입자의 경우 주파수에 따른 위상은 일반 세포와는 현저하게 다르고, 일반 세포와 그래핀 입자의 위상 차이는 주파수가 올라갈수록 증가하는 것을 알 수 있었다. As can be seen from FIG. 5, white blood cells and non-grafted colon cancer cells are similar in phase to each other in frequency, but in graphene particles, the phase according to frequency is significantly different from that of normal cells, It was found that the phase difference of graphene particles increases with increasing frequency.
이와 같은 결과로부터 분리하고자 하는 세포에 그래핀을 부착하여 특정 주파수에서 위상차를 측정함으로써 그래핀이 부착된 세포와 부착되지 않은 세포를 분리 및 구별해 낼 수 있을 것으로 예상할 수 있었다. 그 다음, 백혈구와 그래핀이 결합되어 있지 않은 대장암세포와 상기 실시예처럼 세포 표면에 그래핀을 결합시킨 대장암 세포를 각각 준비하고, 500 kHz 와 10 MHz 두 주파수에서 각 세포의 진폭과 위상을 측정하여 그 결과를 하나의 scatter plot으로 하여 도 6에 나타내었다. From these results, it could be expected that graphene was attached to the cells to be separated and the phase difference at a specific frequency was measured, thereby separating and distinguishing graphene-attached cells from non-attached cells. Next, colonic cancer cells not bound to leukocytes and graphene, and colon cancer cells bound to graphene cells on the cell surface as in the above example were prepared, and the amplitudes and phases of the cells were measured at 500 kHz and 10 MHz frequencies And the results are shown in FIG. 6 as one scatter plot.
도 6 에서 백혈구와 그래핀이 부착되지 않은 대장암세포는 10 MHz에서 위상이 비슷하게 나타나지만 그래핀이 부착된 대장암세포의 위상은 그래핀의 영향으로 위상 변화가 일어나는 것을 확인하였다. 따라서 그래핀을 이용한 위상차를 측정함으로써 세포를 분별할 수 있음이 확인되었다.In FIG. 6, white blood cells and non-grafted colon cancer cells show similar phase at 10 MHz, but the phase of graphene-attached colon cancer cells was found to change due to the influence of graphene. Therefore, it was confirmed that the cells could be discriminated by measuring the phase difference using graphene.
Claims (13)
세포 표면에 존재하는 단백질과 전도성 조절용 조성물 내의 항체와 결합시키는 단계;
상기 항체 표면에 전도성 조절용 조성물 내의 전도성 조절용 입자를 결합시키는 단계; 및
상기 전도성 조절용 입자가 결합된 세포의 주파수 위상을 측정하는 단계; 를 포함하고
상기 세포의 주파수 위상을 측정하는 단계는1 kHz 이상의 주파수에서 측정하는 것을 특징으로 하는 것인, 주파수 위상차를 이용한 세포 분별 방법.
Preparing a sample containing cells to be fractionated;
Binding to a protein present on the cell surface and an antibody in the conductivity modulating composition;
Binding the conductivity modifying particles in the conductive modulating composition to the surface of the antibody; And
Measuring the frequency phase of the cells bound with the conductive modulating particles; Including the
Wherein the step of measuring the frequency phase of the cell is performed at a frequency of 1 kHz or more.
상기 전도성 조절용 입자가 결합된 세포의 전기 전도도는10 mS/m 이상인 것을 특징으로 하는 세포 분별 방법.
The method according to claim 1,
Wherein the conductivity of the cells to which the conductive particles are bound is 10 mS / m or more.
상기 세포는 암, 류마티스성 관절염, 전신 홍반성 루푸스, 제1형 당뇨병, 다발성 경화증, 항호중구 세포질 항체-연관성 혈관염을 포함하는 자가면역질환, 또는 결핵(Tuberculosis), 리스테리아증(Listeriosis), 레지오넬라증(Legionnaires’disease), 칸디다증 (candidiasis), 또는 전염성 단핵구증(infectious mononucleosis)인 것을 특징으로 하는 세포 분별 방법.
The method according to claim 1,
The cell may be an autoimmune disease including cancer, rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, anti-neutrophil cytoplasmic antibody-associated vasculitis, or an autoimmune disease including Tuberculosis, Listeriosis, Wherein the cells are selected from the group consisting of Legionnaires disease, candidiasis, and infectious mononucleosis.
상기 암은 난소암, 유방암, 대장암, 전립선암, 흑색종, 호지킨 림프종, 비호지킨 림프종을 포함하는 림프종, 급성 골수성 백혈병, 만성 골수성 백혈병, 급성 림프구성 백혈병, 만성 림프구성 백혈병을 포함하는 백혈병, 위암, 신장세포암종, 대장암, 결장암, 폐암, 뇌암, 자궁 경부암 또는 식도암인 것을 특징으로 하는 세포 분별 방법.
5. The method of claim 4,
The cancer is selected from the group consisting of leukemia, including ovarian cancer, breast cancer, colorectal cancer, prostate cancer, melanoma, Hodgkin's lymphoma, lymphoma including non-Hodgkin's lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, acute lymphocytic leukemia, , Stomach cancer, kidney cell carcinoma, colon cancer, colon cancer, lung cancer, brain cancer, cervical cancer or esophageal cancer.
상기 세포 표면에 존재하는 단백질은 A33, ABL2, ADAM 10, AFP, ALA, ALIX, ALPL, ApoJ/CLU, ASCA, ASPH(A-IO), ASPH(DOIP), AURKB, B7H3, B7H3, B7H4, BCNP, BDNF, CA125(MUC16), CA-19-9, C-Bir, CD10, CD151, CD24, CD41, CD44, CD46, CD59(MEM-43), CD63, CD63, CD66eCEA, CD81, CD81, CD9, CD9, CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM-CSF, Gro-alpha, HAP, HER3(ErbB3), HSP70, HSPB1, hVEGFR2, iC3b, IL-1B, IL6R, IL6Unc, IL7Ralpha/CD127, IL8, INSIG-2, Integrin, KLK2, LAMN, Mammoglobin, M-CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Mucl, MUC17, MUC2, Ncam, NDUFB7, NGAL, NK-2R(C-21), NT5E (CD73), p53, PBP, PCSA, PCSA, PDGFRB, PIMl, PRL, PSA, PSA, PSMA, PSMA, RAGE, RANK, ReglV, RUNX2, S100-A4, seprase/FAP, SERPINB3, SIM2(C-15), SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, TFF3, TGM2, TIMP-1, TMEM211, Trail-R2, Trail-R4, TrKB(poly), Trop2, TsglOl, TWEAK, UNC93A, VEGFA 또는 wnt-5a(C-16)인 것을 특징으로 하는 세포 분별 방법.
The method according to claim 1,
The protein present on the surface of the cell is A33, ABL2, ADAM10, AFP, ALA, ALIX, ALPL, ApoJ / CLU, ASCA, ASPH (A- IO), ASPH (DOIP), AURKB, B7H3, B7H3, B7H4, BCNP , BDNF, CA125 (MUC16), CA-19-9, C-A, CD10, CD151, CD24, CD41, CD44, CD46, CD59 (MEM-43), CD63, CD63, CD66eCEA, CD81, , CDA, CDADC1, CRMP-2, CRP, CXCL12, CXCR3, CYFRA21-1, DDX-1, DLL4, DLL4, EGFR, Epcam, EphA2, ErbB2, ERG, EZH2, FASL, FLNA, FRT, GAL3, GATA2, GM 2, Integrin, KLK2, LAMN, Mammoglobin, M-IGF-I, IL-1B, IL6R, IL6Unc, IL7Ralpha / CD127, IL8, HGF, HSP70, HSP70, HSP70, HSPB1, HVEGFR2, CSF, MFG-E8, MIF, MISRII, MMP7, MMP9, MUC1, Mucl, MUC17, MUC2, Ncam, NDUFB7, NGAL, NK-2R (C- SPARC, SPC, SPDEF, SPP1, STEAP, STEAP4, SPARC, SPDC, PIMl, PRL, PSA, PSA, PSMA, PSMA, RAGE, RANK, ReglV, RUNX2, S100-A4, seprase / FAP, SERPINB3, SIM2 (C-16), TEF3, TGM2, TIMP-1, TMEM211, Trail-R2, Trail-R4, TrKB (poly), Trop2, TsglOl, TWEAK, UNC93A, VEGFA or wnt-5a A method for cell sorting.
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