KR100891456B1 - Method and apparatus for detecing cellular target materials to bioactive molecules - Google Patents

Abstract

A method and an apparatus for detecting cellular target materials to bioactive molecules are provided to improve analysis sensitivity by determining a high signal-to-background ratio and reduce a data analysis time. A method for detecting cellular target materials to bioactive molecules comprises the following steps of: preparing bioactive molecules and magnetic materials which can be separated by a magnetic field within a living cell; injecting a reaction partner coupled with a marker which can make presence or position images about the reaction partner to the bioactive molecules within the living cell; responding the bioactive molecules and reaction partner within the living cell; and smashing the living cell and applying the magnetic field in order to separate the bioactive molecules and the magnetic materials.

Description

METHOD AND APPARATUS FOR DETECING CELLULAR TARGET MATERIALS TO BIOACTIVE MOLECULES}

The present invention relates to a method and apparatus for detecting an intracellular target material for a physiologically active substance, and more particularly, after reacting a physiologically active substance with a corresponding reaction partner in living cells, applying cell disruption and magnetic field. A method and apparatus for detecting an intracellular target substance for a physiologically active substance, characterized in that by detecting a label separated from the physiologically active substance by determining the reaction partner as a target substance for the physiologically active substance will be.

More specifically, the present invention, after reacting the biologically active material bound to the magnetic material and the corresponding reaction partner in a living cell, and performing cell disruption and magnetic field, and bound to the bioactive material by intracellular reaction The present invention relates to a method and apparatus for easily and quickly and objectively detecting an intracellular target material for a physiologically active substance by quantifying the amount of labeling substance separated with the reaction partner.

Medicines composed of small molecule compounds exhibit efficacy by binding to specific proteins in cells and inhibiting or enhancing their function. The intracellular protein to which the low molecular weight compound binds is called a target protein. However, many of the drugs currently in use are often unknown to their target proteins (Clasy, J. & Walsh, C. Lessons from natural molecules.Nature 432, 829-837). Identifying the target proteins of small molecule compounds during drug development is essential for understanding the efficacy and adverse effects of small molecule compounds [Hart, C. P. Finding the target after screening the phenotype. Drug Discov. Today 10, 613-619 (2005)]. Once the target protein binds to the low molecular compound, the target protein can be easily analyzed, and various derivatives of the low molecular compound can be developed to study structure-activity relationships for optimization of the leading material. Hart, CP Finding the target after screening the phenotype. Drug Discov. Today 10, 613-619 (2005)].

One approach to identifying target proteins for small molecule compounds in drug development is affinity chromatogaraphy [Daub, H., Godl, K., Brehmer, D., Klebl, B., & Muller, G. Evaluation of kinase inhibitor selectivity by chemical proteomics. Assay Drug Develop. Technol. 2, 215-224 (2004). The affinity chromatography method binds a low molecular weight compound to a solid support, and then reacts the low molecular weight compound bound to the solid support with cell lysates to detect a target protein that specifically binds to the low molecular weight compound. How to pay Bantscheff et al. Recently reported a novel target protein of Gleevec, which has been used as a therapeutic agent for chronic myelogenous leukemia using affinity chromatography [Bantscheff, M., et al. Quantitative chemical proteomics reveals mechanisms of action of clinical ABL kinase inhibitors. Nat. Biotechnol. 25, 1035-1044 (2007); WO 2006/134056.

However, affinity chromatography has some disadvantages. First, in order to perform affinity chromatography, cell crushing is inevitable. When cell crushing destroys the cellular context, the natural structure or function of the target protein or protein complex in the living cell is detected. There is a disadvantage of not having it [Tavernier, J., et al. MAPPIT: a cytokine receptor-based two hybrid method in mammalian cells. Clin. Exp. Allergy 32, 1397-1404 (2002). Second, measures such as strengthening the amount of target protein required or expected in large quantities are required for cell lysate containing the target protein. Otherwise, short-lived or short-lived target proteins will not be detected [Tavernier, J., et al. MAPPIT: a cytokine receptor-based two hybrid method in mammalian cells. Clin. Exp. Allergy 32, 1397-1404 (2002).

Therefore, the inventors of the present invention overexpress the target protein in living cells, and transfer the magnetic material bound to the bioactive material (for example, a low molecular weight compound such as a drug) into the living cell, thereby physiologically active in the living cell. After reacting the substance with the corresponding target protein, cell disruption and magnetic field application are performed, and the target protein bound to the bioactive substance is separated and quantified by the intracellular reaction to quantify easily and quickly and objectively. A method and apparatus for detecting intracellular target proteins have been devised.

The present invention is to solve the problems of the prior art as described above, it can be confirmed whether the reaction between the biologically active material and the target material reflecting the living intracellular environment, and bound to the biologically active material by the intracellular reaction It is an object of the present invention to provide a method and apparatus for easily and quickly and objectively detecting an intracellular target substance for a bioactive substance by quantifying the amount of the labeling substance separated with the reaction partner.

In particular, the present invention can determine a relatively high signal-to-background ratio (signal-to-background ratio) through the quantification of the amount of the labeling material to detect intracellular target material for a bioactive material capable of high sensitivity analysis and The purpose is to provide a device.

In addition, an object of the present invention is to provide a method and apparatus for detecting an intracellular target material for a physiologically active substance that can be implemented in a high throughput analysis by reducing the time required for analysis and easy analysis.

Method for detecting the intracellular target material for the bioactive material of the present invention,

Providing a physiologically active substance in living cells with a magnetic substance that can be separated by a magnetic field,

Providing a reaction partner in living cells combined with a labeling material capable of imaging the presence or location of the reaction partner to the bioactive material;

Reacting the bioactive substance with the reaction partner in living cells;

Separating the bioactive material bound to the magnetic material from the cell lysate by performing crushing and magnetic field application of the living cells;

When the bioactive material and the reaction partner are combined by the intracellular reaction, detecting the labeling material separated together with the reaction partner bound to the separated bioactive material;

When the labeling substance is separated and detected, determining the reaction partner as a target material for the bioactive substance.

In the method of an embodiment of the present invention, the method further comprises quantifying the amount of the labeling substance to be separated and detected.

In one embodiment of the present invention, the labeling substance may be a proteinaceous substance. Preferably, the proteinaceous substance is an epitope protein including a myc tag, a FLAG tag, a his tag, an S-tag, or the like. Enzyme protein including glutathione-S-transferase (GST), beta-galactosidase, beta-glucuronidase (GUS), etc. desirable.

Alternatively, the labeling material may be a fluorescent material including a fluorescent protein such as GFP, YFP, CFP and RFP or a tetracysteine fluorescent motif, a luminescent material such as luciferase, or radiation It may be a radioactive material comprising an isotope.

When the label is a proteinaceous material, detection of the label may be performed using an antigen-antibody reaction or by using an enzyme-substrate reaction. For example, the detection of the label may be performed using an ELISA method or a Western blot analysis method.

Specifically, when the labeling substance is a proteinaceous substance, detection may be performed by performing an ELISA or Western blot analysis well known in the art using an antibody that specifically recognizes the same (http: //en.wikipedia). .org / wiki / ELISA). In addition, when the proteinaceous material has an enzyme activity, detection can be performed using enzyme-substrate reactions well known in the art (Sanes, et al. (1986) Use of a recombinant retrovirus to study post-implantation cell linease in mouse embryo.EMBO J. 5, 3133; Lorincz, et al. (1999) Single cell analysis and selection of living retrovirus vector-corrected mucopolysaccharidosis VII cells using a fluorescence-activated cell sorting-based assay for mammalian beta-glucuronidase enzymatic actiity J. Biol. Chem. 274, 657).

In addition, when the labeling substance is a fluorescent substance, detection may be performed by measuring fluorescence by a method well known in the art (Tsien, RY (1998) The green fluorescent protein.Annu. Rev. Biochem. 67, 509 Adams, et al. (2002) New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vio: systhesis and biological applications.J. Am. Chem. Soc. 124, 6063; Griffin, et al. (2000) Fluorescent labeling of recombinant proteins in living cells with FlAsH.Meth.Enzymol.327, 565; Griffin, et al. (1998) Specific covalent labeling of recombinant protein molecules inside live cells.Science 281, 269).

In addition, when the label is a luminescent material, detection can be performed by measuring luminescence by methods well known in the art (Sherf, et al. (1996) Dual-luciferase reporter assay: an advanced co-reporter technology Integrating firefly and Renilla luciferae assay.Promega Notes 57, 2), even if the label is a radioactive material, detection can be performed by measuring radioactivity by methods well known in the art.

In the method of an embodiment of the present invention, the step of quantifying the amount of the labeling substance to be separated and detected may be performed using an ELISA reader, a fluorometer, a luminometer, or a radiation detector. have.

In the method of the preferred embodiment of the present invention, the step of quantifying the amount of the labeling substance to be detected separated may be performed using the following formula:

Where D.M.F. = (Signal value of the sample separated by the magnetic field)-(background value)

        D.L. = (Signal value of cell lysate)-(background value).

In the method of an embodiment of the present invention, the magnetic material means a material that can be separated by applying a magnetic field. Preferably, the magnetic material may be magnetic nanoparticles. For example, the magnetic nanoparticles may be artificially synthesized nanoparticle magnetic particles or magnetic proteins, and the magnetic proteins may be ferritin, bacterioferritin, ferritin-like protein, magnetosome, metal binding protein, or the like. It may include.

In one embodiment method of the present invention, the bioactive material or the reaction partner comprises a nucleic acid consisting of one or more nucleotides, a protein consisting of one or more peptides, amino acids, sugar lipids, low molecular weight compounds and the like. Preferably, the bioactive substance is a drug of a low molecular weight compound, and may be an anti-inflammatory agent or an anticancer agent. In addition, the reaction partner may be a protein, and may be a kinase that performs a phosphorylation reaction in a cell.

Drugs used in the embodiments of the present invention described below include non-steroid indomethacin, ketoprofen, flurbiprofen, naproxen, and ibuprofen. (ibuprofen), ramifenazone, ramifenazone, piroxicam, steroidal cortisone, dexamethasone, fluazacort, celecoxib, rotecoxib, rofecoxib anti-inflammatory agents such as rofecoxib, hydrocortisone, prednisolone, and prednisone, and Dasatinib, cyclophosphamide, actinomycin, and blenomycin. Mycin (bleomycin), daunorubicin, doxorubicin, epirubicin, mitomycin, mitomycin, methodotrexate, fluorouracil, carboplatin , Karmus carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, camptothecin and its derivatives, phenesterine, vinblastine, vincristine anticancer agents such as vincristine, tamoxifen, and piposulfan.

In the method of an embodiment of the present invention, the magnetic material that can be separated by the magnetic field may be directly or indirectly bonded or coated with the bioactive material.

In addition, in a method of a preferred embodiment of the present invention, the step of providing a reaction partner in combination with the labeling material in living cells delivers the labeling material and the base sequences encoding the reaction partner in living cells and live It is characterized by expressing in a cell.

On the other hand, in the method of an embodiment of the present invention, the step of providing a biologically active material bound to the magnetic material in living cells, and the step of providing a reaction partner in combination with the labeling material in living cells Well known methods, for example, using a transducible peptide or fusogenic peptide, a lipid gene carrier (lipid or liposome) or a combination thereof, or using a generally known method such as electroporation or magnetofection (Josepthson, et al. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugate.Bioconjug. Chem. 10, 186; Derfus, et al. (2004) Intracellular delivery of quantum dots for live cell labeling and organelle tracking.Adv. Mater. 16, 961; Frank, et al. (2003) Clinically applicable labeling of mammalian amd stem cells by combinining superp aramagnetic iron oxides and transfection agents.Radiology 228, 480; US Patent No. 2005/0130167; US Patent No. 2005/0271732).

In the method of an embodiment of the present invention, the disruption of the living cells is carried out using a conventional cell disruption buffer solution, magnetic field application, separation of the bioactive material-reaction partner-labeled material conjugate combined with the magnetic material Can be applied using an electromagnet, for example a magnetic field applying device (see magnetic field applying devices disclosed in Korean Patent Nos. 10-0792594 and 10-0862368). In addition, the present invention may use a magnetic material separation method using a permanent magnet, for example, HGMS (High gradient magnetic separation) (US Patent No. 4,247,398 (published Jan. 27, 1981) and Melville, D., F. Paul ,, & S. Roath (1975) Direct magnetic separation of red cells from whole blood.Nature 255: 706).

In one preferred embodiment of the present invention, the magnetic field applying device is a cylindrical core made of non-magnetic magnetic material for fixing the container containing the cell debris and to enhance the strength of the magnetic field, or a plurality of extensions to support the container A bioactive material-reaction partner-label combination combined with the magnetic material is provided by means of a gradient gradient increasing means for concentrating a magnetic field to the container and for strengthening the force of moving and maintaining the magnetic material to the bottom of the container. Can be easily separated. That is, in this case, if only the supernatant is removed from the container containing the cell lysate, the bioactive substance-reaction partner-labeled substance conjugate combined with the magnetic substance can be easily separated. In addition, the permanent magnet or electromagnet is preferably located in close proximity to the cell.

On the other hand, the device for detecting the intracellular target material for the bioactive material of the present invention,

A container for culturing living cells, the reaction partner coupled with a bioactive material bound to a magnetic material that can be separated by a magnetic field, and a labeling material capable of imaging the presence or location of the reaction partner to the bioactive material. Is provided with a vessel for culturing the living cells to react,

Means for disrupting living cells in the vessel,

A magnetic field applying device for applying a magnetic field to the cell lysate crushed by the crushing means, and separating the bioactive material bound to the magnetic material from the cell lysate;

Means for detecting a label that is separated together with the reaction partner bound to the separated bioactive material when the bioactive material and the reaction partner are bound by the intracellular reaction;

When the labeling substance is separated and detected, means for determining the reaction partner as a target material for the bioactive substance.

In one embodiment of the present invention, the apparatus further comprises means for quantifying the amount of the labeling substance to be separated and detected.

In the device of the preferred embodiment of the present invention, the magnetic field applying device is provided with a cylindrical core made of non-magnetic magnetic material for fixing the container containing the cell debris and for enhancing the strength of the magnetic field, or a plurality of extensions supporting the container. A magnetic field gradient increasing means is provided. Thus, the device of the present invention concentrates the magnetic field on the container containing the cell debris and strengthens the force of moving and maintaining the magnetic material to the bottom surface of the container, thereby combining the bioactive material-reaction partner-labeling with the magnetic material. Separation of the substance binder can be facilitated.

The method and apparatus of the present invention can confirm the reaction between the biologically active substance and the target substance reflecting the living cellular environment, and the relatively high signal-to-background ratio through the quantification of the amount of the labeling substance. It is possible to determine the high sensitivity analysis has the advantage.

In addition, the method and apparatus of the present invention can be applied to the quantified analysis by quantifying the detection result of the labeling substance separated by the magnetic field.

In addition, the method and apparatus of the present invention automate the detection and quantification of the labeling substance separated by the magnetic field, thereby reducing the time required for data analysis and facilitating analysis, thereby enabling high throughput analysis.

Hereinafter, the present invention will be described in more detail based on examples. The following examples of the present invention are not intended to limit or limit the scope of the present invention only to embody the present invention. From the detailed description and examples of the present invention, those skilled in the art to which the present invention pertains can easily be interpreted as belonging to the scope of the present invention. References cited in the present invention are incorporated herein by reference.

Example

Example 1

Drug-coated magnetic nanoparticles Intracellular Of target protein  refine

HeLa cells (purchased from ATCC, Cat.No. CCL-2) were passaged at a concentration of 1.5 × 10 ⁶ cells / dish followed by transduction of cDNA library DNA. DNA transduction can be performed using known methods, for example, lipofectamine (purchased from Invitrogen) or Hugene 6 (purchased from Roche). As an example, cDNA library (cDNA library) was used to fusion cDNA derived from HeLa with the green fluorescent protein (EGFP) gene, so that the EGFP-labeled protein is expressed. However, the present invention is that the gene of the epitope protein detectable by the antigen-antibody reaction as described above or the enzyme protein detectable by the enzyme-substrate reaction can be fused with the cDNA to produce a cDNA library. Those skilled in the art will readily understand.

In addition, biotinylated compound (biotinylated compound) is reacted with a streptavidin-bound magnetic nanoparticles and coated with well known methods in the art (Josepthson, et al. (1999) High-efficiency intracellular magnetic labeling with novel superparamagnetic -Tat peptide conjugate.Bioconjug.Chem. 10, 186; Derfus, et al. (2004) Intracellular delivery of quantum dots for live cell labeling and organelle tracking.Adv. Mater. 16, 961; Frank, et al. (2003) Clinically applicable labeling of mammalian amd stem cells by combinining superparamagnetic iron oxides and transfection agents.Radiology 228, 480; US 2005/0130167; US 2005/0271732) were transferred to cells transduced with cDNA.

On the other hand, streptavidin-bound magnetic nanoparticles are manufactured directly by methods well known in the art (US Patent No. 5,665,582; US Patent Publication No. 2003 / 0092029A1) or purchased from a company that manufactures and sells magnetic nanoparticles. Can be used. When the removal of biotinylated drugs that are not bound to magnetic nanoparticles is required, methods well known in the art with regard to magnetic nanoparticle separation, such as HGMS (High gradient magnetic separation) (US Pat. No. 4,247,398; Melville, D., F. Paul ,, & S. Roath (1975) Direct magnetic separation of red cells from whole blood.Nature 255: 706) performed the removal of biotinylated drugs not bound to magnetic nanoparticles.

The cells prepared as above were washed with D-PBS (purchased in Weljin), and then cell lysis buffer [10% glycerol, 50 mM HEPES (pH 8.0), 100 mM KCl, 2 mM EDTA , 0.1% NP-40, 2 mM DTT, 1X protease inhibitor mixture (purchased from Sigma), 10 mM NaF, 0.25 mM NaOVO ₃ ], and then disrupted using pellet pestle. Purification of the target protein in combination with the drug coated on the magnetic nanoparticles is a method well known in the art with respect to magnetic nanoparticle separation as described above, for example, HGMS method (High gradient magnetic separation) (US Pat. No. 4,247,398; Melville, D., F. Paul ,, & S. Roath (1975) Direct magnetic separation of red cells from whole blood.Nature 255: 706). Nonspecifically bound proteins were removed by washing 3 to 5 times using cell disruption buffer during the purification process.

In order to confirm the purified protein, Western blot was performed using anti-EGFP antibody after SDS-PAGE electrophoresis. Anti-EGFP antibodies can be prepared or purchased directly (available from Santa Cruz Biotechnology (Santa Cruz, CA)).

The results according to this example are illustrated in FIG. 1. According to the results of FIG. 1, the target protein specifically bound to the drug coated on the magnetic nanoparticles can be identified (FIG. 1, lane 3). The binding of the drug and target protein thus detected was reconfirmed by competitive binding with non-biotinylated drug (lanes 4 & 5 in FIG. 1).

Example 2

Detection of intracellular target protein using drug-coated magnetic nanoparticles

After PCR amplification of the open reading frame (ORF) of human protein derived kinase and other proteins (Hartley, et al. (2000) DNA cloning using in vitro site-specific recombination.Genome Res. 10, 1788) -1795) was prepared an expression vector capable of expressing a protein in the form of a fusion of green fluorescent protein (EGFP). Human-derived protein ORF used in this example is as follows.

Gene Symbol GenBank Acc. No. Gene Symbol GenBank Acc. No. BIKE NM_017593 CAMK2G NM_172169.1 CAMKK1 NM_172207.2 CAMKK2 NM_153499.2 CDKL5 NM_003159.2 CHEK1 NM_001274 CLK2 NM_003993 CSNK2A1 NM_001895 EPHA2 NM_004431 FGFR2 BC039243 FGFR4 NM_002011 ULK4 / FLJ20574 BC014794.1 HIPK4 NM_144685 IRAK1 BC014963 KIAA1914 NM_001001936 LIMK2 NM_001031801.1 LOC14942 BC028713 MAP3K7 NM_003188 MAPK1 NM_002745 MAPK11 NM_002751 MAPK14 NM_001315 MKNK1 NM_003684.3 NRBP NM_013392 PDGFRB NM_002609 PHKG2 NM_000294 PIM1 NM_002648 PINK1 NM_032409 FLT1 BC039007.1 PIK3C3 NM_002647 PIK4CB NM_002651 PLK1 NM_005030 PLK2 NM_006622 PLK3 NM_004073 PLK4 NM_014264 PRPF4B NM_003913 PTK2 BC028733.2 PTK2 NM_153831 PTK9 BC022344.1 RIOK3 NM_003831 SLC13A3 NM_022829 STK6 NM_003600.2 STK36 NM_015690 TRIB3 BC019363 TRIB3 NM_021158 ULK2 NM_014683 VRK2 NM_006296 VRK3 BC023556 BLK NM_001715 FGR NM_005248 FYN NM_002037 SRC NM_198291 YES1 NM_005433 CDK2 NM_001798 CDK4 BC003644 CDK5 NM_004935 CDK7 NM_001799 CDK9 NM_001261 GSK3B BC000251

HeLa cells were passaged at a concentration of 5,000 to 10,000 cells / well in 96-well plates, followed by transduction of the prepared expression vector DNA. DNA transduction can be performed using known methods, such as lipofectamine (purchased from Invitrogen) or Hugene 6 (purchased from Roche). Magnetic nanoparticles coated with a biotinylated drug were delivered to cells transduced with DNA as in Example 1.

The cells prepared as above were washed with OPTI-MEM I (purchased from Invitrogen) medium and then the cells were disrupted. Cell disruption can be performed using known methods, for example, cell disruption buffer (see Example 1), Passive Lysis Buffer (purchased from Promega), M-PER (purchased from Pierce), and the like. have.

A magnetic field was applied to the cell disruption solution to fix the magnetic nanoparticles, and the supernatant was removed. Alternatively, cell disruption can be performed after applying a magnetic field. As described above, the application of the magnetic field is well known in the art (Korean Patent No. 10-0792594; Korean Patent No. 10-0862368; US Patent No. 4,247,398; Melville, D., F. Paul ,, & S. Roath (1975) Direct magnetic separation of red cells from whole blood.Nature 255: 706).

On the other hand, the target protein bound to the drug coated on the magnetic nanoparticles present in the cell lysate is separated and purified since it exists in an environment mixed with cytoskeleton, organelles, and various other intracellular materials, unlike general affinity chromatography. Is not easy. Therefore, it is important to apply magnetic field to magnetic nanoparticles effectively to separate the magnetic nanoparticles-drug-target protein-labelled substance.

3 shows a magnetic field applying device (see Korean Patent No. 10-0792594) used in one embodiment of the present invention. The magnetic field applying device includes a cylinder 100 for accommodating a well plate 150 having a plurality of wells 152 formed therein, and a core for fixing the well plate 150 and for enhancing the strength of a magnetic field made of a nonmagnetic magnetic material. 140) is installed. Although not shown, the coil is wound several times to several hundred thousand times around the cylinder 100 to form a magnetic field in the region including the well plate 150 when the power is supplied, and a power supply device for supplying power to the coil. Is also provided. The core 140 is composed of a non-magnetic magnetic material that can be magnetized only while a current flows. A phenomenon in which a magnetic field is concentrated toward the core 140 by the installation of the core 140 may occur, causing the core 140 to be near. The strength of the magnetic field is increased. Therefore, when the magnetic field is concentrated toward the core 140 as the core 140 is installed, and the strength of the magnetic field is rapidly increased, the magnetic nanoparticles in the well plate are separated when the well plate 150 is spaced upward. You will be strongly encouraged to move to the center of.

In addition, Figure 4 shows another type of magnetic field applying device (see Korea Patent No. 10-0862368) used in one embodiment of the present invention. The magnetic field applying apparatus 1000a includes a cylinder 100 for accommodating a well plate 150 having a plurality of wells 152 formed thereon, and a protrusion protruding upward with respect to a bottom surface of the cylinder 100. Is provided with a magnetic field gradient increasing means (144) consisting of a plurality of extensions. Although not shown, the coil is wound several times to several hundred thousand times around the cylinder 100 to form a magnetic field in the region including the well plate 150 when the power is supplied, and a power supply device for supplying power to the coil. Is also provided. The magnetic field gradient increasing means 144 increases the magnetic field gradient by a plurality of extensions so that the magnetic nanoparticles in the well 152 of the well plate 150 can be quickly moved to the bottom surface of the well 152. It is possible to strengthen the force to hold the moved magnetic nanoparticles.

As described above, the supernatant was removed while the magnetic field was applied, and the magnetic nanoparticles immobilized by the magnetic field were washed three to four times using a buffer solution and DPBS (purchased in Welljin) used for cell disruption. After removing the magnetic field, the washed magnetic nanoparticles were redispersed in DPBS, and the redispersed magnetic nanoparticle solution was transferred to an ELISA plate. ELISA plates were purchased from materials that bind proteins well (available from Greiner, Corning, Falcon, etc.). The ELISA plate prepared as described above was gently shaken for 2 hours at room temperature, and then the supernatant was removed. Blocking solution (PBS containing 5% skim milk) was added to each well of the ELISA plate, and then reacted with gentle shaking for 1 hour at room temperature, and then the supernatant was removed. A blocking solution in which the anti-GFP antibody was diluted in an appropriate ratio (1:50 to 1: 10,000) was added to each well of the ELISA plate, and then gently shaken for 1.5 hours at room temperature, and the supernatant was removed. Anti-GFP antibody was prepared by using the HRP-bound form or purchased from a manufacturer such as Santa Cruz Biotechnology, Abcam. 1X TBS containing 0.1% Tween 20 (Bio Sesang Co., Ltd., purchased from Seongnam City, Gyeonggi-do) was added to each well, followed by reaction at room temperature for 5 minutes, and the supernatant was removed. This washing reaction was repeated 2-4 times. ELISA ECL solution (purchased from Pierce or Santa Cruz, etc.) was added to each well and the purified target protein was detected using a luminescence detector. LEADSeeker (manufactured by Amersham Bioscience) was used as a luminescence detector.

The detection result of the target protein bound to the magnetic nanoparticles coated in the cell and separated by magnetic field application is illustrated in FIG. 2. When the target protein was purified by binding to the drug, it was detected as a white circle, and black when no target protein was bound to the drug.

Example 3

Numerical method of intracellular target protein using drug-coated magnetic nanoparticles

Transduction of the expression vector DNA into the HeLa cells, delivery of the drug-coated magnetic nanoparticles, and purification of the magnetic nanoparticles and the target protein bound thereto by applying a magnetic field from the lysate of the cells prepared as described in Example 2 Was performed. After redispersing the purified magnetic nanoparticles, they were transferred to an anti-GFP antibody-coated ELISA plate and reacted with gentle shaking for 3 hours at room temperature. ELISA plates coated with anti-GFP antibodies are available from Pierce Biotech, Sigma, et al. At this time, in order to confirm the expression level of the target protein, part of the cell lysate (1/10 to 1/20) was transferred to another ELISA plate and reacted. After washing 1 ~ 3 times with DPBS (purchased in Welljin) containing 0.1% Tween 20, ELISA using HRP-bound anti-GFP antibody was performed as in Example 2.

After subtracting the background value from the measured luminescence value, the following equation can be used to quantify the amount of the labeling substance from the ELISA luminescence value. This is possible.

Where D.M.F. = (Measurement of luminescence of sample separated by magnetic field)-(background)

       D.L. = (Measurement of luminescence of cell lysate)-(background)

That is, if the amount of labeling material is quantified from the ELISA emission value measured using a luminescence detector, the time required for the measurement and data analysis of the purified target protein is reduced, and the analysis is easy, thereby enabling high throughput analysis. Done.

Although the present invention has been described with reference to the above embodiments, the present invention is not limited thereto. Those skilled in the art can make modifications and changes without departing from the spirit and scope of the present invention, and it will be appreciated that such modifications and changes also belong to the present invention.

Figure 1 is a photograph showing the Western blot results of the target protein bound to the drug coated on the magnetic nanoparticles in the cell. Each lane is described as follows. M, marker for molecular weight confirmation; 1, purifying the cell lysate after delivering the FITC-coated magnetic nanoparticles to the cells not transduced with cDNA library DNA; 2, purifying the cell lysate after delivering FITC-coated magnetic nanoparticles to cells transduced with cDNA library DNA; 3. Purification of cell disruption solution after delivery of drug-coated magnetic nanoparticles to cells transduced with cDNA library DNA; 4, purified by delivering a drug-coated magnetic nanoparticles to cells transduced with the cDNA library DNA and then reacting the cell lysate with a non-biotinylated drug competitively; 5, target protein released from the magnetic nanoparticles by delivering the drug-coated magnetic nanoparticles to the cells transduced with the cDNA library DNA and then reacting the cell lysate with the non-biotinylated drug competitively; 6, cDNA library Purified cell lysate after delivery of drug-coated magnetic nanoparticles to cells that are not transduced with DNA.

Figure 2 is a photograph showing the result of detection by ELISA method after purifying the target protein bound to the drug coated on the magnetic nanoparticles in the cell by magnetic field application. When the target protein is purified by binding to the drug, it is detected as a white circle, and black when no target protein is bound to the drug.

3 is a schematic perspective view of a magnetic field applying apparatus used in the method and apparatus of one embodiment of the present invention.

4 is a schematic perspective view of another magnetic field applying device (with magnetic field gradient increasing means) used in the method and apparatus of one embodiment of the present invention.

<Explanation of symbols for main parts of drawing>

100: cylinder 102: center hole

140: core 142: insertion hole

144: means for increasing the magnetic gradient

150: well plate 152: well

190: means of transportation

192: lifting shaft 194: lifting guide

1000a: magnetic field applying device

Claims (10)

Providing a physiologically active substance in living cells with a magnetic substance that can be separated by a magnetic field, Providing a reaction partner in living cells combined with a labeling material capable of imaging the presence or location of the reaction partner to the bioactive material; Reacting the bioactive substance with the reaction partner in living cells; Separating the bioactive material bound to the magnetic material from the cell lysate by performing crushing and magnetic field application of the living cells; When the bioactive material and the reaction partner are combined by the intracellular reaction, detecting the labeling material separated together with the reaction partner bound to the separated bioactive material; When the labeling material is separated and detected, the method comprising the step of determining the reaction partner as the target material for the bioactive material. The method of claim 1, A method for detecting an intracellular target material for a bioactive material further comprising the step of quantifying the amount of the label to be separated and detected. The method of claim 1, The labeling substance is a proteinaceous substance, epitope protein (epitope) protein or method for detecting an intracellular target material for a bioactive material, characterized in that the enzyme protein. The method of claim 1, The labeling material may include a fluorescent protein including a fluorescent protein or tetracysteine fluorescent motif of GFP, YFP, CFP and RFP, a luminescent material such as luciferase, or a radioisotope. A method for detecting an intracellular target material for a bioactive material, characterized in that the radioactive material. The method according to claim 3 or 4, When the marker is a protein, the detection of the marker is carried out using an antigen-antibody reaction or an enzyme-substrate reaction. The method according to claim 1 or 2, The step of quantifying the amount of the labeling material to be detected separated and detected is a method for detecting the intracellular target material for the bioactive material, characterized in that performed using the following formula:

Where D.M.F. = (Signal value of the sample separated by the magnetic field)-(background value)         D.L. = (Signal value of cell lysate)-(background value). The method according to claim 1 or 2, By performing the crushing and magnetic field application of the living cells, the step of separating the bioactive material bound to the magnetic material from the cell lysate is performed using a magnetic field applying device, The magnetic field applying apparatus has a physiology characterized in that it comprises a core made of non-magnetic magnetic material for fixing the container containing the cell lysate and strengthening the strength of the magnetic field, or magnetic field gradient increasing means provided with a plurality of extensions supporting the container. A method for detecting an intracellular target substance for an active substance. A container for culturing living cells, the reaction partner coupled with a bioactive material bound to a magnetic material that can be separated by a magnetic field, and a labeling material capable of imaging the presence or location of the reaction partner to the bioactive material. Is provided with a vessel for culturing the living cells to react, Means for disrupting living cells in the vessel, A magnetic field applying device for applying a magnetic field to the cell lysate crushed by the crushing means, and separating the bioactive material bound to the magnetic material from the cell lysate; Means for detecting a label that is separated together with the reaction partner bound to the separated bioactive material when the bioactive material and the reaction partner are bound by the intracellular reaction; And a means for determining the reaction partner as a target material for the bioactive material when the labeling agent is separated and detected. The method of claim 8, Apparatus for detecting intracellular targets to the bioactive material further comprising means for quantifying the amount of the label to be separated and detected. The method according to claim 8 or 9, The magnetic field applying apparatus has a physiology characterized in that it comprises a core made of non-magnetic magnetic material for fixing the container containing the cell lysate and strengthening the strength of the magnetic field, or magnetic field gradient increasing means provided with a plurality of extensions supporting the container. A device for detecting intracellular targets for active substances.

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