KR20180089849A - Method for monitoring post-translational modification of protein - Google Patents

Abstract

A disclosed method for monitoring post-translational modification of a protein comprises: a step of preparing a first microbead by binding a protein antibody to a surface of a base bead; a step of preparing a second microbead by binding a target protein having a first post-translational modification or a second post-translational modification, which are inversely proportional to each other, to the protein antibody of the first microbead; a step of preparing a third microbead by binding the second microbead to a first post-translational modification antibody that selectively binds to the first post-translational modification of the target protein; a step of preparing a fourth microbead by binding the second microbead to a second post-translational modification antibody that selectively binds to the second post-translational modification of the target protein; a step of measuring impedances of the third microbead and the fourth microbead; and a step of obtaining a ratio of a second difference between the impedance of the fourth microbead and a reference impedance to a first difference between the impedance of the third microbead and the reference impedance.

Description

[0001] METHOD FOR MONITORING POST-TRANSLATIONAL MODIFICATION OF PROTEIN [0002]

The present invention relates to a method for monitoring tau protein, and more particularly, to a method for monitoring post-translational modification of a protein.

Abnormal tau aggregation is a major feature in Alzheimer's disease (AD) and many other neurodegenerative diseases (collectively, tauopathies) (Brandt R, Hundelt M, Shahani N (2005) Tau alteration and neuronal degeneration in tauopathies: mechanisms and models. Biochimica. Et biophysica. Acta. 1739: 331-354). In healthy nerves, tau stabilizes microtubules by promoting growth from the axon and polarizing neurons. When pathologically hyperphosphorylated, the tau is separated from the microtubules and produces insoluble aggregates (Gendron TF, Petrucelli L (2009).) The role of tau in neurodegeneration.

In brains suffering from Alzheimer's disease, abnormally hyperphosphorylated tau and its aggregates are observed as an onset. Thus, excessive phosphorylation is generally considered to be a cause of tau aggregation.

Recent studies have shown that hyperphosphorylation and O-glycosylation in several post translational modifications (PTMs) of tau proteins are inversely related to each other.

Therefore, if the degree of phosphorylation of Tau protein and the degree of O-glycosylation can be measured, it can be a basis for determining the progression rate or prognosis of the disease. It can also be used as a tool for verifying the effects of new drug development.

Therefore, when the tau protein can be accurately detected, it can be helpful in judging the prognosis of neurodegenerative diseases or judging the therapeutic effect.

Currently, tau PET is known as a test method for tau protein. However, since tau PET requires oral administration of a radiological material (contrast agent), there is a limit in the inspection interval and the cost is high. In addition, when a blood test or the like is to be used, the concentration of tau protein is low, and it is difficult to quantitatively analyze it, making reliable and accurate detection difficult.

Also, referring to recent research results, the absolute amount of tau protein or phosphorylated tau protein does not exactly coincide with the patient's disease state due to the difference in the amount of protein in each patient. Therefore, even if an absolute amount of tau protein or phosphorylated tau protein can be detected, it is difficult to use it as an index for diagnosing the condition or prognosis of the patient.

SUMMARY OF THE INVENTION The present invention has been made in view of the above problems, and it is an object of the present invention to provide a method for monitoring post-translational modification of a protein which can increase reliability and is easy to perform.

The method for monitoring post-translational modification of a protein according to an embodiment for realizing the object of the present invention includes the steps of preparing a first microbead by binding a protein antibody to the surface of a base bead, Preparing a second microbead by binding a target protein having a first post-translational modification or a second post-translational modification to the antibody, which is inversely proportional to each other, to prepare a second microbead, And a second post-translational modification antibody that selectively binds the second microbead to a second post-translational modification of the subject protein, To prepare a fourth microbead, and measuring impedances of the third microbead and the fourth microbead, respectively System and a second step of obtaining a ratio of two differences, the impedance and the reference impedance of the fourth micro-bead for the first difference between the impedance and the reference impedance of the third microbeads.

According to one embodiment, the subject protein is tau protein.

According to one embodiment, said post-translational modification is O-glycosylated tau protein, and said second post-translational modification is phosphorylated tau protein.

According to one embodiment, the base bead is a magnetic bead.

According to one embodiment, the reference impedance is an impedance of the first microbead.

According to one embodiment, the reference impedance is an impedance of the second microbead.

According to one embodiment, obtaining the ratio of the second difference to the first difference comprises: at a first point in time, obtaining a ratio of the second difference to the first difference; Obtaining a ratio of the second difference to the first difference at a second time point, and comparing the ratio of the first time point to the ratio of the second time point.

According to one embodiment, measuring the impedance of the third microbead and the fourth microbead is performed by disposing the third microbead or the fourth microbead between the first electrode and the second electrode .

A method for monitoring a post-translational modification of a protein according to an embodiment of the present invention comprises the steps of binding a first post-translational modification antibody selectively binding to a post- Preparing a modified post-translational medium, modifying the second post-translational modification antibody selectively binding to said post-translational modification to a second post-translational modification of said protein of interest in inverse proportion to said post- Of said first post-translational modification medium to physical properties or chemical properties that vary according to the amount of said post-translational modification and said second post-translational modification, Obtaining a first measured value and a second measured value of the post-translational deformation medium for the physical property or chemical property; and determining a first difference between the first measured value and the reference value, And a step of obtaining the value and the ratio of the second difference between the reference value.

According to one embodiment, the first measurement value and the second measurement value are respectively impedances.

According to one embodiment, the first post-translational deformation medium and the second post-translational deformation medium each comprise a phosphor or a light emitter, wherein the first measurement value and the second measurement value respectively correspond to the amounts of the phosphors or phosphors It is an optical measurement.

According to the present invention, by measuring physical properties or chemical properties that vary depending on the amount of post-translationally modified protein having inversely proportional relationship with each other, and using a change in the ratio as an index, Reliable detection results can be obtained regarding the degree of increase / decrease and the degree of increase / decrease.

1 is a flowchart schematically illustrating a method for monitoring post-translational modification of a protein according to an embodiment of the present invention.
FIG. 2 is a graph illustrating a step of determining whether a target protein is increased or decreased using a method for monitoring post-translational modification of a protein according to an embodiment of the present invention.
3A to 3G are cross-sectional views illustrating a method of manufacturing a biosensor having nanogaps usable in a method for monitoring post-translational modification of a protein according to an embodiment of the present invention.

In this application, the terms first, second, etc. may be used to describe various elements, but the elements should not be limited by the terms. The terms are used only for the purpose of distinguishing one component from another. For example, without departing from the scope of the present invention, the first component may be referred to as a second component, and similarly, the second component may also be referred to as a first component.

The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "comprises", "having", and the like are used to specify that a feature, a number, a step, an operation, an element, a part or a combination thereof is described in the specification, But do not preclude the presence or addition of one or more other features, integers, steps, operations, components, parts, or combinations thereof.

How to monitor protein after translation

1 is a flowchart schematically illustrating a method for monitoring post-translational modification of a protein according to an embodiment of the present invention.

Referring to FIG. 1, a microbead 10 combined with a protein antibody 20 capable of binding with a target protein is prepared (S10).

For example, the microbead 10 (base bead) may be a magnetic bead composed of a metal, a polymer, or the like. For example, the diameter of the microbead 10 may be 1 to 5 탆, but it is not limited thereto, and it may be 10 탆 or more depending on the antibody and the detection system.

At least one protein antibody (20) is bound to the surface of the microbead (10). The beads may be tosylated, aminated, or treated with a carboxyl group such that the microbeads 10 can bind to the antibody.

The protein antibody 20 is capable of binding to the protein to be detected. For example, the target protein may be a Tau protein. As the antibody for binding to the tau protein, known ones can be used.

The microbeads 10 combined with the protein antibody 20 may be referred to as first microbeads.

Next, the target protein 30 is bound to the protein antibody 20 of the microbeads 10 (S20). In order to bind the target protein 30 to the protein antibody 20, appropriate incubation and washing may be performed after mixing the microbead 10 and the target protein 30. The protein of interest 30 may be tau and may be derived from a body fluid comprising at least one of blood, plasma, serum, saliva, urine, tears, runny nose, spinal fluid.

The microbead 10 combined with the target protein 30 may be referred to as a second microbead.

Next, a post-translational modification antibody capable of selectively binding in accordance with post-translational modification of the target protein 30 is provided to bind the target protein 30 and the translated post-translational modification antibody (S30-1, S30 -2)

For example, the subject protein 30 has at least two post-translational modifications (first post-translational modification and post-translational modification), wherein the post-translationally modified antibody has at least two .

For example, the subject protein 30 may be phosphorylated tau or O-glycosylated tau. For example, tau proteins can be O-glycosylated or phosphorylated by the following reactions, which are known to have a trade-off between them.

Thus, the post-translationally modified antibody may comprise a first post-translationally modified antibody (42) capable of selectively binding to phosphorylated tau and a second post-translationally modified antibody capable of selectively binding to the O-glycosylated tau 44 are provided, respectively.

Therefore, the number or the ratio of the post-translationally modified antibody binding to the target protein 30 may vary depending on the degree of post-translational modification of the target protein 30, for example, Can be measured by changing the impedance of the antenna.

The microbeads (S30-1) after binding with the first post-translational modification antibody (42) may be referred to as third microbeads, and the microbeads (S30 -2) may be referred to as a fourth microbead.

According to one embodiment of the present invention, the microbead (S20) after being bound to the protein of interest, the impedance of the microbead (S30-1) after being bound to the first post-translational modification antibody (42) And the impedance of the microbeads S30-2 after being bonded to the post-deformed antibody 44 are respectively measured. In order to measure the impedance of the microbead, a biosensor may be used. Preferably, the biosensor may have an electrode structure having a nanogap. This will be described later.

The microbeads (S20) after binding with the target protein and the microbeads after binding with the post-translationally modified antibody may have different impedances. For example, the impedance of the microbead after binding with the post-translationally modified antibody is smaller than the measured impedance of the microbead (S20) after binding with the protein of interest. Also, when the number of post-translationally modified antibodies bound to the protein of interest increases, the difference in impedance of the microbeads (after pre-binding) may increase.

Therefore, the difference between the measured impedance of the microbead (S20) after binding with the target protein and the impedance of the microbead after binding with the post-translationally modified antibody may be a marker for the post- have. However, there is a large change in the amount of protein depending on each patient or time of measurement. Therefore, it is difficult to use only the absolute amount of phosphorylated tau protein as an index to diagnose the condition or prognosis of the patient. Accordingly, in the present invention, by measuring the impedance value by the glycosylated tau having a specific relationship with the phosphorylated tau, and using the ratio thereof as an index, it is possible to determine whether or not the phosphorylated tau is increased or decreased And can be used as an index for diagnosing a patient's condition or prognosis.

In one embodiment, as the reference impedance, the impedance of the microbead S20 after binding with the protein of interest may be used, but this is exemplary and in other embodiments, the microbead after binding to the protein antibody 20 The impedance of step S10 may be used as the reference impedance.

Therefore, in one embodiment of the present invention, the impedances of the post-translationally modified proteins having inversely proportional relations with each other are respectively measured and the change in the ratio is used as an index, And a reliable detection result on the degree of increase and decrease can be obtained.

As the protein antibody and the post-translational modification antibody, those currently available on the market can be used. In addition, although the post-translational antibody does not have selectivity for tau protein, it can be used when there is selectivity for post-translational modification.

For example, antibodies that can selectively bind to phosphorylated tau include, but are not limited to, Phospho-Tau (Ser202, Thr205) Antibody (AT8) MN1020 (Thermo Fisher), Anti-Tau (phosphor S396) antibody [EPR2731] / ab109390 abcam), Phospho-Tau (ser202_ Antibody, # 11834) (Cell signaling), Phospho-Tau (ser396) (PHF13) Mouse mAb, # 9632 (Cell signaling) In addition, antibodies capable of selectively binding to O-glycosylated tau (antibodies capable of binding to various O-glycosylated proteins including tau) include O-GlcNAc (CTD110.6) Mouse mAB, # 9875 Cell signaling, etc. may be used, but these are merely illustrative and the present invention is not limited thereto.

FIG. 2 is a graph illustrating a step of determining whether a target protein is increased or decreased using a method for monitoring post-translational modification of a protein according to an embodiment of the present invention.

1 and 2, a sample containing a tau protein is collected from a subject at a first time point, for example, August, and the impedance of the microbead at step S20 is measured. At step S20, Is coupled with a first post-translational modification antibody capable of binding to phosphorylated tau (P Tau) and a second post-translational modification antibody capable of binding to O-glycosylated tau (Og Tau, respectively), and then the respective impedances are measured . Thereby, the impedance (Zt1) of the reference microbead (microbead not bound to the transformed modified antibody), the impedance (Zp1) caused by the microbead conjugated with the first post-translationally modified antibody, and the binding The impedance (Zo1) value of the microneedle bead is measured, respectively, so that the impedance decrease (Zt1-Zp1) due to the phosphorylated tau and the impedance decrease (Zt1-Zo1) due to the O-glycosylated tau can be obtained .

Next, a sample containing tau protein is taken from a patient at a second time point, for example, September (September), the impedance of the microbead at step S20 is measured, and the microbead at step S20 is measured with the phosphorylated tau (P tau), and a second post-translationally modified antibody capable of binding to O-glycosylated tau (Og Tau), respectively, and then the respective impedances are measured. Thereby, the impedance (Zt2) of the reference microbead (microbead not bound to the transformed modified antibody), the impedance (Zp2) caused by the microbead conjugated with the first post-translationally modified antibody, and the binding (Zt2-Zp2) due to the phosphorylated tau and the impedance decrease (Zt2-Zo2) due to the O-glycosylated tau can be obtained by measuring the impedance (Zo2) .

Considering that it is difficult to directly quantitatively measure the phosphorylated tau as described above, it is necessary to determine the impedance reduction (Zt1-Zp1) due to the phosphorylated tau at the first time point and the phosphorylated tau at the second time point (Zt2-Zp2) caused by the phosphorylated tau is not reliable enough to judge the increase or decrease of the phosphorylated tau.

However, since phosphorylated tau has a mutually exclusive (inverse) relationship with O-glycosylated tau, comparing the ratios of the two can be a reliable detection method.

For example, at a first point in time, the ratio of phosphorylated tau to O-glycosylated tau may be expressed as Zt1-Zp1 / Zt1-Zo1 and at a second point of time, as Zt2-Zp2 / Zt2-Zo2 Can be.

Accordingly, it is possible to determine whether the phosphorylated tau is increased or decreased according to the direction (increase or decrease) of the change in the ratio of phosphorylated tau to O-glycosylated tau, and consequently, the progression of the disease associated with phosphorylated tau or It can be an indicator of improvement. Also, depending on the magnitude of the change, it may be possible to determine the rate or extent of disease progression or improvement.

In the present embodiment, the above method was used using the detection or measurement of phosphorylated tau protein, but the present invention is not limited thereto, and the detection or measurement of all the proteins having post-translational modifications in mutually exclusive relationship similar to the tau protein Can be used for measurement.

Although impedance measurement is used in the above embodiment, the present invention is not limited to this, and similar indices can be obtained by measuring other physical or chemical properties.

For example, the post-translational modification antibody may be combined with a phosphor. In this case, the microbeads (hereinafter, referred to as post-translational modification medium) combined with the post-translational modification antibody include different amounts of phosphors depending on the amount of post-translational modification of the target protein.

Thus, the optical properties of the first post-translational modification medium conjugated with the first post-translational modification antibody and the post-translational modification medium conjugated with the post-translational modification antibody, for example, The intensity of the reflected light and the like can be measured to obtain the first measured value for the first post-translational deformation medium and the second measured value for the post-translational deformation medium. The first measured value may be compared with a reference value to obtain a value (difference) corresponding to a change in the optical property due to the first post-translational deformation. The second measured value may be compared with a reference value, (Difference) corresponding to the change of the optical property by the post-transformation can be obtained. The ratio of the differences may be an indicator of the ratio of the first post-translational modification to the second post-translational modification in the sample comprising the subject protein.

In one embodiment, the phosphor may be associated with the post-translationally modified antibody in advance, before the post-translationally modified antibody binds to the protein of interest, but in another embodiment, And then bound to the post-translationally modified antibody.

In another embodiment, the phosphor may be replaced by a chemiluminescence.

Manufacturing method of biosensor having nano gap

3A to 3G are cross-sectional views illustrating a method of manufacturing a biosensor having nanogaps usable in a method for monitoring post-translational modification of a protein according to an embodiment of the present invention.

Referring to FIG. 3A, an inorganic insulating layer 120 is formed on a base substrate 110. A first metal layer 130 is formed on the inorganic insulating layer 120. A first photoresist pattern 140 is formed on the first metal layer 130.

For example, the base substrate 110 may include silicon, glass, quartz, polymer, and the like.

For example, the inorganic insulating layer 120 may include an insulating material such as silicon oxide or silicon nitride.

The first metal layer 130 may include gold, silver, platinum, chromium, copper, titanium, alloys thereof, and the like. The first metal layer 130 may have a single layer or a laminated structure of different metal layers. In one embodiment, the first metal layer 130 may have a two-layer structure of chrome / gold.

The first photoresist pattern 140 partially covers the first metal layer 130 to partially expose the upper surface of the first metal layer 130.

Referring to FIG. 3B, the first metal layer 130 is etched to form the first electrode 132. The etching is performed by isotropic etching by wet etching. Therefore, the first electrode 132 forms an undercut with respect to the first photoresist pattern 140.

Referring to FIG. 3C, a second metal layer 134 is formed on the exposed upper surface of the first photoresist pattern 140 and the inorganic insulating layer 120. The second metal layer 134 may be formed by deposition such as sputtering, atomic beam evaporation, or the like, and is not formed under the first photoresist pattern 140 having undercuts.

The second metal layer 134 may include gold, silver, platinum, chromium, copper, titanium, alloys thereof, and the like. The second metal layer 134 may have a single layer or a stacked structure of different metal layers. In one embodiment, the second metal layer 134 may have a two-layer structure of chrome / gold.

Referring to FIG. 3D, the first photoresist pattern 140 and the second metal layer 134 disposed thereon are removed. Therefore, a nano gap (NG) may be formed between the first electrode 132 and the remaining second metal layer 134. Since the nano gap is formed by the lift-off after the formation of the undercut and not by the etching using the mask after the exposure of the photolithography process, it can be made smaller than the critical dimension of the photolithography, Process is possible.

Referring to FIG. 3E, a second photoresist pattern 150 is formed on the first electrode 132 and the remaining second metal layer 134. The second photoresist pattern 150 may cover the nanogap (NG) and partially expose the second metal layer 134.

Referring to FIG. 3F, the remaining second metal layer 134 is etched using the second photoresist pattern 159 as a mask to form the second electrode 136 and the contact portion 138.

Referring to FIG. 3G, an organic insulating layer 160 is formed on the first electrode 132, the second electrode 136, and the contact portion 138. The organic insulating layer may include a first opening OP1 exposing the nano gap NG and a second opening OP2 exposing the contact portion 138. [

A microbead having magnetism for sensing may be inserted into the first opening OP1 of the biosensor. For example, when a magnetic bead having magnetic properties is provided on the biosensor and a magnetic substance is disposed under the biosensor, the attraction force in the vertical direction is applied to the micro bead by the magnetic substance. When the magnetic body is moved in the horizontal direction, the micro beads outside the first opening OP1 can be inserted into the first opening OP1 by moving along the magnetic body.

When the microbead is inserted into the first opening OP1, a voltage is applied to the first electrode 132 and the second electrode 136 to measure the impedance of the microbead. The microbeads have different impedances depending on the number of bound antibodies and proteins, and the sensed impedance can be transmitted to an external device, for example, a signal analyzer, etc., through the contact portion 138.

According to the present invention, the size of the electric field can be increased by using a sensor having a nanogap, for example, a nanogap of 1 mu m or less. Therefore, a very low concentration of the protein can be easily and reliably detected.

The biosensor may include an array in which a plurality of electrode pairs having the nanogaps are arranged. For example, the biosensor may include an array in which the electrode pairs having nanogaps are arranged in 10X10, 20X20, 30X30, and the like.

Hereinafter, effects of the embodiments of the present invention will be described with reference to specific experimental examples.

Preparation of Microbeads Bonded with Protein Antibodies

Anti-Tau (Phosphor S262) antibody, ab64193, 50 μl / 250 μl) and a tau protein binding antibody (Thermo Fisher, Dynabead M-280, diameter 2.8 μm, 14203) PBS buffer, placed in an incubator at 37 ° C, and then incubated on a roll mixer for 24 hours.

Next, the magnetic beads bound to the tau protein binding antibody are washed with 0.4% Block ACE (AbD serotec, USA) and blocked with 0.2 M Tris buffer. 30 mg / mL of the microbead solution was stored in PBST (Phosphate Buffered Saline with Tween-20, 0.01% Tween-20) containing 0.4% Block Ace.

Synthesis Example 1 - Preparation of microbeads conjugated with tau protein

The tau protein concentration at 5250 ng / mL was diluted 1/10 with various concentrations (0.5 fg / mL to 50 pg / mL) using 0.1% PBST, and Thiamet G Lt; / RTI >

The tau protein binding antibody-bound microbeads were diluted to 300 μl / ml using 0.1% PBST, and the tau protein (diluent) and the microbeads (diluent) were mixed at a concentration of 1: 2, And reacted in the refrigerator for 22 hours.

Next, the microbeads bound to the tau protein were washed twice with 0.1% PBST and washed twice with PBS (Phosphate Buffered Saline). Thereafter, the beads were washed with PBS at a concentration of 60 μl / ml. < / RTI >

Synthetic example  2O- Glycosylation  The antibody Combined  Micro Bead  Ready

A microbead (300 μl / ml) bound to a tau protein was prepared in the same manner as in Synthesis Example 1.

The microbeads were mixed with 10.4 ng of an antibody capable of binding O-glycosylated proteins (Cell signaling, O-GlcNAc (CTD110.6) Mouse mAB, # 9875) and reacted in the refrigerator for 22 hours.

The microbeads bound to the O-glycosylated tau binding antibody were washed twice with 0.1% PBST, washed twice with PBS, and then the bead concentration was adjusted to 60 μl / ml using PBS Lt; / RTI >

Synthetic example  3 phosphorylation Tau  The antibody Combined  Micro Bead  Ready

A microbead (300 μl / ml) bound to a tau protein was prepared in the same manner as in Synthesis Example 1.

The microbeads were mixed with 10 ng of an antibody capable of binding to phosphorylated tau protein (Thermo Fisher, Phospho-Tau (Ser202, Thr205) Antibody (AT8), MN1020) and reacted in the refrigerator for 22 hours.

The microbeads bound to the phosphorylated tau binding antibody were washed twice with 0.1% PBST, washed twice with PBS, and then diluted with PBS to a bead concentration of 60 μl / ml.

The samples thus obtained were measured for impedance using a biosensor array (10x10 or 20x20) with an electrode interval of 0.7 mu m. In the following table, PBS means a PBS solution containing no microbeads, Neg means a sample that does not bind to a tau protein but contains a microbead having only a protein-binding antibody, and BAT (bead-antibody-tau ) Refers to a sample containing a microbead bound to a tau protein, and BAT2 (bead-antibody-tau-2nd_antibody) refers to a sample obtained by treating a microbead conjugated with a tau protein with O-GlcNAc or AT8. The impedance change rate is a value obtained by dividing the difference between the impedance (Negeg) of Neg and the measurement impedance (BAT or BAT2) by the impedance of Neg (Zneg) multiplied by 100 and the impedance change rate difference is Is defined as a value obtained by subtracting the impedance change rate of BAT.

Table 1 below shows the impedance of samples not treated with Thiamet G for tau protein. Table 1-2 shows the impedance of samples treated with Thiamet G 100uM for tau protein. Respectively.

Table 1-1

Table 1-2

Referring to Tables 1-1 and 1-2, when the concentration of Thiamet G is increased, that is, when the O-glycosylation of tau protein is increased, the difference in impedance change ratio between the microbeads bonded with the O- . Thus, it can be seen that the increase or decrease of O-glycosylation of tau protein can be detected through this. Also, the difference in impedance change rate can be confirmed even at a very low tau protein concentration, for example, 0.5 fg / ml.

Table 2-1 below shows the impedance of samples reacted with O-GlcNAc with microbeads conjugated with Thiamet G-treated tau protein (0.5 pg / ml). Table 2-2 shows the impedance of samples treated with Thiamet G-treated tau protein (0.5 pg / ml), and the impedance change was measured according to the Thiamet G concentration (0uM, 10uM, 30uM, 100uM).

Table 2-1

Table 2-2

Table 3 below shows the differences in BAT and BAT2 impedances (BAT-BAT2 or BAT2-BAT) for the impedance of Neg in Tables 2-1 and 2-2 and thus the difference between the phosphorylated tau (P) and the O- glycosylated Represents the ratio of impedance change due to tau (O).

Table 3

Referring to Table 3, an increase in the concentration of Thiamet G may be regarded as an increase in O-glycosylated tau, that is, a decrease in phosphorylated tau, which is detected from a decrease in the value of P / O .

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the following claims. You will understand.

The present invention can be used for monitoring the prognosis, progress, treatment response, etc. of diseases through protein detection. It can also be applied to a platform for developing therapeutic agents for diseases such as tauopathy.

Claims (13)

Preparing a first microbead by binding a protein antibody to the surface of the base bead;
Preparing a second microbead by binding a target protein having a first post-translational modification or a second post-translational modification, which are inversely proportional to each other, to the protein antibody of the first microbead;
Combining the second microbead with a first post-translational modification antibody that selectively binds to a first post-translational modification of the protein of interest to prepare a third microbead;
Preparing a fourth microbead by combining the second microbead with a second post-translational modification antibody that selectively binds to a second post-translational modification of the protein of interest;
Measuring the impedances of the third microbead and the fourth microbead, respectively; And
And obtaining a ratio of the impedance of the fourth microbead to the second difference of the reference impedance with respect to the first difference between the impedance of the third microbead and the reference impedance. The method according to claim 1, wherein the target protein is a tau protein. 3. The method of claim 2, wherein said post-translational modification is O-glycosylated tau protein and said post-translational modification is phosphorylated tau protein. The method of claim 1, wherein the base bead is a magnetic bead. The method according to claim 1, wherein the reference impedance is an impedance of the first microbead. The method of claim 1, wherein the reference impedance is an impedance of the second microbead. 2. The method of claim 1, wherein obtaining the ratio of the second difference to the first difference comprises:
Obtaining, at a first point in time, a ratio of the second difference to the first difference;
Obtaining a ratio of the second difference to the first difference at a second time point different from the first time point; And
And comparing the ratio at the first time point with the ratio at the second time point. The method of claim 1, wherein measuring the impedance of the third microbead and the fourth microbead comprises:
Wherein the third microbead or the fourth microbead is disposed between the first electrode and the second electrode. Binding a first post-translational modification antibody selectively binding to said post-translational modification to said first post-translational modification of said protein to prepare a post-translational modification medium;
A second post-translational modification antibody that selectively binds to the post-translational modification is bound to a post-translational modification of the subject protein that is in inverse proportion to the post-translational modification, ;
Obtaining a first measured value of said post-translational deformation medium for physical properties or chemical properties that vary according to said amount of said post-translational deformation and said post-translational deformation;
Obtaining a second measurement of said post-translational deformation medium for said physical property or chemical property; And
And obtaining a ratio of a second difference between the second measured value and a second difference to a first difference between the first measured value and a reference value. 10. The method according to claim 9, wherein the target protein is a tau protein. 11. The method of claim 10, wherein the post-translational modification is O-glycosylated tau protein and the post-translational modification is phosphorylated tau protein. 10. The method of claim 9, wherein the first measured value and the second measured value are respectively an impedance. 10. The method of claim 9, wherein the first post-translational modification medium and the second post-translational modification medium each comprise a phosphor or a chemiluminescence, wherein the first measurement value and the second measurement value are respectively Wherein the protein is an optical measurement value according to the amount of the protein.

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