WO2017105146A1 - High-density western blot array analysis method - Google Patents

Abstract

The present invention relates to a high-density western blot array analysis method capable of simultaneously checking whether specific proteins are present in a plurality of samples, or simultaneously checking whether a plurality of proteins are present in a single sample, and according to the analysis method of the present invention, there are advantages of: reducing sample volume; enabling analysis even with a very small amount of antibodies; enabling the time required for testing to be remarkably shortened and a device to be compactly formed without high-priced equipment; and enabling analysis to proceed without restrictions on test formats.

Description

High Density Western Blot Array Analysis Method

The present invention relates to a high-density western blot array analysis method, and more specifically, high-density that can simultaneously determine the presence of a specific protein for a plurality of samples, or at the same time the presence of a plurality of proteins for a single sample Western blot array analysis method.

Western blots are a research technique for detecting proteins expressed by analysis of specific samples.

Western blots are used to verify the presence of proteins in cells, tissues or post-transcriptional modification.

Western blot process consists of SDS-PAGE, protein transcription to Nitrocellulose or PVDF transcription membrane, antibody treatment to target protein, and detection of target protein through antibody labeling. Samples for western blot mainly use protein mixtures in cells, tissues. After crushing the sample through cell lysis, the proteins are separated and used through multiple centrifugation and purification.

In order to efficiently detect a small amount of target, it is necessary to widen the protein in the sample. A commonly used method is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Sodium dodecyl sulfite (SDS) binds to proteins in protein samples, causing them to lose linearity and lose their secondary, tertiary and quaternary structure, and to give proteins high density of negative charges that are attracted toward the anode during electrophoresis. . In other words, the SDS-PAGE is categorized according to size by electrophoresis on SDS-modified proteins on PAG (polyacrylamide gel).

Proteins isolated by SDS-PAGE are embedded in the gel, making it difficult to observe the direct binding of the probe to the protein.

Thus, a transcription operation is performed that allows the protein to be moved onto a thin transcription membrane to facilitate binding with the probe.

Among such transfer operations, the most widely used is electrophoretic transfer.

This uses a principle very similar to electrophoresis, in which two methods have been used conventionally.

One is a semi-dry transfer that separates the gel by placing it horizontally, and the other is a wet transfer method that separates the gel by standing it vertically and filling the buffer.

At this time, the transfer method carefully separates the electrophoresis gel and overlaps with the transfer membrane.

Filter paper and pads are placed on both sides to absorb the buffer sufficiently and protect the gel, while the cathode and anode are placed on both sides.

At this time, since the protein is attracted to the anode, the transcription membrane is located on the anode side.

When voltage is applied, proteins begin to slowly transfer from the gel to the membrane and eventually stick to the transcription membrane.

When the protein is moved over the transcription membrane, a blocking operation is performed to prevent further proteins from attaching to the transcription membrane.

This attaches proteins that are already known and do not react with the probe in all areas except where the sample proteins are attached.

The probe, an antibody, is made from monoclonal lymphocytes and has the property of specifically binding to the target protein.

In other words, probes should be prepared in advance for experiments, and different types of probes should be used depending on the target protein.

Therefore, the conventional western blot has a problem in that an excessive amount of antibody is required and a problem in which an excessive amount of sample is required.

Thus, there was a limit to the number of samples that could be performed at one time.

In addition, the conventional western blot system takes a lot of time for the electrophoresis, there was a problem that the electrophoretic distance for the movement of the sample is long.

Meanwhile, US Patent Publication No. 2011-0028339, which is a prior art, discloses a highly integrated western blot technique.

However, U.S. Patent Publication No. 2011-0028339 requires expensive equipment and has many problems, such as the inability to freely configure an experimental format, in particular, a limited number or type of samples that can be tested at one time. .

(Patent Document 1) US8940232 B2

(Patent Document 2) US7670833 B2

(Patent Document 3) US2011-0028339 A1

One object of the present invention is to overcome the limitations of existing western blots and to implement a highly integrated array technology for running multiple western blots simultaneously.

Another object of the present invention is to provide a gel former, a sample injection unit, and antibody incubation chambers required to implement the above technique.

In order to achieve the above object, an aspect of the present invention comprises the steps of preparing a gel in which a plurality of sample injection grooves are formed in one or more rows; Injecting a sample into each of the sample injection grooves of the gel; Electrophoresis of the gel into which the sample is injected in a direction perpendicular to the heat formed by the sample injection grooves; Transferring a sample of the electrophoretic gel to a membrane; Accommodating the entire membrane onto which the sample has been transcribed into one space formed in the antibody incubation chamber and processing the antibody; And analyzing the membrane treated with the antibody; provides a high density Western blot array analysis method comprising the.

In order to achieve the above object, another aspect of the present invention is to prepare a gel formed so that at least one sample injection slot having a predetermined width is arranged in the longitudinal direction of the width to form one or more rows; Injecting a sample into a sample injection slot of the gel; Electrophoresis of the gel into which the sample is injected in a direction perpendicular to the heat formed by the sample injection slot; Transferring a sample of the electrophoretic gel to a membrane; The membrane onto which the sample has been transferred is mounted in an antibody incubation chamber in which a plurality of antibody injection grooves and a plurality of microchannels are formed so as to match the length of the width of the sample injection slot, and different types of antibodies are formed in the respective antibody injection grooves. Injecting the antibodies into a sample transferred to the membrane along each microchannel; And analyzing the membrane treated with the antibody; provides a high density Western blot array analysis method comprising the.

According to the present invention as described above has the following effects.

First, high density western blots are possible, which reduces the volume of the sample.

Second, there is an advantage that can be analyzed even with a very small amount of antibodies.

Third, there is an advantage that the number of samples that can be tested at one time is greatly increased.

Fourth, there is an advantage that the time required for the test is greatly shortened.

Fifth, there is an advantage that the inspection apparatus is compact because the electrophoretic distance is shortened.

Seventh, it does not require expensive equipment, and there is no restriction in experimental format.

Eighth, since the sealing member is provided between the upper frame and the lower frame, the antibody incubation chamber has an advantage of maintaining airtightness.

1 is a flow chart showing the procedure of the high density western blot array analysis method of the present invention.

2A to 2C are exemplary views of gels used in the high density western blot array analysis method according to the first analysis method of the present invention.

3 and 4 are respectively a perspective view and a front view of a first gel former used in the high density western blot array analysis method according to the first analysis method of the present invention.

5 is a front view of an injection unit used in the high density western blot array analysis method according to the first analysis method of the present invention.

6A and 6B are perspective views of a first antibody incubation chamber used in the high density western blot array analysis method according to the first analysis method of the present invention.

7 is an exemplary diagram of a gel used in the high density western blot array analysis method according to the second analysis method of the present invention.

8 and 9 are respectively a perspective view and a front view of a second gel former used in the high density western blot array analysis method according to the second analysis method of the present invention.

10A is a perspective view illustrating an example of a second antibody incubation chamber used in the high density western blot array analysis method according to the second analysis method of the present invention, and FIG. 10B is a high density western blot array according to the second analysis method of the present invention. It is a perspective view which shows the other example of the 2nd antibody incubation chamber used for the analysis method.

11A is a cross-sectional view taken along the line a-a 'of the second antibody incubation chamber according to the example, and FIG. 11B is a cross section taken along the line a-a' of the second antibody incubation chamber according to the another example. It is a cross section of.

12A is a schematic diagram of an injection unit used in the second antibody incubation chamber according to the above example, and FIG. 12B is a schematic diagram of an injection unit used in the second antibody incubation chamber according to the another example.

13A is a schematic diagram showing an injection unit inserted into a second antibody incubation chamber according to the above example, and FIGS. 13B and 13C illustrate an injection unit inserted into a second antibody incubation chamber according to the other example. As a schematic diagram, FIG. 13B shows a cross section of the portion in which the injection tube is inserted, and FIG. 13C shows a cross section of the portion in which the discharge tube is inserted.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

In this process, the thickness of the lines or the size of the components shown in the drawings may be exaggerated for clarity and convenience of description. In addition, terms to be described later are terms defined in consideration of functions in the present invention, which may vary according to the intention or convention of a user or an operator. Therefore, definitions of these terms should be described based on the contents throughout the specification.

 [First Example ]

One aspect of the present invention provides a high density western blot array analysis method (first analysis method) for simultaneously confirming the presence or absence of a protein that binds a single antibody to a plurality of samples. The first analysis method as described above is a multi sample analysis method, for example, drug, biodrug, RNAi library, antisense RNA library, etc. can be applied to the analysis, HTS (High Throughput Screening) used for in vitro screening Can be used as an alternative to and screening for several target molecules.

Referring to Figure 1, the first analysis method of the present invention comprises the steps of preparing a gel (S1), injecting a sample into the gel (S2), electrophoresis of the sample of the gel (S3) electrophoretic gel Transferring the sample to the membrane (S4), treating the membrane with the antibody (S5) and analyzing the membrane (S6).

 (1-1) Gel Preparation Step (S1)

2A to 2C, in the case of the first analysis method, first, a gel 100 formed to form one or more rows of the sample injection grooves 110 is manufactured (S1).

Each of the plurality of sample injection grooves 110 has a size in which a sample having a volume of several μl, preferably a volume of 0.1 μl to 5 μl, more preferably a volume of 0.5 μl to 2 μl, can be injected.

The sample injection grooves 110 are arranged at intervals of several mm, preferably at intervals of 0.5 mm to 5 mm, more preferably at intervals of 1 mm to 3 mm, and at least 20, preferably at least 50 More preferably, 100 or more may be formed to form one row.

In addition, as described above, a plurality of rows of the sample injection grooves 110 may be provided. In the case of a plurality of rows, the plurality of rows are formed to have a wider interval than the distance at which the sample injected into the sample injection groove is electrophoresed, intervals of several centimeters, preferably intervals of 0.5 to 5 centimeters, Preferably it may be arranged at intervals of 1 cm to 3 cm.

In particular, the sample injection grooves 110 may be tapered in a direction opposite to the direction in which the sample is electrophoretic, as shown in FIGS. 2B and 2C. For example, the sample injection grooves 110 may have a trapezoidal shape.

In one embodiment of the present invention, 101 sample injection grooves arranged at intervals of 1 mm form one row, and four rows of 12 cm × 6 cm sized gels are prepared at 1.2 cm intervals.

The gel as used in the first analysis method of the present invention may be prepared using the first gel former 300 as shown in FIGS. 3 and 4.

3 and 4, the first gel former includes a main body 310 and a cover unit 350.

The main body 310 includes a first plate 311 formed of a flat plate-shaped member and having a rectangular shape, and a second plate 313 provided at a predetermined height at an edge portion of the upper surface of the first plate. The second plate 313 is preferably provided in a 'c' shape, the inner concave formed by the flat plate-shaped first plate 311 and the 'c'-shaped second plate 313 The space having a shape becomes the gel receiving portion 330 into which the gel is injected. As a result, the first plate 311 provides the bottom surface of the gel receiving portion 330, and the second plate 313 provides the side surface of the gel receiving portion 330. In addition, the gel receiving part 330 may be provided with a sealing member (S) to prevent the gel from leaking to the outside, a plurality of clamping device 315 on the upper surface of the second plate 313 is the interval of the crystal It may be prepared as.

The cover unit 350 includes a forming plate 353 having a frame 351 and a plurality of forming protrusions 355. The molding protrusion 355 is formed in a shape protruding on one surface of the molding plate 353, at least a portion of the molding protrusion 355 may be inserted into the gel injected into the gel receiving portion 330 to inject a sample having a volume of several μl. A sample injection groove 110 of a size is formed. The forming protrusions 355 may be formed to form one or more rows at intervals of several mm from each other, and when the plurality of forming protrusions 355 have a plurality of rows, the rows may be arranged at intervals of several centimeters. The forming plate 353 may be integrally formed with the frame 351, but more preferably, may be detachably provided, which may include patterns, intervals, sizes, and numbers of the plurality of forming protrusions 355. It is intended to be replaced with a different back.

The cover unit 350 may be mounted to the main body 310 to be movable between a first position in which the gel accommodating part 330 is opened and a second position in which the gel accommodating part 330 is sealed. More preferably, the cover unit 350 may be connected to be rotatable at one side of the first plate 311. When the cover unit 350 is disposed in the second position to seal the gel receiving portion 330, at least the molding protrusion 355 of the forming plate 353 into the gel injected into the gel receiving portion 330. A portion is inserted to form a sample injection groove.

The clamping device 315 fixes the cover unit 350 to the main body 310 in a state where the cover unit 350 seals the gel accommodating part 330 at the second position. The clamping device 315 maintains the state in which the cover unit 350 keeps the gel accommodating portion 330 closed for a predetermined time so that the gel accommodated in the gel accommodating portion 330 is formed in the shape of the forming protrusion 355. It can be hardened while forming the corresponding sample injection grooves (110).

The first gel former 300 may further include a support unit 370. The support unit 370 has a first support member 371 and a second support member 373, wherein the first support member 373 supports one lower side of the main body 310 and the second support member. The member 373 supports the other lower portion of the main body 310. More preferably, the first support member 371 is fixed to the lower end of the first plate 311 at the connection part side in which the cover unit 350 is rotatably connected to the main body 310, and the second support member 373 is provided. ) Is spaced apart from the first support member 371 and is fixed to the longitudinal lower end of the first plate 311. More preferably, the first support member 371 extends in the lower direction of the first plate 311, and the second support member 373 extends in the upper direction and the lower direction of the first plate 311. The second supporting member 373 extends in the lower direction, and the third supporting member 375 extends in the upper direction. The second supporting member 373 and the third supporting member 375 form a flat plate shape with each other and are supported on the ground so that the main body 310 can stand in the vertical direction. In other words, while the cover unit 350 is fixed by the clamping device 315 in the second position in which the gel receiving part 330 is sealed, the main body 310 is formed of the second supporting member 373 and the The third support member 375 may be erected in a direction perpendicular to the ground. In addition, since the first support member 371 is formed higher than the second support member 373, when the main body 310 is placed on the bottom surface in the horizontal direction, the first plate 311 has an inclination. Meanwhile, the second plate 313 may have a 'c' shape, and each end of the 'c' shape is formed to face a portion where the cover unit 350 and the first plate 311 are connected. In other words, the second plate 313 is formed along the upper surface of both edges in the longitudinal direction of the first plate 321, and is formed in the short width direction of the first plate 321 to have a 'c' shape as a whole. It is done.

Inserting the support film for fixing the gel in the mesh (mesh) form to the gel receiving portion 330, the cover unit 350 is rotated and placed in a second position to seal the gel receiving portion 330, then the clamping The device 315 is used to secure the cover unit 350 in the second position. In the second position where the cover unit 350 seals the gel receiving portion 330 with the support unit 370 lying on the floor, the gel flows into the space between the gel receiving portion 330 and the forming plate 353. Inject. Since the first supporting member 371 is formed longer than the second supporting member 373, the gel slides on the bottom surface of the first plate 311 and flows into the space between the gel receiving portion 330 and the forming plate 353. Of gel is filled. The gel former body 310 may be placed perpendicular to the bottom surface by contacting the second support member 373 and the third support member 375 with the bottom surface. If the gel of the gel housing 330 hardens after a predetermined time, the clamping device 315 is released to move the cover unit 350 to the first position to open the gel housing 330, and then the molding protrusion The gel 100 having the sample injection grooves 110 formed therein so as to correspond to the shape of the 355 is separated from the body 310, and thus the gel as shown in FIG. 2 used in the first analysis method of the present invention ( 100).

 (1-2) Sample injection step (S2)

Next, the sample to be analyzed is injected into the sample injection grooves of the gel prepared as described above (S2). Different samples may be injected into each of the plurality of sample injection grooves, and each sample may be injected in a volume of several μl, preferably in a volume of 0.1 μl to 5 μl, more preferably in a volume of 0.5 μl to 2 μl. Can be. In one embodiment of the present invention, among the 404 sample injection grooves formed in the gel, 384 sample injection grooves are injected with a different volume of 1 μl, and the other 20 sample injection grooves are size markers, respectively. Was injected in a volume of 1 μl.

When a plurality of sample injection grooves are provided in the gel as described above, a sample injection unit 400 as shown in FIG. 5 may be used to simultaneously inject a plurality of samples into the sample injection grooves.

Referring to FIG. 5, the sample injection unit 400 may include a rod unit 410, a second plate 422 connected to the rod unit 410, and a plurality of pins 430 connected to transfer the sample. The first plate 421 may be spaced apart from the two plates 422 to guide the pins 430 to be slidable.

The rod unit 410 is formed to easily hold the sample injection unit 400. The pins 430 are inserted into the sample injection groove of the gel to inject a sample into each of the sample injection grooves. At this time, since the gel made from the first gel former is very weak, the pin 430 may destroy the gel G in some cases when the sample is injected into the sample injection groove formed in the gel. Therefore, even when a predetermined force is applied when the sample is injected into the sample injection groove formed in the gel G, the plurality of pins 430 may be configured to slide in the second plate 422 to damage the gel G. There is no work. In other words, since the first plate 421 and the second plate 422 are spaced apart from each other, the plurality of fins 430 may slide between the spaced intervals 425, thereby causing a gel ( G) may not be destroyed.

 (1-3) electrophoresis step (S3)

After the sample is injected into the sample injection groove of the gel as described above, the gel injected with the sample is electrophoresed (S3). The electrophoresis is a process of separating the proteins of the samples injected into each sample injection groove, it is performed using a horizontal electrophoresis device (not shown). The electrophoresis is carried out in a direction perpendicular to the heat formed by the sample injection grooves for a period of several tens of minutes, preferably 10 minutes to 60 minutes, more preferably 20 minutes to 40 minutes.

 (1-4) Transfer Step (S4)

When the electrophoresis is completed as described above, the sample of the separated gel is transferred to the membrane (S4). The transcription process of such a sample may be performed according to a method commonly used in the art.

 (1-5) Antibody Treatment Step (S5)

After the sample is transferred to the membrane as described above, the membrane is treated with the antibody (S5).

In the case of the first analysis method, the same antibody may be treated to the membrane onto which the sample is transferred. That is, the same one antibody may be processed for a plurality of different samples, and thus the presence or absence of a specific protein for a plurality of different samples may be simultaneously confirmed.

At this time, the antibody to be treated to the membrane may be treated in a volume of several μl to several tens μl, preferably a volume of 0.1 μl to 50 μl, more preferably a volume of 1 μl to 20 μl.

In one embodiment of the present invention by treating the same one antibody to a total of 384 samples, it was confirmed whether the specific protein is present for these 384 samples.

In the first analysis method of the present invention, the antibody may be processed using the first antibody incubation chamber 500 as shown in FIGS. 6A and 6B.

6A and 6B, the first antibody incubation chamber 500 includes an upper frame 510 having holes 512 and 511 and a lower frame 520 provided with a space 521 for accommodating the membrane. It is formed to include.

The upper frame 510 is formed with a first through hole 512 through which the antibody is supplied, and a second through hole 511 through which the waste liquid from which the reaction is completed is discharged, and the upper frame 510 is connected to the lower frame 520. When coupled, the first and second through holes 512 and 511 of the upper frame 510 are in communication with the space 521 of the lower frame 520.

When the membrane on which the sample is transferred is accommodated in one common space 521 as described above, a reaction material including an antibody is injected into the first through hole 512 to react the sample of the transferred membrane with the antibody. When such a reaction is completed, the waste liquid is discharged to the second through hole 511. Thereafter, the first and second through holes 512 and 511 may also be used in the blocking or washing of the membrane through the injection and discharge of the blocking buffer or the washing buffer.

The space portion 521 has a flat portion 550 adjacent to the first through hole 512 on one side thereof and a corner portion 540 adjacent to the second through hole 511 on the other side thereof. The corner portion 540 is formed to meet the first surface 541 and the second surface 542 forming a predetermined angle to each other, which is the corner portion 540 when discharging the waste liquid or the wash buffer solution to the outside It is to be able to discharge the exhaustion by leaving it in place.

Meanwhile, the space 521 may further include a partition 525 as illustrated in FIG. 6B. The partition 525 is used to adjust the volume of the space 521 according to the size of the membrane accommodated in the space 521. It is used to reduce the consumption of the sample.

 (1-6) Analysis step (S6)

When the antibody treatment for the membrane is completed as described above, the membrane is analyzed (S6). The analysis of the membrane may be performed according to a method commonly used in the art, such as through a process of photosensitization and development, or reading with a fluorescence scanner.

 [Second Example ]

Another aspect of the present invention provides a high density western blot array analysis method (second assay method) for simultaneously identifying the presence of proteins binding to each of a plurality of antibodies against a single sample. As described above, the second analysis method is a multi-antibody screening system, which can be actively applied to protein analysis using an antibody library, for example, to protein PTM analysis, and analyzes using several tens to hundreds of antibodies simultaneously as markers. Can be provided.

Referring to Figure 1, the second analysis method of the present invention also comprises the steps of preparing a gel (S1), injecting a sample into the gel (S2), electrophoresis of the sample of the gel (S3) electrophoretic gel Transferring the sample to the membrane (S4), treating the membrane with the antibody (S5) and analyzing the membrane (S6).

 (2-1) Gel Preparation Step (S1)

Referring to FIG. 7, in the second analysis method, first, one or more sample injection slots 210 having a predetermined width are arranged in a length direction of the width to prepare a gel 200 formed to form one or more rows. (S1).

The one or more sample injection slots 210 are each sized to a volume of several μl to several tens of μl, preferably 0.1 μl to 50 μl, and more preferably 1 μl to 20 μl. Have

The sample injection slot 210 has a predetermined width, and has a width of several tens of mm, preferably a width of 10 mm to 80 mm, more preferably a width of 30 mm to 60 mm.

The sample injection slot 210 may be formed so that one sample injection slot 210 forms a row, or two or more sample injection slots 210 may be formed in one row. As described above, when the plurality of sample injection slots 210 form a row, the sample injection slots 210 may be spaced apart from each other by several mm, preferably between 0.5 mm and 5 mm, more preferably between 1 mm and The marker injection grooves 215 may be additionally disposed between the sample injection slots 210 and the sample injection slots 210.

In addition, as described above, one or more rows of the sample injection slots 210 may be provided in plurality. In the case of a plurality of rows, the plurality of rows are formed to have a wider interval than a distance at which the sample injected into the sample injection slot 210 is electrophoresed, and has a spacing of several centimeters, preferably 0.5 to 5 centimeters Spacing, more preferably 1 cm to 3 cm.

In one embodiment of the present invention, four sample injection slots having a width of 45 mm form one row, and four rows of gels having a size of 12 cm × 6 cm are arranged at intervals of 1.2 cm.

The gel 200 as used in the second analysis method of the present invention may be prepared using the second gel former 350 as shown in FIGS. 8 and 9.

8 and 9, the second gel former 350 differs only in the pattern and shape of the forming protrusion 355 ′ of the forming plate 353 ′ provided in the cover unit 350 ′. The configuration is the same as that of the first gel former 300 described in the above [First Embodiment]. Therefore, the rest of the configuration will be omitted by omitting the description of the first gel former 300, and only the pattern and shape of the forming protrusion 355 'specific to the second gel former 350 will be described.

The forming protrusion 355 ′ is formed in the form of a straight line having a width of several tens of mm, preferably a width of 10 mm to 80 mm, more preferably a width of 30 mm to 60 mm, and at least one straight shape is mutually different. It is formed to form one or more rows at intervals of several millimeters, and when there are a plurality of rows formed by the forming protrusions 355 ', each row may be arranged at intervals of several centimeters. The forming plate 355 'may be integrally formed with the frame 351', but more preferably, may be detachably provided, which may include patterns, intervals, and the like of the plurality of forming protrusions 355 '. This is for replacing and changing the size and number.

Also in the case of the second gel former 350, a gel film fixing support film is inserted into the gel accommodating portion 330, and the gel accommodating portion 330 is rotated by rotating the cover unit 350 '. In a second position to seal the cover unit, and then the cover unit 350 ′ is fixed to the second position using the clamping device 130. In the second position where the cover unit 350 'seals the gel containing portion 330 with the support unit 370 lying on the floor, the cover unit 350' is flowable into the space between the gel containing portion 330 and the forming plate 353 '. Inject gel. Since the first supporting member 371 is formed longer than the second supporting member 373, the gel slides the bottom surface of the first plate 311 to the space between the gel receiving portion 330 and the forming plate 353 '. The fluid gel is filled. The gel former body 310 may be placed perpendicular to the bottom surface by contacting the second support member 373 and the third support member 375 with the bottom surface. If the gel of the gel housing 330 hardens after a predetermined time, the clamping device 315 is released to move the cover unit 350 'to the first position, thereby opening the gel housing 330, and then molding. The gel 200 in which the sample injection slot 210 and the marker injection groove 215 are formed is separated from the main body 310 so as to correspond to the shape of the protrusion 355 ′, and thus, the gel 200 is used in the second analysis method of the present invention. To obtain a gel (200).

 (2-2) Sample injection step (S2)

Next, the sample to be analyzed is injected into the sample injection slot of the gel prepared as described above (S2). The one or more sample injection slots may be injected with a volume of several μl to several tens of μl, preferably between 0.1 μl and 50 μl, more preferably between 1 μl and 20 μl, and the sample injection slot In the case of a plurality of samples, different samples may be injected into each sample injection slot. In one embodiment of the present invention, each of the sixteen sample injection slots formed in the gel was injected with a volume of 20 μl.

In the case of this second analysis method, since the number of sample injection slots is not large, a sample injection unit may not be used, unlike in the first embodiment, and a micropipette which is generally used may be used.

 (2-3) electrophoresis step (S3)

After the sample is injected into the sample injection slot of the gel as described above, the gel injected with the sample is electrophoresed (S3). The electrophoresis is a process of separating the proteins of the samples injected into each sample injection slot, it is performed using a horizontal electrophoresis device (not shown). The electrophoresis is performed in a direction perpendicular to the heat formed by the sample injection slots, for a time of several tens of minutes, preferably 10 minutes to 60 minutes, more preferably 20 minutes to 40 minutes.

 (2-4) transfer step (S4)

When the electrophoresis is completed as described above, the sample of the separated gel is transferred to the membrane (S4). The transcription process of such a sample may be performed according to a method commonly used in the art.

 (2-5) Antibody Treatment Step (S5)

After the sample is transferred to the membrane as described above, the membrane is treated with the antibody (S5).

In the case of the second analysis method, a plurality of different antibodies may be processed on a sample separated from one sample injection slot. That is, by processing a plurality of antibodies to one sample, it is possible to determine the presence or absence of several proteins present in one sample at the same time.

However, when there are a plurality of sample injection slots and different samples are respectively injected into the plurality of sample injection slots, the presence or absence of various proteins present in the sample may be simultaneously checked for each of the plurality of samples.

At this time, the plurality of antibodies to be treated in one sample may be treated with a volume of several μl, preferably a volume of 0.1 μl to 5 μl, more preferably a volume of 0.5 μl to 2 μl.

In one embodiment of the present invention by processing 20 different antibodies in one sample to determine whether there are 20 specific proteins in the sample, the presence of 20 specific proteins for each of a total of 16 samples at the same time It was.

In the second analysis method of the present invention, the antibody may be processed using the second antibody incubation chamber 600 as shown in FIGS. 10A, 10B, and 11.

10A, 10B, and 11, the second antibody incubation chamber 600 includes a plurality of antibody injection grooves 651, a plurality of waste fluid discharge grooves 653, and a plurality of microchannels 655. The lower frame 620 is provided with an upper frame 610 and a mounting portion 621 on which the membrane is placed.

10A, 10B, and 11, a plurality of antibody injection grooves 651 and a plurality of waste liquid discharge grooves 653 are formed on the upper surface of the upper frame 610 in a pair of intervals of several mm, It is preferably arranged at intervals of 0.5 mm to 5 mm, more preferably at intervals of 1 mm to 3 mm, so that at least 20, preferably at least 50, more preferably at least 100 are in one row. The antibody injection grooves 651 and the waste liquid discharge grooves 653 are formed at intervals of several centimeters in the direction in which the sample is electrophoresed, preferably at intervals of 0.5 to 5 centimeters, more preferably 1 to 3 centimeters. Can be arranged to correspond to each other in pairs at an interval of cm. In addition, a plurality of microchannels 655 are provided on the lower surface of the upper frame 610. The microfluidic passage 655 may include a first microfluidic pipe 655a connected to the antibody injection groove 651 and the waste liquid discharge groove 653 extending in a direction in which the antibody injection groove 651 extends (vertical direction). Injecting the antibody to connect the third microfluidic pipe 655c and the first microfluidic pipe 655a and the third microfluidic pipe 655c connected to the waste liquid discharge groove 653 in a vertical direction (vertical direction). The first microfluidic pipe 655a and the second microfluid are formed of the second microfluidic pipe 655b formed in a direction perpendicular to the heat (horizontal direction) of the grooves 651 or the waste liquid discharge grooves 653. The fluid pipe 655b and the third microfluidic pipe 655c form a 'U' shape with each other. As a result, the antibody injection groove 651 and the waste liquid discharge groove 653 formed on the upper surface of the upper frame 610 are formed with a 'U'-shaped microchannel 655 formed on the lower surface of the upper frame 610. It is connected to each other to form a large 'U'-shaped flow path as a whole. On the lower surface of the upper frame 610, in order to prevent the antibody injected through the antibody injection groove 651 is diffused to the next other microchannel while passing through the microchannel 655, the microchannel Between the block member 670 may be further provided.

The lower frame 620 is provided with a mounting portion 621 on which the membrane is placed, and the lower frame 620 has an upper surface of the membrane on which the lower surface of the upper frame 610 is placed on the mounting portion 621. And the upper frame 610 to be in close contact with each other. In particular, the mounting portion 621 may be formed as a separate space when the membrane to be placed has a predetermined thickness, the mounting portion 621 when the membrane to be placed is very thin and does not require a substantially separate space. ) May be a predetermined portion existing on the lower frame 620 rather than a space formed separately.

The second antibody incubation chamber 600 may further include a fastener 630 for firmly fixing the coupling of the upper frame 610 and the lower frame 620.

The membrane to which the sample is transferred is accommodated in the space part 621 of the lower frame 621 to be in close contact with the lower surface of the upper frame 610 (if there is a block member 670, in close contact with the block member 670). ), A reaction material including a different kind of antibody is injected into the plurality of antibody injection grooves 611 to react the sample of the transcribed membrane with the antibody.

The antibody injection groove 651 and the waste liquid discharge groove 653 are used for injecting and discharging not only the antibody and its waste liquid but also a cleaning liquid for washing the membrane. The washing liquid injection into the antibody injection groove 651 and the waste liquid suction into the waste liquid discharge groove 653 are performed by the washing unit 700 as shown in FIG. The washing unit 700 includes an injection tube 751 corresponding to the antibody injection groove 651 formed in a plurality of longitudinal directions of the second incubation chamber 600 and a discharge tube 753 corresponding to the waste liquid discharge groove 653. ) Is provided. The plurality of injection tubes 751 formed in the washing unit 700 are formed in the antibody injection groove 651 of the second incubation chamber 600, and the plurality of discharge tubes 753 formed in the washing unit 700 are provided. The waste liquid discharge grooves 653 of the second incubation chamber 600 are respectively inserted. Each of the plurality of injection pipes 751 may be connected to an external cleaning liquid tank (not shown) through a hose, and each of the plurality of discharge pipes 753 may be connected to an external vacuum pump (not shown) and a waste container through a hose. (Not shown).

When the antibody is processed and the reaction between the processed antibody and the sample on the membrane is completed, the injection tubes 751 and the discharge tubes 753 of the washing unit 700 form antibody injection grooves formed in the upper frame 610. 651 and the discharge pipe 753 inserted with the waste liquid discharge grooves 653, and then the waste liquid of the antibody present in the microfluidic passage 655 is first inserted into the waste liquid discharge grooves 653 using a vacuum pump. Inhaled (as above, the waste liquid is discarded into the waste container). Then, when the cleaning liquid stored in the cleaning liquid tank is injected into the antibody injection grooves 651 through the respective injection tubes 751, the injected cleaning liquid is the first microfluidic pipe 655a and the second microfluidic pipe. The unbound antibody and the like present on the membrane are washed while moving to the third microfluidic tube 655c through 655b. When the cleaning is completed as described above, by using the vacuum pump again to suck the waste liquid of the cleaning liquid present in the micro-channel 655 to the discharge tube (753) inserted into the waste liquid discharge groove (653) ( Waste fluid that has been inhaled together is dumped into a waste container).

On the other hand, the upper frame 610 of the second antibody incubation chamber 600, as shown in Figure 10 (b), further provided with a fixing groove 690 to which the cleaning unit 700 can be fixed The fixing groove 690 may include a first fixing groove 691 formed on the plurality of antibody injection grooves 651 and a second fixing groove 693 formed on the plurality of waste liquid discharge grooves 653. Is done. In this case, the antibody injection groove 651 and the waste liquid discharge groove 653 exist inside the first and second fixing grooves 691 and 693, respectively.

When the fixing groove 690 is formed in the upper frame 610 of the second antibody incubation chamber 600, the cleaning unit 700 is inserted into the fixing groove 690 to be inserted into the cleaning unit 700. ) May be provided with a fixed end 750 capable of fixing it, and the injection tube 751 and the discharge tube 753 may be provided at the bottom of the fixed end 750 or disposed therein. When the cleaning unit 700 is inserted into and fixed in the first and second fixing grooves 691 and 693 as described above, the number of the injection pipe 751 and the discharge pipe 753 of the cleaning unit 700 is the number thereof. May be smaller than the antibody injection groove 651 and the waste liquid discharge groove 653, and further, each of the injection tube 751 and the discharge tube 753 may be formed in the antibody injection groove 651 and the waste liquid discharge groove 653. It may not be inserted directly. In this case, when the cleaning unit 700 is fixed to the first and second fixing grooves 691 and 693, the injection tube 751 and the discharge tube 753 are respectively the first fixing groove 691. ) And the second fixing groove 693 are spaced apart from the antibody injection groove 651 and the waste liquid discharge groove 653 at predetermined intervals. The cleaning solution injected into the injection tube 751 is injected into the antibody injection grooves 651 existing in the entire first fixed groove 691 through a space between the injection tube 751 and the antibody injection groove 651. In addition, the waste liquid of the cleaning liquid is also subjected to a suction force by the vacuum pump to the entire second fixing groove 693 through the spaced space between the discharge tube 753 and the waste liquid discharge groove 653, and eventually the second fixed groove. The waste liquid is discharged to the waste liquid discharge grooves 653 which are present throughout.

 (2-6) Analysis step (S6)

When the antibody treatment for the membrane is completed as described above, the membrane is analyzed (S6). The analysis of the membrane may be performed according to a method commonly used in the art, such as through a process of photosensitization and development, or reading with a fluorescence scanner.

Although described above with reference to a preferred embodiment of the present invention, those of ordinary skill in the art various modifications and variations of the present invention without departing from the spirit and scope of the invention described in the claims below It will be appreciated that it can be changed.

[Description of the code]

100: gel 110: sample injection groove

200: gel 210: sample injection slot 215: marker injection groove

300: first gel former 300 ': second gel former 310: the main body

330: gel receiving portion 350, 350 ': cover unit 370: support member

311: first plate 313: second plate 315: clamping device

351: frame 353: forming plate 355: forming projection

351 ': frame 353': forming plate 355 ': forming protrusion

371: first support member 373: second support member 375: third support member

400: injection unit 410: rod unit

421: first plate 422: second plate 430: pin

500: first antibody incubation chamber 525: partition

510: first upper frame 512, 511: first through hole and second through hole

520: first lower frame 521: chamber space portion

530: first fastener 540: corner portion

541: first page 542: second page 550: flat part

600: second antibody incubation chamber

610: upper frame 620: lower frame 621: mounting portion

650: groove pair 651: antibody injection groove 653: waste liquid discharge groove

655: microchannels 655a, 655b, 655c: first, second, and third microfluidic tubes

630: second fastener 670: block member

690: fixing groove 691: first fixing groove 693: second fixing groove

700: cleaning unit 710: frame 750: fixed end

751: injection tube 753: discharge tube

S: sealing member

Claims (24)

1. A high density western blot array analysis method for simultaneously checking the presence or absence of a protein that binds a single antibody to a plurality of samples,

   Using a gel former, preparing a gel in which a plurality of sample injection grooves are formed in one or more rows;

   Injecting a sample into each of the sample injection grooves of the gel;

   Electrophoresis of the gel into which the sample is injected in a direction perpendicular to the heat formed by the sample injection grooves;

   Transferring a sample of the electrophoretic gel to a membrane;

   Accommodating the entire membrane onto which the sample has been transcribed into one space formed in the antibody incubation chamber and processing the antibody; And

   Analyzing the membrane treated with the antibody; high density western blot array analysis method comprising a.
2. The method according to claim 1,

   And wherein the gel comprises one or more rows of 50 or more sample injection grooves.
3. The method according to claim 1,

   And wherein said gel comprises one row of 100 or more sample injection grooves.
4. The method according to claim 2 or 3,

   And wherein said gel comprises a plurality of rows.
5. The method according to claim 1,

   The antibody incubation chamber

   An upper frame having a first through hole for supplying the antibody and a second through hole for discharging the waste fluid; And

   A lower portion coupled to the upper frame while the space portion is formed therein to accommodate the membrane to which the sample is transferred, and the space portion is in communication with the first through holes and the second through holes, respectively. Analytical Method.
6. The method according to claim 5,

   The antibody incubation chamber further comprises a partition for adjusting the volume of the space portion Western blot array analysis method.
7. A high density western blot array analysis method for simultaneously identifying the presence of proteins binding to each of a plurality of antibodies against a single sample,

   Using a gel former, preparing a gel in which one or more sample injection slots having a predetermined width are arranged in the lengthwise direction of the width to form one or more rows;

   Injecting a sample into a sample injection slot of the gel;

   Electrophoresis of the gel into which the sample is injected in a direction perpendicular to the heat formed by the sample injection slot;

   Transferring a sample of the electrophoretic gel to a membrane;

   The membrane onto which the sample has been transferred is mounted in an antibody incubation chamber in which a plurality of antibody injection grooves and a plurality of microchannels are formed so as to match the length of the width of the sample injection slot, and different types of antibodies are formed in the respective antibody injection grooves. Injecting the antibodies into a sample transferred to the membrane along each microchannel; And

   Analyzing the membrane treated with the antibody; high density western blot array analysis method comprising a.
8. The method according to claim 7,

   And wherein said gel comprises two rows of two or more sample injection slots.
9. The method according to claim 8,

   And wherein said gel comprises a plurality of rows.
10. The method according to claim 7,

    The antibody incubation chamber

    A plurality of antibody injection grooves and a plurality of waste liquid discharge grooves are formed in pairs to form one or more rows, and at one end of the plurality of micro-channels formed in a direction perpendicular to the rows of the antibody injection grooves on the bottom surface; An upper frame having other ends connected to the antibody injection groove and the waste liquid discharge groove, respectively; And

    And a lower frame 620 coupled to the upper frame such that a mounting portion on which the sample is transferred is formed, and a lower surface of the upper frame is in close contact with an upper surface of the membrane placed on the mounting portion. Formation of high density western blot array analysis.
11. The method according to claim 10,

    The micro-channel formed on the lower surface of the upper frame 610 is a 'U' shaped high density Western blot array analysis method.
12. The method according to claim 1 or 7,

    The gel former is

    A body having a concave gel receptacle for gel injection; And

    A first position for opening the gel receptacle and a second position for sealing the gel receptacle having a plurality of forming protrusions for forming a plurality of sample injection grooves or one or more sample injection slots in the gel injected into the gel receptacle; And a cover unit mounted to the main body so as to be movable between the high density western blot array analysis methods.
13. The method according to claim 12,

    At least a portion of the forming protrusion is inserted into the gel injected into the gel receiving portion when the cover unit is disposed in the second position.
14. A gel former for preparing gels in western blot systems,

    A body having a concave gel receptacle for gel injection; And

    A first position for opening the gel receptacle and a second position for sealing the gel receptacle having a plurality of forming protrusions for forming a plurality of sample injection grooves or one or more sample injection slots in the gel injected into the gel receptacle; And a cover unit mounted to the main body so as to be movable between them.
15. The method according to claim 14,

    The cover unit includes the cover unit body; And

    And a shaping plate detachably mounted to the cover unit main body and having a plurality of shaping protrusions.
16. The method according to claim 14,

    At least a portion of the forming protrusion is inserted into the gel injected into the gel receiving portion when the cover unit is disposed in the second position.
17. An antibody incubation chamber in which a protein of an electrophoresis gel is transcribed in a western blot system to react an antibody to a membrane transcribed.

    An upper frame having a first through hole for supplying the antibody and a second through hole for discharging the waste fluid; And

    And a lower portion formed therein to accommodate a membrane on which a sample is transferred, and the lower portion coupled to the upper frame while the space portion communicates with the first and second apertures, respectively.
18. The method according to claim 17,

    The antibody incubation chamber further comprises a partition for adjusting the volume of the space.
19. An antibody incubation chamber in which a protein of an electrophoresis gel is transcribed in a western blot system to react an antibody to a membrane transcribed.

    A plurality of antibody injection grooves and a plurality of waste liquid discharge grooves are formed in pairs to form one or more rows, and at one end of the plurality of micro-channels formed in a direction perpendicular to the rows of the antibody injection grooves on the bottom surface; An upper frame having other ends connected to the antibody injection groove and the waste liquid discharge groove, respectively; And

    An incubation chamber including a lower frame coupled to the upper frame such that a mounting portion on which the sample is transferred is formed, and a lower surface of the upper frame adheres to an upper surface of the membrane placed on the mounting portion; .
20. The method according to claim 19,

    The micro-channel formed on the lower surface of the upper frame 'U' shaped incubation chamber.
21. The method according to claim 19,

    The upper surface of the upper frame further comprises a first fixed groove formed on the plurality of antibody injection grooves and a second fixed groove formed on the plurality of waste liquid discharge grooves.
22. A sample injection unit for injecting a plurality of samples into a plurality of grooves,

    Rod unit;

    A second plate connected to the rod unit and connected with a plurality of pins to transfer the sample; And

    And a first plate spaced apart from the second plate and spaced apart from the second plate to guide the pins to be slidable.
23. A cleaning unit for cleaning a plurality of grooves and microchannels,

    A plurality of injection tubes corresponding to the plurality of antibody injection grooves;

    A plurality of discharge tubes corresponding to the plurality of waste liquid discharge grooves; And

    And a plurality of hoses having one end connected to each of the injection pipe and the discharge pipe.
24. The method according to claim 23,

    The washing unit further includes a fixed end,

    And a washing unit disposed below or fixed to the upper end of the injection tube and the discharge tube.

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