JP2004163146A - High-density immunity blot method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-density immunity blot method for reacting a plurality of probes to a membrane transfer article with high density. <P>SOLUTION: In the membrane transfer article 10, a protein sample is developed along a plurality of rails for changing into a solid phase. Reagents a, b, c, d, and so on are dispensed along a different straight line, where distance is separated so that the reagents a, b, c, d, and so on do not interfere mutually with each group 12 of spots. Then, dispensation is made by overlapping on the straight line, where the reagents a, b, c, d, and so on are dispensed with a detection reagent, such as a coloring reagent and a fluorescent reagent, as a secondary reaction reagent to achieve optical detection by developing color in a reactant, where sample protein reacts with the reagents a, b, c, d, and so on of the probe. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
TECHNICAL FIELD The present invention relates to a method for detecting a substance developed and immobilized on a membrane in fields such as clinical, diagnostic, biochemical, and molecular biology.
[0002]
[Prior art]
As a method for detecting a substance which is spread on a membrane and immobilized, there is an immunoblot method. The immunoblot method is generally called a Western blot method, in which a protein sample or the like is developed by electrophoresis, then immobilized on a membrane, and then reacted by using a specific antibody against the target substance as a probe to detect its presence. is there.
[0003]
As a modification to this method, it is also possible to search for a target molecule on a membrane developed with an unknown protein sample, or to search for a molecule that interacts with the target molecule by reacting the target molecule with its specific antibody. Has been done.
[0004]
In order to realize these methods, usually, a reagent containing one kind of probe is added to the whole of the membrane on which the sample has been developed and reacted. Therefore, in order to use a plurality of types of specific probes in parallel, it is necessary to prepare multiple membrane transcripts of the same sample, cut one transcript into strips, or use a chamber divided into a plurality of strips. It is necessary to prepare a separate reaction vessel for each probe, for example, by sandwiching a membrane between them.
[0005]
On the other hand, as a method of dispensing a reagent, there has been proposed a system in which a small amount of a reagent is dispensed by a piezo element so that an antigen-antibody reaction or the like is performed in a minute compartment on the membrane (see Non-Patent Document 1). In the proposed method, a reagent is ejected by a piezo element, and the reagent is dispensed in a “dot” shape at a desired position on the membrane.
[0006]
[Non-patent document 1]
WO98 / 47006 publication
[Problems to be solved by the invention]
As described above, when a plurality of probes are used in parallel for the same sample, it is necessary to prepare a separate reaction tank for each probe. Therefore, in order to prepare membrane transcripts to be used for these, a large number of samples had to be consumed, such as running the same sample in multiple lanes (or in wide lanes). However, for example, when a large number of sera are used as probes for diagnostic purposes or the like, it is very difficult to realize this in consideration of the consumption of the solid-phased sample and the number of physically processed samples.
[0008]
In many cases, these operations are manually performed, and the work efficiency is extremely low.
Therefore, an object of the present invention is to enable a plurality of probes to react with a membrane transcript at high density.
[0009]
[Means for Solving the Problems]
In order to achieve the above object, in the present invention, a plurality of types of reagents are dispensed to each spot on the membrane using a microdispensing method.
That is, in the immunoblotting method of the present invention, regions are different for each spot developed on the membrane and immobilized so that a plurality of types of reagents for detecting substances in the spot do not interfere with each other. This method detects the presence of a target substance or a substance that interacts with the target substance at a high density.
[0010]
Thereby, it is possible to divide the membrane transcript into strips, or to form a small amount of compartments without dividing into separate containers in a special container, and it is possible to dispense a plurality of samples at a high density, thereby facilitating the processing of a large number of samples. The consumption of the membrane transfer material can be greatly reduced, and the use of a small amount dispenser can greatly reduce the labor of a series of operations.
[0011]
Further, in the dispensing method using a piezo element proposed above, the reagent is dispensed at “points” on the membrane. However, in a preferred embodiment of the present invention, such a minute dispensing method is used for the membrane. By using the above-mentioned "linear" dispensing, multiple types of probes can be made to react at high density by continuously dropping multiple types of probes onto each linear micro compartment on a one-dimensional developed membrane transcript in a certain area To do.
[0012]
Thereby, for the sample transfer membrane in which the spot has been developed one-dimensionally, by dispensing a plurality of probes such as antibodies on each line, and preferably further dispensing the detection reagent and the like on those lines, High-density detection can be realized.
[0013]
The first example of the liquid dispensing apparatus used for the microdispensing method is the dispensing method using the piezo element introduced above. In such a liquid dispensing apparatus, a reagent filled in a space connected to a discharge section at the tip is pressed by a driving section having a piezo element to discharge droplets from the discharge section. In that case, the parameter of the control signal may include at least one of the magnitude of the applied voltage to the piezo element, the rise time of the applied voltage, the applied time, and the fall time of the applied voltage.
[0014]
As a similar liquid dispensing apparatus, a liquid dispensing apparatus having a liquid discharging element used in an ink jet type liquid discharging apparatus can be used.
[0015]
As another example of the liquid dispensing device used for the microdispensing method, a dispensing device using a syringe pump can be used. In that case, the parameters of the control signal may include at least one of stroke, speed, and acceleration of the plunger of the syringe pump.
[0016]
Samples immobilized on the membrane include, for example, molecules of proteins, peptides, sugars, lipids, nucleic acids, etc., which are separated and developed in one-dimensional direction by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis or other chromatography. Or a mixture thereof. .
[0017]
Immobilization of the sample on the membrane can be performed by transferring the sample to a membrane after the sample is developed on a gel or other electrophoresis medium.
Examples of the material of the membrane used for solid-phase immobilization include PVDF (polyvinylidene difluoride), nitrocellulose, nylon (registered trademark), and derivatives thereof.
[0018]
In order to detect a substance in a spot that has been spread on a membrane and immobilized thereon, an antibody is used as a substance serving as a probe that binds to a target molecule when the target molecule is an antigen. Generally, a biological substance that specifically reacts with the target molecule can be used. In addition, some antibodies and biological substances can be used in combination.
It is preferable that the dispensing of the reagent is performed only in the region where the spot on the membrane exists. Thereby, waste of the reagent can be omitted.
[0019]
In order to be able to detect the target substance optically, a primary reagent containing a probe that specifically reacts with the target substance as a dispensing reagent, and a secondary reagent for coloring the target substance after reacting with the probe, Preferably. In that case, the primary reagent is dispensed first, and then the secondary reagent is dispensed on the area where the primary reagent has been dispensed. As such a secondary reagent, a coloring reagent or a fluorescent reagent can be used.
[0020]
In addition, as a method of detecting a target molecule by reacting a probe with an appropriate molecule, in addition to dispensing a coloring reagent or a fluorescent reagent, a metal ion is reacted, or a combination of these methods is used. be able to. Examples of such a method include a method using a colloidal gold-labeled antibody, and a method in which a protein or the like having an affinity for a metal ion is introduced into a fluorescent reaction using a Ni ²⁺ chelating enzyme or the like.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
FIG. 1 schematically shows an example of a liquid dispensing apparatus for dispensing a reagent for carrying out the present invention, together with a control system. When the reagent uses a primary reagent and a secondary reagent, the liquid dispensing apparatus can be used for any of the reagents.
[0022]
Reference numeral 1 denotes a liquid dispensing device including a piezo element as a liquid ejection element. Reference numeral 3 denotes a droplet of the reagent discharged from the liquid dispensing device 1, which is dispensed to a membrane 5 held at a lower portion of the liquid dispensing device 1.
[0023]
The membrane 5 is mounted on an XY stage 9 and ejects the reagent as liquid droplets from the liquid dispensing apparatus 1 and moves the membrane 5 along the direction of the lane in which the sample is developed (referred to as X direction). The reagent can be dispensed on the membrane 5 along one straight line by moving the substrate by the -Y stage 9. Repeating dispensing on different straight lines by discharging the reagent from the liquid dispensing apparatus 1 while moving the XY stage 9 in a predetermined amount in the Y direction and moving the XY stage 9 in the X direction in the same manner. Can be.
[0024]
A CCD camera 4 is arranged as an imaging device for capturing an image of a discharge port portion at the tip of the liquid dispensing device 1 and monitoring the dispensing state. The CCD camera 4 can simultaneously image the state of the ejection port portion and the ejected droplet 3. The imaging device is not limited to a CCD camera, and another camera may be used.
[0025]
The CCD camera 4 captures an image of the discharge port at the tip of the liquid dispensing device 1 from the horizontal direction. The image may be taken from an obliquely upward direction with an inclination from the horizontal. However, in order to more accurately monitor the state of the discharge port portion, it is preferable to take an image from the horizontal direction.
[0026]
In order to capture the image of the discharge port portion of the liquid dispensing device 1 more accurately, in this embodiment, the discharge port portion of the liquid dispensing device 1 is placed on the optical axis of the CCD camera 4 so that an image can be taken with transmitted light. The light source 2 is arranged on the opposite side of the CCD camera 4 with respect to. The light source 2 may emit light that is continuous in time, but in this embodiment, a strobe is used. In the case of a strobe, it can be set to emit light in synchronization with the timing at which the droplets 3 are ejected from the liquid dispensing apparatus 1. In this case, even if the camera 4 is operated continuously, the strobe can be used. A clear image is captured only when 2 emits light. Since the clear image is obtained by capturing the images of the droplets 3 sequentially discharged at the same timing, information like a still image can be obtained. Therefore, it is convenient to monitor the state of the droplet 3 and adjust the size of the droplet 3.
[0027]
When a light source that emits continuous light in time is used as the light source 2, the image of the droplet 3 captured by the CCD camera 4 is adopted as an image passing through the same place, so that the droplets sequentially discharged are adopted. The information as if the third image is a still image is obtained.
[0028]
Reference numeral 6 denotes a dispensing control unit that causes a piezo element of the liquid dispensing apparatus 1 to perform a discharging operation by applying a voltage. The timing at which the strobe 2 emits light is based on the timing of the voltage applied from the dispensing control unit 6 to the piezo element of the liquid dispensing device 1, and is synchronized with the voltage application timing. Is controlled so as to emit light after a certain period of time from the ejection of droplets from the liquid crystal.
[0029]
Reference numeral 8 denotes a pressure control mechanism, which adjusts a discharge liquid such as a sample or a reagent filled in the dispensing liquid container 20 containing the discharge liquid of the liquid dispensing apparatus 1 so as to maintain a predetermined pressure.
[0030]
A control computer 7 controls the dispensing control unit 6 to control the dispensing operation. The control computer 7 discharges droplets 3 of a predetermined size based on an image captured by the CCD camera 4. To control. The control computer also controls the driving of the XY stage 9 so that the dispensing operation by the dispensing control unit 6 and the movement of the XY stage 9 are synchronized.
[0031]
The parameters by which the dispensing control unit 6 controls the driving of the piezo element are all of the magnitude of the applied voltage to the piezo element, the applied voltage rise time, the applied time, and the applied voltage fall time, or at least one of them. .
[0032]
In FIG. 2 (A), a plurality of reagents a, b, c, d,... Such as antibodies serving as probes are applied to a membrane transcript 10 obtained by transferring a one-dimensionally developed protein sample (SDS polyacrylamide gel electrophoresis) onto the membrane. Are shown on each of different straight lines.
[0033]
A protein sample is developed and immobilized on the membrane transcript 10 along a plurality of lanes. As shown in an enlarged part of one lane in FIG. 2 (B), different straight lines spaced apart so that the reagents a, b, c, d,. Dispense along. The straight lines in (A) and (B) indicate that the reagent is dispensed along those straight lines.
[0034]
The dispensing of the reagent is performed along the illustrated straight line, but is performed only in the region where the spot 12 exists, and controls the dispensing operation by the dispensing control unit 6 so as not to dispense in the region where the spot 12 does not exist. You can also.
[0035]
In order to perform such dispensing using the liquid dispensing apparatus of FIG. 1, one reagent a is first dispensed to each lane. Next, the reagent is exchanged for b, and the reagent b is dispensed on a straight line at a position separated from the straight line where the reagent a is dispensed so as not to interfere with each other. Similarly, reagents c, d,... Are similarly dispensed.
[0036]
Next, a detection reagent such as a coloring reagent or a fluorescent reagent is dispensed as a secondary reaction reagent so that a reaction between the sample protein and the probe is colored to enable optical detection. At this time, the reagents a, b, c, d,... Are dispensed while being superimposed on the dispensed straight line. The detection reagent may be common to the reagents a, b, c, d,... Or may be unique to each. By dispensing such a secondary reaction reagent, a portion that has reacted with the probe develops a color, as shown by a colored portion 14 shown in black in FIG. 2C, so that it can be detected by an optical microscope. become.
[0037]
Before dispensing the reagents a, b, c, d,..., As necessary, the protein migration lane is used as an index for visualizing the electrophoresis image using a staining reagent or the like to know the subsequent dispensing position. Is also good. As such a method, there is a method in which the whole membrane is treated with a dye such as Coomassie brilliant blue, a protein / sugar-binding fluorescent reagent, or a gold colloid solution to dye a protein or the like.
[0038]
In this embodiment, a liquid dispensing apparatus using a piezo element is used. In the reagent dispensing using the piezo element, the reagent can be dropped on the membrane to a size of about 100 μm in diameter, so that high-density detection can be performed with a large number of probes. However, the ejection element is not limited to the piezo element, and an inkjet type or a syringe type may be used.
[0039]
【The invention's effect】
According to the present invention, for each spot developed and immobilized on a membrane, a plurality of types of reagents for detecting a target substance in the spot are dispensed in different regions so as not to interfere with each other. Because of this, a plurality of probes can be simultaneously formed on the sample electrophoresis image to form independent micro reaction sections.
In addition, if the probe is controlled so as to be added only on the sample migration lane, the probe does not reach the blank portion deviating from the migration lane as in the conventional method, and it is possible to avoid consuming extra reagents. And the realization of the reaction with high efficiency can be expected.
In addition, since a microdispensing apparatus is used, when processing various kinds of specimens such as rare ones, automatic processing of a plurality of reaction systems becomes possible.
[Brief description of the drawings]
FIG. 1 is a block diagram schematically showing an example of a liquid dispensing apparatus for dispensing a reagent for carrying out the present invention, together with a control system.
FIGS. 2A and 2B are diagrams showing the operation of one embodiment, in which FIG. 2A is a schematic plan view showing a state in which a plurality of reagents a, b, c, d,. FIG. 4A is a schematic plan view showing a part of one lane in an enlarged manner, and FIG. 4C is a schematic plan view showing a state after further dispensing a secondary reaction reagent.
[Explanation of symbols]
REFERENCE SIGNS LIST 1 dispensing mechanism using piezo chip 2 strobe 3 droplet 5 target 4 CCD camera 6 dispensing control unit 7 control computer 8 pressure control mechanism 9 XY stage 10 membrane transcript 12 spot 14 coloring part

Claims (4)

For each spot developed on the membrane and immobilized, multiple types of reagents for detecting the target substance in the spot are dispensed in different areas so as not to interfere with each other, and the presence of the target substance Alternatively, an immunoblot method for detecting a substance interacting with a target substance at a high density. The immunoblot method according to claim 1, wherein each of the detection reagents is continuously and linearly dropped onto different lines on the membrane using a microdispensing device. The immunoblotting method according to claim 1 or 2, wherein the dispensing of each detection reagent is performed only in a region where a spot on the membrane exists. The reagent includes a primary reagent containing a probe that specifically reacts with the target substance, and a secondary reagent that develops a color of the target substance after reacting with the probe.First, the primary reagent is dispensed, and then the primary reagent is added. 4. The immunoblotting method according to claim 1, wherein a secondary reagent is dispensed on the dispensed region.

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