JPH0798319A - Detecting method and detecting device for cytokinin and hole slide - Google Patents

Abstract

PURPOSE:To surely detect the low-concentration cytokinin separated by tubular electrophoresis. CONSTITUTION:A sample constituent separated by electrophoresis in a tubule 12 is guided to the outside of the tubule 12 for detecting cytokinin, the sample constituent is brought into contact with at least one cell having the nature changed in shape when reacted with cytokinin, and the cell is observed. This detecting device is provided with a hole slide 14 having recesses 18, 20, 22 storing the cell and an electrolyte, and an optical microscope 16 for observing the cell in the recess 18 of the hole slide 14.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates to methods and apparatus for detecting the presence or absence of cytokines in components of a sample separated by electrophoresis in a capillary or capillary.

[0002]

2. Description of the Related Art By using a capillary electrophoresis method, a sample such as collected blood or a cell culture solution is separated into a plurality of components and these components are specified. The sample is introduced from one end of a thin tube filled with an electrolytic solution, and a high voltage is applied between both ends of the thin tube. The sample in the capillary placed under a high voltage is separated into a plurality of components and appears as a plurality of bands, and migrates in the electrolyte of the capillary toward the other end of the capillary at different speeds. To identify the components, an ultraviolet light beam is passed across the capillary near the other end of the capillary. The component can be specified from the time and the absorbance when the band of the band migrating in the narrow tube sequentially crosses the ultraviolet ray.

[0003]

The sample that can be introduced into the capillary is a small amount (typically several nanoliters). Therefore, it has been difficult to detect components present in low concentrations in a small amount of sample, particularly cytokines such as interleukins in blood and cell culture medium. An object of the present invention is to reliably detect low-concentration cytokines separated by capillary electrophoresis.

[0004]

In order to detect a cytokine, the present invention has the property of guiding the components of a sample separated by electrophoresis in a capillary to the outside of the capillary and changing the shape in response to the cytokine. The component of the sample is brought into contact with at least one cell having the above, and the cell is observed.
The cells are observed with an optical microscope. The cytokine and the cell are, for example, an interleukin and a biodefense cell, respectively.

Further, the cytokine detection apparatus according to the present invention includes at least one cell and an electrolytic solution which have a property of receiving a component of a sample separated by electrophoresis in a narrow tube and having a property of reacting with a cytokine to change its shape. And a light microscope for observing cells in the recess of the hole slide.

Further, the hole slide according to the present invention is
A first recess for accommodating cells and an electrolyte having a property of changing a shape in response to a cytokine, a second recess for accommodating the electrolyte, and both first and second recesses It has a continuous groove and also has an electrode for electrophoresis which is immersed in the electrolytic solution in the second recess. The hole slide further includes a block defining a slope extending from or near an edge of the first recess at an angle with a release surface of the first recess. The hole slide may be made of transparent acrylic resin. The first recess,
The dimensions of the second recess and groove are 15 mm in length,
Width 2 mm and depth 1 mm, length 10 mm, width 10 mm and depth 2 mm, and length 15 mm, width 0.5-1 mm and depth 1 mm.

[0007]

According to the method of the present invention, when the sample component released from the tubule comes into contact with the cells, if the sample component is a cytokine, the cell reacts with the cytokine to change its shape. . Therefore,
By observing the cells under an optical microscope, for example, the presence or absence of low-concentration cytokines in the sample can be clarified. This method is particularly suitable for the detection of interleukin, which is one of the cytokines in a sample using collected blood or cell culture medium as a sample. A ball is suitable.

Further, according to the apparatus of the present invention, the sample components separated by the electrophoresis in the thin tube are guided to the recess of the hole slide in which the cells and the electrolytic solution are placed, and contact the cells there. Therefore, the behavior of the cells can be observed under an optical microscope.

In the hole slide, the first recess and the second recess communicate with each other through the groove, and since the hole slide has an electrode for electrophoresis, both the recess and the groove are connected to each other as a sample component. It can be used as an electrophoretic path for cells, and can be used for observing the cells with an optical microscope. The inclined surface of the block can be used as a fixing surface of an end portion of a capillary used for separating a sample into a plurality of components by electrophoresis, whereby the end portion of the capillary is placed in the first recess. Can be immersed in the electrolyte solution.

[0010]

EXAMPLE A method for detecting a cytokine according to the present invention is to introduce a sample component (hereinafter referred to as “sample component”) separated by electrophoresis in a capillary or a capillary into the outside of the capillary and react with the cytokine. And observing the cells by contacting the sample component with at least one cell (hereinafter, simply referred to as “cell”) having a property of changing the shape.

The cytokines to be detected include, for example, interleukins, and the cells that react with the cytokines and change their shape include, for example, cells in charge of biological defense. The biological defense cells include, for example, lymphocytes, macrophages / monocytes, and fibroblasts that react with them depending on the type of cytokine to be detected, and these cells can be obtained by culturing.

FIG. 1 shows a detection device 10 for carrying out the method according to the invention. The detection device 10 includes a hole slide 14 that receives the components of the sample separated by electrophoresis in the capillaries 12, and an optical microscope 16.

One end of the thin tube 12 has a container (not shown) for containing the sample, for example, the collected blood or cell culture solution.
It is inside. One of a pair of electrodes for electrophoresis (not shown) is immersed in the container. The other end of the thin tube 12 is in the first recess 18 (see FIG. 3) of the hole slide 14 placed on the stage 17 of the optical microscope 16 and is immersed in the electrolyte solution described later.

As shown in FIGS. 2 and 3, the hole slide 14 includes a first recess 18, a second recess 20 spaced from the first recess, and both recesses 18, And a groove 22 continuous with 20. The first recess 18 has at least one that has the property of changing its shape in response to a cytokine.
Two said cells are arranged. Also, the first recess 1
The electrolytic solution is contained in the groove 8, the groove 22, and the second recess 20. Therefore, the inside of the thin tube 12, the both recesses 18 and 20 of the hole slide 14 and the groove 22 are continuous with each other through the electrolytic solution.

The hole slide 14 is provided with a block 23 for attaching the other end of the thin tube 12 thereto. The block 24 has an inclined surface 26 extending from the edge of the first recess 18 or the vicinity thereof at an angle with the release surface of the first recess 18 (the liquid surface of the electrolytic solution). The other end of the thin tube 12 is fixed to the slope 26 of the block 24, and the tip thereof is immersed in the electrolytic solution. Further, the other one 28 of the pair of electrodes for electrophoresis is attached. The illustrated electrode 28 is made of a platinum wire and is immersed in the electrolytic solution in the second recess 20.

Therefore, when a high voltage is applied between both electrodes, both recesses 1 of the inside of the thin tube 12 and the hole slide 14 are
8, 20 and the groove 22 form an electrophoresis path, and the sample moves in the capillary 12 from one end to the other end, and is separated into a plurality of components in the meantime. The separated plurality of components appear as a plurality of bands in the vicinity of the other end of the thin tube 12.

The separated sample components are continuously electrophoresed from the thin tube 12 to the hole slide 14 so that the thin tube 12 is separated.
Is sequentially released into the first recess 18 of the hole slide 14 which is the outside of the second slide, comes into contact with the cells in the first recess 18, and passes through the groove 22 to form the second recess which is a storage space for the electrolytic solution. Place 2
Move to 0. If the sample component is a cytokine,
Since the cells react with the cytokine and change its shape, it can be seen that the cytokine is present in the sample. The behavior of the cells is determined by the optical microscope 16.
Can be observed at.

As long as the hole slide 14 can be observed by the optical microscope 16, its planar shape can be a shape other than the illustrated rectangle, and its material is a resin or material other than the acrylic resin. be able to.

In the illustrated example, the vertical length of the hole slide 14,
The lateral and thickness dimensions are 26 mm, 75 mm and 5 mm.
The first recess 18, the second recess 20 and the groove 22 each have a rectangular planar shape and have a length of 15 m.
m, width 2 mm and depth 1 mm, length 10 mm, width 10 mm and depth 2 mm, and length 15 mm, width 0.5-1 mm and depth 1 mm. The planar shape of the both recesses and the groove can be set to a shape other than the rectangle, and instead of the illustrated example in which the bottoms of the both recesses and the groove are flat surfaces, a surface of another form is formed. , A U-shaped curved surface, for example.

When using the hole slide 14 of the dimensions described above, 10 to 50 μl of a solution containing 1 × 10 ³ to 4 × 10 ⁷ of the above cells in 1 ml is used. As the electrolytic solution, for example, boric acid buffer, Hank's balanced salt solution, or the like can be used.

[Brief description of drawings]

1 is a schematic elevational view of a detection device according to the present invention.

FIG. 2 is a plan view of a hole slide.

FIG. 3 is a vertical sectional view of a hole slide.

[Explanation of symbols]

 10 Detection Device 12 Capillary Tube 14 Hole Slide 16 Microscope 18, 20, 22 First Recess, Second Recess and Groove 24 Block 28 Electrode

Claims (8)

[Claims] 1. A component of the sample separated by electrophoresis in a capillary is brought to the outside of the capillary, and the component of the sample is brought into contact with at least one cell having a property of reacting with a cytokine to change its shape. Observing the cells,
Method for detecting cytokine. 2. The method according to claim 1, wherein the observation of the cells is performed using an optical microscope. 3. The method according to claim 1, wherein the cytokine and the cell are an interleukin and a biodefense cell, respectively. 4. A hole slide having a recess in which at least one cell having a property of undergoing shape change in response to a cytokine, which accepts components of a sample separated by electrophoresis in a capillary tube, and a recess is placed. And a light microscope for observing cells in the recess of the hole slide. 5. A first recess for accommodating cells and an electrolytic solution having a property of changing a shape in response to a cytokine, a second recess for accommodating the electrolytic solution, and first and second And a groove extending to both the recesses, and having an electrophoretic electrode immersed in the electrolytic solution in the second recess. 6. The hole slide of claim 5, including a block defining a slope extending from or near the edge of the first recess at an angle with a release surface of the first recess. 7. A transparent acrylic resin,
The hole slide according to claim 5. 8. The first recess has dimensions of 15 mm length, 2 mm width and 1 mm depth, and the second recess has a length of 10 mm.
Hall slide according to claim 5, characterized in that it has dimensions mm, width 10 mm and depth 2 mm, and the groove has dimensions 15 mm in length, 0.5-1 mm in width and 1 mm in depth.