JPH0587777A - Dyeing method performed using electrophoretic device - Google Patents

Abstract

PURPOSE:To immediately dye fractionated protein or enzyme on a compact and inexpensive electrophoretic dyeing device easy to handle. CONSTITUTION:After serum protein or enzyme is fractionated on the migration stage 7 of an electrophoretic device, an NTB color forming reagent 31 is dropped on a support 17. The support 17 is covered with a transparent flexible water impermeable sheet (e.g., PET film 32) to be held to proper color forming reaction temp.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for staining serum proteins and lactate dehydrogenase fractionated by an electrophoretic device, and more particularly to a method for immediately staining the fractionated protein using the electrophoretic device. The present invention relates to a dyeing method improved so that the dyeing operation can be continuously performed on the electrophoretic device.

[0002]

2. Description of the Related Art Electrophoresis refers to an electrochemical phenomenon in which colloidal particles or polymers having a net charge move in a solution under the influence of an electric current. When this phenomenon began to be studied, a free solution was used as a support for the electric field, and metallurgical applications in which a metal was deposited by discharge in a colloidal solution were pursued, but recently, thin films of cellulose acetate and agarose gel were used. The mainstream is the electrophoresis method in which the material is formed into a support. Biochemical studies on electrophoresis have revealed that various proteins and enzymes in serum have different electrophoretic difficulty depending on their types, and their rapid application in clinical medicine has progressed. For example, when a healthy and normal human serum protein or enzyme is electrophoresed, it is fractionated according to the type of protein, and the fractionation state is useful for diagnosis. However, since it cannot be visually recognized only by fractionation, it is stained and measured with a densitometer. This densitometer has an optical density (opt).
It is a kind of colorimetric quantification device for measuring the oral density, but the color of the stained fraction is recorded by absorbance, and the integrated value obtained by integrating the respective fractional screen products is converted into a percentage and displayed. FIG. 9 is a schematic explanatory view showing the fractionation state, in which the protein fraction and the LDH isozyme fraction are depicted in contrast. An isozyme is an enzyme that exists in the same individual of the same species and has the same reaction mode in principle, but different protein molecule properties. In a broad sense, it refers to an enzyme having different mobility in electrophoresis. LH
D is an enzyme that catalyzes the redox reaction between pyruvic acid and lactic acid that occurs in the glycolytic system. This LHD is a quadruplex of two different subunits each having a molecular weight of 35,000, that is, a combination of a myocardial type (H type) and a skeletal muscle type (M type), and has a molecular weight of about 140,000. LHD _{1 in} Figure 9
For each ～LHD ₅ it was appended a combined state between the H type and M type. Since human LHD is distributed in almost all organs, a large amount of LHD is transmigrated into the serum when those tissues fall into a pathological state. Since the isozyme fraction differs depending on the organ, if the isozyme fraction of each organ is grasped, it can be compared with the normal value and used as a material for clinical judgment. For these reasons, human protein and enzyme fractionation / staining have great medical awareness.

Conventionally, the fractionation of human serum proteins and enzymes by this electrophoresis and the staining of the fractionated proteins and enzymes have been carried out by a method according to the laboratory operation using the instruments shown in FIG. Was there. c is a constant-current / constant-voltage power supply device for passing a current for migration, and is a migration box d
Are connected by a cord e. f is a buffer solution box, g is a staining box, and h is a decolorization box, and tweezers are attached to each. i is a cellulose acetate membrane used as a support, and j is a filter paper for supplying a buffer solution to the cellulose acetate membrane. 1 is a pipette for sampling, and k is a mouthpiece. It is highly skilled to arrange such instruments and materials on a table so that serum can be electrophoresed and fractionated using tweezers and stained to be supplied to a densitometer (not shown). It takes a lot of time and may spill the liquid, which is not suitable for high-efficiency mass processing. In order to solve these problems, the world's first fully automated electrophoresis / staining device was developed by Olympus Corporation, and then Cosmo Co., Ltd., Tsuneko Sangyo Co., Ltd., Helei Laboratory Co., Ltd., and Sankyo Co., Ltd. Developed a fully automated electrophoresis / staining device. FIG. 11 is an explanatory view of the mechanism of the fully-automatic electrophoresis / staining apparatus, and the automatic processing is performed according to the following steps. In step Q, the roll-shaped cellulose acetate membrane shown at the left end of the drawing is fed out, cut into a certain size by a cutter, wetted by a buffer solution wetting portion, and sent out. In step R, the applicator adsorbs the sample (human serum) delivered by the feeder and stamps it on the cellulose acetate membrane (support). In step S, the sample-stamped support (cellulose acetate film) is placed in the migration tank d '.
Electrophoresis is carried out by feeding it to the cell and energizing it. Step T
In the above, the support after the migration / fractionation is suspended in the dyeing / decolorizing box g ', and the dyeing / decoloring / drying is sequentially performed. In step U, the densitometer measures the dyed support.

[0004]

Fractionation and staining of human serum proteins and enzymes using the device shown in FIG. 10 requires a high level of skill, a great deal of time and labor, and a high efficiency of a large number of specimens. Can not be processed in. Further, the fully-automatic electrophoresis / staining device shown in FIG. 11 is difficult to spread to other than large hospitals because the entire mechanical device is large and expensive. The present invention has been made in view of these circumstances, and it is possible to perform post-fractionation staining by electrophoresis much faster and easier than the staining method using the instruments of FIG. It is an object of the present invention to provide a dyeing method which can be performed by using an electrophoresis apparatus which is remarkably simple as compared with the fully automatic equipment.

[0005]

The present inventor has previously described that FIG.
0, which is remarkably more convenient than the zero instruments, and is much smaller and cheaper than the fully automatic equipment shown in Fig. 11, and a semi-automatic portable electrophoresis apparatus was created and filed separately (Patent application 3-2.
31675, hereinafter referred to as the device of the prior application). The present inventor, as a related invention of the electrophoretic device according to the above-mentioned prior application, succeeds to the fractionation operation of the protein by the electrophoretic device without using any other mechanical device, thereby A method was developed to stain the fractionated proteins quickly and easily above. However, the method of the present invention can also be applied to an electrophoretic device other than the electrophoretic device according to the prior application. The method of the present invention is a method for staining serum proteins and enzymes fractionated by an electrophoresis apparatus, in which a smooth horizontal surface is provided on the migration stage of the above electrophoresis apparatus, and the smooth horizontal surface is subjected to an insulating surface treatment to obtain the above smooth A sheet-like support impregnated with a buffer solution is placed on a horizontal surface, and linear electrodes are horizontally arranged in the buffer solution tanks arranged on both sides of the electrophoretic stage through the migration stage. After energization, the serum proteins on the support are electrophoresed and fractionated, then a coloring reagent is dropped on the support, and the upper surface of the support is covered with a transparent and flexible impermeable sheet to develop color. It is characterized in that the reaction is maintained at a suitable temperature.

[0006]

According to the above method, since the electrophoretic stage of the electrophoretic device has a smooth horizontal surface, it is possible to perform staining on the electrophoretic stage without moving the support placed on the electrophoretic stage. Then, since the dyeing solution (coloring reagent) is dropped onto the support and covered with a transparent and flexible water-impermeable sheet, there is no risk of evaporation of the drug solution during the dyeing progress time, and the above-mentioned sheet is transparent. It is possible to visually monitor the progress of dyeing. Further, since the above-mentioned sheet is flexible, it is in close contact with the support to completely prevent evaporation of the drug solution, and because it is flexible, the operation of covering or removing the support is easy. Then, after the coloring reagent is dropped, the coloring reaction temperature is maintained at a suitable coloring temperature (for example, 40 ° C.), so that the coloring reaction proceeds rapidly, and the dyeing work of the efficiency becomes possible. As described above, since the staining method of the present invention is carried out on the electrophoresis apparatus subsequent to the fractionation operation by electrophoresis, it does not require the movement of the support, does not cause loss time, and is particularly necessary for staining. The members are only the above-mentioned flexible transparent sheet and color-developing reagent, and the number of required machinery is extremely small. One of the reasons why the number of required equipments is small is that a part of the electrophoretic device is preliminarily configured so as to be suitable for dyeing.

[0007]

EXAMPLE FIG. 8 shows an example of an electrophoretic device configured to carry out the dyeing method according to the present invention, (A) is a partially cutaway view, and (B) is an external view. Reference numeral 1 denotes a lower body, the top surface of which is constituted by a base plate 1a.
Reference numeral 2 denotes an upper body, which is supported by a hinge 3 in a lid shape that can be freely opened and closed. Reference numeral 19 shown in FIG. (B) is a hook configured to keep the lid-shaped upper body 2 closed. Inside the lower body 1, a temperature control board 4, a constant voltage / constant current power supply device 5a, and a discharging fan 6 are provided.
a and fins 6b are arranged, and a heat radiating hole 1a is provided in the bottom plate of the lower body 1 and a vent hole 1b is provided in the same side plate. The migration stage 7 is installed on the base plate 1a, and the buffer solution tanks 8 are arranged on both sides thereof. The migration stage 7 and the buffer tank 8
Will be described later in detail with reference to FIG. On the other hand, a partition plate 2a is fixed inside the upper body 2, and a high voltage device 5b is installed in a portion covered by the partition plate 2a. Then, direct current (450 V) is conducted to the upper contact 10 attached to the partition plate 2a through the high voltage electric wire 9 (see FIG. 8A). When the lid-shaped upper body 2 is closed, the upper contact 10 is brought into contact with the lower contact 11 so as to be conductive, and is supplied to the electrode in the buffer solution tank 8. This electrode will be described later with reference to FIG. The portion covered with the partition plate 2a is, as shown in FIG.
3a is installed. The opposite surface of the operation control board 13a is exposed to the top surface of the upper body 2 to form the display / operation panel 13 as shown in FIG. The display / operation panel 13 includes an LED lamp 13d for displaying a timer, a timer setting switch 13c, and an LED lamp 13 for displaying an operation.
d, heating / cooling changeover switch 13e, and start /
A stop switch 13f is arranged. The operation and operating method of these lamps and switches will be described later. Reference numeral 12 shown in FIG. 8 (B) denotes the electrophoretic stage 7 described above.
Is a viewing window provided so as to be viewed with the upper body 2 closed, and 20 is a switch connected to the constant voltage constant current power supply device 5. 2A shows a cross section taken along the line AA in the vicinity of the buffer solution tank 8 shown in FIG. 8A, and FIG. 2B shows a cross section in a state where the upper contact 10 and the lower contact 11 are in contact with each other. Show. 7 is a migration stage, the top surface of which is a flat horizontal surface which has been subjected to an electrically insulating surface treatment (in this example, Aflon manufactured by Asahi Glass Co., Ltd. was used). Conventionally, parallel grooves are generally provided on this surface. However, in the present invention, a horizontal smooth surface is formed because a dyeing operation is performed on this surface as described later. A support 1 made of a cellulose acetate film is placed on the migration stage 7.
7 is placed. The buffer solution tanks 8 arranged on both sides of the migration stage 7 are an anode side buffer solution tank 8a and a cathode side buffer solution tank 8
3B, it has a detachable structure as shown in FIG. The buffer solution tank 8 has a topless box-like structure, stores the buffer solution 15 therein, and is covered with a flat plate lid 14a. The flat plate lid 14a is provided with an opening 14b,
It is covered with a transparent inner lid 14c. The above-described structure relating to the buffer solution tank 8 is a configuration for performing a cleaning operation easily and visually observing the support 17 on the migration stage 7 while preventing evaporation of the buffer solution. Opening 14b of the flat plate lid 14a
The curved plate 14d is integrally provided so as to be in contact with an edge of the curved line 14b, curved in an arc shape, and the lower end thereof is immersed in the buffer solution 15 in the buffer solution tank 8. Since the cross section is viewed in FIG. 2A, the bending plate 14d is curved in an arc shape.
When viewed three-dimensionally, the shape is a part of a tubular shape as shown in FIG. As shown in FIG. 2A, the curved plate 14d
Electrodes 16a and 16b made of platinum wire are attached to the lower end of the. These electrodes 16a and 16b are members having the same shape and size as each other.
Is used as an anode, and the electrode 16b is used as a cathode. For the electrodes 16a and 16b described above, FIG.
450 V DC generated in the high-voltage device 5b supplied from the constant-voltage constant-current power supply 5a described in (A) is supplied through the upper contact 10 and the lower contact 11. The upper contact 10 is vertically slidably supported on the partition plate 2a as shown in FIG.
The elastic force is urged downward by 0a. Figure 3
FIG. 3B is a perspective view in which a part of the upper body 2 is cut away so that the contact state between the upper contact 10 and the lower contact 11 can be seen. As can be easily understood from FIGS. 2B and 3B described above, when the upper body 2 is lifted and opened as shown in FIG. 3A, the lower body 1 is disconnected from the high voltage power source. For example, there is no risk of electric shock when the buffer tank 8 is removed as shown by the arrow D in the figure, which is safe. The lower contact 11 and the electrode 16a (16b) are connected by the electric wire 21 shown in FIG. According to such a configuration, since the power supply lines to the electrodes 16a and 16b do not penetrate the buffer solution tank 8, there is no risk of liquid leakage. Wire 2 above
In the case of No. 1, an insulating coating is applied so as not to touch the buffer solution and disturb the electric field.

When using the apparatus of this embodiment, a filter paper 18 moistened with a buffer solution is attached to the concave surface side of the curved plate 14d as shown in FIG. FIG. 5 is a filter paper 1 for convenience of reading.
8 is shown with hatching. This hatching does not mean a cut surface. Note that the hatched portion is indicated by a broken line to indicate that it is hidden. A support (eg, a cellulose acetate film) 17 is provided on the migration stage 7.
2 is mounted and the curved plate 14d (which is integrally provided with the flat plate lid 14a) to which the filter paper 18 is attached as described above is attached to the buffer solution tank 8 as indicated by an arrow E. In the state shown in A), the filter paper 18 attached to the curved plate 14d
Holds the buffer solution by capillarity and adheres to the support (cellulose acetate film) 17. In this way, an energization path from the anode electrode 16a to the cathode electrode 16b via the filter paper 18 and the support 17 is formed. By the above-mentioned energization, the sample (human serum protein or enzyme) on the support (cellulose acetate film) 17 is fractionated by electrophoresis as described later. FIG. 6 is a schematic sectional view of the vicinity of the migration stage 7 described above. The top surface of the electrophoretic stage 7 is a horizontal smooth surface, and is covered with the insulating coating 7a formed by the electric insulating surface treatment as described above. A thermo module is arranged in close contact with the bottom surface of the main body of the electrophoretic stage 7, and the electrophoretic stage 7 can be cooled or heated by switching the energizing direction. Cooling is used in the electrophoretic fractionation operation, absorbs heat generated by energization and prevents temperature rise, and prevents alteration of proteins and enzymes due to overheating. This cooling makes it possible to increase the amount of electricity applied, and the time required for fractionating proteins and enzymes is shortened. In addition, the heating is used to rapidly raise the temperature suitable for dyeing (for example, 40 ° C.) in the dyeing operation after the fractionation and maintain the temperature suitable for dyeing. The above-mentioned cooling / heating switching operation is performed by the heating / cooling switching switch 13e of the display / operation panel 13 shown in FIG. 8 (A). As shown in FIG. 6, the thermo module 22 described above is used.
The aluminum block 23 is sandwiched between the heat dissipation fin 6b and the heat dissipation fin 6b to improve heat transfer. Reference numeral 24 is a heat insulating plate arranged on both sides thereof. Next, a continuous fractionation / staining operation will be described for an example in which the staining method according to the present invention is carried out using the electrophoresis / staining apparatus configured as described above. When this device is connected to a commercial power source (100 volt AC) and the switch shown in FIG. 8B is turned on, the operation display lamp 13d of the display / operation panel 13 shown in FIG. 8 indicates the start of cooling. By this cooling action, the migration stage 7 is cooled to 15 ° C., and thereafter it is kept at 15 ° C. While the cooling action is being advanced, a filter paper 18 wet with a buffer solution is attached to the concave surface side of the curved plate 14d as shown in FIG. 5, while a support body wet with a buffer solution on the migration stage 7 ( Cellulose acetate film 17 is placed. From the state of FIG. 5, the curved plate 14d is inserted into the buffer solution tank 8 (previously filled with buffer solution) as shown by arrow E as shown by arrow E, and the flat plate lid 14a covers the buffer solution tank 8 and 7A, before covering the opening 14b with the inner lid 14c in the state of FIG.
A sample is sampled on the support (cellulose acetate film) 17 as shown in FIG. In the electrophoretic fractionation of human serum proteins and enzymes, it is appropriate to draw a short line (normally 1 microliter) of the sample on the support and at a right angle to the current-carrying direction. In this example, a known sampler 25 having a plurality of sampling leads 25a to 25g is used.
Sampling was performed by stamping on a support with a sample attached to the tip of a to 25 g. As shown in FIG. 2A, the inner lid 14c covers the opening 14b to prevent the buffer solution from evaporating, and the upper body 2 is closed and locked by the hook 19 so as not to be opened as shown in FIG. 8B. .. When the upper body 2 is closed, the upper contact 10 is brought into contact with the lower contact 11 to conduct electricity. The display / operation panel 13 is operated as follows while visually observing the support through the viewing window 12. The timer setting switch 13c is operated to set an arbitrary migration time (energization time). The set time (for example, 10 minutes) is displayed on the timer display lamp 13b. The start / stop switch 13f is pressed to start electrophoresis (energization). When electrophoresis is started, the timer display lamp 13b starts counting down, and the operation display lamp 13d indicates that power is being supplied (during migration). In this case, if there is any abnormality, a safety device (not shown) operates. That is, the energization at a high temperature of 15 ° C. or more, the energization of an overcurrent of 40 mA or more, and the energization of an undercurrent of less than 5 mA are prevented. When energization (electrophoresis) proceeds in a normal state and the displayed value of the timer display lamp 13b becomes 0, a buzzer (not shown) sounds and the operation display lamp 13d indicates the end of electrophoresis. When the electrophoretic fractionation is completed as described above, the upper body 2 is lifted and opened (see FIG. 2 (A)), the flat plate lid 14a and the members fixed thereto, and the inner lid 14c are removed, and the upper body is again removed. 2 is closed, and the heating / cooling changeover switch 1 described above
3e is operated to switch to heating (heating) (Fig. 8 (B)).
reference). During heating, the operation display lamp 13d indicates that the migration stage is being heated, and when the appropriate dyeing temperature (40 ° C.) is reached, the buzzer sounds and the operation display lamp 13d.
Indicates the end of heating. Upper body 2 when heating is completed
1 and open the upper part of the nitrotetrazolium blue (hereinafter abbreviated as NTB) reagent 31 for coloring, as shown in FIG.
It is dropped on the support (cellulose acetate film) 17.
Reference numeral 31a is an NTB reagent being dropped, and reference numeral 31b is an NTB reagent slightly flowing on the support. On the NTB reagent 31b flowing on the support, a 125-micron-thick polyester film (hereinafter abbreviated as PET film) is placed while being careful so that air bubbles do not enter (arrow F). The upper body 2 is closed, an arbitrary time (for example, 15 minutes) is set by the timer setting switch 13c, and the start / stop switch 13f is pressed. As a result, the lactate dehydrogenase fractionated on the support (cellulose acetate) 17 on the migration stage 7 is stained with the NTB reagent. The end of the set time is reported by the operation indicator lamp 13d and the buzzer. When the dyeing is finished, the upper body 2 is opened upward, the PET film is removed, the support (cellulose acetate film) 17 is lifted with tweezers, transferred to a 5% acetic acid solution (not shown), immersed for 10 minutes and washed. Wash with distilled water for 1 minute and dry. This completes the electrophoretic fractionation and staining of lactate dehydrogenase.
As described above, in the dyeing method of this embodiment,
In addition to the electrophoretic device, the PET film 32 (FIG. 1) and the coloring reagent 31 are required for dyeing equipment and materials.
In addition, since the fractionation operation by electrophoresis can be followed by the staining operation, highly efficient work is possible.

[0009]

As described above, the dyeing method of the present invention utilizes the electrophoretic device according to the prior application, and, following the fractionation operation by the device according to the prior application, the electrophoretic device according to the prior application. Since it is carried out on the electrophoretic device, it is possible to stain human serum proteins and enzymes with high efficiency without time loss, and it is economical because there is no need to install a special mechanical device for staining, and the installation area of equipment is economical. Is also saved.

[Brief description of drawings]

FIG. 1 is a perspective view showing an example of a state where a dyeing operation is performed by applying a dyeing method according to the present invention.

FIG. 2 is a cross-sectional view of essential parts showing an embodiment of an electrophoresis / staining device according to the prior application, which is used in the method of the invention.

FIG. 3 is a perspective view shown for explaining the configuration of the electrophoresis / staining apparatus according to the above-mentioned prior application.

FIG. 4 is an exploded perspective view of main parts shown for explaining the configuration of the electrophoresis / staining apparatus according to the above-mentioned prior application.

FIG. 5 is an exploded perspective view of an essential part shown for explaining the configuration of the electrophoresis / staining apparatus according to the above-mentioned prior application.

FIG. 6 is a schematic cross-sectional view of an electrophoretic stage in an example of an electrophoresis / staining device configured to carry out the method of the present invention.

FIG. 7 is an explanatory diagram showing a state in which sampling for fractionation / staining is being performed in one example of the method of the present invention.

FIG. 8 is a perspective view showing an example of an electrophoresis / staining device according to a prior application used in one example of the method of the present invention.

FIG. 9 is a chart showing the fractionation and staining of human serum proteins and enzymes.

FIG. 10 is a perspective view of a conventional electrophoresis / staining apparatus.

FIG. 11 is a schematic explanatory view showing a configuration of a conventional fully-automatic electrophoresis / staining apparatus.

[Explanation of symbols]

1 ... Lower body, 1a ... Base plate, 2 ... Upper body, 3
... Hinges, 4 ... Temperature control board, 5a ... Constant voltage and constant current power supply device, 7 ... Running stage, 8 ... Buffer tank, 9 ... High voltage electric wire, 10 ... Upper contact, 11 ... Lower contact, 12 ... Peep window,
13 ... Display / operation panel, 13a ... Operation control board, 13b ...
Timer display lamp, 13c ... Timer setting switch, 13
d ... operation indicator lamp, 13e ... heating / cooling changeover switch,
13f ... start / stop switch, 14a ... flat plate lid,
14b ... Opening, 14c ... Inner lid, 14d ... Curved plate, 15 ...
Buffer solution, 16a, 16b ... Platinum wire electrode, 17 ... Support (cellulose acetate membrane), 18 ... Filter paper, 20 ... Switch, 21 ... Electric wire, 22 ... Thermo module, 23 ... Aluminum block, 24 ... Heat insulating material, 25 ... Sampler,
25a to 25b ... Sampling lead, 31 ... NTB reagent, 32 ... PET film.

Claims (1)

[Claims] 1. A method for staining serum proteins or enzymes fractionated by an electrophoretic device, wherein a smooth horizontal surface is provided on a migration stage of the electrophoretic device, and the smooth horizontal surface is subjected to an insulating surface treatment to obtain the smooth surface. A sheet-like support impregnated with a buffer solution is placed on a horizontal surface, and linear electrodes are horizontally arranged in buffer solution tanks arranged on both sides of the electrophoretic stage through the migration stage to energize the support. Then, the serum protein on the support was electrophoresed and fractionated, and then a coloring reagent was dropped on the support, and the upper surface of the support was covered with a transparent and flexible impermeable sheet to give a color reaction. A dyeing method performed by using an electrophoresis apparatus, which is characterized by holding at an appropriate temperature.