JP3029364B2 - Separation and purification device and separation and purification method. - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a separation and purification apparatus and a separation and purification method.
The present invention relates to a method for separating and purifying a specific component in a solution comprising an electrophoresis apparatus and a chromatographic apparatus and a method for separating and purifying the same.In particular, a biologically active substance such as an antigen, an antibody, a lectin, It is used for separation and purification of receptors, enzymes, and polynucleotides.

[0002]

2. Description of the Related Art An apparatus using a conventional electrophoresis apparatus is disclosed, for example, in Japanese Patent Application Laid-Open No. 57-198857. This is disadvantageous in that it is not possible to capture only specific components because it is physically captured using a filter. There is known a technique in which a target component is pre-concentrated by electrophoresis as in the technique described in JP-A-58-168957. Although this technique uses an electric capturing means, it has a drawback that it cannot capture only a specific component as in the above-described technique.

[0003] Further, an apparatus in which an electrophoresis apparatus and a chromatograph apparatus are combined is disclosed in JP-A-63-184050, US Pat. PAT. No. 431585, US. PA
T. There is a technique described in Japanese Patent No. 4708752. The technique described in Japanese Patent Application Laid-Open No. 63-184050 simply connects an electrophoresis apparatus and an ion chromatograph mechanically,
It is only a continuous treatment, and the separation of the contained components can be slowed down, but there is a drawback that only the target components cannot be separated.

The above US. PAT. The technique described in 431585 discloses a technique in which an electrophoresis apparatus and an ion chromatograph are integrally connected to each other for processing. However, a specific component can be separated, but a specific component cannot be captured. There is. The US. PAT. 4708
No. 752 discloses a technique in which an electrophoresis apparatus and an ion chromatograph are connected via a multi-port flow path switching valve provided with a special probe, and a contaminated and pressured sample is removed by, for example, mass spectrometry. This was a high vacuum and clean sample condition required for analysis by an analyzer, and was not an apparatus for separating and purifying specific components.

[0005]

SUMMARY OF THE INVENTION
This method is a method of separating substances based on the charge state and molecular size of a substance, and is not used for separation based on the chemical properties of specific components. A sample in which various substances coexist can be analyzed without pretreatment, but the sensitivity is low. There is also a method using an antigen-antibody reaction such as immunoelectrophoresis, but this method is suitable for component analysis, and has a problem that a large amount of sample cannot be separated and purified.

Further, regarding the above-mentioned chromatography,
Generally speaking, since the target component in the mobile phase is separated from other components by the interaction of the target component in the mobile phase and the carrier, by using a carrier that specifically binds to a specific target component, Selectivity can be greatly improved. Accordingly, the sensitivity is high, and excellent results can be obtained for a sample consisting of only a trace component, but the sample is easily affected by coexisting high concentration components.

In the above-mentioned conventional separation and purification techniques, chromatography or electrophoresis is used alone or as described in US Pat. PAT. As in the compound separation and purification apparatus described in Japanese Patent No. 4708752, it is also practiced to take advantage of each apparatus, concentrate the fractionated solution after completing one operation, and perform the other operation.

[0008] Among them, the concentration of the target component is often reduced in the eluate separated by chromatography or electrophoresis alone, so that it is necessary to reconcentrate it, and there is a problem that the recovery rate deteriorates. Further, the complex separation / purification apparatus also has a problem in that the working efficiency is poor and the recovery rate is poor because dilution of the target component occurs.

Furthermore, problems common to the above techniques, since responsible body capacity of the electrophoresis apparatus and an ion chromatograph is fixed, it is difficult corresponding to the case is high or mix very low concentration of the component of interest . For example, separation and purification when the concentration fluctuates greatly like antibodies and hormones.

The present invention has been made in order to solve the above-mentioned problems of the prior art, and it is possible to efficiently and rapidly separate and purify only specific components with high purity in accordance with sample conditions. An object of the present invention is to provide a separation and purification device and a separation and purification method .

[0011]

In order to achieve the above-mentioned object, the structure of the purification / separation apparatus according to the present invention is designed so that the eluent contains a sample.
Connected to the sample injection means for injecting the sample,
Affiliate that captures constant components with a chromatography support
Means for identifying the captured
The crudely purified substance containing the component
To the chromatographic support to release
A liquid sending means for sending syneresis, and containing the released specific component;
The specific component and the specific component
An electrophoresis means for separating components other than the specific component;
Prepared between the chromatography carrier and the electrophoresis carrier.
The sample passed through the chromatography support
Means for discarding the components, and inspecting the separated components.
Detection means for collecting specific components.
And the disposal means moves the chromatography support.
Is characterized in that the amount of waste can be adjusted by
It is.

[0012]

[0013]

[0014] The electrophoretic carrier is characterized in the use of gelled polymers. The chromatography carrier is packed in a storage column, and the storage column is
It is characterized by being detachable. The electrophoresis carrier is filled in a storage container, and the storage container is detachable.
It is characterized by being free. The chromatography carrier is characterized in that any one of an antigen, an antibody, a lectin, a receptor, an enzyme, and a polynucleotide is immobilized. Of the purification separation method according to the present invention
The configuration consists of injecting the sample into the eluent and
Injecting into a chromatographic carrier, said injecting
The specified component in the sample is analyzed by the chromatography support
Capturing by the chromatography
A sample that has passed through the chromatography support due to
Discarding the material, said chromatography carrier
To stop sample disposal and
Forming a sample introduction path to the electrophoresis carrier;
The liquid is trapped by the chromatography support by injection.
Releasing the separated components;
Flow current to separate the introduced sample for each component.
And detecting each of the separated components;
Recovering the specific component.
How to

[0015]

The operation of each of the above technical means is as follows. According to the configuration of the present invention, for example, only specific components are captured by affinity chromatography, the components other than the specific components are discarded, and the captured specific components are desorbed from the chromatography carrier. When the specific components are eluted and separated, high-purity specific components can be quickly separated and purified. Further, by changing the volume of the chromatography carrier or the electrophoresis carrier, the amount of the specific component that can be bound can be changed. Since the amount of waste other than the specific component can be adjusted,
The separation and purification time can be reduced according to the amount of the sample.

[0016] Even if the specific component is eluted and separated by electrophoresis, and the other component is discarded, and then the specific component is captured and separated by chromatography, the high-purity specific component can also be rapidly separated. Can be purified. further,
The specific component is captured and separated by chromatography, non-bonding impurities other than the specific component are removed by electrophoresis, the captured specific component is desorbed from the chromatography carrier, and the high-purity specific component is similarly purified. It can be separated and purified quickly.

Further, a gel can be used as an electrophoresis carrier, and the target component separated in the affinity chromatography step can be concentrated. After the electrophoretic separation, the high-purity target component eluted and separated from the electrophoretic carrier can be confirmed and recovered by, for example, a flow cell spectrophotometer. The carrier capacity can be changed by changing the length and position of the chromatographic separation part, and the capacity of the electrophoretic carrier can be changed by changing the length of the carrier. Can be responded to.

[0018]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a separation and purification apparatus according to one embodiment of the present invention will be described with reference to FIGS. [Embodiment 1] FIG. 1 is a block diagram showing a configuration of a separation / purification apparatus according to one embodiment of the present invention, and FIG. 2 is a sectional view of a separation / purification unit in the separation / purification apparatus according to one embodiment of the present invention. is there.

In FIG. 1, 1 is an eluent, 2 is an automatic dispenser, 3 is a desorbing solution, 4 is a switching valve, 5 is a pump, 6
Is a chromatographic separation unit, 7 is an electrophoresis separation unit, 8
Reference numerals a and 8b denote upper and lower electrodes of the electrophoresis separation section, 9 a waste liquid bottle, 10 a temperature controller, 11 a detector, 12 a recovered liquid, 13 a fraction collector, and 14 a DC power supply.

In FIG. 2, 8a and 8b correspond to FIG.
The upper and lower electrodes of the electrophoresis separation section, 15 is a sample introduction tube,
16 is an upper / lower tube for electrolyte, 17 is an ion exchange tube, 18 is a transfer tube, 19 is an upper electrolyte tank, 20 is an upper electrolyte tank cover, 21 is a position adjustment cylinder, 21a is a position adjustment cylinder screw portion, Reference numeral 22 denotes a position adjustment tube fixing tube, 22a denotes a position adjustment tube fixing tube screw portion, 23 denotes a chromatographic separation unit body, 24a denotes a position adjustment tube fixing member, 24b denotes a position adjustment tube fixing tube fixing member, and 25 denotes waste liquid. Outlet tube, 26 is a chromatography carrier, 27 is a gel holding cap, 2
8 is a gel storage column, 29 is a fixing member, 30 is a cooling water inlet / outlet pipe, 31 is a glass tube, 32 is an electrophoresis carrier, 33 is an electrophoresis separation unit body, 34 is a lower electrolytic solution tank, and 35 is a lower electrolytic solution. , A collection liquid introduction tube,
37 is a collection liquid outlet tube, 38 is a bottom plate of a lower electrolytic solution tank,
Reference numeral 39 denotes an electrophoresis separation unit fixing member, reference numeral 40 denotes an electrolyte, and reference numeral 42 denotes an electrophoresis carrier lower press.

As shown in FIG. 1, a separation and purification apparatus according to one embodiment of the present invention is configured such that a predetermined amount of a sample is sucked up from a sample tube placed on an automatic dispenser 2 using a nozzle, and an eluent is 1 switches to the switching valve 4 and the pump 5 to the chromatographic separation unit 6, but unnecessary components do not combine with the chromatographic separation unit 6. The unbound unnecessary components are collected in the waste liquid bottle 9 via the gap between the chromatography separation unit 6 and the electrophoresis separation unit 7 and discarded. The distance between the gaps can be adjusted by raising or lowering the height of the chromatographic separation unit 6 to thereby control the amount of waste.

After elimination of the gap, the purified component bound to the chromatographic separation section 6 is desorbed from the chromatography carrier by the desorbing liquid 3. Next, when a DC current is passed between the electrodes 8 from the DC power supply 14, the purified components move from the chromatography separation unit 6 to the electrophoresis unit 7, and further purify pure and impure components in the electrophoresis carrier. And will be separated.

The components that have reached the elution end of the electrophoretic carrier are washed with the recovery liquid 12, move in the flow channel, the target components are confirmed by the detector 11, and are collected by the fraction collector 13 in a fixed amount. . During electrophoretic separation,
Since the electrophoresis carrier generates heat, a temperature controller 10 is provided to cool the electrophoresis carrier.

FIG. 2 is a diagram showing in detail a cross section of a separation / purification unit in which a chromatography separation unit and an electrophoresis separation unit are combined in a separation / purification apparatus according to another embodiment of the present invention. The configuration is such that the chromatography separation unit 6 is interposed between the lower electrodes 8a and 8b above the migration separation unit 7.

The separation and purification section is provided with an upper electrolytic solution tank 1
9, a chromatography separation unit body 23, an electrophoresis separation unit body 33, and a lower electrolytic solution tank 34 are provided immediately below. The chromatography separation unit body 23, the electrophoresis separation unit body 33, and the lower electrolytic solution tank 34 are integrally fixed by a fixing member 29 and an electrophoresis separation unit fixing member 39, respectively.

The upper surface of the upper electrolytic solution tank 19 is covered with an upper electrolytic solution cover 20, and an inner hollow portion thereof is filled with an upper electrolytic solution 40a. The upper electrolyte bath cover 2
0 is provided with an upper electrode 8a that penetrates the cover 20 and is immersed in the electrolytic solution 40a. An upper electrolytic solution inlet / outlet tube 16 is provided on the side surface. The entire upper electrolytic solution tank 19 is fixedly supported by a position adjusting cylinder 21.

The position adjusting cylinder 21 is provided with the electrolytic solution tank 19.
An opening 41 for introducing a transfer pipe 18 (described later) is provided in a side surface portion immediately below the fixed support portion, and a through hole 42 communicating with the opening 41 is provided inside the opening 41. Further, the outer peripheral surface of the position adjusting cylinder 21 is
Position adjusting cylinder screwing portion 2 at a predetermined portion close to the fixed support portion
1a is engraved.

Further, at the lower end of the position adjusting cylinder 21,
The chromatography carrier 26 is packed,
Is held by a gel holding column 27 which holds the column with a gel holding cap 27. At the upper end of the gel storage column 28, a storage column opening 43 for introducing a sample into the carrier 26 is provided.

The chromatographic separation unit body 23
Has a hollow portion inside, and the position adjusting cylinder 21 is inserted into the hollow portion and engaged with each other.
The position adjusting cylinder fixing tube 22 has a hollow portion inside, and the inner surface of the hollow portion includes the position adjusting cylinder screwing portion 21.
A position adjusting cylinder fixing tube screwing portion 22a screwed with a is provided. The insertion length of the position adjusting cylinder 21 can be adjusted by screwing and rotating the two screwing portions 21a and 22a. Further, if necessary, the position adjusting cylinder 21 can be taken out of the chromatography separation unit body 23.

After the insertion length of the position adjusting cylinder 21 is adjusted, the position adjusting cylinder 21 is fixed by the position adjusting cylinder fixing member 24a, and the position adjusting cylinder fixing pipe 22 is adjusted by the position adjusting cylinder fixing pipe fixing member 24b. Are fixed respectively. A glass tube 31 is attached to the lower end of the hollow portion of the chromatography separation unit body 23,
The inside thereof is filled with an electrophoretic carrier 32.

The packing 32 is held by the lower support 44 of the electrophoretic carrier. Further, a waste liquid discharge pipe 25 is provided at a side portion of the chromatography separation section body 23.
Is provided. Further, the chromatography separation unit body 23 is fixed to the electrophoresis separation unit body 33 by a plurality of fixing members 29.

The interior of the electrophoresis separation unit 33 forms a cavity, and the inner surface of the cavity is located at a position corresponding to the glass tube 31 filled with the electrophoresis carrier 32. As described above, the plurality of electrophoresis separation unit fixing members 3
9 is fixed to the lower electrolytic solution tank 34. On its side, in order to cool the electrophoretic carrier 32,
A cooling water inlet / outlet pipe 30 is provided.

The lower electrolytic solution tank 34 is provided with the glass tube 3.
1 is held so as to be in contact with an electrophoretic carrier lower retainer 44 at the lower end thereof, the interior thereof is hollow, and a lower electrolytic tank bottom plate 38 is provided at the bottom. The hollow portion is filled with a lower electrolyte 40b. Lower electrode 8b
Penetrates through the bottom plate 38 of the lower electrolytic cell and is immersed in the lower electrolytic solution 40b. A lower electrolytic solution inlet / outlet tube 35 is provided on the side surface, and a recovered liquid introduction tube 36 and a recovered liquid lead-out tube 37 are provided on other side surfaces.

With respect to the separation and purification section configured as described above, the sample introduction pipe 15 descends through the upper electrolytic solution tank cover 20 to become an ion exchange tube 17 in the upper electrolytic solution 40a. . The ion exchange tube 17
However, the sample liquid in the sample introduction tube 15 is provided with conductivity by being in contact with the upper electrolytic solution 40a. The ion exchange tube 17 is thus connected to the upper electrolyte 40a.
Then, it bends upward while maintaining the conductivity, and penetrates the upper electrolyte bath cover 20 again to go out to the outside to become the transfer pipe 18.

The transfer tube 18 that has exited is bent,
The opening 41 of the position adjusting cylinder 21 is inserted through the through hole 42.
To the opening 43 at the upper end of the gel storage column 28.
Is to be reached. Then, the upper electrode 8a
Is the sample liquid, upper electrolyte 40a, ion exchange tube 1
7, the sample solution in the transfer tube 18, the sample solution in the opening 43, the chromatography carrier 26, the electrophoresis carrier 32,
The lower electrode 8b is electrically connected to the lower electrode 8b through the lower electrolytic solution 40b.

The recovered liquid from the recovered liquid introducing tube 36 is used to wash the electrophoretic carrier 32 held by the lower support 44 of the electrophoretic carrier, and the recovered liquid after washing is recovered by the recovered liquid outlet tube 37. It is supposed to be. The above-mentioned sample solution, electrolyte solution, cooling water, recovery solution and the like are sent by a pump (not shown).

Now, the separation and purification unit in which the electrophoresis separation unit and the chromatography separation unit shown in FIG.
The operation of the present invention will be described by replacing the chromatography separation unit 6 and the electrophoresis separation unit 7 with each other. In the above description, the same reference numerals as those used in FIG. 1 indicate the same parts, and therefore, please refer to FIG.

First, the position adjusting cylinder 21 is placed at the upper position, and the sample liquid introduced by the sample introducing pipe 15 is supplied to the ion exchange tube 17 penetrating the upper electrolytic solution tank cover 20 and the upper electrolytic solution bath. An opening 4 at the upper end of the gel storage column 28 passes through a transfer pipe 18 passing through the cover 20.
Reach 3 At this time, the sample liquid is given conductivity by the ion exchange tube 17 being in contact with the upper electrolytic solution 40a.

The sample liquid that has reached the opening 43 is introduced into the chromatography carrier 26 in the gel storage column 28 from the opening 43 and comes into contact with the carrier. The specific components in the sample solution in contact with the carrier and substances very similar thereto bind to the chromatography carrier 26.

As described above, since the position adjusting cylinder 21 is lifted upward, a gap is formed between the gel storage column 28 and the glass tube 31 storing the electrophoretic carrier, and the gap is used to determine the specific position. The sample liquid containing unnecessary components other than the components is discharged through the waste liquid outlet pipe 25.

After that, the position adjusting cylinder 21 is moved as shown by the arrow in the figure, and the gel storage column 28 is inserted into the glass tube 31 for storing the electrophoresis carrier 32, and the gel storage column 28 and the glass tube 31 are connected to each other. When the gap is eliminated, the discharging operation of the sample liquid other than the specific component is stopped. Next, by introducing the elimination liquid 3, the crudely purified substance containing the specific component is released from the chromatography support 26.

Next, the upper and lower electrodes 8a, 8a of the electrophoresis separation section
When a DC voltage is applied to the upper electrode b, the upper electrolyte 40a, the ion exchange tube 17, the sample solution in the transfer tube 18, the sample solution in the opening 43,
Since it is electrically connected to the lower electrode 8b through the sample solution on the chromatography carrier 26, the electrophoresis carrier 32, and the lower electrolyte solution 40b, a direct current flows.

The partially purified substance containing the specific component moves into the electrophoresis carrier 32 by the DC current. The specific component and the other components are separated in the electrophoresis carrier 32 because the moving speed differs depending on the mobility. The component that has reached the lower end of the electrophoresis carrier 32 is
Is washed by the recovery liquid flowing from the recovery liquid outlet pipe 37.
After that, it is confirmed by the detector 11, collected by the fraction collector 13, and the specific component is separated and purified.

As described above, the position adjusting cylinder 21 is moved up and down by screwing and rotating the screwing portion 21a with the screwing portion 22a, taking it out of the chromatography separation unit body 23, and taking out the gel storage column 28. By changing, the amount of the chromatography carrier 26 is changed.

Further, by removing the fixing member 29, the glass tube 31 for accommodating the electrophoresis carrier 32 is taken out together with the chromatographic separation unit body 23, and by replacing the glass tube 31, the glass tube 31 is replaced. Change the amount of 32. Thus, the separation and purification conditions can be changed according to the conditions of the sample solution.

In the configuration of the separation / purification apparatus according to one embodiment of the present invention shown in FIG. 1, electrophoresis is performed after chromatography, but this order is not fixed. In addition, when the impurities are removed by the chromatography, if the impurities are substances that move electrically, electrophoresis can be used.

[Embodiment 2] Next, a separation and purification apparatus according to another embodiment of the present invention will be described. FIG. 3 is a sectional view of a separation / purification unit in a separation / purification apparatus according to another embodiment of the present invention. The separation / purification unit in FIG. 3 performs chromatographic separation following electrophoretic elution separation.

In FIG. 3, the same reference numerals as those in FIG. 2 denote the same parts, and a detailed description thereof will be omitted. Only new reference numerals will be described. 50 is a sample liquid introduction nozzle, 51 is an outer cylinder part, 5
Reference numeral 2 denotes an inner cylinder portion, 53 denotes a recovery liquid switching valve, 54 denotes an overlay liquid, 55 denotes a base portion, 56 denotes a heater coil, and 57 denotes an opening / closing valve.

The separation / purification unit shown in FIG. 3 is one in which an electrophoresis separation unit is provided in the upper part and a chromatography separation unit is provided in the lower part, and the chromatography separation unit and the electrophoresis separation unit are connected and installed. The electrophoresis separation unit is configured such that a double cylinder including an outer tube 51 and an inner tube 52 is disposed on a base 55.

The outer cylindrical portion 51 has an upper part hermetically sealed, and cooling water inlet / outlet pipes 30 provided at upper and lower ends. A sample liquid introduction nozzle 50 is provided to penetrate the outer surface. The inner cylindrical portion 52 is provided with an upper electrode 8a at an upper portion, and the inside thereof is filled with an electrophoretic carrier 32 at a lowermost layer, an overlying solution 54 thereon, and an upper electrolytic solution 40a thereon. Further, since the upper surface of the inner cylindrical portion 52 can be opened, the amount of the electrophoretic carrier 32 can be easily changed.

The upper electrode 8a is connected to the upper electrolyte 40
a. An electrophoretic carrier lower retainer 44 is provided at the lowermost part of the electrophoretic carrier 32 and holds the electrophoretic carrier 32. Further, the overlay liquid 54
The outlet of the sample liquid introduction nozzle 50 that penetrates the outer surface of the outer cylindrical portion 51 projects.

The base 55 has a hollow portion therein, and the lower electrolytic solution 40b is accommodated in the hollow portion.
The lower electrode 40b is immersed in the lower electrolyte 40b. One of the side parts is a lower electrolyte inlet / outlet tube 3
5 is provided, and a collection liquid introduction tube 36 and a collection liquid discharge tube 37 are provided on the other side.

The leading end of the recovery liquid introducing tube 36 cleans the end of the electrophoresis carrier 32 held by the electrophoresis carrier lower retainer 44, and the cleaning liquid is recovered by the recovery liquid outlet tube 37. It has become. The recovered liquid outlet tube 37 is connected to the lower electrolytic solution 4.
0b, and passed through, and the recovery liquid switching valve 53
Is included in the flow path, and comes out of the base 55.

The immersion portion of the recovered liquid outlet tube 37 in the lower electrolytic solution 40b is an ion exchange tube and has conductivity. Thereby, the upper electrode 8a
And the lower electrode 8b, the upper electrolyte solution 40a, the overlay solution 54, the electrophoresis carrier 32, the recovery solution in the ion exchange tube 17, and the ion exchange tube 1
7 and the lower electrolytic solution 40b.

When the recovered liquid in the recovered liquid outlet tube 37 contains a specific component to be separated and purified by switching, the recovery liquid switching valve 53 sends the recovered liquid to the chromatography separation section. Is not contained outside through the collection liquid outlet tube 37. Further, the amount of waste can be adjusted by adjusting the valve opening.

The chromatographic separation unit body 23 provided at the lower part has a hollow portion provided therein, and a chromatographic carrier 26 is accommodated in the hollow portion. A collection liquid containing a specific component in the collection liquid outlet tube 37 is led to the chromatography carrier 26. The chromatographic separation unit body 23
The hollow portion is opened when the electrophoretic separation section is removed, so that the storage amount of the chromatography carrier 26 can be easily changed.

The bottom of the hollow portion is provided with an open / close valve 5.
A collection pipe to the fraction collector 13 is provided through the same. Further, a heater coil 56 is provided to heat the chromatography carrier 26.

Further, the separation and purification unit obtained by combining the electrophoresis separation unit and the chromatography separation unit shown in FIG.
The description will be made by substituting the electrophoresis separation unit 7 and the chromatography separation unit 6 with the above. Therefore, in the above description, the same reference numerals as those in FIG. 1 denote the same parts, and therefore, please refer to FIG.

The electrophoretic carrier 32 is made of polyacrylamide or agarose gel. Generally, those having a small molecular size and those having a large total charge are eluted first. The sample liquid is sent to the sample liquid introduction nozzle 50 using the automatic dispenser 2. The electrophoresis carrier 3
An overlay liquid 54 is layered on 2 in order to suppress the diffusion of the sample liquid. It is preferable that an electrically neutral substance having a high viscosity and a high specific gravity, for example, a 20% sucrose solution is applied on the gel surface on the electrophoresis carrier 32 in a layered manner.

The components eluted and separated from the gel end of the electrophoresis carrier 32 are passed through the ion exchange tube 17 using the eluent 1 sent from the collection solution introducing tube 36, and are passed through the collection solution switching valve 43. Thus, when a specific component is contained, it is sent to the chromatography carrier 26. When the specific component is not included, the specific component is discharged from the collection liquid discharge tube 37, and the discharge amount is adjusted by the valve opening of the valve 53 .

The recovered liquid containing the specific component sent to the chromatography support 26 is combined with the washing liquid and the desorbing liquid introduced from the recovery liquid introduction tube 36 after the specific component is bound to the chromatography support 26. It is eliminated and a specific component is separated. The detector 11 detects the separation of the specific component.
Confirm with.

In this case, although not necessary for the protein, when the polynucleotide is purified by performing double-stranded hybridization, it is heated by the heater coil 56,
When a double-stranded hybrid is formed at 42 ° C. and the single-stranded hybrid is formed, it may be heated to about 90 ° C. and eluted.

Next, an example of separation and purification by the separation and purification apparatus of this embodiment will be described. [Separation and purification example 1] This separation and purification example was carried out using the separation and purification apparatus of [Example 1]. A mixed solution of commercially available conalbumin (molecular weight: 77000-87000) and ovalbumin (molecular weight: 45000) (concentration: 10 mg / m2)
L) was prepared, and it was confirmed by electrophoresis that the protein of the impurity was present in the prepared solution.

An agarose gel on which an anti-conalbumin monoclonal antibody was immobilized was used as the chromatography carrier 26, and a polyacrylamide gel was used as the electrophoresis carrier 32. Antibody immobilization was performed on CNBr-activated agarose gels by conventional techniques.

Next, this gel was packed in a gel storage column 28, and the gel holding cap 27 was used to prevent the gel from leaking. The gel holding cap 27 is covered with a nylon mesh to prevent gel leakage. The polyacrylamide gel of the electrophoresis carrier 32 has a total concentration (%
A gel formed by polymerizing a disk gel composed of a gel having 3.5% of T) and a gelt having T = 6.5% in a glass tube 31 was used.

The gel of T = 3.5% is a gel which is desorbed from the chromatography carrier 26 and concentrates the partially purified product containing the specific component which moves. A gel with T = 6.5%
For separation, large molecules move slowly. The lower end of the gel is flat, and the surface is washed with the recovery liquid flowing from the recovery liquid introduction tube 36.

After the eluted separated components are confirmed by a detector, they are collected by a fraction collector 13. As the detector, a flow cell type spectrophotometer or a fluorometer can be used. For proteins, UV absorbance at 280 nm was suitable. 5 mL of the above protein mixed solution is pH
The mixture was diluted 1/10 with 7.5 0.1M phosphate buffer, and sent to the anti-conalbumin monoclonal antibody-immobilized column at a flow rate of 0.1 mL / min by a constant flow pump.

After the total amount has been sent out, the switching valve 4 (see FIG. 1) is switched, and the eluent 1 containing the above buffer is supplied at a flow rate of 0.
The solution was allowed to flow at 5 mL / min for 2 hours to remove ovalbumin and other impurities. Next, after lowering the position adjusting cylinder 21 until the tip of the gel cap 27 almost contacts the surface of the electrophoretic carrier 32, the switching valve 4 is switched, and the desorbed liquid 2 is discharged at a flow rate of 0.05 mL / min. Allowed to flow for minutes and desorbed. A 0.05 M glycine-hydrochloric acid buffer (pH 2.0) was used as the desorbing solution. Immediately after the detachment, a current was applied to start electrophoresis.

The detached conalbumin moves to the concentrated gel and is further separated on the separation gel. The concentrated gel,
The gel was prepared with a 0.05 M Tris-HCl buffer (pH 6.8), and the separation gel was prepared with a 0.3 M Tris-HCl buffer (pH 8.9). The applied current was a constant current of 45 mA, and protein elution was confirmed by the absorbance (280 nm) of the recovered solution. The recovered liquid is 0.1 M Tr
An is-HCl buffer (pH 8.0) was used. ELIS
Samples in which the presence of conalbumin could be confirmed by Method A were collected.

The purity of this high-purity conalbumin was determined by SDS
-Confirmed by polyacrylamide electrophoresis. It takes about 24 hours for dialysis, concentration, gel filtration, etc. in a normal operation, but in this example, it was completed in about 8 hours.

[Separation and Purification Example 2] In this separation and purification example, an example in which a polynucleotide is separated and purified using the separation and purification apparatus of [Example 2] will be described. A chromatographic carrier was prepared by covalently bonding a 5'-terminal aminohexylphosphoamidated oligonucleotide to a carboxylated dextran with a water-soluble carbodiimide. The immobilization method is described in S.I. S. Gauche, G. F. Musso, New Clay Acids Research, Volume 15, pp. 53
53-5371, (1987).

A 3.5% agarose gel was used as the electrophoresis carrier 32, and DNA was used as the oligonucleotide.
20 base single-stranded DNA chemically synthesized by a synthesizer
(5'-TGCTGCTATGCCTCATCTTC-
3 ′).

Synthetic base single-stranded DNA (5′-ACGACGATACGGAGT) complementary to this single-stranded DNA
AGAAG-3 'of those having the nucleotide sequence) of plasmid pBR322 with the addition of restriction enzyme Sau 3AI degradation fragment mixture was used as a sample solution. First, by electrophoresis,
DNA of a size corresponding to single-stranded DNA of about 20 bases
Was separated and introduced into a chromatography column by changing the flow path.
Binding was performed at 7 ° C.

In this reaction, sufficient efficiency could be ensured by introducing a very low flow rate after introduction into the column. next,
The column capturing the DNA chain is heated to 90 ° C. with a heater coil 5
6 to dissociate the double-stranded DNA, flow a heated buffer, and remove the released complementary base strand DNA by 260 n.
while confirming the absorbance at m.

Further, the recovered DNA strand was subjected to SD
A purity test was performed by S-polyacrylamide electrophoresis, and it was confirmed that the DNA had a length of 20 bases and contained almost no impurities. The time required for this purification was about 12 hours in total for the electrophoresis and the chromatography steps, which was about 1/2 of the conventional time.

As described above, in the present example and the present separation and purification example, as described above, the affinity chromatography method includes, for example, immobilizing an antigen or antibody, a receptor, or a ligand thereof for an immobilized antigen or a protein. For glycoproteins, sugar chain-specific immobilized lectins and the like can be used. In addition, it is also possible to use a reaction with a specific chemical substance or protein such as an enzyme substrate analog.

As described above, in the case of polynucleotides,
A hybridization method based on base chain complementarity can be used. It is also possible to elute a restriction enzyme-degraded fragment of the target polynucleotide chain captured by the immobilized polynucleotide chain and to purify and recover the fragment by electrophoresis.

In addition, the antigen-antibody reaction and the lectin reaction
It is reversible, and the bond can be dissociated by adding pH or a competitive reactant. Affinity chromatography has a concentration effect as well as a specific separation of a specific component. Unwanted impurities can be removed by a washing operation. A gap may be provided between the stationary phase for affinity chromatography and the stationary phase for electrophoresis so that unnecessary impurities can be easily removed by washing and solvent.

[0079]

As described above in detail, according to the present invention, there is provided a separation and purification apparatus capable of efficiently and quickly separating and purifying only specific components with high purity in accordance with sample conditions. Can be provided.

[Brief description of the drawings]

FIG. 1 is a block diagram showing a configuration of a separation and purification device according to one embodiment of the present invention.

FIG. 2 is a sectional view of a separation / purification unit in the separation / purification apparatus according to one embodiment of the present invention.

FIG. 3 is a sectional view of a separation / purification unit in a separation / purification apparatus according to another embodiment of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Eluent 2 Automatic dispenser 3 Desorption liquid 4 Switching valve 5 Pump 6 Chromatography separation part 7 Electrophoresis separation part 8a Upper electrode 8b Lower electrode 9 Waste liquid bottle 10 Temperature controller 11 Detector 12 Recovered liquid 13 Fraction collector 14 DC Power supply 15 Sample liquid introduction tube 16 Upper / lower tube for electrolytic solution 17 Ion exchange tube 18 Transfer tube 19 Upper electrolytic bath 20 Upper electrolytic bath cover 21 Position adjusting tube 21a Position adjusting tube screwing portion 22 Position adjusting tube fixing tube 22a Position Adjusting tube fixing tube screw portion 23 Chromatographic separation unit body 24a Position adjusting tube fixing member 24b Position adjusting tube fixing tube fixing member 25 Waste liquid outlet tube 26 Chromatography carrier 27 Gel holding cap 28 Gel storage column 29 Fixing member 30 Cooling water access pipe 31 Glass pipe 32 Electrophoresis carrier 33 Electrophoresis separation unit body 34 Lower electrolyte solution tank 35 Lower electrolyte solution access tube 36 Recovered solution introduction tube 37 Recovered solution outlet tube 38 Lower electrolyte solution tank bottom plate 39 Electrophoresis separation unit fixing member 40a Upper electrolyte solution 40b Lower electrolyte 41 Opening 42 Through hole 43 Storage column opening 44 Electrophoretic carrier lower holder 50 Sample liquid introduction nozzle 51 Outer cylinder 52 Inner cylinder 53 Recovered liquid switching valve 54 Overlying liquid 55 Base 56 Heater coil 57 Open / close valve

──────────────────────────────────────────────────の Continuing on the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 30/26 G01N 30/48 R 30/48 27/26 315Z (72) Inventor Sadafumi Onuma 882 Ishige, Katsuta, Ibaraki Pref. Address Hitachi, Ltd. Measuring Instruments Division (56) References JP-A-61-243353 (JP, A) JP-A-63-184050 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 30/84 B01D 15/08 B01D 57/02 G01N 27/447 G01N 30/26 G01N 30/48 JICST file (JOIS) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A sample injection means for injecting a sample into an eluent.
Is connected to a specific component in the sample for chromatography.
-Affinity chromatography captured by a carrier
Means and a crudely purified substance containing the captured specific component.
In order to release from the chromatography carrier,
Liquid sending means for sending the desorbed liquid to the chromatography carrier
From the partially purified material containing the released specific component
The specific component and the component other than the specific component are separated by a dynamic carrier.
Electrophoresis means for separation,
Provided between the body and the electrophoresis carrier,
Disposal means for discarding the sample passed through the fee carrier,
Detecting means for detecting each component after the separation;
Collection means for collecting the components, wherein the disposal means is
Move the chromatography carrier to reduce waste
A separation and purification device characterized by being adjustable . Wherein said electrophoretic carrier, separation and purification apparatus according to claim 1, characterized by using a gel polymer. 3. The storage medium according to claim 1, wherein the chromatography carrier is a storage container.
The ram is packed and the storage column is removable
The separation / purification apparatus according to claim 1, wherein: 4. The electrophoretic carrier is filled in a storage container.
The storage container is detachable.
The separation and purification device according to claim 1 . 5. The chromatography carrier, comprising: an antigen;
Antibodies, lectins, receptors, enzymes, separation and purification apparatus according to claim 1, wherein any one of the polynucleotide is characterized in that it is fixed. 6. Injecting a sample into an eluent,
Injecting the liquid into the chromatography support;
The specific components in the injected sample are subjected to the chromatography
Capturing by a carrier, and the chromatography
Pass through the chromatographic support by moving the support
Discarding the cleaned sample;
-Disposal of the sample is stopped by moving the carrier, and
Forming a sample introduction path to the electrophoresis carrier
To the chromatography support by injection of the desorbing solution
Releasing the captured components;
Direct current is applied to the carrier, and the sample introduced is separated for each component
And detecting each of the separated components.
And recovering the specific component.
A separation and purification method characterized by the following .