WO2007099937A1 - Method of filtering solution of protein, etc. and apparatus therefor - Google Patents

Abstract

A method of filtering a solution of protein, etc., in which with respect to proteins of high molecular weight, separation and other treatments can be performed easily, with reduced cost and securely; and an apparatus therefor. Providing a filter enclosing container including a mountable container furnished with a mounting aperture directly or indirectly fittable to one or multiple lines of nozzles through which a gas having been suction discharged by a suction discharge unit capable of suction discharge of designated gas among two or more types of gases can pass, and including a filter enclosed in the mountable container, the filter partitioning so as to allow retention of a liquid in the state of being fitted to nozzles on the side of mounting aperture and being capable of separation of given substance through passage of the liquid, the method comprises the introduction step of introducing therein a solution containing given substance through the mounting aperture; the fitting step of fitting the filter enclosing container having the solution introduced therein to relevant nozzle; and the pressurization filtration step of separating the given substance by carrying out gas discharge from the nozzle into the mounted filter enclosing container.

Description

 Specification

 Protein filtration solution processing method and apparatus

 Technical field

 TECHNICAL FIELD [0001] The present invention relates to a solution filtration treatment method for proteins and the like and an apparatus thereof (a device for automating pretreatment of plasma or serum samples for mass spectrometry and the method thereof), and particularly, plasma or serum samples derived from living bodies such as humans In order to apply a high molecular weight protein such as a mass spectrometer to a mass spectrometer, as a pretreatment, a solution filtration treatment method for proteins and the like that automates a pretreatment for removing or separating a predetermined protein that may interfere with mass spectrometry and its It relates to equipment.

 Background art

 [0002] In the post-genomic era, research on the structure and function (proteomics) of the total protein (proteome) that functions in the living body is prospering worldwide. In particular, the human plasma (plasma) proteome has been considered important as a research tool to find clinical markers useful for disease diagnosis and treatment monitoring. Plasma is a major diagnostic sample, and as a target for human clinical proteome analysis, vigorous clinical markers are being searched for using a mass spectrometer. Plasma contains various “plasma proteins”. Its concentration shows a wide distribution depending on the type of plasma protein. Serum albumin contained in the blood in the amount of 30-50 mg in 1 mL of blood is the most abundant. In addition, blood contains many different immunoglobulins (IgM, IgG, IgA, IgE, IgD). When analyzing trace proteins in plasma, proteins that are contained in large amounts such as albumin interfere with the detection and quantification of trace proteins. Need to be removed by the method. There is an increasing expectation for an analysis method using a mass spectrometer that searches for a novel clinical marker using human plasma as a sample (Non-patent Documents 1, 2, and 3).

[0003] Currently, as an effective method for analyzing human plasma, analysis by a mass spectrometer is mainly performed, and a major protein such as albumin 'IgG (immunoglobulin) in plasma that interferes with mass spectrometry is commercially available. After removing using a column, etc., mass analysis of the passing fraction A method of analyzing with a meter is used (Patent Document 5).

[0004] By the way, column methods such as antibody columns have problems of contamination due to carry-over and difficulty in increasing the amount of analysis and the number of analyzes (throughput). Furthermore, in the clinical field dealing with human plasma, the problem of carry-over and the handling of binoculars have become serious problems, and the column method is not necessarily an appropriate method.

 [0005] On the other hand, as an improvement measure, in order to remove the albumin and IgG, there is a method in which the container containing the human plasma sample is rotated (centrifuged) or shaken to perform the treatment. . In this case, there is a problem that the device structure or the device structure becomes complicated, and it is difficult to manufacture an automation device, and there is a possibility that equipment costs or operation costs may increase.

 [0006] In addition, one of the inventors of the present application has already used a chip in which a chip enclosing a filter is fitted to a dispensing chip mounted on a nozzle, or a chip enclosing a filter is dispensed. A technology has been filed or patented for the technology of attaching to a nozzle without using a tip (Patent Documents 1, 2, 3, and 4). However, these methods are suitable for separating organisms and the like because they are dispensed and filtered by being fitted to a dispensing tip and sucked and discharged from the atmosphere. It may not be appropriate for the separation of such fine substances. This is because for fine substances such as protein molecules, it is necessary to use a filter with a small pore diameter, so that a large pressure is required to allow the liquid to pass through the filter. It is necessary to be able to compress the gas in the cylinder with a small volume. However, when the capacity of the cylinder is increased, there is a problem that it may be difficult to perform suction and discharge of a minute amount of liquid with accurate quantitativeness. In addition, it was necessary to perform treatment in various gases or under various conditions for the same type of gas.

 Disclosure of the invention

 Problems to be solved by the invention

[0007] Therefore, the present invention has been made to solve the above problems, and a first object of the present invention is to provide a simple device, inexpensively and reliably, such as albumin, immunoglobulin, etc. High It is to provide a versatile or versatile solution filtration method for proteins and the like and a device capable of performing various treatments including separation or removal of molecular weight proteins and the like. In addition, the second purpose is that there is no labor and effort to consistently automate the processing of separation or removal of high molecular weight proteins such as albumin and immunoglobulin, and high reliability and reproducibility. The present invention provides a solution filtration method for protein and the like and a device therefor. Furthermore, a third object is to provide a solution filtration method for proteins, etc., and an apparatus for the same, which can accurately and efficiently carry out treatments such as separation or removal of proteins, etc. of polymer materials such as albumin and immune globulin. That is.

 [0008] Patent Document 1: Japanese Patent No. 3630493

 Patent Document 2: WO96Z29602

 Patent Document 3: Japanese Patent Application 2005-144728

 Patent Document 4: # 112005-3251

 Patent Document 5: US Pat. No. 6,660,149

 Non-Patent Document 1: Beckman Coulter, Inc. catalog (PROTEOME LAB IGY, PROTEOME PARTITIONING SOLUTIONS, (BR-9976A)), US, published 20 05

 Non-Patent Document 2: Latest Mass Spectrometry for Life Sciences Kodansha Kenichi Harada, Published 2002

 Non-Patent Document 3: Post-genome 'mass-spectrometry chemistry coterie Published by Toshimitsu Niwa 2002

 Means for solving the problem

[0009] The first invention is directed to one or a plurality of nozzles through which a gas sucked or discharged by a suction discharge unit capable of sucking or discharging a specified gas from two or more kinds of gases can pass. Partitioning the inside of the container so that liquid can be stored in a state of being attached to the nozzle on the side of the opening for mounting, An introduction step of introducing a solution containing the predetermined substance into a filter enclosure having a filter capable of separating the predetermined substance by passing the liquid through the mounting opening, and the filter into which the solution is introduced Enclosed volume A mounting step of mounting a container directly or indirectly on the nozzle, and a pressure filtration step of separating the predetermined substance by discharging the gas from the nozzle to the mounted filter container. It is a solution filtration treatment method for proteins and the like.

 [0010] Here, the "two or more kinds of gases" include, for example, the atmosphere, nitrogen, carbon dioxide, oxygen, argon, or a gas obtained by mixing two or more gases selected at their medium strength in various proportions. is there . Moreover, even if the components of these gases are the same, they belong to different types of gases if their pressure, temperature, etc. are different. For example, the case of compressed gas and rare gas for the same gas.

 [0011] A "filter" allows a liquid to pass through a number of penetrating holes or voids of a predetermined size (pore diameter or average void diameter or length) to separate a given substance in the liquid. A penetrating porous solid for separation and capture or removal. The shape is, for example, a block shape, a thin film shape, a thin plate shape, a film shape, or a plate shape. Filter materials include rubber, silicone, cellulose (including regenerated cellulose), nylon, polyethersulfone and other fibrous materials, resin, metal, ceramics, etc., gels, porous bodies, penetrating pores There are quality and water content. Examples of the thin film carrier include an ultrafiltration membrane that performs ultrafiltration of proteins. Here, the “block shape” includes a cylindrical shape, a prismatic shape, a spherical shape, and the like.

Here, the “predetermined substance” means a molecular weight having a size that can be separated by a predetermined size of the pores or voids of the filter (for example, about twice the size of the pores or voids of the filter) For example, genetic materials such as nucleic acids, biological materials containing biopolymers or low molecules such as proteins, sugars, sugar chains, peptides, pigments, or cells as biological materials , Viruses, plasmids and the like. In the case of protein, for example, in the case of 1000 amino acid molecular force, the pore or void size of the filter is, for example, several nm to several tens nm.

[0013] Alternatively, the predetermined substance may be a predetermined particle capable of adsorbing the substance. Here, “predetermined particles” are micro-sized, for example, 1 μm force and several hundreds of meters, and can be retained by adsorbing a nanosized material, for example, lnm force with a size of 10 nm. As a result, for example, a filter having a pore diameter of about 0.1 μm to several hundred μm can be used. It is possible to separate the predetermined particles and filter the suspension by passing the suspension as a solution in which the predetermined particles are suspended.

 [0014] "Directly or indirectly attachable to the nozzle" means that the mounting opening and the nozzle are directly connected by fitting or screwing or the like, or the nozzle has air permeability. In some cases, the nozzle is indirectly attached to the attachment opening through a nozzle attachment member to be attached, such as a chip or an adapter. The “tip” has a large-diameter pipe and a thin-diameter pipe formed in communication with the large-diameter pipe to be thinner than the large-diameter pipe. It has an opening, and the small diameter tube has a mouth that allows liquid to flow in and out by suction and discharge of gas, and includes, for example, a dispensing tip. The nozzle is mounted by, for example, inserting the nozzle into the mounting opening and fitting the nozzle into the mounting opening.

 The “mountable container” refers to a container that has at least a mounting opening that is mounted on or can be mounted on the member used for suction and discharge, and that can store a liquid therein. Mountable containers include tip-shaped containers. The “chip-shaped container” is a mountable container, and has a mouth portion through which a liquid can be put in and out by suction or discharge of the gas in addition to the mounting opening portion. The “filter enclosure” is a container in which a filter is enclosed in the mountable container, and the filter can store liquid in a state in which the filter is mounted on the nozzle on the mounting opening side. It is provided so as to partition the inside of the container.

[0016] When the mountable container is a chip-shaped container, the filter-enclosed container is called a filter-enclosed chip. The filter is cut between the opening for mounting and the mouth so that the liquid can be stored in the state where the nozzle is mounted on the side of the mounting opening, and by passing the liquid. The predetermined substance can be separated. The tip-shaped container is not limited to having a typical tip shape such as a large diameter tube and a small diameter tube. Further, for example, when the chip-like container has a thick tube and a thin tube, the filter is accommodated in, for example, a portion corresponding to the thick tube or a portion corresponding to a transition portion between the large tube and the thin tube. It is. The volume of the chip-like container is preferably capable of handling a liquid of, for example, about several / z 1 to several hundred 1 or more. Also, the wearable container is integrally formed, You may make it form so that it can divide into two or three. The mouth portion is not necessarily limited to 1, and there may be a plurality of mouth portions. There may be a plurality of thin tubes. The outer diameter of the thin tube may be the same as that of the thick tube so that it can be fitted to the container, and only the inner diameter may be formed thinner than the thick tube. In this case, a plurality of thin tubes may be perforated.

 [0017] The material of the mountable container is preferably a translucent material in order to enable optical observation. Examples of the material of the mountable container include polyethylene, polypropylene, polystyrene, acrylic resin, glass, metal, metal compound, and the like. The size is, for example, a size capable of accommodating several microliters of force and several milliliters of liquid in a thin tube. In the pressurizing step, it is preferable that the mouth portion is located above a container provided outside or inserted into the container.

 [0018] For the "introduction", for example, a dispensing tip is attached to a nozzle capable of suction and discharge, the liquid is sucked from the container, the liquid is transferred to the storage position of the filter-sealed container, and the mounting is performed. This is performed by discharging the liquid into the opening.

 [0019] It should be noted that the volume of the space in which the liquid can be stored in the mountable container in which the filter is sealed is, for example, several microliters or several milliliters. In the case of this example, the liquid storage portion provided outside the filter enclosure can suck the liquid of several microliters or several milliliters into the narrow tube through the mouth of the thin tube. Must be able to be accommodated.

[0020] The solution introduced in the introduction step includes, for example, plasma, and the predetermined particles suspended in the solution include, for example, albumin, immunoglobulin, a2-macroglobulin, Major proteins contained in plasma 'serum such as 1-lipoprotein or _a 1 acidic glycoprotein can be adsorbed and retained.

[0021] Here, plasma refers to a liquid component obtained by removing solid components, ie, blood cells that are cells, from blood. This plasma contains a wide variety of proteins from high to low molecular weight. Proteins contained in protein plasma include immunoglobulins, albumin (molecular weight 66,000 Da), α 1-MG (molecular weight 33,000 Da), j8 2 -MG (molecular weight ll, 800 Da). Furthermore, the immunoglobulins include IgM (molecular weight 900,000 Da), IgG (molecular weight 160,000 Da), IgA (molecular weight 150,000 Da), and the like. Where "Da" is the atomic mass unit da. Represents Noreton is about 1.660 X 10- ²⁷ kg.

 [0022] The second invention includes a desorption step of detaching the filter enclosure from the nozzle cover after the pressure filtration step, and a different type of filter from the filter enclosed in the filter enclosure. A reintroduction step of introducing the filtered solution through the mounting opening into another filter enclosure, and the filter enclosure into which the solution has been introduced into the nozzle. A solution filtration treatment method for proteins and the like, which has a mounting step of mounting directly or indirectly on the filter, and a pressure filtration step of discharging the gas from the nozzle to the mounted filter enclosure.

 [0023] In a third invention, in the introduction step or the reintroduction step, the predetermined substance in the solution is capable of adsorbing or adsorbing the protein, or the predetermined substance in the solution. The protein is a solution filtration method for proteins or the like having predetermined particles, wherein the filter has pores or voids having a pore diameter smaller than the size of the predetermined substance.

[0024] The filter in the reintroduction step is, for example, an ultrafiltration membrane capable of separating proteins or a solution filtration treatment method for proteins such as a microfiltration membrane capable of separating the predetermined particles.

 Here, “ultrafiltration membrane” refers to a porous membrane having a pore diameter in the range of lnm to lOOnm. A “microfiltration membrane” is a membrane used to filter solutes or particles of about 0.01 μm power / zm. The “predetermined particles” are as described in the first invention.

 [0026] A fourth invention is a solution filtration method for proteins, etc., wherein the introduction step, the reintroduction step or the pressure filtration step has a detection step of detecting the amount of liquid introduced into the filter enclosure.

[0027] In a fifth aspect of the invention, one of the two or more types of gases is air, and the introducing step is performed using a dispensing tip attached to the nozzle for suction and discharge of air. Then, the liquid is introduced into the filter enclosure, the attaching step attaches the filter enclosure after the dispensing tip is detached from the nozzle, and the pressure filtration step is a gas other than the atmosphere. Is a solution filtration treatment method for proteins and the like, which is performed by switching to the atmosphere and discharging it to the filter enclosure through the nozzle. [0028] Here, the "dispensing tip" is a tip-shaped container that does not contain a filter capable of separating the predetermined substance, and can suck and discharge liquid into the dispensing tip. It is used for dispensing and transferring liquids.

 [0029] A sixth invention is a solution filtration method for protein or the like, wherein the gas other than the atmosphere is compressed nitrogen gas.

Here, the pressure of the nitrogen gas in the nitrogen gas compression storage is, for example, 10 kgf / cm ^2, and the pressure to be applied to the filter through the nozzle after being reduced in pressure is, for example, 0.1 kgf / cm ² it is to several kgf / cm ^2. Here, “kgf” represents weight kilogram.

 [0031] Further, it is preferable that the pressure value be variable by providing a pressure regulator. Moreover, it is preferable to provide a pressure reducing valve. In this way, the pressure value can be changed based on the purpose of processing, such as when separating predetermined particles as a predetermined substance, or when directly separating proteins, or the structure of the filter sealing container. Optimal processing can be performed.

 [0032] According to a seventh aspect of the present invention, there is provided a solution filtration of protein or the like having a substitution step of replacing the gas remaining in the nozzle with another gas over the pressure filtration step, the introduction step, or the reintroduction step. It is a processing method. Here, the replacement is performed, for example, by supplying a new gas to the nozzle having the residual gas or repeating the suction and discharge.

 [0033] According to an eighth aspect of the present invention, there is provided a suction / discharge portion capable of sucking or discharging two or more kinds of gases having a medium force and a gas sucked or discharged by the suction / discharge portion. One or a plurality of nozzles, a mountable container having a mounting opening that can be mounted directly or indirectly on the nozzle, and enclosed in the mountable container, on the mounting opening side. A filter enclosure having a filter that partitions the inside of the container so that liquid can be stored in the state of being attached to the nozzle, and capable of separating a predetermined substance by passage of the liquid, and a member that can be attached to the nozzle; A solution filtration treatment apparatus for proteins, etc., having a container group having a plurality of containers capable of storing a solution, a specimen, or a reagent, and a moving unit that can move the nozzle head relative to the container group. is there.

[0034] Here, the "suction or discharge of two or more kinds of gas with a specified force" is performed by switching the supply of the gas to the nozzle by a switching unit using a valve. " Examples of the “member that can be attached to the nozzle” include an attachable container having the attachment opening, a filter enclosing container (including a filter enclosing tip), a dispensing tip, an adapter, and the like.

 [0035] The ninth invention relates to the structure or processing of the nozzle or a member that can be attached to the nozzle, and the type, amount, pressure, frequency, time, or position of the gas for suction or discharge of the nozzle force. A protein having a control unit that is controlled based on the type of the solution or the substance contained therein, its concentration, its amount, or the substance condition consisting of the coordinate position including the storage position of the solution, and the processing content of the solution, etc. It is a solution filtration device.

 [0036] Here, the type of substance includes the nature and size of the substance. Further, the amount of the liquid is the amount of liquid in the filter-sealed container or the dispensing tip, or the amount of liquid in the container, and is detected by, for example, a liquid amount detecting means described later. For example, if the control unit detects the amount of the pre-filter solution, mixture solution or suspension stored in the filter enclosure, the solution, mixture solution or suspension solution is detected according to the amount. The desired concentration can be achieved.

 [0037] In a tenth aspect of the invention, the suction / discharge section includes an air suction / discharge section capable of sucking and discharging air through the nozzle, a gas supply section for supplying a gas of a type other than the atmosphere, A protein solution filtration apparatus having a gas supply unit and a switching unit that switches between the air suction and discharge unit and connects to the nozzle. The atmospheric suction / discharge unit is preferably driven by a one-way mechanism. Here, the “one-way mechanism” is a mechanism that can easily perform the forward operation with a small force, and the reverse operation requires a large force and is difficult to execute. For example, there is a mechanism that uses a ball screw or a sliding screw to change the rotational drive to a linear movement of a nut portion that is screwed into the ball screw or the sliding screw.

[0038] More specifically, the atmospheric suction / discharge unit includes, for example, a cylinder and a plunger built in the cylinder so as to be slidable along the axial direction of the cylinder. The ball screw or the sliding screw is coupled to a nut portion that is screwed into a ball screw or a sliding screw having an axial direction parallel to the cylinder, and the ball screw or the sliding screw is rotated in both forward and reverse directions so that the inside of the cylinder is axially driven. Reciprocate along the direction. In this case, the plunger Even if the pressure by the compressed nitrogen gas is strong, the plunger connected to the nut portion hardly moves along the axial direction due to the pressure, so that the influence of the introduction of the compressed nitrogen gas is prevented. Can do.

 [0039] The nozzle has an inlet / outlet through which gas flows in and out at the lower end, communicates with the air suction / discharge unit at the upper end, and communicates with the gas supply unit at the side. The gas inlet is formed. As a result, the structure of the entire apparatus can be simplified, and even when a gas flows in from the inlet, the plunger hardly moves along the axial direction due to the pressure of the gas and maintains a high pressure. It can be introduced as is.

 [0040] An eleventh aspect of the invention is a solution filtration apparatus for proteins, etc., wherein the gas other than the atmosphere is compressed nitrogen gas, and the gas supply unit is a nitrogen gas compression reservoir.

 [0041] In a twelfth aspect of the invention, the predetermined substance is the predetermined substance in the solution is a protein or predetermined particles capable of adsorbing or adsorbing the protein, and the filter includes the predetermined substance. This is a solution filtration apparatus for proteins and the like having pores or voids having pore sizes smaller than the size. Here, the “protein” is, for example, plasma albumin, immunoglobulin, a 1-MG or 13 2 -MG as described above.

 [0042] A thirteenth aspect of the present invention is a solution filtration apparatus for proteins, etc., provided with a desorption part for removing a member attached to the nozzle of the nozzle head from the nozzle.

 [0043] Here, the member attached to the nozzle refers to "a member that can be attached to the nozzle" attached to the nozzle. For example, the attachable container attached to the nozzle, a filter-enclosed container, or a dispenser. Chip, adapter, etc.

 [0044] A fourteenth aspect of the invention is a solution filtration apparatus for proteins, etc., provided with a liquid amount detection means for detecting the amount of liquid in the filter-enclosed container or the container.

 [0045] In the fifteenth aspect of the invention, the control of the type, amount or pressure, number of times, time or position of the suction or discharge of the nozzle force by the control unit is performed by separating or removing a predetermined protein, or It is a solution filtration processing apparatus for proteins and the like, which is performed based on the processing content of either concentration of a protein solution or buffer replacement.

The invention's effect [0046] According to the first invention or the eighth invention, a suction / discharge part capable of sucking or discharging a designated gas from two or more kinds of gases is provided, and the liquid containing a predetermined substance is sucked. The suction / discharge unit sucks and discharges air to the filter enclosure through the nozzle, attaches the filter enclosure to the nozzle, and again discharges and pressurizes the gas by the suction / discharge unit. The predetermined substance can be separated by filtering using the filter.

 [0047] Therefore, by using the nozzle and the suction / ejection unit, it is possible to perform processing of a minute amount of liquid and processing that requires high pressure using the same apparatus, so that a predetermined object in the solution is obtained. Processing with quality filters can be consistently automated. Therefore, since this process can be performed without human intervention, a highly reliable process can be performed. Furthermore, since it is possible to select an appropriate gas, filter, and conditions according to the type of the predetermined substance, it is possible to perform processing under optimum conditions according to the predetermined substance.

 [0048] According to the second invention or the thirteenth invention, after separating the target predetermined substance, the filter enclosure used therein is removed, and the filtrate is reintroduced into a new filter enclosure, It is attached and pressure filtration is performed. Therefore, for example, when performing a separation treatment of rare useful proteins in plasma, once a high-molecular-weight protein such as albumin is separated as a predetermined substance, the substance having a purpose different from that of the substance is used. Rare protein can be isolated. In addition, for example, a substance containing a target substance once adsorbed on a predetermined particle is separated from the predetermined particle, then the substance is dissociated from the predetermined particle, and then the target substance is further separated. Processing can be performed automatically.

 [0049] According to the third invention or the twelfth invention, the predetermined substance is a predetermined particle and protein capable of adsorbing protein, and a filter capable of separating them is used. Therefore, for example, if the liquid is allowed to pass through after being adsorbed to predetermined micro-sized particles but the predetermined particles are not passed through, and separated using a filter, it is relatively low. The target protein can be separated by applying pressure. This makes it possible to separate proteins easily, inexpensively and reliably. In addition, complex processes can be consistently automated.

[0050] According to the fourth invention or the fourteenth invention, the amount of liquid introduced into the filter enclosure is measured. By knowing, for example, by detecting the amount of liquid introduced into the filter enclosure and the amount of liquid in the filter enclosure after filtration, the concentration of the liquid can be known, and the reliability of the process Can be increased.

 [0051] According to the fifth invention or the tenth invention, the suction and discharge of liquid to the dispensing tip attached to the nozzle is performed by switching between the atmosphere and a gas other than the atmosphere to perform suction and discharge or discharge. In addition to being able to be used for discharge, it is possible to perform pressure filtration using a higher pressure gas into a filter enclosure mounted on a nozzle. Therefore, it is possible to consistently perform the filter processing from introduction of the liquid containing the target substance into the filter-sealed container to pressure filtration without human touch, and the reliability is high.

 [0052] According to the sixth invention or the eleventh invention, air and compressed nitrogen gas are used as the gas. Therefore, by using properly a gas suitable for introducing the solution into the container and a gas suitable for pressure filtration, the treatment can be performed efficiently and inexpensively with a simple structure. Nitrogen gas is chemically stable and can be easily obtained at low cost.

[0053] According to the ninth invention or the fifteenth invention, regarding the suction or discharge of the nozzle force, the kind, amount, or pressure, the number of times, the time or the position of the gas is changed to a substance such as the structure of the nozzle. Control is performed based on conditions and processing contents. Therefore, pressure filtration can be performed under conditions suitable for the structure of the nozzle and the processing content. In addition, appropriate processing of the protein solution can be performed by controlling the suction or discharge of the nozzle according to the content of the separation or removal of the specified protein or the concentration or buffer replacement of the protein solution. Can do.

[0054] According to the seventh invention, when gases having different specific gravity are sequentially passed or introduced into the nozzle, if there is a gas remaining in the nozzle, it may be difficult to introduce new gas into the nozzle. . Therefore, the treatment can be smoothly advanced by removing the residual gas before the treatment with a new gas is started.

 BEST MODE FOR CARRYING OUT THE INVENTION

[0055] Next, a protein solution filtration apparatus according to an embodiment of the present invention will be described with reference to the drawings. FIG. 1 (a) shows an overall perspective view of the protein solution filtration apparatus 10 according to the present embodiment. The apparatus 10 includes a main body 11, a nitrogen gas compression reservoir 12 that compresses and stores nitrogen gas, and a pipe line that connects a nozzle (described later) provided in the main body 11 and the nitrogen gas compression reservoir 12. And 13. The main body 11 has a substantially rectangular parallelepiped casing 14, and two side surfaces of the casing 14 are partly cut off, and a lid 14a formed of a transparent plate is fitted into the casing 14 from the outside. The filtration unit 15 can be seen through. A handle 14b is attached to the lid 14a.

 [0056] Further, on the side surface of the housing 14, an operation keyboard 14c is provided on which a user gives instructions, inputs various data, and displays necessary data. However, the main body 11 is not limited to this form. For example, the main body 11 may be used in a state where it is installed in a refrigeration facility without providing the housing 14 of the main body 11.

 FIG. 1 (b) shows a state where the lid 14 a is removed from the casing 14 of the main body 11.

 2, FIG. 3, and FIG. 4 are a perspective view, a plan view, and a side view schematically showing the filtration processing unit 15, respectively.

 In short, the filtration processing unit 15 includes a plunger 17 slidable inside, and performs suction or discharge of the atmosphere, and a plurality of stations (8 stations in this example) arranged in the X-axis direction in the figure. Underneath the cylinder 18 and the cylinder 18 are a nozzle head 16 having eight nozzles 19 each communicating with the cylinder 18, and members that can be attached to the nozzles 19 (including filter enclosing chips described later) ), A container group 22 having a plurality of containers that can store various solutions, specimens, or reagents, and a movement that enables the nozzle head 16 to move in the Y-axis direction and the Z-axis direction in the figure relative to the container group 22 Part (shown in FIGS. 6 and 7). By this moving portion, the nozzle head 16 can move the nozzle 19 to each container of the container group 22 so as to cover the entire area of the container group 22.

[0059] The opening at the upper end of each nozzle 19 communicates with the cylinder 18, and the lower end of the nozzle 19 has an inlet / outlet through which gas can flow in and out, and slightly above the side surface of the nozzle 19, Eight branch pipes 35 communicating with the nitrogen gas compression reservoir 12 through the pipe 13 and the valve 34 have gas inlets connected to each other. The branch pipe 35 or the pipe 13 is flexible. By forming with a rubber tube or the like having a property, the influence of the movement of the nozzle head 16 is absorbed by being deformed with the movement of the nozzle head 16.

 [0060] The valve 34 corresponds to the switching unit, and is, for example, an electromagnetic valve, a stop valve, a gate valve, a check valve, or a cock. As a result, when the atmosphere is sucked and discharged, the influence is not given to the pipe line 13. When an electromagnetic valve is used as the valve 34, a connection cutoff instruction may be given by a control unit (not shown), and a connection or cutoff instruction may be given based on the detection result of the pressure sensor 21.

 The eight nozzles 19 provided in the nozzle head 16 are formed so that the chip-like container can be attached thereto. Each nozzle 19 is hydrodynamically connected to the cylinder 18 and the nitrogen gas compression reservoir 12 described above via the conduit 13, the valve 34 and the branch conduit 35. The valve 34 corresponds to the switching unit, and switches the connection with the nitrogen gas compression store 12. The valve 34, the pipe line 13, and the nitrogen gas compression reservoir 12 are provided separately from the nozzle head 16 and cannot be moved integrally with the nozzle head 16.

 [0062] An elongated plate-like attachment / detachment provided with a plurality of (in this example, eight) through-holes through which the nozzle 19 penetrates below the connection portion of the nozzle 19 with the branch pipe 35. The portion 20 is provided so as to be movable together with the nozzle head 16 so as to be vertically movable with respect to the nozzle head 16. The inner diameter of the through hole is slightly larger than the penetrating nozzle 19 but has an inner diameter smaller than the outer diameter of the upper end opening of the tip-like container to which the nozzle 19 is fitted.

 [0063] On one side surface of the nozzle head 16, there are eight pressure sensors 21 for detecting the pressure inside each nozzle 19, and there is an interval between adjacent nozzles 19 at a position corresponding to each nozzle 19. They are provided at the same interval. Further, as will be described later, the nozzle head 16 is supported by the main body 11 by being coupled to the arms of the moving unit (not shown) on the other side surface of the nozzle head 16.

[0064] The container group 22 includes an ultrafiltration filter tube (Vivspin 2 manufactured by Sartorius) 23 as eight filter-enclosed chips, and a liquid from a filter unit 23 provided below the filter unit 23. Drain tank 24 for receiving various reagents, such as bicarbonate Dispensing tips 25 arranged in two rows for a common buffer and a spare dispensing, for example, a container 26 containing 50 mM ammonium bicarbonate buffer solution, and a limitation as another type of filter enclosing tip. Contains an outer filtration filter unit (Vivaspin 6 from Sartorius) 27, an adapter cap 28 for pressurizing the filter unit 27, and a suspension of albumin and IgG adsorption resin and PBS buffer solution as the predetermined particles. Yes Container 29, for example 70 Sample container 30 containing one or more plasma solutions, Dispensing tips 31 for stirring or non-adsorptive transfer arranged in two rows, 2 for plasma dispensing and spare Dispensing tips 32 arranged in rows are arranged on a processing stage 33 shown in FIG. 4 in a matrix of 12 rows x 8 rows, 8 rows each corresponding to each nozzle 19 of the nozzle head 16. Shi It is also of the.

 [0065] Further, the container group 22 can be moved along the X-axis direction of Fig. 2 to detect the liquid level in the ultrafiltration filter unit 23 and the ultrafiltration filter unit 27. It has a liquid level sensing unit 36 equipped with a CCD image sensor as a means.

 As shown in FIG. 4, the ultrafiltration filter unit 23 has a tip-like container 38 as a mountable container and a filter 39 enclosed in a chip-like container 38. The chip-like container 38 is formed with a thick tube 42 and one or more thin tubes 41 provided on the lower side of the thick tube 42 and having an inner diameter smaller than the inner diameter of the thick tube 42. The transition portion between the thick tube 42 and the thin tube 41 has a step, and the filter 39 is provided above the step. The upper end of the thick tube 42 has a mounting opening 42a that can be mounted indirectly to the nozzle 19 through a pressurizing adapter 40, and the tip of the thin tube 41 is liquidized by discharging the nitrogen gas. It has a mouth part 41a that can flow out. The filter 39 is partitioned so that liquid can be stored between the mounting opening 42a and the mouth portion 41a while being mounted on the nozzle 19 while being mounted on the mounting opening 42a side. ing. Reference numeral 43 denotes a cylindrical member having the same outer diameter as that of the thick tube 42 in which the thin tube 41 is formed.

As shown in FIG. 4, the ultrafiltration filter unit 27 has a chip-like container 45 and a filter 46 sealed inside the chip-like container 45. The chip-like container 45 includes a thick tube 47, one or more thin tubes 48 provided below the thick tube 47 and formed narrower than the inner diameter of the thick tube 47, and the thick tube 47 and the thin tube 48. Have a step at the transition between The filter 46 is provided above the step. The upper end of the thick tube 47 has a mounting opening 47a that can be mounted indirectly to the nozzle 19 via a pressurizing adapter cap 28, and the tip of the thin tube 48 is liquidized by discharging the nitrogen gas. It has a mouth part 4 8a that can flow out. The filter 46 is partitioned between the mounting opening 47a and the mouth 48a so that liquid can be stored in a state of being mounted on the nozzle 19 on the mounting opening 47a side. The

Reference numeral 50 denotes a columnar member in which the thin tube 48 is bored, and has a size substantially the same as the outer diameter of the thick tube 47. Reference numeral 49 denotes a container whose upper end can be fitted to the columnar member 50 and receives liquid discharged from the narrow tube 48.

 In FIG. 4, reference numerals 51, 52, 53, 54, 55, 56 are containers for accommodating the respective dispensing tips provided on the processing stage 33.

[0070] FIG. 5 shows a gap between the nozzle 19 in which an ultrafiltration filter unit 27 and an ultrafiltration filter unit 23 are attached to the nozzle 19 and an air supply port 57 of the nitrogen gas compression reservoir 12. The conceptual diagram of the pneumatic path to be connected is shown. The pipe line 13 includes a pressure regulator 58 that adjusts the pressure so that the maximum pressure becomes 2.7 kgf / cm ² by adjusting the gas amount, a pressure reducing valve 59, and a pressure gauge 60. The pipe 13 is connected to a branch pipe 35 via a valve 34, and the branch pipe 35 is connected to a cylinder 18 and a filter unit 27, which are an air suction / discharge mechanism having a dispensing function.

 [0071] Note that the protein solution filtration apparatus 10 according to the present embodiment has an information processing unit having a CPU (not shown), various memories, a keyboard, various switches, an input means such as a mouse, a display means, or the like. Provided to be connectable to the information processing unit. The information processing unit includes the discharge amount, pressure, frequency, time, or position of the nozzle in the structure of the nozzle, a member attached to the nozzle or a filter-enclosed chip, a solution to be processed, or the solution. A control unit is provided for controlling based on the material condition such as the type, concentration or amount of the substance, or the coordinate position including the storage position, and the processing content for the liquid.

Subsequently, the mechanism of the main body 11 will be described in detail with reference to FIG. 6 and FIG. As described above, the main body 11 has the nozzle head 16 and the container group 22 provided below.

 The nozzle head 16 has eight nozzles 19 and eight cylinders 18 communicating with the nozzles 19 (not shown in FIGS. 6 and 7). Is provided with a plunger 17 slidable therein. The eight plungers 17 are attached to the plunger drive plate 69 at their upper ends. The plunger drive plate 69 is connected to a nut portion 64 that is screwed into the ball screw 65, and in conjunction with the nut portion 64 that moves up and down as the ball screw 65 rotates, the eight plungers 17 Are moved up and down all at once.

 The ball screw 65 is pivotally supported by an inverted L-shaped ball screw support member 68 attached to a head mounting plate 70 to which the nozzle head 16 is attached. A position sensor 96 is provided on the horizontal plate of the ball screw support member 68, and a rod 95 provided on the plunger drive plate 69 so as to protrude upward is provided between the light emitting element and the light receiving element of the position sensor 96. The position of the plunger 17 is detected by blocking.

 The ball screw 65 is rotationally driven by a timing belt (not shown) spanned between the motor shaft 63 of the motor 62 also attached to the head mounting plate 70 and the upper end portion 66 of the ball screw 65. Is done.

 [0076] As shown in Fig. 7 (a), the nozzle 19 on the upper side of the detachable portion 20 is provided with a hole 21a, which communicates with the pressure sensor 21 via a rubber tube 21b. 19 pressure can be measured. In addition, a connecting portion 35a connected to the branch pipe 35 for introducing the compressed nitrogen gas is provided in a portion of the nozzle 19 opposite to the side where the hole 21a is provided.

[0077] As shown in the side view of Fig. 7 (b), the head mounting plate 70 to which the nozzle head 16 is mounted is provided with two guide members 71a, 72a in the vicinity of both edges thereof, It is provided so as to be slidable in the vertical direction by engaging with two guide pillars 73 and 74 extending in the vertical direction (Z-axis direction). The head mounting plate 70 is connected to the head vertical drive plate 77 via springs 75 and 76. When the lower end of the nozzle 19 comes into contact with the bottom of the container or the like, the distance between the head mounting plate 70 and the head vertical drive plate 77 is narrowed and can be detected by a sensor (not shown), and the reaction at the time of contact. Can absorb power. The head vertical drive plate 77 has both edges Two guide members 71b and 72b are provided in the vicinity of the portion, and are respectively supported so as to be slidable in the vertical direction by engaging with two guide columns 73 and 74 extending in the vertical direction. The head upper / lower drive plate 77 is attached to a nut portion 79 that is screwed with a ball screw 78 extending in the vertical direction at the center thereof. The ball screw 78 is rotationally driven by a motor 80 via a coupler 81. The motor 80 is provided on a rack 82, and the rack 82 is provided on a frame 83. The guide pillars 73 and 74 and the ball screw 78, and therefore the nose head 16 as a whole, in the left-right direction (Y-axis direction) with respect to the container group 22 or the housing 14 in the drawing of FIG. It is provided in a frame 83 that is movable.

 Further, along the Y-axis direction and apart from the frame 83, the large vertical plate 86 and the small vertical plate 89 for supporting the track are attached to the casing 14 so as to sandwich the frame 83. It is fixed. As shown in FIGS. 6 and 7, the large vertical plate 86 is provided with rails 87 and 88 along the Y-axis direction, and the wall portion of the frame 83 that faces the large vertical plate 86 is provided. Pieces 84 and 85 are provided at corresponding positions outside 83b and slidably engaged with the rails 87 and 88. Further, the small vertical plate 89 is provided with a rail 90 along the Y-axis direction, and a piece 91 is provided at a corresponding position inside the wall portion 83a facing the small vertical plate 89 of the frame 83. It is provided and slidably engages with the rail 90.

 Further, the large vertical plate 86 is provided with a ball screw 93 extending in the Y-axis direction, and a nut portion 94 is screwed into the ball screw 93, and the frame 83 is formed of the nut portion. It is linked to 94. Further, as shown in FIG. 7A, the ball screw 93 is rotationally driven by a motor 92.

 Subsequently, the operation of the protein solution filtration apparatus 10 will be described with reference to FIG.

 In step S1, the plasma is stored in advance at −80 ° C. (protein concentration: 60-8 Omg / ml) at 500 μl before being subjected to the treatment, and the sample is thawed at room temperature.

[0081] Next, in step S2, the plasma is filtered with a filter having a pore diameter of 0.22 μm (for example, a Millipore Millex GV 0.22 μm syringe filter) using, for example, a dispensing tip attached to the nozzle. A filter-enclosed tip (not shown) enclosed in a tip is attached to the nozzle 19 and subjected to pressure filtration by sucking and discharging using the cylinder 18, etc., and the filtrate 100-400 1 is stored in the sample container 30 of the container group 22. Container 29 contains rubmine and IgG adsorption resin (Amersham Biosciences) and PBS buffer solution, and about 9-10 ml of 50 mM ammonium bicarbonate buffer solution is contained in container 26 of each lane. deep.

 [0082] In step S3, each nozzle 19 of the nozzle head 16 is moved in the Y-axis direction to the position of the dispensing tip 32 for lml, and then moved in the Z-axis direction to thereby move the dispensing tip. 3 Insert the nozzle 19 into the upper end opening of 2, and attach it by pressing it. Next, the nozzle head 16 equipped with the dispensing tip 32 is moved to the position of the container 30, and the branch pipe 35 and the nozzle 19 are connected using the valve 34. Using the cylinder 18, the plasma fluid in the container 30 to 70 1 is aspirated. With the plasma fluid sucked, the nozzle head 16 is moved along the Y-axis direction to the container 29 containing the albumin and IgG adsorption resin and PBS solution, and moved in the Z-axis direction. Then, the plasma is discharged into the container 29 using the cylinder 18 and mixed. Here, in the container 29, 1.2 ml of the albumin IgG adsorption resin is suspended in advance in 2.8 ml of PBS solution.

 [0083] The albumin 'IgG adsorption resin contained in the container 29 is added to 1.2 ml of albumin' IgG adsorption resin (manufactured by Amersham, the amount of slurry is 4 ml) in another filter unit 27 in advance. Then, 2.8 ml PBS solution is added and, for example, centrifugal filtration (1000 g) is performed five times, or the filter unit 27 is attached to the nozzle 19 and the nitrogen gas compression reservoir 12 is used. The resin was washed and equilibrated with PBS in advance by pressure filtration.

[0084] In step S4, the nozzle head 16 in which the plasma, albumin 'IgG adsorption resin, and PBS solution stored in the container 29 are stored and the dispensing tip 32 is attached to each nozzle 19 is The tip 32 is moved in the Y-axis direction to the position of the container 51, which is the original storage position of the tip 32. In the storage position, the dispensing tip 32 is detached by moving the detachment portion 20 downward. The nozzle head 16 to which the dispensing tip 32 has been detached moves in the Y-axis direction to the position of the dispensing tip 31 having a capacity of 4 ml, and the nozzle 19 is moved to the upper end of the dispensing tip 31 in the Z-axis direction. Insert the dispensing tip 31 by inserting it into the tube and pushing it down further. Next, the nozzle head 16 moves to the container 29, and further, the dispensing tip 31 is moved and inserted in the Z-axis direction into the container 29. In this state, at the room temperature (20 to 25 ° C.), the dispensing tip 31 is used to connect the cylinder 18 to the nozzle 19 through the branch line 35 by the valve 34 and repeat the suction and discharge. To stir. Thus, albumin and IgG in plasma are adsorbed on the resin.

 [0086] In step S5, 4 ml of the suspension in which the resin in which the albumin and IgG in the plasma are adsorbed and suspended in the container 29 is suspended is aspirated by the dispensing tip 31 using the cylinder 18. The sample is transferred in the Y-axis direction, placed on the ultrafiltration filter unit 27, and discharged into the ultrafiltration filter unit 27. Next, the nozzle head 16 is moved along the Y-axis to the position of the containers 54 and 53 that accommodate the dispensing tip 31, and the dispensing tip 31 is detached by the detaching portion 20 there.

 [0087] Next, the nozzle head 16 is moved to the position of the pressure adapter cap 28 of the ultrafiltration filter unit 27, the nozzle head 16 is lowered in the Z-axis direction, and the adapter cap 28 is moved to the nozzle. Attach to 19. Next, the nozzle head 16 is moved to the position of the ultrafiltration filter unit 27, the nozzle head 16 is lowered along the Z axis, and the nozzle 19 is moved through the adapter cap 28 to Attach at the upper end of the ultrafiltration filter unit 27 containing the suspension.

Next, the valve 34 is switched to release the connection between the nozzle 19 and the cylinder 18 which is the gas suction / discharge unit, and the nitrogen gas compression reservoir 12 and the conduit 13 are connected. Connecting. Here, by adjusting the pressure regulator 58 and using the pressure reducing valve 59, the suspension in the ultrafiltration filter unit 27 is subjected to pressure filtration (O.lkgf / cm ² ) for 3 minutes, The albumin and IgG adsorbed resin and PBS are separated by a filter 46. These times or discharge amounts are instructed by the control unit. Here, store 2.8 ml of the separated resin-powered IgG elution fraction. Before attaching the adapter cap 28 and the ultrafiltration filter unit 27 to the nozzle 19 of the nozzle head 16, the valve 34 is switched and the nitrogen gas compression reservoir 12 and the conduit 13 are connected. It is preferable to supply nitrogen gas into the nozzle 19 so that the atmosphere in the nozzle is replaced with nitrogen gas.

[0089] In step S6! /, After the resin separation, the detachable part 2 is removed from the nozzle head 16. After the ultrafiltration filter unit 27 is detached using 0, the nozzle head 16 is moved to the position of a new dispensing tip 31 and moved in the Z-axis direction so that the tip of the nozzle is separated. Install dispensing tip 31 by pushing it into tip 31. Further, the valve 34 is switched to connect the cylinder 18 and the nozzle 19 which are atmospheric suction / discharge sections. The nozzle head 16 is transferred to a container 49 provided on the lower side of the ultrafiltration filter unit 27, and 2.8 ml of the filtrate is sucked and transferred to the position of the ultrafiltration filter unit 23. Housed in the outer filtration filter unit 23. In the ultrafiltration filter unit 23, a filter 39 made of a 3 kDa ultrafiltration membrane is enclosed. Next, the valve 34 is switched again, and the nozzle 19 and the nitrogen gas compression reservoir 12 are connected via the pipe line 13.

[0090] Next, in step S7, the ultrafiltration filter unit 23 is concentrated under pressure of 2.7 kgf / cm ² under a temperature condition of 4 ° C to about 0.2 m. At that time, by detecting the liquid level of the liquid stored in the ultrafiltration filter unit 23, the liquid volume is detected by the liquid volume detecting means, and the liquid volume is detected. Pressure is applied accordingly to achieve the concentration. Thereafter, 2.6 ml of 50 mM ammonium bicarbonate solution is added from the container 26, and pressure filtration is performed again. A total of 4 pressurizations are performed, including 3 times of concentration buffer exchange work to this bicarbonate solution. This removes the non-volatile solutes in the solvent that adversely affect analysis (LC MS) by mass spectrometry (MS) using liquid chromatography (LC), leaving only volatile components. In addition, by adjusting the pH to about 9 for trypsin digestion, it is adjusted to the pH required for the enzyme digestion process in the next step.

 [0091] Subsequently, the protein measurement result in the human plasma processed by the protein solution filtration processing device according to the embodiment of the present invention and the same processing are performed, such as installation of the filter-enclosed chip on the centrifugal device. Therefore, stability and repeatability can be improved without centrifugal filtration and manual operation by comparing the measurement results with centrifugal filtration, i.e., centrifugal filtration. The following shows that Takatsuki processing can be performed by this processing equipment.

[0092] The process of step S5 in FIG. 8, that is, the process of removing albumin and IgG from human plasma is based on the manual operation using a centrifuge and the pressure filtration process according to the embodiment of the present invention. Compared. The plasma after the treatment was fragmented into peptides with proteolytic enzymes and measured with a high performance liquid chromatography (LC) mass spectrometer (MS).

[0093] The comparison of the plasma filtration process using the protein solution filtration apparatus according to the embodiment of the present invention and the process result when performed by centrifugal filtration and the manual operation associated therewith is repeated reproduction of the measurement result data. This was done by evaluating the repeatability. That is, three times the results of measuring the protein in plasma treated with the protein solution filtration apparatus according to the embodiment of the present invention with the above-mentioned LS-MS apparatus were overlapped by the alignment method described later, and the peak For each signal peak obtained by the detection process, the relative standard deviation indicates how much the measurement value changed in three measurements, and the result is calculated in the same way. The results were compared with the six measurements obtained by centrifugal filtration and manual processing.

 [0094] When overlaying the results of three measurements, the time axis was aligned using the I-OPAL method (international application PCT / JP2004 / 004621) in order to align the elution time of the LC, which fluctuates with each measurement. The signal peak was detected by the peak detection program of the I-OPAL analysis tool. (The standard substance used as a marker when aligning the time axis in the I OPAL alignment program was added before the sample measurement. Avian egg white lysozyme and three peptide molecule ion signals derived from a-1-antitrypsin present in human plasma.

 [0095] I The correlation coefficient (cosine correlation), which is an index of coincidence when 3 to 6 measurement results obtained by the OPAL method are overlaid, is calculated on the third floor using the apparatus according to the embodiment of the present invention. The measurements were 0.9233, 0.9251, and 0.9445, respectively, while the six measurements that had undergone centrifugal filtration and manual pretreatment were between 0.8816 force and 0.9489, and the average was

0.9269. This indicates that the measurement result obtained by the apparatus according to the embodiment of the present invention is sufficiently reproducible to perform the overlay processing of the measurement data.

[0096] Further, as a result of performing peak detection processing after superposition, the number of signal peaks obtained was 25229 from three measurements using the apparatus according to the embodiment of the present invention, and was manually performed. It was 27475 from 6 measurements, though it was pretreated by. For each obtained signal peak, the relative standard deviation (RSD) indicates how much the peak intensity for each measurement before overlaying by alignment is changed, and the protein according to the embodiment of the present invention. 15.8% when using a solid solution filtration device, and 13.7% when using centrifugal filtration and manual work. In addition, the number of signal peaks in which the RSD exceeded 20% was 6292 when using the apparatus according to the embodiment of the present invention, and 5548 when using the centrifugal filtration and manual work. Yes, all were less than a quarter.

 [0097] From the above results, it is determined that the result of processing the sample using the apparatus according to the embodiment of the present invention has substantially the same reproducibility as that processed by centrifugal filtration and manual work. Is done. FIG. 9 (a) and FIG. 9 (b) show the case where the treatment is performed using the apparatus according to the embodiment of the present invention and the case where the pretreatment is performed by centrifugal filtration and manual operation, respectively. The frequency distribution of RSD values is shown when the signal intensity fluctuations are expressed in relative standard deviation (RSD) by superimposing the results of measurements 6 to 6 times. The horizontal axis is the RSD value divided in 10% increments, with the leftmost region corresponding to 0 to 10% and the rightmost region corresponding to more than 50%. The vertical axis represents the number of signal peaks whose RSD values are in the corresponding range. It can be seen that approximately the same number of distribution shapes can be obtained as those using the device according to the embodiment of the present invention and centrifugal filtration and manual operation. If the device according to the embodiment of the present invention is used, centrifugal filtration and manual operation are obtained. It has been shown that the processing can be performed without the need for the user, and the user's effort can be eliminated or alleviated with a simple device structure and scale.

 The above example is not limited to this example, but the force described only for albumin and IgG as proteins. For example, other immunoglobulins may be used. Further, the filter-enclosed chip is not limited to the illustrated shape, and may have various shapes as described above.

For example, as shown in FIG. 2, only a valve for connecting to and shutting off from the compressed nitrogen gas reservoir is provided corresponding to each nozzle 19, but each branch of the branch pipe 35 is provided. A solenoid valve may be provided for each, and connection and disconnection may be controlled based on the detection result of each pressure sensor corresponding to each cylinder. If an abnormality is detected in the pressure in the nozzle, the valve of the corresponding branch is shut off, the supply of compressed gas to the nozzle is stopped, and an accident is prevented or wasteful processing is performed. You can cancel. Further, the nitrogen gas compression reservoir may be prepared in the number of nozzles connected to each nozzle. Industrial applicability [0100] The present invention relates to a protein filtration treatment method and apparatus. The present invention relates to fields that require handling of in vivo proteins such as immune system, plasma and serum, for example, industrial fields, agricultural fields such as food, agricultural products, and marine products processing, pharmaceutical fields, hygiene, health, immunity, diseases , Medical fields such as genetics, and science fields such as chemistry or biology. The present invention is particularly effective when a series of processes using a large number of reagents and substances are successively executed in a predetermined order.

 Brief Description of Drawings

 [0101] FIG. 1 is a perspective view showing an appearance of a protein solution filtration apparatus according to an embodiment of the present invention.

 FIG. 2 is a perspective view schematically showing a filtration treatment unit of the protein solution filtration apparatus according to the embodiment of the present invention.

 FIG. 3 is a plan view showing a filtration treatment unit of the protein solution filtration apparatus according to the embodiment of the present invention.

 FIG. 4 is a side view schematically showing a filtration treatment unit of the protein solution filtration treatment apparatus according to the embodiment of the present invention.

 FIG. 5 is a conceptual diagram of a pneumatic path of the protein solution filtration apparatus according to the embodiment of the present invention.

 FIG. 6 is a front view showing the mechanism of the main body of the protein solution filtration apparatus according to the embodiment of the present invention.

 FIG. 7 is a side view showing the mechanism of the main body of the protein solution filtration apparatus according to the embodiment of the present invention.

 FIG. 8 is a flowchart of processing according to the embodiment of the present invention.

 FIG. 9 is a frequency distribution diagram of relative standard deviations showing the results of processing using the protein solution filtration processing apparatus according to the embodiment of the present invention, centrifugal filtration, and manual processing. Explanation of symbols

[0102] 10 Protein filtration equipment

 12 Nitrogen gas compression storage (nitrogen gas supply unit, suction discharge unit)

13, 35 pipeline Filter processing section

Nozzle head

Cylinder (Atmospheric suction / discharge unit)

Noznore

Container group

7 Ultrafiltration filter unit (filter enclosure) Valve (switching part)

Claims

The scope of the claims  [1] Can be directly or indirectly attached to one or more nozzles that can pass the gas drawn or discharged by the suction / discharge unit that can suck or discharge the specified gas from two or more gases A mountable container having a suitable opening for opening, and the inside of the container is sealed so that the liquid can be stored in a state of being attached to the nozzle on the side of the mounting opening. An introduction step of introducing a solution containing the predetermined substance into the filter enclosure having a filter capable of separating the predetermined substance by passing the liquid through the mounting opening;  A mounting step of mounting the filter-enclosed container introduced with the solution directly or indirectly to the nozzle;  A solution filtration treatment method for proteins, etc., comprising: a pressure filtration step of discharging the gas from the nozzle to separate the predetermined substance with respect to the filter enclosure mounted.  [2] After the pressure filtration step, a desorption process for detaching the filter enclosure from the nozzle, and another filter enclosure having a different type of filter from the filter enclosed in the filter enclosure A re-introduction step of introducing the solution filtered in the pressure filtration step through the mounting opening, and a mounting step of directly or indirectly mounting the filter-enclosed container into which the solution has been introduced to the nozzle; The method for filtering a solution of protein or the like according to claim 1, further comprising a pressure filtration step of discharging the gas from the nozzle to the attached filter enclosure.  [3] In the introduction step or the reintroduction step, the predetermined substance in the solution is a protein and predetermined particles capable of adsorbing or adsorbing the protein, and the filter is larger than a size of the predetermined substance. The method for filtering a solution of a protein according to claim 1 or 2, wherein the method has a pore or a pore having a pore diameter.  [4] In the introduction step, the reintroduction step, or the pressure filtration step, the detection step of detecting the amount of liquid introduced into the filter enclosure is provided in claim 1 or claim 3! The protein filtration method for protein according to any one of the above. [5] One of the two or more types of gases is the atmosphere, and the introducing step is suction of the atmosphere. And by using a dispensing tip attached to the nozzle to introduce a liquid into the filter sealing container, and the attaching step removes the dispensing tip from the nozzle and then removes the filter. A sealed container is mounted, and the pressure filtration step is performed by switching a gas other than the air to the air through the nozzle while switching to the air. 5. The solution filtration method for proteins and the like according to claim 4.  [6] The protein solution solution filtering method according to [5], wherein the gas other than the atmosphere is compressed nitrogen gas.  [7] The method according to any one of claims 1 to 6, further comprising a replacement step of replacing the gas remaining in the nozzle with another gas in the pressure filtration step, the introduction step, or the reintroduction step. The method for filtering a solution of a protein according to any one of the above.  [8] A suction / discharge section capable of sucking or discharging two or more gases with the specified medium force,  One or more continuous nozzles through which the gas sucked or discharged by the suction / discharge section can pass,  A mountable container having a mounting opening that can be mounted directly or indirectly on the nozzle, and a liquid that is sealed in the mountable container and mounted on the nozzle on the mounting opening side. A filter-sealed container having a filter that partitions the inside of the container so that the liquid can be stored and can separate a predetermined substance by passing the liquid;  A container group having a plurality of containers capable of storing a member, a solution, a specimen, or a reagent that can be attached to the nozzle;  A solution filtration processing apparatus for proteins, etc., having a moving part that allows the nozzle head to move relative to the container group. [9] Regarding the suction or discharge of the nozzle force, the type, amount or pressure, number of times, time or position of the gas are included in the nozzle, the structure of a member that can be attached to the nozzle, the solution to be processed, or there. 9. The control unit according to claim 8, further comprising: a control unit configured to control a substance based on a substance condition including a coordinate position including a type of substance, a concentration thereof, an amount thereof, or a storage position of the solution, and a processing content of the solution. Protein solution processing equipment Place.  [10] The suction / discharge section includes an air suction / discharge section capable of sucking and discharging air through the nozzle, a gas supply section for supplying a gas of a type other than the atmosphere, the gas supply section, 10. The protein etc. solution filtration processing apparatus according to claim 8, further comprising a switching unit that switches between an air suction and discharge unit and connects to the nozzle.  11. The protein solution solution filtering apparatus according to claim 10, wherein the gas other than the atmosphere is compressed nitrogen gas, and the gas supply unit is a nitrogen gas compression reservoir.  [12] The predetermined substance is a predetermined particle in the solution that adsorbs or adsorbs protein and the protein, and the filter has a pore diameter smaller than the size of the predetermined substance. The apparatus for filtering a solution of protein or the like according to claim 8, which has pores or voids.  [13] The protein solution solution treatment apparatus according to any one of claims 8 to 12, wherein a desorption portion for removing a member attached to the nozzle of the nozzle head from the nozzle is provided.  [14] The protein solution filtration process according to any one of claims 8 to 13, wherein a liquid amount detection means for detecting a liquid amount in the filter enclosure or in the container is provided. apparatus.  [15] Regarding the suction or discharge from the nozzle by the control unit, the type, amount or pressure, number of times, time or position of the gas is controlled by separating or removing a predetermined protein or concentrating or removing a protein solution. The protein etc. solution filtration processing apparatus according to claim 8, which is performed based on any of the processing contents of buffer replacement.