EP1468279A1 - Procede et dispositif correspondant et moyen de separation destine a la separation de particules par une electrophorese a ecoulement libre - Google Patents

Procede et dispositif correspondant et moyen de separation destine a la separation de particules par une electrophorese a ecoulement libre

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Publication number
EP1468279A1
EP1468279A1 EP20030729400 EP03729400A EP1468279A1 EP 1468279 A1 EP1468279 A1 EP 1468279A1 EP 20030729400 EP20030729400 EP 20030729400 EP 03729400 A EP03729400 A EP 03729400A EP 1468279 A1 EP1468279 A1 EP 1468279A1
Authority
EP
European Patent Office
Prior art keywords
particles
charge
ffe
medium
separating
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP20030729400
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German (de)
English (en)
Inventor
Christoph Eckerskorn
Hans-Jörg Grill
Gerhard Weber
Peter Weber
David Yost
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tecan Trading AG
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Tecan Trading AG
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Filing date
Publication date
Application filed by Tecan Trading AG filed Critical Tecan Trading AG
Publication of EP1468279A1 publication Critical patent/EP1468279A1/fr
Withdrawn legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44795—Isoelectric focusing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44769—Continuous electrophoresis, i.e. the sample being continuously introduced, e.g. free flow electrophoresis [FFE]

Definitions

  • the invention relates to a method and an apparatus for performing the method and separating means for separating particles in a free-flow electrophoresis device, in particular for separating cells or complexes of the same cell components, organelles or biomolecules, or of parts or complexes thereof, according to the The preamble of independent claims 1, 18, 26 and 27.
  • Free-flow electrophoresis is one of the most promising technologies for the separation of all possible particles [cf. Krivanova L. & Bocek P. (1998) "Continuous Free-Flow Electrophoresis” Electrophoresis 19: 1064-1074].
  • the FFE is the technology of choice for the pre-division of complex protein samples in relation to their different pl value (degree of ionization).
  • the FFE separates cells based on the electrophoretic mobility of the cells.
  • the corresponding principles have already been thoroughly characterized [cf. e.g. Bondy B., Bauer J., Seuffert I. and Weber G.
  • ASECS Antigen-specific electriophoretic cell separation
  • a generic FFE method is described in the international patent application PCT / EP01 / 14408. It is an electrophoresis se method in a device with a separation space through which a separation medium flows, which is delimited by a base and a cover and by these two spacers which are spaced apart from one another.
  • this FFE device comprises a pump for feeding the separation medium, which enters the separation space via media feeds and leaves it again via outlets.
  • the FFE device also includes electrodes for applying an electrical field in the separation medium and sample application points for adding a mixture of particles or analytes to be separated and fractionation points for removing particles separated by the FFE in the separation medium. These separated particles can be sent for analysis or further preparative processing.
  • two or more separate delivery channels of a metering pump are connected to the separation space in the region of the fractionation outlets near the electrodes in order to add medium.
  • U.S. Patent 5,948,231 discloses compounds, methods and devices for performing ultra-fast binding assays in capillary electrophoresis or in other electro-separation techniques such as e.g. in free flow electrophoresis.
  • the object of the present invention is to further improve the preparative or analytical isolation of particles, such as cells, organelles and biomolecules (e.g. protein complexes) and the like, or of bioparticles or biopolymers in the FFE.
  • particles such as cells, organelles and biomolecules (e.g. protein complexes) and the like, or of bioparticles or biopolymers in the FFE.
  • this object is achieved, according to a first aspect, by a free-flow electrophoresis method for separating particles, in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof, with the features of independent claim 1 is proposed.
  • this object is achieved - according to a second aspect - by a free-flow electrophoresis device for carrying out the method for separating particles, in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof , with the features of independent claim 18 is proposed.
  • this object is achieved - according to a third aspect - by separating means for separating particles, in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof in a free-flow electrophoresis device, according to the features of independent claims 26 and 27, respectively.
  • particles are considered to be e.g. all particulate mass units, preferably of biological origin, such as cells, viruses, cell organelles, vesicles, cell nuclei and membranes, and parts or aggregates thereof; also biomolecules, such as lipids, proteins, DNA, RNA and sugar, and complexes or aggregates of biomolecules, such as lipoproteins, glycoproteins, lipopolysaccharides, etc.
  • biomolecules such as lipids, proteins, DNA, RNA and sugar, and complexes or aggregates of biomolecules, such as lipoproteins, glycoproteins, lipopolysaccharides, etc.
  • These particles also include organic and inorganic molecules, such as pharmaceuticals, polymers and the like.
  • a method according to the invention for separating such particles on the basis of the selectively modified surface charge - in particular for separating cells or cell components, organelles or biomolecules, or parts or complexes thereof, using the free-flow electrophoresis technique comprises the following advantages: • Magnetic beads are not suitable for the preparative separation of particles; in addition , only a single type of particle can be isolated with magnetic beads. Magnetic bead applications can therefore be replaced by the FFE according to the invention, if positive and negative charged carriers are used at the same time.
  • the FFE according to the invention allows simultaneous isolation of at least two types of particles with maximum efficiency, because the individual electrophoretic mobility of the particles is increased significantly and selectively.
  • living cells can be isolated qualitatively and quantitatively or on a preparative or analytical scale thanks to the specificity of the antibodies used and the separating power of the FFE.
  • the proposed free flow electrophoresis (FFE) device enables different separation techniques to be carried out with different and selective separation parameters.
  • the proposed FFE method enables not only the isolation but also the selective depletion of particles from a particle mixture, e.g. a depletion of abundant proteins (e.g. separation of serum albumin from body fluids, e.g. blood plasma).
  • a depletion of abundant proteins e.g. separation of serum albumin from body fluids, e.g. blood plasma.
  • Fig. IC are equipped with three epitopes of a second type
  • FIG. 3A the two epitopes of the first type are negatively charged, which makes the net surface charge of the cells strongly negative, and in FIG. 3B the three epitopes of the second type are positively marked, which makes the net surface charge of the cells strongly positive;
  • FIG. 4 shows a basic diagram of a device according to the invention for the simultaneous separation of two charge-marked cell types
  • Fig. 5 is a schematic representation of usable according to the invention
  • FIG. 6 shows schematic top views of devices according to the invention, wherein:
  • 6A and 6D show a CHIEF device with a linear pH gradient and a homogeneous field
  • 6B and 6E show a PZE device with a homogeneous pH value and a homogeneous field
  • Figures 6C and 6F show a CITP device with a pH gradient and a field gradient
  • Figure 1 shows cells to be separated as an example of particles 1, 1 ', 1 "to be separated. These cells can be equipped without specific epitopes (see FIG. 1A). Such cells have a relatively low negative net surface charge and are very difficult to separate in the FFE. However, if the cells have epitopes of a first type 2 '(FIG. 1B) or a second type 2 "(FIG. IC), these cells can be labeled epitope-specifically with charge-carrying molecules or charge carriers.
  • the net Surface charge of particles can be changed selectively by binding charged binding molecules to these particles. This binding can be based on an antigen-antibody interaction.Other interactions which can be used for the inventive modification of the net surface charge of particles for coupling binding molecules to the separating particles also include receptor-ligand, enzyme-substrate and protein-protein interactions, including chelation or binding based on general molecular interactions, be it ionic or based on Van der Waals forces or hydrogen bonds , but a covalent bond can also be used for this purpose.
  • relevant cells can be extracted from body fluids (eg disseminated tumor cells from blood, sputum, ascites, urine, lavage etc.) or from tissue homogenates (eg solid tumors in the kidney, thymus) etc.) or organelles from cell homogenates (eg calciosomes, coated vesicles, endosomes, endopiasmatic reticulum, Golgi cisterns, lysosomes, peroxisomes and mitochondria) and proteins from proteomes or expression systems.
  • body fluids eg disseminated tumor cells from blood, sputum, ascites, urine, lavage etc.
  • tissue homogenates eg solid tumors in the kidney, thymus
  • organelles from cell homogenates eg calciosomes, coated vesicles, endosomes, endopiasmatic reticulum, Golgi cisterns, lysosomes, peroxisomes and mitochondria
  • FIG. 2 shows an epitope-specific antibody and two monomeric or polymeric charge carriers or “charge labels” (cf. “CL” in FIG. 5) in the form of cations 4 or anions 5.
  • a charge carrier 4, 5 is a at a given pH -Value ionized molecule. It can be anionic or cationic, monomeric or polymeric. With the help of such charge carriers 4, 5, the net surface charge of particles can be changed in a targeted manner. Charged particles such as beads can also be used as charge carriers.
  • anionic charge carriers 5 are: polyglutamic acid (PGA); anionic or derivatized proteins such as albumin; anionic polysaccharides such as heparin or alginic acid, polyaspartic acid, polyacrylic acid and polyamic acids with a net negative charge at a usable pH (for example in the range from pH 4 to 10).
  • Anionic polymers with a molecular weight of 500 to about 500,000 Daltons are preferred.
  • Several monomeric or polymeric charge carriers can also be bound to a specific binding molecule in order to further develop the net surface charge of the particles to be separated.
  • Examples of preferred cationic charge carriers 4 include homopolymeric or copolymeric proteins with a preferred molecular weight of 500-5000000 daltons; quaternary ammonium compounds with between 1% and 10% nitrogen content (without counter ion).
  • Commercially available quaternary ammonium polymer compounds include, for example, the products MERQUAT.RTM. (Calgon, Pittsburgh, PA, USA), CELQUAT.RTM. (National Starch and Chemical Corp., Bridgewater, NJ, USA), GAFQUAT.RTM.
  • binding molecule 3 Such an antibody (cf. FIG. 2) is referred to below as binding molecule 3 or “binding molecule”.
  • the bond between a binding molecule 3 and a particle 1 ′, 1 ′′ and the bond between a charge carrier 4, 5 and the binding molecule 3 can be covalent or non-covalent (eg adsorptive, on Van der Waals forces or hydrogen bonds, on one Biotin-streptavidin interaction, etc.)
  • the binding molecule 3 can be, for example, an antibody (cf. FIG.
  • FIG. 3 shows cells to be separated with corresponding epitopes 2 ', 2 ".
  • the two epitopes of the first type 2' are negative in FIG. 3A and the three epitopes of the second type 2" are positively marked in FIG. 3B.
  • the net surface charge of the marked cells is therefore greatly changed, so that these cells behave differently with their specifically changed net surface charge in the FFE.
  • the net surface charge of the cells is preferably changed such that a cell type in a separation medium 8 of a device according to the invention against the cathode 9 and the other cell type moves against the anode 10, as shown in the schematic diagram of Figure 4.
  • the two groups of charge-modified particles 7 ', 7 "and the two charge-modified cell types 7', 7” move away essentially simultaneously of the unmarked or non-charge-modified background cells 7, which move essentially in the middle (or at least at a great distance from the electrodes 9, 10 in the separation medium 8 of the device according to the invention.
  • the arrow indicates the main flow direction of the separation medium 8
  • the various, separate cell types are collected and collected in a collecting area 11 he forwarded for further use. Collecting takes place in a manner known per se via discharge openings arranged in a row.
  • the resolution of the FFE is essentially determined by the migration behavior of the particles 7, 7 ', 7 "and the size and number of the outlet openings.
  • the separation space 14 comprises the space for the separation medium 8 and the two lateral areas 19 near the electrodes for the the focusing cushion 12, 13 formed by the cushion medium 20 (cf. also FIG. 7).
  • one focusing pad 12, 13 is preferably used a likewise flowable, electrically highly conductive separating material, provided in the immediate vicinity of the electrodes 9, 10. Due to its high electrical conductivity, this focusing pad 12, 13 prevents the charge-modified particles 7 ', 7 "from being able to diffuse all the way to the respective electrode.
  • a concentration of the charge-modified particles occurs at the boundary between the separation medium 8 and the pad medium 20 7 ', 7 "as shown in Fig. 7B. Separating medium 8 and cushion medium 20 preferably flow in the same direction.
  • FIG. 5 shows a schematic illustration of separating agents which can be used according to the invention in a possible arrangement and interaction with a particle to be separated.
  • the charge-modified particle 7 ', 7 “here consists of a protein P to which a chain of histidine molecules H (a so-called” His tag ”) has been attached in a manner known per se.
  • a charged binding molecule 6 is bound to this His tag
  • This charged binding molecule 6 consists of nickel-nitrilo-tetraacetic acid (Ni-NTA), which binds to two histidine molecules, here the anionic charge carrier 5 is covalently bound to the nickel-nitrilo-tetraacetic acid, the binding molecule 3.
  • Ni-NTA nickel-nitrilo-tetraacetic acid
  • FIG. 6 shows schematic top views of devices according to the invention.
  • This free-flow electrophoresis device comprises at least one separation space 14 through which a separation medium 8 can flow.
  • This separation space is delimited by a base and a cover and by these two spacers which are spaced apart from one another.
  • This FFE device also includes a metering pump (not shown) for conveying the separating medium 8, which enters the separating space 14 via media feeds 15, 15 'and leaves it again via outlets 16.
  • the FFE device comprises electrodes 9, 10 for applying an electric field in the separation medium 8 as well as sample application points 17 for adding a mixture of particles 7, 7 ', 7 "to be separated and fractionation points 18 for removing particles separated by the FFE in the separation medium 8
  • Two separate delivery channels 15 'of the metering pump are connected to the separating space 14 in the area of the electrode hen fractionation outlets 16 'connected to add medium.
  • These two separate delivery channels of the metering pump are preferably used to form an electrically highly conductive focusing or guide cushion for the charge-modified particles 7 ', 7 "in the regions 19 of the separation medium 8 near the electrodes and are provided with a separate medium container (not shown) for providing an electrical one highly conductive pillow media.
  • the FFE separation technique of continuous isoelectric focusing is based on the different pI value of the particles to be separated.
  • This pI value corresponds to the pH of the surrounding, inhomogeneous medium, against which the particles appear neutral.
  • the FFE of particles due to their different isoelectric point enables the isolation of analytes or particles with the smallest differences in their pI values.
  • the total charge or net surface charge is at the isoelectric point p 1 (ie at the location of the separation medium 8 which has just the pH at which the number of negative and positive charges is the same for a given particle (for example a protein molecule)) of this particle is zero.
  • the focusing effect inherent in the separation medium 8 of a CHIEF device has the effect that a particle l ', l "which diffuses away from the pl automatically receives a (positive or negative) net surface charge again and is moved back to the pl by the electric field.
  • the process of continuous isoelectric focusing is particularly suitable for the micro-preparative to preparative isolation of biopolymers in general and of bioparticles whose biological function or integrity is ensured in the range of the selected pH gradient in the separation medium. This applies in the case of many representatives of viruses, bacteria, cell organelles and membrane domains, as a result of Addition of "osmotic expanders" can ensure the maintenance of an ideal osmotic pressure: By adding uncharged substances (e.g.
  • osmotic pressure is generated for a cell type, ie isomolar conditions are generated (eg for mammalian cells 250-310 mosmol).
  • FIG. 6 shows an inventive device for separating charge-modified particles 7 ', 7 "from non-charge-modified particles 7 in a separation medium 8 which flows in a separation space 14 between two electrodes 9, 10 (large arrow, 8).
  • the separation space 14 comprises the space for the separation medium 8 and two lateral regions 19 near the electrodes for the guide or focusing cushions formed with the cushion medium 20.
  • the electrodes 9, 10 are preferably designed as electrode spaces, from an electrode buffer contacted by an electrical lead 23, 23 ' 24 flow through and have a preferably semi-permeable membrane 25 in relation to the separation space 14.
  • the electrode buffer 24 is introduced into the electrode spaces via separate feed lines 26, 26 '(only partially shown in FIG. 6) and also leaves them via separate outlets 27, 27'.
  • An additional one is preferably used for circulating and, if appropriate, also for cooling the electrode buffer he pumping device (not shown) is used.
  • FIG. 6A shows such a CHIEF device with a linear pH gradient.
  • a separation medium 8 flows in a laminar movement (preferably from the bottom upwards in an inclined separation space) between the two electrodes (large arrow), is braked in the area of the outlet openings by a counterflow of separation medium (small arrow) and leaves the separation space 14 in fractions via the outlet openings.
  • a sample with three particle groups to be separated is added to the separation medium via the sample inlet and moved with the laminar flow of the separation medium. The three particle groups are continuously separated, focused and collected in separate fractions in the pH gradient, which is generated by the electric field generated between the electrodes in the separation medium.
  • the electrical conductivity of the separation medium 8 between the two preferably provided focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is relatively low and homogeneous.
  • the pH gradient is built up in the separation medium 8 between the two focusing pads.
  • FFE separation technique of continuous FFE zone electrophoresis is based on the difference in the value of the electrophoretic mobility of the particles to be separated compared to the separation medium used. FFE zone electrophoresis thus enables the isolation of analytes or particles on the basis of their different sizes and / or shapes and / or net surface charge.
  • the process of continuous FFE-PZE is particularly suitable in the case of the separation of "sensitive" bioparticles and complexes, the separation of which has to meet special requirements for the separation medium. This is particularly the case if the biological function and integrity of these particles should also be guaranteed after the separation. In these cases, special requirements apply: narrowly limited pH range of the separation media, good buffer capacity of the separation media, physiological tolerance of the buffer substances used, minimum contents of various "essential" cations and anions etc. Although the usual cell culture media are not very compatible with all techniques of Apply electrophoresis, the successful separation of cells with FFE-PZE is possible.
  • FIG. 6B shows such a PZE device, in which the samples are separated on the basis of their charge and to a lesser extent on the basis of their shape and size.
  • the electrical conductivity of the Separating medium 8 between the two focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is relatively small and homogeneous.
  • the pH value is the same in the entire separation medium.
  • the FFE separation technique of continuous isotachophoresis is also based on the difference in the value of the electrophoretic mobility of the particles to be separated.
  • the separation takes place in inhomogeneous separation media and offers a better resolution due to an inherent "focusing effect": If individual ponds diffuse away from a separated band of particles (eg proteins) during CITP, these particles enter into one Medium with different electric field strength, which accelerates or slows the particles.
  • the inherent focusing effect means that the slower or faster migrating particles find their way back into the main fraction.
  • This FFE separation technique is suitable in principle for bioparticles and cells whose separation in media without special "essential cations” (eg Mg ++ or Ca ++ ) or ' “essential anions” (eg CI " ), which are responsible for stability and Cell vitality is important, is not possible.
  • essential cations eg Mg ++ or Ca ++
  • essential anions eg CI "
  • FIG. 6C shows such a CITP device in which so-called "discrete spacers" are used.
  • discrete spacers are ions with well-known, defined mobility which crowd in the ISCO between particles to be separated and contribute to the 'separation to proceed efficiently. Precisely defined ampholytes can also be used as such discrete spacers.
  • the electrical conductivity of the separating medium 8 between the two focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is at least partially gradient-like and inhomogeneous.
  • Separating medium 8 is built up between the two focusing pads 12, 13, the areas of the two gradients essentially coinciding and both pH values and the electrical conductivity in the area of the focusing pads 12, 13 are each of different heights.
  • CHIEF and FFE-PZE or CHIEF and CITP are possible.
  • Different separation media are used at the same time in the separation area of the FFE apparatus and thus different separation parameters are used at the same time.
  • the device proposed according to the invention and the method proposed according to the invention allow continuous operating modes to be carried out, in which the separation of the particles 7, 7, ', 7 "is carried out with continuous flow of separation medium, permanent application of the electric field during the entire separation and continuous collection.
  • continuously preparative sample is added continuously with the same separation conditions
  • semi-preparative / analytical discontinuous sample addition with the same separation conditions.
  • the separating medium 8 flows continuously: the sample application takes place when the high voltage is switched off; After stopping the sample application, the medium transport is switched off or greatly reduced or reduced. The high voltage is then switched on until the sample has been separated. After a predetermined time, the voltage is switched off and the separated sample is eluted from the separation chamber with an increased medium flow and collected.
  • FIG. 7 shows the effect of the focusing pillows 12, 13, the boundary of the focusing pillow with the electrically highly conductive pillow medium 20, which runs essentially parallel to the electrode 9, 10, being indicated by dashed lines. If a focusing pad is not produced (FIG. 7A), a normal distribution (hatched) of the particles separated from a rest of a sample by means of the selectively changed net surface charge is obtained.
  • a substantially larger quantity of these particles can be removed in a higher concentration from the FFE device and used for further use;
  • the use of such a focusing pad 12, 13 represents a significant improvement -
  • the use of a focusing pad for the fine separation of identical subunits biomolecules or their complexes appears to be less advantageous.
  • the guide or focusing pads 12, 13 are thus formed by a buffer 20, which has a greatly increased electrical conductivity compared to the separation medium 8.
  • the electrophoretic separating power in the focusing pads 12, 13 decreases so much that the charge-modified particles 7 ', 7 "on the Interface between the separation medium 8 and the focusing pads 12, 13 are focused.
  • EPG epidermaal glycoprotein
  • This EPG is also expressed by tumor cells of epithelial tumors (Moldenhauer G, Momburg F, Möller P, Schwartz R, Hämmerling GJ. Epithelium-specific surface glycoprotein of Mr 34,000 is widely distributed carcinoma marker. BrJ Cancer 1987; 56: 714-21).
  • This monoclonal antibody is modified for immune FFE in two ways:
  • a charge carrier 4, 5 is directly and covalently coupled to the antibody (the binding molecule 3).
  • Streptavidin is covalently coupled to the antiserum. Freely selectable, biotinylated charge carriers 4,5 are then bound to the streptavidin.
  • citrated or heparin whole blood from patients with, for example, breast, ovarian or colon carcinoma is either used directly or prepared as follows:
  • Electrophoresis 16 92-97
  • Peroxysomes are cell organelles that carry out oxidative reactions with molecular oxygen. They generate oxygen peroxide for oxidative purposes.
  • Highly enriched peroxysomes can e.g. from rat liver via an elaborate density gradient centrifugation using the method of Lüers et al. Isolation (Lüers GH, Hartig R, Mohr H, Hausmann M, Fahimi HD, Cremer C, Völkl A. Immuno-isolation of highly purified peroxisomes using magnetic beads and continuous immunomagnetic sorting. Electrophoresis 1998; 19: 1205-210): 1 Homogenization of rat liver using methods known per se.
  • the supernatant is incubated with an antibody modified with charge carriers 4.5 against a membrane protein of the peroxysomes (e.g. PMP70; 70 kDa cytoplasmic membrane protein of peroxysomes, antibodies against the C-terminal, eleven amino acids long, cytoplasmic end).
  • a membrane protein of the peroxysomes e.g. PMP70; 70 kDa cytoplasmic membrane protein of peroxysomes, antibodies against the C-terminal, eleven amino acids long, cytoplasmic end.
  • the separation with immuno-FFE was carried out as by A. Völkl, H. Mohr, G. Weber and H.D. Fahimi described (Electrophoresis 1998, 19, 1140-1144), at a separation medium temperature of 4 ° C and at a pH of 8.0 in an electric field of 100,000 V and 100 mA and a separation medium flow rate of about 5 ml / Fraction per hour.
  • the samples were introduced into the separation chamber at about 2 ml / hour and the separated fractions were collected using a 96-channel peristaltic pump. Each individual separation attempt took about 60 to 90 minutes.
  • Example C Isolation of recombinant proteins
  • vectors which can attach a cassette of 5-10 histidines to the protein to be expressed at the 5 'or 3' end of a construct (His tags, see FIG. 5). These additional histidines are used for later purification of the expressed proteins.
  • Other vectors contain, for example, glutathione-S-transferase as an additional element. These vectors are suitable for the expression of recombinant proteins, for example in E. coli, baculovirus-infected bacteria and mammalian cells. Appropriate methods for producing recombinant proteins are known to the person skilled in the art.
  • Ni-NTA nickel nitrilotriacetic acid
  • the solid phase can be packed in preparative purifications in chromatography columns (e.g. Ni-NTA agarose), for analytical applications (e.g. Ni-NTA) magnetic particles are available.
  • the immune FFE according to the invention shown in FIG. 5, which comprises the following steps, is particularly suitable for the purification of recombinant proteins:
  • Lysis of the cells e.g. yeast, bacteria, mammalian cells
  • Binding of charge carriers 4,5 to the His-tagged proteins Variant 1: Binding of the charge carriers 4, 5 to a binding molecule 3 in the form of Ni-NTA and binding of this charged binding molecule 6 to the His tags; or
  • Variant 2 Binding of the charge carriers 4,5 to a binding molecule 3 in
  • His-tagged proteins can either now be used directly or the His-Tags are cleaved off enzymatically before further use.
  • variant 1 For variant 1: a) detachment of the chelate by known incubation with imidazole and, if necessary, renewed purification of the protein for FFE; b) Known enzymatic cleavage of the His tag with thrombin or exoprotease and, if necessary, renewed purification of the isolated protein for FFE.
  • variant 2 For variant 2: c) Detach the antibodies using methods known per se, for example by changing the salt concentration or the pH. d) Enzymatic cleavage of the His tags using methods known per se. e) Clean again with FFE.
  • the charge label or the charge carriers or the agent carrying the charge carrier can also be additionally marked (e.g. with a dye). This enables detection during or after the FFE.
  • Charge carriers could also be all types of particles (e.g. beads of all types, such as latex, agarose particles, colloids, etc.).
  • Charge carriers can also be virtually any combination of beads and / or antibodies and / or charge-carrying molecules and / or fluorescent markers and / or dyes.
  • Beads can e.g. be colored with fluorescent or other dyes. Beads can carry a fluorescent label (inside or outside), the charge label and the binding molecule (e.g. antibody or Ni-NTA) at the same time.
  • beads that have an enzymatically cleavable bridge, then after cleaning (e.g. cleaning of recombinant proteins with Ni-NTA) the beads can be cleaved from the purified molecule.
  • Beads that are fluorescent and that are provided on the surface with charge carriers and binding molecules are particularly preferred.
  • the separation of particles according to the invention enables concentrated collection or enrichment of biologically relevant particles, the analysis or use of which in diagnostic or therapeutic processes has not previously been possible because of the background of too many related but irrelevant particles present at the same time.
  • the FFE methods and devices presented thus enable isolation and consequently the use of particles naturally present in extremely low concentrations or the provision of their biologically relevant information for use in diagnostic or therapeutic processes.
  • the isolated particles can be used to find and characterize targets for the development of medicaments, targets for use in diagnostics, therapy selection and therapy monitoring both for humans and for animals and plants.

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  • Electrostatic Separation (AREA)

Abstract

L'invention concerne un procédé, un dispositif correspondant et un moyen de séparation destiné à la séparation de particules par une électrophorèse à écoulement libre. Le dispositif d'électrophorèse à écoulement libre comprend un compartiment de séparation (14), une pompe de dosage destinée à transporter un milieu de séparation (8), des électrodes (9, 10) destinées à appliquer un champ électrique au milieu de séparation (8), des points d'alimentation en échantillons (17) destinés à ajouter un mélange de particules à séparer et des points de fractionnement (18) destinés à évacuer des particules (1, 1', 1'') contenues dans le milieu de séparation (8) et séparées par électrophorèse à écoulement libre (FFE). Le procédé d'électrophorèse à écoulement libre (FFE) consiste à prendre un milieu de séparation (8) ; à céder ce milieu de séparation (8) au compartiment de séparation (14) à l'aide de la pompe de dosage par des conduites d'alimentation (15, 15') ; à amener le milieu de séparation (8) à traverser le compartiment de séparation (14) ; à laisser le milieu de séparation (8) s'écouler par des sorties (16) ; à appliquer un champ électrique dans le milieu de séparation (8) ; à associer une particule à séparer (1', 1'') à des porteurs de charge (4, 5) sélectionnés de manière à obtenir des particules de charge modifiée (7', 7'') qui en raison de leur charge de surface nette modifiée de façon sélective présentent dans le procédé d'électrophorèse à écoulement libre un comportement de migration différent de celui des particules de charge non modifiée (7) ; à ajouter un mélange de particules à séparer (1, 1', 1'') au milieu de séparation (8) dans le compartiment de séparation (14) par les points d'alimentation en échantillons (17); à évacuer les particules (1, 1', 1'') contenues dans le milieu de séparation (8) et séparées par électrophorèse à écoulement libre par les points de fractionnement (18); à recueillir les fractions ainsi obtenues et les particules séparées. Le procédé est caractérisé en ce que, entre le milieu de séparation (8) et au moins une électrode (9, 10), des coussins de guidage ou de concentration (12, 13) sont formés par un milieu coussin (20), ce milieu coussin (20) ayant une conductivité électrique nettement plus élevée par rapport au milieu de séparation (8) et est cédé à ces coussins de concentration (12, 13) par des canaux séparés (15').
EP20030729400 2002-01-21 2003-01-20 Procede et dispositif correspondant et moyen de separation destine a la separation de particules par une electrophorese a ecoulement libre Withdrawn EP1468279A1 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
CH872002 2002-01-21
CH87022002 2002-01-21
US35231302P 2002-01-28 2002-01-28
US352313P 2002-01-28
PCT/CH2003/000035 WO2003060504A1 (fr) 2002-01-21 2003-01-20 Procede et dispositif correspondant et moyen de separation destine a la separation de particules par une electrophorese a ecoulement libre

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EP1468279A1 true EP1468279A1 (fr) 2004-10-20

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EP (1) EP1468279A1 (fr)
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WO (1) WO2003060504A1 (fr)

Families Citing this family (2)

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Publication number Priority date Publication date Assignee Title
DE102005020134A1 (de) * 2005-04-29 2006-11-02 Becton, Dickinson And Co. Verfahren und Vorrichtung zur Durchführung eines parallelen und simultanen Mehrfachprozesses der trägerfreien isoelektrischen Fokussierung
AU2007263005B2 (en) * 2006-06-20 2013-07-25 Becton, Dickinson And Company Method and device for separation and depletion of certain proteins and particles using electrophoresis

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EP0103965A2 (fr) * 1982-08-20 1984-03-28 Imperial Chemical Industries Plc Appareil à électrofocaliser
US5284558A (en) * 1990-07-27 1994-02-08 University Of Iowa Research Foundation Electrophoresis-based sequencing of oligosaccharides
DE4139472C1 (fr) * 1991-11-29 1993-03-11 Gerhard Dr. 8011 Kirchheim De Weber
US5630924A (en) * 1995-04-20 1997-05-20 Perseptive Biosystems, Inc. Compositions, methods and apparatus for ultrafast electroseparation analysis
US5993627A (en) * 1997-06-24 1999-11-30 Large Scale Biology Corporation Automated system for two-dimensional electrophoresis
US7037416B2 (en) * 2000-01-14 2006-05-02 Caliper Life Sciences, Inc. Method for monitoring flow rate using fluorescent markers
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See also references of WO03060504A1 *
VÖLKL A. ET AL: "Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis", ELECTROPHORESIS, vol. 18, 1997, pages 774 - 780, XP007901237 *

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WO2003060504A1 (fr) 2003-07-24

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