WO2003060503A1 - Dispositif, utilisation associee et agent de separation pour separer des particules par electrophorese libre - Google Patents

Dispositif, utilisation associee et agent de separation pour separer des particules par electrophorese libre Download PDF

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Publication number
WO2003060503A1
WO2003060503A1 PCT/CH2003/000034 CH0300034W WO03060503A1 WO 2003060503 A1 WO2003060503 A1 WO 2003060503A1 CH 0300034 W CH0300034 W CH 0300034W WO 03060503 A1 WO03060503 A1 WO 03060503A1
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Prior art keywords
particles
charge
ffe
medium
separation
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PCT/CH2003/000034
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German (de)
English (en)
Inventor
Christoph Eckerskorn
Hans-Jörg Grill
Gerhard Weber
Peter Weber
David Yost
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Tecan Trading Ag
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Priority to AU2003201252A priority Critical patent/AU2003201252A1/en
Priority to EP20030729399 priority patent/EP1468278A1/fr
Publication of WO2003060503A1 publication Critical patent/WO2003060503A1/fr

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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 device, the use of the device and separating means for separating particles by means of the free-flow electrophoresis technique, according to the preamble of independent claims 1, 9, 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.
  • a generic FFE device is described in the international patent application PCT / EP01 / 14408. It is an electrophoresis device with a separation space through which a separation medium flows, which is separated by a base and a cover and by these two at a distance spacers held to each other is limited.
  • 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 for further 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 proposing a free-flow electrophoresis device with the features of independent claim 1.
  • this object is achieved - according to a second aspect - by using a method for separating particles, in particular for separating cells, cell components, organelles or biomolecules or parts or complexes thereof, using a free-flow electrophoresis device the features of independent claim 9 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-fiow 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 device for separating such particles based on 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 particle types with maximum efficiency, because the individual electrophoretic mobility of the ponds is increased significantly and selectively.
  • Many FACS applications can be replaced by FFE applications and can be carried out with much cheaper and faster devices, if required also automated.
  • 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.
  • tumor cells occur in the ratio of 1:10 6 to 1: 10 8 in whole blood
  • the white blood cells (2 ml MNC fraction from 10 ml Whole blood) including the tumor cells were separated from the red blood cells using a density gradient and, after appropriate labeling of the tumor cells (incubation with epitope-specific antibodies with "charge labels"), were applied to the FFE for separation. The addition took place at 4 to 10 ml / hour.
  • the purified tumor cells could be obtained in a final volume of 2-8 ml.
  • 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, such as a depletion of abundant proteins (for example separation of serum albumin from body fluids, for example blood plasma). • The setting of particles separated on the electrodes due to their special biological relevance and the resulting substantial losses of these particles can be successfully prevented with a focusing pad made of an electrically highly conductive pad medium.
  • 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-labeled cell types
  • Fig. 5 is a schematic representation of usable according to the invention
  • FIGS. 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
  • 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 and are a relatively low negative net surface charge very difficult to separate in the FFE. However, if the cells have epitopes of a first type 2 '(FIG. IB) or a second type 2 "(FIG. IC), these cells can be epitope-specific with charge-carrying molecules or charge carriers be marked. In general, the net surface charge of particles can thus be selectively changed by binding charged binding molecules to these particles. This binding can be based on an antigen-antibody interaction.
  • Further interactions that can be used for the modification of the net surface charge of particles according to the invention for coupling binding molecules to the particles to be separated also include receptor-ligand, enzyme-substrate and protein-protein interactions. Chelation or a bond based on general molecular interactions, be it ionic or based on Van der Waals forces or on hydrogen bonds, but also a covalent bond can 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 caiciosomes, coated vesicles, endosomes, endoplasmic 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 caiciosomes, coated vesicles, endosomes, endoplasmic reticulum, Golgi cisterns, lysosomes, peroxisomes and mitochondria
  • FIG. 2 shows an epitope-specific antibody and two monomeric or polymeric charge carriers or "charge labe! (cf. "CL” in FIG. 5) in the form of cations 4 or anions 5.
  • a charge carrier 4.5 is a molecule ionized at a given pH. 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, e.g. 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 bonds with between 1% and 10% nitrogen (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 changes in a separation medium 8 of an inventive one Device moved against the cathode 9 and the other cell type against the anode 10, as shown in the schematic diagram of Figure 4.
  • the two groups of charge-modified particles 7 ′, 7 ′′ or the two charge-modified cell types move away essentially simultaneously from the unlabelled or non-charge-modified background cells 7, which are essentially in the middle (or at least a great distance from the electrodes) 9, 10 in the separation medium 8 of the device according to the invention, the arrow indicating the main flow direction of the separation medium 8.
  • the various cell types which are separated from one another are collected in a collection area 11 and sent for further use
  • the resolution of the FFE is essentially determined by the migration behavior of the particles 7, 7 'and the size and number of the discharge openings.
  • the separation space 14 comprises the space for the separation medium 8 and the two lateral regions 19 near the electrodes 19 with the cushion medium 20 formed focusing pillow 12, 13 (cf. also Fig. 7).
  • the mass of the charge-modified particles 7 ', 7 " is fed through the FFE in approximately a normal distribution to the discharge openings, apart from an (unwanted) adhesion of charge-modified particles 7', 7" on the surfaces of the electrodes 9, 10.
  • 7A shows a normal distribution in a schematic cross-sectional view.
  • a focusing pad 12, 13 made of a likewise flowable, electrically highly conductive separating material is preferably 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.
  • 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.
  • This charged binding molecule 6 consists of nickel-nitrilo-tetraacetic acid (Ni-NTA), which binds to the 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 fractionation outlets 16' near the electrodes in order to add medium Particles 7 ', 7 "in the regions 19 of the separating medium 8 near the electrodes and are connected to a separate medium container (not shown) for providing an electrically highly conductive cushion medium.
  • CHIEF CHIEF
  • 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 method of continuous isolectrical focusing is particularly suitable for the micro-preparative to preparative isolation of biopolymers in general and of bioparticles whose biological function or their integrity is ensured in the range of the selected range of the pH gradient in the separation medium.
  • osmotic expanders can ensure the maintenance of an ideal osmotic pressure: by adding uncharged substances (e.g. sugar, as non-ionic, osmotic expander) and / or of salts (eg NaCI, as ionic, osmotic expander) an optimal osmotic pressure for a cell type, ie isomolar conditions (eg for mammalian cells 250-310 mosmol) can be generated.
  • uncharged substances e.g. sugar, as non-ionic, osmotic expander
  • salts eg NaCI, as ionic, osmotic expander
  • 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 semipermeable 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 possibly also for cooling the electrode buffer Pump device (not shown) 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 groups of particles 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.
  • FIG. 6A shows such a CHIEF device with a linear pH gradient.
  • 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 high.
  • the pH gradient is built up in the separation medium 8 between the two focusing pads 12, 13.
  • the FFE separation technique of continuous FFE zone electrophoresis (PZE) 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.
  • 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 separation medium 8 between the two focusing pads 12, 13 - with high conductivity in the regions 19 near the electrodes - is relatively low and homogeneous.
  • the pH value is the same in the entire separation medium.
  • the FFE separation technique of continuous isotachophoresis (CITP) 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.
  • a separated band of particles eg proteins
  • This FFE separation technique is principally suitable for bioparticles and cells, their separation in media without special "essential cations” (eg Mg ++ or Ca ++ ) or "essential anions” (eg Cl " ), which are essential for stability and vitality of cells are important, is not possible.
  • essential cations eg Mg ++ or Ca ++
  • essential anions eg Cl "
  • FIG. 6C shows such a CITP device in which discrete spacers are used.
  • discrete spacers are ions with well-known, defined mobility, which push themselves between particles to be separated in the CITP and contribute to the separation taking place efficiently.
  • 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.
  • an at least partial pH gradient is built up in the separation medium 8 between the two focusing pads 12, 13, the areas of the two gradients essentially coinciding and both the pH values and the electrical conductivity in the area of the focusing pads 12, 13 each being different are high.
  • CHIEF and FFE-PZE or CHIEF and CITP are possible.
  • different separation media can be used simultaneously in the separation area of the FFE apparatus and thus different separation parameters can be 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, 7 "is carried out with a 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 separation 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. It can also be provided that by means of a further metering pump or separate delivery channels of the medium pump - preferably at the end of the FFE device opposite the media feeds 15, 15 '- an auxiliary medium 21 is fed into the separation chamber 14 via auxiliary medium feeds 22 and together with the Separation medium 8 is removed via the fractionation points 18 (see FIGS. 6A to 6C).
  • FIG. 7 shows the effect of the focusing pads 12, 13, the border of the focusing pad with the electrically highly conductive pad medium 20, which runs essentially parallel to the electrode 9, 10, being indicated by dashed lines. If no focusing pad is 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 established. It can also happen that particles adhere to the electrode 9, 10 (not shown), so that in some cases substantial losses occur on these particles, for example because of their particular biological relevance. This can be done with a A focusing pillow 12, 13 made of an electrically highly conductive pillow medium 20 can be successfully prevented (cf. FIG.
  • the focusing pillow 12, 13 behaves like an electrode 9, 10, whereby - thanks to the high electrical conductivity of the cushion medium 20 is prevented that particles can reach the actual electrode 9, 10.
  • a concentration of the isolated particles forms at the cushion boundary, which far exceeds the measure of a normal distribution (shown in broken lines). A much larger amount of these particles in higher concentration can thus be removed from the FFE device and sent for further use via a fractionation point 18 which is positioned precisely in this area.
  • the use of such a focusing pad 12, 13 represents a significant improvement the use of a focusing pad in 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 separation performance is dependent on the field strength and is inversely proportional to the conductivity, the electrophoretic separation performance in the focusing pads 12, 13 decreases so much that the charge-modified particles 7 ', 7 "at the interface between the separation medium 8 and the Focusing pillow 12, 13 can be focused.
  • EPG epidermal glycoprotein
  • Ber-EP4 new monoclonal antibody which distinguishes epithelia from Mesothelia, J Clin Pathol 1990; 43: 213-9.
  • This monoclonal antibody is modified in two ways for the immune FFE: A) A charge carrier 4, 5 is directly and covalently coupled to the antibody (the binding molecule 3). B) 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 is obtained from patients with e.g. Breast, ovarian or colon carcinoma either used directly or prepared as follows:
  • 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 using an expensive density gradient centrifugation according to 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):
  • the resulting pellet then contains the so-called "light microsomal fraction" (including peroxysomes, microsomes and some mitochondria with lysosomes) and thus represents a mixture which also includes the peroxysomes.
  • 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 include e.g. Glutathione-S-transferase as an additional element. These vectors are suitable for the expression of recombinant proteins e.g. in E. coli, baculovirus-infected bacteria and mammalian cells. Appropriate methods for producing recombinant proteins are known to the person skilled in the art.
  • 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
  • His-tagged proteins can either now be used directly or the His-Tags are cleaved off enzymatically before further use.
  • His tags There are several ways to separate the His tags from the proteins: 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) detachment of the antibodies using methods known per se, e.g. by changing the salt concentration or 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 (for example with a dye). This enables detection during or after the FFE.
  • Charge carriers could also be all types of particles (eg 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, for example, be colored with fluorescent or other dyes. Beads can simultaneously carry a fluorescent label (inside or outside), the charge label and the binding molecule (eg antibody or Ni-NTA).
  • beads which have an enzymatically cleavable bridge, then after cleaning (eg 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. Possible measures for maintaining the biological activity or vitality of bioparticles in the various FFE separation techniques are shown in Table 1 below:
  • 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 medicines, targets for use serve in diagnostics, therapy selection and therapy monitoring for humans as well as for animals and plants.
  • the disclosed methods according to the invention are - if appropriate after appropriate adaptation - also suitable for use in a wide variety of areas of chromatography, for example in the affinity chromatography of organic or inorganic molecules and polymers.

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  • Electrostatic Separation (AREA)

Abstract

L'invention concerne un dispositif d'électrophorèse libre, un procédé associé et des agents de séparation pour séparer les particules au moyen d'un dispositif de ce type. Ce dispositif d'électrophorèse libre comporte au moins une chambre de séparation (14) traversée par un agent de séparation (8), cette chambre de séparation étant délimitée par un fond, un couvercle et une entretoise maintenant le couvercle et le fond écartés l'un de l'autre. Ledit dispositif d'électrophorèse libre comprend également les éléments suivants : une pompe de dosage transportant l'agent de séparation (8), lequel est amené dans la chambre de séparation (14) par des conduites d'alimentation (15, 15') et ressort de ladite chambre par des conduites d'écoulement (16) ; des électrodes (9, 10) pour créer un champ électrique dans l'agent de séparation (8) ; des emplacements de chargement d'échantillons (17) pour ajouter un mélange de particules (1, 1', 1'') à séparer ; des emplacements de fractionnement (18) pour évacuer les particules séparées au moyen du dispositif de l'invention dans l'agent de séparation (8). Le dispositif d'électrophorèse libre est pourvu de porteurs de charges (4, 5) sélectionnables pour la combinaison avec des particules (1', 1'') à séparer et pour façonner des particules (7', 7'') à charge modifiée, lesquelles, à cause de leur charge superficielle nette sélectivement modifiée, ont un autre comportement migratoire dans ledit dispositif, sous forme de particules (7) à charge non modifiée. L'invention est caractérisée en ce que ledit dispositif comprend des coussins de guidage et de focalisation (12, 13) entre l'agent de séparation (8) et au moins une électrode (9, 10). Ces coussins de focalisation (12, 13) sont composés d'un milieu (20) doté d'une conductivité électrique très élevée envers l'agent de séparation (8). Enfin, le dispositif de l'invention comporte des canaux (15') séparés pour amener ledit milieu (20) aux coussins de focalisation (12, 13).
PCT/CH2003/000034 2002-01-21 2003-01-20 Dispositif, utilisation associee et agent de separation pour separer des particules par electrophorese libre WO2003060503A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU2003201252A AU2003201252A1 (en) 2002-01-21 2003-01-20 Device, use of said device, and separation agent for separating particles by means of free flow electrophoresis
EP20030729399 EP1468278A1 (fr) 2002-01-21 2003-01-20 Dispositif, utilisation associee et agent de separation pour separer des particules par electrophorese libre

Applications Claiming Priority (4)

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CH86/02 2002-01-21
CH862002 2002-01-21
US35236602P 2002-01-28 2002-01-28
US60/352,366 2002-01-28

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WO2003060503A1 true WO2003060503A1 (fr) 2003-07-24

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2111547B1 (fr) * 2007-01-19 2013-04-10 Becton, Dickinson and Company Électrophorèse à écoulement libre utilisant des milieux de séparation et de stabilisation

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EP0103965A2 (fr) * 1982-08-20 1984-03-28 Imperial Chemical Industries Plc Appareil à électrofocaliser
US4465582A (en) * 1982-05-24 1984-08-14 Mcdonnell Douglas Corporation Continuous flow electrophoresis apparatus
DE4139472C1 (fr) * 1991-11-29 1993-03-11 Gerhard Dr. 8011 Kirchheim De Weber
US5284558A (en) * 1990-07-27 1994-02-08 University Of Iowa Research Foundation Electrophoresis-based sequencing of oligosaccharides
US5948231A (en) * 1995-04-20 1999-09-07 Perseptive Biosystems, Inc. Compositions, methods and apparatus for ultrafast electroseparation analysis
US20010015320A1 (en) * 1997-06-24 2001-08-23 Large Scale Biology Corporation Automated system for two-dimensional electrophoresis
US20010027918A1 (en) * 2000-01-14 2001-10-11 J. Wallace Parce Method for monitoring flow rate using fluorescent markers
WO2001077655A1 (fr) * 2000-04-10 2001-10-18 Invitrogen Corporation Procedes, articles et trousses de marquage de gels de polymeres

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US4465582A (en) * 1982-05-24 1984-08-14 Mcdonnell Douglas Corporation Continuous flow electrophoresis apparatus
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
US5948231A (en) * 1995-04-20 1999-09-07 Perseptive Biosystems, Inc. Compositions, methods and apparatus for ultrafast electroseparation analysis
US20010015320A1 (en) * 1997-06-24 2001-08-23 Large Scale Biology Corporation Automated system for two-dimensional electrophoresis
US20010027918A1 (en) * 2000-01-14 2001-10-11 J. Wallace Parce Method for monitoring flow rate using fluorescent markers
WO2001077655A1 (fr) * 2000-04-10 2001-10-18 Invitrogen Corporation Procedes, articles et trousses de marquage de gels de polymeres

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Title
See also references of EP1468278A1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2111547B1 (fr) * 2007-01-19 2013-04-10 Becton, Dickinson and Company Électrophorèse à écoulement libre utilisant des milieux de séparation et de stabilisation

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AU2003201252A1 (en) 2003-07-30

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