WO1994011078A1 - Immobilisation et manipulation magnetiques d'entites biologiques - Google Patents

Immobilisation et manipulation magnetiques d'entites biologiques Download PDF

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Publication number
WO1994011078A1
WO1994011078A1 PCT/US1993/011087 US9311087W WO9411078A1 WO 1994011078 A1 WO1994011078 A1 WO 1994011078A1 US 9311087 W US9311087 W US 9311087W WO 9411078 A1 WO9411078 A1 WO 9411078A1
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Prior art keywords
vessel
entities
magnetic field
magnetic
ferromagnetic
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PCT/US1993/011087
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English (en)
Inventor
Paul A. Liberti
Yuzhou Wang
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Immunivest Corporation
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Application filed by Immunivest Corporation filed Critical Immunivest Corporation
Publication of WO1994011078A1 publication Critical patent/WO1994011078A1/fr
Priority to US08/931,067 priority Critical patent/US5876593A/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations
    • G01N35/026—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations having blocks or racks of reaction cells or cuvettes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B03C1/031—Component parts; Auxiliary operations
    • B03C1/033—Component parts; Auxiliary operations characterised by the magnetic circuit
    • B03C1/0332—Component parts; Auxiliary operations characterised by the magnetic circuit using permanent magnets
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/025—High gradient magnetic separators
    • B03C1/031—Component parts; Auxiliary operations
    • B03C1/033—Component parts; Auxiliary operations characterised by the magnetic circuit
    • B03C1/034—Component parts; Auxiliary operations characterised by the magnetic circuit characterised by the matrix elements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C1/00—Magnetic separation
    • B03C1/02—Magnetic separation acting directly on the substance being separated
    • B03C1/28—Magnetic plugs and dipsticks
    • B03C1/288—Magnetic plugs and dipsticks disposed at the outer circumference of a recipient
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/18—Magnetic separation whereby the particles are suspended in a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B03—SEPARATION OF SOLID MATERIALS USING LIQUIDS OR USING PNEUMATIC TABLES OR JIGS; MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C—MAGNETIC OR ELECTROSTATIC SEPARATION OF SOLID MATERIALS FROM SOLID MATERIALS OR FLUIDS; SEPARATION BY HIGH-VOLTAGE ELECTRIC FIELDS
    • B03C2201/00—Details of magnetic or electrostatic separation
    • B03C2201/26—Details of magnetic or electrostatic separation for use in medical applications
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70589—CD45
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • This invention relates to the immobilization and manipulation of microscopic biological entities. More particularly, the present invention relates to magnetic labelling and immobilization of microscopic biological entities within an apparatus having an observation path for allowing observation and manipulation of such entities suspended within a fluid medium.
  • Many biological techniques such as are employed in biotechnology, microbiology, clinical diagnostics and treatment, in vitro fertilization, hematology and pathology, require such processes as identification, separation, culturing, or manipulation of a target entity such as a type of cell or microbe within a fluid medium such as blood, other bodily fluids, culture fluids or samples from the environment. It is often desirable to retain viability of the target entity or to culture the target entity.
  • Identification techniques typically involve labelling the target entity with a reagent which can be detected according to a characteristic property. Entities which can be viewed optically such as cells or certain microbes, may be identified using fluorescent MAb's or staining reagents specific to certain classes of cells or microbes. When such identification is done manually or mechanically, as by microscopy, multiple operations involving incubations and washing steps to remove excess labelling reagent are often performed. For example, in the usual method used to identify a subset of T-lymphocytes, such as T-Helper Cells or CD4-positive cells, a mixture of peripheral blood lymphocytes is incubated with a fluorescent MAb directed to CD4-positive cells.
  • T-lymphocytes such as T-Helper Cells or CD4-positive cells
  • the MAb is then given sufficient incubation time to react with the CD4-positive cells.
  • the CD4-positive cells are then washed using multiple centrifugations and can then readily be identified by fluorescent microscopy.
  • direct labeling with MAb's is often impractical due to the expense of obtaining a cell-specific fluorescent Mab and because of reduced signal availability.
  • indirect analysis is common.
  • the target entities are first labeled with a specific non-fluorescent MAb. Excess Mab is washed away. Then, a fluorescent-labeled second reagent such as fluorescent-labeled goat anti-mouse antibody is added to the medium.
  • the medium is allowed to incubate to allow the labelled second reagent to bind with the non-fluorescent MAb and then excess reagent is removed.
  • the target entities may then be identified due to the attachment of the fluorescent secondary reagent to the non- fluorescent biospecific MAb.
  • Such methods are time-consuming, costly, and require considerable quantities of reagents.
  • a greater number of target entities are lost or killed.
  • Accurate microbial analyses employing such methodologies are difficult to achieve because of the small numbers of target entities involved as well as the difficulty of washing away unbound labeling agents.
  • Other methodologies such as flow cytometry (fluorescent activated cell sorting) or field flow fractionation can be used for such analysis and in some instances require fewer manipulations.
  • Manipulation of target entities required by other biological techniques may also involve such processes as insertion of genetic material, organelles, subcellular components, viruses, or other foreign materials or bodies into the target entities. Inserted materials can be labeled prior to insertion so that effects and movements of these materials can be studied during incubation of the medium. In techniques such as transfection, or in vitro fertilization mechanical probes or arms are often used to hold the target entities. Such mechanical holding methods tend to obscure or damage the target entities.
  • Magnetic colloids having particles coated with biospecific compounds which attach to target entities are known to be useful in certain biospecific separation techniques. Reaction rates between such colloidal particles and target entities can be relatively rapid due to fast kinetic activity of the particles and sufficiently large areas of exposed reactant coatings. Magnetic particles in the range of 0.7 to 1.5 microns have been described in U.S. Patent NOS. 3,790,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Certain of these particles are disclosed to be useful solid supports for immunologic reagents, having reasonably good suspension characteristics when mildly agitated.
  • Magnetic particle suspensions presently in commercial use tend to flocculate in time and must be resuspended by agitation prior to use. Such agitation adds another step to any process employing such reagents.
  • Small magnetic particles such as those mentioned above, generally fall into two broad categories: particles that are permanently magnetized; and particles that become magnetic when subjected to a magnetic field.
  • the latter particles are referred to herein as magnetically-responsive particles.
  • Materials displaying magnetically-responsive behavior are sometimes described as superparamagnetic.
  • certain ferromagnetic materials such as magnetic iron oxide crystals, behave in a magnetically-responsive manner when the crystals are less than about 30 nm in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter. The properties of such particles are described in P. Robinson et al., Biotech Bioeng. XV:603-06 (1973).
  • Magnetically-responsive colloidal magnetite is disclosed in U.S. Patent No. 4,795,698 to Owen et al., which relates to polymer-coated, sub-micron size magnetite particles that behave as true colloids.
  • Several devices are known which are used to separate magnetic particles from colloidal suspensions.
  • Examples of such devices are magnetic separators such as the MAIA Magnetic Separator manufactured by Serono Diagnostics, Norwell, MA; the DYNAL MPC-1 manufactured by DYNAL, Inc. , Great Neck, New York; and the BioMag Separator, manufactured by Advanced Magnetics, Inc., Cambridge, Massachusetts.
  • a similar magnetic separator, manufactured by Ciba-Corning Medical Diagnostics, Wampole, MA is provided with rows of bar magnets arranged in parallel and located at the base of the separator. This device accommodates 60 test tubes, with the closed end of each test tube fitting into a recess between two of the bar magnets.
  • the above-described magnetic separators have the disadvantage that the magnetic particles and other impurities tend to form several layers on the inner surface of the sample container where they become entrapped and are difficult to remove even with vigorous washing. These separators are also not capable of establishing monolayers of target entities for microscopic analysis or manipulation.
  • a commercially available high gradient magnetic separator the MACS device made by Miltenyi Biotec GmbH, Gladback, West Germany, employs a column filled with a non-rigid steel wool matrix in cooperation with a permanent magnet.
  • the enhanced magnetic field gradient produced in the vicinity of the steel wool matrix attracts and retains the magnetic particles while the non-magnetic components of the test medium pass through the column.
  • HGMS high-gradient magnetic separation
  • the steel wool matrix of such prior art high-gradient magnetic separation (HGMS) devices often causes non-specific entrapment of biological entities other than the target entities. The entrapped non-magnetic components cannot be removed completely without extensive washing and resuspension of the particles bearing the target substance.
  • the sizes of the columns in many of the prior art HGMS devices require substantial volumes of test media, which poses an impediment to their use in performing various useful laboratory- scale separations.
  • the steel wool matrix may damage sensitive cell types.
  • HGMS affords certain advantages in performing medical or biological analyses based on biospecific affinity reactions involving colloidal magnetic particles
  • the systems developed to date are not particularly suited for immobilization and micromanipulation.
  • collection of microscopic entities upon an irregular structure such as steel wool is not conducive to microscopic observation wherein it is desirable to maintain the subject of interest in the focal plane of a microscope.
  • the convoluted surface of the steel wool would obscure observation of the collected entities. Accordingly, it would be desirable to provide an HGMS apparatus for immobilization and micromanipulation of target entities which is of relatively simple construction and operation and yet maximizes magnetic field gradients, so as to be of practical utility in conducting various laboratory-scale separations and micromanipulations.
  • an apparatus for immobilizing selected microscopic biological entities within a non-magnetic vessel containing a fluid medium.
  • a colloidal suspension of submicroscopic magnetically-responsive particles having a biospecific coating for binding with the selected entities is introduced into the medium, the particles become attached to the selected entities, and are subsequently attracted to a collection structure within or adjacent to the interior of the vessel that produces an intense magnetic field gradient upon application of an external magnetic field by a magnet structure having poles in opposition on either side of the vessel.
  • non-specific binding to some or all of the entities can be employed.
  • the spatial distribution in which entities are collected is related to the shape of the ferromagnetic collection structure and the concentration of magnetically-responsive particles in the fluid medium.
  • the ferromagnetic collection structure provides a collection surface upon which the target entities are immobilized in an orderly manner so that observation and/or manipulation of the immobilized entities is easily, accomplished relative to known techniques.
  • Ferromagnetic collection structures having sufficiently high curvatures, a multi-stranded construction, or sharp edges are capable of producing substantially monolayered one-dimensional spatial distributions of immobilized entities.
  • concentration of magnetically-responsive particles may be selected to facilitate the formation of such a monolayer, for example by controlling the concentration of magnetically-responsive particles suitable to bind with target entities such that the surface area of the bound target entities is commensurate with the surface area of the ferromagnetic collection structure or other collection surface within the magnet separator.
  • a non-magnetic vessel for use in a magnetic immobilization apparatus of the invention preferably has an observation path which allows microscopic observation of the immobilized entities.
  • the observation path includes at least one open or transparent aperture or surface in the non-magnetic vessel for allowing substantially unobscured microscopic observation and physical manipulation, such as microsurgery, of the immobilized entities.
  • the vessel may include at least one surface or aperture that is transparent to at least a portion of the electromagnetic spectrum required for a particular observational technique.
  • the observation path is oriented relative to the collection structure to allow substantially unobscured observation of the immobilized entities.
  • the collection structure is preferably arranged to maintain a plurality of the immobilized entities within an orderly array intersecting the observation path, such as within a focal plane of a microscope.
  • a vessel may further have ports which allow a flow of liquid reagents through the vessel as may be desired in, for example, sequential reaction techniques.
  • the flowthrough vessel also facilitates observation of the immobilized entities by allowing the test medium to be flushed or rinsed. Such flushing or rinsing may be employed to remove an opaque test medium from the vessel while leaving behind the immobilized entities or substances.
  • flushing or rinsing of the test medium allows control of the duration of the separation process so that the test medium may be removed from the vessel after having been placed in the magnetic field for a period of time sufficient for formation of a monolayered array of immobilized entities upon the collection structure and within the observation path.
  • an apparatus may provide for lateral translation or concentration of immobilized entities by the use of a transverse obstruction, or shoulder, which concentrates immobilized entities in the vicinity of the obstruction, or shoulder, as the shoulder is moved laterally along the axis of the collection structure within or out of the field confined to the region between the opposing poles.
  • FIGS. 1A-1C are exploded views of embodiments of a magnetic immobilization apparatus
  • FIGS. 2A and 2B are fragmentary perspective views of ferromagnetic collecting elements suitable for use within the embodiments of FIGS. 1A-1C;
  • FIGS. 3A-3D are perspective views of central troughs suitable for vessels within the embodiments of FIGS. 1A-1C;
  • FIGS. 4A and 4B are overhead perspective views of devices for collecting and concentrating target entities; and FIG. 5 is a schematic diagram of an arrangement for observing and manipulating entities collected within a magnetic immobilization apparatus.
  • target entities within a fluid medium are immobilized for observation, analysis, or manipulation.
  • target entities as may be immobilized hereby include cellular entities and subcellular entities.
  • Magnetic labelling of a target entity is achieved by the use of a colloidal suspension of magnetically-responsive particles having a coating which includes an attachment agent such as a ligand selected to bind to a corresponding receptor of the target entity.
  • the colloidal suspension of magnetically responsive particles is mixed with the fluid medium containing the target entities to form a test medium. Such mixing can be carried out either prior to or after the fluid medium is introduced into a non-magnetic vessel. A magnetic field is then applied to the vessel.
  • the magnetically-labelled target entities Upon application of a magnetic field to the non ⁇ magnetic vessel, the magnetically-labelled target entities tend to migrate toward and adhere to a collection structure or magnetic flux intensifier such as a ferromagnetic wire supported within or adjacent to a vessel containing the test medium.
  • the strength of the magnetic field preferably between about 4 kGauss and 15 kGauss, influences the time required to effect collection. Additionally, the impact and binding forces experienced by the target entities upon the collection structure are determined in part by the strength of the applied magnetic field.
  • the long- range attractive magnetic force acting upon the target entities within the test medium is mainly due to the size of the collection structure and the bulk permeability of the material from which the collection structure is made.
  • the short-range binding forces between the collection structure and the collected target entities is mainly due to the surface geometry of the collection structure.
  • the preferred magnetic particles for use in carrying out this invention are particles that behave as true colloids. Such particles are characterized by their sub-micron particle size, which is generally less than about 200 nanometers (nm) (0.20 microns) , and their stability to gravitational separation from solution for extended periods of time. Such small particles facilitate observation of the target entities via optical microscopy since the particles are significantly smaller than both the target entities and the wavelength range of visible light.
  • Suitable magnetically-responsive particulate materials are composed of a crystalline core of superparamagnetic material surrounded by molecules which may be physically absorbed or covalently attached to the magnetic core and which confer stabilizing colloidal properties. The size of the colloidal particles is sufficiently small that they do not contain a complete magnetic domain, and their Brownian energy exceeds their magnetic moment.
  • colloidal magnetic particles are not readily separable from solution as such even with powerful electromagnets. Rather, a magnetic gradient is required within the test medium in which the particles are suspended in order to achieve separation of the discrete particles.
  • Magnetic particles having the above-described properties can be prepared as described in U.S. Patent No. 4,795,698, the entire disclosure of which is incorporated by reference in the present specification, as if set forth herein in full.
  • the test medium is typically prepared from appropriately prepared fluids, for example, body fluids such as blood, urine, sputum or secretions. It is preferable to add the colloidal magnetic particles to the test medium in a buffer solution.
  • a suitable buffer solution for this purpose comprises a mixture of 5% bovine serum albumin ("BSA") and 95% of a biocompatible phosphate salt solution, optionally including relatively minor amounts of dextrose, sodium chloride and potassium chloride.
  • BSA bovine serum albumin
  • the buffer solution should be isotonic, with a pH of about 7.
  • the BSA protein serves to decrease interactions which tend to interfere with the analysis.
  • the target substance may be added to the test medium before, after or simultaneously with introduction of the magnetic particles.
  • the method of the invention takes advantage of the diffusion controlled solution kinetics of the colloidal magnetic particles, which may be further enhanced by the addition of heat to the test medium.
  • the test medium is usually incubated to promote binding between the receptor and any ligand of interest present therein. Incubation is typically conducted at room temperature or at a temperature slightly above the freezing point of the test medium (i.e., 4°C in an aqueous medium). The period of incubation is normally of short duration (i.e., about 15 minutes) .
  • the test medium may be agitated or stirred during the incubation period to facilitate contact between receptors and ligands.
  • FIG. 1 there is shown an exploded view of a magnetic immobilization apparatus 20a.
  • a non-magnetic vessel such as vessel 22a is provided with a central trough 21a and end pieces 23a and 23b for containing a fluid test medium (not shown) .
  • the vessel 22a is situated between a north pole 24 and a south pole 25 of a device for generating a magnetic field transverse to the longitudinal axis of the vessel 22a.
  • the devices 24 and 25, shown diagrammatically in FIG. 1, may be pole pieces of an electromagnet, two confronting permanent magnets, or parts of a single channel, or a U-shaped, permanent magnet.
  • a ferromagnetic flux intensifier such as collection wire 28a spans between the end pieces 23a and 23b within the vessel 22a with an orientation substantially transverse to the lines of magnetic flux between the poles 24 and 25 respectively.
  • a ferromagnetic wire when used as the collection structure 28a, provides localized intensification of the magnetic flux density commensurate with the curvature of the wire.
  • the two ends of the wire 28a are attached to and are supported between the interior surfaces of the end pieces 23a and 23b so that the wire spans the interior volume of the vessel co- axially with the longitudinal axis of the wire.
  • the two ends of the wire may be supported by, or embedded within the end pieces such that the wire is substantially straight along the entire length thereof between the end pieces.
  • the wire 28a may have a non ⁇ magnetic coating to reduce adhesion of magnetically- responsive particles in the absence of a magnetic field.
  • the immobilization apparatus 20a may be mounted on a stage (not shown) having micromanipulation devices and a suitable microscope to allow an operator to manipulate and/or observe the immobilized target entities.
  • the substantially linear form of the collection surfaces facilitates microscopic observation of the immobilized entities along the length of the collection structure since such a linear array can easily be maintained within the focal plane of a microscope. Direct observation of the immobilized entities can be made along an unobscured optical path extending into the interior of the vessel and intersecting the collection surfaces along either side of the collection structure.
  • the vessel 22a as shown in FIG. 1, has an open top to facilitate access to the interior by such devices as may be used in the practice of cell microsurgery or other biotechnical or chemical investigations.
  • Collection of target entities upon the surface of the collection wire 28a is dominant along the wire surface regions most nearly orthogonal to the lines of magnetic flux, i.e. along the opposite sides 39a and 39a 1 of the wire 28a which face the poles 24 and 25.
  • a non-metallic coating may be applied to the surface of the ferromagnetic collection structure. When such a coating is present upon the ferromagnetic collection structure, the target entities are collected upon a collection surface that is upon the non-metallic coating and coextensive with the ferromagnetic collection structure.
  • the diameter of the collector wire 28a is chosen such that the curvature of the wire 28a approximates the curvature of the target entity, linear monolayers of the target entity, or one- dimensional arrays, tend to form upon the collection wire 28a.
  • target entities such as cells collect upon the wire 28a
  • a cell which collides with another cell already attached to the wire 28a will tend to roll over the attached cell and then attach to an adjacent section of exposed wire.
  • the two attached cells tend to move apart to accommodate attachment of the newly arrived cell to the wire.
  • lymphocytes having a diameter on the order of 10 ⁇ m will tend to collect in linear monolayer arrays upon either side of a .02 mm wire from solutions wherein the number of lymphocytes is limiting.
  • the diameter of the collection wire may be selected in accordance with the diameter of the target entities in order to produce a linear monolayer of immobilized entities.
  • the diameter of the target entities may be such that a corresponding cylindrical collection wire would not have enough bulk to exert a long-range influence upon the magnetic field within the vessel to attract the target entities toward the collection surface.
  • a collection structure having sufficient bulk to produce a long-range magnetic gradient and also having a high curvature surface facing at least one of the magnetic pole faces.
  • the high curvature surface serves to produce a short-range gradient in the vicinity of the collection surface to arrange the entities that have been attracted by the long-range gradient into a linear monolayer upon the collection surface.
  • Such a collection structure may provide enhanced mechanical strength in addition to anisotropic intensification of the magnetic flux density.
  • FIG. 2A is shown in FIG. 2A in the form of a wire 28b.
  • the wire 28b has an elliptical, or ribbon-like, cross section.
  • FIG. 2B Another embodiment of a ferromagnetic collection structure for causing anisotropic flux intensification is shown in FIG. 2B.
  • a composite wire, such as wire 28c has a central strand 30 with two smaller strands 29 parallel to the central strand 30 and positioned along opposite sides 39c and 39c 1 of the central strand 30.
  • a non-magnetic coating 31 provides support for the strand assembly and allows for ease of removal of magnetically-responsive particles in the absence of a magnetic field.
  • FIG. 3A there is shown a central trough 21b for such a vessel.
  • the central trough 21b includes a ferromagnetic collection structure in the form of a wire 28d which is partially embedded in an interior lateral surface of the trough 21b. Collection of target entities in the central trough 21b will predominate along the exposed portion 39d of the surface of collection wire 28d where such entities may easily be observed.
  • FIG. 3B there is shown a central trough 21c having a collection structure in the form of a wire 28e embedded entirely beneath an interior lateral surface 39e of the trough 21c.
  • Collection of target entities in a trough such as trough 21c will predominate along the interior lateral surface 39e of the trough 21c in a line parallel to the wire 28e.
  • Such an arrangement as trough 21c provides a substantially flat collection surface and minimal visual obscuration of the target entities.
  • other geometric configurations of attached or embedded collection wires such as rectilinear grids may be provided to produce a different pattern of target entity collection on the collection structure or on the interior surface of the trough.
  • a central trough 2Id having a flat, narrow collection structure, such as a razor blade 28f, embedded within a side of the trough 2Id with the edge 39f of the blade protruding into the interior of the trough and having a flat side substantially parallel to the bottom of the trough 21d.
  • the edge 39f of collection blade 28f may have a significantly sharper or greater curvature than would be practical in embodiments employing wires suspended within a trough. Such thin wires would be fragile and unlikely to withstand magnetic field variations without substantial deformation.
  • the angular edge 39f of collection element 28f has a high curvature which provides a high magnetic field gradient in the vicinity of the protruding edge thus promoting collection of target entities in a linear monolayer.
  • a vessel such as is shown in FIG. 3D is preferable for such applications in which a linear monolayer of target entities is desired, but where contact with the edge of the collection element would damage the target entities or be otherwise undesirable.
  • the central trough 21e has a flat, narrow collection element 28g embedded within a side of the trough 21e in such a way that the edge of element 28g does not protrude into the interior of the trough. In this case, the target entities are collected on the interior lateral surface 39g along a line adjacent the edge of the element 28g.
  • Each of the trough structures described in connection with FIGS. 3A-3D may be provided with a transparent top member (not shown) or may remain open when used in an immobilization apparatus in order to provide an unobstructed observation path intersecting the collection surface or to provide direct physical access to the immobilized entities as may be desired. Additionally, the central troughs 21b-21e may be partially or entirely transparent to further facilitate such observation. It is often desired to expose target entities to various chemical and biological environments other than the test medium in which such target entities were originally suspended. It is also often desired to rinse or flush the test medium in which the target entities have been immobilized in order to facilitate observation of the entities.
  • FIG. IB A vessel which allows such environmental variation and flushing is shown in FIG. IB.
  • a flowthrough vessel for allowing alteration of the test environment such as flowthrough vessel 22b, has a central trough 2If which may be of such types as troughs 21a-e discussed in connection with FIGS. 1, FIGS. 3A-3D, and FIG. 5B. Attached to the ends of the central trough 21f are end pieces 23c, 23d having one or more pairs of inlet and outlet ports such as ports 48a and 48b.
  • the end pieces 23c, 23d also provide support for a collection wire (not shown) in embodiments employing a wire spanning between the end pieces.
  • the ports 48a and 48b are connected with hoses 46a, 46b which may be further connected to sources of reagents, pumps, valves, and other reagent flow control devices.
  • the flowthrough vessel 22b is provided with a top 40, which may be transparent to provide a clear optical path for microscopic observations.
  • the bottom 41 of the central trough 2If may also be transparent to allow the passage of light through the vessel.
  • the top 40 or any other parts of the vessel may be selectively transparent to portions of the electromagnetic spectrum as may be desired in photobiological investigations.
  • the top 40 and the bottom 41 of the central trough 2If may form a polarizer/analyzer pair for such applications as optical studies of the mechanical properties of transparent membranes.
  • the flowthrough vessel 22b allows sequential exposure to reagents, such as is required by indirect fluorescent MAb labelling, while significantly reducing the loss of target entities as is usual in traditional methods which necessitate multiple washings and centrifugations.
  • the flowthrough vessel 22b further allows for the consumption of a reduced volume of reagents in such sequential processes since target entities are collected in a relatively high concentration and with reduced spatial extent relative to known separation devices.
  • the hoses 46a, 46b may be connected to a reagent recirculation system for further economy of reagent consumption.
  • FIG. 1C A preferred manner of constructing an immobilization vessel is illustrated in FIG. 1C.
  • the vessel 50 includes an upper assembly 52 and a lower assembly 54 which mate along.surfaces 64 and 64' to form a substantially rectangular hollow enclosure.
  • the upper assembly 52 and the lower assembly 54 are both transparent and have substantially flat exterior surfaces so that light may pass through the vessel 50 along a substantially unobstructed and non-distorting optical path.
  • Opposed ends 53a and 53b of the lower assembly 54 have notches or grooves 56a and 56b formed along respective upper edges of the ends 53a and 53b.
  • a ferromagnetic collection structure 58 may be aligned within grooves 56a and 56b and secured therein by an adhesive (not shown) . Additional adhesive is applied to the mating surfaces 64 and 64' of the upper and lower assemblies and the mating surfaces are brought into contact with each other. After the adhesive has set, the protruding portions of the collection structure 58 may be trimmed flush with the exterior of the vessel.
  • semicircular notches 60a, 60a , and 60b may be formed the opposed ends of the upper and ' lower assemblies along the mating surfaces 64, 64 / .
  • inlet port nipple 62a and outlet port nipple 62b may be positioned so that the nipples 62a and 62b are secured within the mated semicircular notches.
  • the flow path is through the hollow inlet nipple 62a, the hollow interior of the enclosure formed by the assemblies 52 and 54, and the hollow outlet nipple 62b.
  • Other methods of assembling an immobilization apparatus, such as injection molding, are contemplated within the scope of the invention.
  • a hollow vessel 70 which may be of any type discussed herein, is supported upon a microscope stage 72.
  • the microscope stage 72 is equipped with a well-known mechanism for translating the vessel 70 in either direction along the axis 73.
  • Magnets 74 and 76 are positioned on either side of the microscope stage to establish an applied magnetic field transverse to the longitudinal axis of the vessel 70.
  • a source of light 78 is provided for projecting a collimated beam 80 of light toward the vessel 70 along an axis that is perpendicular to the longitudinal axis of the vessel and to the applied magnetic field.
  • the vessel 70 has a top and a bottom that are transparent to the beam 80 of light.
  • the top and bottom surfaces of the vessel 70 are substantially flat so that the beam 80 may pass through the top and bottom surfaces substantially undistorted.
  • a ferromagnetic collection structure 82 is positioned within the vessel 70 and in the present instance is supported co-axially of the longitudinal axis of the vessel 70.
  • the ferromagnetic collection structure 82 has a coextensive collection surface upon which magnetically-labeled entities are adhered.
  • the collection surface of the collection structure 82 is oriented relative to the path of beam 80 such that the optical path of the beam 80 intersects the collection surface so that the immobilized entities may be visually observed. Such visual observation may be made, for example, by collecting the light transmitted through the vessel 70 with an objective lens 84 and reflecting the collected light with a mirror 86 toward an eyepiece 88.
  • Lateral translation of target entities along the collection surface may be accomplished within an immobilization apparatus without necessitating physical contact between the target entities and a device such as a micromanipulator.
  • a magnetic field generated between two confronting poles of finite lateral extent possesses a positive lateral gradient toward the region between the poles.
  • magnetically-labeled target entities which have been collected on a ferromagnetic collecting structure such as the wire 28a shown in FIG. 1A will tend to remain between the poles 24 and 25 as the vessel 22a is translated in a direction parallel to the longitudinal axis of the wire 28a.
  • FIG. 4A an alternative collection structure is shown as a collection wire 28h having a reduced diameter at 34 and an enlarged diameter at 35 interconnected by a transverse wall 36 to produce a shoulder therebetween.
  • the collection wire 28h is positioned between confronting opposite magnetic poles 24 and 25 within a vessel (not shown) containing a fluid medium in which are suspended magnetically-labeled target entities.
  • the confinement of magnetic flux lines between poles 24 and 25 will cause collection of target entities along the portion of the outer sides of the wire 28e in the reduced diameter portion 34 which is disposed directly between the poles 24 and 25.
  • the vessel supporting the wire 28h is then laterally translated, such as by translation of a supporting microscope stage (not shown) , in the direction indicated by the arrow 38h so as to move the enlarged portion 35 of the wire 28h into the region between the poles 24 and 25.
  • Lateral translation and congregation of the target entities may also be accomplished using vessels having embedded ferromagnetic collection structures similar to those shown in central troughs 21b-21e shown in FIGS. 3A-3D.
  • a central trough 21i having an embedded ferromagnetic collection element 28i is shown.
  • the central trough 21i further includes a surface irregularity, such as a transverse obstacle or weir 48, forming a shoulder on the interior surface 39i located along the line of collection.
  • target entities collected upon the interior surface 39i of the central trough will tend to congregate at the angular junction between the line of collection and the anterior surface 49 of the shoulder 48.
  • the ability to create dense congregations of target entities without direct mechanical interventions provides unique advantages for such applications as in vitro fertilization or other techniques in which it is desired to propagate a culture of fragile target entities which reproduce at a rate commensurate with their concentration.
  • an immobilization apparatus using a central trough of the type shown in FIG. 3C was constructed and tested.
  • Permanent rare earth alloy magnets made of CRUMAX 355 (a trademark of Crucible Magnetics of Elizabethtown, Kentucky) were used to generate the magnetic field.
  • the magnets were in the form of bar magnets having dimensions of 1.75 inches X 0.375 inches X 0.375 inches with a flux vector parallel to one of the 0.375 dimensions.
  • the magnets were mounted in a yoke made of cold rolled steel.
  • the opposing faces of the magnets were 0.3125 inches apart.
  • the magnetic field strength at each bar magnet face was measured to be 5.8 kGauss.
  • the vessel had outer dimensions of 0.1875 inches X 0.3125 inches X 0.5 inches with the 0.3125 inch dimension fitted between the magnet faces.
  • the wall thickness of the vessel was 0.0625 in.
  • the ferromagnetic collection element was a portion of a razor blade.
  • a dextran-coated magnetic colloid was prepared having a concentration of 0.0212 ⁇ g/ml of Fe. Five ml of the magnetic colloid was incubated with cells of the T-Cell CEM line. The colloid coated the cells via non-specific binding. The final concentration of T- Cells in the mixture was 2,000 cells/ml. Next, 50 ⁇ l aliquots of the cell mixture were inserted into the immobilization vessel and allowed to separate for 5 minutes. Excess cells were flushed away with PBS buffer. A single line of cells collected along the edge of the razor blade were observed with an optical microscope at 3OX magnification.
  • T-Cells were first immobilized as in Example 1 above. After collection, the cells were washed three times in the usual manner with PBS buffer to remove excess magnetic particles from the mixture. Then, 12 ⁇ l of Fitc CD45 T-Cell marker having a concentration of 10 ⁇ g/ml was added to the cell mixture and incubated for five minutes at room temperature. After the labelling step, the cells were washed three times again in PBS buffer. Fluorescently-labelled cells were observed to be aligned with the edge of the razor blade via 60X magnification fluorescent microscopy.
  • the cells were then washed twice in PBS buffer.
  • the immobilization vessel was taken out of the magnetic field to effect re-orientation of the cells and agitated for one minute.
  • the vessel was then placed back into the magnetic field. Surface portions of cells which had previously been in direct contact with neighboring cells surfaces were visible and unstained. Sections of such unstained cell membranes were surgically removed for a comparison of the chemical composition of such sections with comparable sections of stained cell membrane in order to identify chemical cellular communication agents.

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Abstract

On marque des entités biologiques telles que des cellules, des microbes ou leurs composants avec un colloïde magnétique qui contient des particules magnétiques microscopiques dotées d'un revêtement susceptible de liaison biospécifique ou non spécifique avec ces entités. Un dispositif d'immobilisation comporte un récipient amagnétique (70) muni d'une structure de collecte ferromagnétique (82) chargée d'attirer les entités vers une surface de collecte sur laquelle les entités à marquage magnétique sont immobilisées lorsqu'on place le récipient sur un support (72) situé entre deux aimants (74, 76). De préférence, la structure de collecte ferromagnétique (82) comporte un angle vif ou une courbure prononcée qui permet d'intensifier le champ magnétique et de collecter les entités en une couche unique. Le récipient (70) présente un trajet d'observation sans obstacle (80) qui permet d'observer et/ou de manipuler les entités immobilisées. La structure de collecte ferromagnétique (82) peut être disposée selon différents motifs bidimensionnels pour donner la configuration de collecte souhaitée. De plus, ce dispositif peut comporter des orifices d'entrée et de sortie (84, 86) qui permettent à un flux de réactif liquide de traverser le récipient (70) afin de laver ou de colorer les entités immobilisées. Le support (72) peut subir une translation de façon qu'un épaulement placé sur la structure de collecte (82) concentre les entités collectées par le déplacement de cet épaulement dans une direction transversale par rapport au champ magnétique.
PCT/US1993/011087 1990-09-26 1993-11-16 Immobilisation et manipulation magnetiques d'entites biologiques WO1994011078A1 (fr)

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WO1997046882A1 (fr) * 1996-06-07 1997-12-11 Immunivest Corporation Separation magnetique au moyen de gradients externes et internes
US5741714A (en) * 1995-07-18 1998-04-21 Immunivest Corporation Detection of bound analyte by magnetic partitioning and masking
US5834197A (en) * 1994-05-11 1998-11-10 Genera Technologies Limited Methods of capturing species from liquids and assay procedures
EP0970365A1 (fr) * 1997-03-25 2000-01-12 Immunivest Corporation Appareil permettant la capture et l'analyse d'entites particulaires et techniques correspondantes
US6136182A (en) * 1996-06-07 2000-10-24 Immunivest Corporation Magnetic devices and sample chambers for examination and manipulation of cells
WO2001017687A2 (fr) * 1999-09-03 2001-03-15 Miltenyi Biotec Gmbh Procedes de modification de cellules selectionnees dans une colonne de separation magnetique de cellules
US6241894B1 (en) * 1997-10-10 2001-06-05 Systemix High gradient magnetic device and method for cell separation or purification
WO2001071034A2 (fr) * 2000-03-22 2001-09-27 President And Fellows Of Harvard College Procede et appareil pour l'analyse biologique magnetique et la manipulation paralleles
EP1144073A1 (fr) * 1998-11-30 2001-10-17 Immunivest Corporation Appareil et procedes de separation magnetique
DE10136472A1 (de) * 2001-07-23 2003-02-20 Inst Chemo Biosensorik Vorrichtung und Verfahren für den Nachweis lebender Mikroorganismen, eukaryotischer Zellen oder Organellen
US6660159B1 (en) 1996-06-07 2003-12-09 Immunivest Corporation Magnetic separation apparatus and methods
US6790366B2 (en) 1996-06-07 2004-09-14 Immunivest Corporation Magnetic separation apparatus and methods
US6890426B2 (en) 1996-06-07 2005-05-10 Immunivest Corporation Magnetic separation apparatus and methods
EP1574259A1 (fr) * 2004-03-11 2005-09-14 Christian Doppler Labor für spezifische Adsorptionstechnologien in der Medizin Procédé et dispositif servant à détecter de microparticules marquées
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US7666308B2 (en) * 1996-06-07 2010-02-23 Veridex, Llc. Magnetic separation apparatus and methods
US8263387B2 (en) 2009-06-10 2012-09-11 Cynvenio Biosystems, Inc. Sheath flow devices and methods
EP2701851A1 (fr) * 2011-04-29 2014-03-05 Becton, Dickinson and Company Systèmes fluidiques d'immobilisation et de collecte de particules en continu et leurs procédés d'utilisation
WO2014082752A1 (fr) * 2012-11-30 2014-06-05 Hochschule Trier Dispositif pour la séparation de microparticules magnétiques ou magnétisables d'une suspension au moyen d'une séparation magnétique à gradient élevé
US9404916B2 (en) 2008-09-20 2016-08-02 University College Cardiff Consultants Limited Use of a protein kinase inhibitor to detect immune cells, such as T cells
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease

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US5622831A (en) * 1990-09-26 1997-04-22 Immunivest Corporation Methods and devices for manipulation of magnetically collected material
US5834197A (en) * 1994-05-11 1998-11-10 Genera Technologies Limited Methods of capturing species from liquids and assay procedures
US5741714A (en) * 1995-07-18 1998-04-21 Immunivest Corporation Detection of bound analyte by magnetic partitioning and masking
US5993665A (en) * 1996-06-07 1999-11-30 Immunivest Corporation Quantitative cell analysis methods employing magnetic separation
US5985153A (en) * 1996-06-07 1999-11-16 Immunivest Corporation Magnetic separation apparatus and methods employing an internal magnetic capture gradient and an external transport force
WO1997046882A1 (fr) * 1996-06-07 1997-12-11 Immunivest Corporation Separation magnetique au moyen de gradients externes et internes
US6013188A (en) * 1996-06-07 2000-01-11 Immunivest Corporation Methods for biological substance analysis employing internal magnetic gradients separation and an externally-applied transport force
US7666308B2 (en) * 1996-06-07 2010-02-23 Veridex, Llc. Magnetic separation apparatus and methods
US6136182A (en) * 1996-06-07 2000-10-24 Immunivest Corporation Magnetic devices and sample chambers for examination and manipulation of cells
US6890426B2 (en) 1996-06-07 2005-05-10 Immunivest Corporation Magnetic separation apparatus and methods
US6790366B2 (en) 1996-06-07 2004-09-14 Immunivest Corporation Magnetic separation apparatus and methods
US6660159B1 (en) 1996-06-07 2003-12-09 Immunivest Corporation Magnetic separation apparatus and methods
WO1998055867A1 (fr) * 1996-07-16 1998-12-10 Immunivest Corporation Detection d'un analysat lie, effectuee par separation magnetique et par masquage
US6623983B1 (en) 1997-03-25 2003-09-23 Immunivest Corporation Apparatus and methods for capture and analysis of particulate entities
EP0970365A4 (fr) * 1997-03-25 2001-01-17 Immunivest Corp Appareil permettant la capture et l'analyse d'entites particulaires et techniques correspondantes
EP0970365A1 (fr) * 1997-03-25 2000-01-12 Immunivest Corporation Appareil permettant la capture et l'analyse d'entites particulaires et techniques correspondantes
US6241894B1 (en) * 1997-10-10 2001-06-05 Systemix High gradient magnetic device and method for cell separation or purification
EP1144073A1 (fr) * 1998-11-30 2001-10-17 Immunivest Corporation Appareil et procedes de separation magnetique
EP1144073A4 (fr) * 1998-11-30 2004-07-14 Immunivest Corp Appareil et procedes de separation magnetique
WO2001017687A3 (fr) * 1999-09-03 2002-01-17 Miltenyi Biotec Gmbh Procedes de modification de cellules selectionnees dans une colonne de separation magnetique de cellules
US6468432B1 (en) 1999-09-03 2002-10-22 Miltenyi Biotec Gmbh Methods of modification of selected cells in a magnetic cell separation column
JP2003508073A (ja) * 1999-09-03 2003-03-04 ミルテニィ バイオテック ゲーエムベーハー 磁性細胞分離カラムにおける選択された細胞の改変方法
WO2001017687A2 (fr) * 1999-09-03 2001-03-15 Miltenyi Biotec Gmbh Procedes de modification de cellules selectionnees dans une colonne de separation magnetique de cellules
WO2001071034A3 (fr) * 2000-03-22 2002-08-15 Harvard College Procede et appareil pour l'analyse biologique magnetique et la manipulation paralleles
WO2001071034A2 (fr) * 2000-03-22 2001-09-27 President And Fellows Of Harvard College Procede et appareil pour l'analyse biologique magnetique et la manipulation paralleles
DE10136472A1 (de) * 2001-07-23 2003-02-20 Inst Chemo Biosensorik Vorrichtung und Verfahren für den Nachweis lebender Mikroorganismen, eukaryotischer Zellen oder Organellen
US7285412B2 (en) 2001-07-27 2007-10-23 Surface Logix Inc. Device for magnetic immobilization of cells
US7169578B2 (en) 2001-07-27 2007-01-30 Surface Logix, Inc. Cell isolation and screening device and method of using same
US7169577B2 (en) 2001-07-27 2007-01-30 Surface Logix, Inc. Cell isolation and screening device and method of using same
EP1574259A1 (fr) * 2004-03-11 2005-09-14 Christian Doppler Labor für spezifische Adsorptionstechnologien in der Medizin Procédé et dispositif servant à détecter de microparticules marquées
US10336808B2 (en) 2007-03-26 2019-07-02 Dako Denmark A/S MHC peptide complexes and uses thereof in infectious diseases
US10030065B2 (en) 2007-07-03 2018-07-24 Dako Denmark A/S MHC multimers, methods for their generation, labeling and use
US10611818B2 (en) 2007-09-27 2020-04-07 Agilent Technologies, Inc. MHC multimers in tuberculosis diagnostics, vaccine and therapeutics
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
US9404916B2 (en) 2008-09-20 2016-08-02 University College Cardiff Consultants Limited Use of a protein kinase inhibitor to detect immune cells, such as T cells
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease
US10369204B2 (en) 2008-10-02 2019-08-06 Dako Denmark A/S Molecular vaccines for infectious disease
US8263387B2 (en) 2009-06-10 2012-09-11 Cynvenio Biosystems, Inc. Sheath flow devices and methods
EP2701851A4 (fr) * 2011-04-29 2015-01-28 Becton Dickinson Co Systèmes fluidiques d'immobilisation et de collecte de particules en continu et leurs procédés d'utilisation
EP2701851A1 (fr) * 2011-04-29 2014-03-05 Becton, Dickinson and Company Systèmes fluidiques d'immobilisation et de collecte de particules en continu et leurs procédés d'utilisation
WO2014082752A1 (fr) * 2012-11-30 2014-06-05 Hochschule Trier Dispositif pour la séparation de microparticules magnétiques ou magnétisables d'une suspension au moyen d'une séparation magnétique à gradient élevé

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