WO1997020214A1 - Extraction et marquage de materiaux biologiques par perles ou liposomes magnetiques et fluorescents - Google Patents

Extraction et marquage de materiaux biologiques par perles ou liposomes magnetiques et fluorescents Download PDF

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Publication number
WO1997020214A1
WO1997020214A1 PCT/GB1996/002919 GB9602919W WO9720214A1 WO 1997020214 A1 WO1997020214 A1 WO 1997020214A1 GB 9602919 W GB9602919 W GB 9602919W WO 9720214 A1 WO9720214 A1 WO 9720214A1
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WIPO (PCT)
Prior art keywords
agent
target
core
binding
agents
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PCT/GB1996/002919
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English (en)
Inventor
Andrew Charles Pinder
Rosemary Georgina Clarke
David Cunliffe
Original Assignee
The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
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Application filed by The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland filed Critical The Minister Of Agriculture, Fisheries And Food In Her Britannic Majesty's Government Of The United Kingdom Of Great Britain And Northern Ireland
Priority to AU76363/96A priority Critical patent/AU7636396A/en
Publication of WO1997020214A1 publication Critical patent/WO1997020214A1/fr

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Classifications

    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • CCHEMISTRY; METALLURGY
    • C08ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08FMACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F292/00Macromolecular compounds obtained by polymerising monomers on to inorganic materials
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326Magnetic particles
    • G01N33/5434Magnetic particles using magnetic particle immunoreagent carriers which constitute new materials per se
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00Magnetic particle immunoreagent carriers
    • G01N2446/20Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00Magnetic particle immunoreagent carriers
    • G01N2446/40Magnetic particle immunoreagent carriers the magnetic material being dispersed in the monomer composition prior to polymerisation
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00Magnetic particle immunoreagent carriers
    • G01N2446/60Magnetic particle immunoreagent carriers the magnetic material being dispersed in a medium other than the main solvent prior to incorporation into the polymer particle
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00Magnetic particle immunoreagent carriers
    • G01N2446/60Magnetic particle immunoreagent carriers the magnetic material being dispersed in a medium other than the main solvent prior to incorporation into the polymer particle
    • G01N2446/64Magnetic material dispersed in oil drop
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2446/00Magnetic particle immunoreagent carriers
    • G01N2446/60Magnetic particle immunoreagent carriers the magnetic material being dispersed in a medium other than the main solvent prior to incorporation into the polymer particle
    • G01N2446/66Magnetic material dispersed in surfactant

Definitions

  • the present invention relates to methods having utility for the extraction of target materials present as part of a larger source material, and their fluorescent labelling for detection purposes.
  • the invention further relates to agents for use in these methods, methods for preparation of these agents, and kits employing them.
  • target materials e.g. biological materials such as pathogenic organisms
  • target DNA can be labelled specifically using a fluorescent nucleotide probe.
  • fluorescent nucleotide probe Another commonly used technique is ' immunofluorescence' whereby antibodies conjugated to a fluorescent agent may be specifically bound to a target antigenic material
  • Such specific labelling techniques are particularly useful when the target material is initially present m only small quantities in a sample material e.g. for detecting specific bacterial contaminants m food.
  • the target can be more readily detected and quantified.
  • one useful technique is immunofluorescence flow cytometry. Using this technique it is possible to detect specific organisms in a sample material down to levels of 10 /ml (Pinder & McClelland 1994; J Microscopy 176: 17-22) .
  • Japanese patent application JP 04285857 A published in 1992 discloses ferromagnetic particles having fluorescently labelled antibody coupled thereto. Suitable antigens can be assayed by contacting them with the antibody and detecting the fluorescence intensity.
  • the present invention provides, methods and kits with one or more improved properties compared with any of these prior art technologies .
  • a detection agent for detecting target biological material present m a sample material comprising: (a) a core material; (b) binding means peripherally disposed away from the surface of the core material for binding to the target; (c) magnetic means present within the core material (d) and a fluorescent dye present withm the core material characterised m that the agent is less than 1 ⁇ m m diameter.
  • the agents may be produced as monodispersed particles having uniform properties (size, fluorescence, magnetic content) .
  • the target material is a cellular organism, and more preferably it s a single cell e.g. a bacterium.
  • the invention is applied to sample materials wherein the target material is present only m a relatively small concentration e.g. as a contaminant, or as a minor component of a larger population.
  • the sample material is a foodstuff, which may be prepared such as to increase the ease of extraction e.g. by liquefaction or dilution.
  • the size of the agent is important in determining whether it will be suitable for flow cytometry. Thus preferably the size of agent is approximately 10% of the size of the target.
  • the agent's diameter is less than 500 nm, more preferably less than 200 nm or less than 100 nm, but most preferably approximately lOOnm (i.e. within 20%, plus or minus) .
  • the size of the agent is important because it is one factor which will determine how many agents will bind to the target.
  • the diameter of the core material of the agent is selected such that in use at least 2 agents are bound per target, and more preferably at least 5 agents are bound per target. This will allow the easy discrimination between the high accumulated fluorescence of the target bacteria and the much weaker signal from an unbound bead in solution.
  • the size and the fluorescence loading of the core of the agent is such that a single target having one (but preferably more) agents bound thereto is readily distinguishable from a single agent, or from an unlabelled target, using fluorescence-detection and, where required, light scatter flow cytometry.
  • the fluorescent dyes and magnetic particles may be present as a dispersion within the core. Alternatively they may be present in a void within the core.
  • the use of a labelled core has the advantage that a high loading of fluorescent dye can be achieved compared to direct fluorescent labelling of antibodies, and background leakage of the fluorescent label can be virtually eliminated: this provides a high contrast system when the beads and complexes are detected.
  • the binding means must be suitably disposed away from the core in order to be able to effectively bind the target e.g. in the form of one or more peripherally attached antibodies.
  • the core of the detection agent is composed of a polymeric material, e.g. latex, for retaining the magnetic and fluorescent elements of the agent, and for attachment of the binding means.
  • the core is a liposome core.
  • polymer cores may be more robust to storage and more widely applicable than liposomes, under some circumstances liposomes may easier to prepare and load with fluorescent dye.
  • the agents of the invention can be readily detected individually by flow cytometry.
  • the binding means is an antibody capable of specific binding to the target material or an antigen thereon; antibodies thus used ' could be polyclonal but are preferably monoclonal in order to aid consistency of binding.
  • target-binding antibodies are attached to each particle such as to increase the chances of successful binding of the particle to the target.
  • target-binding antibodies may be attached directly to the particles by conjugation methods well known to those skilled in the art e.g. by using carbodiimide activation. It may be desirable to attach a further connecting group between the two, such as Protein A.
  • biotin may be incorporated into the lipid bilayer (e.g. in the form of Biotin-X-dipalmityl phosphatidyl ethanolamine) . This can then be used to anchor target-binding antibodies
  • the surface and/or sample material are 'blocked' with a suitable agent e.g. serum proteins.
  • the magnetic means allows extraction of the bound target from the sample material by use of magnetic force e.g. by methods analogous to those presently used in non fluorescent magnetic beads such as Dynabeads (TM) .
  • the magnetic means comprises a paramagnetic material such as magnetite.
  • fluorescent dye any fluorescent material or functional group.
  • materials and groups are well known to those skilled in the art e.g. dan ⁇ yl chloride (UV excitable), fluorescein isothiocyanate.
  • a method for detecting a target material present m a sample material comprising the steps of: (a) combining agent as described above with the sample under conditions whereby the agent can bind the target, (b) extracting agent and target/agent complex from the sample using magnetic force, (c) detecting the target/agent complex by means of the fluorescent dye associated therewith.
  • the target/agent complex is detected by fluorescence- detection flow cytometry, for instance by methods analogous to those described in Pinder & McClelland 1994, supra.
  • fluorescence- detection flow cytometry for instance by methods analogous to those described in Pinder & McClelland 1994, supra.
  • a less sophisticated optical system is required to detect and quantify the targets. This will allow the use of cheaper more rugged detection instruments, thereby increasing the attractiveness of the method to industry and encouraging 'pro ⁇ active' intervention in manufacturing processes. Such intervention is presently difficult because the widely used detection techniques e.g. culture techniques, take too long to perform.
  • the use of luorescence-detection flow cytometry m conjunction with the agents of the present invention may allow not only detection of target, but also estimation or quantification of it, thus allowing the severity of contamination to be assessed.
  • kits suitable for use comprising 'cores' of a size as discussed above in relation to the agents of the present invention, containing magnetic means and a fluorescent dye, and being adapted to be attached to an antibody capable of binding a target material in a sample material .
  • the beads may be adapted for target antibody attachment by having a surface of activatable carboxyl groups, or by having an intermediate antibody capable of specifically binding a target- specific antibody, or by other means discussed above or such as may occur to those skilled in the art.
  • kits will allow users to readily prepare the agents of the present invention using target antibodies of their choice.
  • the polymer beads can be prepared by the emulsion polymerisation of monomers in the presence of an aqueous dispersion of colloidal magnetite.
  • the fluorescent dye can be incorporated in the polymerisation process as discussed in more detail below.
  • the invention makes available agents, methods, processes and kits for the detection of target materials which address many, and preferred forms all, of the problems of the prior art.
  • the relative cheapness and simplicity of the agents, and the methods of their use, means that they can be used as part of regular quality control or investigative testing programmes.
  • the invention is particularly suited to the food industry wherein the facility for rapid testing for bacterial contaminants will reduce the possibility of any infected foodstuffs leaving the factory.
  • Fig 1 Shows the size distribution of the latex beads prepared in accordance with Example 1 as judged by light scatter.
  • Fig 2 Shows a schematic scheme for the conjugation of Protein A to the beads .
  • EXAMPLE 1 PROCESS FOR MANUFACTURE OF MAGNETIC/FLUORESCENT POLYMER PARTICLES
  • Polymer particles were prepared by the emulsion polymerisation of monomers in the presence of an aqueous dispersion of colloidal magnetite and fluorescein.
  • the magnetite (' ferrofluid' ) was prepared by the co-precipitation of iron(II) and iron (III) hydroxides. These ultra-fine magnetite particles were then coated in a double layer of surfactant: the primary layer was a long chain fatty acid (oleic) chemically adsorbed on to the magnetite surface; the secondary layer was either a fatty acid salt, long chain sulphonic acid or sulphate acid salt, (sodium dodecyl sulphate and sodium dodecylbenzene sulphonate were investigated) . Agents were selected such as to avoid later extensive demulsification (creaming) in the presence of the monomer (styrene or styrene/methacrylic acid) .
  • the fluorescent dye fluorescein was incorporated during the latex polymerisation process. This dye, unlike rhodamine dyes, does not leech out significantly. Dansyl chloride was also used, being prepared by the method of Rembaum et al (1976) Macromolecules 9, 238, but it was found that the resulting dye had a relatively low solubility in the monomer mixture.
  • the detailed polymerisation method was based on that of Naguchi et al (1993) J. Appl. Poly, Sci. 48, 1539 and Yana ⁇ e et al (1993) J. Appl. Polym. Sci. 50, 765 and was as follows:
  • ACVA ' -Azobis-4 -cyanopentanoic
  • the particle surface was characterised by a method was based on that of Vanderhoff et al (1970) In 'Clean Surfaces: Their Preparation and Characterisation for Interfacial Studies', ed. G Goldfmger. Marcel Dekker, New York.
  • To determine the carboxyl content of the polymer particles the latex was cleaned by contact with a bed of mixed ion exchange resms (IR 120 PLUS and IRA 410 Aldrich Chemical Co) for 12 hours. This removed surfactant molecules from the latex and converted the carboxyl groups on the particles' surface into the acid form.
  • the solid ⁇ content of the latex was determined by taking a sample of known mass and evaporating to dryness. Another sample was then taken and titrated with sodium hydroxide solution (0.5 mM) while purging with nitrogen. The end point was determined by measuring the conductivity as the sodium hydroxide was added.
  • the active carboxy groups of the synthesi ⁇ ed particles were used for covalent Protein A and antibody attachment, as shown schematically in Fig 2.
  • Alternative 'bridging' molecules to Protein A were al ⁇ o tried (e.g. intermediate antibodies) - however such molecules lead to beads which were more prone to clumping and therefore Protein A was adopted for the standard preparation.
  • the beads as prepared have carboxyl groups exposed on their surface. These groups were activated using the carbodiimide (CDI) activation procedure (Lea et al (1990) in “Flow Cytometry and Sorting” Eds. Melamed, Lidmo and Mendelsohn; Wiley-Liss, New York) .
  • CDI carbodiimide
  • Thi ⁇ allowed a slow removal of the ⁇ urfactant from the beads and covalent attachment of the Protein A.
  • Bead ⁇ were wa ⁇ hed three time ⁇ in 0.01 M Hepes, pH 7.0 °C and stored in the same containing 0.1% BSA.
  • ATTACHMENT OF ANTIBODY A solution of ammonium sulphate- precipitated tissue culture supernatant containing Salmonella- specific IgG antibody was added to the beads. After mixing, absorption of the IgG to the protein A was allowed overnight at 4°C with gentle rotation. The supernatant was then removed and the beads washed twice in PBS pH 7.0, containing 0.1 % BSA and stored in the same. In order to minimise clumping, 0.05% v/v Tween-20 (TM) was sometimes used.
  • TM v/v Tween-20
  • Samples containing bacteria were prepared in phosphate buffered saline at pH 7.4 (PBS) . To each 1 ml of sample, 50 ⁇ l of prepared particles were added. This mixture was then rotated gently for 20-30 minutes at room temperature to allow attachment of the beads to the antigen.
  • PBS phosphate buffered saline at pH 7.4
  • the extraction efficiency of the beads at different cell concentrations is shown in Table 3.
  • the target-binding specificity of the particles was found to be improved when the surface of the particles was 'blocked' with mouse serum (5% BSA for 3 hours) . This was found to reduce (relatively) the non-specific binding of non-target organisms to the latex beads (Table 4 ]
  • Table 4 Effect of blocking procedures on the percentage extraction of bacteria at 10 4 cells/ml.
  • FLOW CYTOMETRY This was carried out essentially as described in Pinder & McClelland 1994 (supra) the content of which document is incorporated herein by reference. In this study samples were analysed on the basis of narrow angle ( ⁇ 20°) forward light scatter, wide angle (90°) light scatter, and red and green fluorescence. The sample was introduced into the flow cytometer at speeds of between 5 and 30 1/mm, depending on the exact concentration of the sample (resuspended beads) . From the data it is possible to distinguish between unlabelled bacteria, bacteria labelled with beads and unassociated beads. It is also possible to obtain a value for the concentration of each population.
  • ADDITIONAL STAINING OF BACTERIA fluorescent antibody labels do not distinguish between viable and non-viable target organisms. Viability was assessed with a secondary staining procedure using proprietary ChemChrome sta (Chemunex) . Essentially only bacteria with intact outer membranes and active intracellular esterases fluoresce green as a result of the procedure. The use of this sta , m conjunction with green fluorescence flow cytometry, can increase the certainty of the result ⁇ obtained using the fluorescem/light scatter detection alone.
  • liposome based agents were also tested. Liposomes were produced using the reverse-phase separation technique, a modification of the procedures described m Szoka and Papahad opoulos (1978) Proc Natl Acad Sci USA 75(9) : 4194 4198, and Siebert et al (1995) Anal Chim Acta 311: 309-318.
  • the ratio of dipalmityl phosphatidyl choline: cholesterol: dipalmityl phosphatidyl glycerol: Biotm-X-dipalmityl phosphatidyl ethanolamine was 5 : 5 : 0.5 : 0.01.
  • Carboxyfluorescem (2.5 mM) was encapsulated withm the liposome. Magnetite may also be encapsulated using methods analogous to those above, or as used De Cuyper and Joiau (1988) Eur Biophys J 15: 311-319.
  • Liposomes were filtered sequentially through 3, 0.4 and 0.22 ⁇ m pore size filters.
  • Antibodies were conjugated to mouse anti-S typhimurium antibody (43 ⁇ g/ml)using streptavidin (27 ⁇ g/ml) and goat anti-mouse IgM-biotm (42 ⁇ g/ml) . This was mixed with liposomes (diluted 1:10) . Conjugation of the antibodies was confirmed using a test strip containing (first) strips of bound S. typhimurium lipopolysaccharide and then (second) goat anti biotin. Binding of the liposomes to these strip ⁇ was confirmed using UV to visualise the liposomes. The protocol used produced optimum binding (i.e.
  • liposome ⁇ localised the first strip.
  • the liposomes v/ere found to be stable (no fluorescence leakage) to the shear forces induced by passage through the flow cytometer.
  • bacteria with bound liposome ⁇ could be readily distinguished from unbound liposomes by their different light scatter characteristics, and from unlabelled bacteria by their difrerent fluorescent characteristics.

Abstract

Agent de détection servant à détecter des matériaux cibles, particulièrement des matériaux biologiques tels que cellules bactériennes, présents dans un échantillon (par exemple alimentaire), cet agent comprenant un moyen de liaison (par exemple un anticorps) pouvant se fixer sur la cible, un moyen magnétique d'extraction de la cible et un colorant fluorescent pour le marquage de la cible. Les agents sont de diamètre inférieur à 1 νm, de préférence de l'ordre de 100 nm. Sont également décrits des procédés d'utilisation selon lesquels les cellules, chacune étant marquée par plusieurs agents à perles polymères, sont détectés par cytométrie de flux par détection de fluorescence. L'invention concerne également des nécessaires destinés à la mise en oeuvre de cette méthode, et des procédés de fabrication des nécessaires et des agents.
PCT/GB1996/002919 1995-11-29 1996-11-28 Extraction et marquage de materiaux biologiques par perles ou liposomes magnetiques et fluorescents WO1997020214A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU76363/96A AU7636396A (en) 1995-11-29 1996-11-28 Extraction and labelling of biological materials with magnetic and fluorescent beads or liposomes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9524393.7 1995-11-29
GBGB9524393.7A GB9524393D0 (en) 1995-11-29 1995-11-29 Extraction and labelling of materials

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WO1997020214A1 true WO1997020214A1 (fr) 1997-06-05

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1210461A1 (fr) * 1999-08-21 2002-06-05 John S. Fox Detection a grande sensibilite de biomolecules, au moyen de particules magnetiques
FR2832507A1 (fr) * 2001-11-20 2003-05-23 Stago Diagnostica Methode de detection d'analyte(s) a l'aide de particules magnetiques colloidales
WO2006079016A1 (fr) * 2005-01-20 2006-07-27 Luminex Corporation Mircrospheres magnetiques et applications basees sur la fluorescence
WO2007143615A3 (fr) * 2006-06-02 2008-03-20 Luminex Corp Systèmes et procédés pour effectuer des mesures sur un ou plusieurs matériaux
US8296088B2 (en) 2006-06-02 2012-10-23 Luminex Corporation Systems and methods for performing measurements of one or more materials
US8747677B2 (en) 2010-04-09 2014-06-10 Luminex Corporation Magnetic separation device

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4230685A (en) * 1979-02-28 1980-10-28 Northwestern University Method of magnetic separation of cells and the like, and microspheres for use therein
US4369226A (en) * 1979-03-19 1983-01-18 California Institute Of Technology Polyglutaraldehyde synthesis and protein bonding substrates
US4783336A (en) * 1981-07-01 1988-11-08 Yeda Research And Development Company, Ltd. Polyacrolein-type microspheres
JPH03137022A (ja) * 1989-10-19 1991-06-11 Tdk Corp 結合性官能基を有する磁気微粒子およびその製造方法
US5076950A (en) * 1985-12-20 1991-12-31 Syntex (U.S.A.) Inc. Magnetic composition for particle separation
US5320906A (en) * 1986-12-15 1994-06-14 Vestar, Inc. Delivery vehicles with amphiphile-associated active ingredient
WO1994013751A1 (fr) * 1992-12-11 1994-06-23 Minnesota Mining And Manufacturing Company Microbilles collantes contenant des fragments oligomeres ou polymeres hydrophiles pendants
US5393527A (en) * 1993-01-04 1995-02-28 Becton, Dickinson And Company Stabilized microspheres and methods of preparation
US5395688A (en) * 1987-10-26 1995-03-07 Baxter Diagnostics Inc. Magnetically responsive fluorescent polymer particles

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4230685A (en) * 1979-02-28 1980-10-28 Northwestern University Method of magnetic separation of cells and the like, and microspheres for use therein
US4369226A (en) * 1979-03-19 1983-01-18 California Institute Of Technology Polyglutaraldehyde synthesis and protein bonding substrates
US4783336A (en) * 1981-07-01 1988-11-08 Yeda Research And Development Company, Ltd. Polyacrolein-type microspheres
US5076950A (en) * 1985-12-20 1991-12-31 Syntex (U.S.A.) Inc. Magnetic composition for particle separation
US5320906A (en) * 1986-12-15 1994-06-14 Vestar, Inc. Delivery vehicles with amphiphile-associated active ingredient
US5395688A (en) * 1987-10-26 1995-03-07 Baxter Diagnostics Inc. Magnetically responsive fluorescent polymer particles
JPH03137022A (ja) * 1989-10-19 1991-06-11 Tdk Corp 結合性官能基を有する磁気微粒子およびその製造方法
WO1994013751A1 (fr) * 1992-12-11 1994-06-23 Minnesota Mining And Manufacturing Company Microbilles collantes contenant des fragments oligomeres ou polymeres hydrophiles pendants
US5393527A (en) * 1993-01-04 1995-02-28 Becton, Dickinson And Company Stabilized microspheres and methods of preparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
NOGUCHI H. ET AL: "Preparation and Characterisation by Thermal Analysis of Magnetic Latex Particles", JOURNAL OF APPLIED POLYMER SCIENCE, 1993, NEW YORK US, XP002027479 *
PATENT ABSTRACTS OF JAPAN vol. 015, no. 345 (C - 0864) 3 September 1991 (1991-09-03) *
YANASE N ET AL: "PREPARATION OF MAGNETIC LATEX PARTICLES BY EMULSION POLYMERIZATION OF STYRENE IN THE PRESENCE OF A FERROFLUID", JOURNAL OF APPLIED POLYMER SCIENCE, vol. 50, no. 5, 5 November 1993 (1993-11-05), pages 765 - 776, XP000464169 *

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* Cited by examiner, † Cited by third party
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EP1210461A4 (fr) * 1999-08-21 2005-11-23 John S Fox Detection a grande sensibilite de biomolecules, au moyen de particules magnetiques
EP1210461A1 (fr) * 1999-08-21 2002-06-05 John S. Fox Detection a grande sensibilite de biomolecules, au moyen de particules magnetiques
US7332353B2 (en) 2001-11-20 2008-02-19 Diagnostica Stago Method for detecting analyte(s) using magnetic colloidal particles
FR2832507A1 (fr) * 2001-11-20 2003-05-23 Stago Diagnostica Methode de detection d'analyte(s) a l'aide de particules magnetiques colloidales
WO2003044532A1 (fr) * 2001-11-20 2003-05-30 Diagnostica Stago Methode de detection d'analyte(s) a l'aide de particules magnetiques colloidales
US8283037B2 (en) 2005-01-20 2012-10-09 Luminex Corporation Magnetic microspheres for use in fluorescence-based applications
US8968874B2 (en) 2005-01-20 2015-03-03 Luminex Corporation Magnetic microspheres for use in fluorescence-based applications
CN101650998A (zh) * 2005-01-20 2010-02-17 卢米尼克斯股份有限公司 微球、包含其的微球群、以及分析试验样品的方法
US7718262B2 (en) 2005-01-20 2010-05-18 Luminex Corporation Magnetic microspheres for use in fluorescence-based applications
EP2244270A3 (fr) * 2005-01-20 2011-02-23 Luminex Corporation Microsphères magnétiques et applications basées sur la fluorescence
WO2006079016A1 (fr) * 2005-01-20 2006-07-27 Luminex Corporation Mircrospheres magnetiques et applications basees sur la fluorescence
US9645142B2 (en) 2005-01-20 2017-05-09 Luminex Corporation Magnetic microspheres for use in fluorescence-based applications
CN101650998B (zh) * 2005-01-20 2013-02-06 卢米尼克斯股份有限公司 微球、包含其的微球群、以及分析试验样品的方法
US8568881B2 (en) 2005-01-20 2013-10-29 Luminex Corporation Magnetic microspheres for use in fluorescence-based applications
US9376613B2 (en) 2005-01-20 2016-06-28 Luminex Corporation Magnetic microspheres for use in fluorescence-based applications
US8798951B2 (en) 2006-06-02 2014-08-05 Luminex Corporation Systems and methods for performing measurements of one or more materials
EP2765423A1 (fr) 2006-06-02 2014-08-13 Luminex Corporation Systèmes et procédé pour réaliser des mesures sur un ou plusieurs matériaux
US8889347B2 (en) 2006-06-02 2014-11-18 Luminex Corporation Systems and methods for performing measurements of one or more materials
WO2007143615A3 (fr) * 2006-06-02 2008-03-20 Luminex Corp Systèmes et procédés pour effectuer des mesures sur un ou plusieurs matériaux
US8296088B2 (en) 2006-06-02 2012-10-23 Luminex Corporation Systems and methods for performing measurements of one or more materials
US10451541B2 (en) 2006-06-02 2019-10-22 Luminsex Corporation Systems and methods for performing measurements of one or more materials
US11125678B2 (en) 2006-06-02 2021-09-21 Luminex Corporation Systems and methods for performing measurements of one or more materials
US8747677B2 (en) 2010-04-09 2014-06-10 Luminex Corporation Magnetic separation device

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