EP1342088A1 - Verfahren für die magnetisierung der chemischen und biologischen markierungen - Google Patents

Verfahren für die magnetisierung der chemischen und biologischen markierungen

Info

Publication number
EP1342088A1
EP1342088A1 EP01999812A EP01999812A EP1342088A1 EP 1342088 A1 EP1342088 A1 EP 1342088A1 EP 01999812 A EP01999812 A EP 01999812A EP 01999812 A EP01999812 A EP 01999812A EP 1342088 A1 EP1342088 A1 EP 1342088A1
Authority
EP
European Patent Office
Prior art keywords
markers
particles
magnetic particles
biological
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01999812A
Other languages
English (en)
French (fr)
Inventor
Frédéric BUFFIERE
Christine Betremieux
Laetitia Gaillard
Gérard OVLAQUE
Christophe Vinzia
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Diagast SAS
Original Assignee
Diagast SAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Diagast SAS filed Critical Diagast SAS
Publication of EP1342088A1 publication Critical patent/EP1342088A1/de
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the invention relates to a method of magnetizing chemical or biological markers as well as to the use of said biological markers in a biological analysis test.
  • Immunological analysis tests then typically relate to the search, in blood or in a blood component, for antigens present on the surface of red blood cells using magnetized anti-erythrocyte antibodies (for example erythrocyte typing) or the anti-erythrocyte antibody research using magnetized red cells on which specific antigens are present and / or attached.
  • magnetized anti-erythrocyte antibodies for example erythrocyte typing
  • anti-erythrocyte antibody research using magnetized red cells on which specific antigens are present and / or attached.
  • This type of process uses magnetic particles, the surface of which has been functionalized so as to form bonds with a specific marker.
  • the invention therefore aims to remedy all of these drawbacks by proposing in particular a method of direct magnetization of particulate and / or figured elements without the intermediary of molecules recognizing for example structures of antigen and / or antibody type and this without masking and / or modifying the structures to be implemented, for example during a biological analysis test.
  • the invention provides a method of magnetizing chemical or biological markers using magnetic particles, said method comprising the steps consisting in: - activating the magnetic particles so as to modify their state of surface ; - bringing the activated magnetic particles into contact with the markers so as to create non-specific bonds between them.
  • the invention proposes the use of magnetized biological markers by the implementation of such a method as reagents or analytes in a biological analysis test.
  • the method makes it possible to magnetize markers using magnetic particles by creating non-specific bonds between them.
  • the markers can be chemical, for example in molecular form, or biological, for example in cellular form, and the magnetic particles are for example beads of compatible polymers which are loaded with a magnetic material.
  • a marker is in contact with its immediate environment via its surface and, in the case of a cellular element, through its membrane.
  • the cell has, in a way, an arsenal of possible links with various surrounding molecular structures, such as magnetic particles.
  • the cell is able to establish links with elements as well so:
  • a receptor structure of the membrane is recognized by an effector which is specific to it (for example in the case of hormone-receptor bonds or antigen-antibody bonds);
  • This cell membrane the first meeting structure of the cell, will therefore be the site of important interactions both for the cell and for the external environment. It carries on its surface the majority of molecular structures for identifying the cell, but also structures capable of specifically or not binding to foreign molecules.
  • the markers are red blood cells whose cell membrane is the support for the antigenic structures defining the various blood groups which are sought before any blood transfusion.
  • the process according to the invention uses the large areas of phospholipids and / or proteins present on the surface of a red cell, said zones not directly involved in the definition of blood groups and phenotypes.
  • the red cells thus magnetized have the double property of being attracted under the effect of a magnetic field and of carrying on their surface the antigenic structures mentioned above. -above.
  • the magnetized red blood cell will therefore be able to retain its expression properties of an antigenic structure while being mobile.
  • markers and in particular red blood cells, are treated in such a way that their magnetic susceptibility is greatly increased, thus allowing them to migrate in a magnetic field created by a permanent magnet or an electromagnet.
  • magnetic marking is not done by means of a magnetized probe molecule but by the use of particles interacting in a non-specific way with the red blood cell membrane so as to create a multitude of weak intensity bonds between the red blood cell surface and magnetic particles.
  • magnetic particles are used which have the characteristics of having a very high homogeneity in size, in particular less than a micron and for example around 200 nm, a high load of ferromagnetic material for example around 75% by mass and a rather hydrophobic surface finish.
  • the size of the markers is greater than that of the particles used to allow transfer into the magnetic field.
  • These particles are attached to the surface of the red blood cell, for example by the intermediary of bovine serum albumin so as to create multiple non-specific bonds and low intensities between the surface of the red blood cell and the particles.
  • the fixation takes place in two stages, the first consists in the activation of the particles so as to modify their surface state and the second is the bringing into contact of these activated particles with a suspension of red blood cells treated or not by proteolytic enzymes, so as to create non-specific bonds between particles and red cells.
  • Activation can be carried out either immediately before contacting the marker or during manufacture.
  • the red cells thus obtained are attracted by a magnetic field and can thus be used directly or, in a variant, treated with solutions of enzymes generally encountered in immuno-hematological tests.
  • An embodiment of the method for magnetizing red cells without damaging the antigens which they carry is described below, in which the activation of the particles is carried out using a sticky substance comprising a solution of bovine albumin.
  • Particles of type P201 from the company Ademtech are placed in the presence of a solution of bovine albumin at 0.1% (weight / volume) in PBS buffer pH 7.2. After an incubation of thirty minutes at room temperature and with shaking (proscribe any magnetic shaking), the particles in suspension are attracted by a magnet and the supernatant devoid of particles is eliminated.
  • the “glued” particle pellet can be used directly during the red blood cell awareness phase.
  • the activation of the particles can be carried out, optionally in addition to the action of a sticky substance, using a wetting agent or detergent such as cholic acid or Tween 20® so to modify the surface state of said particles.
  • this activation can be carried out using electromagnetic radiation, such as gamma or UV radiation, which are known to modify plastic-type surfaces.
  • Second step raising red blood cells
  • the globular suspension buffered LISS Low lonic Strenght Solution
  • the suspension After having perfectly homogenized the suspension (check that there are no more particle aggregates), it is incubated for 30 minutes at room temperature with stirring soft but homogeneous (the entire reaction volume must be set in motion).
  • the red cells are then washed with a PBS buffer pH 7.4 (two washings by centrifugation, three minutes at 500 g).
  • the pellet of sensitized red cells can then be taken up at the concentration for carrying out the analysis using a LISS buffer.
  • the ratio between the quantity of particles used and the quantity of red blood cells is between 600 and 1000 so as to obtain sufficient magnetization without risking degrading the antigens present on the surface of the red blood cell.
  • the surface occupied by the particles typically represents around 10% of the total surface of the red blood cell membrane.
  • red cells sensitized by paramagnetic particles then have the double property of being attracted by a magnetic field and also of possessing on their surface blood antigens (group and phenotype). They can then be used as a reaction support and vector for transporting the antigen-antibody couple in an immunological analysis test.
  • the red cells thus obtained can be used in tests of RAI type (Search for Irregular Agglutinins) either directly as a reagent or as an analyte, or undergo treatment with proteolytic enzymes such as papain to perform a so-called enzymatic analysis.
  • RAI type Search for Irregular Agglutinins
  • red cells having ferromagnetic particles on their surface giving them a paramagnetic property can be driven under the action of a magnetic force towards the reactive zone of a display device so as to allow the detection of directed antibodies. against antigenic determinants present on the surface of red blood cells. It is also possible to treat directly using the method described the red blood cells as an analyte of a blood sample to make them paramagnetic, and thus allow the migration of said red blood cells to an area capable of detecting the antigens that 'they support.
  • particulate elements for example antibodies
  • magnetized antibodies they can be used for training said red cells for example for their grouping.
  • chemical markers can be treated so as to be made paramagnetic by means of a method analogous to that presented above.
  • Such magnetized markers then have the dual property of being attracted under the effect of a magnetic field and of retaining a functional surface to allow the coupling of all kinds of chemical or biological molecules.
  • the method of the invention allows direct magnetization of markers, and in particular of figured elements, without the use of covalently coupled molecules.
  • the interaction between the particles and the markers is not final. There is therefore a possibility of finding the markers in their initial state after desorption of the particles. This desorption step can be carried out under mild conditions which do not alter the surface of the marker.
  • the method also has the advantage of the speed and simplicity of setting up the interaction between the markers and the particles, simply under the influence of the probabilities of encountering the elements which have to interact.
  • the particles used are non-organic products whose shelf life is very long and incommensurate with that of particles functionalized with organic products which are known to be highly unstable over time.

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
EP01999812A 2000-12-08 2001-12-07 Verfahren für die magnetisierung der chemischen und biologischen markierungen Withdrawn EP1342088A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR0016026A FR2817967B1 (fr) 2000-12-08 2000-12-08 Procede de magnetisation de marqueurs chimiques ou biologiques
FR0016026 2000-12-08
PCT/FR2001/003887 WO2002046758A1 (fr) 2000-12-08 2001-12-07 Procede de magnetisation de marqueurs chimiques ou biologiques

Publications (1)

Publication Number Publication Date
EP1342088A1 true EP1342088A1 (de) 2003-09-10

Family

ID=8857446

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01999812A Withdrawn EP1342088A1 (de) 2000-12-08 2001-12-07 Verfahren für die magnetisierung der chemischen und biologischen markierungen

Country Status (5)

Country Link
US (1) US20040063163A1 (de)
EP (1) EP1342088A1 (de)
AU (1) AU2002217216A1 (de)
FR (1) FR2817967B1 (de)
WO (1) WO2002046758A1 (de)

Families Citing this family (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2359689B1 (de) 2002-09-27 2015-08-26 The General Hospital Corporation Mikrofluid-Vorrichtung zur Zelltrennung, sowie Verwendung derselben
US20050266433A1 (en) * 2004-03-03 2005-12-01 Ravi Kapur Magnetic device for isolation of cells and biomolecules in a microfluidic environment
FR2869996B1 (fr) * 2004-05-05 2006-12-15 Diagast Soc Par Actions Simpli Utilisation de ferrofluides pour le phenotypage sanguin et applications derivees
US20070026413A1 (en) * 2005-07-29 2007-02-01 Mehmet Toner Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070026417A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070026415A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070026414A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
EP1874920A4 (de) * 2005-04-05 2009-11-04 Cellpoint Diagnostics Vorrichtungen und verfahren zur anreicherung und veränderung zirkulierender tumorzellen und anderer partikel
US20070196820A1 (en) 2005-04-05 2007-08-23 Ravi Kapur Devices and methods for enrichment and alteration of cells and other particles
US20070059680A1 (en) * 2005-09-15 2007-03-15 Ravi Kapur System for cell enrichment
US20070026416A1 (en) * 2005-07-29 2007-02-01 Martin Fuchs Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US8921102B2 (en) 2005-07-29 2014-12-30 Gpb Scientific, Llc Devices and methods for enrichment and alteration of circulating tumor cells and other particles
US20070059782A1 (en) * 2005-09-13 2007-03-15 Graham Henry A Magnetic particle tagged blood bank reagents and techniques
US9488665B2 (en) * 2005-09-13 2016-11-08 Chrome Red Technologies, Llc Magnetic particle tagged reagents and techniques
US20070059719A1 (en) * 2005-09-15 2007-03-15 Michael Grisham Business methods for prenatal Diagnosis
US20070059718A1 (en) * 2005-09-15 2007-03-15 Mehmet Toner Systems and methods for enrichment of analytes
US20070059774A1 (en) * 2005-09-15 2007-03-15 Michael Grisham Kits for Prenatal Testing
US20070059716A1 (en) * 2005-09-15 2007-03-15 Ulysses Balis Methods for detecting fetal abnormality
US20070059781A1 (en) * 2005-09-15 2007-03-15 Ravi Kapur System for size based separation and analysis
US20070059683A1 (en) * 2005-09-15 2007-03-15 Tom Barber Veterinary diagnostic system
FR2963108B1 (fr) 2010-07-21 2017-06-23 Diagast Procede magnetique d'immunodiagnostic et kit pour la mise en evidence de complexe anticorps/antigene de groupe/phenotype sanguin

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US4935147A (en) * 1985-12-20 1990-06-19 Syntex (U.S.A.) Inc. Particle separation method
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DE68919565T2 (de) * 1988-07-20 1995-06-29 Olympus Optical Co Immuntestverfahren unter Verwendung magnetischer Markerteilchen.
AU4746590A (en) * 1988-12-28 1990-08-01 Stefan Miltenyi Methods and materials for high gradient magnetic separation of biological materials
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JP3996644B2 (ja) * 1996-06-07 2007-10-24 イムニベスト・コーポレイション 外部及び内部勾配を有する磁気分離
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See also references of WO0246758A1 *

Also Published As

Publication number Publication date
FR2817967A1 (fr) 2002-06-14
FR2817967B1 (fr) 2003-02-28
US20040063163A1 (en) 2004-04-01
WO2002046758A1 (fr) 2002-06-13
AU2002217216A1 (en) 2002-06-18

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