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WO2007051459A2 - Method for the selective particle-bound enrichment of biomolecules or bioparticles - Google Patents

Method for the selective particle-bound enrichment of biomolecules or bioparticles Download PDF

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Publication number
WO2007051459A2
WO2007051459A2 PCT/DE2006/001931 DE2006001931W WO2007051459A2 WO 2007051459 A2 WO2007051459 A2 WO 2007051459A2 DE 2006001931 W DE2006001931 W DE 2006001931W WO 2007051459 A2 WO2007051459 A2 WO 2007051459A2
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biomolecules
binding
bound
target molecule
particle
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PCT/DE2006/001931
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German (de)
French (fr)
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WO2007051459A3 (en
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Dieter Scholz
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    • 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
    • G01N33/5432—Liposomes or microcapsules

Definitions

  • the invention relates to the use of immobilized micro-beads for the enrichment of biomolecules and bioparticles.
  • Fields of application of the invention are biotechnology, medicine and molecular biology research.
  • FACS fluorescence activated cell sorting
  • Magnetic separation processes have been used more and more for more than 10 years to detect and isolate particles / cells.
  • the separation is carried out by a strong magnetic field.
  • the immuno-magnetic separation successfully commercialized by, among others, Dynal and Miltenyi Biotec, has become established as a simple, relatively inexpensive method of cell separation.
  • the magnetic separation has proven itself for the isolation of relatively rare cell types, eg for the isolation of fetal cells from the maternal blood for prenatal diagnosis.
  • Another application is the detection of tumor cells in the blood after surgical removal of the primary tumor to initiate further treatments.
  • syngeneic CD34 + peripheral blood stem cells are now routinely obtained from patients for reimplantation suffering from certain malignancies of the blood-forming system. This is preferably done by the purification of leukocytes with specific antibodies coupled to magnetic beads. For cell fractionation during the blood / cell donation several systems are available that use the different size and specific gravity of the blood cells for a separation in the gravitational field (centrifugation).
  • PCT / DE 001371 describes a process of cascade filtration with which several substances to be separated from mixtures or large volumes are separated / enriched in one operation by using microparticles of different sizes to which different specific "catcher" molecules are coupled are.
  • the invention has for its object to develop a method with the small amounts of substances to be detected are bound to small surfaces, which are then available for further analytical or other purposes.
  • This object has been achieved by attaching a second capture principle to the microbead surface without or via a spacer which binds the complex microparticle biomolecule to a surface.
  • a second capture principle to the microbead surface without or via a spacer which binds the complex microparticle biomolecule to a surface.
  • As catcher principle everything that is useful for temporary or permanent fixation, usually biotin-avidin complex or even antibody antigen or molecule interactions with sufficiently large adhesion (eg via hydrophobic interactions) is suitable.
  • the combination of cascade filtration with the described microparticle fixation provides an easy-to-use system with which small amounts of a target substance are permanently or temporarily fixed from substance mixtures for further processing.
  • the target biomolecule can also be detached from the complex carrier-microbead target molecule by other chemical or physical effects.
  • the method is suitable, inter alia, for detection systems of biomolecules in liquids such as blood, excretions and secrete but also in food, environment and the process control of biotechnological production processes.
  • liquids such as blood, excretions and secrete but also in food, environment and the process control of biotechnological production processes.
  • the complex microparticle biomolecule is immobilized on a solid surface for further work steps.

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  • 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)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

The invention relates to the use of immobilized microbeads for enriching biomolecules and bioparticles. Disclosed is a method for the selective, particle-bound enrichment of biomolecules or bioparticles with the aid of at least two different binding processes. The invention applies to the fields of biotechnology, medicine, and molecular biology research.

Description

Verfahren zur selektiven partikelgebundenen Anreicherung von Biomolekülen oder BiopartikelnProcess for the selective particle-bound enrichment of biomolecules or bioparticles

Beschreibungdescription

Die Erfindung betrifft die Verwendung von immobilisierten Mikro-Beads für Anreicherung von Biomolθkülen und Biopartikeln. Anwendungsgebiete der Erfindung sind die Biotechnologie, die Medizin und die molekularbiologische Forschung.The invention relates to the use of immobilized micro-beads for the enrichment of biomolecules and bioparticles. Fields of application of the invention are biotechnology, medicine and molecular biology research.

Die Isolierung von biologischen Stoffen und Partikeln aus komplexen Stoffgemischen gewinnt eine immer größere Bedeutung. Neben der Kombination von Trennverfahren auf Grundlage der physikalischen Stoffbesonderheiten werden in zunehmendem Maße spezifische Liganden (z.B. Antikörper) als Fänger- bzw. Tracer-Molekül verwendet. Diese Fänger/Tracer erkennen und binden ausschließlich das Zielobjekt. Um die Stofftrennung vorzunehmen, muss das fangende Prinzip auf einen festen Körper gebracht werden, die das Abtrennen des Fänger-Zielobjekt-Komplexes ermöglicht. Die Abtrennung erfolgt mechanisch oder nach Waschen des Komplexes durch spezielle Flüssigkeiten.The isolation of biological substances and particles from complex mixtures is becoming increasingly important. In addition to the combination of physical substance specificity separation methods, specific ligands (e.g., antibodies) are increasingly being used as the scavenger and tracer molecules, respectively. These catchers / tracers only recognize and bind the target object. In order to make the separation of substances, the catching principle must be brought to a solid body, which allows the separation of the catcher-target complex. The separation is carried out mechanically or after washing the complex by special liquids.

Die Zellanalyse und Zellseparation wird seit Jahrzehnten mittels Fluorescence Activated Cell Sorting (FACS) vorgenommen. Es ist die Methode der Wahl für die Analytik spezifischer Zellpopulationen durch Oberflächenmarker. Probleme bereitet die FACS-Anwendung für die Isolierung großer Zellzahlen für den therapeutischen Einsatz. Das die Zellen enthaltende Medium muss stark verdünnt werden, die Trennzeit ist relativ lange für größere Zellmengen, und es bereitet Probleme die aseptischen Bedingen einzuhalten. Das Verfahren insgesamt verursacht erhebliche Kosten.Cell analysis and cell separation has been performed for decades using fluorescence activated cell sorting (FACS). It is the method of choice for the analysis of specific cell populations by surface markers. The FACS application is preparing for the isolation of large numbers of cells for therapeutic use. The medium containing the cells must be heavily diluted, the separation time is relatively long for larger cell volumes, and there are problems to meet the aseptic conditions. The process as a whole causes considerable costs.

Zur Erkennung und Isolierung von Partikeln/Zellen werden seit mehr als 10 Jahren magnetische Trennverfahren im wachsenden Maße eingesetzt. Dazu werden entweder die Fängermoleküle mit Eisen beladen, bzw. einen Eisenkern enthaltende Mikropartikel werden mit dem Fängermolekül beschichtet. Die Abtrennung erfolgt durch ein starkes magnetisches Feld. Die immunmagnetische Separation, erfolgreich kommerzialisiert u.a. durch Dynal und Miltenyi Biotec, hat sich als einfache, relativ kostengünstige Methode der Zelltrennung etabliert. Besonders im Vergleich zur Fow-Zytometrie hat sich die magnetische Separation für die Isolierung von relativ seltenen Zelltypen bewährt, so z.B. für die Isolierung fetaler Zellen aus dem mütterlichen Blut für die pränatale Diagnostik. Eine weitere Anwendung ist der Nachweis von Tumorzellen im Blut nach chirurgischer Entfernung des Primärtumors zur Einleitung weiterer Behandlungen.Magnetic separation processes have been used more and more for more than 10 years to detect and isolate particles / cells. To either the catcher molecules are loaded with iron, or an iron core containing microparticles are coated with the catcher molecule. The separation is carried out by a strong magnetic field. The immuno-magnetic separation, successfully commercialized by, among others, Dynal and Miltenyi Biotec, has become established as a simple, relatively inexpensive method of cell separation. Especially in comparison to Fow cytometry, the magnetic separation has proven itself for the isolation of relatively rare cell types, eg for the isolation of fetal cells from the maternal blood for prenatal diagnosis. Another application is the detection of tumor cells in the blood after surgical removal of the primary tumor to initiate further treatments.

Für therapeutische Zwecke werden heute routinemäßig syngene CD34+- periphere Blutstammzellen von Patienten für eine Reimplantation gewonnen, die an bestimmten malignen Erkrankungen des Blut-bildenden Systems leiden. Das geschieht vorzugsweise durch die Aufreinigung von Leukozyten mit spezifischen Antikörpern, die an Magnetic Beads gekoppelt sind. Für die Zellfraktionierung während der Blut/Zell-Spende stehen mehrere Systeme zur Verfügung, die die unterschiedliche Größe und spezifische Dichte der Blutzellen für eine Trennung im Schwerefeld (Zentrifugation) nutzen.For therapeutic purposes, syngeneic CD34 + peripheral blood stem cells are now routinely obtained from patients for reimplantation suffering from certain malignancies of the blood-forming system. This is preferably done by the purification of leukocytes with specific antibodies coupled to magnetic beads. For cell fractionation during the blood / cell donation several systems are available that use the different size and specific gravity of the blood cells for a separation in the gravitational field (centrifugation).

In PCT/DE 001371 wird ein Verfahren der Kaskaden-Filtration beschrieben, mit welchem in einem Arbeitsgang mehrere zu trennenden Stoffe aus Gemischen oder großen Volumina getrennt/angereichert werden, indem Mikropartikel unterschiedlicher Größe verwendet werden, an die verschiedene spezifische „Fänger"-Moleküle gekoppelt sind.PCT / DE 001371 describes a process of cascade filtration with which several substances to be separated from mixtures or large volumes are separated / enriched in one operation by using microparticles of different sizes to which different specific "catcher" molecules are coupled are.

Der Erfindung liegt die Aufgabe zugrunde, ein Verfahren zu entwickeln mit dem geringe Mengen zu detektierender Stoffe auf kleine Oberflächen gebunden werden, die dann für weitere analytische oder andere Zwecke zur Verfügung stehen. Diese Aufgabe wurde dadurch gelöst, dass ein zweites Fängerprinzip an die Mikrobead-Oberfläche ohne oder über einen Spacer angebracht wird, der den Komplex Mikropartikel-Biomolekül an eine Oberfläche bindet. Als Fänger-Prinzip eignet sich alles, was zum temporären oder permanenten Fixieren zweckvoll ist, üblicherweise Biotin-Avidin-Komplex oder auch Antikörper-Antigen oder Molekülinteraktionen mit ausreichend großer Adhäsion (z.B. über hydrophobe Wechselwirkungen).The invention has for its object to develop a method with the small amounts of substances to be detected are bound to small surfaces, which are then available for further analytical or other purposes. This object has been achieved by attaching a second capture principle to the microbead surface without or via a spacer which binds the complex microparticle biomolecule to a surface. As catcher principle, everything that is useful for temporary or permanent fixation, usually biotin-avidin complex or even antibody antigen or molecule interactions with sufficiently large adhesion (eg via hydrophobic interactions) is suitable.

Durch die Kombination von Kaskaden-Filtration mit der beschriebenen Mikropartikel-Fixation wird ein einfach zu handhabendes System bereit gestellt, mit dem aus Stoffgemischen geringe Mengen einer Zielsubstanz permanent oder temporär für eine weitere Bearbeitung fixiert wird.The combination of cascade filtration with the described microparticle fixation provides an easy-to-use system with which small amounts of a target substance are permanently or temporarily fixed from substance mixtures for further processing.

Bei Verwendung von Fängerantikörpern kann z.B. durch pH-Wert-Senkung das Zielmolekül aus der Antikörperbindung selektiv gelöst werden. Bei Kopplung des Fängermoleküls über einen geeigneten Spacer, kann das Ziel-Biomolekül auch durch andere chemische oder physikalische Einwirkungen von dem Komplex Träger-Microbead-Zielmolekül abgelöst werden.When using capture antibodies, e.g. by lowering the pH of the target molecule from the antibody binding can be selectively solved. When the capture molecule is coupled via a suitable spacer, the target biomolecule can also be detached from the complex carrier-microbead target molecule by other chemical or physical effects.

Das Verfahren eignet sich unter anderem für Nachweissysteme von Biomolekülen in Flüssigkeiten wie Blut, Se- und Exkreten aber auch in Lebensmitteln, Umwelt und der Prozesskontrolle von biotechnologischen Herstellungsverfahren. Insbesondere für diagnostische Zwecke ist es von Vorteil, wenn der Komplex Mikropartikel-Biomolekül für weitere Arbeitsschritte ein einer festen Oberfläche immobilisiert wird.The method is suitable, inter alia, for detection systems of biomolecules in liquids such as blood, excretions and secrete but also in food, environment and the process control of biotechnological production processes. In particular, for diagnostic purposes, it is advantageous if the complex microparticle biomolecule is immobilized on a solid surface for further work steps.

Die Erfindung soll nachfolgend durch Ausführungsbei'spiele näher erläutert werden.The invention will be explained in more detail by Ausführungsbei ' games.

Ausführungsbeispiele: EXAMPLES

Claims

Patentansprüche claims 1. Verfahren zur selektiven partikelgebundenen Anreicherung von Biomolekülen oder Biopartikeln durch die Verwendung von mindestens zwei unterschiedlichen Bindungsmethoden.1. A method for the selective particle-bound enrichment of biomolecules or bioparticles by the use of at least two different binding methods. 2. Verfahren nach Anspruch 1 , dadurch gekennzeichnet, dass eine Bindungsmethode dazu dient, das Zielmolekül zu erkennen und zu binden und die zweite Bindungsmethode, das gebundene Zielmolekül an eine feste Oberfläche temporär oder permanent zu fixieren.2. The method according to claim 1, characterized in that a binding method is used to recognize the target molecule and to bind and the second binding method to fix the bound target molecule to a solid surface temporarily or permanently. 3. Verfahren nach Anspruch 1 und 2, dadurch gekennzeichnet, dass in einem ersten Schritt die Partikel in das Stoffgemisch verbracht werden, um das Zielmolekül zu binden und nach Ablauf dieser3. The method according to claim 1 and 2, characterized in that in a first step, the particles are spent in the mixture to bind the target molecule and after expiration of this Reaktion in einem zweiten Schritt dieses Reaktionsgemisch mit einer festenReaction in a second step, this reaction mixture with a solid Oberfläche in Kontakt gebracht wird, welche über eine zweiteSurface is brought into contact, which has a second Bindungsmethode das gebundene Zielmolekül fixiert.Binding method fixes the bound target molecule. 4. Verfahren nach Anspruch 1 - 3, dadurch gekennzeichnet, dass für selektive Bindung der Zielmoleküle vorzugsweise Antikörper eingesetzt werden, die über bekannte Verfahren direkt oder über geeignete Spacer kovalent an die Partikeloberfläche gebunden werden.4. The method according to claim 1 - 3, characterized in that preferably antibodies are used for selective binding of the target molecules, which are bound by known methods directly or via suitable spacers covalently to the particle surface. 5. Verfahren nach Anspruch 1 - 3, dadurch gekennzeichnet, dass für die Bindung des Partikels und Zielmoleküis an die Oberfläche Substanzen zum Einsatz kommen, die eine ausreichend stabile Fixierung an der festen Oberfläche gewährleisten. 5. The method according to claim 1 - 3, characterized in that substances are used for the binding of the particle and Zielmoleküis to the surface, which ensure a sufficiently stable fixation on the solid surface. 6. Verfahren nach Anspruch 5, dadurch gekennzeichnet, dass vorzugsweise Biotin-Streptavidin für die Fixierung von Partikel , und Zielmolekül verwendet wird.6. The method according to claim 5, characterized in that preferably biotin-streptavidin is used for the fixation of particles, and target molecule. 7. Verfahren nach Anspruch 1 - 6, dadurch gekennzeichnet, dass die so immobilisierten Biomoleküle an der festen Oberfläche direkt über bekannte, z.B. immunchemische Verfahren nachgewiesen oder durch geeignete Verfahren abgelöst und separat weiter bearbeitet werden.Process according to claims 1 - 6, characterized in that the immobilized biomolecules on the solid surface are directly accessible via known, e.g. Immunochemical methods are detected or replaced by suitable methods and processed separately. 8. Verwendung des Verfahrens nach Anspruch 1 - 7, zum Nachweis von Biomolekülen in der biologischen Forschung, der medizinischen Diagnostik, in Lebens- und Futtermittelindustrie, in der Umwelt und in der Prozesskontrolle biotechnologischer Herstellungsverfahren. 8. Use of the method according to claim 1-7, for the detection of biomolecules in biological research, medical diagnostics, in food and feed industry, in the environment and in the process control of biotechnological production processes.
PCT/DE2006/001931 2005-11-03 2006-11-03 Method for the selective particle-bound enrichment of biomolecules or bioparticles Ceased WO2007051459A2 (en)

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DE102005052957.7 2005-11-03
DE200510052957 DE102005052957A1 (en) 2005-11-03 2005-11-03 Process for the selective particle-bound enrichment of biomolecules or bioparticles

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WO2007051459A3 WO2007051459A3 (en) 2007-07-26

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1312277C (en) * 1987-12-18 1993-01-05 Richard C. Sutton Avidin- and biotin-immobilized reagents, analytical elements and methods of use
FI20002623A7 (en) * 2000-11-30 2002-05-31 Inno Trac Diagnostics Oy Bioanalytical assay method
JP4087200B2 (en) * 2002-09-17 2008-05-21 ユニバーサル・バイオ・リサーチ株式会社 Particle complex and method for producing the particle complex
GB0228914D0 (en) * 2002-12-11 2003-01-15 Dynal Biotech Asa Particles

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DE102005052957A1 (en) 2007-05-10

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