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EP2564195A1 - Détermination d'interactions entre une substance ou un mélange de substances et une cible - Google Patents

Détermination d'interactions entre une substance ou un mélange de substances et une cible

Info

Publication number
EP2564195A1
EP2564195A1 EP11716236A EP11716236A EP2564195A1 EP 2564195 A1 EP2564195 A1 EP 2564195A1 EP 11716236 A EP11716236 A EP 11716236A EP 11716236 A EP11716236 A EP 11716236A EP 2564195 A1 EP2564195 A1 EP 2564195A1
Authority
EP
European Patent Office
Prior art keywords
substance
target
sample
buffer solution
mixture
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Withdrawn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
EP11716236A
Other languages
German (de)
English (en)
Inventor
Hinnerk Boriss
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.)
Sovicell GmbH
Original Assignee
Sovicell GmbH
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 Sovicell GmbH filed Critical Sovicell GmbH
Publication of EP2564195A1 publication Critical patent/EP2564195A1/fr
Withdrawn legal-status Critical Current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
    • 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
    • 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
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the present invention relates to a method for determining interactions between a substance or mixture of substances and a potential target and to a device which can be used in such a method.
  • a target is known to be a target compound or a target receptor to which, for example, can bind active or harmful substances.
  • targets to be investigated in particular biological targets such as proteins, - -
  • Enzymes antibodies, biological membranes or whole cells of interest.
  • a method for determining binding constants and distribution coefficients on membranes is known from DE 198 14 775.
  • solid-supported membranes are contacted with an aqueous solution of the substance to be assayed and incubated for a time sufficient to bind the solute to the membrane surface. Thereafter, the solid supported membranes are separated from the solution (e.g., by sedimentation).
  • the concentration of the substance to be investigated is determined in the remaining solution (supernatant) or optionally also in the separated solid-supported membranes by suitable measuring methods, namely for several different proportions of solid-supported membrane to substance to be examined.
  • the method according to the invention serves to determine interactions between a substance or a mixture of substances and a target.
  • a substance or a mixture of substances are suitable as target.
  • targets but also synthetic or semi-synthetic receptors of interest, such as lipid membrane model systems, as described in DE 198 14 775.
  • the substances or substance mixtures to be investigated may be, in particular, active ingredient molecules for pharmaceutical applications. Of course, it is also possible to investigate the effects of pollutants and pollutant mixtures on biological targets such as cells.
  • a method according to the invention always comprises at least the following steps:
  • the first and the second sample are incubated with different amounts of substance of the target, whereas, however, all sample containers in the incubation contain the same amount of buffer solution and preferably the same amount of substance to be examined substance or the substance mixture to be examined.
  • the total volume of the samples to be incubated (volume of the buffer solution + volume of the carrier together with the target immobilized thereon) varies.
  • the inventive method therefore provides much more reliable information about the interactions between a substance or a mixture of substances and a target, as is the case with conventional methods.
  • the support on which the target is immobilized is preferably a support that is insoluble in aqueous solution. It may consist of organic or inorganic material, in the latter case in particular metal oxides such as silica and alumina, silicates, aluminates, borates or zeolites are mentioned. Metals and precious metals can also be used in principle.
  • the carrier is particularly preferably particulate, ie in the form of particles. These particles usually have sizes in the ⁇ range, but may optionally be at least partially nanoscale.
  • the support in particular the particles, may be non-porous or even porous, the latter generally being associated with a significant increase in the usable support surface area (an "outer” surface formed by the outside of the support may still be added) usable “inner” surface in the form of pore walls added).
  • Targets can be immobilized on the outer as well as possibly also on the inner surface of the carrier in the usual way.
  • the preparation of immobilized target carriers of this kind is known to the person skilled in the art and described in detail, for example, for lipid membrane model systems as targets in the earlier DE 100 48 822 of the Applicant.
  • lipid membranes or lipid bilayers are immobilized as a target on a particulate carrier. These preferably surround the carrier particles at least partially, preferably completely.
  • the lipids are brain tissue lipids, i. to those that occur in the brain tissue of animals or humans.
  • said brain tissue lipids are derived from brain tissue extracts.
  • the corresponding lipids or extracts are known to the person skilled in the art and can be obtained commercially from various companies.
  • the separation of the carrier can be carried out, for example, by filtration or centrifugation and, if appropriate, subsequent decantation of the buffer solution.
  • carrier particles with magnetic properties can also be used. These can easily be separated from the buffer solution by applying a magnetic field.
  • aqueous solutions containing saline such as are customary and commercially available for biological applications, are used as the buffer solution.
  • a distribution coefficient and / or a binding constant is usually determined to determine interactions between the substance under investigation or the substance mixture investigated and the target.
  • the necessary mathematical instruments are known from the literature.
  • concentration values measured for different molar amounts of the target are required, which can be obtained according to the invention by measuring the concentration of the substance or of the substance mixture in the supernatant of the first and the second sample container. Basically, however, the error rate decreases the more concentration values can be used for the determination.
  • At least one further sample of the substance or the substance mixture is particularly preferred with the sample container containing at least one further buffer solution, preferably immobilized on a solid, preferably particulate carrier 1 - 10 additional buffer solution containing sample containers, incubated.
  • the further sample or the further samples are incubated with in each case different amounts of substance of the target, which in particular also depend on the corresponding target substance quantities in the first and in the second sample container. - - divorce.
  • the sample containers always contain the same amount of buffer solution during incubation as the first and the second sample container.
  • concentration of the substance or of the substance mixture in the respective supernatant is determined relative to at least one reference sample.
  • a reference sample is, in particular, a sample which contains only buffer solution and the substance or substance mixture to be investigated.
  • a device comprises a first and a second sample container, each containing a target immobilized on a solid, particulate carrier substance and buffer solution, wherein the first and the second sample container contain different amounts of substance of the target but the same amount of buffer solution.
  • the device may in particular be a microtiter plate.
  • the sample containers are correspondingly preferably the wells of a microtiter plate.
  • the substance samples can be incubated with a target.
  • the method according to the invention can be carried out particularly well with the aid of a microtiter plate which comprises at least a first and a second cavity, each containing a target immobilized on a solid, particulate carrier substance (as described above) and buffer solution.
  • the first and the second cavity contain different amounts of substance of the target but the same amount of buffer solution.
  • the wells of the microtiter plate thus form the sample containers.
  • microtiter plates are usually rectangular and consist of plastics such as polystyrene, polypropylene or polyvinyl chloride, for very special applications also made of glass. They contain a variety of - - wells which are usually isolated in rows and columns, the exact dimensions (length ⁇ width ⁇ height) are preferably 127.76 mm ⁇ 85.48 mm ⁇ 14.35 mm are a variety of formats, which usually always the same base area, but in part have a variable height, for example, there are microtiter plates
  • microtiter plate according to the invention can be present in all these constitutional and form variants.
  • the microtiter plate according to the invention preferably also has at least one further cavity, preferably between 1 and 10 further cavities, which are filled with the same amount of buffer solution as the first and the second cavity.
  • This at least one further cavity or one or a part of the further cavities can serve as the reference already mentioned above. In this case, they are or will be filled to perform the method according to the invention with the same amount of substance to be examined substance or substance mixture to be examined as the first and the second cavity, but are free of the immobilized target.
  • the at least one further cavity or one or a part of the further cavities can also be used to determine additional - -
  • Concentration values serve to lower the mentioned error rate when determining a distribution coefficient and / or a binding constant. In this case, they contain not only the same amount of buffer solution as the first and the second cavity nor a defined amount of substance of the immobilized target, but must deviate from the corresponding amounts of substance in the first and in the second cavity. To carry out the method according to the invention, they are then likewise filled with the same amount of substance to be examined substance or substance mixture to be examined as the first and the second cavity.
  • the sample containers are individual containers which are arranged removably on a holder.
  • the individual vessels may be, for example, simple Eppendorf -Tu bes, the holder to a frame or a shelf.
  • the holder preferably has the dimensions of a microtiter plate. Reference is made to the corresponding statements on the possible dimensions of a microtiter plate according to the invention.
  • the individual vessels can also be a container made of glass, in particular a glass vial. For example, silanized glass vessels are particularly suitable for - -
  • HSA human serum albumin
  • the HSA was fixed on the surface of silica beads as a carrier suspended in buffer solution (HSA suspension on carrier). - -
  • HSA human serum albumin
  • [A] stands for the concentration of unbound test substance
  • [B] for the concentration of HSA
  • [AB] for the concentration of the HSA test substance complex.
  • the concentration of unbound test substance can also be described as the product of the relative proportion of unbound test substance f u and sum of free and bound concentration of HSA:
  • / b stands for the relative proportion of bound ligand.
  • the binding parameter K D can thus be determined exactly and robustly.
  • measurement-related outliers can be easily recognized.
  • the unconformity method for outlier detection can be applied in linear regression.
  • the quality of the adaptation of the measured data to the binding model can be checked well, since the axis distance of the regression model should be zero. Deviations from this point to errors in the experiment, or indicate that the simple non-cooperative binding model is insufficient to describe the data.
  • the reciprocal of the free concentration versus the ligand concentration can be plotted.
  • the intercept of this plot corresponds to the reference concentration in the test system without ligand. If the reference concentration thus determined agrees with the reference concentration determined by measurement technology, then a good model adaptation is present.
  • the binding constant can then be determined from the regression of the quotient b / / u against the free receptor concentration.
  • the reciprocal of the slope corresponds to the binding constant K D.
  • the binding constants can be determined to membranes, if the molarity of Membraniipide is known.
  • the membrane affinity can be determined as a measure of the binding to membranes.
  • the membrane affinity (MA) is - defined as the ratio of the concentration of a test substance in the lipid to the concentration of the test substance in the buffer: c (lipid)
  • the membrane affinity corresponds to the slope of the regression model plus 1. Since the axis section of the regression model corresponds to the volume in the assay (volume of the buffer plus volume of the lipid), this model also has an internal quality control.
  • the brain lipids were fixed on the surface of silica beads as carriers suspended in buffer solution (supported lipid suspension).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Endocrinology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détermination d'interactions entre une substance ou un mélange de substances et une cible. Selon le procédé, au moins un premier et un deuxième échantillon de la substance ou du mélange de substances sont mis à incuber avec la cible immobilisée sur un support solide. L'incubation s'effectue respectivement dans un récipient à échantillon. Le premier et le deuxième échantillon sont mis en incubation avec des quantités de matière cible différentes, tous les récipients à échantillon contenant par contre la même quantité de solution tampon lors de l'incubation. Après incubation, le support solide conjointement avec la cible immobilisée dessus et éventuellement la substance qui y est liée sont séparés des solutions tampons. La concentration de la substance ou du mélange de substances est ensuite mesurée dans le surnageant respectif. L'invention concerne également un dispositif permettant de mettre un tel procédé en œuvre. Ce dispositif comprend au moins un premier et un deuxième récipient à échantillon contenant respectivement une cible immobilisée sur une substance porteuse particulaire solide ainsi qu'une solution tampon, le premier et le deuxième récipient à échantillon contenant des quantités de matière cible différentes, mais la même quantité de solution tampon.
EP11716236A 2010-04-27 2011-04-20 Détermination d'interactions entre une substance ou un mélange de substances et une cible Withdrawn EP2564195A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102010018965A DE102010018965A1 (de) 2010-04-27 2010-04-27 Bestimmung von Wechselwirkungen zwischen einer Substanz oder einem Substanzgemisch und einem Target
PCT/EP2011/056324 WO2011134860A1 (fr) 2010-04-27 2011-04-20 Détermination d'interactions entre une substance ou un mélange de substances et une cible

Publications (1)

Publication Number Publication Date
EP2564195A1 true EP2564195A1 (fr) 2013-03-06

Family

ID=44168290

Family Applications (1)

Application Number Title Priority Date Filing Date
EP11716236A Withdrawn EP2564195A1 (fr) 2010-04-27 2011-04-20 Détermination d'interactions entre une substance ou un mélange de substances et une cible

Country Status (4)

Country Link
US (1) US20130203181A1 (fr)
EP (1) EP2564195A1 (fr)
DE (1) DE102010018965A1 (fr)
WO (1) WO2011134860A1 (fr)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE102014115088A1 (de) 2014-10-16 2016-04-21 Sovicell Gmbh Bestimmung von Bindungskonstanten mittels Gleichgewichtsverlagerung

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19814775C2 (de) 1998-04-02 2001-04-19 Nimbus Biotechnologie Gmbh Verfahren zur Bestimmung der Lipid-Bindungskonstanten von Substanzen in wässriger Lösung an Oberflächen aus amphiphilen Molekülen
DE10048822A1 (de) 2000-09-29 2002-04-18 Nimbus Biotechnologie Gmbh Verfahren zur Immobilisierung von Lipidschichten
EP1658499B1 (fr) * 2003-08-16 2007-10-03 Bayer HealthCare AG Determination de fractions libres
DE102004038873A1 (de) * 2004-08-05 2006-02-23 Nimbus Biotechnologie Gmbh Bestimmung von Bindungsparametern
DE102008007673A1 (de) * 2008-01-25 2009-07-30 Nimbus Biotechnologie Gmbh Bestimmung der Permeabilität von Substanzen durch die Blut-Hirn-Schranke
EP2249156A1 (fr) * 2009-04-29 2010-11-10 Aterovax Analyse enzymatique pour la détermination quantitative de l'activité phospholipase A1 ou A2 dans un échantillon basée sur l'utilisation d'une phase solide enrobée d'un substrat marqué au fluorochrome

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
KIM YUMEE ET AL: "A phospholipase A-2 kinetic and binding assay using phospholipid-coated hydrophobic beads", ANALYTICAL BIOCHEMISTRY, ACADEMIC PRESS INC, NEW YORK, vol. 250, no. 1, 1 January 1997 (1997-01-01), pages 109 - 116, XP002546382, ISSN: 0003-2697, DOI: 10.1006/ABIO.1997.2200 *

Also Published As

Publication number Publication date
WO2011134860A1 (fr) 2011-11-03
DE102010018965A1 (de) 2011-10-27
US20130203181A1 (en) 2013-08-08

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