EP1782045A1

EP1782045A1 - Determination binding parameters

Info

Publication number: EP1782045A1
Application number: EP05769619A
Authority: EP
Inventors: Johannes Schmitt; Joachim Noeller; Thomas Mueller
Original assignee: Nimbus Biotechnologie GmbH
Current assignee: Nimbus Biotechnologie GmbH
Priority date: 2004-08-05
Filing date: 2005-07-29
Publication date: 2007-05-09
Also published as: WO2006015736A1; DE102004038873A1; US20080003590A1

Abstract

The invention relates to a method for determining binding parameters between at least one type of substance and at least one binding partner by using at least one luminescent probe and an appropriate kit. For carrying out the inventive method, a solid body which is at least partially loaded with at least one binding partner and which is incubated with the testable substance is used. After separation of the solid body and a non-bound substance, the concentration of the testable substance is determined by measuring the modulation or weakening of luminescence signals of at least one probe by the non-bound substance in a supernatant.

Description

description

Determination of binding parameters

The present invention relates to a method for determining binding parameters and to a kit which is suitable for carrying out this method.

The determination of binding parameters between a substance and a binding partner is of considerable importance for, for example, pharmacology, medicine, biotechnology and biochemistry. It allows insights as to how molecules (for example biological molecules such as, for example, peptides, proteins, nucleic acids, in particular biologically active substances or active substances) interact with biological membranes, as they are transported within cells, and signal action can unfold. The knowledge of binding parameters is therefore particularly advantageous for the development of therapeutic agents and other novel drugs [Böhm, HJ, Klebe, G., Kubinyi, H., "Drug Design, Spectrum", Akademischer Verlag, 1st edition, Heidelberg (1996)]. In order to determine binding parameters, it is currently generally necessary to separate the bound and free fraction of the substance to be investigated from each other after the binding process. This separation step can be carried out, for example, by using a semipermeable membrane (dialysis), by ultrafiltration or ultracentrifugation. Subsequently, the concentration of the substance in the free fraction or in the bound fraction can be determined. So conclusions can be drawn on the binding affinity of the substance to the binding partner. The disadvantage here is that after the separation usually further transfer steps of the phases with bound or unbebunde¬ nem proportion are required. This will not meet the requirements of a high-throughput screening (HTS) procedure that will allow hundreds to thousands of substances to be tested daily. To simplify the separation of free and bound fraction of a substance, a binding partner (for example a protein or a lipid membrane) can be immobilized on a solid. The separation can be carried out in this case by sedimentation, filtration or by a simple low-speed centrifugation. However, at least one further transfer step of liquids for determining the concentration of the substance is also required here.

In order to avoid the costly transfer of free and bound part of the substance to be examined after separation, various approaches have already been pursued to carry out a determination of binding parameters without separation of substance and binding partner using so-called marker substances. By way of example, the use of fluorescent or radioactive substance substances which bind in specific regions of the binding partner to be investigated permits the quantification of the binding of the substances to be investigated via displacement of the fluorescent [DE Epps, TJ Raub, FJ Kezdy "A General, Wide-Range Spectrofluorometric Method for Measuring Site-Specific Affinities of Drugs toward Human Serum Albumin ", Analytical Biochemistry 227, 342-350 (1995)] or radioactive marker [N. Bosworth and P. Towers" Scintillation proximity assay "Nature 341, 167-168 (1989)]. Without the use of marker substances, the determination of binding parameters can usually only be realized in special cases. This includes the investigation of protein bonds in which intrinsic, detectable properties of the protein after the. Change bond. An example of this is the binding of substances to human serum albumin [DE Epps, TJ Raub, V. Caiolfa, A. Chlari, M. Zamal] Determination of the Affinity of Drugs for Serum Albumin by Measurement of the Quenching of the Intrinsic Tryptophan Fluorescence of the protein J. Pharm Pharmacol 51, 41-48 (1999)]. However, this is only possible in the case of very strongly binding substances and leads in many cases to defects due to the UV activity of the substances to be investigated Such an approach has also been described in the literature for the determination of the binding parameters for the α1-acidic glycoprotein and also for membrane-bound proteins [A.K. Johansen, N.-P. Wiillase, G. Sager "Fluorescence studies of β-adrenergic ligand binding to α1-acid glycoprotein with 1-anilino-8-naphthalenesulfonates, isoprenalines, adrenalines and propranolol "Biochemical Pharmacology" 43 (4), 725-729, (1992); R. Liu, A. Siemiarcuk , FJ Sharom "Intrinsic fluorescen ce of the P-glycoprotein multidrug transporter: sensitivity of tryptophan residues to binding of drugs and nucleotides "Biochemistry 39 (48): 14927-14938 (2000)]. Again, these are special cases that can not be generalized. Further possibilities are the modification of the binding partner itself [JP Vilardaga, M. Bünemann, C. Krasel, M. Castro, MJ Lohse "Measurement of the millisecond activation switch and protein-coupled receptors in living cells" Nat. Biotechnol., 21 (7), 807-812 (2003)], with one or more fluorescence probes, preferably in the vicinity of the binding pockets, in which the binding causes a change in the fluorescence signal Another approach uses the so-called surface plasmon resonance (SPR surface plasmon - A -

[See, for example: P. Mistrik, F. Moreau, JM Allen "BlaCore analysis of leptin-leptin receptor interaction: evidence for 1: 1 stoichiometry" Anal. Biochem. 327 (2), 271-277 (2004) However, the currently available devices also do not allow a high sample throughput.Neutral nuclear magnetic resonance spectroscopy (NMR), paramagnetic electron resonance spectroscopy (EPR) [E. Paula, S. Schreier "Molecular and Physicochemical Aspects of Local Anasthetic-Membrane Interaction" Brazilian Journal of Medical and Biological Research 29, 877-894 (1996)] and Differential Scanning Calorimetry (DSC) [V. Plotnikov, A. Rochalski, M. Brandts, JF Brandts, S. Williston, V. Frasca, LN Un "An autosampling differential scanning calorimeter instrument for studying molecular interactions" Assay Drug Dev Technol. 1, 83-90 (2002) Although these are very precise and provide further information beyond simple binding, they only allow the determination of fewer substances per day.

The previous methods for determining binding parameters thus require either complex separation and / or transfer steps in order to realize a separation of bound and unbound portions of the substance to be investigated, or are only applicable to certain cases. They therefore do not or only insufficiently do justice to a generally usable HTS process. Furthermore, conventional methods are generally less user-friendly and prevent easy automation.

The invention thus has the object of providing a method and a corresponding kit in which the described disadvantages are avoided. In particular, the method should enable a generally applicable determination of binding parameters, which is not limited to special cases. The procedure should be without be difficult to perform and be suitable for a high Probendurch¬ rate.

This object is achieved by a method as described in claim 1. Dependent claims 2 to 16 relate to preferred embodiments of the process. The claim 17 is concerned with a kit for determining binding parameters. Preferred embodiments of this kit are mentioned in the dependent claims 18 to 26. By reference, the wording of all claims is hereby made the content of the description.

The method according to the invention for determining binding parameters between at least one substance and at least one binding partner uses at least one luminescent probe. In this case, luminescence is used as a generic term in particular for fluorescence and phosphorescence, but also, for example, for electrochemical, thermoluminescent and chemiluminescence. It is first provided at least one solid, which is at least partially loaded with at least one binding partner. The substance or substances to be investigated are brought together with this solid, so that interactions between the substance and the binding partner, in particular bonds, can form. This incubation of the solid with the at least one substance is followed by a separation of the solid, which can now carry the bound substance, and non-bound substance in a reaction vessel in sediment and supernatant. To determine the substance concentration in the supernatant, a modulation or attenuation of luminescence signals of the at least one probe by unbound substance in the supernatant is measured. This requires the overlap of the absorption spectra of the at least one substance with excitation and / or emission spectra of the at least one probe. The method according to the invention thus utilizes the so-called internal filter effect, in which the light absorption of substances can be determined indirectly via a weakening of luminescence intensities of suitable probes. This phenomenon is based on the fact that the intensity of light rays, which are absorbed on the way to a suitable probe or on the way from the probe to a detector of other dissolved substances, that is to say are thus attenuated. The overlap of excitation or emission wavelengths with the absorption band of the substance to be investigated leads to a modulation or weakening of the luminescence intensity proportional to the concentration in the measurement at suitable wavelengths (ie in the range of the absorption band of the substance) examining substances in the supernatant. This effect is thus particularly advantageously suitable for determining the concentration of the substance to be examined in the supernatant according to the method according to the invention.

The internal filter effect has already been exploited for various analytical methods. For example, WO 94/17388 describes the use of the internal filter effect for the determination of ions in liquids by means of a sensor membrane. US Pat. No. 4,822,746 deals generally with the use of the internal filter effect for determining a ligand binding, in which case ligands are to be understood as meaning ions, substances or the like. The ligand to be detected is hereby made accessible for analysis by means of a suitable (color) reaction. Furthermore, US Pat. No. 4,654,300 describes the use of such a quenching effect in immunoassays. In each of these cases, the internal filter effect is used for analytical purposes. However, this effect is in no case caused directly or exclusively by the substance to be investigated, as provided for in the process according to the invention. In addition to the internal filter effect, the method according to the invention requires a spatial separation of bound and free substance within a reaction vessel, a separation into sediment and supernatant taking place for this separation. The sediment comprises the solid body with binding partner and optionally with bound substance. The supernatant contains the unbound substance. Hier¬ by a compartmentalization in a predominantly liquid phase and a substantially solid phase is achieved. This separation of bound and unbound fraction of the substance to be investigated within a vessel requires no further transfer steps for the separation of sediment and supernatant. Rather, the concentration of the substance to be examined in the supernatant can be determined directly in the reaction vessel with the aid of the internal filter effect.

To carry out the process according to the invention, two alternatives are possible in principle, which can optionally be combined with one another. In the first alternative, the luminescent probe is located in the sediment during the measurement. In the second alternative, the luminescent probe is in the supernatant during the measurement.

According to the first alternative, the luminescent probe is attached to the solid, which may be loaded with binding partner. For this purpose, the solid is preferably provided with suitable probes before immobilization of the binding partner. Furthermore, it may also be preferred that the solid itself has Lumineszenzeigen¬ schaften.

In a preferred embodiment of the method, in particular according to this alternative, for the inventive method for a solid is largely without specific and / or largely without unspecific binding affinity to be examined for Substance provided as a so-called bond-passive solid and / or weite¬ rer solid with specific binding affinity to the substance as a so-called binding active solid. In this case, the solid with specific binding affinity is generally loaded with the at least one binding partner or carries the binding partner in immobilized form. The bond-passive solids are largely inert or inert to unspecific binding of the substance molecules, whereby the inertization can take place via a covalent and / or non-covalent saturation of reactive groups. They are generally used to determine the initial concentration, ie the concentration of substance before binding to the binding partner. The binding-active solids with the immobilized binding partner are used to determine the substance concentration after binding. To clarify this process, reference is made to FIG. 1.

In a preferred embodiment of this alternative of the process according to the invention, probe-labeled substances or solids are added to the binding-active and bond-passive solids, which therefore no longer necessarily have to be labeled by means of a probe. The added probe-labeled substances or solids then enable a determination of the substance concentration by means of the internal filter effect. This process requires a largely identical sedimentation behavior of specially marked substances or solids and the binding active and binding passive solids.

According to the second alternative of the method according to the invention, the probe is found in the supernatant during the measurement. Here, the detection of the substance concentration by means of the internal Filter¬ effect in the largely liquid phase, ie the supernatant, which are in the supernatant luminescent probes as particles, which follows do not sediment. This is preferably achieved in that the luminescent particles have a density comparable to the aqueous phase, that is, for example, with water or buffer. Preferably, these particles are still sufficiently large to scatter ultraviolet and visible light. So they preferably have optically scattering properties. Due to these light-scattering properties of the particles or of the probes, the luminescence signal, in particular the exciting light, does not reach the sedimented solid, and a uniform optical path length is established. For clarification of this method, reference is made to FIG. 2.

In a particularly preferred embodiment of this alternative of the method, the addition of the luminescent probes, in particular of the particles, takes place after the binding and spatial separation or separation of the solid. On the other hand, it is also possible to provide the luminescent particles or probes together with the solid and the controls in the reaction vessels, and to add the substance to be investigated at the end. This embodiment requires that the scattering and luminescent particles remain in suspension during the sedimentation of the solid.

The two described basic alternatives for carrying out the method according to the invention differ in particular in that the luminescent particles or probes remain in the supernatant in the second alternative of the method according to the invention and are not in the sediment or the solid phase after separation. Furthermore, in the second alternative, the use of bond passive solid is not necessary, but possible. The spatial separation of bound and free substance fraction within the reaction space or the reaction However, the vessel for both alternatives is the basic prerequisite for carrying out the process.

The measured luminescence signals preferably represent concentration-proportional quantities with the aid of which the quantification of the binding takes place. Preferably, the luminescence intensities of the bond-passive solid with and without substance in the supernatant are measured for this purpose. The determination of the concentration-proportional variable results from the Lambert-Beer law:

C = C 'before binding ⁼ Og I' Q / V = ε -C ⁽ J

Here, T _{0 is} the luminescence intensity in the reaction vessel with buffer solution and I 'is the luminescence intensity in the reaction vessel with substance solution. To determine the binding, the solid is occupied by the binding partner, and the binding-active solid is separated from the free substance fraction by, for example, sedimentation or centrifugation. It is preferably again the measurement of a reference without substance and the concentration-proportional size of the substance in the supernatant is in accordance with the Lambert-Beer law

C * = C * _nac h binding = lθg I VI * = S Cd determined. By comparing the concentrations before and after the binding, the binding can be quantified.

In a particularly preferred embodiment, the at least one luminescent probe exhibits fluorescent and / or phosphorescent properties. In this case, the excitation of the respective fluorescence or phosphorescence either takes place with one or more defined wavelengths, or excitation spectra are recorded. Furthermore, as a probe, a luminescent probe can be used which emits a signal without prior excitation of light. For example, for this purpose, a probe with electro-, thermo- and / or chemiluminescence can be used. However, the use of probes with fluorescence and phosphorescence is particularly This is preferably a procedure which has been established in laboratory practice and which is very easy to handle.

In a further preferred embodiment of the method according to the invention, a single probe or a single probe type is used. On the other hand, it may also be preferable to use a combination of different probes. Such a combination of probes may preferably be coupled to one another via the so-called Förster energy transfer.

In a preferred embodiment of the method according to the invention, the solid or solids consist of a glass, ceramic, silicate, metal and / or polymer material or of combinations of these materials. Very particular preference is given to solids in the form of beads, so-called beads, which are preferably porous beads. Very particular preference is given to such beads, preferably porous beads, of silicate material.

In a preferred embodiment of the method according to the invention, the binding partner is at least one lipid membrane. This may be a simple lipid layer, a so-called monolayer, or a double lipid layer, a so-called bilayer. The composition of such lipid layers can be chosen freely depending on the desired application. For example, such lipid layers can be largely homogeneously structured, or different lipids, for example, can be used as constituents of the membrane layers, as a result of which, for example, the conditions of a native membrane can be adjusted. Furthermore, it may also be preferred that the lipid membrane is a so-called biomembrane, which is produced in particular from natural membranes. The composition of such a biomembrane may of course vary. Such lipid membranes or biomembranes are immobilized on a solid according to the inventive method. In this case, these membranes (monolayer or bilayer) can be covalently fixed. However, it is particularly preferred if these lipid layers are not covalently fixed, so that they reflect natural conditions of a lipid membrane particularly well. With the aid of such immobilized lipid layers, the binding parameters of a substance to be examined with lipid layers can be analyzed with particular advantage, as is of interest for many aspects in the investigation of potential active substances or the like.

In another preferred embodiment, according to the invention, peptides, proteins, carbohydrates, surfactants, steroids, polymers, nucleotides, oligonucleotides, DNA and / or RNA can be used as binding partners. In a particularly preferred manner immobilized proteins are used as binding partners, in particular serum proteins, for example human serum albumin, enzymes or antibodies. In accordance with the method according to the invention, the binding parameters of a substance to be examined with one or more of these binding partners can be investigated hereby.

In a further preferred embodiment, the solid laden with binding partner may have further components. For example, another protein or several proteins may be fixed on or within a lipid membrane which is immobilized on the solid. In particular, it may be proteins reconstituted in the lipid membrane. Furthermore, carbohydrates, surfactants, steroids, polymers, nucleotides, oligonucleotides, DNA and / or RNA or even peptides can be immobilized on the solid as further components. This is particularly preferably done in conjunction with a lipid membrane, in particular a lipid bilayer. It may be, for example, a reconstituted ligand act, for example a protein. By integration of one or more proteins, carbohydrates or other further components within a lipid membrane, in particular a lipid bilayer, native conditions of a lipid membrane can be adjusted with particular advantage, so that with the aid of such relatively complex binding partners on the basis of a lipid membrane a largely natural binding behavior of the substance to be investigated can be nach¬ provided. On the other hand, other combinations, such as proteins and carbohydrates, may also be immobilized as a binding partner on the solid.

In principle, all substance classes come into question as substances to be investigated. In particular, potential active substances, in particular potential pharmaceutical and / or biological active substances, which can be used for therapy and / or diagnosis of various diseases, can be investigated with the aid of the method according to the invention. These may be, for example, peptides or proteins. In particular, it is also possible to investigate low molecular weight substances, as frequently occurring in the case of potential pharmaceutical active substances. Furthermore, with the method according to the invention, for example, potential environmental toxins can be investigated, which can have different compositions or can be assigned to different substance classes.

A particular advantage of the method according to the invention is that the separation into sediment and supernatant takes place without any particular expense. As a result of the separation, bound and free fractions of the substance to be investigated are spatially separated from one another, so that the determination of the concentration in the supernatant within the reaction vessel can be carried out by utilizing the internal filter effect. It is particularly preferred that the separation is carried out by sedimentation and / or centrifugation, in particular by low-speed centrifugation. Above all, a simple sedimentation has the advantage that no expenditure on apparatus is required for this and that the separation into sediment and supernatant takes place without further action. Carrying out the separation with the aid of a centrifuging, in particular a low-speed centrifugation, has the advantage that the separation process can thereby be accelerated and optionally intensified, which may be advantageous depending on the desired application.

The process according to the invention can be carried out within conventional reaction vessels. Particularly preferred here is the use of microtiter plates, ie in particular the use of the wells of a microtiter plate as a reaction vessel. In this case, microtiter plates of conventional dimensions are suitable, it being possible, for example, to use microtiter plates with 384 wells (cavities) which on the one hand provide a sufficient volume within the individual cavity and on the other hand permit a high sample throughput. In addition, the method according to the invention can of course also be carried out in other suitable constructions.

In a particularly preferred embodiment, the measurement of the luminescence signals takes place at one or more discrete wavelengths, in particular excitation and emission wavelengths. On the other hand, however, it is also possible to measure entire spectra and draw conclusions about the modulation of the respective signals of specific wavelengths.

In a preferred embodiment, the excitation of the probes takes place at a certain wavelength by irradiation "from above", ie the excitation takes place on the side of the supernatant.Of course, this need not necessarily be the upper side of the reaction. examples For example, in a centrifuge, this can mean, for example, a radiation from the side. This exciting wavelength passes through the supernatant and is attenuated by light absorption of the unbound substances in the supernatant. This has the effect that only a reduced proportion of the irradiated signal to the probe in the sediment or in the supernatant, depending on the alternative of the method, meets, and thus the luminescence emitted by the probe Lumi¬ neszenzsignal is reduced in comparison with corresponding controls. When using luminescent probes which emit a signal without prior excitation, the radiated signal will usually be recorded on the side of the supernatant, since this phenomenon of the internal filter effect can be exploited in a corresponding manner.

In a further preferred embodiment, the measurement of the emitted signal, that is to say the emitted radiation of the probe, takes place on the side of the sediment, that is to say without passing through the supernatant. The excitation with a suitable excitation wavelength is also preferably carried out on this side (measurement "from below"), thereby achieving an altered luminescence intensity after binding of the substance to be investigated to the binding partner in the sediment The type of measurement thus serves, above all, to standardize the measurement results, which can preferably be used to standardize the luminescence intensity of the measurement "from above", so that the luminescence modulation can be corrected by the substance bound to the solid. Preferably, especially in the case of the first alternative of the method according to the invention, a measurement takes place both "from above" and "from below". On the other hand, it may also be preferred that the measurement takes place exclusively by measuring the emitted radiation and preferably also by excitation of the probes "from below", for example when it comes to the determination of relatively high concentrations In the described second alternative of the method according to the invention, the excitation and / or measurement of the emitted radiation preferably takes place "from above." For this purpose, either the excitation or emission spectra can be determined, or the luminescence signals can be determined in one or more embodiments Several discrete excitation and emission wavelengths can be determined, but of course combinations of the various possibilities are also possible.

In a preferred manner, the determination of the substance concentration in the supernatant takes place by determination of concentration-proportional quantities, as has already been described. Alternatively or in combination, it may be preferred that the substance concentration is determined on the basis of calibration lines. For this purpose, calibration lines are advantageously taken before and / or after the binding in which, in the presence of the luminescent probes, for example the particle according to the second alternative of the method, the concentration of the substance is varied and according to Lambert's invention. Law c "= log I ¹ VI" = ε cd = α c the proportionality constant α is determined. In this case, l " _{0 are} the luminescent intensity, in particular the fluorescence and / or phosphorus intensity, of the buffer solution and I" the luminescence intensity, in particular the fluorescence and / or phosphorescence intensity, of the substance solution. On the other next to or as an alternative to the concentration da¬ proportional sizes c _"VO r bond and c" _na ch binding determined, and the binding can be quantified by comparing two parameters.

The invention further includes a kit for determining binding parameters, which comprises at least one solid and at least one luminescent probe. The probe preferably has fluorescent decorating and / or phosphorescent properties and is during the measurement according to the described method in Sedi¬ ment or in the supernatant. It is also possible to use probes with luminescent properties without light excitation, in particular electrical, thermal and / or chemiluminescence properties.

Preferably, the solid is at least partially loaded with at least one binding partner. On the other hand, it may also be preferred that the solid is provided in the kit of the invention without further Bela¬ tion, so that the user gets the opportunity to make a load with suitable binding partners themselves. As a result, a greater flexibility of the possible applications of the kit according to the invention is achieved, so that the user can easily adapt the components of the kit according to the invention to the respective questions of the experiment to be carried out. Especially in the case where the solid is provided without loading with a binding partner in the kit, further components can be contained in the kit which can be used by the user for loading or immobilization with binding partners, for example suitable buffers , Linker molecules or the like.

In a preferred embodiment of the kit, the solid is loaded with a lipid layer, preferably with a lipid bilayer. Loading with a lipid monolayer may also be preferred. The immobilization takes place, for example, via covalent forces. However, immobilization via noncovalent forces, for example via lipophilic interactions, in particular van der Waals interactions, and / or electrostatic interactions, is particularly preferred. This has the advantage that in this way native conditions of a membrane can be imitated particularly well. With regard to a suitable immobilization of lipid layers, reference is made to WO 99/51984. In a further preferred embodiment of the kit according to the invention, the solid laden with binding partner is provided by a solid with immobilized proteins. Particular preference is given here to serum proteins immobilized on the solid, for example human serum albumin, enzymes or antibodies. The solid-immobilized proteins can be immobilized, for example, directly or via suitable linker molecules on the solid.

In a further preferred embodiment, the binding partner is formed by a lipid layer which has reconstituted proteins within the layer. In this way, for example, binding affinities between the substance and the binding partner can be analyzed, the binding partner in the narrower sense being the protein which is offered in its natural environment, namely the lipid layer or a lipid bilayer, to the substance for binding. Hereby, the natural conditions of binding reactions can be adjusted in a particularly advantageous manner. The proteins as binding partners, which are immobilized either on and / or within a lipid layer on the solid, are preferably membrane-bound and / or transmembrane proteins, in particular serum proteins, preferably human serum albumin.

In a preferred embodiment of the kit according to the invention, the solid body with the at least one luminescent probe is ver¬ see. This embodiment of the kit according to the invention is above all suitable for carrying out the method according to the first alternative described above. In another preferred embodiment of the kit according to the invention, the probe has optically scattering properties and is not coupled to the solid. This embodiment of the kit according to the invention is primarily used for management of the inventive method according to the above beschrie¬ benen second alternative suitable.

It is particularly advantageous to provide the solid, which is at least partially loaded with at least one binding partner, in pre-pipetted microtiter plates with, for example, 96, 384 and 1536 well (cavities) format, in which the solid-immobilized binding partner, if appropriate the probe, and the buffer are already contained so that the user only has to add the substance and optionally the probe.

With regard to further features of the various components of the inventive kit, reference is made to the above description.

The use of luminescence-labeled solids and / or scattering and luminescent particles according to the method according to the invention in conjunction with the solid-supported bond between an immobilized binding partner and a substance to be investigated is a generally applicable method for determining binding parameters Concentration-proportional quantities can advantageously be carried out directly after separation into sediment and supernatant by simple sedimentation or the like. Here, the substance to be examined serves as a marker itself, so to speak. As a result, a concentration change after binding is detected directly and exclusively by the substance to be investigated and becomes accessible by modulation of the luminescent signal. The method according to the invention therefore meets the requirements of an automated and user-friendly high-throughput screening method, as is preferably used in the pharmaceutical industry, in a particularly advantageous manner. The described features and further features of the invention will become apparent from the following description of examples in conjunction with the dependent claims and the figures. In this case, the individual features can be implemented individually or in combination with each other.

In the figures shows

1 shows a schematic representation of the measuring principle according to the first alternative of the method according to the invention;

2 shows a schematic representation of the measuring principle according to the second alternative of the method according to the invention;

FIG. 3: concentration dependence of the concentration-proportional size log F ₀ (λ) / F {λ) = e (Λ) cd for warfarin;

4: Correlation of the membrane affinities logMA measured according to the invention with the values from conventional membrane affinity measurements using filter assays;

FIG. 5: Correlation of the K _d values measured according to the invention

(Dissociation constant) of HSA binding with values from conventionally determined dissociation constants using filter assays;

FIG. 6: Concentration dependence of the concentration-proportional size log Fo (λ) IF (λ) = e (Λ) cd in strongly scattering media for warfarin. Fig. 1 shows a schematic representation of the measuring principle of the inventive method in the form of the first alternative Ver¬ procedure in a preferred embodiment. In this case, the measurement of the fluorescence of the passivated solids gives the intensities I ₀ '(passivated solid plus buffer), and I' (passivated solid plus buffer plus substance) provides the concentration-proportional variable c ' _VO r _B _and nd- The measurement of the fluorescence of Bonding-active solid provides the intensities I ₀ * (bond-active solid plus buffer), and I * (bond-active solid plus buffer plus substance) yields the concentration-proportional size c ^* after binding. FIG. 2 shows in a comparable manner Measuring principle of the inventive method according to the second alternative in a preferred embodiment. In this case, I _{0 is} the fluorescence intensity of the buffer solution, l _VO r Bin _du ng "the fluorescence intensity of the substance solution and l _na ch Bi _n tion" the fluorescence intensity after the binding event and compartmentalization in sediment and supernatant.

Furthermore, the implementation of the method according to the invention is described on the basis of the affinity determination of warfarin on a lipid membrane, the determination of the albumin binding of carbamazepine as well as on the albumin binding of warfarin in strongly scattering media.

Examples

1. Preparation of Amino Carriers

2 g of a porous silicate material (Nucleoprep 300-12, Macherey-Nagel, Düren) are dissolved in a silane solution consisting of 9.2 ml of N- (2-aminoethyl) -3-aminopropyltrimethoxysilane and 243 μl of concentrated acetic acid in 450 ml of deionized water, and added for three hours. sam rotates, thereafter, the silicate material is sedimented, washed three times with deionized water and dried at 80 ⁰ C.

2. Preparation of fluorescence-active carriers

1 mg of 3-indoleacetic acid is dissolved in 0.5 ml of dimethylformamide, and to this solution are added 10 mg of N-hydroxysuccinimide (NHS) and 12 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, also in 0.5 ml Dissolved dimethylformamide given. This solution is stirred for at least 2 hours, and then 200 mg of the aminocarrier prepared in 1. are added. This suspension is rotated overnight and washed three times with PBS (phosphate buffered saline, 10 mM sodium phosphate, 0.9% sodium chloride, pH 7.4) and transferred to carbonate buffer.

100 μl of a solution of fluorescein isothiocyanate (FITC) in a carbonate buffer (1.33 mg / ml) are added to 100 mg of the support in 900 μl of carbonate buffer (pH 9.5) and rotated overnight. To remove unreacted FITC, it is first washed twice with carbonate buffer and then three times with PBS.

3. Production of Fluorescence-Active and Bonding-Passable Carriers (Fl-Ref)

For the 100 mg of the fluorescence-active carrier from 2., 1 ml of a solution of polyethylene glycol bisglycidyl ether (Mn about 526) in PBS (3% (w / v)) is added and rotated on the overhead head over night. Finally, it is washed three times with PBS. 4. Reconstitution of Lipid Membranes on Fluorescent Active Carriers (FI-EiPC)

For the reconstitution of lipid membranes, first 1 g of a carrier prepared according to 2. 2 treated rotator in an overhead solution with 25 mg of polystyrene sulfonate, sodium salt in 25 ml of water and then washed three times with water and PBS.

60 ml of a vesicle solution of 5 mg / ml EiPC (lipid extract from egg yolk) in PBS (preparation by homogenization in the homogeniser EmulsiFlex-C5 from AVESTIN) are added to this support and incubated overnight. To remove excess lipid, wash three times with PBS and then store the carrier in PBS. The determination of the lipid concentration is carried out by drying defined suspension volumes, removing the lipid from the solid by means of an organic solvent and subsequent HPLC analysis.

5. Immobilization of Human Serum Albumin (HSA) on Fluorescecent Carriers (FI-HSA)

For the immobilization of HSA, 100 mg of HSA, dissolved in 10 ml of phosphate buffer (20 mM, pH 7.4), are added to 1 g of a carrier prepared according to 2, and rotated over the head for at least four hours. To remove free protein, it is washed with phosphate buffer, 1 M NaCl in phosphate buffer and finally with PBS. Quantification of protein immobilization is performed by supernatant analysis. The suspension is adjusted and stored in a desired protein concentration in PBS. 6. Determination of the concentration-proportional size of warfarin

200 mg of the binding-passivated and fluorescence-active carrier prepared according to 3. are brought to a total volume of 1 ml with PBS. 30 μl each of these suspensions are filled into different wells of a 96 well microtiter plate (Greiner UV-Star). By adding defined volumes of warfarin and PBS, ten different warfarin concentrations between 0 and 2 E-4 mol / L are produced in a total volume of 300 μl. After mixing the cavities by resuspension, the solid is separated from the supernatant by sedimentation. In a plate reader (TECAN Safire), the excitation spectra in the "top-read" mode are measured in all ten cavities between 250 and 480 nm at an emission wavelength of 530 nm and according to the Lambert-Beer law log F ₀ (A) IF (A) = e (A) cd is calculated, the excitation spectrum of the buffer solution corresponding to Fo (A) A plot of the concentration-dependent variable log F _o / F at the maximum of the warfarin absorption of 310 nm as a function of the concentration (FIG ) shows a linear course.

7. Determination of the Membrane Affinity of Warfarin in the 96-well Form

To determine the membrane affinity, 200 mg of the FI-EiPC carrier prepared in 4 ° C. with PBS were brought to a total volume of 1 ml, likewise 200 mg of a Fl-Ref prepared according to 3 were adjusted to 1 ml with PBS.

To determine the concentration-dependent quantities, the following volumes were added to the wells of a 96-well UV plate (Greiner UV-Star): Before binding

Cavity Reference 1: 100 μl Fl-Ref suspension + 200 μl PBS;

Cavity Substance 1: 100 μl Fl-Ref suspension + 170 μl PBS + 30 μl warfarin solution;

After binding

Cavity Reference 1 ': 100 μl FI-EiPC suspension + 200 μl PBS;

Cavity substance 1 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

Warfarinlösung;

where the final concentration of warfarin in the wells is 70 μM. After mixing the cavities by resuspension, the solid is separated from the supernatant by sedimentation. In order to determine the concentration-proportional size c _before binding, in the "top-read" mode excitation spectra between 250 and 480 nm at an emission wavelength of 530 nm are taken from the cavities Reference 1 and Substance 1 and according to c _before binding. Fov (Λ) / Fv (Λ) are offset, where F _0v (Λ) and F _M (A) are the excitation spectra of the cavities Reference 1 and Substance 1. To determine the concentration-proportional variable C after binding, the above procedure is used to stimulate supply spectra of the wells reference 1 'and substance 1' men and aufgenom¬ according _to c F _O n (Λ) / F _n (Λ) is calculated. Here, F _On (Λ) and F _n (A) are given by the excitation spectra of the cavities Reference 1 'and Substance 1'. With the lipid volumes Vii _Pid and a total volume V total in the cavities Reference 1 'and Substance 1', the membrane affinity of warfarin according to

^"Total C _V ~ C after binding or binding log MA = log

»Lipid Cnach binding receive. From a triple determination a value of 1.7 + 0.3 was obtained, which is in good agreement with values from conventional binding assays.

8. Determination of the membrane affinity in the 96-well format

In order to determine the membrane affinity, 200 mg of the FI-EiPC carrier prepared in 4 ° C. with PBS were brought to a total volume of 1 ml, likewise 200 mg of a Fl-Ref prepared according to 3 were adjusted to 1 ml with PBS.

To determine the concentration-dependent quantities, the following volumes were pipetted into the wells of a 96-well UV plate (Greiner UV-Star):

Before binding

Cavity Reference 1: 100 μl Fl-Ref suspension + 200 μl PBS;

Cavity substance 2: 100 μl Fl-Ref suspension + 170 μl PBS + 30 μl

Benzocainsäure;

Cavity Substance 3: 100 μl Fl-Ref Suspension + 170 μl PBS + 30 μl

propranolol;

Cavity Substance 4: 100 μl Fl-Ref suspension + 170 μl PBS + 30 μl imipramine solution;

Well substance 5: 100 μl Fl-Ref suspension + 170 μl PBS + 30 μl indomethacin solution;

Cavity Substance 6: 100 μl Fl-Ref suspension + 170 μl PBS + 30 μl metoprolol solution; After binding

Cavity Reference 1 ': 100 μl FI-EiPC suspension + 200 μl PBS;

Cavity substance 1 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

Warfarinlösung;

Cavity substance 2 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

Benzocainsäure;

Cavity substance 3 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

propranolol;

Cavity substance 4 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

Irnipraminlösung;

Well substance 5 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

indomethacin;

Cavity substance 6 ': 100 μl FI-EiPC suspension + 170 μl PBS + 30 μl

metoprolol;

wherein the substance concentration in the cavities is 70 μM. After mixing the cavities by resuspension, the solid is separated from the supernatant by sedimentation. To determine the concentration-proportional variable Cv _O r Bi _n d _un g,) are both in the "top-read" -MO- dus and in the "bottom-read" mode excitation spectra from 250 to 480 nm at an emission wavelength of 530 nm of the cavities Reference 1 and substance i and the spectra recorded "from above" are normalized by division by the spectra recorded correspondingly "from the bottom" and according to c _VO r _B i _n d _un g _, i = log Fov (Λ) / Fvj (Λ), where F _0v (Λ) and F _vj (Λ) are the normalized excitation spectra (praise / lunts) of the cavities reference 1 and substance i. To determine the concentration-proportional variable c _na c _{h B} i _n _n g _,! In accordance with the above procedure, excitation spectra of the cavities 1 'and substance i' are recorded and _calculated according to c _naC h _B iπd _un g _, i = log Fon (Λ) / Fni (Λ). Here are Fon (Λ) and F _ni (Λ) is given by the normalized excitation spectra (l _O ben / l th _Un) of the cavities Reference 'and substance i'. With the lipid volume V | _iP id and a total volume V _ge s _amt in the cavities reference 1 'and substance i', the membrane affinity logMAj of the substance i is obtained in accordance with

»Total Cvor bond, i Cnach bond, i log MA | = log

Vijpid Cnach bond, i

The good agreement of the logMA values with the comparative values obtained from a heterogeneous filter assay is shown in FIG. 4.

9. Determination of albumin binding of carbamazepine in the 384-well format

To determine the binding of albumin, 200 mg of the FI-HSA carrier prepared in the 5th preparation were applied to a total volume of 1 ml with PBS, likewise 200 mg of an Fl-ref prepared according to 3 were adjusted to 1 ml with PBS.

To determine the concentration-dependent quantities, the following volumes were pipetted into the wells of a 384-well UV plate (Greiner UV plate):

Before binding

Cavity Reference 1: 35 μl Fl-Ref suspension + 65 μl PBS;

Well 1: 35 μl Fl-Ref suspension + 55 μl PBS + 10 μl carbamazepine solution;

After binding

Well Reference 1 ': 35 μl FI-HSA suspension + 65 μl PBS;

Well 1 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl carbamazepine solution; wherein the final concentration of carbamazepine in the wells is 70 μM. After mixing the cavities by resuspension, the solid is separated from the supernatant by sedimentation. In order to determine the concentration-proportional size c _before binding, excitation spectra between 250 and 480 nm at an emission wavelength of 530 nm are recorded by the cavities Reference 1 and Substance 1 in the "top-read" mode and according to c _VO r F _0v (Λ) / F _v (Λ), where Fov (Λ) and F _V (Λ) are the excitation spectra of the cavities Reference 1 and Substance 1. In order to determine the concentration-proportional quantity C after binding, excitation spectra of the cavities reference 1 'and substance 1' are recorded in accordance with the above procedure and according to c _naC h F _0n (A) ZF _n (A) is calculated. Here F _On (Λ) and F _n (A) are given by the excitation spectra of the cavities reference V and substance 1 '. With the concentration of HSA in the cavities Reference 1 'and the substance 1' C _HSA calculated neglecting saturation effects, the dissociation constant K _d for Carba¬ mazepin according to

CHSA C after binding κ _d =

Cvor Bond "Cnach Bond

The obtained K _d value of 1.7 E-4 [mol / l] agrees well with the values from conventional binding assays.

10. Determination of albumin binding in 384-well format

For the determination of the albumin binding, 200 mg of the FI-HSA carrier prepared in the 5th step are mixed with PBS to a total volume of 1 ml. 200 mg of a Fl-Ref prepared according to 3. were adjusted to 1 ml with PBS.

Before binding

Cavity Reference 1: 35 μl Fl-Ref suspension + 65 μl PBS;

Cavity Substance 1: 35 μl of Fl-Ref suspension + 55 μl of PBS + 10 μl of phenylbutazone solution;

Cavity Substance 2: 35 μl of Fl-Ref suspension + 55 μl of PBS + 10 μl of carbamazepine solution;

Cavity Substance 3: 35 μl Fl-Ref suspension + 55 μl PBS + 10 μl diclofenac solution;

Cavity Substance 4: 35 μl Fl-Ref suspension + 55 μl PBS + 10 μl Keptone solution;

Cavity Substance 5: 35 μ Fl-Ref suspension + 55 μl PBS + 10 μl furo- semid solution;

Cavity Substance 6: 35 μl Fl-Ref suspension + 55 μl PBS + 10 μl

Warfarinlösung;

After binding

Well Reference 1 ': 35 μl FI-HSA suspension + 55 μl PBS;

Well 1 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl phenylbutazone solution;

Cavity substance 2 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl carbamazepine solution;

Well 3 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl diclofenac solution;

Well substance 4 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl ketophosphate solution; Well substance 5 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl furo- semid solution;

Cavity substance 6 ': 35 μl FI-HSA suspension + 55 μl PBS + 10 μl warfarin solution;

wherein the substance concentration in the cavities is 70 μM. After mixing the cavities by resuspension, the solid is separated from the supernatant by sedimentation. To determine the konzentra¬ tion proportional size C prior Bindu _n g ,! In both the "top-read" mode and the "bottom-read" mode, excitation spectra between 250 and 480 nm are recorded at an emission wavelength of 530 nm by the cavities Reference 1 and Substance i and recorded from the top Spectra are normalized by division by the spectra recorded correspondingly "from below" and are calculated according to c _VO r _B i _nd u _n g, i = log Fo _v (Λ) / F _V i (Λ), where F _Ov (Λ) and F _V j (Λ) are the normalized excitation spectra (Uen / lu _n th) of the cavities reference 1 and substance i. To determine the concentration magnitude proportional c _nd ung _for Bi ,! In accordance with the above procedure, excitation spectra of the cavities reference 1 'and substance i' are recorded and according to c _na c _h F _On (Λ) / F _ni (Λ) is calculated. Here F _On (Λ) and F _n i (Λ) are given by the normalized excitation spectra (W _n / Ute _n ) of the cavities reference 1 'and substance i'. With the concentration of HSA in the cavities Reference 1 'and Substance i' C _H S _A , the dissociation constant K _d , i of the substance i is obtained, neglecting saturation effects

CHSA ^' C _nac h bond, i

Kd, i ⁼

Cvor bond ,! "Cf ₁₃ Ch bond, i

The good agreement of the K _d values with the comparative values obtained from a heterogeneous filter assay is shown in FIG. 5. 11. Determination of the concentration-proportional size in scattering media

10 μl of a suspension of europium-labeled polystyrene microparticles (diameter 1 μm, Molecular Probes, Eugene, OR, USA) are introduced into each of a plurality of wells in a 384 well microtiter plate. By adding defined volumes of warfarin and PBS, five different levels of warfarin between 0 and 7E-5 mol / l are produced in a total volume of 100 μl. The final concentration of polystyrene particles is 0.05%. In a plate reader (TECAN Safire), the excitation spectra are measured in the "toρ-read" mode in all five cavities between 230 and 580 nm at an emission wavelength of 620 nm (lag time 100 μs, integration time 400 μs) and according to Lambert-Beer law log F ₀ (A) ZF (A) = e (A) cd calculated with each other, whereby the excitation spectrum of the buffer solution corresponds to F ₀ (A) A plot of the concentration-dependent variable log F _o / F at the maximum the warfarin absorption of 310 nm as a function of concentration (FIG. 6) shows a linear course.

12. Determination of albumin binding of warfarin in strongly scattering media

To determine the albumin binding in strongly scattering media wer¬ the three wells of a 384 well microtiter plate commercially erhält¬ Lichem TRANSIL ^® HSA (NIMBUS Biotechnology GmbH Leipzig) as follows filled

Well Buffer 1: 90 μl PBS;

Cavity Reference 1: 80 μl PBS + 10 μl warfarin solution; Cavity substance 1: 35 ul TRANSIL ^® suspension HSA + 45 ul PBS + 10 ul Warfarinlösung;

wherein the warfarin concentration in the cavities Reference 1 and Sub¬ stanz 1 is 70 uM. After thorough mixing and sedimentation of the solid, 10 μl of a suspension of europium-labeled polystyrene microparticles (diameter 1 μm, Molecular Probes, Eugene, OR, USA) are added. In a plate reader (TECAN Safire), the excitation spectra are measured in the "top-read" mode in all three cavities between 230 and 580 nm at an emission wavelength of 620 nm (lag time 100 μs, integration time 400 μs) and according to Lambert-Beer law log F ₀ (Λ) / F (Λ) = e (Λ) -cd, with the excitation spectrum corresponding to the buffer solution F ₀ (M) Concentrations in the substance and Referenzkavität determined and the K _d according to

CfHSA Csubstanzi

Kd, i =

CReferenzi "CSubstanzi

to K _d = 1, 7E-5 [mol / l], which is in good agreement with the results of conventional methods.

Claims

claims

1. A method for determining binding parameters between at least one substance and at least one binding partner using at least one luminescent probe, comprising the method steps:

Providing at least one solid which is at least partially loaded with at least one binding partner, incubating the solid with the at least one substance,

Separation of solid and unbound substance in a reaction vessel in sediment and supernatant and measurement of a modulation of luminescence signals of at least one probe by unbound substance in the supernatant to determine the substance concentration in the supernatant, wherein absorption spectra of at least ei¬ NEN substance with excitation and / or emission spectra of the at least one probe overlap.

2. The method according to claim 1, characterized in that the probe is in the sediment during the measurement.

3. The method according to claim 1 or claim 2, characterized gekenn¬ characterized in that the probe is attached to loaded with binding partner Fest¬ body and / or applied to unloaded solid.

4. The method according to any one of the preceding claims, characterized in that the solid itself has Lumineszenzeigen¬ properties.

5. The method according to any one of the preceding claims, characterized in that the probe is in the supernatant during the measurement, wherein preferably the probe has optically streu¬ end properties.

6. The method according to any one of the preceding claims, characterized in that solids are provided substantially without specific and / or substantially no non-specific binding affinity to the substance as binding passive solid and / or solid with specific binding affinity to the substance as a binding active solid.

7. The method according to any one of the preceding claims, characterized in that the luminescent probe has fluorescent and / or phosphorescent properties.

8. The method according to any one of the preceding claims, characterized in that a single probe or a combination of probes is used.

9. The method according to any one of the preceding claims, characterized in that the solid body is a glass, ceramic, silicate, metal and / or polymeric material, in particular in the form of beads, preferably porous beads.

10. The method according to any one of the preceding claims, characterized in that the binding partner is at least one lipid membrane, biomembrane, peptide, protein, enzyme, carbohydrate, surfactant, steroid, polymer, nucleotide, oligonucleotide, DNA and / or RNA.

11. The method according to any one of the preceding claims, characterized in that the separation by sedimentation and / or centrifugation, in particular low-speed centrifugation, er¬ follows.

12. The method according to any one of the preceding claims, characterized in that the reaction vessel is the cavity of a microtiter terplatte.

13. The method according to any one of the preceding claims, characterized in that the luminescence signals are measured at one or more meh¬ discrete wavelengths and / or as a spectrum.

14. The method according to any one of the preceding claims, characterized in that for the measurement, the luminescence signals are recorded after passing through the supernatant.

15. The method according to any one of the preceding claims, characterized in that for the measurement, in particular for normalization of the measurement, the luminescence signals are recorded without passing through the Cl- supernatant.

16. The method according to any one of the preceding claims, characterized in that the determination of the substance concentration in the supernatant by determining concentration-proportional magnitudes and / or with the aid of calibration lines.

17. Kit for the determination of binding parameters according to a method according to one of the preceding claims, comprising at least one of the following components: at least one solid and at least one luminescent probe.

18. Kit according to claim 17, characterized in that the Festkör¬ by at least partially loaded with at least one binding partner.

19. Kit according to claim 18, characterized in that the solid binder laden with solids is a solid with immobilisier¬ ter lipid layer, preferably lipid bilayer, in particular with non-covalently immobilized lipid layer.

20. Kit according to claim 18 or claim 19, characterized in that the charged with binding partner solid is a solid with immobilized proteins.

21. Kit according to any one of claims 18 to 20, characterized in that the charged with binding partner solid is a solid with reconstituted in a lipid layer proteins.

22. Kit according to claim 20, characterized in that the proteins are at least one serum protein, in particular human serum albumin.

23. Kit according to any one of claims 17 to 22, characterized gekennzeich¬ net, that the solid body is provided with the probe.

24. Kit according to any one of claims 17 to 23, characterized in that the probe has optically scattering properties.

25. Kit according to any one of the preceding claims 17 to 24, da¬ characterized in that solid, in particular solid with immobilized binding partner, and preferably buffer in pre-pipetted microtiter plates, in particular in a 96, 384 and / or 1536 well (wells) format.

26. Kit according to any one of claims 17 to 25, characterized in that the components of the kit have at least one of the features according to one of claims 1 to 16.