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Method for determining the binding behavior of ligands which specifically bind to target moleculesRelated Patent Categories: Liquid Purification Or Separation, Processes, ChromatographyMethod for determining the binding behavior of ligands which specifically bind to target molecules description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060201886, Method for determining the binding behavior of ligands which specifically bind to target molecules. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application is a divisional of U.S. application Ser. No. 10/478,025, the entire disclosure whereof is expressly incorporated by reference herein, which is a U.S. National Stage of International Application No. PCT/EP02/05543, filed May 21, 2002, which claims priority under 35 U.S.C. .sctn. 119 of German Patent Application No. 101 25 258.7, filed May 23, 2001. BACKGROUND OF THE INVENTION [0002] 1. Field of the Invention [0003] The invention relates to a method for determining the binding behavior of ligands which specifically bind to target molecules at at least one binding site. [0004] 2. Discussion of Background Information [0005] DE 198 14 775 A1 discloses a method for determining the binding constants of dissolved agents and substances on surfaces constituted by amphiphilic molecules. Therein, layers of amphiphilic molecules are bonded to a solid carrier so that a solid-supported membrane is formed. A defined quantity of this solid-supported membrane is contacted with a mobile phase. The mobile phase contains a defined quantity of substances which are to be examined with respect to lipid binding. After an incubation, the solid-supported membranes are separated from the mobile phase. The concentrations of the substances in the mobile phase or the solid-supported membrane are determined. The method can be carried out with solid-supported membranes and substances that are present in different quantity ratios. From the determined concentrations, the binding constant can be calculated. [0006] From WO 99/50669 A1 there is known a method for screening a compound library to determine the relative affinity of a plurality of putative ligands to a target receptor in relation to an indicator compound by means of frontal chromatography. Therein, the target receptors are immobilized on the solid phase of a chromatographic column. The compound library is applied onto the chromatographic column and equilibrated therewith at least partially. Subsequently, the indicator compound is applied and its break through time, i.e., the time until it appears in the effluent of the chromatographic column, is determined by means of mass spectrometry. The break through time of the indicator compound is affected by its affinity, the affinity of ligands present to the target receptor, and the number of target receptors which are not occupied by the ligands. From the change in the break through time in comparison to the known break through time of the indicator compound, it can be determined whether compounds of the compound library have an affinity to the target receptors, and whether this affinity is higher or lower than that of the indicator compound. [0007] A disadvantage of this method is that the target receptors are invariably immobilized. They are not present in the native state, whereby it is possible to only a limited extent to say something about the binding behavior on native target receptors. A further disadvantage of the method is that in kinetic studies, it leads to incorrect results regarding the binding behavior between ligands and the indicator compound. If a ligand and the indicator compound had the same affinities to the target receptor, the affinity of the ligand would be determined as too low if the ligand shows slower binding kinetics relative to the indicator compound. A further disadvantage is that the quantity of the indicator compound which is bound or unbound, respectively, in the binding equilibrium state cannot be determined. [0008] WO 01/22078 A1 relates to a method for the identification of an active chemical substance from a mixture of active and inactive chemical substances, where a target substance is added to the mixture. Complexes formed of the active substance and the target substance are separated from the mixture. For identification, the active chemical substance may be released from the complexes and identified by, for example, mass spectrometry. Alternatively, the active substance may also be identified by comparing a chromatogram or a mass spectrum, respectively, of each of the mixture of active and inactive substances and the mixture after separation of the complex. A disadvantage thereof is that it must be possible to determine each of the substances of the mixture. [0009] WO 97/43301 A2 relates to a method for characterizing the members of a combinatorial library which bind to a certain domain. Therein, the domain is mixed with the members of the combinatorial library to permit a binding of the members to the domain. The formed complexes are separated from the unbound members. The bound members are eluted from the complexes and analyzed by mass spectrometry. Therein, it is possible to elute more weakly bound members separately from more strongly bound members. A disadvantage thereof is that each of the members of the mixture must be determinable by mass spectrometry. [0010] From WO 96/22530 A1 and U.S. Pat. No. 5,891,742 A there is known a method for determining a compound which binds to a target compound from a mixture of compounds. Therein, the target compound is contacted with the mixture of compounds. Unbound compounds are separated from formed complexes of the compounds with the target molecules. The formed complexes are analyzed by mass spectrometry. It is of disadvantage therein that all compounds and complexes, respectively, of the binding assay must be determinable by mass spectrometry. [0011] From WO 00/00823 A1 there is known a method for identifying a ligand which covalently binds to a target molecule. In this method, a target molecule is contacted with several compounds of a substance library. The compound which forms a covalent bond with a chemically reactive group of the target molecule is identified by mass spectrometry. For this purpose, the conjugate formed by the covalent bond may be fragmented. The bound compound may as well be released by cleaving a disulfide bond. For identification, the mass spectroscopic characteristics of the compounds to be determined or of the fragment to be determined, respectively, must be known in this method. [0012] From WO 99/45150 A1 there is known a method for determining the relative binding affinity to a target substance of compounds of a combinatorial mixture. Therein, a first complex of the target substance and a standard compound binding thereto is contacted with the combinatorial mixture of compounds, whereby second complexes are formed from these compounds and the target substance. The mixture is analyzed by mass spectrometry. From the quantity of first and second complexes contained therein, the relative binding affinity can be determined. Also in this assay, it must be possible to analyze all compounds which potentially form complexes with the target molecules, and the complexes formed therefrom, respectively. [0013] From WO 00/47999 A1 there is known a method for screening complex biological samples for a ligand which binds to a target protein. Therein, the target protein is mixed with the complex biological sample and incubated under conditions which permit the formation of complexes of ligands with the target protein. Formed ligand-target protein complexes are separated and caused to dissociate. Released ligands can be analyzed by mass spectrometry following the separation of the target protein. The separation is effected in each case by means of size exclusion processes. Furthermore, WO 00/47999 A1 discloses the use of a reference substance and of a known competitive ligand. The competitive ligand serves to determine whether a ligand binds specifically or non-specifically to the selected target protein. For this purpose, the competitive ligand is mixed with the complex biological sample, the process is carried out as described, and it is determined whether the mass spectroscopic signal of the ligand is affected by the presence of the competitive ligand. The method discloses a comparison of the binding assays in the presence and absence of the competitive ligand. However, all bound ligands are invariably determined by mass spectrometry. [0014] Moreover, it is generally known form the prior art to indirectly determine the binding of unlabeled ligands to a target molecule by means of ligands which are labeled with a marker substance. Therein, the unlabeled ligands are incubated with the target molecules together with a defined portion of labeled ligands. The subsequent determination of the quantity of the marker substance which is bound to the target molecules through the labeled ligands allows the determination of the binding behavior of the ligands which are bound to the target molecules. [0015] The methods known in the prior art make it necessary to directly or indirectly quantify either the bound or the unbound ligands. With direct quantification, this may involve a substantial expenditure, depending on the ligand. For quantification, the employed detection system must be calibrated for each of the ligands to be quantified. This is necessary also for ligands of which it is not known whether they show an affinity to the target molecules. The indirect quantification by means of labeled ligands involves substantial expenditure as well. Initially it is necessary to label the ligands with a marker substance so that the labeled ligands still have sufficient affinity to the target molecules. Frequently, various labeling methods with various marker substances must be tested for this purpose. Often, only radioactive marker substances prove to be suitable. Handling these marker substances and the ligands labeled therewith is dangerous, and because of the required safety precautions, it involves substantial expenditure. Furthermore, not every ligand is suitable for labeling by a certain desired method. This may necessitate a search for suitable ligands. [0016] Object of the present invention is the elimination of the disadvantages of the prior art. In particular, there is to be provided a method which makes it possible to determine, with little expenditure, the specific binding behavior of any ligands to any desired target molecules, unaffected to a large extent by the kinetics of the binding of the ligands to the target molecules. SUMMARY OF THE INVENTION [0017] In accordance with the invention, there is provided a method for determining the binding behavior of ligands which specifically bind to target molecules at at least one binding site, which method comprises the following steps: [0018] (a) preparation of a first mixed phase wherein the ligands in a concentration K1 are contacted with markers, in a concentration K2, which specifically bind to the target molecules, and with the target molecules in a concentration K3, and of a second mixed phase wherein the markers are contacted with the target molecules, wherein after the contacting, the first and second mixed phases are incubated under identical conditions which permit a binding of the ligands and the markers to the target molecules, [0019] (b) separation of the bound markers GM1 contained in the first mixed phase from the first mixed phase, and of the bound markers GM2 contained in the second mixed phase from the second mixed phase, and [0020] (c1) determination of a concentration K4 of the unbound markers in the first mixed phase, and of a concentration K5 of the unbound markers in the second mixed phase, wherein a concentration K6 is determined from the concentration K5, which concentration K6 corresponds to the concentration of the unbound markers in the second mixed phase under the assumption that the markers in the concentration K2 have been contacted therein with the target molecules in the concentration K3, and determination of the binding behavior of the ligands from the ratio between the concentrations K6 and K4, or [0021] (c2) determination of an quantity M1 of the bound markers GM1 and of a quantity M2 of the bound markers GM2, wherein a quantity M3 is determined form the quantity M2, which quantity M3 corresponds to the quantity of the bound markers in the second mixed phase under the assumption that the markers in the concentration K2 have been contacted therein with the target molecules in the concentration K3, and determination of the binding behavior of the ligands from the ratio of the quantities M3 and M1, wherein the markers are present in a native form, and the determination of the concentrations K4 and K5 or of the quantities M2 and M1 is effected by means of mass spectrometry. [0022] The binding behavior is to be construed to mean any behavior which characterizes the specific binding of the ligands to the target molecules at at least one binding site. It may be a relative binding behavior. In this case, the binding behavior indicates whether the ligands bind better or worse than the markers to the target molecules, and optionally, to which extent they bind thereto. It may as well be an absolute binding behavior. In this case, the affinity of the ligands to the target molecules, or the quantity of ligands which are bound to the target molecules are determined. The determination of the binding behavior may as well consist of a mere approximation of the binding behavior. [0023] The term mixed phase comprises both a pure solution and a suspension. The mixed phase may contain membrane structures such as, e.g., vesicles. The target molecules may be present in solution, suspension, immobilized or embedded in membrane structures, in particular, vesicles. Further, instead of the target molecules, the ligands may be immobilized. The target molecules or the ligands may be present immobilized on, e.g., the wall of a vessel or on particles. Further, in the case of target molecules which are embedded in vesicles, the vesicles may be immobilized, e.g., by binding to a solid phase. The conditions according to lit. a are selected so that a binding of the ligands and the markers to the target molecules can take place, in particular, as regards temperature and duration of the incubation. The incubation may be carried out until a binding equilibrium is at least almost reached. [0024] Markers are understood to be known, quantifiable agents which at the employed concentrations K2 and K3 and under the conditions according to lit. a, bind specifically to the target molecules. The binding of the markers to the target molecules is inhibited by the binding of the ligands. This may occur due to, e.g., an allosteric inhibition of the binding, or because the markers and the ligands compete for the binding to the binding site of the ligands on the target molecules. The concentration K6 or the quantity M3, respectively, may be determined by calculation or graphically from the binding properties of the markers and the concentration K5 or the quantity M2, respectively. The concentration K6 is that concentration of unbound marker, and the quantity M3 is that quantity of bound marker, which would be present in the second mixed phase after the incubation if the markers at the concentration K2 had been contacted therein with the target molecules at the concentration K3. From the ratio between the concentrations K6 and K4 or the quantities M3 and M1, respectively, the quantity of the markers whose binding to the target molecules is inhibited, and therefrom the binding behavior of the ligands, may be determined. The ratio in the sense of the invention may be both a relation and a difference. Both the markers and the ligands and the target molecules may be of a uniform type, or may differ from each other. [0025] The separation according to lit. b may take place, for example, by centrifugation of formed complexes. The complexes may contain target molecules and ligands, target molecules and markers, or target molecules, ligands and markers. It is to be understood that instead of a concentration, a quantity, and instead of a quantity, a concentration may be determined. Continue reading about Method for determining the binding behavior of ligands which specifically bind to target molecules... 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