Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings

Definitions

• the present invention relates to a method for the determination of stoichiometry of binding between two binding partners, such as, for example, a receptor and a ligand.
• the number of binding sites involved when two molecules interact i.e. the binding stoichiometry, is many times of fundamental interest, since it is related to molecular function.
• the binding stoichiometry can be measured directly, for instance by determining the molecular weight of the formed complex, or it can be measured by indirect methods provided that the concentrations of both interacting molecules, or binding partners, are known.
• Prior art indirect methods for determining stoichiometry of binding typically use spectrophotometric methods, such as UV or NIR absorption spectrometry, or fluorescence-based detection for determining the total concentration of a molecule.
• US 6,025, 142 A discloses determination of the stoichiometry and affinity of the binding of the fluorophore 8-anilino- l -naphthalene sulfonate (ANS) to urokinase-type plasminogen activator (u-PA) by titrating fixed concentrations of u-PAR with ANS up to a concentration of 100 ⁇ .
• the theoretical fluorescence of a molar solution if all were bound to u-PAR was calculated by titration of a protein concentration
• US 2005-0037377 A discloses a method for determining the binding affinity and/or stoichiometry of a binding complex between a binding factor and a probe using fluorescence techniques.
• the method comprises: (a) labeling the probe with a fluorophore; (b) incubating the labeled probe with a factor or a group of factors which may bind the labeled probe to form a binding complex; (c) separating the binding complex and the free probe into different fractions; (d) subjecting each fraction from step (c) to fluorescence polarization measurement under conditions wherein the binding complex produces a fluorescence pattern different from that of the free probe, thereby allowing detection of the binding complex; and (e) determining binding affinity and/ or stoichiometry between the probe and the binding factor.
• the binding complex formation is monitored by fluorescence polarization detection.
• the volume of the titrant required to achieve the maximum signal changes is utilized to calculate the dissociation constant and the binding stoichiometry of the protein-ligand complex according to the theoretical relationships developed herein. Specifically, the interaction of avidin with a chromophoric biotin analogue, 2-(4'- hydroxyazobenzene)benzoic acid, was studied by following the absorption signal of their interaction at 500 nm.
• binding stoichiometry is determined based on determination of active molecule concentrations rather than total molecule
• a method for determining binding stoichiometry for the interaction between a first molecular species and a second molecular species forming a complex between them comprises the steps of:
• concentrations of the first molecular species and the second molecular species wherein the initial active concentration of the second molecular species is selected to be sufficient to cause saturation of the binding of the second molecular species to the first molecular species;
• Steps a) to c) above may then be performed by providing a plurality of solutions, wherein each solution contains a fixed predetermined concentration of the first molecular species and a varying predetermined concentration of the second molecular species.
• the free active concentration of the second molecular species is determined for each solution, and the respective differences between initial active concentration and free active concentration of the second molecular species are calculated.
• the saturation level for this difference (between initial active concentration and free active concentration of the second molecular species) can be determined, which is then used in step d).
• the method comprises the steps of:
• concentration and the free active concentration of the second molecular species to the difference between the initial active concentration and the free active concentration of the first molecular species, or vice versa;
• Determination of active concentration is preferably performed using an interaction analysis sensor, which typically comprises a sensing surface supporting a specific binding partner to the molecular species whose active concentration is to be determined. After contacting the sensing surface with the molecular species, the association/ dissociation process at the surface is monitored.
• an interaction analysis sensor typically comprises a sensing surface supporting a specific binding partner to the molecular species whose active concentration is to be determined.
• the determination of at least initial active concentration comprises contacting the solution with a sensor surface at varying flow rates under conditions of at least partial mass transport limitation, whereby the use of a calibration standard will not be required.
• Figure 1 is a diagram showing a plot of (Btot - Bfree) versus Btot for a simulated procedure according to an embodiment of the method of the present invention.
• the present invention relates to the determination of the stoichiometry of binding between two interacting molecules, for example a receptor and a ligand, such that a complex between the molecules is formed.
• the method is based on determining initial active concentrations of the interacting molecules and active concentrations of free (non-complexed) molecules of one or both molecules after complex formation has been initiated, and based on the resulting data determining the binding stoichiometry for the interaction.
• interacting molecule pairs include antibody/ antigen.
• the determination of the stoichiometry of the binding between two molecules A and B which may interact to form a complex AB comprises the following steps:
• molecules A and B are interchangeable, i.e., instead, molecule B may be in a fixed active concentration and titrated with varying active concentrations of molecule A.
• the stoichiometry can be determined by studying the ratio of molecules in the complex formed when incubating fixed concentrations of molecules A and B in solution, i.e. by calculating the ratio (Btot- Bfree) / (Atot-Afree). This will provide a "snapshot" of complex stoichiometry for these conditions which, however, may differ from the step determination since sites of different affinity on molecule A need not be populated at the same time.
• measurements may be performed on two or more different mixtures of molecules A and B to obtain a more accurate stoichiometry value.
• the determination of active concentration of molecules A and B is preferably performed using an interaction analysis sensor, typically a biosensor.
• an interaction analysis sensor typically a biosensor.
• biosensor- based determination of active concentration is described in, for example Karlsson, R., et al. ( 1993) J. Immunol. Methods 166( l):75-84; Richalet-Secordel, P. M., et al. ( 1997) Anal Biochem. 249(2): 165-73; and Sigmundsson K., et al. (2002) Biochemistry
• the interaction analysis sensor typically comprises a sensing surface (s) having immobilized thereon a specific binding partner for the molecule whose active concentration is to be determined.
• CFCA Calibration-Free Concentration Analysis
• a biosensor is typically based on label-free techniques, detecting a change in a property of a sensor surface, such as mass, refractive index or thickness of the immobilized layer.
• Typical biosensors for the purposes of the present invention are based on mass detection at the sensor surface and include especially optical methods and piezoelectric or acoustic wave methods.
• Representative sensors based on optical detection methods include those that detect mass surface concentration, such as sensors based on reflection-optical methods, including e.g. evanescent wave-based sensors including surface plasmon resonance (SPR) sensors, frustrated total reflection (FTR) sensors, and waveguide sensors, including e.g. reflective interference
• Piezoelectric and acoustic wave sensors include surface acoustic wave (SAW) and quartz crystal microbalance (QCM) sensors.
• SAW surface acoustic wave
• QCM quartz crystal microbalance
• Biosensor systems based on SPR and other detection techniques are commercially available today.
• Exemplary such SPR-biosensors include the flow-through-cell-based Biacore ® systems (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and ProteOnTM XPR system (Bio-Rad Laboratories, Hercules, CA, USA) which use surface plasmon resonance for detecting interactions between molecules in a sample and molecular structures immobilized on a sensing surface or surfaces.
• the progress of binding directly reflects the rate at which the interaction occurs. Injection of sample is usually followed by a buffer flow during which the detector response reflects the rate of dissociation of the complex on the surface.
• a typical output from the system is a graph or curve describing the progress of the molecular interaction with time, including an association phase part and a dissociation phase part. This binding curve, which is usually displayed on a computer screen, is often referred to as a "sensorgram".
• Biacore ® systems it is thus possible to determine in real time without the use of labeling, and often without purification of the substances involved, not only the presence and concentration of a particular molecule, or analyte, in a sample, but also additional interaction parameters, including kinetic rate constants for association (binding) and dissociation in the molecular interaction as well as the affinity for the surface interaction.
• additional interaction parameters including kinetic rate constants for association (binding) and dissociation in the molecular interaction as well as the affinity for the surface interaction.
• the Biacore ® systems as well as analogous sensor systems, measure the active analyte concentration as distinct from the total concentration of the analyte.
• active it is the choice of ligand on the sensor surface that defines the kind of activity being measured. While e.g. standard protein concentration analysis using a calibration curve may be used, the Biacore ® systems (and analogous sensor systems) permit assessment of protein (and e.g. other macromolecule) concentration by a calibration-free method, which is often referred to as calibration-free concentration analysis (CFCA).
• CFCA calibration-free concentration analysis
• the method relies on changes in binding rates of analyte to a target (ligand) immobilized on a surface with varying flow rates under conditions of partial or total mass transport and does, as mentioned, not require standards of known
• the observed binding rate must be at least partially limited by transport.
• the concentration is obtained by running the binding experiments at at least two different flow rates and fitting the data to a model describing the process, e.g. a two-compartment model (Myszka, D. G., et al. ( 1998) Biophys. J. 75, 583-594, and Schank-Retzlaff, M. L. and Sligar, S. G. (2000) Anal Chem. 72, 4212-4220).
• a model describing the process e.g. a two-compartment model (Myszka, D. G., et al. ( 1998) Biophys. J. 75, 583-594, and Schank-Retzlaff, M. L. and Sligar, S. G. (2000) Anal Chem. 72, 4212-4220).
• a model describing the process e.g. a two-compartment model (Myszka, D. G., et al
• the binding of analyte to surface-attached ligand in a controlled flow system is represented by the sum of two processes, transport of analyte to the surface and molecular interaction with the immobilized ligand.
• the molecular interaction is described by the rate constants k a and kd, while transport of analyte to and from the surface is described by the mass transport constants k m and k- m (also referred to as k t and k-t) .
• a protein solution is injected at least twice (different flow rates) over the surface with immobilized interaction partner.
• the binding phases of the sensorgrams obtained from such an experiment are fitted to a bi-molecular
• the interaction model with mass transfer term in which the active concentration is a fitted parameter.
• the fitting is preferably global, i.e. the interaction model is fitted simultaneously to multiple binding curves (sensorgrams).
• the value of the mass transport coefficient is introduced as a constant, which, as described above, may be calculated from the dimensions of the flow cell, the diffusion coefficient of the protein and the flow rate used.
• the response increase dR/dt at the sensor surface given by bound protein is proportional to the mass transport constant k t and the active concentration, i.e.
• the diffusion coefficient D is a function of the size and shape of the molecule and the frictional resistance offered by the viscosity of the solvent in question.
• the diffusion coefficient is inversely proportional to the radius and thus proportional to the cube root of the molecular weight.
• the diffusion coefficient is relatively insensitive to the molecular weight.
• concentrations of molecules A and B are determined using CFCA and sensing surfaces with immobilized binding partner to molecules A and B, respectively.
• a number of solution mixtures are then prepared from the stock solutions which contain a fixed concentration of molecule A and varying concentrations of the molecule B.
• the initial concentrations of molecules A and B (Atot and Btot,
• the concentrations before any interaction has taken place may be calculated from the volumes of stock solutions used.
• the active concentrations of molecule B in the different mixtures are determined using a sensing surface(s) with immobilized binding partner to molecule B and either (i) CFCA, or (ii) a standard or calibration curve (prepared using the active concentration determined by CFCA for the stock solution of B). Based on the results of the concentration
• the binding stoichiometry for the molecular interaction may then be determined as described further above.
• the different solution mixtures are prepared before being injected into the biosensor instrument, or, optionally, solutions of the respective interactants in known active concentrations may be injected into the biosensor instrument to be mixed in predetermined proportions within the instrument, as described in, for example, WO 2008/033073 (the full disclosure of which is incorporated by reference herein) .
• a determination of stoichiometry according to the invention may, for example, be performed using a BiacoreTM system, e.g. a Biacore ® T100 (GE Healthcare Bio- Sciences AB, Uppsala, Sweden), wherein a micro-fluidic system passes samples and running buffer through four individually detected flow cells (one by one or in series).
• a BiacoreTM system e.g. a Biacore ® T100 (GE Healthcare Bio- Sciences AB, Uppsala, Sweden)
• a micro-fluidic system passes samples and running buffer through four individually detected flow cells (one by one or in series).
• sensor chip may, for example, be used Series S Sensor Chip CM5 (GE Healthcare Bio-Sciences AB) which has a gold-coated surface with a covalently carboxymethyl- modified dextran polymer hydrogel.
• the output from the instrument is a "sensorgram” which is a plot of detector response (measured in "resonance units", RU) as a function of time.
• An increase of 1000 RU corresponds to an increase of mass on the sensor surface of approximately 1 ng/ mm 2 .
• the dedicated BlAevaluation Software and Biacore T100 Software 2.0 may be used, which includes a module for calibration-free concentration analysis (CAFC).
• a procedure for determining the number of binding sites on a molecule A for molecule B using a Biacore ® T100 may be performed as follows:
• Aim to immobilize between 25 and 100 RU/kDa That is, if molecule A has a molecular weight of 50 kDa, immobilize between 1250 and 5000 RU.
• a typical injection time could be 3 minutes and a typical flow rate 10 ⁇ /min.
• Use a report point set 10 seconds after injection stop as response value.

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