JP4751387B2 - Methods for detecting molecular surface interactions - Google Patents

Description

  The present invention relates to the detection of molecular interactions at the surface of a solid support, and more particularly, an analyte present in a sample based on a complex medium such as a body fluid, and an immobilized binder to the analyte, Relates to a method for measuring the interaction between.

  Various analytical techniques have been used to characterize the interactions between molecules, particularly in the context of assays aimed at detecting and interacting with biomolecules. For example, antibody-antigen interactions have fundamental importance in many fields, including biology, immunology and pharmacology. Many analytical techniques in this regard include the binding of a “ligand” such as an antibody to a solid support followed by contacting the ligand with an “analyte” such as an antigen. Yes. Following contact of the ligand with the analyte, several properties that are indicative of the interaction, such as the ability of the ligand to bind to the analyte, are measured. After measurement of the interaction, the free ligand is “regenerated” so that the ligand-analyte pair can be dissociated to allow the ligand surface to be reused for subsequent analytical measurements. Is often desired.

Analytical sensor systems that can monitor such molecular interactions in real time are of increasing interest. These systems are often based on optical biosensors and are usually referred to as interaction analysis sensors or biospecific interaction analysis sensors. Sensing Representative of such biosensor system, Biacore (Biacore AB) (Uppsala, Sweden) Biacore sold by ^{(R) (Biacore (R)} ) is a measuring device, which is a molecule in a sample Surface plasmon resonance (SPR) is used to detect interactions between molecular structures immobilized on a sensing surface. Using the Biacore ^(R) system, the presence and concentration of specific molecules in the sample in real time without using labeling, as well as additional interaction parameters for molecular interactions such as association rate constants and dissociation The rate constant can be measured.

  However, if the analyte is present in a complex medium such as plasma, the measurement is usually a molecule other than the analyte that is present in the plasma and interacts with the solid surface to produce non-specific binding. Will be significantly disturbed by species. This leads to measurement results that are usually difficult to interpret, especially for low molecular weight analytes.

  An object of the present invention is to solve the above-described problems in detecting the interaction between an analyte and a solid phase surface when the analyte is present in a complex medium.

  These and other objectives and advantages have been immobilized on a solid support surface when the analyte is present in a complex medium containing one or more molecular species that can interact with the solid support surface. Provided by a novel method for measuring the interaction between a binding agent or ligand and an analyte. In accordance with the present invention, a solid support surface when the monitored solid support surface is first contacted with a complex medium containing no analyte and then immediately contacted with a complex medium containing the analyte. The influence of nonspecific binding on the measurement results can be at least substantially reduced. All non-specific binding of the complex medium to the solid support has thereby occurred to at least a substantial extent before contact between the analyte-containing medium and the surface and was then detected at the surface All additional changes can therefore largely be attributed to binding with the analyte.

  Thus, in one aspect, the present invention is immobilized on an analyte and solid support surface by contacting a liquid sample containing the analyte with the solid support surface and detecting a binding event at the surface. A method for measuring an interaction with a binding agent is provided, wherein the sample is based on a complex medium containing at least one molecular species other than an analyte capable of interacting with a solid support surface, the method Is characterized by comprising contacting the surface with a sample medium that does not contain the analyte before contacting the complex medium containing the analyte with the solid support surface.

  In another aspect, the present invention provides a method for measuring the propensity of a drug to bind to plasma, comprising: (i) in a complex medium containing plasma, and (ii) in a non-complex medium. Measuring the binding of the drug present to the immobilized binding agent to the drug and comparing the binding of the drug in the plasma-containing medium to the binding of the drug in an uncomplicated medium, wherein Measurement of drug binding is performed according to the first method aspect described above.

  In yet another aspect, the present invention provides an analytical system for studying molecular interactions, which includes computer processing means including program code means for performing the steps of the method.

  In yet another aspect, the present invention relates to a computer comprising program code means stored on a computer readable medium or executed on an electrical or optical signal for performing the steps of the method. Providing program products.

  These and other aspects of the invention will be apparent upon reference to the attached drawings and the following detailed description.

As mentioned above, the present invention relates to a method for reducing or eliminating the non-specific effects of non-analyte components of complex sample media in assays or studies relating to the detection of binding events on a solid support surface. . Surface binding interactions can be characterized by many different interaction analysis techniques. Recently, label-free biosensor technology has become a powerful tool for such interaction analysis. Commercially available biosensors include the aforementioned Biacore ^(R) system device that allows monitoring of surface interactions in real time based on surface plasmon resonance (SPR).

The phenomenon of SPR is well known, SPR occurs when light is reflected under certain conditions at the interface between two media of different refractive indices, and the interface is a metal film, typically silver or gold It would be enough to say that it is covered by. Biacore ^(R) This medium is device sample and a glass of a sensor chip in contact with the sample by microfluidic (microfluidic) flow system. This metal film is a thin layer of gold on the chip surface. SPR causes a decrease in reflected light intensity at a specific reflection angle. The angle of the minimum reflected light intensity varies depending on the refractive index close to the surface on the opposite side from the reflected light ^(the sample side in the via core ^(R) system).

A schematic diagram of the Biacore ^(R) system is shown in FIG. The sensor chip 1 has a gold film 2 supporting a capture molecule (ligand) 3, eg an antibody, exposed to a sample stream with an analyte 4 (eg antigen) via a flow channel 5. . The monochromatic p-polarized light 6 from the light source 7 is coupled to the glass / metal interface 9 where this light is totally reflected by the prism 8. The intensity of the reflected light beam 10 is detected by the optical detection unit 11.

As molecules in the sample bind to the capture molecules on the sensor chip surface, the concentration, and hence the change in surface and refractive index of the SPR response, is detected. Plotting the response to time during the course of the interaction provides a quantitative measure of the progress of the interaction. Such a plot is usually called a sensorgram. In the Biacore ^(R) system, the SPR response value is expressed in resonance units (RU). 1 RU represents a change of 0.00001 ° in angle of minimum reflected light intensity and is roughly equivalent to a concentration change of about 1 pg / mm ² on the sensor surface for many proteins. When a sample containing an analyte comes into contact with the sensor surface, capture molecules (ligands) bound to the sensor surface interact with the analyte in a stage called “association”. This stage is shown as an increase in RU on the sensorgram when the sample first comes into contact with the sensor surface. Conversely, “dissociation” usually occurs when the sample stream is displaced, for example, by a buffer stream. This stage is shown as a slow RU drop on the sensorgram when the analyte dissociates from the surface-bound ligand.

  A representative sensorgram (binding curve) of the reversible interaction at the sensor chip surface is shown in FIG. 2, where the sensing surface holds immobilized capture molecules (eg, antibodies) that interact with the analyte in the sample. ing. The vertical axis (y-axis) shows the response (here in resonance units RU) and the horizontal axis (x-axis) shows the time (here in seconds s). Initially, a buffer is passed over the sensing surface to provide a baseline response A in the sensorgram. An increase in signal due to analyte binding is observed during sample injection. Part B of this binding curve is usually referred to as the “binding phase”. Eventually a steady state condition is reached where the resonance signal is flat at C. At the end of sample injection, the sample is replaced with a continuous flow of buffer and the decrease in signal reflects the dissociation or release of the analyte from the surface. This portion D of the binding curve is usually called the “dissociation phase”. The form of this binding / dissociation curve provides valuable information about the kinetics of the interaction and the height of the resonance signal represents the surface concentration (ie, the response resulting from the interaction is the mass concentration on the surface Related to changes).

Assuming a reversible reaction between analyte A and surface bound (immobilized) capture molecule B (mass transfer is not limited) (first order kinetics):

（ここで Γ は結合したアナライトの濃度であり、Γ_max はこの表面の最大結合容量であり、ｋ_ass は結合速度定数であり、ｋ_diss は解離速度定数であり、そしてＣはバルクのアナライト濃度である）この式をならべかえると次の等式が与えられる：

全ての濃度が同じ単位で測定された場合はこの等式は次のように書き換えることができる：

（ここでＲはRUで表した応答である。）積分型では、この等式は次のようになる：

The rate of change of the surface concentration of A during the analyte injection is the following equation:

(Where Γ is the concentration of bound analyte, Γ _max is the maximum binding capacity of this surface, k _ass is the binding rate constant, k _diss is the dissociation rate constant, and C is the bulk analyte. Rearranging this equation (which is the light density) gives the following equation:

If all concentrations are measured in the same units, this equation can be rewritten as:

(Where R is the response in RU). In the integral form, this equation is:

そして積分型では次のようになる：

The dissociation rate can be expressed as:

And for the integral type:

Affinity is expressed by the association constant K _A = k _ass / k _diss or the dissociation constant K _D = k _diss / k _ass .

  For a more detailed description for measuring the kinetics of molecular interactions, see, for example, Karlsson, R. and Faelt, A. (1997) Journal of Immunological Methods, 200, 121-133.

  By the way, if the sample is a “complex medium” that includes one or more molecular species that may interact with the sensor surface in addition to the analyte, the presence of such molecular species The resulting non-specific binding significantly interferes with the measurement on the surface, especially when the analyte of interest is a low molecular weight compound (usually an organic having a molecular weight of about 100 to about 1,000, typically about 500 to about 800) It is easy to see that when it is a compound (sometimes called a “small molecule”), it can give binding curves that are difficult to interpret.

  According to the present invention, when the analyte is present in a complex medium on the sensor surface, the analyte-containing sample medium is brought into contact with the complex sample medium not containing the analyte before contacting the surface. It has been found that the binding of the analyte to the immobilized binder can be measured successfully, even for low molecular weight molecules. By doing so, non-specific binding to the sensor surface occurs primarily when the surface comes into contact with a complex medium that does not contain an analyte. If the sample is then contacted with the sensor surface, this non-specific binding process will be at least substantially reduced and sample measurement will be hindered at least to a much lesser extent, at the same time as the following example Will be demonstrated.

Preferably, the sensor surface is contacted with the analyte-containing sample medium and then again contacted with a complex medium that does not contain the analyte, and the dissociation of the analyte from the immobilized binding agent to the analyte is reduced to the binding agent. Occur in the same environment as the combination of. Therefore, referring to the above Biacore ^(R) system, the liquid injection sequence of the present invention is different from the prior art methods <buffer solution>, <analyte-containing sample medium>, <buffer solution><Analyte-free sample medium>, <Analyte-containing sample medium>, <Analyte-free sample medium>.

  The complex media on which the sample is based can be selected from a number of media that contain one or more analytes of interest. Examples of complex media include body fluids such as cerebrospinal fluid, saliva, breast milk, urine, bile, serum, plasma, tears, and homogenized biopsy samples, and cell culture media, cell lysates, crude plant extracts , Extracted or dissolved foodstuffs, liquid foodstuffs such as beverages (milk, fruit juice, beer, etc.).

  Depending on the particular complex medium to be analyzed, the test sample may be a collected original sample or a dilution of the original sample with a suitable diluent. In general, the content of complex media in the sample is about 1 to about 100% (v / v), usually about 10 to about 100% (v / v), especially about 30 to about 100% (v / v). For example, about 30 to about 50% (v / v)

  For example, if the complex sample medium is based on human plasma or serum, the analyte-containing medium can be based on plasma or serum collected from the patient after administering the drug, and then the sample medium that does not contain the analyte Can be based on plasma or serum collected from the same patient prior to drug administration.

  Thus, the method of the present invention provides a reaction rate and affinity constant (binding constant, dissociation constant, binding rate constant and dissociation rate constant) for the molecular interaction between an analyte in a complex medium and an immobilized binder for the analyte. Can be used for measurement. Of course, the method of the present invention can also be used to determine the concentration of one or more analytes in a complex medium. Apart from contacting the binder-supporting surface with the complex medium containing the analyte, and possibly after, contacting the surface with the complex medium without the analyte, the reaction rate and Simultaneously with the affinity constant, the analyte concentration can be measured by a known method.

  In a variation of the invention, the method is used to compare the plasma binding propensity (eg, affinity) of various drugs. More specifically, the binding of the drug to the immobilized receptor, particularly in the form of the binding curve described above, (i) a buffer with the immobilized receptor, (i) a drug dissolved in a buffer, and (ii) a buffer plus plasma. It is measured by contacting the drug dissolved in the liquid and then the binding level, or preferably the binding curve, is compared. If the drug binds to plasma, the resulting binding to the immobilized receptor in the presence of plasma will be substantially reduced compared to the drug present only in the buffer. Measurements at one drug concentration will often be sufficient.

While the above method is implemented in several aspects of the Biacore ^(R) system, the present invention provides techniques for detecting binding interactions on many other solid support surfaces, such as radiolabels, Chromophore, fluorophore, marker for scattered light, electrochemical activity marker (eg, field effect transistor based on potentiometry), electro-stimulated emission, magnetic activity marker, It will be appreciated that it can be used in connection with techniques that rely on labels such as thermoactive markers, chemiluminescent moieties or transition metals, as well as so-called label-free detection systems. However, real-time detection systems are preferred, especially when based on chemical sensor or biosensor technology.

  Biosensors either directly couple to solid physicochemical transducers or use components for molecular recognition by mobile carrier beads / particles coupled to the transducer (eg, layers with immobilized antibodies) As a device, it is defined in a broad sense. Such sensors are typically based on label-free techniques, such as detection of mass change, refractive index or immobilization layer thickness, but there are also sensors that rely on some type of labeling. Typical sensor detection techniques include, but are not limited to, mass detection methods such as optical, thermo-optical and piezoelectric or acoustic (eg surface acoustic waves (SAW) and quartz crystal microbalances). (Including QCM); and electrochemical methods such as potentiometry, electrical conductivity measurement, current measurement and capacitance / impedance methods. Typical methods for optical measurement methods are those that detect mass surface concentration, eg, reflective optical methods that include both external and internal reflection methods, which include angle, wavelength, polarization, or phase resolved. Evanescent wave ellipsometry and evanescent wave spectroscopy (EWS, or internal reflection spectroscopy), both of which enhance the evanescent field via surface plasmon resonance (SPR) Brewster refraction angle measurement method, critical refraction angle measurement method, leaky total reflection (FTR), scattering total internal reflection (STIR), which can include scattering enhancement labels, optical waveguide sensor; external Reflection imaging, imaging based on evanescent waves, eg critical angle-resolved averaging, Brewster angle-resolved imaging, SPR angle Solution imaging etc. are mentioned. In addition, in combination with photometric and imaging / microscopy “self” or reflection methods, eg surface-sensitized Raman spectroscopy (SERS), surface-sensitized resonance Raman spectroscopy (SERRS), evanescent wave fluorescence (TIRF) And phosphorescence emission methods, as well as waveguide interferometers, waveguide leakage mode spectroscopy, reflection interference spectroscopy (RIfS), transmission interferometry, holography spectroscopy, and atomic force microscopy (AFR).

What is currently on the market is a bisensor system, especially based on SPR. Examples of so the SPR biosensor includes the above Biacore ^(R) instrument. Biacore ^(R) A detailed behavior of the technical aspects and SPR instruments discussion will be found in U.S. Patent 5,313,264. More detailed information regarding matrix coatings for biosensor sensing surfaces is given, for example, in US Pat. Nos. 5,242,828 and 5,436,161. Furthermore, detailed discussion of the technical aspects of the biosensor chips associated with Biacore ^(R) instrument is found in U.S. Patent 5,492,840. The entire disclosures of the above US patents are incorporated herein by reference.

In a flow cell the method of the present invention, for example, it is often would conveniently be carried out in type used in the above Biacore ^(R) instrument. Other flow cells that can be used in the present invention are also well known to those skilled in the art and need not be described herein.

  As used herein, the expression “solid support” should be interpreted broadly and one or more binding agents can be immobilized and their molecular interactions detected by a selected detection system. It means any solid (soft or hard) substrate that can be made. The substrate can be biological, non-biological, organic, inorganic or mixtures thereof and has any convenient shape such as disc, sphere, circle, particle, string, precipitate, gel , Sheets, tubes, spheres, containers, capillaries, pads, sections, films, plates, slides and the like. The substrate surface can have any two-dimensional structure and includes, for example, steps, ridges, bumps, terraces, etc., and the surface of the layer of material may be different from the remaining surface of the substrate.

  In the following examples, various aspects of the present invention are disclosed more specifically and non-limitingly for purposes of illustration.

  This example demonstrates the interaction between iophenoxy acid (α-ethyl-3-hydroxy-2,4,6-triiodohydro-cinnamic acid) and immobilized human serum albumin (HSA) in a saliva-containing buffer. 1 illustrates the use of the method of the invention to measure. Iofenoxy acid is known to bind HAS but not IgG and is therefore used as a reference. Salivary components bind nonspecifically to both HAS and IgG.

Measuring equipment Biacore ^(R) 3000 equipment (Biacore, Uppsala, Sweden) was used. The instrument is based on surface plasmon resonance (SPR) detection on the gold surface, and samples and transfer buffers are passed one by one or continuously through four individually detected flow cells (labeled Fc1 to Fc4). A micro-fluidic system is used. The sensor chip was a series CM5 certified grade (Biacore, Uppsala, Sweden) having a gold coated surface with a covalently bound carboxymethyl modified dextran polymer hydrogel. The output from this instrument is a “sensorgram” plotting the detector response (measured in “resonance units”, RU) as a function of time. An increase of 1000 RU corresponds to a mass increase of about 1 ng / mm ² on the sensor surface.

  A prototype TRIINJECT command was used to inject two liquids, A and B, in the order <liquid A> <liquid B> <liquid A> (Andersson, Karl, et al. (1999) PROTEINS: Structure, Function, and Genetics 37, 494-498).

Three flow channels, Fc1, Fc2 and Fc3, of the immobilized via core ^(R) 3000 device of the sensor chip were used. Immobilization of anti-myoglobin IgG (self-made reagent) and human serum albumin (HSA) (Sigma-Aldrich, Missouri, USA) using amine coupling kit (Biacore, Uppsala, Sweden) according to the manufacturer's instructions did. The running buffer was 10 mM sodium acetate buffer pH 5.0 (Biacore, Uppsala, Sweden). The sensor chip was immobilized as follows:
Fc1: Blank
Fc2: Approximately 8600 RU IgG (for reference)
Fc3: Approximately 11000 RU human serum albumin (HSA)
Fc4: Approximately 9000 RU human serum albumin (HSA)

  While sensorgrams recorded Fc1 to Fc4, only sensorgrams recording Fc2 and Fc3 were used for analysis.

Measurement of non-specific binding of saliva to IgG and HAS The following two buffers were prepared:
HBS-EP (Biacore, Uppsala, Sweden) containing 1% HBS : 1% DMSO (Riedel de Haen, Seels, Germany).
HBSS 1 % : HBS-EP (Biacore) containing 1% DMSO (Ridel de Haen) and 30% saliva (provided by healthy men).

Biacore ^(R) 3000 using a tri inject (triinject) command device, the liquid A is HBS1%, and HBS1% or HBSS1% of liquid B is either a continuous flow cell 1 3 (Fc1, Fc2 Fc3). That is, the injection order was <HBS 1%><HBS1%><HBS1%> and <HBS 1%><HBSS1%><HBS1%>, respectively. The transfer buffer used during the preparation and washing steps was HBS-EP (Biacore, Uppsala, Sweden). The resulting sensorgram for the HSA surface (Fc3) is shown as an overlay plot in FIG. The dotted line indicated by A in the figure shows the sensorgram when the liquid B in the triinjection is HBSS 1%, and the solid line indicated by B in the figure is the liquid B in the triinjection 1% HBS. The sensorgram is shown for.

  During section 10 in FIG. 3, the instrument prepares for injection with transfer buffer. Categories 11 and 13 are <Liquid A> in the triinject, in this case 1% HBS. Section 12 is the actual sample injection, ie <Liquid B> in the triinjection. 12A corresponds to 1% HBSS and 12B corresponds to 1% HBS. Section 14 is washing with transfer buffer. The sudden shift in sensorgram response level (RU) in each segment movement reflects the difference in refractive index in the bulk of each liquid, and the more relaxed change in response level between each segment is Corresponds to specific or non-specific interactions. When comparing sections 12A and 12B, section 12A has a significantly greater slope than 12B, indicating that saliva components in 12A have bound to the surface.

  In a similar experiment, a sample injection of 1% HBSS was “embedded” in 1% HBSS, ie <Liquid A> and <Liquid B> were both 1% HBSS. Sensorgrams from two such injections are shown as overlay plots in FIG. 4 (signal from flow cell 3 as in FIG. 3), with sections 21 through 23 corresponding to sections 11 through 13 in FIG. As shown in FIG. 4, there is an early large drift in section 21 due to rapid non-specific component binding. At the time of movement from segment 21 to 22, this float decreases. In particular, the float between sections 22 is lower than that between sections 12, which means that the signal is non-specific to the same extent if the surface has already been preincubated with non-specific components when the sample is injected. It is shown that it is not affected by dynamic coupling.

Measurement of interaction between iophenoxy acid and HAS in 30% saliva
50 μM iofenoxy acid (Sigma Aldrich, Missley, USA) in 1% HBSS was used as <Liquid B> and 1% HBSS as <Liquid A>, and triinjection was performed on Fc1, Fc2 and Fc3 Went to the series. Detection was performed with Fc3 (HSA-surface) and Fc2 (IgG-surface). The sensorgram obtained from Fc3 was “corrected” by subtracting the reference Fc2 sensorgram. The resulting reference subtraction sensorgram (Fc3-Fc2) is shown as an overlay plot in FIG. The solid line indicated by E in the figure shows the sensorgram when the liquid B in the triinjection is 50 μM iophenoxy acid in 1% HBSS, and the dotted line indicated by F in the figure is in the triinjection. The sensorgram when the liquid B of HBSS is 1% is shown.

  In FIG. 5, sections 31, 32F and 33 are 1% HBSS and section 32E is 50 μM iodophenic acid in 1% HBSS. The slope between segments 31-33 is due to saliva components that bind nonspecifically to IgG (Fc2) at a lower rate than to HSA (Fc3). However, the effect of non-specific binding is stable when iophenoxy acid is injected in section 32. As can be seen from sections 32E and 32F, the binding of iophenoxy acid can be easily quantified.

  To correct for non-specific binding differences between IgG and HAS, the sensorgram from the buffer injection (F in FIG. 5) is derived from the sensorgram from the iophenoxy acid injection (E in FIG. 5). Can be deducted. The binding of iophenoxy acid to HSA was characterized 3 times with 1% HBSS and 3 times with 1% HBS, and the sensorgram obtained in this way is shown in FIG. The sensorgram of saliva-containing buffer is shown by the dotted line (D), while the sensorgram of pure buffer is shown by the solid line (C). As shown in FIG. 6, sensorgram D (saliva-containing buffer) is consistent with sensorgram C (pure buffer), as this method characterizes molecular interactions in complex matrices. It shows that it is convenient.

  Therefore, pre-incubation with saliva components that non-specifically bind the HSA and IgG surfaces prior to contacting the surface with the sample will prevent non-determination from sample media that may interfere with the measurement of iophenoxy acid interaction. Specific binding is minimized. To that end, the method of the present invention is particularly suitable for minimizing potential non-specific binding effects when measuring how small molecules bind to ligands in complex sample media.

  The invention is not limited to the specific embodiments of the invention described above, but it is to be understood that the scope of the invention is established by the appended claims.

1 is an illustrative side view of an SPR-based biosensor system. FIG. Figure 2 is a sensorgram showing the detector response over time for the interaction between the analyte and the immobilized binding agent for the analyte. (I) Sensorgrams for injection of saliva containing buffer embedded during injection of pure buffer at dotted line, and (ii) Overlay plot of sensorgram for sequential injection of pure buffer at solid line. 2 is an overlay plot of two sensorgrams, each representing three sequential injections of saliva-containing buffer. (I) a sensorgram for injecting iofenoxy acid in a saliva-containing buffer embedded during the injection of saliva-containing buffer with a solid line, and (ii) a sensorgram for sequential injection of the corresponding saliva-containing buffer with a dotted line It is an overlay plot. The dotted lines are three corrected overlay plots obtained by subtracting the sensorgram corresponding to sensorgram (ii) of FIG. 5 from the sensorgram corresponding to sensorgram (i) of FIG. The solid line is the corresponding sensorgram for the injection of iophenoxy acid in pure buffer.

Claims (9)

  Measure the interaction between the analyte and the binding agent immobilized on the solid support surface by contacting the liquid sample containing the analyte with the solid support surface and detecting a binding event at the surface. A method based on surface plasmon resonance, wherein the sample is based on a complex medium containing at least one molecular species other than an analyte capable of interacting with a solid support surface, the method comprising: Said method comprising contacting the surface with an analyte-free sample medium prior to contacting the solid support surface.   The method according to claim 1, characterized in that after contacting the surface of the solid support and the sample containing the analyte, the sample is again brought into contact with the sample medium not containing the analyte, and the dissociation of the analyte from the surface is measured. The method described.   The method according to claim 1, wherein at least one of an association constant, dissociation constant, association rate constant, and dissociation rate constant of the interaction is measured.   The method according to claim 1, wherein the concentration of the analyte in the sample is measured.   The method according to claim 1, wherein the complex medium is a body fluid.   The complex medium is selected from cell culture media, cell lysates, crude plant extracts, liquid foodstuffs, extracted foodstuffs and dissolved foodstuffs. The method in any one of.   The sample is based on plasma or serum collected from the patient after administering the drug to the patient, and the sample medium without the analyte is based on plasma or serum collected from the patient prior to administering the drug. The method according to claim 1, characterized in that:   8. A method according to any of claims 1 to 7, characterized in that the solid support surface contains a sensor surface and binding to the surface causes a measurable change in the properties of the sensor surface.   A method for measuring the plasma binding propensity of a drug comprising: (i) said drug to an immobilized binding agent for the drug in a complex medium containing plasma, and (ii) in a non-complex medium. And measuring the binding of the drug in the plasma-containing medium, and comparing the binding of the drug in the plasma-containing medium to the binding of the drug in the uncomplex medium. The above method, which is performed according to the method according to any one of 1 to 5 and 8.

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