+

WO2009037660A2 - Analyse de la liaison de molécules liées par une membrane cellulaire - Google Patents

Analyse de la liaison de molécules liées par une membrane cellulaire Download PDF

Info

Publication number
WO2009037660A2
WO2009037660A2 PCT/IB2008/053790 IB2008053790W WO2009037660A2 WO 2009037660 A2 WO2009037660 A2 WO 2009037660A2 IB 2008053790 W IB2008053790 W IB 2008053790W WO 2009037660 A2 WO2009037660 A2 WO 2009037660A2
Authority
WO
WIPO (PCT)
Prior art keywords
molecules
cell membrane
membrane bound
specific binding
target cell
Prior art date
Application number
PCT/IB2008/053790
Other languages
English (en)
Other versions
WO2009037660A3 (fr
Inventor
Electra Gizeli
Michail Saitakis
Original Assignee
Foundation For Research And Technology Hellas
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Foundation For Research And Technology Hellas filed Critical Foundation For Research And Technology Hellas
Publication of WO2009037660A2 publication Critical patent/WO2009037660A2/fr
Publication of WO2009037660A3 publication Critical patent/WO2009037660A3/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/02—Analysing fluids
    • G01N29/032—Analysing fluids by measuring attenuation of acoustic waves
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
    • G01N29/22—Details, e.g. general constructional or apparatus details
    • G01N29/30—Arrangements for calibrating or comparing, e.g. with standard objects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/024—Mixtures
    • G01N2291/02466—Biological material, e.g. blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/02—Indexing codes associated with the analysed material
    • G01N2291/028—Material parameters
    • G01N2291/02818—Density, viscosity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2291/00—Indexing codes associated with group G01N29/00
    • G01N2291/04—Wave modes and trajectories
    • G01N2291/042—Wave modes
    • G01N2291/0422—Shear waves, transverse waves, horizontally polarised waves

Definitions

  • the invention relates to the field of analysing the binding of cell membrane bound molecules, in samples of whole cells, to specific binding molecules.
  • the analysis may, for example, be an analysis as to whether the cell membrane bound molecules bind to the specific binding molecules or an analysis of (e.g. calculation of) kinetic and/or affinity parameters relating to the binding of the cell membrane bound molecules to the specific binding molecules.
  • cell membrane bound molecules and “cell membrane proteins” includes molecules and proteins respectively which are bound to cell membranes directly or via other molecules.
  • Cell membrane proteins are typically adhered to the cell membrane either by spanning the cell membrane and including a central hydrophobic region, or via a covalent bond to one or more lipid molecules which are inserted into the cell membrane, or by adhesion (e.g. covalent attachment) to other cell membrane molecules.
  • Cell membrane proteins have great biological and biotechnological significance. Around 30% of all proteins encoded by the genome are cell membrane proteins. Cell membrane proteins mediate interactions between cells and extracellular components or other cells. Cell membrane proteins can also be important drug targets. Cells are in constant communication with their environment via cell membrane bound molecules. The binding of cell membrane molecules to specific ligands attached to the surface of other cells is pivotal in numerous physiological and developmental conditions such as leukocyte adhesion and rolling, and cell mediated immune reactions. Accordingly, it is desirable to be able to study the properties of membrane proteins whilst they remain attached to the cell membranes of whole cells. This enables properties of membrane protein interactions to be studied in their native environment, without requiring the proteins to be purified and separated from the membranes of whole cells.
  • Optical biosensors such as Surface Plasmon Resonance (SPR) devices, have not been effective due to the relatively large cell mass present in the sensor's evanescent field which produces a bulk response that is not sensitive to the number of membrane receptor/surface immobilized ligand complexes.
  • SPR Surface Plasmon Resonance
  • the present invention makes use of liquid-phase acoustic wave sensors which are sensitive to visco-elastic changes occurring close to the sensing surface, as well as mass coupling.
  • the invention aims to provide improved or alternative methods for analyzing the binding of cell membrane bound molecules to specific binding molecules whilst the cell membrane bound molecules remain bound to the membranes of whole, and preferably live, cells.
  • Some embodiments of the invention seek to calculate two-dimensional kinetic and/or affinity parameters relating to the binding of cell membrane bound molecules, in samples of whole cells, to specific binding molecules.
  • a method of analysing the binding of cell membrane bound molecules to specific binding molecules comprising the steps of:
  • the method may comprise the step of determining whether the cell membrane bound molecules bind to the specific binding molecules, using the monitored signal.
  • the method may comprise the step of analysing one or more kinetic and/or affinity parameters relating to the cell membrane bound molecules within the measurement sample of whole cells, using the monitored signal.
  • the analysis of one or more kinetic and/or affinity parameters may be a calculation of one or more kinetic and/or affinity parameters relating to the binding of the cell membrane bound molecules within the measurement sample of whole cells to the specific binding molecules, using the monitored signal.
  • At least one of the one or more kinetic and/or affinity parameters is a two- dimensional kinetic and/or affinity parameter.
  • the one or more kinetic and/or affinity parameters may be kinetic and/or affinity parameters of the binding of the cell membrane bound molecules to the specific binding molecules.
  • the one or more kinetic and/or affinity parameters may comprise or consist of the two-dimensional association rate (k a ) of a cell membrane molecule bound with the specific binding molecules, the two-dimensional dissociation rate (k d ) or the two-dimensional binding affinity (K A ) of a cell membrane bound molecule with the specific binding molecules.
  • the method may comprise the step of determining properties of the cell membrane bound molecules which affect their interaction with the specific binding molecules. These properties can be deduced from the monitored signal.
  • the method may comprise the step of analysing (e.g. calculating) the rate of diffusion of the target cell-bound molecule within the cell membrane. (This two-dimensional rate of diffusion within a membrane should not be confused with the rate of diffusion in solution).
  • the method may comprise the step of determining whether further molecules compete with the specific binding molecules to bind the cell membrane bound molecules.
  • the method may alternatively, or additionally, comprise the step of analysing (e.g. calculating) one or more kinetic and/or affinity parameters relating to the interaction between the cell membrane bound molecules within the measurement sample of whole cells and further molecules, using the monitored signal.
  • the further molecules may, for example, be cell membrane bound molecules with which the target cell membrane bound molecules interact, or extracellular molecules which compete with the specific binding molecules to bind the target cell membrane bound molecules.
  • the step of calculating one or more kinetic and/or affinity parameters relating to cell membrane bound molecules using the monitored signal may optionally disregard the monitored signal for a period of time after cells were first brought into contact with the sensing surface. This enables changes in the monitored signal due to the diffusion of cells through the liquid to the sensing surface and their initial adherence to the sensing surface to be disregarded.
  • the signal is monitored whilst changes in the monitored signal result predominantly (preferably substantially entirely) from the formation of bonds between the specific binding molecules and the target cell membrane bound molecules.
  • the step of calculating one or more kinetic and/or affinity parameter may comprise the step of determining the exponent, for a suitable base (such as 10 or e), of an exponential change in the monitored signal resulting from the binding of target cell membrane bound molecules to the specific binding molecules.
  • the analysis of the binding of cell membrane bound molecules to specific binding molecules may be an analysis of changes affecting the binding of cell membrane bound molecules to specific binding molecules.
  • the method may employ two different samples of whole cells each of which comprises target cell membrane bound molecules, wherein the samples of whole cells are different such that a different rate of binding of target cell membrane bound molecules to specific binding molecules arises when the two different samples of whole cells are introduced to equivalent sensing surfaces having specific binding molecules attached thereto.
  • the method may comprise the step of bringing a reference sample of whole cells having cell membranes with target cell membrane bound molecules bound thereto into contact with the sensing surface of a liquid-phase acoustic wave sensor which generates an acoustic wave and produces a signal which is related to energy losses of the acoustic wave, which acoustic wave sensor has a sensing surface comprising specific binding molecules which are operable to form specific bonds with target cell membrane bound molecules, and monitoring the signal while specific bonds are formed between the specific binding molecules and the target cell membrane bound molecules of the second sample of whole cells.
  • the reference sample of whole cells may be used, for example, as a control or for calibration or comparative purposes, and may be brought into contact with the sensing surface of a liquid-phase acoustic wave sensor before, after, or simultaneously to the bringing of the measurement sample of whole cells into contact with the sensing surface of a liquid-phase acoustic wave sensor.
  • a plurality of signals measured while specific bonds are formed between specific binding molecules and target cell membrane bound molecules of samples of whole cells may be used to prepare calibration data for use when analysing the monitored signal while specific bonds are formed between the specific binding molecules and the target cell membrane bound molecules of the measurement sample of whole cells.
  • the method may be used to analyse the binding of cell membrane bound molecules in the reference sample of whole cells to specific binding molecules by comparing the monitored signal while specific bonds are formed between the specific binding molecules and the target cell membrane bound molecules of the reference sample of whole cells and the monitored signal while specific bonds are formed between the specific binding molecules and the target cell membrane bound molecules of the reference sample of whole cells.
  • the measurement and reference samples of whole cells may be brought into contact with the sensing surface of the same liquid-phase acoustic wave sensor or different liquid-phase acoustic wave sensors.
  • the method may be repeated using three or more than three samples of cells each of which comprises target cell membrane bound molecules, wherein the samples of whole cells are different such that a different rate of binding of target cell membrane bound molecules to specific binding molecules arises when the different samples of whole cells are introduced to equivalent sensing surfaces having specific binding molecules attached thereto.
  • An array of liquid-phase acoustic wave sensors each of which comprises an individual sensing surface, or a liquid-phase acoustic wave sensor comprising a plurality of separate detection regions and associated measurement channels for providing separate signals, within the same sensing surface using microfluidic devices, may be provided to analyse the binding of a plurality of samples (e.g. a plurality of measurement samples) of whole cells to specific binding molecules simultaneously.
  • a plurality of samples e.g. a plurality of measurement samples
  • the or each acoustic wave sensor may also be operable to carry out other measurements (i.e. non-acoustic measurements) to analyse the binding of whole cells to specific binding molecules.
  • the acoustic wave sensor may also be used as an optical sensor, for example, a Surface Plasmon Resonance sensor, or an electrochemical sensor.
  • the acoustic wave sensor may also be operable to carry out non-acoustic measurements (for example, optical or electrochemical measurements), and the method may comprise the step of monitoring an additional signal, derived from a non- acoustic measurement (for example, an optical or electrochemical measurement).
  • a non-acoustic measurement for example, the optical or electrochemical measurement
  • the additional signal may be derived from a non- acoustic (for example, optical or electrochemical) measurement indicative of the formation of specific bonds with target cell membrane bound molecules.
  • the additional signal may be derived from a non-acoustic (for example, optical or electrochemical) measurement which is sensitive to the amount of material (e.g. the number or mass of whole cells) on or adjacent to the sensing surface.
  • the additional signal may be derived from a non-acoustic (for example, optical or electrochemical) measurement which is independent of the amount of material (e.g. the number or mass of whole cells) on or adjacent to the sensing surface.
  • the monitored signal and the additional signal can be analysed to better determine one or more kinetic and/or affinity parameters relating to the cell membrane bound molecules within the measurement sample of whole cells.
  • the samples of whole cells e.g.
  • the measurement and reference samples of whole cells may differ in that they include different numbers of cells with the same average number of target cell membrane bound molecules.
  • the samples of cells may differ in that they include cells with different numbers of target cell membrane bound molecules and/or different surface densities of target cell membrane bound molecules which are operable to bind the specific binding molecules.
  • the samples of cells may have been subject to different chemical treatments which affect the number of target cell membrane bound molecules which are operable to bind the specific binding molecules.
  • the samples of cells may have been cultured differently to affect the number of target cell membrane bound molecules which are operable to bind the specific binding molecules.
  • the samples of cells may differ in that they include cells with target cell membrane bound molecules in different conformations with different affinities for the specific binding molecules.
  • the samples of cells will be selected such that in each case different surface concentrations of the target cell membrane bound molecules will be provided.
  • the samples of cells may differ in that they include cells which have been treated differently prior to binding to the specific binding molecules. This facilitates testing to establish whether a particular treatment, e.g. a particular cell growth regime or an interaction between a cell and a test agent leads to a change in the affinity and/or kinetics of a cell membrane bound molecule, for example because the growth regime or interaction affects the structure of the cell membrane bound molecule.
  • a particular treatment e.g. a particular cell growth regime or an interaction between a cell and a test agent leads to a change in the affinity and/or kinetics of a cell membrane bound molecule, for example because the growth regime or interaction affects the structure of the cell membrane bound molecule.
  • the samples of cells may differ in that they include different amounts of a reagent which affects the binding of target cell membrane bound molecules to specific binding molecules.
  • the reagent may bind with the target cell membrane bound molecules.
  • the reagent may be another cell membrane bound molecule which binds with the target cell membrane bound molecules.
  • the reagent may compete with the specific binding molecules to bind the target cell membrane bound molecules.
  • the reagent may compete with the target cell membrane bound molecules to bind the specific binding molecules.
  • the reagent may be operable to modify the target cell membrane bound molecules, for example, the target cell membrane bound molecules may comprise receptors and the reagent may be a ligand for the receptors.
  • the reagent may be a test agent and the method may be a method of determining whether the reagent interacts with the target cell membrane bound molecules or the specific binding molecules to cause a change in one or more kinetic and/or affinity parameters of the cell membrane bound molecule.
  • the monitored signal is substantially independent of the mass within the penetration depth of the sensor.
  • the acoustic wave has a penetration depth of less than 500nm and more preferably less than 200nm from the sensing surface. More preferably still, the acoustic wave has a penetration depth of less than 100nm from the sensing surface.
  • the monitored signal should depend on the formation of specific bonds between cell membrane bound molecules and specific binding molecules and should not be affected by signal changes due to the presence of the cell mass in the sensing area
  • the penetration depth is the distance from the sensing surface within which the amplitude of liquid oscillation decays to 1/e of the value of the amplitude of oscillation at the sensing surface.
  • the monitored signal will typically be related to the energy loss or dissipation of the acoustic wave which is generated, for example, the monitored signal may be related to the amplitude of the generated acoustic wave.
  • the liquid-phase acoustic wave sensor may be a Bulk Acoustic Wave type device, such as a Quartz Crystal Microbalance or Thickness Shear Mode resonator.
  • the monitored signal will typically be related to the energy dissipation of the wave generated by the acoustic wave sensor.
  • the liquid-phase acoustic wave sensor may be an acoustic wave sensor which generates a shear wave; such Surface Acoustic Wave type devices can employ interdigitated transducers to generate a shear wave, such as a Love wave, Surface Skimming Bulk Wave, Acoustic Plate Mode, Bleustein-Gulyaev wave or Surface Transverse Wave.
  • the monitored signal will typically be related to the energy loss or dissipation of the surface acoustic wave which is generated, for example, the monitored signal may be related to the amplitude of the generated surface acoustic wave.
  • the liquid-phase acoustic wave sensor may be an acoustic wave sensor using a thin membrane to excite an acoustic wave in a configuration known as Flexural Plate Wave or Lamb wave device.
  • the shear acoustic wave sensor may be a non-1 DT based device such as a device employing an electromagnetically excited shear acoustic wave.
  • the specific binding molecules are typically biological macromolecules, such as proteins, for example antibodies. However, the specific binding molecules may be other organic molecules, for example ligands for cell receptor proteins.
  • the specific binding molecules may be immobilised on the sensing surface via intervening molecules, e.g. via a surfacing binding layer of molecules (such as a monolayer). The specific binding molecules may be bound to whole cells immobilised on the sensing surface.
  • the target cell membrane bound molecules are typically cell membrane molecules attached to the outside of cell membranes or located within cell membranes and operable to form bonds with extracellular specific binding molecules.
  • the target cell membrane bound molecules may be cell membrane receptors and the specific binding molecules may be ligands for the cell membrane receptors.
  • the cells are whole live cells.
  • a method of measuring the concentration of cell membrane bound molecules on a cell surface which are available to bind to surface-immobilised specific binding molecules comprising the steps of:
  • the step of comparing the monitored signals comprises comparing the rate of change of the monitored signals, optionally with normalisation.
  • the monitored signals will typically be related to the energy loss or dissipation of the acoustic wave which is generated.
  • the monitored signals may be related to the amplitude of the generated acoustic wave.
  • the step of comparing the monitored signals may comprise comparing the rate of amplitude change of the monitored signals normalised with respect to the absolute value of the amplitude change.
  • step (i) is carried out in respect of a plurality of different reference samples of cells having cell membranes with a known concentration of target cell membrane bound molecules bound to the surface thereof and available to bind to surface-immobilised specific binding molecules and the plurality of resulting monitored signals are compared to determine the concentration of target cell membrane bound molecules and available to bind to surface-immobilised specific binding molecules.
  • the plurality of resulting monitored signals may be used to provide a calibration curve.
  • the plurality of different reference samples of cells may have different concentrations of target cell membrane bound molecules bound to the surface thereof.
  • the plurality of different reference samples of cells may comprise different concentrations of cells with the same known concentration of target cell membrane bound molecules bound to the surface thereof.
  • the plurality of different reference samples of cells may have target cell membrane bound molecules with different availabilities to bind the said immobilised specific binding molecules, for example, because they have different configurations or have bound different concentrations of a competing molecule.
  • the monitored signal is substantially independent of the mass within the penetration depth of the sensor, enabling different numbers of target cell membrane bound molecules to be bound to the specific binding molecules by varying the number of cells within the samples.
  • the method may comprise the step of measuring the number of cells which have adhered to the sensing surface to determine the number of target cell membrane bound molecules per cell.
  • the method may comprise the step of determining the availability of the target cell membrane bound molecules to bind the said immobilised specific binding molecules.
  • the invention also extends in a third aspect to a method of assessing the availability of the target cell bound molecules in a sample of whole cells to bind surface- immobilised specific binding molecules, measuring the concentration of cell membrane bound molecules on the surface of the treated cells which are available to bind to surface-immobilised specific binding molecules by a method according to the second aspect of the present invention, determining the concentration of cell membrane bound molecules on the surface of the treated cells and thereby determining the availability of the target cell membrane bound molecules of the treated sample of whole cells to bind the said immobilised specific binding molecules.
  • the method may comprise the step of treating a sample of whole cells prior to assessing the availability of the target cell bound molecules in the sample of treated cells to bind to surface-immobilised specific binding molecules and thereby assessing the effect of the treatment on the availability of the target cell bound molecules in the sample of treated cells to bind to surface-immobilised specific binding molecules.
  • the treatment may comprise the addition of a test agent, to determine whether the test agent affects the availability of the target cell membrane bound molecules to bind the said immobilised specific binding molecules, for example by affecting the conformation of the immobilised specific binding molecules or by competing with the immobilised specific binding molecules to bind the target cell membrane bound molecules.
  • a method according to any one of the first three aspects of the invention may be part of a method of studying the interactions of cell membrane bound molecules and/or ligands for cell membrane bound molecules.
  • a method according to any one of the first three aspects of the invention may be part of a method of studying interactions of immune system molecules.
  • a method according to any one of the first three aspects of the invention may be part of a method of screening potential treatment agents.
  • a method according to any one of the first three aspects of the invention may be part of a diagnostic method.
  • a method according to any one of the first three aspects of the invention may be part of a method of screening surface structures which are candidates for use in synthetic transplants.
  • Figure 1 is a schematic perspective view of a Love wave sensor
  • Figure 2 is a schematic cross-section through a Love wave sensor including a cell having cell membrane proteins
  • Figure 3 is a graph of the change in amplitude (constituting the monitored signal) as a function of the number of bound cells during the binding of untreated, mild acid treated and from high density culture LG2 cells. Cell densities were calculated with an average accuracy of 10%;
  • Figure 4 is a graph of amplitude change as a function of time during the interaction of LG2 cells (2.5*10 5 cells/ml) with the surface-immobilized anti-HLA antibody. Arrows (a) and (b) indicate the corresponding times for the addition of the cell suspension (10 ⁇ l min-1 ) and buffer (50 ⁇ l min-1 ), respectively.
  • the binding curve is divided in two phases. The first one ( ⁇ 500 s after addition) represents the time required for cells to reach the surface and form initial tethers and is cell-diffusion limited. The second phase (shaded rectangle) depicts the formation of HLA/anti-HLA bonds;
  • Figure 5 is an optical microscopy photograph of LG2 cells on the biosensor surface after an acoustic experiment. Cytoplasmic protrusions are formed as a result of the formation of cell-surface contact points. (Scale bar: 15 ⁇ m);
  • Figure 6 is a graph showing real-time amplitude/time binding curves when various LG2 cell suspensions were added to immobilized anti-HLA antibody. Arrows (a) and (b) indicate the times for the addition of the cell suspension and buffer, respectively. In order from top to bottom the traces show the results for cell suspensions with concentrations of 6.0*10 4 /ml, 1.0 ⁇ 10 5 /ml, 1.2 ⁇ 10 5 /ml, 1.5 ⁇ 10 5 /ml, 2.0 ⁇ 10 5 /ml, 2.4 ⁇ 10 5 /ml, 3.0 ⁇ 10 5 /ml, and 6.0*10 5 /ml; and
  • Figure 7 is a plot of ⁇ (dA/dt)/ ⁇ A, where (dA/dt) is the rate of amplitude (A) change derived from real-time curves and ⁇ (dA/dt)/ ⁇ A the slope of (dA/dt) against A, versus the total number of cell-attached HLA-A2 molecules available for binding, i.e. C for the experiments illustrated in Figure 6.
  • the two-dimensional rate constants k a (in ⁇ m 2 s "1 per molecule) and k d (in s "1 ) can be derived from the slope and intercept with the ordinate, respectively, while the ratio of k a /k d is a measure of the two-dimensional binding affinity K A (in ⁇ m 2 per molecule).
  • FIG. 1 is a schematic diagram of a Love wave sensor, shown generally as 1 , which functions as the liquid-phase acoustic wave sensor.
  • the sensor comprises a quartz substrate 2, with interdigitated transducers (not shown) deposited thereon.
  • a polymer guiding layer 4 is formed on the quartz substrate between the interdigitated transducers.
  • a gold layer 6 is deposited on the polymer guiding layer to facilitate immobilisation of specific binding molecules.
  • a Protein G layer 8 is adhered to the gold layer and antibodies 10, functioning as the specific binding molecules, are attached to the Protein G layer and thereby immobilised on the sensing surface formed by the gold layer and immobilised proteins. Details of the construction of a Love wave sensor and formation of the antibody layer are described below.
  • an oscillating electric potential is applied to the Love wave sensor via the IDTs to create a shear-horizontal surface acoustic wave.
  • the phase and amplitude of the wave which are related to the acoustic velocity and energy, respectively, are measured as a function of time through electrical connections to the IDTs and recorded for use in subsequent calculations.
  • the polymer guiding layer serves as an acoustic waveguide by localizing the acoustic energy of the wave close to the sensing surface. All sensing occurs within the volume in contact with the device surface in which there is significant displacement as a result of the acoustic wave/liquid coupling.
  • the acoustic wave sensor has a penetration depth of approximately 50 nm when in contact with pure water and increases with the square root of the liquid viscosity. The vast majority of the mass of cells which bind to the sensing surface is outside of the penetration depth.
  • whole live cells 12 including a target membrane protein 14 with which the immobilised antibodies can form specific bonds are brought into contact with the surface of the Love wave sensor.
  • the approximate penetration depth of the acoustic wave sensor is shown by dashed line 16.
  • the cells diffuse to the sensing surface and bind to the sensing surface by virtue of the specific binding interaction between the antibodies and the target membrane proteins. Specific bonds form between the antibodies and the target membrane proteins over a period of time (typically tens of minutes).
  • the amplitude of the acoustic waves is correlated directly to the formation of specific bonds between the antibodies and the target membrane proteins and is not significantly affected by the mass of the cells.
  • the amplitude is monitored while each of a plurality of samples of whole cells including different number of otherwise equivalent cells are brought into contact with sensing surfaces. Accordingly, by monitoring the amplitude of the acoustic wave while specific bonds are formed between antibodies and target membrane proteins, and comparing the change in amplitude with time for samples of whole cells with different numbers of cells, and therefore different maximum target membrane protein concentrations on the sensing surfaces, kinetic parameters concerning the interaction between the antibodies and the target membranes can be derived.
  • the rate of change of amplitude, ⁇ (dA/dt), divided by the change of amplitude relative to a reference value, ⁇ A, is plotted against C, the two dimensional surface density of cell membrane HLA molecules.
  • the 2-dimensional association and dissociation rate constants k a and k d are calculated from the slope of this plot and the intercept with the ordinate respectively.
  • the two-dimensional binding affinity K A can then be calculated as the ratio k a /k d .
  • this method allows the calculation of kinetic and affinity parameters of cell membrane bound molecules in situ whilst they remain bound to the membranes of live cells.
  • the membrane receptor protein used was the HLA-A2 molecule, the most common class I Major Histocompatibility Complex allele in human populations, expressed in the B-lymphoblastoid LG2 cell line.
  • the natural function of HLA-A2 is to present short endogenous peptides (8-1 1 residues) to the T cell receptors (TCRs) of T lymphocytes, which can trigger immune response.
  • the specific binding molecules used in this example are surface immobilized anti-HLA-A2 monoclonal antibodies BB7.2; oriented on the surface through the F c fragment (Saha et al., 2003; Fahnestock et al., S. R., 1986).
  • the antibody is specific for the ⁇ chain (Parham and Brodsky, 1981 ) of HLA-A2 when the latter exists in a heterotrimer form consisting of ⁇ chain/ ⁇ 2 -microglobulin/peptide (Hogan and Brown, 1992).
  • the heterotrimer form of the HLA-A2 molecules function as the target cell membrane bound molecules.
  • HLA-A2 molecules on the cell membrane can be found in a heterodimeric form, i.e. just ⁇ -chain/ ⁇ 2 -microglobulin, or as single ⁇ -chains (Matko et al., 1994); ⁇ chains in the last two forms are not recognized by BB7.2.
  • HLA-A2 heterotrimers Samples of cells containing different numbers of HLA-A2 heterotrimers were prepared using LG2 cells prepared under three different conditions: as untreated cells, as mild acid-treated and as untreated from high density cultures. Mild acid treatment removes bound peptides from the HLA groove (van der Burg et al., 1995), so treated cells have low heterotrimer numbers. In addition, cells grown in high density cultures will display high heterotrimer numbers on the cell surface (Matko et al., 1994). Using an indirect quantitative immunofluorescence assay and flow cytometry (described further below), the number of HLA-A2 heterotrimers (hereafter referred to as HLA or HLA-A2) on the cell surface was calculated.
  • the values were: 3.7-5.7*10 5 HLA-A2 molecules per untreated LG2 cell (12 experiments), 1-10*10 4 molecules per mild-acid treated LG2 cell (6 experiments) and 9.7-10.0> ⁇ 10 5 per LG2 cell from a high density culture (4 experiments).
  • the HLA molecules on the cell membrane would interact with the immobilized IgG through the 7 nm long extracellular part of HLA-A2 molecules (Bjorkman et al., 1997). Taking into account cell membrane thickness (6-10 nm), it is clear that the vast majority of the cell mass will be outside the penetration depth (cell diameter 14.4 ⁇ 2.2 ⁇ m). For membrane HLA-A2, the calculated total mass-per-area values in the acoustic experiments are very low to cause a detectable phase change due to mass coupling. Instead, amplitude response is related to acoustic energy dissipated from the sensor surface to the liquid interface. Apparently, the cell membrane, in which the HLA molecules are embedded, acts as an effective damper by adsorbing energy through the HLA- A2/antibody bonds, hence the observed amplitude change.
  • control experiments which included the injection of HLA non-expressing K562 cells over the antibody-modified sensor surface and the addition of anti-HLA pretreated LG2 cells, also proved the specificity of the observed signal change to the formation of HLA/anti- HLA bonds.
  • the acoustic biosensor was found to be sensitive to the addition of as few as 6*10 3 cells ml "1 or 156 cells immobilized on the sensor surface, which corresponds to a total mass of HLA molecules per sensor surface 0.6 pg mm "2 . This damping related detection is lower than the mass related detection of SPR for soluble molecules (100- 1000 pg mm "2 ).
  • (dA/dt) is the rate of amplitude (A) change derived from real-time graphs
  • C the concentration of the soluble analyte
  • k a and k d the association and dissociation rate constants, respectively.
  • the three dimensional molar concentration C is replaced by the two dimensional HLA-A2 surface density C 2D , which should reflect the number of cell membrane HLA molecules available for binding
  • k a , k d are the corresponding 2D rate constants.
  • N HLA being the total number of HLA molecules on the cell surface, f their fractional mobility, and S ce ⁇ the cell surface.
  • S ce ⁇ was multiplied by the surface roughness factor of 1.8, resulting in a final value of 1 172.6 ⁇ m 2 .
  • HLA HLA-A2 receptors
  • the total number of HLA receptors per cell remains constant and the only variable is the total number of applied cells. This implies that 374 molecules ⁇ m "2 is the maximum [HLA] that can be presented to the sensor's surface and will correspond to the maximum coverage of the surface by cells. In practice, the maximum coverage was observed at cell suspensions equal or higher to 6x10 5 cells/ml (C max ), as detected acoustically through ⁇ A measurements. For cell suspensions lower than C max , the HLA surface density per cell will still remain 374 molecules ⁇ m "2 ; however, the effective [HLA] sensed by the sensor will be a function of the coverage of the sensor surface by the cells, i.e.
  • an acoustic wave sensor can be employed for assaying the binding of receptor-bearing cells, i.e. leukemic cells expressing class I Major Histocompatibility Complex (MHC) molecules, to a specific monoclonal antibody immobilized on the sensor surface.
  • the measured signal (amplitude) changed as a function of time upon addition of viable cells to the modified sensor surface.
  • the time course of the change in the measured signal was found to correlate directly to the number of cell-membrane molecules specifically attached to the immobilized antibodies. This finding allowed for the calculation of two- dimensional kinetics and affinity parameters.
  • acoustic damping to the number of cell/surface specific bonds provides a unique sensing mechanism for investigating membrane interactions.
  • the proposed label-free and non invasive acoustic biosensor could be further applied to characterize various membrane-associated events. Examples of interactions which could be studied include T cell receptor/MHC and MHC/antigenic peptide interactions using whole cells, thereby avoiding the need for membrane protein purification and/or reconstitution.
  • ⁇ (dA/dt)/ ⁇ A is proportional to C, the total surface density of cell bound molecules which are available to interact with an immobilised ligand.
  • the total surface density of cell bound molecules which are available to interact with an immobilised ligand within a measurement sample of whole cells having an unknown total surface density of cell bound molecules which are available to interact with an immobilised ligand can be determined from a calibration curve of ⁇ (dA/dt)/ ⁇ A.
  • a calibration curve of ⁇ (dA/dt)/ ⁇ A can be determined either by using reference samples having different concentrations of cells, each of which has the same the total surface density of cell bound molecules which are available to interact with an immobilised ligand, or by using different reference samples of cells having different total surface densities of cell bound molecules which are available to interact with an immobilised ligand.
  • ⁇ (dA/dt)/ ⁇ A is a function of the total surface density of cell bound molecules which are available to interact with an immobilised ligand
  • a comparison of ⁇ (dA/dt)/ ⁇ A between two samples with the same total surface density of cell bound molecules but which have been treated differently enables changes in cell bound molecules which affect their availability to interact with an immobilised ligand, e.g. conformation changes or the binding of competing molecules, to be detected or quantified.
  • the invention can be applied to the study of cell membrane molecules and their ligands.
  • the invention can be employed to calculate the two- dimensional kinetics and affinity of various molecular interactions that mediate physiological and developmental conditions.
  • One specific example is the study of the interactions of leukocyte homing receptors. These receptors determine the arrest and extravasation of neutrophils and other leukocytes in response to infection.
  • leukocytes carrying the receptors are introduced to vicinity of the sensor surface where the ligands would be immobilized.
  • This setup resembles the physiological condition as the ligands are normally expressed on the surface of the endothelial cells that form blood vessels.
  • Various pumping rates can be employed to resemble the physiological blood flows.
  • This example can be extended to the study of receptors that mediate tumor metastasis, since the mechanism of action is similar.
  • Another example application is the study of immunologically important interactions.
  • Such interactions include T cell receptor/major histocompatibility complex molecules, NK receptors and their ligands and Toll-like receptors and their ligands.
  • T cell receptor/major histocompatibility complex molecules include T cell receptor/major histocompatibility complex molecules, NK receptors and their ligands and Toll-like receptors and their ligands.
  • NK receptors and their ligands have been extensively studied using soluble molecules.
  • immobilized ligands are provided on the sensor surface and cells carrying the receptors are introduced to the vicinity of the sensor surface. The real-time monitoring of the bond formation between cell and substrate provides information on the extent of the interaction.
  • the mechanism of activation can be investigated using various ligands at the sensor surface or a different extent of immobilization or even a different type of immobilization (i.e. insertion of ligands into supported lipid bilayers in order to be laterally mobile).
  • the invention can be also applied to the screening of pharmacological agents.
  • One particular field that can benefit is the screening and analysis of therapeutic antibodies.
  • the invention can be used to investigate the two-dimensional kinetics and affinity for the interactions of therapeutic antibodies and their molecular targets on the surface of particular cell types, i.e. tumor cells.
  • the experimental procedure can comprise immobilizing the antibody of interest on the sensor surface and then adding the cell type that presents the molecular target of the antibody at its surface. In that way, the efficacy of the therapeutic antibody can be easily evaluated.
  • Another example of application of the invention to drug screening is the field of viral attachment and entry. Viruses specifically attach to and enter in certain cell types. They do that via their viral receptors that recognize and bind their ligands on the surface of these cell types.
  • the invention can be employed to study the molecular interaction events of viral attachment.
  • the viral receptors can be immobilized on the sensor surface and various cell types applied over the surface.
  • This experimental setup can yield the cell type specificity of viruses as well as the kinetics and affinity of the molecular interaction of viral receptors and their ligands.
  • Potential inhibitors can be added in real-time during the monitoring of the interaction of viral receptors and their target cells. The successful inhibitors will result in lowering the monitored signal change since no binding would occur. Besides being added in real-time, potential inhibitors can be used to treat the target cells prior to addition over the sensor surface.
  • Diagnostics is a another field which could benefit from the current invention.
  • the invention can be applied to detect various types of molecules or cells in blood or other biological fluids.
  • Certain molecules i.e. antibodies, specific receptors/ligands
  • Another example of the use of the invention in diagnostics can be immunophenotyping, i.e. the identification of antigens of blood groups or the major histocompatibilty complex with the use of antibodies immobilized on the sensor surface. This can become a fast screening assay of blood cells for the antigens that determine blood transfusions and organ transplantations.
  • the invention can be also applied to the screening of biocompatible surfaces that are used in synthetic transplants.
  • the working example can be to prepare sensor surface as in the transplants and then apply certain cell types. This will allow checking for the way and extent cells interact with the transplant surface, a factor that usually determines the efficacy of biocompatible surfaces.
  • certain molecules are usually attached on the surface of transplants. Such molecules, which can be certain receptors or growth factors, are usually the bio-active part of the transplant.
  • the invention can be used to measure interactions between the immobilized molecules and their target cells. This can yield the kinetics and affinity of the molecular interaction of the immobilized molecules and their ligands in an environment close to the physiological conditions, i.e. the cell surface.
  • 1 10 MHz quartz devices were fabricated on 0.5-mm thick Y-cut piezoelectric quartz crystals.
  • the interdigitated transducers composed of a 210-nm thick Cr/Au (10/200 nm) electrode, consisted of 80 pairs of split fingers with a periodicity of 45 ⁇ m.
  • the devices were coated with a 0.7 ⁇ m thick poly(methylmethacrylate) (PMMA) (Aldrich) layer on top of which 20 nm of gold were sputtered using a BAL-TEC SCD 050 sputter coater.
  • PMMA poly(methylmethacrylate)
  • the acoustic devices were mounted on a special holder and liquid was pumped through on the area between the IDTs using a peristaltic pump (Gilson) and a flow-through cell.
  • the flow cell was sealed on the surface by using a custom- made rubber gasket exposing a sensing area of 12 mm 2 .
  • a Hewlett-Packard 4195A network analyzer was used to monitor the amplitude and phase of the wave and LabVIEW (National Instruments) software for collecting acoustic data. Experiments were run at least in triplicates. Following the end of acoustic experiments, the biosensor surfaces were observed under a Nikon Eclipse E800 microscope and photographs were taken with an attached Nikon Coolpix E5400 camera. Cells on the sensor surface were counted from at least three different areas.
  • the EBV-transformed human B-lymphoblastoid cell line LG2 HLA-A * O2O1 Y
  • the chronic myelogenous leukemic K562 cells HLA-A " B " C "
  • RPMI 1640 GEBCO Inc.
  • 1 mg L "1 gentamycin and 10% of fetal bovine serum was used as culture medium.
  • Culture flasks were kept in humidified 5% CO 2 atmosphere at 37° C. Medium was exchanged every 2-3 days. The cell density was 3-8*10 5 cells ml "1 in normal cultures and 2-3*10 6 cells ml "1 in high density cultures.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Mycology (AREA)
  • Acoustics & Sound (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analyzing Materials By The Use Of Ultrasonic Waves (AREA)

Abstract

La présente invention concerne un procédé d'analyse de la liaison de molécules liées par une membrane cellulaire à des molécules de liaison spécifique à l'aide d'un dispositif de détection acoustique à phase liquide pour surveiller le changement d'un signal tandis que des molécules liées par une membrane cellulaire forment des liaisons spécifiques avec des molécules de liaison immobilisées sur la surface de détection du dispositif de détection acoustique à phase liquide.
PCT/IB2008/053790 2007-09-17 2008-09-17 Analyse de la liaison de molécules liées par une membrane cellulaire WO2009037660A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0718073.0 2007-09-17
GB0718073A GB0718073D0 (en) 2007-09-17 2007-09-17 Analysing the binding of cell membrane bound molecules

Publications (2)

Publication Number Publication Date
WO2009037660A2 true WO2009037660A2 (fr) 2009-03-26
WO2009037660A3 WO2009037660A3 (fr) 2009-07-30

Family

ID=38659065

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IB2008/053790 WO2009037660A2 (fr) 2007-09-17 2008-09-17 Analyse de la liaison de molécules liées par une membrane cellulaire

Country Status (2)

Country Link
GB (1) GB0718073D0 (fr)
WO (1) WO2009037660A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2515111A1 (fr) 2011-04-19 2012-10-24 SAW instruments GmbH Procédé analytique permettant d'étudier la liaison des ligands analytes aux cellules

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE0403139D0 (sv) * 2004-12-23 2004-12-23 Nanoxis Ab Device and use thereof

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2515111A1 (fr) 2011-04-19 2012-10-24 SAW instruments GmbH Procédé analytique permettant d'étudier la liaison des ligands analytes aux cellules
WO2012143463A1 (fr) 2011-04-19 2012-10-26 Saw Instruments Gmbh Procédé analytique pour étudier la liaison d'analytes ligands à des cellules

Also Published As

Publication number Publication date
GB0718073D0 (en) 2007-10-24
WO2009037660A3 (fr) 2009-07-30

Similar Documents

Publication Publication Date Title
Chunta et al. Low-density lipoprotein sensor based on molecularly imprinted polymer
FI118061B (fi) Menetelmä ja bioanturi analyysiä varten
Liu et al. Quartz crystal biosensor for real-time monitoring of molecular recognition between protein and small molecular medicinal agents
Uludağ et al. Piezoelectric sensors based on molecular imprinted polymers for detection of low molecular mass analytes
Chung et al. Additive assay of cancer marker CA 19-9 by SPR biosensor
Seo et al. Polydiacetylene liposome microarray toward facile measurement of platelet activation in whole blood
Zhou et al. A supported lipid bilayer-based lab-on-a-chip biosensor for the rapid electrical screening of coronavirus drugs
Kowalczyk et al. Parallel SPR and QCM-D quantitative analysis of CD9, CD63, and CD81 tetraspanins: a simple and sensitive way to determine the concentration of extracellular vesicles isolated from human lung cancer cells
US20120198920A1 (en) Analytical method and sensor
EP1766398B1 (fr) Detection des interactions superficielles moleculaires
US20110086368A1 (en) Method for immune response detection
Mazouzi et al. Biosensing extracellular vesicle subpopulations in neurodegenerative disease conditions
Zhang et al. Determining binding sites of drugs on human serum albumin using FIA-QCM
Liu et al. Quartz crystal biosensor for real-time kinetic analysis of interaction between human TNF-α and monoclonal antibodies
Mitsakakis et al. Quantitative determination of protein molecular weight with an acoustic sensor; significance of specific versus non-specific binding
Saitakis et al. Probing the interaction of a membrane receptor with a surface-attached ligand using whole cells on acoustic biosensors
Damiati Acoustic biosensors for cell research
CA2110705A1 (fr) Methodes et capteurs pour determiner les marqueurs de l'infarctus du myocarde
JP6954988B2 (ja) 表面プラズモン共鳴による複雑な生体試料中の活動濃度及び／又は動力学相互作用定数の測定方法
US7563587B2 (en) Method and kit for cell analyte assay
WO2009037660A2 (fr) Analyse de la liaison de molécules liées par une membrane cellulaire
Saitakis et al. Measurement of two-dimensional binding constants between cell-bound major histocompatibility complex and immobilized antibodies with an acoustic biosensor
TW201245715A (en) Analytical method to investigate binding of analyte ligands to cells
Gianneli et al. Novel QCM-based method to predict in vivo behaviour of nanoparticles
Zhang et al. QCM–FIA with PGMA coating for dynamic interaction study of heparin and antithrombin III

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08807711

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 08807711

Country of ref document: EP

Kind code of ref document: A2

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载