1. Field of the Invention
The present invention relates generally to binding assays such as immunoassays and, more specifically, to the use of force generated from an ultrasonic power source to characterize specific binding interactions and to differentiate specific and nonspecific binding interactions in such assays.
2. Description of the Related Art
A remarkable ability has developed in nature for molecular recognition through the use of multiple noncovalent bonds, i.e., van der Waals, hydrogen, ionic and hydrophobic interactions, which possess a high degree of spatial and orientational specificity. Molecular recognition plays a central role in catalysis (Enzyme Structure and Mechanism, Alan Fersht, W.H. Freeman and Company, New York, 1985), cellular behavior (Bongrand, P. Physical Basis of Cell-Cell Adhesion, CRC Press: Boca Raton, Fla., 1988), the immunological response (Eisen, H. N. Immunology, 3rd Ed., Harpers and Row Publishers: New York, 1990) and many other biological processes. The binding energy of molecular recognition interactions span at least two logs in magnitude, resulting in weak reversible interactions and interactions as strong as a covalent bond. Examples of specific molecular recognition include interactions between ligands and receptors, between enzymes and substrates, between chelators and ions, and between polynucleic acids and complementary strands. The magnitude of molecular interactions found in nature range from very weak to very strong.
Information regarding the characteristics of particular molecular interactions has enormous practical utility. For example, knowledge about the binding affinity between ligands and receptors can be used in developing screening assays to identify pharmaceutical compounds that mimic or inhibit a specific interaction. Although the structure and binding properties of molecular recognition systems can be measured, the forces involved in intermolecular interaction have remained largely unknown. Recently, it has been demonstrated that binding forces between molecular entities may be measured or characterized by conducting experiments wherein molecular entities are allowed to bond and wherein the force that is required to cause the molecular entities to separate from each other is measured. A theoretical framework for analyzing the behavior of single bonds in response to an applied force is still under development. At this time, it is clear that the binding force observed for a loading rate is determined by the binding potential of a specific molecular interaction. A theoretical framework relating these parameters is set forth in Merkell et al, xe2x80x9cEnergy Landscapes of Receptor-Ligand Bonds Explored with Dynamic Force Spectroscopyxe2x80x9d. Nature, Vol. 397 (1999), pp. 50-53, in Bell, G. I., xe2x80x9cModels for the Specific Adhesion of Cells to Cellsxe2x80x9d, Science, 200, (1978), pp 618-627, and in Evans, E. et al, xe2x80x9cDynamic Strength of Molecular Adhesion Bondsxe2x80x9d, Biophysical Journal, 72, (1997) pp 1541-1555, all incorporated herein by reference.
Various methods have been developed for measuring binding forces. For example, micropipettes have been used in conjunction with optical microscopy to measure the interaction forces between ligands bound to model cells. In this technique one molecule is attached to a cell held in a micropipette and the other molecule is attached to another cell held in a second micropipette, allowing the two molecules to bond and then exerting a force on the cantilever that gradually increases until the molecules separate. The use of micropipettes is described by Evans, E. et al, xe2x80x9cDynamic Strength of Molecular Adhesion Bondsxe2x80x9d, Biophysical Journal, 72, (1997) pp 1541-1555.
Magnetically derived forces may also be used to apply force to intermolecular bonds. In this technique, one molecular entity is bound to a surface and the other molecular entity is bound to a magnetic or paramagnetic bead. Force is applied to the intermolecular bond by applying a magnetic field that pulls on the magnetic bead. As discussed below, magnetic force has been used as a way of separating bound components in immunoassays. However, the magnetic force that can be delivered to a binding site by current methods is only about 2-5 pN, which not strong enough for separating many of the binding interactions that one would typically want to study.
Binding forces between molecules can be measured by atomic force microscopy by attaching one molecule to a surface and the other molecule to an atomic force microscope cantilever, allowing the two molecules to bond and then exerting a force on the cantilever that gradually increases until the molecules separate. The use of atomic force microscopy to study intermolecular forces is described, for example, in the following patents, publications, and patent applications incorporated herein by reference: U.S. Pat. No. 5,363,697 to Nakagawa; U.S. Pat. No. 5,372,930 to Colton et al; Florin E.-L. et al, xe2x80x9cAdhesion Forces Between Individual Ligand-Receptor Pairsxe2x80x9d Science 264 (1994). pp 415-417; Lee, G. U et al, xe2x80x9cSensing Discrete Streptavidin-Biotin Interactions with Atomic Force Microscopyxe2x80x9d Langmuir, vol. 10(2), (1994) pp 354-357; Dammer U. et al xe2x80x9cSpecific Antigen/Antibody Interactions Measured by Force Microscopyxe2x80x9d Biophysical Journal Vol. 70 (May 1996) pp 2437-2441; Chilikoti A. et al, xe2x80x9cThe Relationship Between Ligand-Binding Thermodynamics and Protein-Ligand Interaction Forces Measured by Atomic Force Microscopyxe2x80x9d Biophysical Journal Vol. 69 (November 1995) pp 2125-2130; Allen S. et al, xe2x80x9cDetection of Antigen-Antibody Binding Events with the Atomic Force Microscopexe2x80x9d Biochemistry, Vol.36, No.24 (1997) pp 7457-7463; and Moy V. T. et al, xe2x80x9cAdhesive Forces Between Ligand and Receptor Measured by AFMxe2x80x9d Colloids and Surfaces A: Physicochemical and Engineering Aspects 93 (1994) pp 343-348, and U.S. patent application Ser. No. 09/074,541 for xe2x80x9cApparatus and Method for Measuring Internolecular Interactions by Atomic Force Microscopyxe2x80x9d, filed May 8, 1998. This method is useful for measuring intermolecular forces of individual molecules but is slow and impractical to be used as a sensor due to the small active area that is sensed by the atomic force microscope cantilever.
Knowledge about specific binding interactions, particularly antibody-antigen interactions has led to the development of assays that exploit specific binding in determining the presence or absence of particular molecular species in test samples or in the environment. Many different types of assays are based on the specific binding of an analyte of interest (that is, whatever chemical species one is trying to detect with the assay) with one or more molecules that have a binding affinity for the analyte. In the class of techniques typically known as immunoassays, for example, detectable tags or labels are attached to antibodies that specifically bind to an analyte, and the presence of the analyte in a test sample is detected by detecting the formation of labeled antibody-analyte complexes or by measuring the amount of labeled antibody that remains unbound. Other types of binding molecules such as chelators, strands of polynucleic acids and receptors may also be used in binding assays.
In a conventional solid phase assay, molecules that have a binding affinity for an analyte are immobilized onto a solid surface, and the surface is exposed to a test sample. The analyte, if present in the test sample, binds to the immobilized binding member. Various methods have been used to generate a macroscopically observable signal to indicate that such binding has occurred. For example, a labeled reagent that binds to the analyte or that binds to the binding member-analyte complex (as in, for example, a sandwich assay) may be added to the test sample. Various types of labels including radioactive, enzymatic, fluorescent and infrared-active have been used for creating labeled reagents.
Magnetically-active beads have been used as labels in immunoassays. See, for example, the following U.S. patents and patent applications, incorporated herein by reference: U.S. Pat. No. 5,445,970 to Rohr and U.S. Pat. No. 5,445,971 to Rohr, and U.S. patent application Ser. No. 09/008,782 for xe2x80x9cForce Discrimination Assayxe2x80x9d by Gil U Lee, filed on Jan. 20, 1998.
In binding assays of all types, including those that use magnetically-active beads, a persistent problem is the occurrence of false positive results. False positives may be caused by nonspecific binding of labeled reagents to the surface, by cross-reactivity of a labeled reagent with compounds that are analogs of the analyte or by gravitational settling of a labeled reagent onto the surface of the surface. Each of these events can cause an excess of labeled reagent to remain on an assay surface. False positives results may be reduced by applying a force to a surface that is sufficient to remove undesirable or excess labeled reagent but that is not sufficient to disrupt the specific binding that is being measured in the assay. Methods of force differentiation previously described include centrifugation, hydrodynamics and magnetic force transduction. For example, in an assay using magnetically-active beads, magnetic force may be applied to remove beads that settle onto the surface due to gravitational force. However, magnetic force alone is often insufficient to dislodge beads that bind to the surface by nonspecific binding or by cross-reactivity with an analog of the analyte. As described in U.S. patent application Ser. No. 09/008,782, nonspecific binding of labeled reagents to a surface can be reduced by chemically modifying the surface to reduce nonspecific adhesive forces.
Centrifugation may be used to apply force to rupture bonds between molecular entities. A disadvantage of centrifugation is that it is difficult to make an accurate calculation of the amount of force that is delivered to a binding site. Centrifugal force must be applied over an extended period of time (during acceleration and deceleration) and the force has components of torque caused by the acceleration and deceleration.
Hydrodynamic forces may also be used to apply force to rupture bonds between molecular entities. A use of hydrodynamic force to study receptor-mediated adhesion is described in Cozen-Roberts et al, xe2x80x9cReceptor-Mediated Adhesion Phenomenaxe2x80x9d Biophys. J. 58 (1990), pp 107-125, incorporated herein by reference. This technique has the disadvantages that it produces off-axis forces and requires a complex flow cell arrangement.
Ultrasonic force has used commercially for a wide variety of industrial and medical purposes including imaging, welding, cleaning, and dispersing solids in a liquid medium. In the field of solid phase assays, the use of ultrasonic force has, up until now, been limited to enhancing the reactivity of a solid phase binder (see, for example, Chen et al, xe2x80x9cUltrasound-Accelerated Immunoassay as Exemplified by Enzyme Immunoassay of Choriogonadotropinxe2x80x9d, Clinical Chemistry, 30, (1984), pp 1446-1451 or Tarcha et al, xe2x80x9cAbsorption-enhanced Solid-Phase Immunoassay Method Via Water-Swellable Poly(acrylamide)Microparticlesxe2x80x9d Journal of Immunological Methods, 125 (1989) pp243-249 or dissociating binder-ligand complexes so that the amount of ligand can be measured or so that the binder can be reused (see, for example, U.S. Pat. No. 4,615,984 to Stoker, incorporated herein by reference, and Haga et al, xe2x80x9cEffect of Ultrasonic Irradiation on the Dissociation of Antigen-Antibody Complexes. Application to Homogeneous Enzyme Immunoassayxe2x80x9d, Chem. Pharm. Bull. 35(9) (1987), pp 3822-2830).
Thus, it is an object of the present invention to provide a method of characterizing binding forces between binding members wherein the force that is applied can be varied and is strong enough to separate intermolecular complexes.
Further, it is a object of the present invention to provide a method of characterizing binding forces between binding members wherein the force that is applied is oriented primarily in the direction of the molecular interaction and wherein off-axis or tangential forces are minimized.
Further, it is a object of the present invention to provide a method for characterizing binding forces between binding members that does not require complex and expensive apparatus.
Further, it is an object of the present invention to provide a method for characterizing binding forces between binding members that can test a large number of binding members simultaneously and quickly.
It has now been discovered that force generated from an ultrasonic power source can be used in an assay to measure or characterize molecular interactions, such as binding affinities of ligands and receptors. This is done by attaching a binding member to a bead or other particle that can be observed in real time, for example, through a microscope, and then allowing the particle-bound binding member to bind with a surface-bound binding member to form a complex. The presence of complexes on the surface is detected by observing the presence of immobilized particles on the surface. Ultrasonic force is then applied, and the movement or lack of movement of the particles, indicating dissociation or lack of dissociation of the complexes may be observed by microscopy or other methods of detection. Alternatively, the ultrasonic force may be applied at a strength level that is insufficient to separate binding members from each other (while dislodging particles bound to the surface by nonspecific interactions) and then gradually increased to the point where the surface-bound binding member and the particle-bound binding member separate and the particles become mobile on the surface. By this same method, the binding strength of different compounds can be measured and compared simultaneously by providing a surface having the different binding members attached to spatially distinguishable areas and by observing on which areas of the surface the particles remain bound as the strength of the ultrasonic force is increased. It is also possible to measure the binding strength of different compounds simultaneously by attaching each different compound to a different distinguishable class of particle and then observing which classes of particles remain bound as the strength of the ultrasonic force is increased.
Therefore, in one aspect, a device and method are provided to measure the binding forces of a first binding member with a second binding member. In this embodiment, a surface is provided that has a first binding member attached thereto, and one or more particles are provided that have a second binding member attached thereto. A reaction vessel is provided for exposing the surface to the particles whereby, if the first binding member has a binding affinity for the second binding member, a complex is formed between individual first binding members and individual second binding members and the particles thereby become immobilized with respect to the surface. An ultrasonic force means is operatively disposed with respect to the surface for applying a variable ultrasonic force onto the surface and a means is provided for monitoring the position of the particles with respect to the surface, particularly as the intensity of the ultrasonic force is varied, so that the intensity level at which the complex breaks can be noted. In an alternative embodiment, the surface can include spatially addressable subregions, with each subregion having a different surface-bound binding member attached thereto. This embodiment of the device can be used to measure the binding forces of a plurality of different surface-bound binding members. In another alternative embodiment the binding forces of a plurality of different particle-bound binding members may be measured by attaching each type of binding member to a different distinguishable class of particle.
It is also an object of the present invention to provide a binding assay (that is, an assay using binding interactions to determine the presence or absence of an analyte) wherein false positive results are minimized by removing labeled molecular entities that are not bound by specific binding interactions.
It has also been discovered that ultrasonic force can be used in a binding assay to dislodge and remove labeled compounds that adhere nonspecifically to a surface or that become bound due to cross-reactivity with an analog of an analyte. By the dislodging and removal of labeled compounds that are not bound by specific binding reactions, false positive results can be greatly reduced and the sensitivity of a binding assay can be improved.
Therefore, according to another aspect, the present invention is an assay device and method for detecting the presence or amount of an analyte in a test sample. In this embodiment, a surface is provided that has immobilized binding members that bind specifically to an analyte attached thereto, a reaction vessel for exposing the surface to the test sample, and a labeled reagent that, when contained in the test sample and exposed to the surface, becomes immobilized with respect to the surface specific ally in relation to the amount of the analyte in the test sample. An ultrasonic force means is operatively disposed with respect to the surface for applying an ultrasonic force onto the surface for dislodging any of the labeled reagent that binds non-specifically to the surface or that becomes immobilized on the surface of the surface due to cross-reactivity with an analog of the analyte, and a means is provided for detecting the amount of labeled reagent that remains immobilized with respect to the surface after the ultrasonic force is applied. In one embodiment, the labeled reagent of the assay device is in the form of a plurality of particles that have second binding members attached thereto, wherein the second binding members are capable of undergoing a selective binding interaction in relation to the amount of the analyte in the test sample, and the assay device includes a means to observe the particles during and after the application of the ultrasonic force. In an alternative embodiment, the surface can include spatially addressable subregions, with each subregion having a different surface-bound binding member attached thereto so that a plurality of analytes can be detected simultaneously in one assay.
It has also been discovered that ultrasonic force may be used in a xe2x80x9ctwo-beadxe2x80x9d assay, (that is, an assay using binding interactions between two or more types of particles to determine the presence or absence of an analyte). In this embodiment, ultrasonic force is used to dislodge particles that bind to each other by nonspecific binding.