Ultrasonic force differentiation assay

Chemical apparatus and process disinfecting – deodorizing – preser – Process disinfecting – preserving – deodorizing – or sterilizing – Using sonic or ultrasonic energy

Reexamination Certificate

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C134S001000, C134S184000, C180S020000, C180S002100, C180S193000, C180S193000, C204S157420, C204S157620, C209S214000, C210S222000, C210S223000, C210S695000, C422S062000, C422S069000, C422S091000, C422S105000, C422S128000, C422S236000, C436S518000, C436S526000, C436S528000, C436S534000, C436S536000, C436S806000, C436S824000

Reexamination Certificate

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06368553

ABSTRACT:

BACKGROUND OF THE INVENTION
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, 3
rd
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, “Energy Landscapes of Receptor-Ligand Bonds Explored with Dynamic Force Spectroscopy”. Nature, Vol. 397 (1999), pp. 50-53, in Bell, G. I., “Models for the Specific Adhesion of Cells to Cells”, Science, 200, (1978), pp 618-627, and in Evans, E. et al, “Dynamic Strength of Molecular Adhesion Bonds”, 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, “Dynamic Strength of Molecular Adhesion Bonds”, 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, “Adhesion Forces Between Individual Ligand-Receptor Pairs” Science 264 (1994). pp 415-417; Lee, G. U et al, “Sensing Discrete Streptavidin-Biotin Interactions with Atomic Force Microscopy” Langmuir, vol. 10(2), (1994) pp 354-357; Dammer U. et al “Specific Antigen/Antibody Interactions Measured by Force Microscopy” Biophysical Journal Vol. 70 (May 1996) pp 2437-2441; Chilikoti A. et al, “The Relationship Between Ligand-Binding Thermodynamics and Protein-Ligand Interaction Forces Measured by Atomic Force Microscopy” Biophysical Journal Vol. 69 (November 1995) pp 2125-2130; Allen S. et al, “Detection of Antigen-Antibody Binding Events with the Atomic Force Microscope” Biochemistry, Vol.36, No.24 (1997) pp 7457-7463; and Moy V. T. et al, “Adhesive Forces Between Ligand and Receptor Measured by AFM” Colloids and Surfaces A: Physicochemical and Engineering Aspects 93 (1994) pp 343-348, and U.S. patent application Ser. No. 09/074,541 for “Apparatus and Method for Measuring Internolecular Interactions by Atomic Force Microscopy”, 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.

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