Chemistry: analytical and immunological testing – Involving an insoluble carrier for immobilizing immunochemicals
Reexamination Certificate
1998-03-20
2001-06-26
Chin, Christopher L. (Department: 1641)
Chemistry: analytical and immunological testing
Involving an insoluble carrier for immobilizing immunochemicals
C385S012000, C385S015000, C385S025000, C385S123000, C385S129000, C385S130000, C422S051000, C422S051000, C422S082050, C422S082080, C422S082110, C435S006120, C435S007900, C435S287100, C435S287200, C435S287900, C435S288700, C435S808000, C436S164000, C436S172000, C436S510000, C436S524000, C436S527000, C436S065000, C436S805000, C436S814000, C436S817000, C436S818000
Reexamination Certificate
active
06251688
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an apparatus and a method for measuring binding between two molecular components resembling biologically active molecules or fragments thereof, such as and without limitation, binding between a specific protein and a specific nucleotide or G-protein. In one embodiment, the affinity of said binding is modulated by the binding of one molecular component to a third molecule. It is the sense of the present invention that said third molecule does not compete with binding between the first two components, but rather said third molecule induces a change in one of the first two components which results in a change in affinity between said molecular components. In another embodiment, measurement of binding between a protein component and its intended DNA response element is in itself of value. The apparatus and method of the present invention are of a type known by those skilled in the art as evanescent sensor fluorometry, and represents an improvement upon the apparatus and method described in U.S. patent application Ser. No. 08/616,576 entitled Surface Treatment and Light Injection Method and Apparatus which is assigned to the assignee of the present invention. As such, the apparatus described in the present invention can be equally well applied to immunoassay, which was the application toward which the apparatus of the previous patent application was directed.
The method of the present invention has particular relevance to study of the effect of certain test compounds, such as and without limitation, hormone mimics, on biological signal transduction which is mediated by binding of biological receptors and/or regulatory molecules to subsequent molecules such as and without limitation DNA molecules, involved in the transduction mechanism. The word “receptor” is defined for purpose of this invention according to the definition appearing in
Illustrated Dictionary of Immunology,
edited by Julius M. Cruse and Robert E. Lewis and published by CRC Press, Boca Raton, 1995, p.258, ISBN 0-8493-4557-X: “A molecular configuration on a cell or macromolecule that combines with molecules that are complementary to it.” The term “regulatory molecule” is defined “a molecule which, upon binding to a specific complementary molecule, initiates a sequence of events resulting in regulation of a biological process.”
In a first embodiment, the apparatus and method utilizing the principles of the invention are adapted for use as a screening tool for recognizing the presence of estrogen mimics in a sample. In a second embodiment the apparatus and method utilizing the principles of the invention are adapted for measuring estrogen receptor content in a tissue biopsy sample and evaluating in vitro the probable response of cancer cells, of a type present in that tissue biopsy sample, to certain pharmacologic agents which act through receptor binding. In a third embodiment, the apparatus and method utilizing the principles of the invention are adapted for evaluating the competency of the p53 protein present in a tissue sample.
2. Background to the Invention
Many biological processes are regulated by the binding of regulatory molecules such as hormones, neurotransmitters or cytokines to specific biological receptor molecules. Upon binding to the regulatory molecule, the receptor activates the next step in a signal transduction mechanism by itself binding to another molecular component of the transduction mechanism such as a nuclear response element or G-protein. The affinity with which this second stage of receptor binding occurs, or in some cases, whether or not this second stage binding occurs at all, is affected by the binding of the regulatory molecule to the receptor. A review of such mechanisms can be found in an article entitled “Mechanisms of Signal Transduction: Sex Hormones, Their Receptors and Clinical Utility” by James L. Wittliff and Wolfgang Raffelsberger, which appeared in
Journal of Clinical Ligand Assay,
Volume 18, Number 4, Winter, 1995. This text is fully and completely incorporated herein by reference, word for word and paragraph for paragraph.
There are many benefits which derive from the study of both the binding of receptors to regulatory molecules and the second stage binding of the receptors to another component of the signal transduction mechanism. Such study can assist in the design of drugs which exert their biological effect through binding to biological receptors. It can also lead to recognition of compounds in the environment which have the capacity to disrupt important biological regulatory mechanisms by virtue of the ability of such molecules to bind to molecular receptors. It is believed that the presence of such molecules in the environment plays a role in the development of a variety of disease types including cancer, immune dysfunctions, and reproductive problems.
Current methods used for studying these binding phenomena are described in the previously cited review. Because the methods require physical separation of bound from unbound molecules, the methods are quite time consuming and do not have the capacity to provide real-time data while binding is occurring between a receptor and a regulatory molecule or between receptors and another component of the signal transduction mechanism. The reliance of current methods on radiolabeled ligands also limits the circumstances under which such measurements can be made. The apparatus and method of the present invention overcomes the limitations of the prior art by removing the need to separate bound from unbound molecules prior to performing a measurement, with the consequence that real time binding between components can be monitored and association and dissociation constants and equilibrium constants can be calculated far more quickly and easily.
The apparatus of the invention is a type of evanescent fiber optic sensor. Evanescent fiber optic sensors provide a method whereby a molecule bearing a fluorescent tag can be directly monitored as it binds to a binding partner attached to an optical fiber. Light traveling through an optical fiber at or near the critical angle is totally internally reflected so that it does not excite fluorescence in the surrounding solution. Total internal reflection does, however, produce an evanescent field which extends about 1000 angstroms from the surface of the fiber. This means that fluorescence of molecules binding to the surface of the fiber can be excited without exciting fluorescence of unbound molecules in the surrounding solution. Therefore measurement of binding can be made without the necessity for physical separation of bound from unbound molecules. Evanescent sensors based upon measurement at a certain time of fluorescent antigen bound to antibodies on the fiber have been used to perform immunoassays by calculating concentrations of antigen in a solution. These have been reported in literature and patents and are thoroughly described in the book
Biosensors with Fiber Optics,
Donald L. Wise and Lemuel B. Wingard, Jr. Editors; Humana Press, Clifton, N.J., 1991. This text is fully and completely incorporated herein by reference, word for word and paragraph for paragraph. The immunoassay-based evanescent sensors of the prior art do not utilize data collected continuously by the sensor over a time period to perform the assay. Rather a single point in time is defined for taking a single measurement from the sensor and a standard curve is prepared relating such single point measurements to concentration of antigen in the solution. The prior art is therefore directed toward assay of a specific compound in a sample rather than assessment of the kinetic and binding parameters describing the interaction between a component in the sample and a component attached to the sensor waveguide surface.
3. Background to the Evanescent Sensor Apparatus of the Invention
The essential feature of an evanescent biosensor, is confinement of the measurement area to the surface of the waveguide by taking advantage of the evanescent fiel
Downward James G.
Erb Judith L.
Erb-Downward John R.
Ulrich Otho
Chin Christopher L.
IA, Inc.
Kohn & Associates
LandOfFree
Method and apparatus for measurement of binding between a... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method and apparatus for measurement of binding between a..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method and apparatus for measurement of binding between a... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2466603