Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-03-20
2001-10-09
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C385S012000, C385S123000, C385S129000, C422S051000, C422S082050, C422S082080, C422S082110, C435S007900, C435S287100, C435S287200, C435S288700, C435S808000, C436S065000, C436S164000, C436S165000, C436S172000, C436S510000, C436S518000, C436S527000, C436S805000, C436S814000, C436S811000, C436S815000, C436S817000
Reexamination Certificate
active
06300082
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to an apparatus and a methodology for measurement of the effect of certain test compounds, such as and without limitation, hormone mimics, on biological signal transduction at the level of biological receptors and their binding 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.”
In one embodiment the apparatus and methodology 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 methodology utilizing the principles of the invention are adapted for use as a method 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. The fiber optic biosensor described herein provides information which reflects the biological impact of the sample. It also has the potential for elucidating mechanisms of receptor action in that the sensor provides a means of obtaining kinetic information on receptor binding events as the binding process occurs.
2. Background of 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. 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, 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.
Evanescent fiber optic sensors provide a method whereby a plurality of molecules of interest bearing an optical tag can be directly monitored as they bind to binding partners attached to an optical fiber. An optical tag may comprise molecules belonging to that class of chemicals which interact with light in a manner so as to alter a characteristic of light received from said sensor by means such as and without limitation, absorbance, fluorescence, luminescence, or polarization; or which produces a second chemical which interacts with light in said manner, such as and without limitation, an enzyme having action producing or destroying a fluorescent, absorbing, luminescent or polarizing compound. The molecular tag may be chemically attached to said plurality of molecules or it may be chemically attached to a plurality of a second type of molecule such as and without limitation, an antibody, having affinity for said molecule of biological interest. Light traveling through an optical fiber at or near the critical angle is totally internally reflected so that it does not significantly effect unbound tagged molecules 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 a response from molecules bound to the surface of the fiber can be excited by light totally internally reflected within the fiber, without exciting a response from 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 which measure concentrations of antigen in a solution based upon measurement at a certain time of an amount of fluorescent antigen in the sample which is bound to antibodies on the fiber. Such sensors have been reported in literature 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 use of an evanescent sensor having the membrane receptor for acetyl choline bound to its surface to explore the binding of cholinergic ligands has been previously reported by Kim B. Rogers, J. J. Valdes, and Mohyee E. Elderfrawi in an article entitled
Acetylcholine receptor fiberoptic evanescent fluorosensor
, appearing in the Nov. 1, 1989 issue of Analytical Biochemistry,
182
(2):353-9. Membrane receptors for neurotransmitters function differently than receptors for hormones in that membrane receptors do not utilize subsequent binding to a nuclear response element in signal transduction. The transduction process for membrane receptors involves subsequent alteration in transport of ions through channels in the membrane. Use of hormone receptors as reagents in conjunction with an evanescent sensing apparatus possessing a feature resembling a nuclear response element for said hormone receptor has not, to our knowledge, been previously described.
3. Background of the Estrogen Receptor Embodiment
A general background is provided which pertains to the specific embodiment employing the estrogen receptor and the estrogen response element as a means of testing compounds for estrogenic or anti-estrogenic activity. This embodiment is described for the specific application of identification of environmental estrogen mimics and for the specific application of in vitro assessment of the probable efficacy of specific anti-estrogenic compounds, such as and without limitation, tamoxifen, in the treatment of cancer.
The Problems Posed by Estrogen Mimics
During the past 50 years there. has been a marked increase in the number of abnormalities relating to the human reproductive system. Endometriosis, once thought to be a rare condition, has become the most common diagnostic entity in gynecology today. The incidence of female breast cancer is now 1 in 8. A retrospective analysis of data since the 1940's revealed that the sperm count of
Downward, IV James G.
Erb Judith L.
Erb-Downward John R.
Wittliff James L.
Chin Christopher L.
IA, Inc.
Kohn & Associates
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