Electricity: measuring and testing – Particle precession resonance – Spectrometer components
Reexamination Certificate
1999-11-02
2001-10-23
Arana, Louis (Department: 2862)
Electricity: measuring and testing
Particle precession resonance
Spectrometer components
C422S067000
Reexamination Certificate
active
06307372
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to the field of chemical screening, and in particular to the screening of various chemicals for biological or other activity. More specifically, the invention relates to assays where nuclear magnetic resonance is employed as a screening tool.
Modern chemistry has proven to be effective in creating a wide assortment of chemicals that may be useful for a variety of applications including drug therapy, industrial manufacturing, painting, and the like. A recent development in the field of modern chemistry is that of combinatory chemistry where vast libraries of chemical compounds may rapidly be created. For example, combinatorial chemistry techniques are described in U.S. Pat. No. 5,503,805, the complete disclosure of which is herein incorporated by reference.
One significant challenge for the modern chemist is the ability to find useful applications for the vast libraries of chemicals now being created. For example, the drug discovery industry is currently expending significant resources to identify chemicals that may be used as drugs to treat a wide variety of ailments. In order to determine whether a chemical will be useful for a particular application, the chemical needs to be tested in a particular environment to determine if the chemical is active under certain conditions, often referred to as screening. This can be particularly challenging if the number of chemicals to be screened is large. For example, Wilhelm Stahl, “What is the Future of High Throughput Screening?”
Journal of Biomolecular Screening,
Volume 4, Number 3, pp. 117-118, 1999; Anthony M. Maffia III et. al, “Miniaturization of a Mammalian Cell-Based Assay: Luciferase Reporter Gene Readout in a 3 Microliter 1536-Well Plate”,
Journal of Biomolecular Screening,
Volume 4, Number 3, pp. 137-142, 1999; and Laura Abriola et al., “Digital Imaging as a Detection Method for a Fluorescent Protease Assay in 96-Well and Miniaturized Assay Plate Formats”,
Journal of Biomolecular Screening,
Volume 4, Number 3, pp. 121-127, 1999, the complete disclosures of which are herein incorporated by reference, describe the challenges associated with the demand to increase high throughput screening.
Various types of assays have been developed to screen chemicals for activity at the cellular level. For example, some assays that have been utilized include cell reporter assays, such as lawn assays, direct binding assays, and the like. Further, various types of chemical handling devices and automated equipment have been developed to increase the throughput or efficiency of the screening process. For example, many assays utilize multiwell plates, such as standard 96 well plates where 96 chemicals may be screened in parallel. Other equipment used to facilitate such assays includes robots for plate and chemical handling, plate readers employing CCD cameras, and the like. Merely by way of example, useful apparatus and equipment are described in U.S. Pat. No. 5,722,470 and in copending U.S. patent application Ser. No. 08/834,803, filed Apr. 3, 1997, the complete disclosures of which are herein incorporated by reference.
Even so, there is still a need for other techniques to screen for biological or other activity in a high throughput manner, particularly in view of the large chemical libraries now being created. Further, a need also exists for new assays and associated screening techniques that may be used to screen chemicals for certain types of activity. Hence, the invention is related to alternative assays and screening techniques that are particularly adapted for high throughput screening and/or for providing alternative avenues to screen for certain types of activity.
SUMMARY OF THE INVENTION
The invention utilizes nuclear magnetic resonance to evaluate a wide variety of assays. In one embodiment, a screening method is provided where an assay is performed where one or more chemicals and/or entities are present in a mixture to produce an outcome. A static magnetic field B
0
is applied to the mixture along with one or more RF magnetic pulses. Resulting FID or echo signals are measured and evaluated to evaluate the outcome of the assay. In this way, nuclear magnetic resonance may be employed to evaluate the outcome of a wide variety of assays.
In one aspect, the assay is performed in a plate having a plurality of wells. The mixture in each well includes a different chemical and/or entity, and spatially dependent B
0
gradient fields are applied to the wells. FID or echo signals are then measured and evaluated from each of the wells. In this way, multiple outcomes may be evaluated using nuclear magnetic resonance in a high throughput manner. Optionally, the wells may be arranged in a three-dimensional array to increase the throughput of the screening procedure. For example, multiple plates may be stacked on top of each other and then screened using nuclear magnetic resonance.
In another aspect, the entities in the mixture may comprise cells which each produce a different test product. Each test product may produce a detectable nuclear magnetic resonance signal that is dependent on the degree of interaction of the test compound with the mixture. For example, the cells may produce a test product with a chemical shift that is different from that of water protons. In another aspect, the assay proceeds by providing the mixture with a test compound that causes a detectable nuclear magnetic resonance signal to be produced to indicate whether the test compound is biologically active in the mixture.
In one particular aspect, a test compound may be combined with at least one cell in an aqueous medium. Biological activity of the test compound is indicated when the test compound binds to a receptor on the cell surface or within the cell. In so doing, an enzyme is produced that either directly or indirectly affects the relaxation properties of water protons to indicate that the test compound is biologically active. For example, the enzyme may modify the relaxation properties of water protons by cleaving a probe molecule in the mixture. In this way, the exposure of a paramagnetic atom in the probe molecule to water protons may be increased to thereby decrease the T1 relaxation rate of the water protons. As one specific example, the enzyme may comprise beta-galactosidiase, and the probe molecule may comprise a chelated gadolinium atom that is complexed with a galactose molecule. In this manner, the galactosidiase will cause the galactose molecule to be cleaved from the chelated gadolinium.
In another aspect of the method, a test compound may be provided in an aqueous medium and mixed with a chemical or entity that when reacting with the test compound changes the pH of the medium. The change in pH is measurable using nuclear magnetic resonance to indicate that the test compound is biologically active. For example, the change in pH of the medium may cause a change to the ionization of polymer molecules that are also in the medium. As a result, the viscosity of the medium is changed, thereby affecting nuclear magnetic resonance relaxation properties, spin density and/or the diffusion coefficient of the water protons of the aqueous medium. As another example, the change in pH of the medium may cause a change to the configuration of gel particles that are included within the medium. The configuration change of the gel particles affects nuclear magnetic resonance relaxation properties, spin density and/or the diffusion coefficient of the water protons of the medium. In one aspect, the medium may include a pH sensitive contrast agent that causes the nuclear magnetic resonance relaxation properties of water protons or other nuclei of the medium to be changed in a manner that is reflective of pH. In this way, a change in pH affects the contrast agent that in turn affects the nuclear magnetic resonance relaxation properties of water protons or other nuclei. pH sensitive contrast agents may contain lanthanide metals including Ce, Pr, Nd, Sm, Eu, Gd, Db, Dy, Ho, Er, Tm, or Yb, or other paramagnetic
Star-Lack Joshua M.
Sugarman Jeffrey H.
Arana Louis
Glaxo Wellcome Inc.
Townsend and Townsend / and Crew LLP
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