Chemistry: analytical and immunological testing – Optical result – With claimed manipulation of container to effect reaction or...
Reexamination Certificate
1999-12-23
2001-10-30
Beisner, William H. (Department: 1744)
Chemistry: analytical and immunological testing
Optical result
With claimed manipulation of container to effect reaction or...
C436S172000, C422S105000, C422S105000, C435S004000, C435S029000, C435S033000, C435S288400, C435S288700, C435S297500
Reexamination Certificate
active
06309889
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to the field of assays, and in particular to techniques to facilitate the performance of such assays in a high throughput manner. In one particular aspect, the invention relates to the organization of solid supports and/or organisms, such as cells, that are used in performing such assays.
The creation of large chemical libraries has led to the use of a wide variety of assays to evaluate such libraries. As is known in the art, such chemical libraries may be created using a variety of synthesizing techniques, such as those described in U.S. Pat. Nos. 5,503,805 and 5,708,153, the complete disclosures of which are herein incorporated by reference. Typically, such synthesizing processes utilize solid supports, such as small resin beads, onto which the chemicals are synthesized.
Because of the immense size of such libraries, it is desirable to find ways to evaluate the chemicals in a high throughput manner. However, this can be difficult due to the nature of many existing assay formats. For example, some assays involve the use of living mammalian cells or other organisms. This can be especially challenging because of the difficulty in handling and/or organizing the solid supports and the cells, as well as because of the need to provide nutrients to keep the cells viable.
Merely by way of example, one type of assay is a cell reporter assay utilizing cells that produce a signal upon activation of a receptor. Hence, if a chemical that is released from a solid support activates the receptor, a signal is produced. Standard detectors may then used to detect the signal. With such assays, the ability to screen large numbers of reactions while also maintaining cell viability can be especially challenging.
Another type of assay is a “bead marking” assay where libraries of compounds are released from beads and permitted to diffuse to inducible cells where they interact with target receptors and induce the cells to produce and secrete an enzyme. The enzyme diffuses back to the beads and “marks” them by metabolizing a substrate covalently attached to the beads. Such an assay is described generally in copending U.S. patent application Ser. No. 08/758,307, filed Mar. 12, 1996, now U.S. Pat. No. 5,958,703, the complete disclosure of which is herein incorporated by reference.
To enhance such an assay as a drug discovery tool, it would be desirable to minimize the number of falsely marked beads. For example, a bead can be falsely marked if it comes into physical contact with a cell. Another way is if a cell that was induced by a compound from a positive bead marks a bead with an inactive compound through excessive diffusion of a compound or enzyme.
Another group of assays that can pose challenges when utilized in a high throughput manner are competition assays that are based on the interference of the binding of two ligands. High throughput screening of such assays while maintaining cell viability can be difficult.
Hence, the invention is related to techniques to facilitate the performance of various assays that utilize solid supports and/or other organisms, such as cells, in an efficient manner. In this way, the invention provides for the screening of large chemical libraries in a high throughput manner using a wide assortment of assay formats.
SUMMARY OF THE INVENTION
The invention provides exemplary devices and methods to facilitate the performance of assays. In one embodiment, one such device comprises a holding member having a top surface, a bottom surface, and a plurality of holding locations that are adapted to hold one or more articles, such as one or more solid supports and/or cells. When within the holding locations, the articles are preferably disposed below the top surface. A membrane is positioned above the top surface of the holding member, and a pressure system is provided to apply positive pressure to the membrane to force the membrane against the top surface of the holding member. In this way, a seal may be provided between the holding locations, with the membrane being spaced apart from the articles.
In one aspect, the pressure system comprises a housing that defines a chamber sized to receive the holding member and the membrane. The housing has an inlet port to permit a fluid, such as a gas or a liquid, to be supplied into the chamber to force the membrane against the top surface of the holding member. In another aspect, the housing may further include an outlet port to permit fluids to be evacuated from the chamber.
In one particular aspect, the housing further comprises an upper window, a lower window, and a spacer between the upper window and the lower window. Further, the inlet port extends through the upper window, and the holding member is configured to fit within the spacer. With such a configuration, the membrane may be coupled to the top window so as to be spaced apart from the holding member until operation of the pressure system to force the membrane against the top surface. In this way, the holding member may be loaded with solid supports and then placed onto the lower window while being framed by the spacer. The upper window may then be placed onto the spacer, with the membrane being spaced apart from the holding member.
Conveniently, seals may be positioned between the upper window and the spacer and the lower window and the spacer. Also, a vent may be formed within the lower window. In one particular aspect, an upper frame may be disposed above the upper window and a lower frame may be disposed below the lower window. Further, at least one securing mechanism may be provided to secure the upper frame to the lower frame.
In one aspect, the upper and lower windows may be constructed of an acrylic plastic that permits the transmission of ultraviolet light down to at least 270 nm. In this way, chemicals on the solid supports may be released by ultraviolet photolysis while disposed within the device.
In yet another aspect, the holding mechanism comprises a plate, and the holding locations comprise an array of wells formed in the plate. Conveniently, the wells may define a volume in the range from about 0.1 nl to about 100 nl, and more preferably from about 1 nl to about 25 nl. In still another aspect, an organism is coupled to the membrane. For example, the organism may comprise mammalian cells, insect cells, plant cells, bacteria, yeast and the like. In another aspect, the membrane is selectively permeable, e.g., based on molecular weight. In this way, nutrients and gases may be provided to the organisms while the membrane is forced against the plate.
In another embodiment, the housing comprises an upper window having the inlet port and an outlet port, a spacer that is adapted to receive the holding member, and a lower plate having a vent. Conveniently, at least one clamping device may be provided to clamp the spacer between the upper window and the lower plate. In one aspect, the housing may have a thickness that is less than about 1.5 cm to permit the device to be used with a fluorescence or a luminescence microscope. However, it will be appreciated that the devices may be constructed to be larger depending on the objective lens of the microscope.
The invention further provides an exemplary method for organizing items, such as solid supports, cells and the like. According to the method, items that each have at least one associated chemical or biological component (such as a cell that is configured to produce and secrete an enzyme) are placed into a plurality of holding locations within a holding member such that the items are disposed below a top surface of the holding member. A membrane is then forced onto the top surface of the holding member to cover the holding locations and to provide seal between the holding locations. In this way, the membrane is kept spaced apart from the items while still providing a seal to prevent or reduce the chances of cross contamination between the holding locations.
Depending on the nature of the assay to be performed, each holding location may receive a single item or multip
Cutler Thomas A.
Kelly Andrew J. G.
Lalonde Guy
Wagstrom Christopher R.
Beisner William H.
Glaxo Wellcome Inc.
Townsend and Townsend / and Crew LLP
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