High throughput cell-based assay kits
First Claim
Patent Images
1. A method of detecting a marker protein of a cell, comprising the steps of:
- (a) culturing the cell on a filter plate, wherein the filter plate has;
(i) a low background phosphorescence; and
(ii) pores which permit the cell to be cultured on the filter plate and to be washed in situ;
(b) contacting the cell with a reagent comprising;
(i) a ligand which specifically binds to the marker protein; and
(ii) a lanthanide chelate comprising a lanthanide ion and a chelating agent, wherein the chelating agent is bound to the ligand;
(c) washing the cell with vacuum assist to remove reagent which is not bound to the marker protein to provide a washed cell; and
(d) observing the washed cell for a sufficient time to detect fluorescence associated with the lanthanide ion which is at least two-fold greater relative to any background fluorescence, wherein detection of the fluorescence associated with the lanthanide ion indicates the presence of the marker protein.
0 Assignments
0 Petitions
Accused Products
Abstract
A new assay is described that incorporates the time saving features of a lanthanide chelate-conjugated ligand and a filter plate on which cells can be both cultured and washed in situ with vacuum assist. Loss of cells during washing steps is significantly curtailed through the use of the cell-culture-compatible filter plates, and the high endogenous fluorescence backgrounds characteristic of cell-based assays is avoided by use of time-resolved fluorescence. The assay described herein permits the screening of large libraries of compounds and/or recombinant proteins for either toxicity or stimulation of differentiation.
42 Citations
43 Claims
-
1. A method of detecting a marker protein of a cell, comprising the steps of:
-
(a) culturing the cell on a filter plate, wherein the filter plate has;
(i) a low background phosphorescence; and
(ii) pores which permit the cell to be cultured on the filter plate and to be washed in situ;
(b) contacting the cell with a reagent comprising;
(i) a ligand which specifically binds to the marker protein; and
(ii) a lanthanide chelate comprising a lanthanide ion and a chelating agent, wherein the chelating agent is bound to the ligand;
(c) washing the cell with vacuum assist to remove reagent which is not bound to the marker protein to provide a washed cell; and
(d) observing the washed cell for a sufficient time to detect fluorescence associated with the lanthanide ion which is at least two-fold greater relative to any background fluorescence, wherein detection of the fluorescence associated with the lanthanide ion indicates the presence of the marker protein. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 28, 42, 43)
-
-
23. A method of detecting a marker protein of a cell, comprising the steps of:
-
(a) culturing the cell on a filter plate, wherein the filter plate has;
(i) a low background phosphorescence; and
(ii) pores which permit the cell to be cultured on the filter plate and to be washed in situ;
(b) contacting the cell with a first ligand which specifically binds to the marker protein;
(c) washing the cell with vacuum assist to remove first ligand which is not specifically bound to the marker protein;
(d) contacting the cell with a reagent comprising;
(i) a second ligand which specifically binds to the first ligand; and
(ii) a lanthanide chelate comprising a lanthanide ion and a chelating agent, wherein the chelating agent is bound to the second ligand;
(e) washing the cell with vacuum assist to remove reagent which is not specifically bound to the first ligand to provide a washed cell; and
(f) observing the washed cell for a sufficient time to detect fluorescence associated with the lanthanide ion which is at least two-fold greater relative to any background fluorescence, wherein detection of the fluorescence associated with the lanthanide ion indicates the presence of the marker protein. - View Dependent Claims (24, 25, 26, 29)
-
-
27. A test kit, comprising:
-
(a) a ligand for a marker protein;
(b) a source of lanthanide ion; and
(c) a filter plate suitable for culturing cells, having;
(1) a low background phosphorescence; and
(2) pores which permit cells to be cultured on the filter plate and to be washed in situ. - View Dependent Claims (30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41)
-
Specification