Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2002-05-28
2004-10-19
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007210, C435S007900, C435S188000, C435S287200, C436S063000, C436S532000, C436S823000
Reexamination Certificate
active
06806058
ABSTRACT:
BACKGROUND
The gel microdrop (GMD) secretion assay involves encapsulating cells in a biotinylated matrix, followed by capture and detection of cell-secreted molecules with fluorescent markers (17-34). This technology differs from other encapsulation methods in that the small size of the microdrop (e.g., <50 &mgr;m diameter) creates a defined microenvironment around the cell without impeding diffusion of nutrients, antibodies, or nucleic acid probes into the microdrops, or diffusion of secreted products. Furthermore, microdrops can readily be analyzed using flow cytometry and sub-populations can be detected. The number of occupied cells in each microdrop preparation depends on the number of cells used for encapsulation and is approximated by Poisson statistics for single cell encapsulation (19). To obtain microdrops having a high probability of initially containing 0 or 1 cells, an experimental protocol has been developed in which 1-1.5 million cells are encapsulated in 20 million microdrops, resulting in approximately 5-10% single cell occupation . . . . The emulsion is transiently cooled, causing the drops to gel. Once gelled, the microdrops are physically distinct and robust and can be removed from the oil into an aqueous medium by low speed centrifugal separation. Since the microdrop agarose matrix is a permeable semi-solid support, immunochemical procedures can be performed on encapsulated cells.
SUMMARY OF THE CLAIMED INVENTION
The invention provides methods of analzying a secreted protein. Such method entail encapsulating a cell in a microdrop wherein the microdrop comprises matrix component molecules, first biotin molecules linked to the matrix component molecules, capture molecules with affinity for a molecule secreted by the cell linked to second biotin molecules, and streptavidin linking the first and second biotin molecules, the first biotin molecules and the matrix component molecules being in a molar ratio of less than 0.85, preferably 0.01 to 0.2, and optionally 0.02-0.2 moles biotin per mole matrix component molecules. The molecule is secreted from the cell and binds to the capture molecules is thereby retained within the microdrop. The secreted molecule is then detected. In some methods the concentration of the first biotin molecules in the microdrop is less than or equal to 42 micromolar.
In some methods, the encapsulating step encapsulates a plurality of cells in the microdrop. In some methods, the encapsulating step encapsulates a single cell in the microdrop. In some methods, the secreted molecule is a protein, hormone, or carbohydrate. In some methods, the streptavidin and capture molecules are encapsulated into the microdrops at the same time as the cell. In some methods, the streptavidin and captured molecules are incorporated into the microdrop after the encapsulating step. In some methods, the microdrop further comprises second capture molecules with affinity for a second secreted molecule, the second capture molecules being linked to additional copies of the second biotin molecules. In some methods, the cell secretes the second molecule. In some methods, the detecting step is performed by contacting the microdrop with a detection reagent having affinity for the secreted molecule, and detecting binding of the detection reagent to the secreted molecule. In some methods, the detection reagent is labeled. In some methods, a signal of the labeled detection reagent bound to the secreted molecule is proportional to the number of copies of the secreted molecule within the microdrop. In some methods, the detecting step is performed by contacting the microdrop with a first detection reagent having specific affinity for the secreted molecule and a second detection reagent having specific affinity for the second secreted molecule, wherein the first and second detection reagents are differentially labeled. In some methods, the cell secretes a third secreted molecule, and the microdrop further comprises third capture molecules with affinity for the third secreted molecule, the third capture molecules being linked to additional copies of the second biotin molecules, and the method further comprises contacting the microdrop with first, second, third and fourth detection reagents having specific affinity for the secreted molecule, the second secreted molecule, the third secreted molecule and a cell surface marker respectively, and the detecting step detects the secreted protein, the second secreted protein, the third secreted protein and the cell surface marker. In some methods, the detection step is performed by contacting the microdrop with a first detection reagent having affinity for the secreted molecule and a second detection reagent having affinity for a cell surface marker, and the first and second detection reagents are differentially labeled. In some methods, the detection reagent and the capture molecules bind to different epitopes on the secreted molecule. In some methods, the matrix component is agarose. In some methods, the secreted protein is an antibody. In some methods, the secreted protein is an antibody of IgG isotype and the capture molecules are antibodies specific for the IgG isotype. In some methods, the secreted protein is a cytokine. Some methods further comprise inducing the cell to secrete the secreted molecule. In some methods, the inducing is performed after the encapsulating step. In some methods, the inducing is performed before the encapsulating step.
In some methods, the cell comprises a vector comprising a nucleic acid segment encoding the secreted protein, the segment being operably linked to one or more regulatory DNA segments that effect expression of the secreted protein. In some methods, the secreted protein is naturally secreted by the cell. Some methods further comprise a step of propagating the cell to form a cell line after the detecting step. In some methods, the cell was obtained from a patient. In some methods, the cell is a cytotoxic T-cell. Some methods further comprise introducing a population of cells resulting from propagating the cell into the patient. In some methods, the cell is a stem cell. In some methods, the patient is suffering from an autoimmune disease and the cell is a Th2 cell. In some methods, the cell is an islet cell secreting insulin and the patient is in a prodromal period prior to onset of clinical symptoms. Some methods further comprise treating the cell with IL-10 during the propagating step. Some methods further comprise separating the cells from other cells using a cell sorter based on a fluorescent signal resulting from binding of a fluorescently labeled detection reagent to the secreted molecule. In some methods, the cell secretes first and second proteins, and the matrix comprises first and second capture molecules with affinity for the first and second proteins respectively, and the analyzing step comprises contacting the cell with first and second detection reagents that bind to the first and second secreted proteins, and detecting the first and second proteins from signal of the first and second detecting reagents bound to the first and second secreted proteins.
The invention provides methods of analyzing a population of cells. Such methods entail encapsulating a population of cells in microdrops. The cells are contacted with a first detection reagent for a first marker, and a second detection reagent for a second marker, wherein the first marker is a secreted protein and the second marker is a cell surface protein or a second secreted protein. Cells having both the first and second markers are detected. In some methods, at least some microdrops encapsulate single cells. Some methods further comprise separating the microdrops encapsulating the cells from unoccupied microdrops on a Percoll, polysucrose, sodium diatrizoate, or iodixanol gradient. Some method further comprise forming an array of microdrops encapsulating the cells attached to a solid support, and wherein the detecting is performed using a scanning fluorescent, colorimetric, chemiluminescent detector. Some methods further co
Akselband Yevgenya
Jesperson Diana
McGrath Patricia
Moen Phillip T.
Trnovsky Jan
Cheu Jacob
Le Long V.
One Cell Systems, Inc.
Townsend and Townsend / and Crew LLP
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