Secretions of proteins by encapsulated cells
AssigneeOne Cell Systems, Inc.
InventorsDiana Jesperson, Yevgenya Akselband, Patricia McGrath, Jan Trnovsky, Phillip T. Moen
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Abstract
The invention provides methods of analzying a secreted protein from a cell encapsulated in a microdrop. The microdrop is formulated with biotinylated matrix molecules at a reduced ratio of biotin to matrix molecules compared with previous formulations. The reduced ratio is advantageous for improving the resolution of detection and allows simultaneous detection of multiple secreted proteins and/or multiple cell surface markers. The invention further provides inter alia methods of isolating IgG isotype antibodies that have switched from IgM isotype.
Claims106
A method of analyzing a secreted protein, comprising:
- 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 moles biotin per mole matrix component molecules, wherein the molecule is secreted from the cell and binds to the capture molecules thereby being retained within the microdrop;
- and
- detecting the secreted molecule.
A method of analyzing a population of cells, comprising
- encapsulating a population of cells in microdrops;
- contacting the cells 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;
- detecting cells having both the first and second markers.
A method of analyzing a population of cells, comprising:
- encapsulating a population of cells expressing proteins in microdrops, and wherein the microdrops comprise matrix component molecules, first biotin molecules linked to matrix component molecules, at least first capture molecules having affinity for an epitope in at least one type of secreted protein;
- the first capture molecules being linked to second biotin molecules, and streptavidin, linking the first and second biotin molecules;
- whereby proteins are secreted from the cells and proteins having affinity for the first capture molecules are captured within the microdrops;
- analyzing the secreted proteins within the microdrops.
A population of microdrops encapsulating cells, and the microdrops comprise matrix component molecules, first biotin molecules linked to the matrix component molecules, capture molecules with affinity for a protein 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 molecules being in a molar ratio of less than 0.85 moles biotin per mole matrix component molecules.
In a method of analyzing a protein secreted by a cell in which the cell is encapsulated with a microdrop comprising biotinylated agarose the improvement wherein the molar ratio of biotin to agarose is less than 0.85 moles biotin per mole agarose.
A method of analyzing a secreted protein, comprising:
- encapsulating a cell in a microdrop wherein the microdrop comprises matrix component molecules, Ni2+NTA linked to the matrix component molecules, capture molecules with affinity for a molecule secreted by the cell linked to a hexahistidine tag, wherein the molecule is secreted from the cell and binds to the capture molecules thereby being retained within the microdrop;
- contacting the cell with a detection reagent that specifically binds to the secreted molecule wherein the detection reagent.
A method of analyzing a secreted protein, comprising:
- encapsulating a cell in a microdrop wherein the microdrop comprises matrix component molecules linked to biotin, streptavidin linked to Ni2+ and capture molecules with affinity for a molecule secreted by the cell linked to a hexahistidine tag, wherein the molecule is secreted from the cell and binds to the capture molecules thereby being retained within the microdrop;
- contacting the cell with a detection reagent that binds to the secreted molecule wherein the detection reagent.
A method of preparing an antibody of IgG isotype, comprising;
- culturing a population of cells secreting antibodies of IgM isotype under conditions whereby one or more of the cells can undergo isotype switching to IgG isotype;
- encapsulating the population of cells in microdrops including a capture reagent specific for antibodies of IgG isotype, whereby microdrops containing a cell secreting an antibody of IgG isotype capture the secreted antibody of IgG isotype within the cells;
- detecting one or more microdrops containing a cell secreting an antibody of IgG isotype.
A method of screening a population of cells for a sub-population having a desired property, comprising
- (a) encapsulating the population of cells in microdrops at a first ratio of average number of cells per occupied by microdrop;
- (b) screening the encapsulated cells to identify a first sub-population of microdrops encapsulated cells having the desired property;
- (c) isolating the cells from the microdrops resulting from the screening step;
- (d) encapsulating the cells from the screening step at a second ratio of average number of cells per occupied microdrop, the second ratio being smaller than the first ratio;
- (e) screening the encapulsated cells to identify a second sub-population of microdrops encapsulating cells having the desired property.
A kit for making microdrops, comprising of
- matrix component molecules linked to biotin, in a molar ratio of biotin to matrix molecules of less than 0.85 moles biotin per mole matrix component molecules
A kit for detecting an antibody of IgG isotype, comprising biotinylated matrix molecules
- a capture molecule that specifically binds to the isotypic region of an IgG antibody;
- a detection molecule that specifically binds to the IgG antibody at a different site than the capture molecule.
Dependencies are inferred from the claim text.