Secretions of proteins by encapsulated cells

• US 20050019839A1
• Filed: 08/13/2004
• Published: 01/27/2005
• Est. Priority Date: 05/26/2001
• Status: Active Grant

• Current Assignee: Once Cell Systems Incorporated
• Original Assignee: Once Cell Systems Incorporated
• Inventors: McGrath, Patricia, Akselband, Yevgenya, Jespersen, Diana, Trnovsky, Jan, Moen, Phillip T.
• Granted Patent: US 7,556,928 B2
• Time in Patent Office: Days
• US Class Current: 435/7.200
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/52 Constant or Fc region; Isotype
• CPC Class Codes: G01N 33/5432 Liposomes or microcapsules
• CPC Class Codes: G01N 33/5436 with ligand physically entrapped within the solid phase liposomes G01N33/5432; immunological test elements G01N33/54386
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/56972 White blood cells
• CPC Class Codes: Y10S 436/823 Immunogenic carrier or carrier per se

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.

Claims

1. 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.

2. The method of claim 1, wherein the molar ratio is 0.01 to 0.2.

3. The method of claim 1, wherein the molar ratio is 0.02 to 0.2.

4. The method of claim 1, wherein the concentration of the first biotin molecules in the microdrop is less than or equal to 42 micromolar.

5-84. -84. canceled.

85. 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.

86. The method of claim 85, wherein the cells are cultured in the presence of an agent that stimulates isotype switching.

87. The method of claim 85, wherein the capture reagent is an antibody to the IgG isotype.

88. The method of claim 85, wherein the detecting comprises contacting the microdrops with a detection reagent that specifically binds to the captured antibody at a different site than the capture reagent.

89. The method of claim 85, wherein the detection reagent is an anti-idiotypic antibody.

90. The method of claim 85, wherein the anti-idiotypic antibody is fluorescently labelled.

91. The method of claim 85, further comprising isolating the microdrop that has captured the cell secreting the IgG antibody;

92. The method of claim 85, wherein the population of cells are encapsulated in microdrops such that at least some microdrops encapsulate a plurality of cells;

and the method isolates a microdrop that has captured a plurality of cells, one or more of which secretes IgG antibody.

93. The method of claim 85, further comprising isolating the plurality of cells.

94. The method of claim 93, further comprising encapsulating the plurality of cells in microdrops under conditions whereby at least some microdrops encapsulate a single one of the plurality of cells, wherein the microdrops include a capture reagent specific for antibodies of IgG isotype, whereby microdrops containing a cell secreting an antibody of IgG isotype capture the secreted antibody within the microdrops;

detecting one or more microdrops containing a cell secreting an antibody of IgG isotype.

95. The method of claim 85, wherein the capture molecules are antigen molecules that specifically bind to the secreted antibody of IgG isotype.

96. 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.

97. The method of claim 96, further comprising repeating steps (c)-(e) at a further ratio of average number of cells per occupied microdrops to isolate a further sub-population of microdrops.

98. The method of claim 96, further comprising isolating a microdrop encapsulating a single cell from the second sub-population.

99. The method of claim 96, wherein the sub-population of cells having the desired property is less than 0.01% of the population of cells before the method is performed.

100-106. -106. canceled.