Isolating cells expressing secreted proteins
A method of detecting and isolating cells that produce a secreted protein of interest (POI), for example, an antibody, comprising: a) providing a eukaryotic cell comprising (i) a nucleic acid encoding the POI, and (ii) a nucleic acid encoding a cell surface capture molecule, which comprises a membrane anchor and is capable of binding the POI; (b) culturing the cell under conditions in which the POI and cell surface capture molecule are expressed, and a POI-cell surface capture molecule complex is formed intracellularly and displayed on the cell surface; c) detecting the surface-displayed POI by contacting the cells with a detection molecule, which binds the POI; and d) isolating cells based on the detection molecule.
1. A method of detecting or isolating a eukaryotic cell that produces an antibody, comprising:
(a) transfecting an eukaryotic cell with (i) a nucleic acid encoding an antibody heavy chain, (ii) a nucleic acid encoding an antibody light chain, and (iii) a nucleic acid encoding a cell surface capture molecule, which comprises a membrane anchor and is capable of binding the antibody;
(b) culturing the cell under conditions in which the antibody and cell surface capture molecule are expressed, and an antibody-cell surface capture molecule complex is formed intracellularly and displayed on the cell surface;
(c) contacting the cell with a detection molecule, which binds to the antibody; and
(d) detecting or isolating the cell.
2. The method of claim 1 , wherein the cell is detected in step (d) by flow cytometry.
3. The method of claim 1 , wherein the membrane anchor is a transmembrane anchor or a GPI link.
4. The method of claim 1 , wherein the detection molecule comprises two molecules that bind each other and are differentially labeled.
5. The method of claim 1 , wherein the detection molecule is linked to a solid support or particle.
6. The method of claim 1 , further comprising contacting the cell with a blocking molecule that binds the cell surface capture molecule or the antibody to block the adherence of the antibody secreted from the expressing cell to a neighboring cell.
7. The method of claim 1 , wherein the nucleic acid encoding the cell surface capture molecule is introduced into the cell before the nucleic acid encoding the antibody heavy chain and the nucleic acid encoding the antibody light chain.
8. The method of claim 7 , wherein the nucleic acid encoding the cell surface capture molecule is stably integrated in the cell.
9. The method of claim 7 , wherein the nucleic acid encoding the antibody heavy chain and the nucleic acid encoding the antibody light chain are introduced into the cell simultaneously.
10. The method of claim 9 , wherein the nucleic acid encoding the antibody heavy chain and the nucleic acid encoding the antibody light chain introduced into the cell are included in the same vector or plasmid.
11. The method of claim 9 , wherein the nucleic acid encoding the antibody heavy chain and the nucleic acid encoding the antibody light chain introduced into the cell are included in different vectors or plasmids.
12. The method of claim 1 , wherein the nucleic acid encoding the cell surface capture molecule is introduced into the cell after the nucleic acid encoding the antibody heavy chain and the nucleic acid encoding the antibody light chain.
13. The method of claim 1 , wherein the nucleic acid encoding the cell surface capture molecule is introduced into the cell simultaneously with the nucleic acid encoding the antibody heavy chain and the nucleic acid encoding the antibody light chain.