Methods of culturing and characterizing antibody secreting cells
This disclosure relates to methods of culturing and characterizing antibody secreting cells. In certain embodiments, this disclosure relates to methods of isolating antibody secreting cells, e.g., long lived plasma cells, replicating the isolated cells in growth media disclosed herein, and determining the nucleic acids sequences in the cells that encode the produced antibodies.
1. A method of identifying cells that produce antibodies that bind to an antigen comprising
isolating antibody secreting cells from a sample, providing isolated single antibody secreting cells;
mixing the isolated antibody secreting cells with secretions of allogeneic mesenchymal stromal/stem cells and exogenously added A-proliferation-inducing ligand (APRIL) under conditions such that isolated antibody secreting cells replicate providing replicated antibody secreting cells; and
identifying antibodies that specifically bind to the antigen being secreted from the cells indicating the presence of antigen binding antibody secreting cells in the sample.
2. The method of claim 1 wherein mixing the isolated antibody secreting cells with secretions of allogeneic mesenchymal stromal/stem cells and exogenously added A-proliferation-inducing ligand (APRIL) is under hypoxic conditions.
3. The method of claim 1 , wherein the sample is blood, bone marrow, or product derived therefrom.
4. The method of claim 1 , wherein the antibody secreting cells have a cell surface profile of CD19(−), CD38(hi), and CD138(+).
5. The method of claim 1 , wherein the antibody secreting cells have a cell surface profile of CD19+, CD27hi, and CD38hi.
6. The method of claim 1 , wherein the antigen is a vaccine antigen or autoimmune antigen.
7. A method of producing a hybridoma that produces antibodies that bind to an antigen comprising:
isolating antibody secreting cells from a sample providing separated single antibody secreting cells in a plurality of separate areas,
mixing the separated single antibody secreting cells with secretions of allogeneic mesenchymal stromal/stem cells and exogenously added A-proliferation-inducing ligand (APRIL) under conditions such that separated single cells replicate providing replicated homogenous antibody secreting cells; and
identifying homogenous antibody secreting cells that produce antibodies that specifically bind to the antigen; and
fusing a homogenous antibody secreting cell identified to produce antibodies that specifically bind the antigen with an immortalized cell providing hybridoma cells that produce antibodies that specifically bind to the antigen.