Expert: Shweta Mishra - 6/15/2009

Question
outline the hybridoma method for the production of a monoclonal antibody. Thanks a lot

Answer
1- The mice is immunized with the antigen against which the antibody is to be produced. When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.

2- The antibody producing spleen B cells are then fused with myeloma cells to form HYBRIDOMAS (poyethylene glucol is used to fuse cells) which are immortal and hence are capable of producing antibodies indefinitely.
Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion. A week before cell fusion, myeloma cells are grown to 8-azuguanine. Cells must have high viability and rapid growth. The HAT medium allows only the fused cells to survive in culture.

3- Once the hybridomas have grown on the selection medium, this is followed by growing the hybridomas further by tissue culture methods using feeder cells, or by the Mouse Ascites  method where these hybridomas are injected into mouse peritoneal cavity and after sometime the mouse cavity is filled with fluid containing these hybridomas (therefore called  ascites method).