Cloning, Subcloning, Isotyping and Cryopreservation
Hybridoma cloning and subcloning
Each positive hybrid that is producing the specific antibody of interest will be cloned and subcloned to maintain the stability and monoclonal character of the hybridoma. Cloning and subcloning are performed using the limiting dilution technique; hybrids are cloned at one cell per well and subcloned at 0.3 cell per well in one 96-well microliter plate each.
Cloning plates are weaned off HAT-IMDM selection media and replaced by HT-IMDM selection media, and subcloning plates are then weaned to base IMDM culture media only.
As subclones test positive for relevant antibodies, one subclone is selected, saved and now called a monoclonal antibody. Each monoclonal antibody supernate is isotyped for immunoglobulin subtype, which is needed to determine the type of purification technique to perform and how it will be used in assays.
Monoclonal antibody cell lines are then cryopreserved in liquid nitrogen for future use. A minimum of six vials, at 5 million to 6 million cells per vial, of each cell line are frozen in 10 percent dimethyl sulfoxide-supplemented culture medium. Cell lines may be stored indefinitely in liquid nitrogen, with periodic thawing to regrow them and freeze fresh vials.