Hybridoma cell lines secreting monoclonal antibodies against foot-and-mouth disease virus (FMDV). II. Cloning conditions.
Three methods of cloning hybridoma cells--picking colonies from the masterplate, limit dilution cloning, and cloning in semi-solid medium over macrophage (m phi) feeder layers--were compared. Cloning in semi-solid medium was found to be the most efficient and reliable, especially with our relatively slow growing anti-foot-and-mouth disease virus (FMDV) antibody secreting hybridoma cells. The optimum culture dish for this cloning was the 6-well (6W) dish (well diameter 1.5 cm), while the optimum cloning procedure was 10 to 10(2) hybridoma cells in 1% (w/v) methylcellulose in Dulbecco's minimal essential medium (DMEM) supplemented with 50% (v/v) conditioned medium, 10% (v/v) foetal or donor calf serum and 2 mM glutamine, over a 24-48 h old culture of syngeneic m phi in each well of the 6W plate. This could, however, produce problems in that colony formation was sometimes 'loose', and confident picking of individual colonies was impaired. Such a problem could be avoided by using a solid agar interface of 1% (w/v) agar Noble in DMEM or RPMI 1640 medium between the feeder cells and the hybridoma cells in semi-solid medium.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000914:Antibodies, Viral / Q000276:immunology*; D005537:Aphthovirus / Q000276:immunology*; D002460:Cell Line; D002999:Clone Cells / Q000378:metabolism*; D003470:Culture Media; D046508:Culture Techniques / Q000379:methods*; D006825:Hybridomas / Q000378:metabolism*; D051379:Mice
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D003470:Culture Media