After an investigator specifies their needs and provides an immunogen, the Lymphocyte Culture Center will then perform all steps to produce a hybridoma.  Contact staff to determine strategy and costs.

• Investigator prepares and characterizes immunogen
• Center staff immunize animals with approved protocols
• Center staff collect sera according to approved protocols
• Staff develop ELISA for screening cell culture supernatants
• Construct hybridoma by fusion

PROTOCOL:

• Immunize mouse
• Isolate spleen cells
• wash once in Iscove's MDM and mix with washed SP2/0-Ag14 myeloma cells [Shulman, et al. (1978), Nature 276:269] at a 5:1 ratio (spleenocyte to myeloma)
• pellet cells and aspirate media
• fuse cells by the stepwise addition of 50% polyethylene glycol (PEG 4000, Gibco) over one minute
• Dilute PEG dropwise with Iscove's MDM
• Pellet cells and gently wash once in Iscove's MDM containing 15% fetal bovine serum, hypoxanthine (H) and thymidine (T)
• Resuspend cells in HT medium, transfer to a petri dish and incubate at 37°C in an incubator at 5% CO₂/95% air for one hour
• Resuspend cells in HT medium and plate into 96-well tissue culture plates at a density of approximately 2-4 x 10⁵ cells/well
• Feed cultures after 24 hours with HT medium containing aminopterin (HAT medium) and maintain in this media for two weeks with periodic feeding
• Macroscopic colonies usually appear within 7-10 days following the fusion and supernatants are screened for specific antibody
• Antibody positive hybridomas are cloned twice by limiting dilution
• Store cells from each stage of hybridoma selection (parental, primary, secondary) in liquid nitrogen cell banks