Hybridoma Construction

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Hybridoma Construction

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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