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RESEARCH DIVISION Laboratory Manual

 


 

Polyclonal Antibodies

Immunization of Mice:

Injections should be made at intervals of at least two weeks.. We have been using either of the two following adjuvants with success: Freund adjuvant and MPL+TDM adjuvant ( RIBI ImmunoChem Research, Inc. ), the latter being less hazardous than the first.

  1. 1st injection: 15 - 50 ug Antigen in 200 ul adjuvant, injected subcutaneously.
  2. 2nd injection: 2 weeks later; boost with 15 - 50 ug of Antigen in adjuvant. (If time permits, boost again a month later).
  3. 7 - 10 days later bleed the mouse and test serum using the assay which will be used for screening. If titre is not high enough, boost again two weeks after previous boost.

Serum Preparation:

  1. After collecting, blood should be allowed to clot for 60 min . at 37C or O.N at 4C.
  2. Separate the clot from the sides of the tube (ringing) using a pasteur pipette. Place clot at 4C O.N.
  3. Spin at 10000 x g for 10 min. at 4C to separate the serum.
  4. Serum can be stored at -20C after adding Glycerol to 50%.

If monoclonal antibodies are desired, once you have a "good" polyclonal and a reliable screening method proceed to the next step.

Rabbits:

Rabbits are immunized essentially as described above. Injections:

  1. First in Freund's complete 100-200ug/1ml injection, two sites (over each shoulder).
  2. Second 1 month later in Freund's INCOMPLETE as above.
  3. Bleed 2 weeks later. Boost 2 weeks later again (2 + 2 week cycle).
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998