Adaped from David Bowtell (bowtell@ariel.ucs.unimelb.edu.au)
Polyclonal Antibody
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 37 C or O.N at 4 C.
2) Separate the clot from the sides of the tube (ringing) using a pasteur pipette. Place clot at 4 C O.N.
3) Spin at 10,000 x g for 10 min. at 4 C to separate the serum.
4) Serum can be stored at -20 C 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.
Immunization of 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).
Monoclonal Antibody
If titre is positive do one of the following 2 weeks later:
I - Boost tail vein with 20 ug- 50 ug of Antigen in PBS and proceed with fusion on day 4.
OR
II - Boost subcutaneously with 50 ug - 100 ug of Antigen in PBS and proceed with fusion 4 days later.
OR
III - Boost 3 days in a row with 15 ug Ag in PBS and proceed with fusion on 4th day.
