The immunogen is diluted to 0.5 ml with sterile saline and combined with 0.5 ml
of the appropriate adjuvant.
The antigen and adjuvant are mixed thoroughly to
form a stable emulsion that is injected beneath the skin of the rabbit
(subcutaneously) in the area around the shoulders and intra-muscularly into the
large muscle of the rear legs. (That is about 1/4 of the antigen being used in
This provides enhanced immune response from the sustained
presentation of the immunogen.
Blood is collected from the central ear artery with a 19-gauge needle and
allowed to clot and retract at 37°C for 1h followed by 12h at 4°C. The clotted
blood is then decanted and clarified by centrifugation at 10,000 RPM for 10
Week 0 : Bleed 3-5 ml (yields 1-2 ml pre-immune serum) Immunize with 500 µg / rabbit
antigen in CFA.
Week 2 : Immunize with 250 µg antigen / rabbit in IFA.
Week 5 : Immunize with 250 µg antigen / rabbit in IFA.
Week 6 : Test bleed 1-2 ml. ELISA and Western blotting (WB) on appropriate cells /
tissue. If the ELISA OD is < 2.0 at a serum dilution of 1:1000, and OD <1.0 at a
serum dilution of 1:3000, the antibody titer is considered low, and we will
immunize again with 250 µg antigen / rabbit in CFA.
Week 7 : 1st Production bleed 10-15 ml if test was positive. ELISA and WB test
again if previous tests were negative.
Week 8 : 2nd Production bleed 10-15 ml. Immunize with 250 µg antigen/rabbit in
IFA. ELISA and WB test again if previous tests were negative. The continuation
of the project needs to be discussed if negative.
Week 9 : 3rd Production bleed 10-15 ml.
Week 10 : 4th Production bleed 10-15 ml. Immunize with 200 µg antigen / rabbit in
Week 11 : Terminating bleed 60 -100 ml / rabbit. Protein G purification of bleeds. Average
yield of purified Ig fraction is 80 -100 mg.
Peptide affinity purifications (diluted serum through non-phospho-peptide first
and flow-through phospho-peptide column). The yield can differ
significantly between antibodies.
Final ELISA, Dot Blot and WB (if applicable) QC test.
Note:Protocols are subject to change.
The rabbits can be maintained on a ten-week cycle of immunisations and bleeds as
required by the project. The results of testing will determine whether the
programme should be continued, modified, or terminated. Extra bleeds can be
taken upon customer’s requirement of extended project with extra charge.