Procedure

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 each area).
This provides enhanced immune response from the sustained presentation of the immunogen.


Bleeds

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 minutes.

Schedule
• Week 0 : Bleed 1-2 ml (yields 0.5-1 ml pre-immune serum) Immunize with 250-350mg/rabbit antigen in CFA.
• Week 2 : Immunize with 250-350 mg antigen / rabbit in CFA.
• Week 4 : Immunize with 100-150 mg antigen / rabbit in IFA.
• Week 5 : Immunize with 100-150 mg antigen / rabbit in IFA.
• Week 6 : Test bleed 0.5-1 ml If ELISA data OD value，1：4000 lower than 2.0， 1：6000 lower than 1.0，re-immunize antigen / rabbit in CFA
• Week 7 : 1st Production bleed 3-5 ml. Immunize with 100-150 mg antigen / rabbit in IFA. ELISA test again if previous tests were negative. The continuation of the project needs to be discussed if negative at this stage
• Week 8 : 2nd Production bleed 3-5 ml. Immunize with 100-150 mg antigen /rabbit in 0.9% NaCl-H2O.
• Week 9 : 3rd Production bleed 3-5 ml. Immunize with 100-150 mg antigen / rabbit in 0.9% NaCl-H2O.
• Week 10 : Terminating bleed 40-60 ml/rabbit. A total of 60-80ml/rabbit is collected.

Sera is then shipped to customer or purified either using Protein A/G or Ammonium Sulphate. Peptide affinity purification can be added at this stage.

Note
The rabbits can be maintained on a 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.