You Recieve:
Peptide and phospho peptide 2-3mg each.
0.5-1ml of pre-immune sera and antisera per rabit.
Project report with ELISA results and dot blot.
Non-phospho antibody 3-5ml.
1-5mg of affinity purified phospho-specific antibody.
Service Includes:
Immunization of 2 rabits.
2x (0.5-1ml) pre-immunization samples.
2x (0.5-1ml) post-immunization samples.
Synthesis of peptides and KLH conjugation.
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.
Protocols are subject to change.