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PHOSPHO SPECIFIC POLYCLONAL ANTIBODY SERVICE

SUBJECT - Japanese White Rabbits:

  • Adjuvant - Complete Freund’s Adjuvant (CFA) initially, followed by Incomplete Freund’s Adjuvant (IFA) for all subsequent injections.
  • Immunogen - KLH conjugated phospho-peptide. 50-100 µg immunogen is used for each immunisation.
  • THE SERVICE PROVIDED BY AMSBIO

    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 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 PBS.
  • 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:

    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.

    Protocols are subject to change.
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