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SERVICE |
DESCRIPTION |
| 1
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Subject |
Japanese White Rabbits
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| 2
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Adjuvant |
Complete Freund's Adjuvant (CFA) initially, followed by Incomplete Freund's
Adjuvant (IFA) for all subsequent injections
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| 3
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Immunogen |
KLH conjugated phospho-peptide. 50-100 µg immunogen is used for each
immunisation.
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| 4
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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.
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| 5
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Bleeds |
Blood is collected from the central ear artery with a 19-gauge needle and
allowed to clot and retract at 37°C overnight. The clotted blood is then
refrigerated for 24 hours before the serum is decanted and clarified by
centrifugation at 2,500 RPM for 20 minutes.
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| 6
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Schedule |
| Week 0 |
Bleed 10 ml (yields 5-8 ml pre–immune serum) Immunize with 200
µg/rabbit antigen in CFA. |
| Week 2 |
Immunize with 100 µg/rabbit antigen in CFA. |
| Week 4 |
Immunize with 100 µg/rabbit antigen in IFA. |
| Week 5 |
Test bleed 20-30 ml.
ELISA and Western blotting (WB) on appropriate cell / tissue. Immunize with 100
µg/rabbit antigen in PBS.
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| Week 6 |
1st Production bleed 20-30 ml if test positive. Immunize with 100
µg/rabbit antigen in PBS. ELISA and WB test again if previous tests were
negative.
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| Week 7 |
2nd Production bleed 20-30 ml. Immunize with 100 µg/rabbit antigen in PBS.
ELISA and WB test again if previous tests were negative. The continuation of
the project needs to be discussed if negative.
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| Week 8 |
3rd Production bleed 20-30 ml. Immunize with 100 µg/rabbit antigen in PBS.
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| Week 9 |
4th Production bleed 20-30 ml. Immunize with 100 µg/rabbit antigen in PBS.
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| Week 10 |
Terminating bleed 100-120 ml. Protein G purification of pooled bleeds. Average
yield of purified Ig fraction is 100-150 mg.
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| Week 11 |
Peptide affinity purifications (phospho-peptide first and then non-
phospho-peptide column). The yield is significantly different between
antibodies.
Final ELISA and WB QC test.
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| 5
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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
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